U.S. patent application number 10/584831 was filed with the patent office on 2008-05-22 for polypeptides for modulating binding of trp channel proteins and trp- associated proteins.
This patent application is currently assigned to NoNO Inc.. Invention is credited to Michael P. Belmares, Jonathan David Garman, Michael Tymianski.
Application Number | 20080119412 10/584831 |
Document ID | / |
Family ID | 34710254 |
Filed Date | 2008-05-22 |
United States Patent
Application |
20080119412 |
Kind Code |
A1 |
Tymianski; Michael ; et
al. |
May 22, 2008 |
Polypeptides for Modulating Binding of Trp Channel Proteins and
Trp- Associated Proteins
Abstract
This invention relates to inhibitors and modulators of the
binding between a transcient receptor potential (TRP) channel
protein and a TRP-associated binding protein. In particular
inhibitors and modulators of the binding between the TRPM7 channel
protein and PDZ-domain containing TRP-associated proteins. These
modulators and inhibitors are used to control Ca2+ transport
through TRPM7 channel proteins and reduce damage to mammalian
cells, including brain and spinal cord cells, after ischemia or
traumatic injury.
Inventors: |
Tymianski; Michael;
(Ontario, CA) ; Garman; Jonathan David; (San Jose,
CA) ; Belmares; Michael P.; (San Jose, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
NoNO Inc.
399 Bathrust Street, Toronto, Ontario
CA
Arbor Vita Corporation
Sunnyvale
CA
|
Family ID: |
34710254 |
Appl. No.: |
10/584831 |
Filed: |
December 22, 2004 |
PCT Filed: |
December 22, 2004 |
PCT NO: |
PCT/CA04/02193 |
371 Date: |
May 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60532169 |
Dec 23, 2003 |
|
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|
Current U.S.
Class: |
514/1.2 ;
514/17.4; 514/17.7; 514/8.3; 530/326; 530/327; 530/328 |
Current CPC
Class: |
C07K 2319/10 20130101;
C12N 2799/022 20130101; C07K 14/705 20130101; G01N 2500/00
20130101; C07K 14/47 20130101; A61K 38/00 20130101; C12N 15/1138
20130101 |
Class at
Publication: |
514/14 ; 514/15;
514/16; 530/326; 530/327; 530/328 |
International
Class: |
A61K 38/10 20060101
A61K038/10; A61K 38/08 20060101 A61K038/08; C07K 7/06 20060101
C07K007/06 |
Claims
1. An inhibitor comprising an isolated, recombinant or synthetic
polypeptide that inhibits binding between a TRP channel protein and
a TRP-associated protein.
2. The inhibitor of claim 1, wherein the TRP-associated protein
comprises at least one PDZ domain.
3. The inhibitor of claim 2, wherein the TRP-associated protein
comprising at least one PDZ domain is selected from the group
consisting of RIM-2, Mint 1, INADL, Syntrophin 1 alpha, SITAC-18,
LIM mystique, ZO-1, PAR3L, MAST2, PAR3, and novel serine
protease.
4. The inhibitor of claim 1, wherein the TRP channel protein is
human TRPM7 or mouse TRPM7.
5. The inhibitor of claim 2, wherein the C-terminus of the
polypeptide comprises a PDZ-Ligand sequence.
6. The inhibitor of claim 5, wherein the PDZ-Ligand sequence
comprises the amino acid sequence X-L/I/V-X-L/V/A.
7. The inhibitor of claim 6, wherein the PDZ-Ligand sequence is
STNSVRLML [SEQ ID NO:260] or ATNSVRLML [SEQ ID NO:384].
8. The inhibitor of claim 5, wherein the C-terminus of the
polypeptide further comprises a cell membrane transduction
domain.
9. The inhibitor of claim 8, wherein the cell membrane transduction
domain is selected from the group consisting of HIV tat, Drosophila
antennapedia, herpes simplex virus VP22, anti-DNA CDR2, anti-DNA
CDR3, polyarginine and penetratin.
10. The inhibitor of claim 9, wherein the cell-membrane
transduction domain is HIV tat YGRKKRRQRRR [SEQ ID NO:257].
11. The inhibitor of claim 10, wherein the inhibitor comprises the
amino acid sequence YGRKKRRQRRRSTNSVRLML [SEQ ID NO:258] or
YGRKKRRQRRRATNSVRLML [SEQ ID NO:380].
12. A pharmaceutical composition comprising the inhibitor of claim
1 and a physiologically acceptable carrier, diluent or
excipient.
13. An inhibitor comprising a nucleic acid sequence capable of
inhibiting the expression of a TRP channel protein post
transcriptionally.
14. The inhibitor of claim 13, wherein the TRP channel protein is
TRPM7.
15. The inhibitor of claim 14, wherein the nucleic acid sequence is
selected from the group consisting of nucleotides 5152-5172 of
Genbank accession #AY032951, nucleotides 5023-5043 of Genbank
accession #AY032951 and nucleotides 1318-1338 of Genbank accession
#AY032951.
16. The inhibitor of 15, wherein the nucleic acid sequence is
coupled to a delivery system selected from the group consisting of
an adenovirus vector and an adeno-associated virus vector.
17. The inhibitor of claim 16, wherein the nucleic acid sequence is
coupled to an adenovirus vector and comprises the nucleic acid
sequence GAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAA
CGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAC
CACTCGGATCCGAGTGCATGACTGGTGAATTTCAAGAGAATTCACC
AGTCATGCACTCTTTTTGGAAAAGCTT [SEQ ID NO:381].
18. A pharmaceutical composition comprising an inhibitor of claim
13 and a physiologically acceptable carrier, diluent or
excipient.
19. A method of treating mammalian cell injury, comprising
introducing a modulator of binding between a TRP channel protein
and a TRP channel associated protein into a cell.
20. The method of claim 19, wherein the cell is a damaged
neuron.
21. The method of claim 19, wherein the modulator is a
polypeptide.
22. The method of claim 19, wherein the modulator is a fusion
polypeptide.
23. The method of claim 19, wherein the modulator is a small
interfering RNA.
24. The method of claim 19, wherein the TRP channel protein is
TRPM7.
25. The method of claim 24, wherein the modulator has a C-terminal
amino acid sequence of LML.
26. A method of reducing the damaging effect of ischemia or
traumatic injury to the brain or spinal cord in a mammal, said
method comprising treating said mammal with a non-toxic,
damage-reducing, effective amount of a modulator of binding between
a TRP channel protein and a TRP channel associated protein.
27. The method of claim 26, wherein the TRP-associated protein
comprises at least one PDZ domain.
28. The method of claim 27, wherein the TRP-associated protein
comprising at least one PDZ domain is selected from the group
consisting of RIM-2, Mint 1, INADL, Syntrophin 1 alpha, SITAC-18,
LIM mystique, ZO-1, PAR3L, MAST2, PAR3, and novel serine
protease.
29. The method of claim 26, wherein the TRP channel protein is
TRPM7.
30. The method of claim 26, wherein the modulator is a
polypeptide.
31. The method of claim 30, wherein the C-terminus of the
polypeptide comprises a PDZ-Ligand sequence.
32. The method of claim 31, wherein the PDZ-Ligand sequence
comprises the amino acid sequence X-L/I/V-X-L/V/A.
33. The method of claim 32, wherein the PDZ-Ligand sequence is
STNSVRLML [SEQ ID NO:260] or ATNSVRLML [SEQ ID NO:384].
34. The method of claim 31, wherein the C-terminus of the
polypeptide further comprises a cell membrane transduction
domain.
35. The method of claim 34, wherein the cell membrane transduction
domain is selected from the group consisting of HIV tat, Drosophila
antennapedia, herpes simplex virus VP22, anti-DNA CDR2, anti-DNA
CDR3, polyarginine and penetratin.
36. The method of claim 35, wherein the cell-membrane transduction
domain is HIV tat YGRKKRRQRRR [SEQ ID NO:257].
37. The method of claim 36, wherein the inhibitor comprises the
amino acid sequence YGRKKRRQRRRSTNSVRLML [SEQ ID NO:258] or
YGRKKRRQRRRATNSVRLML [SEQ ID NO:380].
38. The method of claim 26, wherein the modulator is a small
interfering RNA.
39. A method of controlling the concentration of
Ca.sup.2+-dependent signaling molecules in the vicinity of ion
channel pores of cells in vivo to prevent the diffusion of toxic
amounts of said Ca.sup.2+ influx to prevent the triggering of
neurotoxic phenomena, said method comprising administering an
effective, non-toxic amount of a modulator of TRP channel proteins
or cellular protein interaction domains that effect said TRP
channel protein interactions.
40. A method for determining whether a test compound modulates
binding between a TRP channel protein and a PDZ domain-containing
polypeptide, comprising: i. contacting a TRP channel PDZ-Ligand
sequence with a PDZ domain-containing polypeptide; and ii.
measuring the amount of complex formed between the TRP channel
PDZ-Ligand sequence and the PDZ domain-containing polypeptide.
41. The method of claim 40, wherein the PDZ domain is a PDZ domain
selected from the group consisting of RIM-2, Mint 1, INADL,
Syntrophin 1 alpha, SITAC-18, LIM mystique, ZO-1, PAR3L, MAST2,
PAR3, and novel serine protease.
Description
FIELD OF THE INVENTION
[0001] This invention relates to methods of reducing the damaging
effect of an injury to mammalian cells by treatment with compounds
which reduce cell death or dysfunction, including cellular damage
following episodes of tissue ischemia, trauma, epilepsy, and acute
or chronic degeneration. The invention discloses methods of
treating these disorders by administering inhibitors that disrupt
protein-protein interactions involved in these disorders, screening
methods to identify such inhibitors and specific compositions
useful for treating these disorders.
BACKGROUND
[0002] Every year, stroke and neurotrauma afflict approximately
660,000 and 350,000 North American individuals, respectively, and
about 175,000 stroke and 52,000 trauma victims will die
(Stroke--American Stroke Association Web Site, 2000; Centers for
Disease Control and Prevention, Traumatic injury in the United
States: An interim report to Congress, Centers for Disease Control
and Prevention, 2001). 4.5 million people live with Alzheimer's
disease, with the prevalence expected to triple by 2050 (Hebert et
al. (2003) Arch. Neurol. 60:1119-1122). Epilepsy is the third most
common neurological disorder after stroke and Alzheimer's disease.
It affects 2.3 million Americans of all ages. Approximately 181,000
new cases of seizures and epilepsy occur each year. One in every 10
Americans will experience a seizure at some point in their lives.
Three percent will eventually develop epilepsy (Epilepsy Foundation
of America Web Site,
www.epilepsyfoundation.org/answerphone/statistics.cfm). Thus
stroke, CNS trauma, neurodegenerative illnesses and epilepsy are
each disorders of major public health significance. Moreover,
cardiovascular disorders, pulmonary diseases, and endocrine
illnesses such as diabetes count as among the most common causes of
non-neurological morbidity and mortality worldwide. Common to all
of these common illnesses is damage to cells in target tissues in
the nervous, cardiovascular or endocrine system.
[0003] The public health consequences of these disorders are
significant. For example, in 1998, $3.4 billion was paid in 1999 to
just those Medicare beneficiaries that were discharged from
short-stay hospitals, not including the long term care for
>1,000,000 people that reportedly have functional limitations or
difficulty with activities of daily living resulting from stroke
(Heart and Stroke Statistics--2004 Update, American Heart
Association, 2004). At this time, no therapeutics are available to
reduce brain damage resulting from stroke, and this major disorder
can be used as an example for the basis of the current invention,
though the field of the invention obviously applies to other
disorders involving mammalian cell injury.
[0004] Stroke is characterized by neuronal cell death in areas of
ischemia, brain hemorrhage or trauma. Many lines of evidence have
demonstrated that this cell death is triggered by glutamate
over-excitation of neurons, leading to increased intracellular
Ca.sup.2+ and increased nitric oxide due to an increase in nNOS
(neuronal nitric oxide synthase) activity. Excitotoxicity is the
process by which L-glutamate, the major excitatory neurotransmitter
in the mammalian CNS, damages neurons (Olney (1969) Science
164:719-721; Olney and Sharpe (1969) Science 166:386-388). It is
established as a predominant neurotoxic mechanism in acute
neurological disorders such as stroke, epilepsy and traumatic
nervous system injuries (reviewed in Rothman and Olney (1987) TINS
10:299-302; Choi et al. (1988) Neuron 8:623-634; Coyle and
Puttfarcken (1993) Science 262:689-695; Lipton and Rosenberg (1994)
N. Engl. J. Med. 330:613-622; Hardingham and Bading (2003) Trends.
Neurosci. 26:81-89). In brain ischemia, excitotoxic activation of
postsynaptic glutamate receptors triggers downstream pathways
implicated in subsequent neuronal death (reviewed in Lipton (1999)
Physiol. Rev. 79:1431-1568). Of these, Ca.sup.2+ influx through
N-methyl-D-aspartate (NMDA) glutamate receptors was the process
consistently revealed as a key event. In these studies, blocking
NMDA receptors permitted neurons destined to die from anoxia to
survive (Rothman (1983) Science 220:536-537; Goldberg et al. (1987)
J. Pharmacol. Exp. Ther. 243:784-791), and animal research
suggested that ischemic brain damage could be treated by this
approach (Simon et al., (1984) Science 226:850-852). However,
blocking NMDA receptors may be detrimental to animals and humans
(Fix et al. (1993) Exp. Neurol. 123:204-215; Davis et al. (2000)
Stroke 31:347-354; Ikonomidou et al. (2000) Proc. Natl. Acad. Sci.
U.S.A. 97:12885-12890). Moreover, though blocking excitotoxicity
was effective in laboratory models of disease, clinical trials of
anti-excitotoxic therapies (AET) have generally failed to benefit
patients (Davis et al., (1997) Lancet 349:32, Davis et al. (2000)
Stroke 31:347:354; Morris et al. (1999) J. Neurosurg 91:737-743;
Lees et al. (2000) Lancet 355:1949-1954). The reason for this, in
the face of a clear role for excitotoxicity in acute neurological
disorders, has remained a mystery (Birmingham (2002) Nat. Med. 8:5;
Ikonomidou and Turski (2002) Lancet Neurol. 1:383-386).
[0005] The present invention relates to our discovery that
processes other than excitotoxicity are responsible for neuronal
damage in conditions such as stroke. In neurons exposed to oxygen
glucose deprivation (OGD), AET unmasks a lethal cation current
I.sub.OGD mediated by TRPM7, a member of the transient receptor
potential (TRP) cation channel superfamily (Nadler et al. (2001)
Nature 411:590-595). In OGD, I.sub.OGD is activated by reactive
oxygen/nitrogen species (ROS), permitting Ca.sup.2+ uptake that
further stimulates ROS and I.sub.OGD activation. Blocking I.sub.OGD
or suppressing TRPM7 expression prevents anoxic neuronal death even
in the absence of AET, indicating that TRPM7 is an essential
mediator of anoxic death. This work defines a new paradigm for
understanding anoxic neuronal damage in which excitotoxicity is a
subset of a larger framework of mammalian cell injury that involves
TRP cation channels.
SUMMARY
[0006] The present invention relates to the treatment of
cytodestructive disorders such as tissue ischemia or brain damage
resulting from stroke, epilepsy, neurodegenerative conditions or
traumatic brain and spinal cord injuries by modulating specific
protein:protein interactions including, but not limited to, PDZ
domain:PDZ Ligand (PL) interactions that involve, directly or
indirectly, members of the TRP cation channel family, and are
involved in mediating these clinical disorders. Methods for
identifying specific therapeutics that modulate the specific
protein:protein interactions involved in these disorders are also
provided. Compounds and compositions for treating these neuronal
disorders are also disclosed.
[0007] Methods of identifying the cellular PDZ proteins that are
bound by TRP channels and associated proteins are provided herein
after speaking with Dr. M. Tymianski (MT) it was determined that
although we would remove specific reference to PLC, we would keep
general statements regarding TRP channels and associated proteins.
Methods are also provided to identify inhibitors that are high
affinity for TRP-specific interactions as well as TRP-associated
protein interactions. Other methods are provided to determine
selectivity of inhibition, both against the different TRP channels,
TRP-associated proteins and the PDZs that can bind them. Methods
for delivering peptide inhibitors to cells such as neuron cells are
also disclosed.
[0008] One class of pharmaceutical compositions that are provided
include a pharmaceutical composition comprising an isolated,
recombinant or synthetic polypeptide inhibitor that inhibits
binding between a TRP channel and a PDZ protein with a
physiologically acceptable carrier, diluent or excipient, wherein
the polypeptide comprises a C-terminal amino acid sequence of
X-L/V/I-X-V/L/A. In certain embodiments, the C-terminal amino acid
sequence of the polypeptide is XLML (SEQ ID NO:1). These
compositions can be used to inhibit binding between a TRP channel
and various PDZ proteins, including, for example, but not limited
to, NNOS, LIM, KIAA1095, HEMBA1003117, AIPC, KIAA1526, DVL1, DVL2,
DVL3, PTPL1, ZO-1, ZO-2, ZO-3, KIAA1719, Mupp1, INADL, Shank 3,
NENT1, MINT2, MAGI1, MAGI2, MAGI3, NeDLG, syntenin, PSD-95, hDLG,
PAR3, MAST1, MAST2, AF6, SIP1, LIM mystique, HTRA2, TIP-1,
KIAA0316, PICK1, RIM-2, INADL, Syntrophin 1 alpha, SITAC-18, PAR3L,
MAST2, and NSP [novel serine protease].
[0009] The polypeptides in these compositions can be of varying
lengths. In a certain embodiment such polypeptides are 3-20 amino
acids in length. In other embodiments, the polypeptides are fusion
polypeptides, which include the C-terminal amino acid sequence of
the PL polypeptide and a segment of a transmembrane transporter
sequence that is effective to facilitate transport of the
polypeptide into the desired cell type, for example a neuronal cell
(also known as a cell-membrane transduction domain).
[0010] Another class of pharmaceutical compositions may also
include an isolated, recombinant or synthetic polypeptide and a
physiologically acceptable carrier, diluent or excipient, wherein
the polypeptide is 3-20 amino acids in length and inhibits binding
between a TRP channel and a PDZ protein. The polypeptides in some
of these compositions are 3-8 amino acids in length. Exemplary
sequences of such polypeptides include LML
[0011] Still other pharmaceutical compositions include a fusion
polypeptide that inhibits binding between a TRP channel and a PDZ
protein and a physiologically acceptable carrier, diluent or
excipient. In certain embodiments, the fusion polypeptide inhibitor
in these compositions is a fusion of (i) a 9 amino acid segment
that has a C-terminal sequence and (ii) an amino acid segment of a
transmembrane transporter that is effective to transport the
polypeptide into a neuron or other affected cell. In other
embodiments the fusion polypeptide inhibitor comprises (i) a 3-8
amino acid segment that has a C-terminal sequence and (ii) an amino
acid segment of a transmembrane transporter that is effective to
transport the polypeptide into a neuron or other affected cell. In
yet other embodiments the fusion polypeptide inhibitor comprises
(i) a 9-20 amino acid segment that has a C-terminal sequence and
(ii) an amino acid segment of a transmembrane transporter that is
effective to transport the polypeptide into a neuron or other
affected cell.
[0012] The polypeptide inhibitors in the foregoing pharmaceutical
compositions can be used in a variety of therapies, including
treatment of a number of neurological disorders. Examples of such
disorders include, but are not limited to, stroke, ischemia,
myocardial ischemia, glaucoma, Parkinson's disease, Huntington's
disease, Alzheimer's disease, epilepsy and inherited ataxias. The
inhibitors can also be used in the preparation of medicaments for
use in the treatment of neurological disorders.
[0013] Still other pharmaceutical compositions include small
inhibitory RNA ("siRNA") sequences that can specifically reduce the
activity of a TRP cation channel family member and protect against
cell death from OGD. Specific inhibitory sequences and methods of
identifying siRNA sequences are described within.
[0014] Also provided are methods for determining whether a test
compound modulates binding between a PDZ protein and a TRP channel.
Certain of these methods involve contacting a PDZ-domain containing
polypeptide and a PDZ-Ligand ("PL") containing peptide having at
least the C-terminal 3 amino acids of the TRP channel in the
presence of the test compound. In certain embodiments, the PDZ
proteins in these screening methods may be selected from the group
consisting of DLG1, DLG2, KIAA0973, NeDLG, Outer membrane protein,
Syntrophin alpha 1, TIP1, TIP2, INADL, KIAA0807, KIAA1634,
Lim-Mystique, LIM-RIL, MAGI1, MAGI2, NH-ERF1, NH-ERF2, Syntrophin
beta-1 and Syntrophin gamma-1, RIM-2, Mint 1, Syntrophin 1 alpha,
SITAC-18, ZO-1, PAR3L, MAST2, PAR3, and NSP [novel serine protease]
The amount (Susan do you think that amount a broader term than
concentration? OK) of complex formed between the PDZ-domain
polypeptide and the PL peptide is then determined. The test
compound is identified as a potential inhibitor of binding between
the PDZ protein and the TRP channel if a lower amount of the
complex is detected in the presence of the test compound relative
to the concentration of the complex in the absence of the test
compound. Another assay can be conducted using compounds identified
in the initial screen to determine whether the identified compound
mitigates against a condition associated with a neuronal or
ischemic disorder. Examples of such assays include necrosis assays,
apoptosis assays, caspase assays, cytochrome c assays and cell
lysis assays.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a phylogenetic tree of TRP channels
[0016] FIG. 2. shows the domain structures of different TRP channel
sub-families
[0017] FIG. 3 demonstrates that siRNA knockdown of TRPM7 reduces
Reactive Oxygen Species (ROS) production, calcium uptake and cell
death under prolonged oxygen-glucose deprivation (OGD).
[0018] FIG. 4 shows the loss of neuroprotection and blockage of
Ca.sup.2+ accumulation by glutamate and Ca.sup.2+ channel
inhibitors with extended OGD.
[0019] FIG. 5 shows the delay of calcium uptake in the presence of
glutamate and Ca.sup.2+ channel inhibitors and the effect of
extracellular calcium concentration on OGD survival.
[0020] FIG. 6 shows the effect of Gd.sup.3+ on calcium uptake in
the presence of glutamate and Ca.sup.2+ channel inhibitors
[0021] FIG. 7 demonstrates that Gd.sup.3+ can permit survival of
neurons destined to die from prolonged OGD, and that superoxide and
nNOS inhibition are synergistic while inhibitors of ROS pathways
outside of superoxide and nitric oxide show no benefit in OGD
survival.
[0022] FIG. 8 shows the results of rtPCR experiments demonstrating
the effect of siRNA treatment of TRPM7 on other TRPM channels using
different constructs.
[0023] FIGS. 9A, 9B, 9C, AND 9D show the results of titrations of
the Tat-TRPM7 peptide with RIM-2 d2, INADL d3, ZO-1 d2, and Par3
d3.
[0024] FIGS. 10A AND 10B show the results of titrations of Peptide
#1829 with ZO-1 d2 and Peptide #1839 with INADL d3.
[0025] FIG. 11 shows the adenoviral construct. The TRPM7
siRNA-pAdTrack.sup.(1) (containing the hairpin under the H1
promoter and GFP under a CMV promoter as well as adeno
recombination sequences was cotransfected with pAdEasy (containing
viral sequences) into HEK cells and the cells selected with
kanamycin. Recombined virus containing the TRPM7 siRNA hairpin and
GFP sequences was then be produced from these cells. (Reference
(1): T. C. He, S. Zhou, L. T. da Costa, J. Yu, K. W. Kinzler, and
B. Vogelstein. A simplified system for generating recombinant
adenoviruses. Proc. Natl. Acad. Sci. U.S.A 95 (5):2509-2514,
1998).
[0026] FIG. 12 shows the effects of infecting primary cultured
neurons on TRPM7 mRNA by RT-PCR. Cultured cortical neurons plated
in 12 well plates (106 cells/well) were infected at the time of
plating with the corresponding adenoviral constructs and the RNA
was harvested 5 days later. RT-PCR was performed on equivalent
amounts of RNA using primers for TRPM7 or .beta.-actin.
[0027] FIG. 13 shows the effect of oxygen glucose deprivation (OGD)
for the indicated duration on neuronal cell death in the presence
(MCN(+)) or absence (MCN(-)) of the combination of MK-801 (10
.mu.M), CNQX (10 .mu.M) and nimodipine (2 .mu.M), denoted as MCN,
antagonists of NMDA and AMPA/kainate glutamate receptors and L-type
Ca.sup.2+ channels, respectively. The fraction of dead cells was
determining by dividing the number of neurons expressing GFP which
became stained with propidium iodide 20 h after OGD by the number
of neurons expressing GFP at the beginning of the experiment. Data
for each culture was obtained from counting cells in 3 high power
microscope fields per experiment.
[0028] FIG. 14 shows the effect of treating the cultures with
Tat-9cTRPM7. The sequence of Tat-9cTRPM7 is:
[Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Ser-Thr-Asn-Ser-Val-Arg-Leu--
Met-Leu] or [YGRKKRRQRRR-STNSVRLML], whereby the first 11 residues
correspond to the cell membrane transduction domain of the human
immunodeficiency virus type 1 (HIV-1) Tat protein and the last 9
residues correspond to the last 9 amino acids of the C-terminus of
human TRPM7 (accession Q96QT4). We predict similar results with a
Tat-conjugated peptide encoding the last 9 residues of the mouse
TRPM7 C-terminus (YGRKKRRQRRR-ATNSVRLML; accession Q923J1). (A)
Neuronal survival at 20 h in the indicated concentrations of
Tat-9cTRPM7 in the absence of excitotoxic challenge. (B) Neuronal
survival 20 h after challenging the cultures for 1 h with the
indicated concentration of NMDA. Tat-9cTRPM7 was applied
immediately after the NMDA challenge.
BRIEF DESCRIPTION OF THE TABLES
[0029] TABLE 1 shows classifications of genetically encoded and
non-encoded amino acids. Column 1 shows the classification, column
2 shows genetically encoded amino acids, and column 3 genetically
non-encoded amino acids.
[0030] TABLE 2 shows glutamate receptors with PL sequences. Column
1 shows the names of the receptor, column 2 shows the GenBank
number, column 3 shows the C-terminal 20mer sequence, column 4
shows the C-terminal 4mer sequence, column 5 shows which sequences
are PLs, and column 6 shows internal PL ID.
[0031] TABLE 3 shows the number of TRP genes in C. elegans,
Drosophila melangaster, mice, and humans. Column 1 shows the
subfamily, column 2 shows the number in C. elegans, column 3 shows
the number in Drosophila melanogaster, column 4 shows the number in
mice, and column 5 shows the number in humans.
[0032] TABLE 4 shows the nomenclature of the mammalian TRP
superfamily. Column 1 shows the name of the protein, column 2 shows
the group number, column 3 shows former names of the protein, and
column 4 shows the GenBank accession numbers.
[0033] TABLE 5 shows the sequences of PDZ domains cloned to produce
GST-PDZ fusion proteins. Column 1 shows the gene name, column 2
shows the GenBank G1 or Accession number, column 3 shows the domain
number, and column 4 shows the sequence fused to the GST
construct.
[0034] TABLE 6 shows PDZs predicted to interact with TRPM7
[0035] TABLE 7 shows PDZs demonstrated to interact with specific PL
proteins.
[0036] TABLE 8 shows PDZ domains which bind the Tat-TRPM7 peptide
in G0 or G3 assays. Column 1 shows the name of the PDZ protein,
column 2 shows the domain number, column 3 shows the GenBank number
for the gene encoding the PDZ protein, column 4 shows whether the
literature indicates expression in the nervous system or brain, and
column 5 shows the literature reference for column 5.
[0037] TABLE 9 shows the results of titrations of the Tat-TRPM7
peptide with RIM2 (177.4), Mint 1 (d1,d2) (36.5a), TIP1 d1 (54.10),
Mint1 d1 (146.5), Mint1 d2 (147.2), INADL d3 (96.3), MUPP1 d3
(108.3), Syntrophin 1 alpha d1 (52.5), SITAC-18 d1 (122.2),
SITAC-18 d2 (123.2), LIM Mystique d1 (232.1), ZO-1 d2 (241.3),
PAR3L d3 (406.1), MAST2 d1 (174.6), PAR3 d3 (278.1), and KIAA1284
d1 (191.2), Column 1 shows the PDZ protein and domain number,
column 2 shows the EC50 in .mu.M, column 3 shows the error of fit
for the EC.sub.in in .mu.M, and column 4 shows the OD.sub.max 450
nm). ND=not determined.
[0038] TABLE 10 shows the results of titration of peptides with
ZO-1 d2 and INADL d3. Column 1 shows peptide #, column 2 shows
peptide sequence, column 3 shows SEQ ID NO of the peptide, column 4
shows the PDZ protein and domain number, column 5 shows the EC50 in
.mu.M, column 6 shows the error of fit for the EC.sub.in in .mu.M,
and column 7 shows the OD.sub.max 450 nm). ND=not determined.
[0039] TABLE 11 shows the results of titrations or binding assays
of peptides with RIM-2 d1, INADL d3, ZO-1 d2, PAR3 d3, and
syntrophin 1 alpha d1. Column 1 shows PDZ domain bound, Column 2
shows peptide sequence, and Column 3 shows SEQ ID NO.
DETAILED DESCRIPTION
I. Definitions
[0040] "Polypeptide," "protein" and "peptide" are used
interchangeably herein and include a molecular chain of amino acids
linked through peptide bonds. The terms do not refer to a specific
length of the product. Thus, "peptides," "oligopeptides," and
"proteins" are included within the definition of polypeptide. The
terms include post-translational modifications of the polypeptide,
for example, glycosylations, acetylations, phosphorylations and the
like. In addition, protein fragments, analogs, mutated or variant
proteins, fusion proteins and the like are included within the
meaning of polypeptide. The polypeptide, protein and peptides may
be in cyclic form or they may be in linear form (Piserchio et al.
Chem. Biol. (2004) 111:469-473; Li et al., Bioorg. Med. Chem. Lett.
(2004) 14:13855-1388; Baruch et al. Biochemistry (2003)
42:2797-2805; Harris et al., Biochemistry (2001) 40:5921-5930).
[0041] A "fusion protein" or "fusion polypeptide" as used herein
refers to a composite protein, i.e., a single contiguous amino acid
sequence, made up of two (or more) distinct, heterologous
polypeptides which are not normally fused together in a single
amino acid sequence. Thus, a fusion protein can include a single
amino acid sequence that contains two entirely distinct amino acid
sequences or two similar or identical polypeptide sequences,
provided that these sequences are not normally found together in
the same configuration in a single amino acid sequence found in
nature. Fusion proteins can generally be prepared using either
recombinant nucleic acid methods, i.e., as a result of
transcription and translation of a recombinant gene fusion product,
which fusion comprises a segment encoding a polypeptide of the
invention and a segment encoding a heterologous protein, or by
chemical synthesis methods well known in the art.
[0042] A "fusion protein construct" as used herein is a
polynucleotide encoding a fusion protein.
[0043] As used herein, the term "PDZ domain" refers to protein
sequence (i.e., modular protein domain) of approximately 90 amino
acids, characterized by homology to the brain synaptic protein
PSD-95, the Drosophila septate junction protein Discs-Large (DLG),
and the epithelial tight junction protein ZO1 (ZO1). PDZ domains
are also known as Discs-Large homology repeats ("DHRs") and GLGF
repeats. PDZ domains generally appear to maintain a core consensus
sequence (Doyle, D. A., 1996, Cell 85: 1067-76).
[0044] PDZ domains are found in diverse membrane-associated
proteins including members of the MAGUK family of guanylate kinase
homologs, several protein phosphatases and kinases, neuronal nitric
oxide synthase, and several dystrophin-associated proteins,
collectively known as syntrophins.
[0045] Exemplary PDZ domain-containing proteins and PDZ domain
sequences are shown in TABLE 5. The term "PDZ domain" also
encompasses variants (e.g., naturally occurring variants) of the
sequences of TABLE 5 (e.g., polymorphic variants, variants with
conservative substitutions, and the like). Typically, PDZ domains
are substantially identical to those shown in TABLE 5, e.g., at
least about 70%, at least about 80%, or at least about 90% amino
acid residue identity when compared and aligned for maximum
correspondence.
[0046] As used herein, the term "PDZ protein" refers to a naturally
occurring protein containing a PDZ domain. Exemplary PDZ proteins
include, but are not limited to NNOS, LIM, KIAA1095, HEMBA1003117,
AIPC, KIAA1526, DVL1, DVL2, DVL3, PTPL1, ZO-1, ZO-2, ZO-3,
KIAA1719, Mupp1, INADL, Shank 3, MINT1, MINT2, MAGI1, MAGI2, MAGI3,
NeDLG, syntenin, PSD-95, hDLG, PAR3, MAST1, MAST2, AF6, SIP1, LIM
mystique, HTRA2, TIP-1, KIAA0316, PICK1, RIM-2, INADL, Syntrophin 1
alpha, SITAC-18, PAR3L, MAST2, and NSP [novel serine protease] and
those listed in TABLE 5.
[0047] As used herein, the term "PDZ-domain polypeptide" refers to
a polypeptide containing a PDZ domain, such as a fusion protein
including a PDZ domain sequence, a naturally occurring PDZ protein,
or an isolated PDZ domain peptide.
[0048] As used herein, the term "PL protein" or "PDZ Ligand
protein" refers to a naturally occurring protein that forms a
molecular complex with a PDZ-domain, or to a protein whose
carboxy-terminus, when expressed separately from the full length
protein (e.g., as a peptide fragment of 4-25 residues, e.g., 8, 10,
12, 14 or 16 residues), forms such a molecular complex. The
molecular complex can be observed in vitro using the A assay or G
assay described infra, or in vivo. Exemplary TRP channel PL
proteins listed in TABLE 4 and TRP associated proteins listed in
TABLE 2 are demonstrated to bind specific PDZ proteins. This
definition is not intended to include anti-PDZ antibodies and the
like.
[0049] As used herein, the terms "NMDA receptor," "NMDAR," or "NMDA
receptor protein" refer to a membrane associated protein that is
known to interact with NMDA. The term thus includes the various
subunit forms, including for example, those listed in TABLE 2. The
receptor can be a non-human mammalian NMDAR (e.g., mouse, rat,
rabbit, monkey) or a human NMDAR, for example.
[0050] As used herein, the term "NMDAR-PL" or "NMDA receptor-PL"
refers to a NMDA receptor that forms a molecular complex with a PDZ
domain or to a NMDAR protein whose carboxy-terminus, when expressed
separately from the full length protein (e.g., as a peptide
fragment of 4-25 residues, e.g., 8, 10, 12, 14 or 16 residues),
forms such a molecular complex.
[0051] As used herein, the term "TRP channel" refers to an ion
channel protein of the transient receptor potential family of
proteins.
[0052] As used herein, the term "TRP associated proteins" refers to
proteins that interact physically or functionally with TRP [ion]
channel proteins. MT states that he will soon have data that
demonstrates binding between TRPM7 and NMDAR (a TRP associated
protein).
[0053] As used herein, the term "ion channel" refers to an ion
channel protein, which could refer singularly or collectively to
different ion channels, including TRP channels, NMDA Receptor
channels, or other ion channels.
[0054] As used herein, "oxygen-glucose deprivation" or "OGD" refers
to a change in the cellular environment that results in lower than
normal access to oxygen and/or glucose.
[0055] As used herein, a "PL sequence" refers to the amino acid
sequence of the C-terminus of a PL protein (e.g., the C-terminal 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or 25 residues)
("C-terminal PL sequence") or to an internal sequence known to bind
a PDZ domain ("internal PL sequence").
[0056] As used herein, a "PL peptide" is a peptide having a
sequence from, or based on, the sequence of the C-terminus of a PL
protein. Exemplary PL peptides (biotinylated) are listed in TABLE 2
and TABLE 4.
[0057] As used herein, a "PL fusion protein" is a fusion protein
that has a PL sequence as one domain, typically as the C-terminal
domain of the fusion protein. An exemplary PL fusion protein is a
tat-PL sequence fusion.
[0058] As used herein, the term "PL inhibitor peptide sequence"
refers to PL peptide amino acid sequence that (in the form of a
peptide or PL fusion protein) inhibits the interaction between a
PDZ domain polypeptide and a PL peptide (e.g., in an A assay or a G
assay).
[0059] As used herein, a "PDZ-domain encoding sequence" means a
segment of a polynucleotide encoding a PDZ domain. In various
embodiments, the polynucleotide is DNA, RNA, single stranded or
double stranded.
[0060] As used herein, the terms "antagonist" and "inhibitor," when
used in the context of modulating a binding interaction (such as
the binding of a PDZ domain sequence to a PL sequence), are used
interchangeably and refer to an agent that reduces the binding of
the, e.g., PL sequence (e.g., PL peptide) and the, e.g., PDZ domain
sequence (e.g., PDZ protein, PDZ domain peptide).
[0061] As used herein, the terms "agonist" and "enhancer," when
used in the context of modulating a binding interaction (such as
the binding of a PDZ domain sequence to a PL sequence), are used
interchangeably and refer to an agent that increases the binding of
the, e.g., PL sequence (e.g., PL peptide) and the, e.g., PDZ domain
sequence (e.g., PDZ protein, PDZ domain peptide).
[0062] The terms "isolated" or "purified" means that the object
species (e.g., a polypeptide) has been purified from contaminants
that are present in a sample, such as a sample obtained from
natural sources that contain the object species. If an object
species is isolated or purified it is the predominant
macromolecular (e.g., polypeptide) species present in a sample
(i.e., on a molar basis it is more abundant than any other
individual species in the composition), and preferably the object
species comprises at least about 50 percent (on a molar basis) of
all macromolecular species present. Generally, an isolated,
purified or substantially pure composition comprises more than 80
to 90 percent of all macromolecular species present in a
composition. Most preferably, the object species is purified to
essential homogeneity (i.e., contaminant species cannot be detected
in the composition by conventional detection methods), wherein the
composition consists essentially of a single macromolecular
species.
[0063] The term "recombinant" when used with respect to a
polypeptide refers to a polypeptide that has been prepared be
expressing a recombinant nucleic acid molecule in which different
nucleic acid segments have been joined together using molecular
biology techniques.
[0064] The term "synthesized" when used with respect to a
polypeptide generally means that the polypeptide has been prepared
by means other than simply purifying the polypeptide from naturally
occurring sources. A synthesized polypeptide can thus be prepared
by chemical synthesis, recombinant means, or by a combination of
chemical synthesis and recombinant means. Segments of a synthesized
polypeptide, however, may be obtained from naturally occurring
sources.
[0065] The term "biological function" or "biological activity" in
the context of a cell, refers to a detectable biological activity
normally carried out by the cell, e.g., a phenotypic change such as
proliferation, cell activation, excitotoxicity responses,
neurotransmitter release, cytokine release, degranulation, tyrosine
phosphorylation, ion (e.g., calcium) flux, metabolic activity,
apoptosis, changes in gene expression, maintenance of cell
structure, cell migration, adherence to a substrate, signal
transduction, cell-cell interactions, and others described herein
or known in the art.
[0066] As used herein, the terms "peptide mimetic,"
"peptidomimetic," and "peptide analog" are used interchangeably and
refer to a synthetic chemical compound which has substantially the
same structural and/or functional characteristics of a PL
inhibitory or PL binding peptide of the invention. The mimetic can
be either entirely composed of synthetic, non-natural analogues of
amino acids, or is a chimeric molecule of partly natural peptide
amino acids and partly non-natural analogs of amino acids. The
mimetic can also incorporate any amount of natural amino acid
conservative substitutions as long as such substitutions also do
not substantially alter the mimetic's structure and/or inhibitory
or binding activity. As with polypeptides of the invention which
are conservative variants, routine experimentation will determine
whether a mimetic is within the scope of the invention, i.e., that
its structure and/or function is not substantially altered. Thus, a
mimetic composition is within the scope of the invention if it is
capable of binding to a PDZ domain and/or inhibiting a PL-PDZ
interaction.
[0067] Polypeptide mimetic compositions can contain any combination
of nonnatural structural components, which are typically from three
structural groups: a) residue linkage groups other than the natural
amide bond ("peptide bond") linkages; b) non-natural residues in
place of naturally occurring amino acid residues; or c) residues
which induce secondary structural mimicry, i.e., to induce or
stabilize a secondary structure, e.g., a beta turn, gamma turn,
beta sheet, alpha helix conformation, and the like.
[0068] A polypeptide can be characterized as a mimetic when all or
some of its residues are joined by chemical means other than
natural peptide bonds. Individual peptidomimetic residues can be
joined by peptide bonds, other chemical bonds or coupling means,
such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters,
bifunctional maleimides, N,N=-dicyclohexylcarbodiimide (DCC) or
N,N=-diisopropylcarbodiimide (DIC). Linking groups that can be an
alternative to the traditional amide bond ("peptide bond") linkages
include, e.g., ketomethylene (e.g., --C(.dbd.O)--CH.sub.2-- for
--C(.dbd.O)--NH--), aminomethylene (CH.sub.2--NH), ethylene, olefin
(CH.dbd.CH), ether (CH.sub.2--O), thioether (CH.sub.2--S),
tetrazole (CN.sub.4--), thiazole, retroamide, thioamide, or ester
(see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino
Acids, Peptides and Proteins, Vol. 7, pp 267-357, A Peptide
Backbone Modifications, Marcel Dekker, NY).
[0069] A polypeptide can also be characterized as a mimetic by
containing all or some non-natural residues in place of naturally
occurring amino acid residues. Nonnatural residues are well
described in the scientific and patent literature; a few exemplary
nonnatural compositions useful as mimetics of natural amino acid
residues and guidelines are described below.
[0070] Mimetics of aromatic amino acids can be generated by
replacing by, e.g., D- or L-naphylalanine; D- or L-phenylglycine;
D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or 4-pyreneylalanine;
D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or
L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or
L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine;
D-(trifluoromethyl)-phenylalanine; D-p-fluorophenylalanine; D- or
L-p-biphenylphenylalanine; K- or L-p-methoxybiphenylphenylalanine;
D- or L-2-indole(alkyl)alanines; and, D- or L-alkylainines, where
alkyl can be substituted or unsubstituted methyl, ethyl, propyl,
hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl,
or a non-acidic amino acids. Aromatic rings of a nonnatural amino
acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,
benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic
rings.
[0071] Mimetics of acidic amino acids can be generated by
substitution by, e.g., non-carboxylate amino acids while
maintaining a negative charge; (phosphono)alanine; sulfated
threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can
also be selectively modified by reaction with carbodiimides
(R.dbd.N--C--N--R.dbd.) such as, e.g.,
1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or
glutamyl can also be converted to asparaginyl and glutaminyl
residues by reaction with ammonium ions.
[0072] Mimetics of basic amino acids can be generated by
substitution with, e.g., (in addition to lysine and arginine) the
amino acids ornithine, citrulline, or (guanidino)-acetic acid, or
(guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrile
derivative (e.g., containing the CN-moiety in place of COOH) can be
substituted for asparagine or glutamine. Asparaginyl and glutaminyl
residues can be deaminated to the corresponding aspartyl or
glutamyl residues.
[0073] Arginine residue mimetics can be generated by reacting
arginyl with, e.g., one or more conventional reagents, including,
e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or
ninhydrin, preferably under alkaline conditions.
[0074] Tyrosine residue mimetics can be generated by reacting
tyrosyl with, e.g., aromatic diazonium compounds or
tetranitromethane. N-acetylmidizol and tetranitromethane can be
used to form O-acetyl tyrosyl species and 3-nitro derivatives,
respectively.
[0075] Cysteine residue mimetics can be generated by reacting
cysteinyl residues with, e.g., alpha-haloacetates such as
2-chloroacetic acid or chloroacetamide and corresponding amines; to
give carboxymethyl or carboxyamidomethyl derivatives. Cysteine
residue mimetics can also be generated by reacting cysteinyl
residues with, e.g., bromo-trifluoroacetone,
alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl
2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4
nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole.
[0076] Lysine mimetics can be generated (and amino terminal
residues can be altered) by reacting lysinyl with, e.g., succinic
or other carboxylic acid anhydrides. Lysine and other
alpha-amino-containing residue mimetics can also be generated by
reaction with imidoesters, such as methyl picolinimidate, pyridoxal
phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic
acid, O-methylisourea, 2,4, pentanedione, and
transamidase-catalyzed reactions with glyoxylate.
[0077] Mimetics of methionine can be generated by reaction with,
e.g., methionine sulfoxide. Mimetics of proline include, e.g.,
pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy
proline, dehydroproline, 3- or 4-methylproline, or
3,3,-dimethylproline. Histidine residue mimetics can be generated
by reacting histidyl with, e.g., diethylprocarbonate or
para-bromophenacyl bromide.
[0078] Other mimetics include, e.g., those generated by
hydroxylation of proline and lysine; phosphorylation of the
hydroxyl groups of seryl or threonyl residues; methylation of the
alpha-amino groups of lysine, arginine and histidine; acetylation
of the N-terminal amine; methylation of main chain amide residues
or substitution with N-methyl amino acids; or amidation of
C-terminal carboxyl groups.
[0079] A component of a natural polypeptide (e.g., a PL polypeptide
or PDZ polypeptide) can also be replaced by an amino acid (or
peptidomimetic residue) of the opposite chirality. Thus, any amino
acid naturally occurring in the L-configuration (which can also be
referred to as the R or S, depending upon the structure of the
chemical entity) can be replaced with the amino acid of the same
chemical structural type or a peptidomimetic, but of the opposite
chirality, generally referred to as the D-amino acid, but which can
additionally be referred to as the R- or S-form.
[0080] The mimetics of the invention can also include compositions
that contain a structural mimetic residue, particularly a residue
that induces or mimics secondary structures, such as a beta turn,
beta sheet, alpha helix structures, gamma turns, and the like. For
example, substitution of natural amino acid residues with D-amino
acids; N-alpha-methyl amino acids; C-alpha-methyl amino acids; or
dehydroamino acids within a peptide can induce or stabilize beta
turns, gamma turns, beta sheets or alpha helix conformations. Beta
turn mimetic structures have been described, e.g., by Nagai (1985)
Tet. Lett. 26:647-650; Feigl (1986) J. Amer. Chem. Soc.
108:181-182; Kahn (1988) J. Amer. Chem. Soc. 110:1638-1639; Kemp
(1988) Tet. Lett. 29:5057-5060; Kahn (1988) J. Molec. Recognition
1:75-79. Beta sheet mimetic structures have been described, e.g.,
by Smith (1992) J. Amer. Chem. Soc. 114:10672-10674. For example, a
type VI beta turn induced by a cis amide surrogate,
1,5-disubstituted tetrazol, is described by Beusen (1995)
Biopolymers 36:181-200. Incorporation of achiral omega-amino acid
residues to generate polymethylene units as a substitution for
amide bonds is described by Banerjee (1996) Biopolymers 39:769-777.
Secondary structures of polypeptides can be analyzed by, e.g.,
high-field 1H NMR or 2D NMR spectroscopy, see, e.g., Higgins (1997)
J. Pept. Res. 50:421-435. See also, Hruby (1997) Biopolymers
43:219-266, Balaji, et al., U.S. Pat. No. 5,612,895.
[0081] As used herein, "peptide variants" and "conservative amino
acid substitutions" refer to peptides that differ from a reference
peptide (e.g., a peptide having the sequence of the
carboxy-terminus of a specified PL protein) by substitution of an
amino acid residue having similar properties (based on size,
polarity, hydrophobicity, and the like). Thus, insofar as the
compounds that are encompassed within the scope of the invention
are partially defined in terms of amino acid residues of designated
classes, the amino acids may be generally categorized into three
main classes: hydrophilic amino acids, hydrophobic amino acids and
cysteine-like amino acids, depending primarily on the
characteristics of the amino acid side chain. These main classes
may be further divided into subclasses. Hydrophilic amino acids
include amino acids having acidic, basic or polar side chains and
hydrophobic amino acids include amino acids having aromatic or
apolar side chains. Apolar amino acids may be further subdivided to
include, among others, aliphatic amino acids. The definitions of
the classes of amino acids as used herein are as follows:
[0082] "Hydrophobic Amino Acid" refers to an amino acid having a
side chain that is uncharged at physiological pH and that is
repelled by aqueous solution. Examples of genetically encoded
hydrophobic amino acids include Ile, Leu and Val. Examples of
non-genetically encoded hydrophobic amino acids include t-BuA.
[0083] "Aromatic Amino Acid" refers to a hydrophobic amino acid
having a side chain containing at least one ring having a
conjugated electron system (aromatic group). The aromatic group may
be further substituted with groups such as alkyl, alkenyl, alkynyl,
hydroxyl, sulfanyl, nitro and amino groups, as well as others.
Examples of genetically encoded aromatic amino acids include Phe,
Tyr and Trp. Commonly encountered non-genetically encoded aromatic
amino acids include phenylglycine, 2-naphthylalanine,
.beta.-2-thienylalanine,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,
4-chloro-phenylalanine, 2-fluorophenyl-alanine,
3-fluorophenylalanine and 4-fluorophenylalanine.
[0084] "Apolar Amino Acid" refers to a hydrophobic amino acid
having a side chain that is generally uncharged at physiological pH
and that is not polar. Examples of genetically encoded apolar amino
acids include Gly, Pro and Met. Examples of non-encoded apolar
amino acids include Cha.
[0085] "Aliphatic Amino Acid" refers to an apolar amino acid having
a saturated or unsaturated straight chain, branched or cyclic
hydrocarbon side chain. Examples of genetically encoded aliphatic
amino acids include Ala, Leu, Val and Ile. Examples of non-encoded
aliphatic amino acids include Nle.
[0086] "Hydrophilic Amino Acid" refers to an amino acid having a
side chain that is attracted by aqueous solution. Examples of
genetically encoded hydrophilic amino acids include Ser and Lys.
Examples of non-encoded hydrophilic amino acids include Cit and
hCys.
[0087] "Acidic Amino Acid" refers to a hydrophilic amino acid
having a side chain pK value of less than 7. Acidic amino acids
typically have negatively charged side chains at physiological pH
due to loss of a hydrogen ion. Examples of genetically encoded
acidic amino acids include Asp and Glu.
[0088] "Basic Amino Acid" refers to a hydrophilic amino acid having
a side chain pK value of greater than 7. Basic amino acids
typically have positively charged side chains at physiological pH
due to association with hydronium ion. Examples of genetically
encoded basic amino acids include Arg, Lys and His. Examples of
non-genetically encoded basic amino acids include the non-cyclic
amino acids ornithine, 2,3-diaminopropionic acid,
2,4-diaminobutyric acid and homoarginine.
[0089] "Polar Amino Acid" refers to a hydrophilic amino acid having
a side chain that is uncharged at physiological pH, but which has a
bond in which the pair of electrons shared in common by two atoms
is held more closely by one of the atoms. Examples of genetically
encoded polar amino acids include Asx and Glx. Examples of
non-genetically encoded polar amino acids include citrulline,
N-acetyl-lysine and methionine sulfoxide.
[0090] "Cysteine-Like Amino Acid" refers to an amino acid having a
side chain capable of forming a covalent linkage with a side chain
of another amino acid residue, such as a disulfide linkage.
Typically, cysteine-like amino acids generally have a side chain
containing at least one thiol (SH) group. Examples of genetically
encoded cysteine-like amino acids include Cys. Examples of
non-genetically encoded cysteine-like amino acids include
homocysteine and penicillamine.
[0091] As will be appreciated by those having skill in the art, the
above classification are not absolute--several amino acids exhibit
more than one characteristic property, and can therefore be
included in more than one category. For example, tyrosine has both
an aromatic ring and a polar hydroxyl group. Thus, tyrosine has
dual properties and can be included in both the aromatic and polar
categories. Similarly, in addition to being able to form disulfide
linkages, cysteine also has apolar character. Thus, while not
strictly classified as a hydrophobic or apolar amino acid, in many
instances cysteine can be used to confer hydrophobicity to a
peptide.
[0092] Certain commonly encountered amino acids which are not
genetically encoded of which the peptides and peptide analogues of
the invention may be composed include, but are not limited to,
.beta.-alanine (b-Ala) and other omega-amino acids such as
3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr),
4-aminobutyric acid and so forth; .alpha.-aminoisobutyric acid
(Aib); s-aminohexanoic acid (Aha); .delta.-aminovaleric acid (Ava);
N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline
(Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG);
N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine
(Cha); norleucine (Nle); 2-naphthylalanine (2-Nal);
4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine
(Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine
(Phe(4-F)); penicillamine (Pen);
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);
.beta.-2-thienylalanine (Thi); methionine sulfoxide (MSO);
homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric
acid (Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine
(Phe(pNH.sub.2)); N-methyl valine (MeVal); homocysteine (hCys) and
homoserine (hSer). These amino acids also fall conveniently into
the categories defined above.
[0093] The classifications of the above-described genetically
encoded and non-encoded amino acids are summarized in TABLE 1,
below. It is to be understood that TABLE 1 is for illustrative
purposes only and does not purport to be an exhaustive list of
amino acid residues which may comprise the peptides and peptide
analogues described herein. Other amino acid residues which are
useful for making the peptides and peptide analogues described
herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook
of Biochemistry and Molecular Biology, CRC Press, Inc., and the
references cited therein. Amino acids not specifically mentioned
herein can be conveniently classified into the above-described
categories on the basis of known behavior and/or their
characteristic chemical and/or physical properties as compared with
amino acids specifically identified.
TABLE-US-00001 TABLE 1 Genetically Classification Encoded
Genetically Non-Encoded Hydrophobic Aromatic F, Y, W Phg, Nal, Thi,
Tic, Phe(4-Cl), Phe(2-F), Phe(3-F), Phe(4-F), Pyridyl Ala,
Benzothienyl Ala Apolar M, G, P Aliphatic A, V, L, I t-BuA, t-BuG,
MeIle, Nle, MeVal, Cha, bAla, MeGly, Aib Hydrophilic Acidic D, E
Basic H, K, R Dpr, Orn, hArg, Phe(p-NH.sub.2), DBU, A.sub.2BU Polar
Q, N, S, T, Y Cit, AcLys, MSO, hSer Cysteine-Like C Pen, hCys,
p-methyl Cys
[0094] As used herein, a "detectable label" has the ordinary
meaning in the art and refers to an atom (e.g., radionuclide),
molecule (e.g., fluorescein), or complex, that is or can be used to
detect (e.g., due to a physical or chemical property), indicate the
presence of a molecule or to enable binding of another molecule to
which it is covalently bound or otherwise associated. The term
"label" also refers to covalently bound or otherwise associated
molecules (e.g., a biomolecule such as an enzyme) that act on a
substrate to produce a detectable atom, molecule or complex.
Detectable labels suitable for use in the present invention include
any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Labels useful in the present invention include biotin for staining
with labeled streptavidin conjugate, magnetic beads (e.g.,
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, Texas red,
rhodamine, green fluorescent protein, enhanced green fluorescent
protein, and the like), radiolabels (e.g., .sup.3H, .sup.125I,
.sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., hydrolases,
particularly phosphatases such as alkaline phosphatase, esterases
and glycosidases, or oxidoreductases, particularly peroxidases such
as horseradish peroxidase, and others commonly used in ELISAs),
substrates, cofactors, inhibitors, chemiluminescent groups,
chromogenic agents, and calorimetric labels such as colloidal gold
or colored glass or plastic (e.g., polystyrene, polypropylene,
latex, etc.) beads. Patents teaching the use of such labels include
U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Means of detecting such labels
are well known to those of skill in the art. Thus, for example,
radiolabels and chemiluminescent labels may be detected using
photographic film or scintillation counters, fluorescent markers
may be detected using a photodetector to detect emitted light
(e.g., as in fluorescence-activated cell sorting). Enzymatic labels
are typically detected by providing the enzyme with a substrate and
detecting the reaction product produced by the action of the enzyme
on the substrate, and colorimetric labels are detected by simply
visualizing the colored label. Thus, a label is any composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical means. The label
may be coupled directly or indirectly to the desired component of
the assay according to methods well known in the art.
Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to
the molecule. The ligand then binds to an anti-ligand (e.g.,
streptavidin) molecule which is either inherently detectable or
covalently bound to a signal generating system, such as a
detectable enzyme, a fluorescent compound, or a chemiluminescent
compound. A number of ligands and anti-ligands can be used. Where a
ligand has a natural anti-ligand, for example, biotin, thyroxine,
and cortisol, it can be used in conjunction with the labeled,
naturally occurring anti-ligands. Alternatively, any haptenic or
antigenic compound can be used in combination with an antibody. The
molecules can also be conjugated directly to signal generating
compounds, e.g., by conjugation with an enzyme or fluorophore.
Means of detecting labels are well known to those of skill in the
art. Thus, for example, where the label is a radioactive label,
means for detection include a scintillation counter, photographic
film as in autoradiography, or storage phosphor imaging. Where the
label is a fluorescent label, it may be detected by exciting the
fluorochrome with the appropriate wavelength of light and detecting
the resulting fluorescence. The fluorescence may be detected
visually, by means of photographic film, by the use of electronic
detectors such as charge coupled devices (CCDs) or photomultipliers
and the like. Similarly, enzymatic labels may be detected by
providing the appropriate substrates for the enzyme and detecting
the resulting reaction product. Also, simple calorimetric labels
may be detected by observing the color associated with the label.
It will be appreciated that when pairs of fluorophores are used in
an assay, it is often preferred that they have distinct emission
patterns (wavelengths) so that they can be easily
distinguished.
[0095] As used herein, the term "substantially identical" in the
context of comparing amino acid sequences, means that the sequences
have at least about 70%, at least about 80%, or at least about 90%
amino acid residue identity when compared and aligned for maximum
correspondence. An algorithm that is suitable for determining
percent sequence identity and sequence similarity is the FASTA
algorithm, which is described in Pearson, W. R. & Lipman, D.
J., 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 2444. See also W. R.
Pearson, 1996, Methods Enzymol. 266: 227-258. Preferred parameters
used in a FASTA alignment of DNA sequences to calculate percent
identity are optimized, BL50 Matrix 15: -5, k-tuple=2; joining
penalty=40, optimization=28; gap penalty=-12, gap lengthpenalty=-2;
and width=16.
[0096] As used herein, the terms "test compound" or "test agent"
are used interchangeably and refer to a candidate agent that may
have enhancer/agonist, or inhibitor/antagonist activity, e.g.,
inhibiting or enhancing an interaction such as PDZ-PL binding. The
candidate agents or test compounds may be any of a large variety of
compounds, both naturally occurring and synthetic, organic and
inorganic, and including polymers (e.g., oligopeptides,
polypeptides, oligonucleotides, and polynucleotides), small
molecules, antibodies (as broadly defined herein), sugars, fatty
acids, nucleotides and nucleotide analogs, analogs of naturally
occurring structures (e.g., peptide mimetics, nucleic acid analogs,
and the like), and numerous other compounds. In certain embodiment,
test agents are prepared from diversity libraries, such as random
or combinatorial peptide or non-peptide libraries. Many libraries
are known in the art that can be used, e.g., chemically synthesized
libraries, recombinant (e.g., phage display libraries), and in
vitro translation-based libraries. Examples of chemically
synthesized libraries are described in Fodor et al., 1991, Science
251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al.,
1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710;
Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;
Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926;
Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426;
Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al.,
1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993,
Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO
93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA
89:5381-5383. Examples of phage display libraries are described in
Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990,
Science, 249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol.
227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et
al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318
dated Aug. 18, 1994. In vitro translation-based libraries include
but are not limited to those described in PCT Publication No. WO
91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc.
Natl. Acad. Sci. USA 91:9022-9026. By way of examples of nonpeptide
libraries, a benzodiazepine library (see e.g., Bunin et al., 1994,
Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use.
Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA
89:9367-9371) can also be used. Another example of a library that
can be used, in which the amide functionalities in peptides have
been permethylated to generate a chemically transformed
combinatorial library, is described by Ostresh et al. (1994, Proc.
Natl. Acad. Sci. USA 91:11138-11142).
[0097] The term "specific binding" refers to binding between two
molecules, for example, a ligand and a receptor, characterized by
the ability of a molecule (ligand) to associate with another
specific molecule (receptor) even in the presence of a plurality of
other diverse molecules, i.e., to show preferential binding of one
molecule for another in a heterogeneous mixture of molecules.
Specific binding of a ligand to a receptor is also evidenced by
reduced binding of a detectably labeled ligand to the receptor in
the presence of excess unlabeled ligand (i.e., a binding
competition assay).
[0098] As used herein, a "plurality" of PDZ proteins (or
corresponding PDZ domains or PDZ fusion polypeptides) has its usual
meaning. In some embodiments, (Susan our US associate has cautioned
us to remove phrases such as "in one embodiment" as the applicant
may be required to include all of these features as limitations in
the claims. I am not sure if you agree, it is now our practice to
use the phrase "in certain embodiments" OK with me the plurality is
at least 5, and often at least 25, at least 40, or at least 60
different PDZ proteins. In some embodiments, the plurality is
selected from the list of PDZ polypeptides listed in TABLE 5. In
some embodiments, the plurality of different PDZ proteins are from
(i.e., expressed in) a particular specified tissue or a particular
class or type of cell. In some embodiments, the plurality of
different PDZ proteins represents a substantial fraction (e.g.,
typically at least 50%, more often at least 80%) of all of the PDZ
proteins known to be, or suspected of being, expressed in the
tissue or cell(s), e.g., all of the PDZ proteins known to be
present in neurons. In some embodiments, the plurality is at least
50%, usually at least 80%, at least 90% or all of the PDZ proteins
disclosed herein as being expressed in a particular cell.
[0099] When referring to PL peptides (or the corresponding
proteins, e.g., corresponding to those listed in TABLE 2, or
elsewhere herein) a "plurality" may refer to at least 5, at least
10, and often at least 25 PLs such as those specifically listed
herein, or to the classes and percentages set forth supra for PDZ
domains.
[0100] The term "cytodestructive disorder" generally refers to a
disorder in which an injury to a tissue is sustained that results
in the destruction or dysfunction of cells. Specific examples of
such disorders include, but are not limited to, tissue ischemia,
thermal burns, electrical burns, burns sustained from contact with
caustic solutions, poisonings, endocrine and metabolic
derangements, nutritional deficiencies, exposure to solar or
ionizing radiation, malignant transformation, and mechanical tissue
deformation such as might occur in tissue trauma.
[0101] The term "neurological disorder," "neurological injury",
"neurological disease" and other related terms generally refers to
a disorder correlated with some type neuronal insult or neuronal
cell death. Specific examples of such disorders include, but are
not limited to, stroke, ischemic stroke, glaucoma, retinal
ischemia, Parkinson's disease, Huntington's disease, Alzheimer's
disease, epilepsy, inherited ataxias and motor neuron diseases.
Further examples of such disorders include, but are not limited to,
disorders of synaptic transmission in the brain, such disorders
resulting in impairments of learning, memory, neuropsychiatric and
mood disorders, and congenital disorders of mentation.
[0102] A "stroke" has the meaning normally accepted in the art and
generally refers to neurological injury resulting from impaired
blood flow regardless of cause. Potential causes include, but are
not limited to, embolism, hemorrhage and thrombosis. An "ischemic
stroke" refers more specifically to a type of stroke that is of
limited extent and caused due to blockage of blood flow.
[0103] A difference in general is typically considered to be
"statistically significant" if the difference is less than
experimental error. Thus a difference is considered statistically
significant if the probability of the observed difference occurring
by chance (the p-value) is less than some predetermined level. As
used herein a "statistically significant difference" can refer to a
p-value that is <0.05, preferably <0.01 and most preferably
<0.001.
II. General
[0104] The present inventors have identified interactions between
PDZ proteins and proteins that contain a PL motif that are involved
in various biological functions in different types of cells. Some
of these interactions involve PDZ:PL protein interactions between
proteins that have important roles in neuronal cells. As such,
modulation of these interactions has direct implications for the
treatment of various disorders of mammalian cell injury, including,
but not limited to, neurological disorders such as stroke and brain
ischemia.
[0105] The inventors have identified distinct strategies for
treating various neurological disorders based on PDZ:PL
interations. One strategy is based upon the finding that disrupting
the activity of a member of the TRP cation channel family, is
protective for cellular damage induced by OGD (oxygen-glucose
deprivation) or anoxia. The inventors have determined common
structural features of a class of polypeptides that are effective
in disrupting the interaction between a member of the TRP cation
channel family and PDZ domain-containing polypeptides. These
polypeptides are thus useful in treating disorders associated with
OGD. The second strategy is based upon the recognition that several
types of ion channels can be grouped and regulated by PDZ proteins,
and that disruption of these interactions can modulate the
structure of these complexes and provide protection against damage
resulting from OGD.
[0106] The current inventors have thus identified compounds that
inhibit the interactions between these different proteins, as well
as developed methods for designing additional compounds. One
general class of inhibitors is those that mimic the carboxy
terminus of a PL protein and thus interfere with the ability of the
carboxy terminus of the PL protein to bind its cognate PDZ protein.
Another general class of inhibitors includes the PDZ domain from a
PDZ protein that is involved in an interaction that is to be
disrupted. These inhibitors bind the PL protein that is the cognate
ligand for the PDZ protein of interest and thus prevent binding
between the PL protein and PDZ protein. Because the PDZ:PL protein
interactions that are described herein are involved in the
biological activity of mammalian, and especially neuronal cells,
the inhibitors that are provided can be used to inhibit PDZ:PL
protein interactions for the treatment of neurological disorders
such as stroke, ischemia, Parkinson's disease, Huntington's
disease, Alzheimer's disease, epilepsy, inherited ataxias, motor
neuron diseases as well as myocardial ischemia, retinal ischemia
and glaucoma. Methods for determining whether a test compound acts
a modulator of a particular PDZ protein and PL protein binding pair
are also described.
[0107] For those PDZ proteins containing multiple PDZ domains, the
methods that are provided can be utilized to determine to which
specific domain(s) a particular PL protein of interest binds. The
methods can thus be utilized to identify or design inhibitors that
have increased selectivity for a particular PDZ domain. The methods
that are disclosed can also be used to identify inhibitors with
high binding affinity. Because TRP channels play a key regulatory
role in many cell types, an initial set of studies were undertaken
to determine which PDZ proteins bind to the PL of TRPM7. These
analyses were conducted using the A and G assays described in
detail below. Without intending to be limiting, the PDZ proteins
identified as being able to bind TRPM7 are listed in TABLE 6.
[0108] The C-terminal sequences of the various TRP channels that
contain a PL sequence are listed in TABLE 4. Because the C-terminal
region of the PL protein is the most common region that binds to
PDZ proteins, agents that include similar amino acid motifs can be
used to inhibit binding between TRP proteins and the PDZ proteins
that bind to them. As described in greater detail below, for
example, certain classes of peptide inhibitors typically include at
least 2 contiguous amino acids from the C-terminus of the TRP
proteins listed in TABLE 4, but can include 3-20 or more contiguous
amino acids from the C-terminus.
[0109] Additional studies identify the structural motifs common to
the polypeptides capable of inhibiting the interaction between
TRPM7 and PDZ domains. One class of compounds are polypeptides that
have the following characteristics: 1) a length of about 3-20 amino
acids (although somewhat longer polypeptides can be used), and 2) a
C-terminal consensus sequence of X-L/I/V-X-V/L/A (the slash
separates different amino acids that can appear at a given
position). These polypeptides also typically have IC.sub.50 values
of less than 50 .mu.M.
[0110] The inventors have also found that the C-termini of several
MAGUK (membrane associated Guanylate Kinases) are themselves PL
sequences and thus can bind PDZ proteins. Accordingly, another
class of inhibitors are those that disrupt binding between the PL
sequences of these PDZ proteins and their PDZ binding partners.
These proteins are involved in organizing signaling complexes
associated with excitotoxic, anoxic or cytodestructive cellular
death. Interactions of this type thus provide another therapeutic
target for treatment of various neurological diseases.
[0111] Although the foregoing classes of inhibitors are based upon
the C-terminal sequences of PL proteins that bind a PDZ protein, as
alluded to above, another class of inhibitors includes polypeptides
that include all or a part of a PDZ domain that binds to the PL
sequence of a TRP channel protein or TRP associated protein.
Because inhibitors in this class typically include most or the
entire PDZ domain, polypeptide inhibitors in this class typically
are at least 50-70 amino acids in length.
[0112] The various classes of polypeptide inhibitors just described
can also be fusion proteins. These generally include a PL inhibitor
peptide sequence such as those just listed that is fused to another
sequence that encodes a separate protein domain. One specific
example of an inhibitory fusion protein is one in which a PL
sequence (e.g., it might simply be easier to reinsert the list cite
Table 10 and Table 11) is coupled to a transmembrane transporter
peptide. As described in greater detail infra, a variety of
different transmembrane transporter peptides can be utilized.
[0113] Although certain classes of inhibitors such as those just
described are polypeptides, other inhibitors are peptide mimetics
or variants of these polypeptides as described in greater detail
infra. Regardless of type, the inhibitors typically had IC.sub.50
values less than 50 .mu.M, 25 .mu.M, 10 .mu.M, 0.1 .mu.M or 0.01
.mu.M. In general the inhibitors typically have an IC.sub.50 value
of between 0.1-1 .mu.M. These inhibitors can be formulated as
pharmaceutical compositions and then used in the treatment of
various neurological disorders such as those listed above.
[0114] The following sections provide additional details regarding
the identification of PDZ:PL interactions in neuron cells, the
structural characteristics of inhibitors that disrupt these
interactions and treatment methods utilizing such inhibitors.
III. Identification of Candidate PL Proteins and Synthesis of
Peptides
[0115] A PL protein (short for PDZ Ligand protein), such as the TRP
proteins described herein, is a protein (or a C-terminal fragment
thereof) that can bind PDZ proteins via its carboxy terminus. PDZ
proteins, in turn, are proteins with PDZ domains, which are domains
common to three prototypical proteins: post synaptic density
protein -95 (PSD-95), Drosophila large disc protein and Zonula
Occludin 1 protein (see, e.g., Gomperts et al., 1996, Cell
84:659-662; see also, Songyang et al., 1997, Science 275:73; and
Doyle et al., 1996, Cell 88:1067-1076). Certain classes of PDZ
proteins contain three PDZ domains, one SH3 domain and one
guanylate kinase domain. As described in greater detail herein, PL
proteins have certain carboxy terminal motifs that enable these
proteins to functions as ligands to PDZ proteins. When these
carboxy terminal regions are referred to, the positioning of the
carboxy terminal residues are sometimes referred to herein by a
numbered position, which is illustrated in the following
scheme:
TABLE-US-00002 Position: -3 -2 -1 0 (C-terminal)
[0116] Certain PDZ domains are bound by the C-terminal residues of
PDZ-binding proteins. To identify TRP channels and TRP associated
proteins containing a PL motif, the C-terminal residues of
sequences were visually inspected to identify sequences that bind
to PDZ-domain containing proteins (see, e.g., Doyle et al., 1996,
Cell 85, 1067; Songyang et al., 1997, Science 275, 73). TABLES 2
and 4 list these proteins, and provide corresponding C-terminal
sequences and GenBank accession numbers.
TABLE-US-00003 TABLE 2 Glutamate Receptors with PL Sequences
internal PL Name GI# PL ID NMDAR1 307302 X AA216 NMDAR1-1 292282 X
AA216 NMDAR1-4 472845 X AA216 NMDAR1-3b 2343286 X AA216 NMDAR1-4b
2343288 X AA216 NMDAR1-2 11038634 NMDAR1-3 11038636 NMDAR2C 6006004
X AA180 NMDAR3 560546 X AA34.1 NMDAR3A 17530176 NMDAR2B 4099612 X
NMDAR2A 558748 X AA34.2 NMDAR2D 4504130 X GluR2 3287973 X GluR3
481504 X GluR1 1169961 X GluR5 729597 X GluR6 2492627 X GluR7
12729188 X
[0117] A. Preparation of Peptides
[0118] 1) Chemical Synthesis
[0119] Technique for the preparation of peptides and peptide
analogues of the current invention are well known in the art. For
example, the peptides may be prepared in linear form using
conventional solution or solid phase peptide syntheses and cleaved
from the resin followed by purification procedures (Creighton,
1983, Protein Structures And Molecular Principles, W.H. Freeman and
Co., N.Y.). Suitable procedures for synthesizing the peptides
described herein are well known in the art. The composition of the
synthetic peptides may be confirmed by amino acid analysis or
sequencing (e.g., the Edman degradation procedure and mass
spectroscopy).
[0120] In addition, analogues and derivatives of the peptides can
be chemically synthesized. The linkage between each amino acid of
the peptides of the invention may be an amide, a substituted amide
or an isostere of amide. Nonclassical amino acids or chemical amino
acid analogues can be introduced as a substitution or addition into
the sequence. Non-classical amino acids include, but are not
limited to, the D-isomers of the common amino acids, .alpha.-amino
isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,
.gamma.-Abu, .epsilon.-Ahx, 6-amino hexanoic acid, Aib, 2-amino
isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
.beta.-alanine, fluoro-amino acids, designer amino acids such as
.beta.-methyl amino acids, C.alpha.-methyl amino acids,
N.alpha.-methyl amino acids, and amino acid analogues in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0121] Synthetic peptides of defined sequence (e.g., corresponding
to the carboxyl-termini of the indicated proteins) can be
synthesized by any standard resin-based method (see, e.g., U.S.
Pat. No. 4,108,846; see also, Caruthers et al., 1980, Nucleic Acids
Res. Symp. Ser., 215-223; Horn et al., 1980, Nucleic Acids Res.
Symp. Ser., 225-232; Roberge, et al., 1995, Science 269:202). The
peptides used in the assays described herein were prepared by the
FMOC (see, e.g., Guy and Fields, 1997, Meth. Enz. 289:67-83;
Wellings and Atherton, 1997, Meth. Enz. 289:44-67). In some cases
(e.g., for use in the A and G assays of the invention), peptides
were labeled with biotin at the amino-terminus by reaction with a
four-fold excess of biotin methyl ester in dimethylsulfoxide with a
catalytic amount of base. The peptides were cleaved from the resin
using a halide containing acid (e.g. trifluoroacetic acid) in the
presence of appropriate antioxidants (e.g. ethanedithiol) and
excess solvent lyophilized.
[0122] 2) Recombinant Synthesis
[0123] If the peptide is composed entirely of gene-encoded amino
acids, or a portion of it is so composed, the peptide or the
relevant portion may also be synthesized using conventional
recombinant genetic engineering techniques. For recombinant
production, a polynucleotide sequence encoding a linear form of the
peptide is inserted into an appropriate expression vehicle, i.e., a
vector which contains the necessary elements for the transcription
and translation of the inserted coding sequence, or in the case of
an RNA viral vector, the necessary elements for replication and
translation. The expression vehicle is then transfected into a
suitable target cell which will express the peptide. Depending on
the expression system used, the expressed peptide is then isolated
by procedures well-established in the art. Methods for recombinant
protein and peptide production are well known in the art (see,
e.g., Maniatis et al., 1989, Molecular Cloning A Laboratory Manual,
Cold Spring Harbor Laboratory, N.Y.; and Ausubel et al., 1989,
Current Protocols in Molecular Biology, Greene Publishing
Associates and Wiley Interscience, N.Y.).
[0124] A variety of host-expression vector systems may be utilized
to express the peptides described herein. These include, but are
not limited to, microorganisms such as bacteria transformed with
recombinant bacteriophage DNA or plasmid DNA expression vectors
containing an appropriate coding sequence; yeast or filamentous
fungi transformed with recombinant yeast or fungi expression
vectors containing an appropriate coding sequence; insect cell
systems infected with recombinant virus expression vectors (e.g.,
baculovirus) containing an appropriate coding sequence; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus or tobacco mosaic virus) or transformed
with recombinant plasmid expression vectors (e.g., Ti plasmid)
containing an appropriate coding sequence; or animal cell
systems.
[0125] The expression elements of the expression systems vary in
their strength and specificities. Depending on the host/vector
system utilized, any of a number of suitable transcription and
translation elements, including constitutive and inducible
promoters, may be used in the expression vector. For example, when
cloning in bacterial systems, inducible promoters such as pL of
bacteriophage .lamda., plac, ptrp, ptac (ptrp-lac hybrid promoter)
and the like may be used; when cloning in insect cell systems,
promoters such as the baculovirus polyhedron promoter may be used;
when cloning in plant cell systems, promoters derived from the
genome of plant cells (e.g., heat shock promoters; the promoter for
the small subunit of RUBISCO; the promoter for the chlorophyll a/b
binding protein) or from plant viruses (e.g., the 35S RNA promoter
of CaMV; the coat protein promoter of TMV) may be used; when
cloning in mammalian cell systems, promoters derived from the
genome of mammalian cells (e.g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the vaccinia
virus 7.5 K promoter) may be used; when generating cell lines that
contain multiple copies of expression product, SV40-, BPV- and
EBV-based vectors may be used with an appropriate selectable
marker.
[0126] In cases where plant expression vectors are used, the
expression of sequences encoding the peptides of the invention may
be driven by any of a number of promoters. For example, viral
promoters such as the 35S RNA and 19S RNA promoters of CaMV
(Brisson et al., 1984, Nature 310:511-514), or the coat protein
promoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be
used; alternatively, plant promoters such as the small subunit of
RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al.,
1984, Science 224:838-843) or heat shock promoters, e.g., soybean
hsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol.
6:559-565) may be used. These constructs can be introduced into
planleukocytes using Ti plasmids, Ri plasmids, plant virus vectors,
direct DNA transformation, microinjection, electroporation, etc.
For reviews of such techniques see, e.g., Weissbach &
Weissbach, 1988, Methods for Plant Molecular Biology, Academic
Press, NY, Section VIII, pp. 421-463; and Grierson & Corey,
1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch.
7-9.
[0127] In one insect expression system that may be used to produce
the peptides of the invention, Autographa californica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express the
foreign genes. The virus grows in Spodoptera frugiperda cells. A
coding sequence may be cloned into non-essential regions (for
example the polyhedron gene) of the virus and placed under control
of an AcNPV promoter (for example, the polyhedron promoter).
Successful insertion of a coding sequence will result in
inactivation of the polyhedron gene and production of non-occluded
recombinant virus (i.e., virus lacking the proteinaceous coat coded
for by the polyhedron gene). These recombinant viruses are then
used to infect Spodoptera frugiperda cells in which the inserted
gene is expressed. (e.g., see Smith et al., 1983, J. Virol. 46:584;
Smith, U.S. Pat. No. 4,215,051). Further examples of this
expression system may be found in Current Protocols in Molecular
Biology, Vol. 2, Ausubel et al., eds., Greene Publish. Assoc. &
Wiley Interscience.
[0128] In mammalian host cells, a number of viral based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, a coding sequence may be ligated to an
adenovirus transcription/translation control complex, e.g., the
late promoter and tripartite leader sequence. This chimeric gene
may then be inserted in the adenovirus genome by in vitro or in
vivo recombination. Insertion in a non-essential region of the
viral genome (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing peptide in infected
hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci.
USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may
be used, (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.
USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864;
Panicali et al., 1982, Proc. Natl. Acad. Sci. USA
79:4927-4931).
[0129] Other expression systems for producing linear peptides of
the invention will be apparent to those having skill in the
art.
[0130] B. Purification of Peptides and Peptide Analogues
[0131] The peptides and peptide analogues of the invention can be
purified by art-known techniques such as high performance liquid
chromatography, ion exchange chromatography, gel electrophoresis,
affinity chromatography and the like. The actual conditions used to
purify a particular peptide or analogue will depend, in part, on
factors such as net charge, hydrophobicity, hydrophilicity, etc.,
and will be apparent to those having skill in the art. The purified
peptides can be identified by assays based on their physical or
functional properties, including radioactive labeling followed by
gel electrophoresis, radioimmuno-assays, ELISA, bioassays, and the
like.
[0132] For affinity chromatography purification, any antibody which
specifically binds the peptides or peptide analogues may be used.
For the production of antibodies, various host animals, including
but not limited to rabbits, mice, rats, etc., may be immunized by
injection with a peptide. The peptide may be attached to a suitable
carrier, such as BSA or KLH, by means of a side chain functional
group or linkers attached to a side chain functional group. Various
adjuvants may be used to increase the immunological response,
depending on the host species, including but not limited to
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacilli Calmette-Guerin) and Corynebacterium
parvum.
[0133] Monoclonal antibodies to a peptide may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include but are not
limited to the hybridoma technique originally described by Koehler
and Milstein, 1975, Nature 256:495-497, the human B-cell hybridoma
technique, Kosbor et al., 1983, Immunology Today 4:72; Cote et al.,
1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030 and the
EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). In
addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A.
81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et
al., 1985, Nature 314:452-454) by splicing the genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. Alternatively, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce peptide-specific single chain antibodies.
[0134] Antibody fragments which contain deletions of specific
binding sites may be generated by known techniques. For example,
such fragments include but are not limited to F(ab').sub.2
fragments, which can be produced by pepsin digestion of the
antibody molecule and Fab fragments, which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragments.
Alternatively, Fab expression libraries may be constructed (Huse et
al., 1989, Science 246:1275-1281) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity for the peptide of interest.
[0135] The antibody or antibody fragment specific for the desired
peptide can be attached, for example, to agarose, and the
antibody-agarose complex is used in immunochromatography to purify
peptides of the invention. See, Scopes, 1984, Protein Purification:
Principles and Practice, Springer-Verlag New York, Inc., N.Y.,
Livingstone, 1974, Methods Enzymology Immunoaffinity Chromatography
of Proteins 34:723-731.
[0136] For the peptides used in the present invention, cleavage
from resin and lyophilization was followed by peptides being
redissolved and purified by reverse phase high performance liquid
chromatography (HPLC). One appropriate HPLC solvent system involves
a Vydac C-18 semi-preparative column running at 5 ml per minute
with increasing quantities of acetonitrile plus 0.1%
trifluoroacetic acid in a base solvent of water plus 0.1%
trifluoroacetic acid. After HPLC purification, the identities of
the peptides are confirmed by MALDI cation-mode mass spectrometry.
As noted, exemplary biotinylated peptides are provided in TABLE
2.
IV. TRP Channels A. Nomenclature
Taken from C. Montell, et al. A Unified Nomenclature for the
Superfamily of TRP Cation Channels. Mol. Cell 9 (2):229-231,
2002
[0137] The TRP superfamily includes a diversity of
non-voltage-gated cation channels that vary significantly in their
selectivity and mode of activation. Nevertheless, members of the
TRP superfamily share significant sequence homology and predicted
structural similarities. Until recently most of the genes and
proteins that comprise the TRP superfamily have had multiple names
and, in at least one instance, two distinct genes belonging to
separate subfamilies have the same name. Moreover, there are many
cases in which highly related proteins that belong to the same
subfamily have unrelated names. Therefore, to minimize confusion, a
unified nomenclature has been accepted for the TRP superfamily.
[0138] The unified TRP nomenclature focuses on three subfamilies
(TRPC, TRPV, and TRPM) that bear significant similarities to the
founding member of this superfamily, Drosophila TRP, and which
include highly related members in worms, flies, mice, and humans
(TABLE 3). Members of the three subfamilies contain six
transmembrane segments, a pore loop separating the final two
transmembrane segments, and similarity in the lengths of the
cytoplasmic and extracellular loops. In addition, the charged
residues in the S4 segment that appear to contribute to the voltage
sensor in voltage-gated ion channels are not conserved. The
TRP-Canonical (TRPC) subfamily (formerly short-TRPs or STRPs) is
comprised of those proteins that are the most highly related to
Drosophila TRP. The TRPV subfamily (formerly OTRPC), is so named
based on the original designation, Vanilloid Receptor 1 (VR1), for
the first mammalian member of this subfamily (now TRPV1). The name
for the TRPM subfamily (formerly long-TRPs or LTRPs) is derived
from the first letter of Melastatin, the former name (now TRPM1) of
the founding member of this third subfamily of TRP-related
proteins. Based on amino acid homologies, the mammalian members of
these three subfamilies can be subdivided into several groups each
(TABLE 4 and FIG. 1).
TABLE-US-00004 TABLE 3 Number of TRP Genes in Worms (C. elegans),
Flies (Drosophila melanogaster), Mice, and Humans Subfamily Worms
Flies Mice Humans TRPC 3 3 7 .sup. 6.sup.a TRPV 5 2 5 5 TRPM 4 1 8
8 .sup.aTRPC2 is a pseudogene and is not counted.
TABLE-US-00005 TABLE 4 Nomenclature of the Mammalian TRP
Superfamily Former Name Group Names Accession Numbers TRPC
Subfamily TRPC1 1 TRP1 (CAA61447), (AAA93252) TRPC1 TRPC2 2 TRP2
(X89067), (AAD17195), (AAD17196), TRPC2 (AAG29950), (AAG29951),
(AAD31453), (CAA06964) TRPC3 3 TRP3 (AAC51653) TRPC3 TRPC4 4 TRP4
(CAA68125), (BAA23599) TRPC4 TRPC5 4 TRP5 (AAC13550), (CAA06911),
(CAA06912) TRPC5 TRPC6 3 TRP6 NP_038866 TRPC6 TRPC7 3 TRP7
(AAD42069), NP_065122 TRPC7 TRPV Subfamily TRPV1 1 VR1 (AAC53398)
OTRPC1 TRPV2 1 VRL-1 (AAD26363), (AAD26364), (BAA78478) OTRPC2 GRC
TRPV3 (not assigned) TRPV4 2 OTRPC4 (AAG17543), (AAG16127),
(AAG28027), VR-OAC (AAG28028), (AAG28029), TRP12 (CAC20703) VRL-2
TRPV5 3 ECaC1 (CAB40138) CaT2 TRPV6 3 CaT1 (AAD47636) ECaC2
(CAC20416) CaT-L (CAC20417) TRPM Subfamily TRPM1 1 Melastatin
(AAC13683), (AAC80000) TRPM2 2 TRPC7 (BAA34700) LTRPC2 TRPM3 1
KIAA1616 (AA038185) LTRPC3 TRPM4 3 TRPM4 (H18835) LTRPC4 TRPM5 3
MTR1 (AAF26288) LTRPC5 TRPM6 4 Chak2 (AF350881) TRPM7 4 TRP-PLIK
(AAF73131), (AY032951) Chak1 LTRPC7 TRPM8 2 TRP-p8 (AC005538)
[0139] The numbering system for the mammalian TRPC, TRPV, and TRPM
proteins takes into account the order of their discovery and, in as
many cases as possible, the number that has already been assigned
to the genes and proteins (TABLE 4). In the case of the TRPV
proteins, the numbering system is also based in part on the
groupings of the TRPV proteins. New members of each subfamily will
maintain the same root name and, with the exception of TRPV3, will
be assigned the next number in the sequence. Currently, TRPV3 is
unassigned to maintain the TRPV1/TRPV2 and TRPV5/TRPV6 groupings
and so that the former OTRPC4 could be renamed TRPV4. The next TRPV
protein will be designated TRPV3.
[0140] B. Current Knowledge on TPP Channel Function. (Taken
Directly from Clapham (2003) Nature 426:517-524,
[0141] Transient receptor potential (TRP) channels were first
described in Drosophila, where photoreceptors carrying trp gene
mutations exhibited a transient voltage response to continuous
light. They have been called store-operated channels (SOCs), but
this description is theoretical and related to a poorly understood
phenomenon. All TRP channels are putative six-transmembrane (6TM)
polypeptide subunits that assemble as tetramers to form
cation-permeable pores (FIG. 2). In general, they are almost
ubiquitously expressed and most have splice variants. So most cells
have a number of TRP channel proteins.
[0142] FIG. 2 emphasizes the diversity of TRP cytoplasmic
domains.
[0143] The selectivity filter (light blue and inset) is formed by
amino acids that dip into the bilayer (pore loops), one contributed
from each of the four subunits. S5 has been removed to emphasize
the link between the S6 gating helix and the TRP C-terminal
polypeptide chain. The TRP box is EWKFAR (SEQ ID NO:2) in TRPC, but
is less conserved in TRPV and TRPM. CC indicates a coiled-coil
domain. Ankyrin repeats (AnkR) range from 0 to 14 in number (3 or 4
in TRPV and TRPC, 14 in ANKTM; not shown). Numbers on the right
indicate range in length. CIRB, putative calmodulin- and
IP3R-binding domain; EF hand, canonical helix-loop-helix
Ca.sup.2+-binding domain; PDZ, amino acids binding PDZ domains;
PLIK, phospholipase-C-interacting kinase, an atypical protein
kinase intrinsic to the TRPM6 and TRPM7 polypeptide chains; Nudix,
NUDT9 hydrolase protein homologue binding ADP ribose.
The TRPC (Canonical TRP) Subfamily
[0144] All mammalian TRPC proteins appear to be analogous to the
TRP involved in Drosophila phototransduction, in that they function
as receptor-operated channels. They are activated by stimulation of
G-protein-coupled receptors (GPCRs) and receptor tyrosine
kinases.
[0145] TRPC1, the first mammalian TRP reported, forms heteromeric
channels with TRPC4 and/or TRPC5. The properties of the
heteromultimers are distinct from those of TRPC4 and TRPC5
homomultimers. TRPC5, but not TRPC1, is present in hippocampal
growth cones and modulates neurite extension. Mice lacking TRPC4
have defects in agonist-induced vasoregulation and lung
microvascular permeability.
[0146] TRPC3, TRPC6 and TRPC7 proteins share 75% identity, have
relatively low selectivity for Ca.sup.2+ over Na.sup.+, and are
sensitive to the intracellular concentration of Ca.sup.2+
([Ca.sup.2+]i). Diacylglycerol (DAG) analogs potentiate their
activity, but not through protein kinase C activation. TRPC3 has
been investigated extensively as a putative
inositol-1,4,5-trisphosphate (InsP3) receptor (IP3R)-binding SOC,
with conflicting results. All of the TPRC3, TRPC6 and TRPC7
subfamily are highly expressed in smooth and cardiac muscle cells,
making them candidates for the receptor-activated nonselective
cation channels known to exist in these sites. In support of this
idea, TRPC6 is an essential part of the 1-adrenoreceptor-activated
cation channel in rabbit portal vein myocytes. They may also have
roles in the regulation of vascular tone, airway resistance and
cardiac function. TRPC2 appears to be a pseudogene in humans, but
its rat orthologue encodes an important sensor localized to
neuronal microvilli in the its rat orthologue encodes an important
sensor localized to neuronal microvilli in the vomeronasal organ.
Trpc2-deficient mice display abnormal mating behavior, consistent
with a role for this channel in pheromone signaling.
The TRPV (Vanilloid Receptor, osm9-Like) Subfamily
[0147] TRPV1 was identified by expression cloning using the `hot`
pepper-derived vanilloid compound capsaicin as a ligand. TRPV1 is a
Ca.sup.2+-permeant channel that is potentiated by heat
(>43.degree. C.) and decreased pH, and inhibited by
intracellular phosphatidylinositol-4,5-bisphosphate (PIP2). Its
thermal sensitivity is enhanced by bradykinin and nerve growth
factor, which appear to act via phospholipase C (PLC) to hydrolyse
PIP2, releasing inhibition of the channel. Trpv1-/- mice are
defective in nociceptive, inflammatory and hypothermic responses to
vanilloid compounds, supporting the interpretation that TRPV1
contributes to acute thermal nociception and hyperalgesia after
tissue injury. TRPV1 also participates in mechanically evoked
purinergic signaling by the bladder urothelium.
[0148] TRPV2, which is 50% identical to TRPV1, may mediate
high-threshold (>52.degree. C.) noxious heat sensation, perhaps
through lightly myelinated A nociceptors. Interestingly, TRPV2
translocates from intracellular pools upon insulin growth factor
stimulation of transfected cells. Stretch reportedly increases
TRPV2 translocation, and cardiac-specific transgene expression of
TRPV2 results in Ca.sup.2+-overload-induced cardiomyopathy. But it
is not surprising that overexpression of a Ca.sup.2+-permeant
channel induces cardiomyopathy, because such TRP channels are
deleterious to many cells, including neurons. In fact, the
mechanism of pain-relieving topical capsaicin is due, in part, to
neuronal cell death.
[0149] Increased temperature also activates TRPV3 (>31.degree.
C.) and TRPV4 (>25.degree. C.). The neuronal distribution of
TRPV3 overlaps with TRPV1, raising the interesting possibility that
they may heteromultimerize. TRPV3 is also highly expressed in skin,
tongue and the nervous system, possibly explaining the activity of
`warm-sensitive` neurons. The effect of temperature on rates of
biological processes is expressed as the 10.degree. C. temperature
coefficient1: Q10=rate(T+10.degree. C.)/rate(T). Most ion channels
and enzymes have gating Q10 values of 3-5, but the Q10 of TRPV3
gating is >20, and for TRPV1 and TRPV4 it is estimated to be
10-20. TRPV4 current is potentiated by hypotonicity (cell
swelling). Trpv4-/- mice have a marginally impaired renal response
to hypertonicity, probably due to abnormal central control of
antidiuretic hormone secretion. Hypotonicity increases
TRPV4-mediated current in primary afferent nociceptive nerve
fibers, an effect that is enhanced by the hyperalgesic inflammatory
mediator prostaglandin E2. Expressed TRPV4 may be gated by
epoxyeicosatrienoic acids.
[0150] TRPV5 and TRPV6 comprise a subfamily of homomeric and
heteromeric channels found in transporting epithelia of the kidney
and intestine. They show strong inwardly rectifying currents and
are the most Ca.sup.2+-selective TRP channels (permeability ratio
PCa/PNa>100), suggesting that they mediate Ca.sup.2+ uptake.
Both are inactivated by [Ca.sup.2+]I; TRPV6 shows voltage-dependent
intracellular Mg.sup.2+ blockade.
The TRPM (Melastatin) Subfamily and TRPA
[0151] TRPM1 (melastatin) was initially identified as a transcript
that showed decreased expression in highly metastatic versus
non-metastatic melanoma cells.
[0152] TRPM2 forms a Ca.sup.2+-permeant channel that is gated by
binding of ADP ribose (EC.sub.50 100 .mu.M) and nicotinamide
adenine dinucleotide (NAD; 1 mM) to a carboxy-terminal NUDT9 Nudix
hydrolase domain. ADP ribose is a breakdown product of NAD, CD38,
cyclic ADP ribose (a Ca.sup.2+-release messenger) and protein
de-acetylation (O-acetylated ADP ribose), but the TRPM2 domain
itself is an ineffective hydrolase. The channel is regulated by
signaling pathways responsive to H2O2 and tumor-necrosis factor,
suggesting that it may act as a sensor of intracellular
oxidation/reduction, possibly during the oxidative burst of
neutrophils.
[0153] TRPM3 forms a Ca.sup.2+-permeant nonselective channel that
is constitutively active when heterologously expressed. Its
activity is increased by hypotonicity (200 mOsm per liter). TRPM3
is expressed primarily in kidney distal-collecting-duct epithelium
and in the central nervous system.
[0154] TRPM4 and TRPM5 are the only monovalent-selective ion
channels of the TRP family. They are widely distributed and may
account for observed Ca.sup.2+-activated 20-30 pS nonselective
channel activities. They are activated through GPCRs coupled to
PLC-dependent endoplasmic reticular Ca.sup.2+ release, perhaps by
direct Ca.sup.2+ binding to the channel. However, relatively high
[Ca.sup.2+]i is required to activate these channels, suggesting
that they localize close to sites of Ca.sup.2+ release or that
other modulators are important. Although their instantaneous I-V
relationships are linear, the fraction of open channels increases
at positive potentials. This voltage dependence is not mediated by
divalent cation binding, suggesting an intrinsic voltage-sensing
mechanism.
[0155] TRPM5 is found in cells expressing taste receptors. In an in
vivo study in TrpM5-/- mice, it was shown that taste receptors T1R
and T2R share a common signaling pathway involving PLC2 and TRPM5,
to produce sweet, umami and bitter taste sensations. The authors
concluded that InsP3, Ca.sup.2+ and thapsigargin-mediated store
depletion did not activate TRPM5. However, it is possible that PIP2
or other molecules modulate its sensitivity to [Ca.sup.2+]i.
[0156] TRPM6 and TRPM7 are unique among ion channels because they
also contain functional kinase domains. TRPM7 passes little inward
current under physiological conditions, is permeant to both
Ca.sup.2+ and Mg.sup.2+, and is inhibited by 0.6 mM intracellular
free Mg.sup.2+. In contrast to other GPCR-activated TRP channels,
TRPM7 current increases slowly under whole-cell recording
conditions and is inactivated by PIP2 hydrolysis by PLCbeta or
PLCgamma. The function of the kinase domain is poorly understood
and its substrates have not been identified. The kinase domain, in
contrast to original reports, is not required for channel
activation. The catalytic core of the kinase domain is similar to
that of other eukaryotic protein kinases and to enzymes with
ATP-grasp domains. The sensitivity of TRPM7 to physiological Mg-ATP
levels has been suggested to have a central role in metabolic
sensing or to serve as a mechanism to adjust cellular Mg.sup.2+
homeostasis. But a spontaneous human mutation in TRPM6 results in
familial hypomagnesaemia with secondary hypocalcemia, suggesting
that TRPM6 may be important for Mg.sup.2+ uptake in the kidney and
intestine.
[0157] TRPM8 was identified as a messenger RNA that was upregulated
in prostatic and other cancers. Its sensory role was recognized
when it was isolated by expression cloning of a menthol receptor
from trigeminal neurons. TRPM8 is a nonselective, outwardly
rectifying channel that can be activated by cold (8-28.degree. C.)
and enhanced by `cooling` compounds such as menthol and icilin.
TRPM8 is widely expressed, but thought to function specifically as
a thermosensor in TrkA+, small-diameter primary sensory
neurons.
[0158] ANKTM1, a Ca.sup.2+-permeant, nonselective channel
homologous to Drosophila painless, is distinguished by 14
amino-terminal ankyrin repeats. It is activated by noxious cold
temperature (<15.degree. C.) but bears little similarity to
menthol-sensitive TRPM8. It is found in a subset of nociceptive
sensory dorsal root ganglion neurons, in the company of
capsaicin-sensitive TRPV1, but not TRPM8. Interestingly, the
Drosophila orthologue of ANKTM1 responds to warming (>27.degree.
C.) rather than to cooling when expressed in Xenopus oocytes. These
observations are consistent with sensitivity to the surrounding
membrane environment, but might also be reconciled if the lowest
energy state of the mammalian channel is the open configuration.
Other TRP channels have not been systematically tested for
temperature sensitivity, but such a comparison would clarify this
issue.
The TRPP (Polycystin) and TRPML (Mucolipin) Subfamilies
[0159] Polycystic kidney disease proteins PKD2, PKD2L1 and PKD2L2
are 6TM Ca.sup.2+-permeant channels called TRPP2, TRPP3 and TRPP5,
respectively. The much larger TRPP1, polycystin-REJ and
polycystin-1L1 proteins are 11TM proteins that contain a C-terminal
6TM TRP-like channel domain. TRPP1 is not known to form a channel
by itself, but it complexes with TRPP2 to form a
Ca.sup.2+-permeable nonselective cation channel 70. Autosomal
dominant polycystic kidney disease is caused by mutations in TRPP1
or TRPP2, leading to alterations in the polarization and function
of cyst-lining epithelial cells. Trpp1-/- and Trpp2-/- mice die in
utero with cardiac septal defects and cystic changes in nephrons
and pancreatic ducts. The mouse ortholog of TRPP3 is deleted in krd
mice, resulting in defects in the kidney and retina.
[0160] TRPP proteins have another role in development. Normal body
asymmetry appears to arise from leftward extracellular flow
generated by motor-protein-dependent rotation of monocilia on the
ventral surface of the embryonic node. Motile monocilia generate
nodal flow, and non-motile TRPP2-containing cilia sense nodal flow,
initiating an asymmetric Ca.sup.2+ signal at the left nodal border.
TRPP1 and TRPP2 both appear to be targeted to primary cilia cells
of renal epithelia, where the channel complex is gated by fluid
flow.
[0161] The mucolipins (MCOLN1, MCOLN2 and MCOLN3) are 6TM channels
that are probably restricted to intracellular vesicles. Mutations
in MCOLN1 (TRPML1) are associated with mucolipidosis type IV, a
neurodegenerative lysosomal storage disorder. The defect appears to
be in sorting or transport in the late endocytic pathway. Mutations
in a Caenorhabditis elegans TRPML1 homologue, cup-5, cause excess
lysosome formation and apoptosis in all cell types. TRPML3 is
present in the cytoplasm of hair cells and the plasma membrane of
stereocilia. TRPML3 is mutated in the varitint-waddler mouse,
resulting in deafness and pigmentation defects.
TRP Channel Related Disorders
[0162] Modulation of TRP channel protein levels, protein
interactions, and function may be used to treat disorders
including, but not intended to be limit to:
[0163] Defects in osmoregulation, including brain edema, abdominal
ascites, pulmonary effusion, pericardial effusion and bruising
[0164] Defects in olfaction
[0165] Defects in taste sensation
[0166] Defects in hearing
[0167] Peripheral pain syndromes, including painful neuromas,
denervation pain, and pain due to averrent innervation
[0168] Central pain syndromes, including thalamic pain, denervation
pain, hyperpathia, and hyperalgesia
[0169] Defect in thermoregulation and thermal sensation
(nociception)
[0170] Visual defects, including defects in phototransduction,
glaucoma, retinal ischemia, idiopathic retinopathies, and macular
degeneration
[0171] Defects of vascular tone, including hypertension,
orthostatic hypotension and defects of microvascular regulation
including pulmonary, brain, myocardial edemas, and abdominal
ascites
[0172] Defects of airway resistance, including asthma
[0173] Defects of myocardial function, including cardiomyopathies,
myocardial ischemia, and cardiac arrhythmias
[0174] Defects of bladder and ureteral function
[0175] Defects of renal function.
[0176] Defects of the immune system and inflammatory response
[0177] Defects of tissue senescence/aging
[0178] Defects of the endocrine system and metabolism, including
familial hypomagnesemia with secondary hypocalcemia, diabetes and
mucolipidosis type IV
[0179] Cancer, including prostate [cancer] and melanoma
[0180] Congenital malformations, including atrial septal defects,
polycystic kidneys, congenital retinopathies and congenital
deafness
[0181] Defects in skin pigmentation
V. PDZ Protein and PL Protein Interactions
[0182] TABLES 6, 7, and 8 list PDZ proteins and other PL proteins
which the current inventors have identified as binding to one
another. This column provides the gene name for the PDZ portion of
the GST-PDZ fusion that interacts with the PDZ-ligand to the left.
Many of the genes listed in TABLE 5 express more than one amino
acid sequence, depending on alternative exon splicing and single
amino acid changes. It is understood in the art that many
alternatively spliced and point mutated forms of the same gene may
exist in nature. As indicated supra, all peptides were biotinylated
at the amino terminus and the amino acid sequences correspond to
the C-terminal sequence of the gene name listed in column 1.
[0183] TABLE 5 lists the sequences of the PDZ domains cloned into a
vector (PGEX-3X vector) for production of GST-PDZ fusion proteins
(Pharmacia). More specifically, the first column (left to right)
entitled "Gene Name" lists the name of the gene containing the PDZ
domain. The second column labeled "GI or Acc#" is a unique Genbank
identifier for the gene used to design primers for PCR
amplification of the listed sequence. The next column labeled
"Domain#" indicates the Pfam-predicted PDZ domain number, as
numbered from the amino-terminus of the gene to the
carboxy-terminus. The last column entitled "Sequence fused to GST
Construct" (SEQ ID NO:s) provides the actual amino acid sequence
inserted into the GST-PDZ expression vector as determined by DNA
sequencing of the constructs.
TABLE-US-00006 TABLE 5 Sequences of PDZ Domains Cloned to Produce
GST-PDZ Fusions SEQ GI or ID Gene Name Acc. # Domain # Sequence
fused to GST construct NO: AF6 430993 1
LRKEPEIITVTLKKQNGMGLSIVAAKGAGQDKLGIY 3
VKSVVKGGAADVDGRLAAGDQLLSVDGRSLVGLSQE RAAELMTRTSSVVTLEVAKQG AIPC
12751451 1 LIRPSVISIIGLYKEKGKGLGFSIAGGRDCIRGQMG 4
IFVKTIFPNGSAAEDGRLKEGDEILDVNGIPIKGLT
FQEAIHTFKQIRSGLFVLTVRTKLVSPSLTNSS AIPC 12751451 3
QSENEEDVCFIVLNRKEGSGLGFSVAGGTDVEPKSI 5
TVHRVFSQGAASQEGTMNRGDFLLSVNGASLAGLAH
GNVLKVLHQAQLHKDALVVIKKGMDQPRPSNSS AIPC 12751451 2
GISSLGRKTPGPKDRIVMEVTLNKEPRVGLGIGACC 6
LALENSPPGIYIHSLAPGSVAKMESNLSRGDQILEV
NSVNVRHAALSKVHAILSKCPPGPVRLVIGRHPNPK VSEQEMDEVIARSTYQESKEANSS AIPC
12751451 4 LGRSVAVHDALCVEVLKTSAGLGLSLDGGKSSVTGD 7
GPLVIKRVYKGGAAEQAGIIEAGDEILAINGKPLVG LMHFDAWNIMKSVPEGPVQLLIRKHRNSS
ALP 2773059 1 REEGGMPQTVILPGPAPWGFRLSGGIDFNQPLVITR 8
ITPGSKAAAANLCPGDVILAIDGFGTESMTHADAQD RIKAAAHQLCLKIDRGETHLWSPNSS
APXL1 13651263 1 ILVEVQLSGGAPWGFTLKGGREHGEPLVITKIEEGS 9
KAAAVDKLLAGDEIVGINDIGLSGFRQEAICLVKGS HKTLKLVVKRNSS CARD11 12382772
1 SVGHVRGPGPSVQHTTLNGDSLTSQLTLLGGNARGS 10
FVHSVKPGSLAEKAGLREGHQLLLLEGCIRGERQSV
PLDTCTKEEAHWTIQRCSGPVTLHYKVNHEGYRK CARD14 13129123 1
RRPARRILSQVTMLAFQGDALLEQISVIGGNLTGIF 11
IHRVTPGSAADQMALRPGTQIVMVDYEASEPLFKAV
LEDTTLEEAVGLLRRVDGFCCLSVKVNTDGYKR CARD14 13129123 1
ILSQVTMLAFQGDALLEQISVIGGNLTGIFIHRVTP 12
GSAADQMALRPGTQIVMVDYEASEPLFKAVLEDTTL EEAVGLLRRVDGFCCLSVKVNTDGYKRL
CASK 3087815 1 TRVRLVQFQKNTDEPMGITLKMNELNHCIVARIMHG 13
GMIHRQGTLHVGDEIREINGISVANQTVEQLQKMLR EMRGSITFKIVPSYRTQS CNK1
3930780 1 LEQKAVLEQVQLDSPLGLEIHTTSNCQHFVSQVDTQ 14
VPTDSRLQIQPGDEWQINEQVVVGWPRKNMVRELLR EPAGLSLVLKKIPIP Cytohesin
3192908 1 QRKLVTVEKQDNETFGFEIQSYRPQNQNACSSEMFT 15 binding
TLICKIQEDSPAHCAGLQAGDVLANINGVSTEGFTY Protein KQWDLIRSSGNLLTIETLNG
Densin 16755892 1 RCLIQTKGQRSMDGYPEQFCVRIEKNPGLGFSISGG 16
ISGQGNPFKPSDKGIFVTRVQPDGPASNLLQPGDKI
LQANGHSFVHMEHEKAVLLLKSFQNTVDLVIQRELT V DLG 6 AB053303 1
PTSPEIQELRQMLQAPHFKGATIKRHEMTGDILVAR 17 splice
IIHGGLAERSGLLYAGDKLVEVNGVSVEGLDPEQVI variant 2
HILAMSRGTIMFKWPVSDPPVNSS DLG 6, 14647140 1
PTSPEIQELRQMLQAPHFKALLSAHDTIAQKDFEPL 18 splice
LPPLPDNIPESEEAMRIVCLVKNQQPLGATIKRHEM variant 1
TGDILVARIIHGGLAERSGLLYAGDKLVEVNGVSVE
GLDPEQVIHILAMSRGTIMFKVVPVSDPPVNSS DLG1 475816 1
IQVNGTDADYEYEEITLERGNSGLGFSIAGGTDNPH 19
IGDDSSIFITKIITGGAAAQDGRLRVNDCILQVNEV DVRDVTHSKAVEALKEAGSIVRLYVKRRN
DLG1 475816 2 IQLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIE 20
GGAAHKDGKLQIGDKLLAVNNVCLEEVTHEEAVTAL KNTSDFVYLKVAKPTSMYMNDGN DLG1
475816 1, 2 VNGTDADYEYEEITLERGNSGLGFSIAGGTDNPHIG 21
DDSSIFITKIITGGAAAQDGRLRVNDCILQVNEVDV
RDVTHSKAVEALKEAGSIVRLYVKRRKPVSEKIMEI
KLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEG
GAAHKDGKLQIGDKLLAVNNVCLEEVTHEEAVTALK NTSDFVYLKVAKPTSMYMNDGYA DLG1
475816 3 ILHRGSTGLGFNIVGGEDGEGIFISFILAGGPADLS 22
GELRKGDRIISVNSVDLRAASHEQAAAALKNAGQAV TIVAQYRPEEYSR DLG2 12736552 3
IEGRGILEGEPRKVVLHKGSTGLGFNIVGGEDGEGI 23
FVSFILAGGPADLSGELQRGDQILSVNGIDLRGASH
EQAAAALKGAGQTVTIIAQHQPEDYARFEAKIHDLN SS DLG2 12736552 1
ISYVNGTEIEYEFEEITLERGNSGLGFSIAGGTDNP 24
HIGDDPGIFITKIIPGGAAAEDGRLRVNDCILRVNE VDVSEVSHSKAVEALKEAGSIVRLYVRRR
DLG2 12736552 2 IPILETVVEIKLFKGPKGLGFSIAGGVGNQHIPGDN 25
SIYVTKIIDGGAAQKDGRLQVGDRLLMVNNYSLEEV
THEEAVAILKNTSEVVYLKVGKPTTIYMTDPYGPPN SSLTD DLG5 3650451 1
GIPYVEEPRHVKVQKGSEPLGIS1VSGEKGGIYVSK 26
VTVGSIAHQAGLEYGDQLLEFNGINLRSATEQQARL IIGQQCDTITILAQYNPHVHQLRNSSLTD
DLG5 3650451 2 GILAGDANKKTLEPRVVFIKKSQLELGVHLCGGNLH 27
GVFVAEVEDDSPAKGPDGLVPGDLILEYGSLDVRNK
TVEEVYVEMLKPRDGVRLKVQYRPEEFIVTD DVL1 2291005 1
LNIVTVTLNMERHHFLGISIVGQSNDRGDGGIYIGS 28
IMKGGAVAADGRIEPGDMLLQVNDVNFENMSNDDAV RVLREIVSQTGPISLTVAKCW DVL2
2291007 1 LNIITVTLNMEKYNFLGISLVGQSNERGDGGIYIGS 29
IMKGGAVAADGRIEPGDMLLQVNDMNFENMSNDDAV RVLRDIVHKPGPIVLTVAKCWDPSPQNS
DVL3 6806886 1 IITVTLNMEKYNFLGISIVGQSNERGDGGIYIGSIM 30
KGGAVAADGRIEPGDMLLQVNEINFENMSNDDAVRV LREIVHKPGPITLTVAKCWDPSP EBP50
3220018 2 QQRELRPRLCTMKKGPSGYGFNLHSDKSKPGQFIRS 31
VDPDSPAEASGLRAQDRIVEVNGVCMEGKQHGDVVS AIRAGGDETKLLVVDRETDEFFKNSS
EBP50 3220018 1 GIQMSADAAAGAPLPRLCCLEKGPNGYGFHLHGEKG 32
KLGQYIRLVEPGSPAEKAGLLAGDRLVEVNGENVEK
ETHQQVVSRIRAALNAVRLLVVDPETDEQLQKLGVQ
VREELLRAQEAPGQAEPPAAAEVQGAGNENEPREAD KSHPEQRELRN EBP50 3220018 1, 2
GIQMSADAAAGAPLPRLCCLEKGPNGYGFHLHGEKG 33
KLGQYIRLVEPGSPAEKAGLLAGDRLVEVNGENVEK
ETHQQVVSRIRAALNAVRLLVVDPETDEQLQKLGVQ
VREELLRAQEAPGQAEPPAAAEVQGAGNENEPREAD
KSHPEQRELRPRLCTMKKGPSGYGFNLHSDKSKPGQ
FIRSVDPDSPAEASGLRAQDRIVEVNGVCMEGKQHG DVVSAIRAGGDETKLLVVDRETDEFFK
EBP50 3220018 1 QMSADAAAGAPLPRLCCLEKGPNGYGFHLHGEKGKL 34
GQYIRLVEPGSPAEKAGLLAGDRLVEVNGENVEKET
HQQVVSRIRAALNAVRLLVVDPETDEQLQKLGVQVR
EELLRAQEAPGQAEPPAAAEVQGAGNENEPREADKS HPEQRELRNSS ELFIN 1 2957144 1
LTTQQIDLQGPGPWGFRLVGGKDFEQPLAISRVTPG 35
SKAALANLCIGDVITAIDGENTSNMTHLEAQNRIKG CTDNLTLTVARSEHKVWSPLVTNSSW
ENIGMA 561636 1 IFMDSFKVVLEGPAPWGFRLQGGKDFNVPLSISRLT 36
PGGKAAQAGVAVGDWVLSIDGENAGSLTHIEAQNKI RACGERLSLGLSRAQPV ERBIN
8923908 1 QGHELAKQEIRVRVEKDPELGFSISGGVGGRGNPFR 37
PDDDGIFVTRVQPEGPASKLLQPGDKIIQANGYSFI NIEHGQAVSLLKTFQNTVELIIVREVSS
FLJ00011 10440352 1 KNPSGELKTVTLSKMKQSLGISISGGIESKVQPMVK 38
IEKIFPGGAAFLSGALQAGFELVAVDGENLEQVTHQ
RAVDTIRRAYRNKAREPMELVVRVPGPSPRPSPSD FLJ11215 11436365 1
EGHSHPRVVELPKTEEGLGFNIMGGKEQNSPIYISR 39
IIPGGIADRHGGLKRGDQLLSVNGVSVEGEHHEKAV ELLKAAQGKVKLVVRYTPKVLEEME
FLJ12428 BC012040 1 PGAPYARKTFTIVGDAVGWGFVVRGSKPCHIQAVDP 40
SGPAAAAGMKVCQFVVSVNGLNVLHVDYRTVSNLIL TGPRTIVMEVMEELEC FLJ12615
10434209 1 GQYGGETVKIVRIEKARDIPLGATVRNEMDSVIISR 41
IVKGGAAEKSGLLHEGDEVLEINGIEIRGKDVNEVF DLLSDMHGTLTFVLIPSQQIKPPPA
FLJ21687 10437836 1 KPSQASGHFSVELVRGYAGFGLTLGGGRDVAGDTPL 42
AVRGLLKDGPAQRCGRLEVGDLVLHINGESTQGLTH
AQAVERIRAGGPQLHLVIRRPLETHPGKPRGV FLJ31349 AK055911 1
PVMSQCACLEEVHLPNIKPGEGLGMYIKSTYDGLHV 43
ITGTTENSPADRSQKIHAGDEVTQVNQQTVVGWQLK NLVKKLRENPTGVVLLLKKRPTGSFNFTP
FLJ32798 AK057360 1 IDDEEDSVKIIRLVKNREPLGATIKKDEQTGAIIVA 44
RIMRGGAADRSGLIHVGDELREVNGIPVEDKRPEEI IQILAQSQGAITFKIIPGSKEETPS
GORASP 2 13994253 1, 2 MGSSQSVEIPGGGTEGYHVLRVQENSPGHRAGLEPF 45
FDFIVSINGSRLNKDNDTLKDLLKANVEKPVKMLIY
SSKTLELRETSVTPSNLWGGQGLLGVSIRFCSFDGA
NENVWHVLEVESNSPAALAGLRPHSDYIIGADTVMN
ESEDLFSLIETHEAKPLKLYVYNTDTDNCREVIITP
NSAWGGEGSLGCGIGYGYLHRIPTRPFEEGKKISLP
GQMAGTPITPLKDGFTEVQLSSVNPPSLSPPGTTGI
EQSLTGLSISSTPPAVSSVLSTGVPTVPLLPPQVNQ
SLTSVPPMNPATTLPGLMPLPAGLPNLPNLNLNLPA
PHIMPGVGLPELVNPGLPPLPSMPPRNLPGIAPLPL
PSEFLPSFPLVPESSSAASSGELLSSLPPTSNAPSD
PATTTAKADAASSLTVDVTPPTAKAPTTVEDRVGDS TPVSEKPVSAAVDANASESP GORASP 2
13994253 2 NENVWHVLEVESNSPAALAGLRPHSDYIIGADTVMN 46
ESEDLFSLIETHEAKPLKLYVYNTDTDNCREVIITP NSAWGGEGSLGCGIGYGYLHRIPTR
GORASP 2 13994253 1 MGSSQSVEIPGGGTEGYHVLRVQENSPGHRAGLEPF 47
FDFIVSINGSRLNKDNDTLKDLLKANVEKPVKMLIY
SSKTLELRETSVTPSNLWGGQGLLGVSIRFCSFDGA NE GORASP 1 29826292 2
RASEQVWHVLDVEPSSPAALAGLRPYTDYVVGSDQI 48
LQESEDFFTLIESHEGKPLKLMVYNSKSDSCREVTV TPNAAWGGEGSLGCGIGYGYLHRIPTQ
GORASP 1 29826292 1 MGLGVSAEQPAGGAEGFHLHGVQENSPAQQAGLEPY 49
FDFIITIGHSRLNKENDTLKALLKANVEKPVKLEVF
NMKTMRVREVEWPSNMWGGQGLLGASVRFCSFRRAS E GORASP 1 29826292 1, 2
MGLGVSAEQPAGGAEGFHLHGVQENSPAQQAGLEPY 50
FDFIITIGHSRLNKENDTLKALLKANVEKPVKLEVF
NMKTMRVREVEVVPSNMWGGQGLLGASVRFCSFRRA
SEQVWHVLDVEPSSPAALAGLRPYTDYVVGSDQILQ
ESEDFFTLIESHEGKPLKLMVYNSKSDSCREVTVTP
NAAWGGEGSLGCGIGYGYLHRIPTQPPSYHKKPPGT
PPPSALPLGAPPPDALPPGPTPEDSPSLETGSRQSD
YMEALLQAPGSSMEDPLPGPGSPSHSAPDPDGLPHF
METPLQPPPPVQRVMDPGFLDVSGISLLDNSNASVW
PSLPSSTELTTTAVSTSGPEDICSSSSSHERGGEAT
WSGSEFEVSFLDSPGAQAQADHLPQLTLPDSLTSAA
SPEDGLSAELLEAQAEEEPASTEGLDTGTEAEGLDS QAQISTTE GRIP 1 4539083 6
IYTVELKRYGGPLGITISGTEEPFDPIIISSLTKGG 51
LAERTGAIHIGDRILAINSSSLKGKPLSEAIHLLQM AGETVTLKIKKQTDAQSA GRIP 1
4539083 1 VVELMKKEGTTLGLTVSGGIDKDGKPRVSNLRQGGI 52
AARSDQLDVGDYIKAVNGINLAKFRHDEIISLLKNV GERVVLEVEYE GRIP 1 4539083 3
HVATASGPLLVEVAKTPGASLGVALTTSMCCNKQVI 53
VIDKIKSASIADRCGALHVGDHILSIDGTSMEYCTL
AEATQFLANTTDQVKLEILPHHQTRLALKGPNSS GRIP 1 4539083 7
IMSPTPVELHKVTLYKDSDMEDFGFSVADGLLEKGV 54
YVKNIRPAGPGDLGGLKPYDRLLQVNHVRTRDFDCC LVVPLIAESGNKLDLVISRNPLA GRIP 1
4539083 4 IYTVELKRYGGPLGITISGTEEPFDPIIISSLTKGG 55
LAERTGAIHIGDRILAINSSSLKGKPLSEAIHLLQM AGETVTLKIKKQTDAQSA GRIP 1
4539083 5 IMSPTPVELHKVTLYKDSDMEDFGFSVADGLLEKGV 56
YVKNIRPAGPGDLGGLKPYDRLLQVNHVRTRDFDCC LVVPLIAESGNKLDLVISRNPLA GTPase
2389008 1 SRGCETRELALPRDGQGRLGFEVDAEGFVTHVERFT 57 activating
FAETAGLRPGARLLRVCGQTLPSLRPEAAAQLLRSA enzyme PKVCVTVLPPDESGRP
Guanine 6650765 1 CSVMIFEVVEQAGAIILEDGQELDSWYVILNGTVEI 58 exchange
SHPDGKVENLFMGNSFGITPTLDKQYMHGIVRTKVD factor
DCQFVCIAQQDYWRILNHVEKNTHKVEEEGEIVMVH HEMBA 10436367 2
PRETVKIPDSADGLGFQIRGFGPSVVHAVGRGTVAA 59 1000505
AAGLHPGQCIIKVNGINVSKETHASVIAHVTACRKY RRPTKQDSIQ HEMBA 10436367 1
LENVIAKSLLIKSNEGSYGFGLEDKNKVPIIKLVEK 60 1000505
GSNAEMAGMEVGKKIFAINGDLVFMRPFNEVDCFLK SCLNSRKPLRVLVSTKP HEMBA
7022001 1 EDFCYVFTVELERGPSGLGMGLIDGMHTHLGAPGLY 61 1003117
IQTLLPGSPAAADGRLSLGDRILEVNGSSLLGLGYL RAVDLIRHGGKKMRFLVAKSDVETAKKI
hShroom 18652858 1 IYLEAFLEGGAPWGFTLKGGLEHGEPLIISKVEEGG 62
KADTLSSKLQAGDEVVHINEVTLSSSRKEAVSLVKG SYKTLRLVVRRDVCTDPGH HSPC227
7106843 1 NNELTQFLPRTITLKKPPGAQLGFNIRGGKASQLGI 63
FISKVIPDSDAHRAGLQEGDQVLAVNDVDFQDIEHS KAVEILKTAREISMRVRFFPYNYHRQKE
HTRA 3 AY040094 1 FLTEFQDKQIKDWKKRFIGIRMRTITPSLVDELKAS 64
NPDFPEVSSGIYVQEVAPNSPSQRGGIQDGDIIVKV
NGRPLVDSSELQEAVLTESPLLLEVRRGNDDLLFS HTRA 4 AL576444 1
NKKYLGLQMLSLTVPLSEELKMHYPDFPDVSSGVYV 65
CKVVEGTAAQSSGLRDHDVIVNINGKPITTTTDVVK ALDSDSLSMAVLRGKDNLLLTV INADL
2370148 3 PGSDSSLFETYNVELVRKDGQSLGIRIVGYVGTSHT 66
GEASGIYVKSIIPGSAAYHNGHIQVNDKIVAVDGVN
IQGFANHDVVEVLRNAGQVVHLTLVRRKTSSSTSRI HRD INADL 2370148 8
PATCPIVPGQEMIIEISKGRSGLGLSIVGGKDTPLN 67
AIVIHEVYEEGAAARDGRLWAGDQILEVNGVDLRNS SHEEAITALRQTPQKVRLVVY INADL
2370148 2 LPETVCWGHVEEVELINDGSGLGFGIVGGKTSGVVV 68
RTIVPGGLADRDGRLQTGDHILKIGGTNVQGMTSEQ VAQVLRNCGNSVRMLVARDPAGDIQSPI
INADL 2370148 6 PNFSHWGPPRIVEIFREPNVSLGISIVVGQTVIKRL 69
KNGEELKGIFIKQVLEDSPAGKTNALKTGDKILEVS
GVDLQNASHSEAVEAIKNAGNPVVFIVQSLSSTPRV IPNVHNKANSS INADL 2370148 7
PGELHIIELEKDKNGLGLSLAGNKDRSRMSIFVVGI 70
NPEGPAAADGRMRIGDELLEINNQILYGRSHQNASA IIKTAPSKVKLVFIRNEDAVNQMANSS
INADL 2370148 5 LSSPEVKIVELVKDCKGLGFSILDYQDPLDPTRSVI 71
VIRSLVADGVAERSGGLLPGDRLVSVNEYCLDNTSL AEAVEILKAVPPGLVHLGICKPLVEFIVTD
INADL 2370148 1 IWQIEYIDIERPSTGGLGFSVVALRSQNLGKVDIFV 72
KDVQPGSVADRDQRLKENDQILAINHTPLDQNISHQ
QAIALLQQTTGSLRLIVAREPVHTKSSTSSSE INADL 2370148 4
NSDDAELQKYSKLLPIHTLRLGVEVDSFDGHHYISS 73
IVSGGPVDTLGLLQPEDELLEVNGMQLYGKSRREAV SFLKEVPPPFTLVCCRRLFDDEAS
KIAA0313 7657260 1 HLRLLNIACAAKAKRRLMTLTKPSREAPLPFILLGG 74
SEKGFGIFVDSVDSGSKATEAGLKRGDQILEVNGQN
FENIQLSKAMEILRNNTHLSITVKTNLFVFKELLTR LSEEKRNGAP KIAA0316 6683123 1
IPPAPRKVEMRRDPVLGFGFVAGSEKPWVRSVTPGG 75
PSEGKLIPGDQIVMINDEPVSAAPRERVIDLVRSCK ESILLTVIQPYPSPK KIAA0340
2224620 1 LNKRTTMPKDSGALLGLKVVGGKMTDLGRLGAFITK 76
VKKGSLADVVGHLRAGDEVLEWNGKPLPGATNEEVY NIILESKSEPQVEIIVSRPIGDIPRIHRD
KIAA0380 2224700 1 RCVIIQKDQHGFGFTVSGDRIVLVQSVRPGGAAMKA 77
GVKEGDRIIKVNGTMVTNSSHLEVVKLIKSGAYVAL TLLGS KIAA0382 7662087 1
ILVQRCVIIQKDDNGFGLTVSGDNPVFVQSVKEDGA 78
AMRAGVQTGDRIIKVNGTLVTHSNHLEVVKLIKSGS YVALTVQGRPPGNSS KIAA0440
2662160 1 SVEMTLRRNGLGQLGFHVNYEGIVADVEPYGYAWQA 79
GLRQGSRLVEICKVAVATLSHEQMIDLLRTSVTVKV VIIPPH KIAA0545 14762850 1
LKVMTSGWETVDMTLRRNGLGQLGFHVKYDGTVAEV 80
EDYGFAWQAGLRQGSRLVEICKVAVVTLTHDQMIDL LRTSVTVKVVIIPPFEDGTPRRGW
KIAA0559 3043641 1 HYIFPHARIKITRDSKDHTVSGNGLGIRIVGGKEIP 81
GHSGEIGAYIAKILPGGSAEQTGKLMEGMQVLEWNG
IPLTSKTYEEVQSIISQQSGEAEICVRLDLNML KIAA0613 3327039 1
SYSVTLTGPGPWGFRLQGGKDFNMPLTISRITPGSK 82
AAQSQLSQGDLVVAIDGVNTDTMTHLEAQNKIKSAS YNLSLTLQKSKNSS KIAA0858
4240204 1 FSDMRISINQTPGKSLDFGFTIKWDIPGIFVASVEA 83
GSPAEFSQLQVDDEIIAINNTKFSYNDSKEWEEAMA KAQETGHLVMDVRRYGKAGSPE
KIAA0902 4240292 1 QSAHLEVIQLANIKPSEGLGMYIKSTYDGLHVITGT 84
TENSPADRCKKIHAGDEVIQVNHQTVVGWQLKNLVN ALREDPSGVILTLKKRPQSMLTSAPA
KIAA0967 4589577 1 ILTQTLIPVRHTVKIDKDTLLQDYGFHISESLPLTV 85
VAVTAGGSAHGKLFPGDQILQMNNEPAEDLSWERAV DILREAEDSLSITVVRCTSGVPKSSNSS
KIAA1202 6330421 1 RSFQYVPVQLQGGAPWGFTLKGGLEHCEPLTVSKIE 86
DGGKAALSQKMRTGDELVNINGTPLYGSRQEALILI KGSFRILKLIVRRRNAPVS KIAA1222
6330610 1 ILEKLELFPVELEKDEDGLGISIIGMGVGADAGLEK 87
LGIFVKTVTEGGAAQRDGRIQVNDQIVEVDGISLVG
VTQNFAATVLRNTKGNVRFVIGREKPGQVSE KIAA1284 6331369 1
KDVNVYVNPKKLTVIKAKEQLKLLEVLVGIIHQTKW 88
SWRRTGKQGDGERLVVHGLLPGGSAMKSGQVLIGDV
LVAVNDVDVTTENIERVLSCIPGPMQVKLTFENAYD VKRET KIAA1389 7243158 1
TRGCETVEMTLRRNGLGQLGFHVNFEGIVADVEPFG 89
FAWKAGLRQGSRLVEICKVAVATLTHEQMIDLLRTS VTVKVVIIQPHDDGSPRR KIAA1415
7243210 1 VENILAKRLLILPQEEDYGFDIEEKNKAVVVKSVQR 90
GSLAEVAGLQVGRKIYSINEDLVFLRPFSEVESILN QSFCSRRPLRLLVATKAKEIIKIP
KIAA1526 5817166 1 PDSAGPGEVRLVSLRRAKAHEGLGFSIRGGSEHGVG 91
IYVSLVEPGSLAEKEGLRVGDQILRVNDKSLARVTH
AEAVKALKGSKKLVLSVYSAGRIPGGYVTNH KIAA1526 5817166 2
LQGGDEKKVNLVLGDGRSLGLTIRGGAEYGLGIYIT 92
GVDPGSEAEGSGLKVGDQILEVNGRSFLNILHDEAV RLLKSSRHLILTVKDVGRLPHARTTVDE
KIAA1620 10047316 1 LRRAELVEIIVETEAQTGVSGINVAGGGKEGIFVRE 93
LREDSPAARSLSLQEGDQLLSARVFFENFKYEDALR LLQCAEPYKVSFCLKRTVPTGDLALR
KIAA1719 1267982 5 IQTTGAVSYTVELKRYGGPLGITISGTEEPFDPIVI 94
SGLTKRGLAERTGAIHVGDRILAINNVSLKGRPLSE AIHLLQVAGETVTLKIKKQLDR
KIAA1719 1267982 6 ILEMEELLLPTPLEMHKVTLHKDPMRHDFGFSVSDG 95
LLEKGVYVHTVRPDGPAHRGGLQPFDRVLQVNHVRT
RDFDCCLAVPLLAEAGDVLELIISRKPHTAHSS KIAA1719 1267982 2
IHTVANASGPLMVEIVKTPGSALGISLTTTSLRNKS 96
VITIDRIKPASVVDRSGALHPGDHILSIDGTSMEHC SLLEATKLLASISEKVRLEILPVPQSQRPL
KIAA1719 1267982 1 ITVVELIKKEGSTLGLTISGGTDKDGKPRVSNLRPG 97
GLAARSDLLNIGDYIRSVNGIHLTRLRHDEIITLLK NVGERVVLEVEY KIAA1719 1267982
3 IQIVHTETTEVVLCGDPLSGFGLQLQGGIFATETLS 98
SPPLVCFIEPDSPAERCGLLQVGDRVLSINGIATED
GTMEEANQLLRDAALAHKVVLEVEFDVAESV KIAA1719 1267982 1
ILDVSLYKEGNSFGFVLRGGAHEDGHKSRPLVLTYV 99
RPGGPADREGSLKVGDRLLSVDGIPLHGASHATALA TLRQCSHEALFQVEYDVATP KIAAI719
1267982 4 QFDVAESVIPSSGTFHVKLPKKRSVELGITISSASR 100
KRGEPLIISDIKKGSVAHRTGTLEPGDKLLAIDNIR LDNCPMEDAVQILRQCEDLVKLKIRKDEDN
LIM 12734250 1 MALTVDVAGPAPWGFRITGGRDFHTPIMVTKVAERG 101 mystique
KAKDADLRPGDIIVAINGESAEGMLHAEAQSKIRQS PSPLRLQLDRSQATSPGQT LIM
3108092 1 SNYSVSLVGPAPWGFRLQGGKDFNMPLTISSLKDGG 102 protein
KAAQANVRIGDVVLSIDGINAQGMTHLEAQNKIKGC TGSLNMTLQRAS LIMK1 4587498 1
TLVEHSKLYCGHCYYQTVVTPVIEQILPDSPGSHLP 103
HTVTLVSIPASSHGKRGLSVSIDPPHGPPGCGTEHS
HTVRVQGVDPGCMSPDVKNSIHVGDRILEINGTPIR NVPLDEIDLLIQETSRLLQLTLEHD
LIMK2 1805593 1 PYSVTLISMPATTEGRRGFSVSVESACSNYATTVQV 104
KEVNRMHISPNNRNAIHPGDRILEINGTPVRTLRVE EVEDAISQTSQTLQLLIEHD LIM-RIL
1085021 1 IHSVTLRGPSPWGFRLVGRDFSAPLTISRVHAGSKA 105
SLAALCPGDLIQAINGESTELMTHLEAQNRIKGGHD HLTLSVSRPE LU-1 U52111 1
VCYRTDDEEDLGIYVGEVNPNSIAAKDGRIREGDRI 106
IQINGVDVQNREEAVAILSQEENTNISLLVARPESQ LA MAGI 1 3370997 2
IPATQPELITVHIVKGPMGFGFTIADSPGGGGQRVK 107
QIVDSPRCRGLKEGDLIVEVNKKNVQALTHNQVVDM LVECPKGSEVTLLVQRGGNSS MAGI 1
3370997 5 IPDYQEQDIFLWRKETGFGFRILGGNEPGEPIYIGH 108
IVPLGAADTDGRLRSGDELICVDGTPVIGKSHQLVV
QLMQQAAKQGHVNLTVRRKVVFAVPKTENSS MAGI 1 3370997 4
IPGVVSTVVQPYDVEIRRGENEGFGFVIVSSVSRPE 109
AGTTFAGNACVAMPHKIGRIIEGSPADRCGKLKVGD
RILAVNGCSITNKSHSDIVNLIKEAGNTVTLRIIPG DESSNAEFIVTD MAGI 1 3370997 1
IPSELKGKFIHTKLRKSSRGFGFTVVGGDEPDEFLQ 110
IKSLVLDGPAALDGKMETGDVIVSVNDTCVLGHTHA
QVVKIFQSIPIGASVDLELCRGYPLPFDPDGIHRD MAGI 1 3370997 3
QATQEQDFYTVELERGAKGFGFSLRGGREYNMDLYV 111
LRLAEDGPAERCGKMRIGDEILEINGETTKNMKHSR AIELIKNGGRRVRLFLKRG Magi 2
2947231 1 REKPLFTRDASQLKGTFLSTTLKKSNMGFGFTIIGG 112
DEPDEFLQVKSVIPDGPAAQDGKMETGDVIVYINEV
CVLGHTHADVVKLFQSVPIGQSVNLVLCRGYP Magi 2 2947231 3
HYKELDVHLRRMESGFGFRILGGDEPGQPILIGAVI 113
AMGSADRDGRLHPGDELVYVDGIPVAGKTHRYVIDL MHHAARNGQVNLTVRRKVLCG Magi 2
2947231 4 EGRGISSHSLQTSDAVIHRKENEGFGFVIISSLNRP 114
ESGSTITVPHKIGRIIDGSPADRCAKLKVGDRILAV
NGQSIINMPHADIVKLIKDAGLSVTLRIIPQEEL Magi 2 2947231 2
LSGATQAELMTLTIVKGAQGFGFTIADSPTGQRVKQ 115
ILDIQGCPGLCEGDLIVEINQQNVQNLSHTEVVDIL
KDCPIGSETSLIIHRGGFF Magi 2 2947231 5
LSDYRQPQDFDYFTVDMEKGAKGFGFSIRGGREYKM 116
DLYVLRLAEDGPAIRNGRMRVGDQIIEINGESTRDM THARAIELIKSGGRRVRLLLKRGTGQ
Magi 2 2947231 6 HESVIGRNPEGQLGFELKGGAENGQFPYLGEVKPGK 117
VAYESGSKLVSEELLLEVNETPVAGLTIRDVLAVIK HCKDPLRLKCVKQGGIJHR MAGI 3
10047344? 2 ASSGSSQPELVTIPLIKGPKGFGFAIADSPTGQKVK 118
MILDSQWCQGLQKGDIIKEIYHQNVQNLTHLQVVEV LKQFPVGADVPLLILRGGPPSPTKTAKM
MAGI 3 10047344 5 QNLGCYPVELERGPRGFGFSLRGGKEYNMGLFILRL 119
AEDGPAIKDGRIHVGDQIVEINGEPTQGITHTRAIE LIQAGGNKVLLLLRPGTGLIPDHGLA
MAGI 3 10047344 3 LYEDKPPNTKDLDVFLRKQESGFGFRVLGGDGPDQS 120
IYIGAIIPLGAAEKDGRLRAADELMCIDGIPVKGKS
HKQVLDLMTTAARNGHVLLTVRRKIFYGEKQPEDDS MAGI 3 10047344 1
PSQLKGVLVRASLKKSTMGFGFTIIGGDRPDEFLQV 121
KNVLKDGPAAQDGKIAPGDVIVDINGNCVLGHTHAD VVQMFQLVPVNQYVNLTLCRGYPLPDDSED
MAGI 3 10047344 4 PAPQEPYDVVLQRKENEGFGFVILTSKNKPPPGVIP 122
HKIGRVIEGSPADRCGKLKVGDHISAVNGQSIVELS HDNIVQLIKDAGVTVTLTVIAEEEHHGPPS
MAST1 4589589 1 GLRSPITIQRSGKKYGFTLRAIRVYMGDTDVYSVHH 123
IVWHVEEGGPAQEAGLCAGDLITHVNGEPVHGMVHP EVVELILKSGNKVAVTTTPFEN MAST2
3882334 1 ISALGSMRPPIIIHRAGKKYGFTLRAIRVYMGDSDV 124
YTVHHMVWHVEDGGPASEAGLRQGDLITHVNGEPVH GLVHTEVVELILKSGNKVAISTTPLENSS
MAST3 3043645 1 LCGSLRPPIVIHSSGKKYGFSLRAIRVYMGDSDVYT 125
VHHVVWSVEDGSPAQEAGLRAGDLITHINGESVLGL
VHMDVVELLLKSGNKISLRTTALENTSIKVG MAST4 2224546 1
PHQPIVIHSSGKNYGETIRAIRVYVGDSDIYTVHHI 126
VWNVEEGSPACQAGLKAGDLITHINGEPVHGLVHTE VIELLLKSGNKVSITTTPF MGC5395
BC012477 1 PAKMEKEETTRELLLPNWQGSGSHGLTIAQRDDGVF 127
VQEVTQNSPAARTGVVKEGDQIVGATIYFDNLQSGE VTQLLNTMGHHTVGLKLHRKGDRSPNSS
MINT1 2625024 1 SENCKdVFIEKQKGEILGVVIVESGWGSILPTVIIA 128
NMMHGGPAEKSGKLNIGDQIMSINGTSLVGLPLSTC QSIIKGLKNQSRVKLNIVRCPPVNSS
MINT1 2625024 1, 2 SENCKDVFIEKQKGEILGVVIVESGWGSILPTVIIA 129
NMMHGGPAEKSGKLNIGDQIMSINGTSLVGLPLSTC
QSIIKGLENQSRVKLNIVRCPPVTTVLIRRPDLRYQ
LGFSVQNGIICSLMRGGIAERGGVRVGHRIIEINGQ
SVVATPHEKIVHILSNAVGEIHMKTMPAAMYRLL MINT1 2625024 2
LRCPPVTTVLIRRPDLRYQLGFSVQNGIICSLMRGG 130
IAERGGVRVGHRIIEINGQSVVATPHEKIVHILSNA VGEIHMKTMPAAMYRLLNSS MINT3
3169808 1 HNGDLDHFSNSDNCREVHLEKRRGEGLGVALVESGW 131
GSLLPTAVIANLLHGGPAERSGALSIGDRLTAINGT
SLVGLPLAACQAAVRETKSQTSVTLSIVHCPPVT MINT3 3169808 2
PVTTAIIHRPHAREQLGFCVEDGIICSLLRGGIAER 132
GGIRVGHRIIEINGQSVVATPHARIIELLTEAYGEV HIKTMPAATYRLLTGNSS MINT3
3169808 1 LSNSDNCREVHLEKRRGEGLGVALVESGWGSLLPTA 133
VIANLLHGGPAERSGALSIGDRLTAINGTSLVGLPL
AACQAAVRETKSQTSVTLSIVHCPPVTTAIM MPP1 189785 1
RKVRLIQFEKVTEEPMGITLKLNEKQSCTVARILHG 134
GMIHRQGSLHVGDEILEINGTNVTNHSVDQLQKAMK ETKGMISLKVIPNQ MPP2 939884 1
PVPPDAVRMVGIRKTAGEHLGVTFRVEGGELVIARI 135
LHGGMVAQQGLLHVGDIIKEVNGQPVGSDPRALQEL LRNASGSVILKILPNYQ MPP3
21536463 1 NIDEDFDEESVKIVRLVKNKEPLGATIRRDEHSGAV 136
VVARIMRGGAADRSGLVHVGDELREVNGIAVLHKRP DEISQILAQSQGSITLKIIPATQEEDR
MUPP1 2104784 5 WEAGIQHIELEKGSKGLGFSILDYQDPIDPASTVII 137
IRSLVPGGIAEKDGRLLPGDRLMFVNDVNLENSSLE EAVEALKGAPSGTVRIGVAKPLPLSPEE
MUPP1 2104784 12 LQGLRTVEMKKGPTDSLGISIAGGVGSPLGDVPIFI 138
AMMHPTGVAAQTQKLRVGDRIVTICGTSTEGMTHTQ AVNLLKNASGSIEMQVVAGGDVSV MUPP1
2104784 2 PVHWQHMETIELVNDGSGLGFGIIGGKATGVIVKTI 139
LPGGVADQHGRLCSGDHILKIGDTDLAGMSSEQVAQ VLRQCGNRVKLMIARGAIEERTAPT
MUPP1 2104784 3 QESETFDVELTKNVQGLGITIAGYIGDKKLEPSGIF 140
VKSITKSSAVEHDGRIQIGDQIIAVDGTNLQGFTNQ QAVEVLRHTGQTVLLTLMRRGMKQEA
MUPP1 2104784 11 KEEEVCDTLTIELQKKPGKGLGLSIVGKRNDTGVFV 141
SDIVKGGIADADGRLMQGDQILMVNGEDVRNATQEA VAALLKCSLGTVTLEVGRIKAGPFHS
MUPP1 2104784 8 LTGELHMIELEKGHSGLGLSLAGNKDRSRMSVFIVG 142
IDPNGAAGKDGRLQIADELLEINGQILYGRSHQNAS SIIKCAPSKVKIIFIRNKDAVNQ MUPP1
2104784 13 LGPPQCKSITLERGPDGLGFSIVGGYGSPHGDLPIY 143
VKTVFAKGAASEDGRLKRGDQIIAVNGQSLEGVTHE EAVAILKRTKGTVTLMVLS MUPP1
2104784 6 RNVSKESFERTINIAKGNSSLGMTVSANKDGLGMIV 144
RSIIHGGAISRDGRIAIGDCILSINEESTISVTNAQ ARAMLRRHSLIGPDIKITYVPAEHLEE
MUPP1 2104784 10 LPGCETTIEISKGRTGLGLSIVGGSDTLLGAIIIHE 145
VYEEGAACKDGRLWAGDQILEVNGIDLRKATHDEAI NVLRQTPQRVRLTLYRDEAPYKE MUPP1
2104784 7 LNWNQPRRVELWREPSKSLGISIVGGRGMGSRLSNG 146
EVMRGIFIKHVLEDSPAGKNGTLKPGDRIVEVDGMD
LRDASHEQAVEAIRKAGNPWFMVQSIINRPRKSPLP SLL MUPP1 2104784 9
LSSFKNVQHLELPKDQGGLGIAISEEDTLSGVIIKS 147
LTEHGVAATDGRLKVGDQILAVDDEIVVGYPIEKFI SLLKTAKMTVKLTIHAENPDSQ MUPP1
2104784 1 QGRHVEVFELLKPPSGGLGFSVVGLRSENRGELGIF 148
VQEIQEGSVAHRDGRLKETDQILAINGQALDQTITH QQAISILQKAKDTVQLVIARGSLPQLV
MUPP1 2104784 4 LNYEIVVAHVSKFSENSGLGISLEATVGHHFIRSVL 149
PEGPVGHSGKLFSGDELLEVNGITLLGENHQDVVNI LKELPIEVTMVCCRRTVPPT NeDLG
10863920 2 ITLLKGPKGLGFSIAGGIGNQHIPGDNSIYITKIIE 150
GGAAQKDGRLQIGDRLLAVNNTNLQDVRHEEAVASL KNTSDMVYLKVAKPGSLE NeDLG
10863920 1 IQYEEIVLERGNSGLGFSIAGGIDNPHVPDDPGIFI 151
TKIIPGGAAAMDGRLGVNDCVLRVNEVEVSEVVHSR AVEALKEAGPVVRLVVRRRQN NeDLG
10863920 3 ILLHKGSTGLGFNIVGGEDGEGIFVSFILAGGPADL 152
SGELRRGDRILSVNGVNLRNATHEQAAAALKRAGQS
VTIVAQYRPEEYSRFESKIHDLREQMMNSSMSSGSG SLRTSEKRSLE NeDLG 10863920 1,
2 YEEIVLERGNSGLGFSIAGGIDNPHVPDDPGIFITK 153
IIPGGAAAMDGRLGVNDCVLRVNEVEVSEVVHSRAV
EALKEAGPVVRLVVRRRQPPPETIMEVNLLKGPKGL
GFSIAGGIGNQHIPGDNSIYITKIIEGGAAQKDGRL
QIGDRLLAVNNTNLQDVRHEEAVASLKNTSDMVYLK VAKPGSL Neurabin II AJ401189 1
RVERLELFPVELEKDSEGLGISIIGMGAGADMGLEK 154
LGIFVKTVTEGGAAHRDGRIQVNDLLVEVDGTSLVG
VTQSFAASVLRNTKGRVRCRFMIGRERPGEQSEV NOS1 642525 1
QPNVISVRLFKRKVGGLGFLVKERVSKPPVIISDLI 155
RGGAAEQSGLIQAGDIILAVNGRPLVDLSYDSALEV LRGIASETHVVLILRGPE novel
7228177 2 PSDTSSEDGVRRIVHLYTTSDDFCLGFNIRGGKEFG 156 PDZ
LGIYVSKVDHGGLAEENGIKVGDQVLAANGVRFDD1 gene
SHSQAVEVLKGQTHIMLTIKETGRYPAYKEM novel 7228177 1
EANSDESDIIHSVRVEKSPAGRLGFSVRGGSEHGLG 157 PDZ
IFVSKVEEGSSAERAGLCVGDKITEVNGLSLESTTM gene
GSAVKVLTSSSRLHMMVRRMGRVPGIKESKEK novel 1621243 1
DKIKKFLTESHDRQAKGKAITKKKYIGIRMMSLTSS 158 serine
KAKELKDRHRDFPDVISGAYIIEVIPDTPAEAGGLK protease
ENDVIISINGQSVVSANDVSDVIKRESTLNMVVRRG NEDIMITV Numb BP AK056823 2
YRPRDDSFHVILNKSSPEEQLGIKLVRKVDEPGVFI 159
FNALDGGVAYRHGQLEENDRVLAINGHDLRYGSPES AAHLIQASERRVHLVVSRQVRQRSPD
Numb BP AK056823 3 PTITCHEKVVNIQKDPGESLGMTVAGGASHREWDLP 160
IYVISVEPGGVISRDGRIKTGDILLNVDGVELTEVS RSEAVALLKRTSSSIVLKALEVKEYEPQ
Numb BP AK056823 1 PDGEITSIKINRVDPSESLSIRLVGGSETPLVHIII 161
QHIYRDGVIARDGRLLPRDIILKVNGMDISNVPHNY AVRLLRQPCQVLWLTVMREQKFRSR Numb
BP AK056823 4 PRCLYNCKDIVLRRNTAGSLGFCIVGGYEEYNGNKP 162
FFIKSIVEGTPAYNDGRIRCGDILLAVNGRSISGMI HACLARLLKELKGRITLTIVSWPGTFL
outer 7023825 1 LLTEEEINLTRGPSGLGFNIVGGTDQQYVSNDSGIY 163 membrane
VSRIKENGAAALDGRLQEGDKILSVNGQDLKNLLHQ DAVDLFRNAGYAVSLRVQHRLQVQNGIHS
p55T 12733367 1 PVDAIRILGIHKRAGEPLGVTFRVENNDLVIARILH 164
GGMIDRQGLLHVGDIIKEVNGHEVGNNPKELQELLK NISGSVTLKILPSYRDTITPQQ PAR3
8037914 2 GKRLNIQLKKGTEGLGFSITSRDVTIGGSAPIYVKN 165
ILPRGAAIQDGRLKAGDRLIEVNGVDLVGKSQEEVV SLLRSTKMEGTVSLLVFRQEDA PAR3
8037914 1 IPNFSLDDMVKLVEVPNDGGPLGIHVVPFSARGGRT 166
LGLLVKRLEKGGKAEHENLFRENDCIVRINDGDLRN
RRFEQAQHMFRQAMRTPIIWFHWPAANKEQYEQ PAR3 8037914 3
PREFLTFEVPLNDSGSAGLGVSVKGNRSKENHADLG 167
IFVKSIINGGAASKDGRLRVNDQLIAVNGESLLGKT
NQDAMETLRRSMSTEGNKRGMIQLIVASRISKCNEL KSNSS PAR3L 18874467 2
ISNKNAKKIKIDLKKGPEGLGFTVVTRDSSIHGPGP 168
IFVKNILPKGAAIKDGRLQSGDRILEVNGRDVTGRT QEELVAMLRSTKQGETASLVIARQEGH
PAR3L 18874467 3 ITSEQLTFEIPLNDSGSAGLGVSLKGNKSRETGTDL 169
GIFIKSIIHGGAAFKDGRLRMNDQLIAVNGESLLGK
SNHEAMETLRRSMSMEGNIRGMIQLVILRRPERP PAR3L 18874467 1
IPRTKDTLSDMTRTVEISGEGGPLGIHVVPFFSSLS 170
GRILGLFIRGIEDNSRSKREGLFHENECIVKINNVD
LVDKTFAQAQDVFRQAMKSPSVLLHVLPPQNR PAR6 2613011 1
PETHRRVRLHKHGSDRPLGFYIRDGMSVRVAPQGLE 171
RVPGIFISRLVRGGLAESTGLLAVSDEILEVNGIEV
AGKTLDQVTDMMVANSHNLIVTVKPANQRNNV PAR6 1353716 1
IPVSSIIDVDILPETHRRVRLYKYGTEKPLGFYIRD 172 beta
GSSVRVTPHGLEKVPGIFISRLVPGGLAQSTGLLAV
NDEVLEVNGIEVSGKSLDQVTDMMIANSRNLIITVR PANQRNNRIHRD PAR6 13537118 1
IDVDLVPETHRRVRLHRHGCEKPLGFYIRDGASVRV 173 GAMMA
TPHGLEKVPGIFISRMVPGGLAESTGLLAVNDEVLE
VNGIEVAGKTLDQVTDMMIANSHNLIVTVKPANQRN NVV PDZ-73 5031978 3
PEQIMGKDVRLLRIKKEGSLDLALEGGVDSPIGKVV 174
VSAVYERGAAERHGGIVKGDEIMAINGKIVTDYTLA
EADAALQKAWNQGGDWIDLVVAVCPPKEYDD PDZ-73 5031978 2
IPGNRENKEKKVFISLVGSRGLGCSISSGPIQKPGI 175
FISHVKPGSLSAEVGLEIGDQIVEVNGVDFSNLDHK
EAVNVLKSSRSLTISIVAAAGRELFMTDEF PDZ-73 5031978 1
RSRKLKEVRLDRLHPEGLGLSVRGGLEFGCGLFISH 176
LIKGGQADSVGLQVGDEIVRINGYSISSCTHEEVIN LIRTKKTVSIKVRHIGLIPVKSSPDEFH
PDZK1 2944188 2 RLCYLVKEGGSYGFSLKTVQGKKGVYMTDITPQGVA 177
MRAGVLADDHLIEVNGENVEDASHEEVVEKVKKSGS RVMFLLVDKETDKREFIVTD PDZK1
2944188 3 QFKRETASLKLLPHQPRIVEMKKGSNGYGFYLRAGS 178
EQKGQIIKDIDSGSPAEEAGLKNNDLVVAVNGESVE
TLDHDSVVEMIRKGGDQTSLLWDKETDNMYRLAEFI VTD PDZK1 2944188 2, 3,
RLCYLVKEGGSYGFSLKTVQGKKGVYMTDITPQGVA 179 4
MRAGVLADDHLIEVNGENVEDASHEKVVEKVKKSGS
RVMFLLVDKETDKRHVEQKIQFKRETASLKLLPHQP
RIVEMKKGSNGYGFYLRAGSEQKGQIIKDIDSGSPA
EEAGLKNNDLVVAVNGESVETLDHDSVVEMIRKGGD
QTSLLVVDKETDNMYRLAHFSPFLYYQSQELPNGSV
KEAPAPTPTSLEVSSPPDTTEEVDHKPKLCRLAKGE
NGYGFHLNAIRGLPGSFIKEVQKGGPADLAGLEDED
VIIEVNGVNVLDEPYEKVVDRIQSSGKNVTLLVCGK PDZK1 2944188 4
PDTTEEVDHKPKLCRLAKGENGYGFHLNAIRGLPGS 180
FIKEVQKGGPADLAGLEDEDVIIEVNGVNVLDEPYE KVVDRIQSSGKNVTLLVGKNSS PDZK1
2944188 1 LTSTFNPRECKLSKQEGQNYGFFLRIEKDTEGHLVR 181
VVEKCSPAEKAGLQDGDRVLRINGVFVDKEEHMQVV DLVRKSGNSVTLLVLDGDSYEKAGSHEPS
PICK1 4678411 1 LGIPTVPGKVTLQKDAQNLIGISIGGGAQYCPCLYI 182
VQVFDNTPAALDGTVAAGDEITGVNGRSIKGKTKVE VAKMIQEVKGEVTIHYNKLQADPKQGM
PIST 98374330 1 SQGVGPIRKVLLLKEDHEGLGISITGGKEHGVPILI 183
SEIHPGQPADRCGGLHVGDAILAVNGVNLRDTKHKE AVTILSQQRGEIEFEVVYVAPEVDSD
prlL16 1478492 2 TAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLT 184
INRIFKGAASEQSETVQPGDEILQLGGTAMQGLTRF EAWNIIKALPDGPVTIVIRRKSLQSK
prlL16 1478492 1 IHVTILHKEEGAGLGFSLAGGADLENKVITVHRVFP 185
NGLASQEGTIQKGNEVLSINGKSLKGTTHHDALAIL RQAREPRQAVIVTRKLTPEEFIVTD
prlL16 1478492 1, 2 IHVTILHKEEGAGLGFSLAGGADLENKVITVHRVFP 186
NGLASQEGTIQKGNEVLSINGKSLKGTTHHDALAIL
RQAREPRQAVIVTRKLTPEAMPDLNSSTDSAASASA
ASDVSVESTAEATVCTVTLEKMSAGLGFSLEGGKGS
LHGDKPLTINRIFKGAASEQSETVQPGDEILQLGGT
AMQGLTRFEAWNIIKALPDGPVTIVIRRKSLQSK PSAP 6409315 1
IREAKYSGVLSSIGKIFKEEGLLGFFVGLIPHLLGD 187
VVFLWGCNLLAHFINAYLVDDSVSDTPGGLGNDQNP
GSQESQALAIRSYTKFVMGIAVSMLTYPFLLVGDLM
AVNNCGLQAGLPPYSPVFKSWIHCWKYLSVQGQLFR GSSLLFRRVSSGSCFALE PSD95
3318652 1, 2, EGEMEYEEITLERGNSGLGFSIAGGTDNPHIGDDPS 188 3
IFITKIIPGGAAAQDGRLRVNDSILFVNEVDVREVT
HSAAVEALKEAGSIVRLYVMRRKPPAEKVMEIKLIK
GPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAH
KDGRLQIGDKILAVNSVGLEDVMHEDAVAALKNTYD
VVYLKVAKPSNAYLSDSYAPPDITTSYSQHLDNEIS
HSSYLGTDYPTAMTPTSPRRYSPVAKDLLGEEDIPR
EPRRIVIHRGSTGLGFNIVGGEDGEGIFISFILAGG
PADLSGELRKGDQILSVNGVDLRNASHEQAAIALKN AGQTVTIIAQYKPE PSD95 3318652 2
HVMRRKPPAEKVMEIKLIKGPKGLGFSIAGGVGNQH 189
IPGDNSIYVTKIIEGGAAHKDGRLQIGDKILAVNSV
GLEDVMHEDAVAALKNTYDVVYLKVAKPSNAYL PSD95 3318652 3
REDIPREPRRIVIHRGSTGLGFNIVGGEDGEGIFIS 190
FILAGGPADLSGELRKGDQILSVNGVDLRNASHEQA AIALKNAGQTVTIIAQYKPEFIVTD
PSD95 3318652 1 LEYEeITLERGNSGLGFSIAGGTDNPHIGDDPSIFI 191
TKIIPGGAAAQDGRLRVNDSILFVNEVDVREVTHSA AVEALKEAGSIVRLYVMRRKPPAENSS
PSMD9 9184389 1 RDMAEAHKEAMSRKLGQSESQGPPRAFAKVNSISPG 192
SPASIAGLQVDDEIVEFGSVNTQNFQSLHNIGSVVQ HSEGALAPTILLSVSM PTN-3 179912
1 QNDNGDSYLVLIRITPDEDGKFGFNLKGGVDQKMPL 193
VVSRINPESPADTCIPKLNEGDQIVLINGRDISEHT HDQVVMFIKASRESHSRELALVIRRRAVRS
PTN-4 190747 1 IRMKPDENGRFGFNVKGGYDQKMPVIVSRVAPGTPA 194
DLCVPRLNEGDQVVLINGRDIAEHTHDQVVLFIKAS CERHSGELMLLVRPNA PTPL1 515030
2 GDIFEVELAKNDNSLGISVTGGVNTSVRHGGIYVKA 195
VIPQGAAESDGRIHKGDRVLAVNGVSLEGATHKQAV ETLRNTGQVVHLLLEKGQSPTSK PTPL1
515030 1 PEREITLVNLKKDAKYGLGFQIIGGEKMGRLDLGIF 196
ISSVAPGGPADFHGCLKPGDRLISVNSVSLEGVSHH
AAIEILQNAPEDVTLVISQPKEKISKVPSTPVHL PTPL1 515030 4
ELEVELLITLIKSEKASLGFTVTKGNQRIGCYVHDV 197
IQDPAKSDGRLKPGDRLIKVNDTDVTNMTHTDAVNL LRAASKTVRLVIGRVLELPRIPMLPH
PTPL1 515030 3 TEENTFEVKLFKNSSGLGFSFSREDNLIPEQINASI 198
VRVKKLFAGQPAAESGKIDVGDVILKVNGASLKGLS
QQEVISALRGTAPEVFLLLCRPPPGVLPEIDT PTPL1 515030 5
MLPHLLPDITLTCNKEELGFSLCGGHDSLYQVVYIS 199
DINPRSVAAIEGNLQLLDVIHYVNGVSTQGMTLEEV NRALDMSLPSLVLKATRNDLPV RGS 3
18644735 1 VCSERRYRQITIPRGKDGFGFTICCDSPVRVQAVDS 200
GGPAERAGLQQLDTVLQLNERPVEHWKCVELAHEIR SCPSEIILLVWRMVPQVKPG RGS12
3290015 1 RPSPPRVRSVEVARGRAGYGFTLSGQAPCVLSCVMR 201
GSPADFVGLRAGDQILAVNEINVKKASHEDVVKLIG KCSGVLHMVIAEGVGRFESCS Rho-GAP
10 50345878 1 SEDETFSWPGPKTVTLKRTSQGFGFTLRHFIVYPPE 202
SAIQFSYKDEENGNRGGKQRNRLEPMDTIFVKQVKE
GGPAFEAGLCTGDRIIKVNGESVIGKTYSQVIALIQ NSDTTLELSVMPKDED Rhophilin
AY082588 1 SAKNRWRLVGPVHLTRGEGGFGLTLRGDSPVLIAAV 203
IPGSQAAAAGLKEGDYIVSVNGQPCRWWRHAEVVTE LKAAGEAGASLQVVSLLPSSRLPS
Rhophilin- AF268032 1 ISFSANKRWTPPRSIRFTAEEGDLGFTLRGNAPVQV 204 like
HFLDPYCSASVAGAREGDYIVSIQLVDCKWLTLSEV
MKLLKSFGEDEIEMKVVSLLDSTSSMHNKSAT RIM2 12734165 1
TLNEEHSHSDKHPVTWQPSKDGDRLIGRILLNKRLK 205
DGSVPRDSGAMLGLKVVGGKMTESGRLCAFITKVKK
GSLADTVGHLRPGDEVLEWNGRLLQGATFEEVYNII LESKPEPQVELVVSRPIG SEMCAP 3
5889526 2 QEMDREELELEEVDLYRMNSQDKLGLTVCYRTDDED 206
DIGIYISEIDPNSIAAKDGRIREGDRIIQINGIEVQ NREEAVALLTSEENKNFSLLIARPELQLD
SEMCAP 3 5889526 1 QGEETKSLTLVLHRDSGSLGFNIIGGRPSVDNHDGS 207
SSSEGIFVSKIVDSGPAAKEGGLQIHDRIIEVNGRD
LSRATHDQAVEAFKTAKEPIVVQVLRRTPRTKMFTP semcap2 7019938 1
ILAHVKGIEKEVNVYKSEDSLGLTITDNGVGYAFIK 208
RIKDGGVIDSVKTICVGDHIESINGENIVGWRHYDV AKKLKELKKEELFTMKLIEPKKAFEI
serine 2738914 1 RGEKKNSSSGISGSQRRYIGVMMLTLSPSILAELQL 209 protease
REPSFPDVQHGVLIHKVILGSPAHRAGLRPGDVILA
IGEQMVQNAEDVYEAVRTQSQLAVQIRRGRETLTLY V Shank 1 6049185 1
ILEEKTVVLQKKDNEGFGFVLRGAKADTPIEEFTPT 210
PAFPALQYLESVDEGGVAWQAGLRTGDFLIEVNNEN
VVKVGHRQVVNMIRQGGNHLVLKVVTVTRNLDPDDN SS Shank 2 7025450 1
ILKEKTVLLQKKDSEGFGFVLRGAKAQTPIEEFTPT 211
PAFPALQYLESVDEGGVAWRAGLRMGDFLIEVNGQN
VVKVGHRQVVNMIRQGGNTLMVKVVMVTRHPDMDEA VQNSS Shank 3 * 1
SDYVIDDKVAVLQKRDHEGFGFVLRGAKAETPIEEF 212
TPTPAFPALQYLESVDVEGVAWRAGLRTGDFLIEVN
GVNVVKVGHKQVVALIRQGGNRLVMKVVSVTRKPEE DG sim to lig 22477649 2
SNSPREEIFQVALHKRDSGEQLGIKLVRRTDEPGVF 213 of numb
ILDLLEGGLAAQDGRLSSNDRVLAINGHDLKYGTPE px2 LAAQIIQASGERVNLTIARPGKPQPG
sim to lig 22477649 3 IQCVTCQEKHITVKKEPHESLGMTVAGGRGSKSGEL 214 of
numb PIFVTSVPPHGCLARDGRIKRGDVLLNINCIDLTNL px2
SHSEAVAMLKASAASPAVALKALEVQIVEEAT Similar to 14286261 1
MGLGVSAEQPAGGAEGFHLHGVQENSPAQQAGLEPY 215 GRASP65
FDFIITIGHSRLNKENDTLKALLKANVEKPVKLEVF
NMKTMRVREVEVVPSNMWGGQGLLGASVRFCFRRAS E Similar to 14286261 2
RASEQVWHVLDVEPSSPAALAGLRPYTDYVVGSDQI 216 GRASP65
LQESEDFFTLIESHEGKPLKLMVYNSKSDSCRESGM
WHWLWVSTPDPNSAPQLPQEATWHPTTFCSTTWCPT T Similar to 21595065 1
ISVTDGPKFEVKLKKNANGLGFSFVQMEKESCSHLK 217 Protein-
SDLVRIKRLFPGQPAEENGAIAAGDIILAVNGRSTE Tyrosine-
GLIFQEVLHLLRGAPQEVTLLLCRPPPGA Phosphatase Homolog SIP1 2047327 1
QPEPLRPRLCRLVRGEQGYGFHLHGEKGRRGQFIRR 218
VEPGSPAEAAALRAGDRLVEVNGVNVEGETHHQVVQ RIKAVEGQTRLLWDQETDEELRRRNSS
SIP1 2047327 2 PLRELRPRLCHLRKGPQGYGFNLHSDKSRPGQYIRS 219
VDPGSPAARSGLRAQDRLIEVNGQNVEGLRHAEVVA SIKAREDEARLLVVDPETDEHFKRNSS
SITAC 18 8886071 1 PGVREIHLCKDERGKTGLRLRKVDQGLFVQLVQANT 220
PASLVGLRFGDQLLQIDGRDCAGWSSHKAHQVVKKA SGDKIVVVVRDRPFQRTVTM SITAC 18
8886071 2 PFQRTVTMHKDSMGHVGFVIKKGKIVSLVKGSSAAR 221
NGLLTNHYVCEVDGQNVIGLKDKKIMEILATAGNVV TLTIIPSVIYEHIVEFIV SNPC IIa
20809633 1 SLERPRFCLLSKEEGKSFGFHLQQELGRAGHVVCRV 222
DPGTSAQRQGLQEGDRILAVNNDVVEHEDYAVVVRR IRASSPRVLLTVLARHAHDVARAQ
SYNTENIN 2795862 2 LRDRPFERTITMHKDSTGHVGFIFKNGKITSIVKDS 223
SPARNGLLTEHNICEINGQNVIGLKDSQIADILSTS GTVVTITIMPAFIFEHMNSS SYNTENIN
2795862 1 LEIKQGIREVILCKDQDGKIGLRLKSIDNGIFVQLV 224
QANSPASLVGLRFGDQVLQINGENCAGWSSDKAHKV LKQAFGEKITMRIHRD Syntrophin
1145727 1 QRRRVTVRKADAGGLGISIKGGRENKMPILISKIFK 225 1 alpha
GLAADQTEALFVGDAILSVNGEDLSSATHDEAVQVL KKTGKEVVLEVKYMKDVSPYFK
Syntrophin 476700 1 PVRRVRVVKQEAGGLGISIKGGRENRMPILISKIFP 226 beta 2
GLAADQSRALRLGDAILSVNGTDLRQATHDQAVQAL KRAGKEVLLEVKFIRE Syntrophin
9507162 1 EPFYSGERTVTIRRQTVGGFGLSIKGGAEHNIPVVV 227 gamma 1
SKISKEQRAELSGLLFIGDAILQINGINVRKCRHEE VVQVLRNAGEEVTLTVSFLKRAPAFLKL
Syntrophin 9507164 1 SHQGRNRRTVTLRRQPVGGLGLSIKGGSEHNVPVVI 228 gamma
2 SKIFEDQAADQTGMLFVGDAVLQVNGIHVENATHEE VVHLLRNAGDEVTITVEYLREAPAFLK
TAX2-like 3253116 1 RGETKEVEVTKTEDALGLTITDNGAGYAFIKRIKEG 229
protein SIINRIEAVCVGDSIEAINDHSIVGCRHYEVAKMLR ELPKSQPFTLRLVQPKRAF
TIAM1 4507500 1 HSIHIEKSDTAADTYGFSLSSVEEDGIRRLYVNSVK 230
ETGLASKKGLKAGDEILEINNRAADALNSSMLKDFL SQPSLGLLVRTYPELE
TIAM2 6912703 1 PLNVYDVQLTKTGSVCDFGFAVTAQVDERQHLSRIF 231
ISDVLPDGLAYGEGLRKGNEIMTLNGEAVSDLDLKQ MEALFSEKSVGLTLIARPPDTKATL TIP1
2613001 1 QRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFSED 232
KTDKGIYVTRVSEGGPAEIAGLQIGDKIMQVNGWDM
TMVTHDQARKRLTKRSEEVVRLLVTRQSLQK TIP2 2613003 1
RKEVEVFKSEDALGLTITDNGAGYAFIKRIKEGSVI 233
DHIHLISVGDMIEAINGQSLLGCRHYEVARLLKELP RGRTFTLKLTEPRK TIP33 2613007 1
HSHPRVVELPKTDEGLGFNVMGGKEQNSPIYISRII 234
PGGVAERHGGLKRGDQLLSVNGVSVEGEHHEKAVEL LKAAKDSVKLVVRYTPKVL TIP43
2613011 1 LSNQKRGVKVLKQELGGLGISIKGGKENKMPILISK 235
IFKGLAADQTQALYVGDAILSVNGADLRDATHDEAV QALKRAGKEVLLEVKYMREATPYVK
Unknown 22382223 1 IQRSSIKTVELIKGNLQSVGLTLRLVQSTDGYAGHV 236 PDZ
IIETVAPNSPAAIADLQRGDRLIAIGGVKITSTLQV LKLIKQAGDRVLVYYERPVGQSNQGA
Vartul 1469875 1 ILTLTILRQTGGLGISIAGGKGSTPYKGDDEGIFIS 237
RVSEEGPAARAGVRVGDKLLEVNGVALQGAEHHEAV
EALRGAGTAVQMRVWRERMVEPENAEFIVTD Vartul 1469875 4
RELCIQKAPGERLGISIRGGARGHAGNPRDPTDEGI 238
FISKVSPTGAAGRDGRLRVGLRLLEVNQQSLLGLTH
GEAVQLLRSVGDTLTVLVCDGFEASTDAALEVS Vartul 1469875 3
LEGPYPVEEIRLPRAGGPLGLSIVGGSDHSSHPFGV 239
QEPGVFISKVLPRGLAARSGLRVGDRILAVNGQDVR
DATHQEAVSALLRPCLELSLLVRRDPAEFIVTD Vartul 1469875 2
PLRQRHVACLARSERGLGFSIAGGKGSTPYRAGDAG 240
IFVSRIAEGGAAHRAGTLQVGDRVLSINGVDVTEAR HDHAVSLLTAASPTIALLLEREAGG
Vartul 1469875 1, 2 TLTILRQTGGLGISIAGGKGSTPYKGDDEGIFISRV 241
SEEGPMRAGVRVGDKLLEGIFVSRIAEGGAAHRAGT
LQVGDRVLSINGVDVTEARHDHAVSLLTAASPTIAL LLERE X-11 beta 3005559 2
IPPVTTVLIKRPDLKYQLGFSVQNGIICSLMRGGIA 242
ERGGVRVGHRIIEINGQSVVATAHEKIVQALSNSVG EIHMKTMPAAMFRLLTGQENSSL X-11
beta 3005559 1 IHFSNSENCKELQLEKHKGEILGVVVVESGWGSILP 243
TVILANMMNGGPAARSGKLSIGDQIMSINGTSLVGL
PLATCQGIIKGLKNQTQVKLNIVSCPPVTTVLIKRN SS ZO-1 292937 1
IWEQHTVTLHRAPGFGFGIAISGGRDNPHFQSGETS 244
IVISDVLKGGPAEGQLQENDRVAMVNGVSMDNVEHA FAVQQLRKSGKNAKITIRRKKKVQIPNSS
ZO-1 292937 2 ISSQPAKPTKVTLVKSRKNEEYGLRLASHIFVKEIS 245
QDSLAARDGNIQEGDWLKJNGTVTENMSLTDAKTLI ERSKGKLKMVVQRDRATLLNSS ZO-1
292937 3 IRMKLVKFRKGDSVGLRLAGGNDVGIFVAGVLEDSP 246
AAKEGLEEGDQILRVNNVDFTNIIREEAVLFLLDLP KGEEVTILAQKKKDVFSN ZO-2
12734763 1 IQHTVTLHRAPGFGFGIAISGGRDNPHFQSGETSIV 247
ISDVLKGGPAEGQLQIENDRVAMVNGVSMDNVEHAF AVQQLRKSGKNAKITIRRKKKVQIPNSS
ZO-2 12734763 3 HAPNTKMVREKKGDSVGLRLAGGNDVGIFVAGIQEG 248
TSAEQEGLQEGDQILKVNTQDFRGLVREDAVLYLLE IPKGEMVTILAQSRADVY ZO-2
12734763 2 RVLLMKSRANEEYGLRLGSQIFVKEMTRTGLATKDG 249
NLHEGDIILKINGTVTENMSLTDARKLIEKSRGKLQ LVVLRDS ZO-3 10092690 3
RGYSPDTRVVRFLKGKSIGLRLAGGNDVGIFVSGVQ 250
AGSPADGQGIQEGDQILQVNDVPFQNLTREEAVQFL
LGLPPGEEMELVTQRKQDIFWKMVQSEFIVTD ZO-3 10092690 1
IPGNSTIWEQHTATLSKDPRRGFGIAISGGRDRPGG 251
SMVVSDVVPGGPAEGRLQTGDHIVMVNGVSMENATS
AFAIQILKTCTKMANITVKRPRRIHLPAEFIVTD ZO-3 10092690 2
QDVQMKPVKSVLVKRRDSEEFGVKLGSQIFIKHITD 252
SGLAARHRGLQEGDLILQINGVSSQNLSLNDTRRLI EKSEGKLSLLVLRDRGQFLVNIPNSS *:
No GI number for this PDZ domain containing protein - it was
computer cloned at Arbor Vita Corpration. using rat Shank3 seq
against human genomic clone AC000036. In silico spliced together
nt6400-6496, 6985-7109, 7211-7400 to create hypothetical human
Shank3.
[0184] As discussed in detail herein, the PDZ proteins listed in
TABLE 5 are naturally occurring proteins containing a PDZ domain.
Thus, one aspect of the present invention is directed to the
detection and modulation of interactions between a PDZ protein and
PL protein pair listed in TABLE 6 or 7 or 8. As used herein the
phrase "protein pair" or "protein binding pair" when used in
reference to a PDZ protein and PL protein refers to a PL protein
and PDZ protein such as those listed in TABLES 6 or 7 or 8 which
bind to one another. It should be understood that TABLES 6, 7, and
8 are set up to show that certain PL proteins bind to a plurality
of PDZ proteins.
[0185] The interactions like those summarized in TABLES 6, 7, and 8
can occur in a wide variety of cell types. Examples of such cells
include neuronal, hematopoietic, stem, muscle, epidermal,
epithelial, endothelial, and cells from essentially any tissue such
as liver, lung, placenta, uterus, kidney, ovaries, testes, stomach,
colon and intestine. Because the interactions disclosed herein can
occur in such a wide variety of cell types, these interactions can
also play an important role in a variety of biological
functions.
[0186] Thus, for example, in certain embodiments of the invention,
the PL proteins and/or the PDZ protein to which it binds are
expressed in the nervous system (e.g., neurons). In an embodiment,
the PL proteins of the invention bind a PDZ protein that is
expressed in neurons. In various embodiments, the PL protein is
highly expressed in neuronal cells. In still other instances the PL
proteins and/or the PDZ protein to which it binds are expressed in
non-neural cells (e.g., in hematopoietic cells).
[0187] In various embodiments of the invention, the PL protein is
expressed or up-regulated upon cell activation (e.g., in stimulated
neurons), upon entry into mitosis (e.g., up-regulation in rapidly
proliferating cell populations), or in association with cell
death.
[0188] A. Detection of PDZ Domain-Containing Proteins
[0189] As noted supra, the present inventors have identified a
number of PDZ protein and NMDAR PL protein interactions and a
number of PDZ protein and TRP PL protein interactions that can play
a role in modulation of a number of biological functions in a
variety of cell types. A comprehensive list of PDZ
domain-containing proteins was retrieved from the Sanger Centre
database (Pfam) searching for the keyword, "PDZ". The corresponding
cDNA sequences were retrieved from GenBank using the NCBI "entrez"
database (hereinafter, "GenBank PDZ protein cDNA sequences"). The
DNA portion encoding PDZ domains was identified by alignment of
cDNA and protein sequence using CLUSTALW. Based on the DNA/protein
alignment information, primers encompassing the PDZ domains were
designed. The expression of certain PDZ-containing proteins in
cells was detected by polymerase chain reaction ("PCR")
amplification of cDNAs obtained by reverse transcription ("RT") of
cell-derived RNA (i.e., "RT-PCR"). PCR, RT-PCR and other methods
for analysis and manipulation of nucleic acids are well known and
are described generally in Sambrook et al., (1989) Molecular
Cloning: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring
Harbor Laboratory hereinafter, "Sambrook"); and Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publishing and
Wiley-Interscience, New York (1997), as supplemented through
January 1999 (hereinafter "Ausubel").
[0190] Samples of cDNA for those sequences identified through the
foregoing search were obtained and then amplified. In general a
sample of the cDNA (typically, 1/5 of a 20 .mu.l reaction) was used
to conduct PCR. PCR was conducted using primers designed to amplify
specifically PDZ domain-containing regions of PDZ proteins of
interest. Oligonucleotide primers were designed to amplify one or
more PDZ-encoding domains. The DNA sequences encoding the various
PDZ domains of interest were identified by inspection (i.e.,
conceptual translation of the PDZ protein cDNA sequences obtained
from GenBank, followed by alignment with the PDZ domain amino acid
sequence). TABLE 5 shows the PDZ-encoded domains amplified, and the
GenBank accession number of the polynucleotides encoding the
PDZ-domain containing proteins. To facilitate subsequent cloning of
PDZ domains, the PCR primers included endonuclease restriction
sequences at their ends to allow ligation with pGEX-3X cloning
vector (Pharmacia, GenBank XXI13852) in frame with glutathione-S
transferase (GST).
[0191] TABLE 5 lists the proteins isolated for use in the
aforementioned assays.
[0192] B. Production of Fusion Proteins Containing PDZ-Domains
[0193] GST-PDZ domain fusion proteins were prepared for use in the
assays of the invention. PCR products containing PDZ encoding
domains (as described supra) were subcloned into an expression
vector to permit expression of fusion proteins containing a PDZ
domain and a heterologous domain (i.e., a glutathione-S transferase
sequence, "GST"). PCR products (i.e., DNA fragments) representing
PDZ domain encoding DNA was extracted from agarose gels using the
"sephaglas" gel extraction system (Pharmacia) according to the
manufacturer's recommendations.
[0194] As noted supra, PCR primers were designed to include
endonuclease restriction sites to facilitate ligation of PCR
fragments into a GST gene fusion vector (pGEX-3X; Pharmacia,
GenBank accession #XXU13852) in-frame with the glutathione-S
transferase coding sequence. This vector contains a IPTG inducible
lacZ promoter. The pGEX-3X vector was linearized using Bam HI and
Eco RI or, in some cases, Eco RI or Sma I, and dephosphorylated.
For most cloning approaches, double digestion with Bam HI and Eco
RI was performed, so that the ends of the PCR fragments to clone
were Bam HI and Eco RI. In some cases, restriction endonuclease
combinations used were Bgl II and Eco RI, Bam HI and Mfe I, or Eco
RI only, Sma I only, or BamHI only. When more than one PDZ domain
was cloned, the DNA portion cloned represents the PDZ domains and
the cDNA portion located between individual domains. DNA linker
sequences between the GST portion and the PDZ domain containing DNA
portion vary slightly, dependent on which of the above described
cloning sites and approaches were used. As a consequence, the amino
acid sequence of the GST-PDZ fusion protein varies in the linker
region between GST and PDZ domain. Protein linker sequences
corresponding to different cloning sites/approaches are shown
below. Linker sequences (vector DNA encoded) are bold, PDZ domain
containing gene derived sequences are in italics.
TABLE-US-00007 1) GST-BamHI/BamHI-PDZ domain insert Gly--Ile-PDZ
domain insert 2) GST-BamHI/BgIII-PDZ domain insert Gly-Ile-PDZ
domain insert 3) GST-EcoRI/EcoI-PDZ domain insert
Gly-Ile-Pro-Gly--Asn-PDZ domain insert (SEQ ID NO:253) 4)
GST--SmaI/SmaI-PDZ domain insert Gly-Ile-Pro-PDZ domain insert
[0195] The PDZ-encoding PCR fragment and linearized pGEX-3X vector
were ethanol precipitated and resuspended in 10 .mu.l standard
ligation buffer. Ligation was performed for 4-10 hours at 7.degree.
C. using T4 DNA ligase. It will be understood that some of the
resulting constructs include very short linker sequences and that,
when multiple PDZ domains were cloned, the constructs included some
DNA located between individual PDZ domains.
[0196] The ligation products were transformed in DH5.alpha. or
BL-21 E. coli bacteria strains. Colonies were screened for presence
and identity of the cloned PDZ domain containing DNA as well as for
correct fusion with the glutathione S-transferase encoding DNA
portion by PCR and by sequence analysis. Positive clones were
tested in a small scale assay for expression of the GST/PDZ domain
fusion protein and, if expressing, these clones were subsequently
grown up for large scale preparations of GST/PDZ fusion
protein.
[0197] GST-PDZ domain fusion protein was overexpressed following
addition of IPTG to the culture medium and purified. Detailed
procedure of small scale and large scale fusion protein expression
and purification are described in "GST Gene Fusion System" (second
edition, revision 2; published by Pharmacia). In brief, a small
culture (3-5 ml) containing a bacterial strain (DH5.alpha., BL21 or
JM109) with the fusion protein construct was grown overnight in
LB-media at 37.degree. C. with the appropriate antibiotic selection
(100 .mu.g/ml ampicillin; a.k.a. LB-amp). The overnight culture was
poured into a fresh preparation of LB-amp (typically 250-500 ml)
and grown until the optical density (OD) of the culture was between
0.5 and 0.9 (approximately 2.5 hours). IPTG (isopropyl
.beta.-D-thiogalactopyranoside) was added to a final concentration
of 1.0 mM to induce production of GST fusion protein, and culture
was grown an additional 1.5-2.5 hours. Bacteria were collect by
centrifugation (4500 g) and resuspended in Buffer A- (50 mM Tris,
pH 8.0, 50 mM dextrose, 1 mM EDTA, 200 .mu.M
phenylmethylsulfonylfluoride). An equal volume of Buffer A+ (Buffer
A-, 4 mg/ml lysozyme) was added and incubated on ice for 3 min to
lyse bacteria. An equal volume of Buffer B (10 mM Tris, pH 8.0, 50
mM KCl, 1 mM EDTA. 0.5% Tween-20, 0.5% NP40 (a.k.a. IGEPAL CA-630),
200 .mu.M phenylmethylsulfonylfluoride) was added and incubated for
an additional 20 minutes. The bacterial cell lysate was centrifuged
(.times.20,000 g), and supernatant was added to glutathione
SEPHAROSE 4B beads (Pharmacia, Cat. #17-0765-01) previously swelled
(rehydrated) in 1.times. phosphate-buffered saline (PBS). The
supernatant-glutathione SEPHAROSE bead slurry was poured into a
column and washed with at least 20 bed volumes of 1.times.PBS. GST
fusion protein was eluted off the glutathione SEPHAROSE beads by
applying 0.5-1.0 ml aliquots of 5 mM glutathione and collected as
separate fractions. Concentrations of fractions were determined
using BioRad Protein Assay (Cat. #500-0006) according to
manufacturer's specifications. Those fractions containing the
highest concentration of fusion protein were pooled and dialyzed
against 1.times.PBS/35% glycerol. Fusion proteins were assayed for
size and quality by SDS gel electrophoresis (PAGE) as described in
"Sambrook." Fusion protein aliquots were stored at minus 80.degree.
C. and at minus 20.degree. C.
[0198] C. Classification of PDZ Domain-Containing Proteins
[0199] The PDZ proteins identified herein as interacting with
particular PL proteins can be grouped into a number of different
categories. Thus, as described in greater detail below, the methods
and reagents that are provided herein can be utilized to modulate
PDZ interactions, and thus biological functions, that are regulated
or otherwise involve the following classes of proteins. It should
be recognized, however, that modulation of the interactions that
are identified herein can be utilized to affect biological
functions involving other protein classes.
[0200] 1) Protein Kinases
[0201] A number of protein kinases contain PDZ domains. Protein
kinases are widely involved in cellular metabolism and regulation
of protein activity through phosphorylation of amino acids on
proteins. An example of this is the regulation of signal
transduction pathways such as T cell activation through the T cell
Receptor, where ZAP-70 kinase function is required for transmission
of the activation signal to downstream effector molecules. These
molecules include, but are not limited to KIAA0303, KIAA0561,
KIAA0807, KIAA0973, and CASK.
[0202] 2) Guanalyte Kinases
[0203] A number of guanalyte kinases contain PDZ domains. These
molecules include, but are not limited to Atrophin 1, CARD11,
CARD14, DLG1, DLG2, DLG5, FLJ12615, MPP1, MPP2, NeDLG, p55T, PSD95,
ZO-1, ZO-2, and ZO-3.
[0204] 3) Guanine Exchange Factors
[0205] A number of guanine exchange factors contain PDZ domains.
Guanine exchange factors regulate signal transduction pathways and
other biological processes through facilitating the exchange of
differently phosphorylated guanine residues. These molecules
include, but are not limited to GTPase, Guanine Exchange, KIAA0313,
KIAA0380, KIAA0382, KIAA1389, KIAA1415, TIAM1, and TIAM2.
[0206] 4) LIM PDZ's
[0207] A number of LIM proteins contain PDZ domains. These
molecules include, but are not limited to .alpha.-Actinin 2,
ELFIN1, ENIGMA, HEMBA 1003117, KIAA0613, KIAA0858, KIAA0631, LIM
Mystique, LIM protein, LIM-RIL, LIMK1, LIMK2, and LU-1.
[0208] 5) Proteins Containing Only PDZ Domains
[0209] A number of proteins contain PDZ domains without any other
predicted functional domains. These include, but are not limited to
26s subunit p27, AIPC, Cytohesion Binding Protein, EZRIN Binding
Protein, FLJ0011, FLJ20075, FLJ21687, GRIP1, HEMBA1000505,
KIAA0545, KIAA0967, KIAA1202, KIAA1284, KIAA1526, KIAA1620,
KIAA1719, MAGI1, Novel PDZ gene, Outer Membrane, PAR3, PAR6,
PAR6.gamma., PDZ-73, PDZK1, PICK1, PIST, prIL16, Shank1, SIP1,
SITAC-18, Syntenin, Syntrophin .gamma.2, TIP1, TIP2, and TIP43.
[0210] 6) Tyrosine Phosphatases
[0211] A number of tyrosine phosphatases contain PDZ domains.
Tyrosine phosphatases regulate biological processes such as signal
transduction pathways through removal of phosphate groups required
for function of the target protein. Examples of such enzymes
include, but are not limited to PTN-3, PTN-4, and PTPL1.
[0212] 7) Serine Proteases
[0213] A number of serine proteases contain PDZ domains. Proteases
affect biological molecules by cleaving them to either activate or
repress their functional ability. These enzymes have a variety of
functions, including roles in digestion, blood coagulation and
lysis of blood clots. These include, but are not limited to Novel
Serine Protease and Serine Protease.
[0214] 8) Viral Oncogene Interacting Proteins that Contain PDZ
Domains
[0215] A number of TAX interacting proteins contain PDZ domains.
Many of these also bind to multiple viral oncoproteins such as
Adenovirus E4, Papillomavirus E6, and HBV protein X. These TAX
interacting proteins include, but are not limited to AIPC,
Connector Enhancer, DLG1, DLG2, ERBIN, FLJ00011, FLJ11215,
HEMBA1003117, INADL, KIAA0147, KIAA0807, KIAA1526, KIAA1634, LIMK1,
LIM Mystique, LIM-RIL, MUPP1, NeDLG, Outer Membrane, PSD95, PTN-4,
PTPL-1, Syntrophin .gamma.1, Syntrophin .gamma.2, TAX2-like
protein, TIP2, TIP1, TIP33, and TIP43.
[0216] 9) Other PDZ-Domain Containing Proteins
[0217] A number of proteins containing RA and/or RHA and/or DIL
and/or IGFBP and/or WW and/or L27 and/or SAM and/or PH and/or DIX
and/or DIP and/or Dishevelled and/or LRR and/or Hormone 3 and/or C2
and/or RPH3A and/or zf-TRAF and/or zf-C3HC4 and/or PID and/or
NO_Synthase and/or Flavodoxin and/or FAD binding and/or NAD binding
and/or Kazal and/or Trypsin and/or RBD and/or RGS and/or GoLoco
and/or HR1 and/or BR01 domains contain PDZ domains. These include,
but are not limited to AF6, APXL-1, MAGI1, DVL1, DVL2, DVL3,
KIAA0417, KIAA0316, KIAA0340, KIAA0559, KIAA0751, KIAA0902,
KIAA1095, KIAA1222, KIAA1634, MINT1, NOS1, RGS12, Rhophilin-like,
Shank 3, Syntrophin 1.alpha., Syntrophin .beta.2, and
X11.beta..
[0218] D. Assays for Detection of Interactions Between PDZ-Domain
Polypeptides and TRP PL Proteins and TRP Associated Proteins
[0219] Two complementary assays, termed A and G, were developed to
detect binding between a PDZ-domain polypeptide and candidate PDZ
ligand. In each of the two different assays, binding is detected
between a peptide having a sequence corresponding to the C-terminus
of a protein anticipated to bind to one or more PDZ domains (i.e. a
candidate PL peptide) and a PDZ-domain polypeptide (typically a
fusion protein containing a PDZ domain). In the A assay, the
candidate PL peptide is immobilized and binding of a soluble
PDZ-domain polypeptide to the immobilized peptide is detected (the
A assay is named for the fact that in one embodiment an avidin
surface is used to immobilize the peptide). In the G assay, the
PDZ-domain polypeptide is immobilized and binding of a soluble PL
peptide is detected (The G assay is named for the fact that in one
embodiment a GST-binding surface is used to immobilize the
PDZ-domain polypeptide). Preferred embodiments of these assays are
described in detail infra. However, it will be appreciated by
ordinarily skilled practitioners that these assays can be modified
in numerous ways while remaining useful for the purposes of the
present invention.
[0220] 1) A Assay Detection of PDZ-Ligand Binding Using Immobilized
PL Peptide.
[0221] In one aspect, the invention provides an assay in which
biotinylated candidate PL peptides are immobilized on an avidin
coated surface. The binding of PDZ-domain fusion protein to this
surface is then measured. In a preferred embodiment, the PDZ-domain
fusion protein is a GST/PDZ fusion protein and the assay is carried
out as follows:
[0222] (1) Avidin is bound to a surface, e.g. a protein binding
surface. In one embodiment, avidin is bound to a polystyrene 96
well plate (e.g., Nunc Polysorb (Cat. #475094) by addition of 100
.mu.l per well of 20 .mu.g/ml of avidin (Pierce) in phosphate
buffered saline without calcium and magnesium, pH 7.4 ("PBS",
GibcoBRL) at 4.degree. C. for 12 hours. The plate is then treated
to block nonspecific interactions by addition of 200 .mu.l per well
of PBS containing 2 g per 100 ml protease-free bovine serum albumin
("PBS/BSA") for 2 hours at 4.degree. C. The plate is then washed 3
times with PBS by repeatedly adding 200 .mu.l per well of PBS to
each well of the, plate and then dumping the contents of the plate
into a waste container and tapping the plate gently on a dry
surface.
[0223] (2) Biotinylated PL peptides (or candidate PL peptides, e.g.
see TABLE 2 and TABLE 4) are immobilized on the surface of wells of
the plate by addition of 50 .mu.l per well of 0.4 .mu.M peptide in
PBS/BSA for 30 minutes at 4.degree. C. Usually, each different
peptide is added to at least eight different wells so that multiple
measurements (e.g. duplicates and also measurements using different
(GST/PDZ-domain fusion proteins and a GST alone negative control)
can be made, and also additional negative control wells are
prepared in which no peptide is immobilized. Following
immobilization of the PL peptide on the surface, the plate is
washed 3 times with PBS.
[0224] (3) GST/PDZ-domain fusion protein (prepared as described
supra) is allowed to react with the surface by addition of 50 .mu.l
per well of a solution containing 5 .mu.g/ml GST/PDZ-domain fusion
protein in PBS/BSA for 2 hours at 4.degree. C. As a negative
control, GST alone (i.e. not a fusion protein) is added to
specified wells, generally at least 2 wells (i.e. duplicate
measurements) for each immobilized peptide. After the 2 hour
reaction, the plate is washed 3 times with PBS to remove unbound
fusion protein.
[0225] (4) The binding of the GST/PDZ-domain fusion protein to the
avidin-biotinylated peptide surface can be detected using a variety
of methods, and detectors known in the art. In one embodiment, 50
.mu.l per well of an anti-GST antibody in PBS/BSA (e.g. 2.5
.mu.g/ml of polyclonal goat-anti-GST antibody, Pierce) is added to
the plate and allowed to react for 20 minutes at 4.degree. C. The
plate is washed 3 times with PBS and a second, detectably labeled
antibody is added. In one embodiment, 50 .mu.l per well of 2.5
.mu.g/ml of horseradish peroxidase (HRP)-conjugated polyclonal
rabbit anti-goat immunoglobulin antibody is added to the plate and
allowed to react for 20 minutes at 4.degree. C. The plate is washed
5 times with 50 mM Tris pH 8.0 containing 0.2% Tween 20, and
developed by addition of 100 .mu.l per well of HRP-substrate
solution (TMB, Dako) for 20 minutes at room temperature (RT). The
reaction of the HRP and its substrate is terminated by the addition
of 100 .mu.l per well of 1 M sulfuric acid and the optical density
(O.D.) of each well of the plate is read at 450 nm.
[0226] (5) Specific binding of a PL peptide and a PDZ-domain
polypeptide is detected by comparing the signal from the well(s) in
which the PL peptide and PDZ domain polypeptide are combined with
the background signal(s). The background signal is the signal found
in the negative controls. Typically a specific or selective
reaction will be at least twice background signal, more typically
more than 5 times background, and most typically 10 or more times
the background signal. In addition, a statistically significant
reaction will involve multiple measurements of the reaction with
the signal and the background differing by at least two standard
errors, more typically four standard errors, and most typically six
or more standard errors. Correspondingly, a statistical test (e.g.
a T-test) comparing repeated measurements of the signal with
repeated measurements of the background will result in a p-value
<0.05, more typically a p-value <0.01, and most typically a
p-value <0.001 or less.
[0227] As noted, in an embodiment of the A assay, the signal from
binding of a GST/PDZ-domain fusion protein to an avidin surface not
exposed to (i.e. not covered with) the PL peptide is one suitable
negative control (sometimes referred to as B1). The signal from
binding of GST polypeptide alone (i.e. not a fusion protein) to an
avidin-coated surface that has been exposed to (i.e. covered with)
the PL peptide is a second suitable negative control (sometimes
referred to as B2. Because all measurements are done in multiples
(i.e. at least duplicate) the arithmetic mean (or, equivalently,
average) of several measurements is used in determining the
binding, and the standard error of the mean is used in determining
the probable error in the measurement of the binding. The standard
error of the mean of N measurements equals the square root of the
following: the sum of the squares of the difference between each
measurement and the mean, divided by the product of (N) and (N-1).
Thus, in one embodiment, specific binding of the PDZ protein to the
plate-bound PL peptide is determined by comparing the mean signal
("mean S") and standard error of the signal ("SE") for a particular
PL-PDZ combination with the mean B1 and/or mean B2.
[0228] 2) G Assay-Detection of PDZ-Ligand Binding Using Immobilized
PDZ-Domain Fusion Polypeptide
[0229] In one aspect, the invention provides an assay in which a
GST/PDZ fusion protein is immobilized on a surface (G assay). The
binding of labeled PL peptide (e.g., as listed in TABLE 2 and TABLE
4) to this surface is then measured. In a preferred embodiment, the
assay is carried out as follows:
[0230] (1) A PDZ-domain polypeptide is bound to a surface, e.g. a
protein binding surface. In a preferred embodiment, a GST/PDZ
fusion protein containing one or more PDZ domains is bound to a
polystyrene 96-well plate. The GST/PDZ fusion protein can be bound
to the plate by any of a variety of standard methods known to one
of skill in the art, although some care must be taken that the
process of binding the fusion protein to the plate does not alter
the ligand-binding properties of the PDZ domain. In one embodiment,
the GST/PDZ fusion protein is bound via an anti-GST antibody that
is coated onto the 96-well plate. Adequate binding to the plate can
be achieved when:
[0231] a. 100 .mu.l per well of 5 .mu.g/ml goat anti-GST polyclonal
antibody (Pierce) in PBS is added to a polystyrene 96-well plate
(e.g., Nunc Polysorp) at 4.degree. C. for 12 hours.
[0232] b. The plate is blocked by addition of 200 .mu.l per well of
PBS/BSA for 2 hours at 4.degree. C.
[0233] c. The plate is washed 3 times with PBS.
[0234] d. 50 .mu.l per well of 5 .mu.g/ml GST/PDZ fusion protein)
or, as a negative control, GST polypeptide alone (i.e. not a fusion
protein) in PBS/BSA is added to the plate for 2 hours at 4.degree.
C.
[0235] e. the plate is again washed 3 times with PBS.
[0236] (2) Biotinylated PL peptides are allowed to react with the
surface by addition of 50 .mu.l per well of 20 .mu.M solution of
the biotinylated peptide in PBS/BSA for 10 minutes at 4.degree. C.,
followed by an additional 20 minute incubation at 25.degree. C. The
plate is washed 3 times with ice cold PBS.
[0237] (3) The binding of the biotinylated peptide to the GST/PDZ
fusion protein surface can be detected using a variety of methods
and detectors known to one of skill in the art. In one embodiment,
100 .mu.l per well of 0.5 .mu.g/ml streptavidin-horseradish
peroxidase (HRP) conjugate dissolved in BSA/PBS is added and
allowed to react for 20 minutes at 4.degree. C. The plate is then
washed 5 times with 50 mM Tris pH 8.0 containing 0.2% Tween 20, and
developed by addition of 100 .mu.l per well of HRP-substrate
solution (TMB, Dako) for 20 minutes at room temperature (RT). The
reaction of the HRP and its substrate is terminated by addition of
100 .mu.l per well of 1 M sulfuric acid, and the optical density
(O.D.) of each well of the plate is read at 450 um.
[0238] (4) Specific binding of a PL peptide and a PDZ domain
polypeptide is determined by comparing the signal from the well(s)
in which the PL peptide and PDZ domain polypeptide are combined,
with the background signal(s). The background signal is the signal
found in the negative control(s). Typically a specific or selective
reaction will be at least twice background signal, more typically
more than 5 times background, and most typically 10 or more times
the background signal. In addition, a statistically significant
reaction will involve multiple measurements of the reaction with
the signal and the background differing by at least two standard
errors, more typically four standard errors, and most typically six
or more standard errors. Correspondingly, a statistical test (e.g.
a T-test) comparing repeated measurements of the signal with
-repeated measurements of the background will result in a p-value
<0.05, more typically a p-value <0.01, and most typically a
p-value <0.001 or less. As noted, in an embodiment of the G
assay, the signal from binding of a given PL peptide to immobilized
(surface bound) GST polypeptide alone is one suitable negative
control (sometimes referred to as B1). Because all measurement are
done in multiples (i.e. at least duplicate) the arithmetic mean
(or, equivalently, average.) of several measurements is used in
determining the binding, and the standard error of the mean is used
in determining the probable error in the measurement of the
binding. The standard error of the mean of N measurements equals
the square root of the following: the sum of the squares of the
difference between each measurement and the mean, divided by the
product of (N) and (N-1). Thus, in one embodiment, specific binding
of the PDZ protein to the platebound peptide is determined by
comparing the mean signal ("mean S") and standard error of the
signal ("SE") for a particular PL-PDZ combination with the mean
B1.
[0239] i) G1 Assay, G2 Assay, and G3 Assay
[0240] Three specific modifications of the specific conditions
described supra for the G assay are particularly useful. The
modified assays use lesser quantities of labeled PL peptide and
have slightly different biochemical requirements for detection of
PDZ-ligand binding compared to the specific assay conditions
described supra. For convenience, the assay conditions described in
this section are referred to as the G1 assay, the G2 assay, and the
G3 assay, with the specific conditions described in the preceding
section on G assays being referred to as the G0 assay. The G1 assay
is identical to the G0 assay except at step (2) the peptide
concentration is 10 .mu.M instead of 20 .mu.M. This results in
slightly lower sensitivity for detection of interactions with low
affinity and/or rapid dissociation rate. Correspondingly, it
slightly increases the certainty that detected interactions are of
sufficient affinity and half-life to be of biological importance
and useful therapeutic targets.
[0241] The G2 assay is identical to the G0 assay except that at
step (2) the peptide concentration is 1 .mu.M instead of 20 .mu.M
and the incubation is performed for 60 minutes at 25.degree. C.
(rather than, e.g., 10 minutes at 4.degree. C. followed by 20
minutes at 25.degree. C.). This results in lower sensitivity for
interactions of low affinity, rapid dissociation rate, and/or
affinity that is less at 25.degree. C. than at 4.degree. C.
Interactions will have lower affinity at 25.degree. C. than at
4.degree. C. if (as we have found to be generally true for
PDZ-ligand binding) the reaction entropy is negative (i.e. the
entropy of the products is less than the entropy of the reactants).
In contrast, the PDZ-PL binding signal may be similar in the G2
assay and the G0 assay for interactions of slow association and
dissociation rate, as the PDZ-PL complex will accumulate during the
longer incubation of the "G'' assay." Thus comparison of results of
the G2 assay and the G0 assay can be used to estimate the relative
entropies, enthalpies, and kinetics of different PDZ-PL
interactions. (Entropies and enthalpies are related to binding
affinity by the equations delta G=RT in (Kd)=delta H-T delta S
where delta G, H, and S are the reaction free energy, enthalpy, and
entropy respectively, T is the temperature in degrees Kelvin, R is
the gas constant, and Kd is the equilibrium dissociation constant).
In particular, interactions that are detected only or much more
strongly in the G0 assay generally have a rapid dissociation rate
at 25.degree. C. (t.sub.1/2<10 minutes) and a negative reaction
entropy, while interactions that are detected similarly strongly in
the "G'' assay" generally have a slower dissociation rate at
25.degree. C. (t.sub.1/2>10 minutes). Rough estimation of the
thermodynamics and kinetics of PDZ-PL interactions (as can be
achieved via comparison of results of the G0 assay versus the G2
assay as outlined supra) can be used in the design of efficient
inhibitors of the interactions. For example, a small molecule
inhibitor based on the chemical structure of a PL that dissociates
slowly from a given PDZ domain (as evidenced by similar binding in
the G2 assay as in the G0 assay) may itself dissociate slowly and
thus be of high affinity.
[0242] The G3 assay is identical to the G0 assay with the following
exceptions. The peptides are typically present at 0.1 .mu.M rather
than 20 .mu.M. The peptides are also pre-incubated with the
HRP-streptavidin prior to adding to the assay plate. In the G0
assay, free peptide is incubated with the PDZ proteins prior to the
addition of the HRP-streptavidin. Thus, for the G0 assay one can
lose signal if the bound peptide dissociates from the PDZ protein
prior to the addition of the HRP-streptavidin. In the G3 modified
assay the HRP-streptavidin/peptide complex is added to the plate in
one step, thus increasing the likelihood that all the bound peptide
will be bound to HRP-streptavidin. The G3 modified assay increases
the chance of observing weak interactions.
[0243] In this manner, variation of the temperature and duration of
step (2) of the G assay can be used to provide insight into the
kinetics and thermodynamics of the PDZ-ligand binding reaction and
into design of inhibitors of the reaction.
[0244] With any of the assays, peptides should be titrated to find
the optimal concentration for which the signal to noise ratio is in
the appropriate range over the entire collection of PDZ domains
tested.
[0245] 3) Assay Variations
[0246] As discussed supra, it will be appreciated that many of the
steps in the above-described assays can be varied, for example,
various substrates can be used for binding the PL and
PDZ-containing proteins; different types of PDZ containing fusion
proteins can be used; different labels for detecting PDZ/PL
interactions can be employed; and different ways of detection can
be used.
[0247] The PL protein used in the assay is not intended to be
limited to a 20 amino acid peptide. Full length or partial protein
may be used, either alone or as a fusion protein. For example, a
GST-PL protein fusion may be bound to the anti-GST antibody, with
PDZ protein added to the bound PL protein or peptide.
[0248] The PDZ-PL detection assays can employ a variety of surfaces
to bind the PL and PDZ-containing proteins. For example, a surface
can be an "assay plate" which is formed from a material (e.g.
polystyrene) which optimizes adherence of either the PL protein or
PDZ-containing protein thereto. Generally, the individual wells of
the assay plate will have a high surface area to volume ratio and
therefore a suitable shape is a flat bottom well (where the
proteins of the assays are adherent). Other surfaces include, but
are not limited to, polystyrene or glass beads, polystyrene or
glass slides, and the like.
[0249] For example, the assay plate can be a multiwell plate. The
term multiwell plate when used herein refers to a multiwell assay
plate, e.g., having between about 30 to 200 individual wells,
usually 96 wells. Alternatively, high density arrays can be used.
Often, the individual wells of the multiwell plate will hold a
maximum volume of about 250 .mu.l. Conveniently, the assay plate is
a 96 well polystyrene plate (such as that sold by Becton Dickinson
Labware, Lincoln Park, N.J.), which allows for automation and high
throughput screening. Other surfaces include polystyrene multiwell
ELISA plates such as that sold by Nunc Maxisorp, Inter Med,
Denmark. Often, about 50 .mu.l to 300 .mu.l, more preferably 100
.mu.l to 200 .mu.l, of an aqueous sample comprising buffers
suspended therein will be added to each well of the assay
plate.
[0250] The detectable labels of the invention can be any detectable
compound or composition which is conjugated directly or indirectly
with a molecule (such as described above). The label can be
detectable by itself (e.g., radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, can catalyze a
chemical alteration of a substrate compound or composition which is
detectable. The preferred label is an enzymatic one which catalyzes
a color change of a non-radioactive color reagent.
[0251] Sometimes, the label is indirectly conjugated with the
antibody. One of skill is aware of various techniques for indirect
conjugation. For example, the antibody can be conjugated with
biotin and any of the categories of labels mentioned above can be
conjugated with avidin, or vice versa (see also A and G assay
above). Biotin binds selectively to avidin and thus, the label can
be conjugated with the antibody in this indirect manner. See,
Ausubel, supra, for a review of techniques involving biotin-avidin
conjugation and similar assays. Alternatively, to achieve indirect
conjugation of the label with the antibody, the antibody is
conjugated with a small hapten (e.g. digoxin) and one of the
different types of labels mentioned above is conjugated with an
anti-hapten antibody (e.g. anti-digoxin antibody). Thus, indirect
conjugation of the label with the antibody can be achieved.
[0252] Assay variations can include different washing steps. By
"washing" is meant exposing the solid phase to an aqueous solution
(usually a buffer or cell culture media) in such a way that unbound
material (e.g., non-adhering cells, non-adhering capture agent,
unbound ligand, receptor, receptor construct, cell lysate, or HRP
antibody) is removed therefrom. To reduce background noise, it is
convenient to include a detergent (e.g., Triton X) in the washing
solution. Usually, the aqueous washing solution is decanted from
the wells of the assay plate following washing. Conveniently,
washing can be achieved using an automated washing device.
Sometimes, several washing steps (e.g., between about 1 to 10
washing steps) can be required.
[0253] Various buffers can also be used in PDZ-PL detection assays.
For example, various blocking buffers can be used to reduce assay
background. The term "blocking buffer" refers to an aqueous, pH
buffered solution containing at least one blocking compound which
is able to bind to exposed surfaces of the substrate which are not
coated with a PL or PDZ-containing protein. The blocking compound
is normally a protein such as bovine serum albumin (BSA), gelatin,
casein or milk powder and does not cross-react with any of the
reagents in the assay. The block buffer is generally provided at a
pH between about 7 to 7.5 and suitable buffering agents include
phosphate and TRIS.
[0254] Various enzyme-substrate combinations can also be utilized
in detecting PDZ-PL interactions. Examples of enzyme-substrate
combinations include, for example:
[0255] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g. orthophenylene diamine [OPD] or
3,3',5,5'-tetramethyl benzidine hydrochloride [TMB]) (as described
above).
[0256] (ii) alkaline phosphatase (AP) with para-Nitrophenyl
phosphate as chromogenic substrate.
[0257] (iii) .beta.-D-galactosidase (.beta. D-Gal) with a
chromogenic substrate (e.g. p-nitrophenyl-.beta.-D-galactosidase)
or fluorogenic substrate
4-methylumbelliferyl-.beta.-D-galactosidase.
[0258] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see
U.S. Pat. Nos. 4,275,149 and 4,318,980, both of which are herein
incorporated by reference.
[0259] Further, it will be appreciated that, although, for
convenience, the present discussion primarily refers antagonists of
PDZ-PL interactions, agonists of PDZ-PL interactions can be
identified using the methods disclosed herein or readily apparent
variations thereof.
[0260] E. Detecting PDZ-PL Interactions
[0261] The present inventors were able in part to identify the
interactions summarized in TABLES 6, 7, and 8 by developing new
high throughput screening assays which are described supra. Various
other assay formats known in the art can be used to select ligands
that are specifically reactive with a particular protein. For
example, solid-phase ELISA immunoassays, immunoprecipitation,
Biacore, Fluorescence Polarization (FP), Fluorescence Resonance
Energy Transfer (FRET) and western blot assays can be used to
identify peptides that specifically bind PDZ-domain polypeptides.
As discussed supra, two different, complementary assays were
developed to detect PDZ-PL interactions. In each, one binding
partner of a PDZ-PL pair is immobilized, and the ability of the
second binding partner to bind is determined. These assays, which
are described supra, can be readily used to screen for hundreds to
thousands of potential PDZ-ligand interactions in a few hours. Thus
these assays can be used to identify yet more novel PDZ-PL
interactions in neuronal cells. In addition, they can be used to
identify antagonists of PDZ-PL interactions (see infra).
[0262] In various embodiments, fusion proteins are used in the
assays and devices of the invention. Methods for constructing and
expressing fusion proteins are well known. Fusion proteins
generally are described in Ausubel et al., supra, Kroll et al.,
1993, DNA Cell. Biol. 12:441, and Imai et al., 1997, Cell
91:521-30. Usually, the fusion protein includes a domain to
facilitate immobilization of the protein to a solid substrate ("an
immobilization domain"). Often, the immobilization domain includes
an epitope tag (i.e., a sequence recognized by a antibody,
typically a monoclonal antibody) such as polyhistidine (Bush et al,
1991, J. Biol Chem 266:13811-14), SEAP (Berger et al, 1988, Gene
66:1-10), or M1 and M2 flag (see, e.g., U.S. Pat. Nos. 5,011,912;
4,851,341; 4,703,004; 4,782,137). In an embodiment, the
immobilization domain is a GST coding region. It will be recognized
that, in addition to the PDZ-domain and the particular residues
bound by an immobilized antibody, protein A, or otherwise contacted
with the surface, the protein (e.g., fusion protein), will contain
additional residues. In some embodiments these are residues
naturally associated with the PDZ-domain (i.e., in a particular
PDZ-protein) but they may include residues of synthetic (e.g.,
poly(alanine)) or heterologous origin (e.g., spacers of, e.g.,
between 10 and 300 residues).
[0263] PDZ domain-containing polypeptide used in the methods of the
invention (e.g., PDZ fusion proteins) of the invention are
typically made by (1) constructing a vector (e.g., plasmid, phage
or phagemid) comprising a polynucleotide sequence encoding the
desired polypeptide, (2) introducing the vector into an suitable
expression system (e.g., a prokaryotic, insect, mammalian, or cell
free expression system), (3) expressing the fusion protein and (4)
optionally purifying the fusion protein.
[0264] In one embodiment, expression of the protein comprises
inserting the coding sequence into an appropriate expression vector
(i.e., a vector that contains the necessary elements for the
transcription and translation of the inserted coding sequence
required for the expression system employed, e.g., control elements
including enhancers, promoters, transcription terminators, origins
of replication, a suitable initiation codon (e.g., methionine),
open reading frame, and translational regulatory signals (e.g., a
ribosome binding site, a termination codon and a polyadenylation
sequence. Depending on the vector system and host utilized, any
number of suitable transcription and translation elements,
including constitutive and inducible promoters, can be used.
[0265] The coding sequence of the fusion protein includes a PDZ
domain and an immobilization domain as described elsewhere herein.
Polynucleotides encoding the amino acid sequence for each domain
can be obtained in a variety of ways known in the art; typically
the polynucleotides are obtained by PCR amplification of cloned
plasmids, cDNA libraries, and cDNA generated by reverse
transcription of RNA, using primers designed based on sequences
determined by the practitioner or, more often, publicly available
(e.g., through GenBank). The primers include linker regions (e.g.,
sequences including restriction sites) to facilitate cloning and
manipulation in production of the fusion construct. The
polynucleotides corresponding to the PDZ and immobilization regions
are joined in-frame to produce the fusion protein-encoding
sequence.
[0266] The fusion proteins of the invention may be expressed as
secreted proteins (e.g., by including the signal sequence encoding
DNA in the fusion gene; see, e.g., Lui et al, 1993, PNAS USA,
90:8957-61) or as nonsecreted proteins.
[0267] In some embodiments, the PDZ-containing proteins are
immobilized on a solid surface. The substrate to which the
polypeptide is bound may in any of a variety of forms, e.g., a
multiwell dish, a test tube, a dipstick, a microcentrifuge tube, a
bead, a spinnable disk, and the like. Suitable materials include
glass, plastic (e.g., polyethylene, PVC, polypropylene,
polystyrene, and the like), protein, paper, carbohydrate, lipid
monolayer or supported lipid bilayer, and other solid supports.
Other materials that may be employed include ceramics, metals,
metalloids, semiconductive materials, cements and the like.
[0268] In some embodiments, the fusion proteins are organized as an
array. The term "array," as used herein, refers to an ordered
arrangement of immobilized fusion proteins, in which particular
different fusion proteins (i.e., having different PDZ domains) are
located at different predetermined sites on the substrate. Because
the location of particular fusion proteins on the array is known,
binding at that location can be correlated with binding to the PDZ
domain situated at that location. Immobilization of fusion proteins
on beads (individually or in groups) is another particularly useful
approach. In one embodiment, individual fusion proteins are
immobilized on beads. In one embodiment, mixtures of
distinguishable beads are used. Distinguishable beads are beads
that can be separated from each other on the basis of a property
such as size, magnetic property, color (e.g., using FACS) or
affinity tag (e.g., a bead coated with protein A can be separated
from a bead not coated with protein A by using IgG affinity
methods). Binding to particular PDZ domain may be determined;
similarly, the effect of test compounds (i.e., agonists and
antagonists of binding) may be determined.
[0269] Methods for immobilizing proteins are known, and include
covalent and non-covalent methods. One suitable immobilization
method is antibody-mediated immobilization. According to this
method, an antibody specific for the sequence of an "immobilization
domain" of the PDZ-domain containing protein is itself immobilized
on the substrate (e.g., by adsorption). One advantage of this
approach is that a single antibody may be adhered to the substrate
and used for immobilization of a number of polypeptides (sharing
the same immobilization domain). For example, an immobilization
domain consisting of poly-histidine (Bush et al, 1991, J. Biol Chem
266:13811-14) can be bound by an anti-histidine monoclonal antibody
(R&D Systems, Minneapolis, Minn.); an immobilization domain
consisting of secreted alkaline phosphatase ("SEAP") (Berger et al,
1988, Gene 66:1-10) can be bound by anti-SEAP (Sigma Chemical
Company, St. Louis, Mo.); an immobilization domain consisting of a
FLAG epitope can be bound by anti-FLAG. Other ligand-antiligand
immobilization methods are also suitable (e.g., an immobilization
domain consisting of protein A sequences (Harlow and Lane, 1988,
Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory;
Sigma Chemical Co., St. Louis, Mo.) can be bound by IgG; and an
immobilization domain consisting of streptavidin can be bound by
biotin (Harlow & Lane, supra; Sigma Chemical Co., St. Louis,
Mo.). In a preferred embodiment, the immobilization domain is a GST
moiety, as described herein.
[0270] When antibody-mediated immobilization methods are used,
glass and plastic are especially useful substrates. The substrates
may be printed with a hydrophobic (e.g., Teflon) mask to form
wells. Preprinted glass slides with 3, 10 and 21 wells per 14.5
cm.sup.2 slide "working area" are available from, e.g., SPI
Supplies, West Chester, Pa.; also see U.S. Pat. No. 4,011,350). In
certain applications, a large format (12.4 cm.times.8.3 cm) glass
slide is printed in a 96 well format is used; this format
facilitates the use of automated liquid handling equipment and
utilization of 96 well format plate readers of various types
(fluorescent, calorimetric, scintillation). However, higher
densities may be used (e.g., more than 10 or 100 polypeptides per
cm.sup.2). See, e.g., MacBeath et al, 2000, Science
289:1760-63.
[0271] Typically, antibodies are bound to substrates (e.g., glass
substrates) by adsorption. Suitable adsorption conditions are well
known in the art and include incubation of 0.5-50 .mu.g/ml (e.g.,
10 .mu.g/ml) mAb in buffer (e.g., PBS, or 50 to 300 mM Tris, MOPS,
HEPES, PIPES, acetate buffers, pHs 6.5 to 8, at 4.degree. C.) to
37.degree. C. and from 1 hr to more than 24 hours.
[0272] Proteins may be covalently bound or noncovalently attached
through nonspecific bonding. If covalent bonding between the fusion
protein and the surface is desired, the surface will usually be
polyfunctional or be capable of being polyfunctionalized.
Functional groups which may be present on the surface and used for
linking can include carboxylic acids, aldehydes, amino groups,
cyano groups, ethylenic groups, hydroxyl groups, mercapto groups
and the like. The manner of linking a wide variety of compounds to
various surfaces is well known and is amply illustrated in the
literature.
[0273] F. Results of PDZ-PL Interaction Assays
[0274] TABLE 6 shows the predicted results of assays in which
specific binding will be detected between TRPM7 and PDZ proteins
using the G3 assay described herein TABLE 8 shows PDZ domains
demonstrated to interact with Tat-TRPM7 peptide. TABLE 7 shows PDZs
that have been demonstrated to interact with PLs involved in
excitotoxicity.
TABLE-US-00008 TABLE 6 PDZs predicted to interact with TRPM7 TRPM7
NNOS, LIM, KIAA1095, HEMBA1003117, AIPC, KIAA1526, DVL1, DVL2,
DVL3, PTPL1, ZO-1, ZO-2, ZO-3, KIAA1719, Mupp1, INADL, Shank 3,
MINT1, MINT2, MAGI1, MAGI2, MAGI3, NeDLG, syntenin, PSD-95, hDLG,
PAR3, MAST1, MAST2, AF6, SIP1, LIM mystique, KIAA0751, HTRA1,
HTRA2, TIP-1, KIAA0316, PICK1,
TABLE-US-00009 TABLE 7 PDZs demonstrated to interact with specific
PL proteins PL PDZs that interact NMDA Receptor 2s PSD95, DLG1,
DLG2, NeDLG, KIAA0973, Outermembrane Protein, syntrophin alpha 1,
TIP1, TIP2, MAGI1, MAGI2, syntrophin beta 1, syntrophin gamma1,
LIM-RIL, KIAA1634, KIAA0807 NMDA Receptor 1s NeDLG, DLG1, PTPL1,
PSD-95, MAGI3, AMPA Rs PLC beta, gamma EBP50, NNOS internal
TABLE-US-00010 TABLE 8 PDZ domains demonstrated to interact with
Tat-TRPM7 peptide Nervous System/ Brain PDZ Protein Domain GenBank
# Expression Reference RIM-2 1 12734165 yes Mukasa et al. (2004)
Brain Pathol. 14: 34-42 Mint 1 1 and 2 2625024 yes Ho et al. (2003)
Proc. Natl. Acad. Sci. U.S.A. 100: 1409-1414 INADL 3 2370148 yes
Philipp et al. (1997) FEBS Lett. 413: 243-248 Syntrophin 1 alpha 1
1145727 yes Connors et al. (2004) J. Biol. Chem. 279: 28387-28392
g45700023 g34455039 SITAC-18 2 8886071 yes Borrell-Pages et al.
(2000) Mol. Biol. Cell 11: 4217-4225 LIM mystique 1 12734250 no
ZO-1 2 292937 yes Poliak et al. (2002) J. Cell Biol. 159: 361-372
PAR3L 3 18568347 yes Gao et al. (2002) Gene 294: 99-107 MAST2 1
3882334 yes g47035747 PAR3 3 8037914 yes Poliak et al. (2002) J.
Cell Biol. 159: 361-372 NSP [novel serine 1 1621243 yes g46953413
protease]
TABLE-US-00011 TABLE 9 results of titrations of the Tat-TRPM7
peptide error of fit, PDZ EC50, uM EC50, uM ODmax (450 nm) RIM2
(177.4) 0.047 0.005 2.10 Mint 1 (d1, d2) (36.5a) 0.180 0.016 1.90
TIP1 d1 (54.10) <1 ND 1.82 Mint1 d1 (146.5) ND ND 0.33 Mint1 d2
(147.2) <2 uM ND 0.76 INADL d3 (96.3) 0.120 0.015 1.90 MUPP1 d3
(108.3) 0.290 0.037 2.06 Syntrophin 1 alpha d1 0.160 0.017 2.05
(52.5) SITAC-18 d1 (122.2) <1 uM ND 1.70 SITAC-18 d2(123.2)
0.090 0.013 1.95 LIM Mystique d1 (232.1) 0.090 0.015 2.10 ZO-1 d2
(241.3) 0.033 0.003 2.20 PAR3L d3 (406.1) 0.100 0.017 1.97 MAST2 d1
(174.6) 0.110 0.008 2.10 PAR3 d3 (278.1) 0.018 0.002 2.50 KIAA1284
d1 (191.2) >2 uM ND 0.63
[0275] ZO-1 is a PDZ protein that is known to associate with cell
surface proteins, junctions and cytoskeletal proteins. TRPC4 has
also been shown to colocalize with ZO-1 (Song, X. et. Al. Glia.
2004, Nov. 11 [Epub] PMID 15540229). We have demonstrated that
TRPM7 interacts with the second PDZ domain of ZO-1. Without being
bound by mechanism, we propose that interruption of TRPM7/ZO-1 d2
interaction may reduce the efficiency Ca.sup.2+ pumping activity of
TRPM7/TRP during oxygen/glucose deprivation or ischemia, resulting
in protection from ischemia, especially neuroprotection.
[0276] MINT1 is a PDZ protein containing 2 PDZ domains and a PTB
domain. MINT1 has been shown to associate with KIR2, an inward
rectifying potassium channel (Leonoudakis D. et. al. J. Biol. Chem.
2004 279(21):22331-46). MINT1 has also been demonstrated to affect
transport of NMDA Receptor 2B to the post-synaptic density of
neurons (Scorza et al. Epilepsy Res. 2003 57(1): 49-57), and to
play a role in transient global ischema in the mouse hippocampus
(Nishimura, H. J. Cereb. Blood Flow Metab. 2000 20(10): 1437-45).
We have disclosed herein that TRPM7 binds to MINT1. Without being
bound by specific mechanism, we propose that disruption of this
interaction with a peptide therapeutic or small molecule will
reduce excitotoxicity and result in neuroprotection from ischemic
damage.
[0277] Partitioning-defective homolog 3 (PAR3) is a PDZ protein
with three PDZ domains. It is known to localize to tight junctions
form a complex that regulates signaling through cdc42/Rac and
Protein Kinases C and A (Lin, D. Nat. Cell Biol. 2 (8), 540-547
(2000)). We have disclosed that TRPM7 interacts with PAR3. Without
being bound by specific mechanism, we propose that disruption of
this interaction with a peptide therapeutic or small molecule will
reduce excitotoxicity and result in neuroprotection from ischemic
damage.
[0278] RIM2 is a PDZ protein known to be involved with secretory
vesicle exocytosis and vesicle transport (Fukuda, M. Genes Cells.
2004 September; 9(9):831-42). We have disclosed that TRPM7
interacts with RIM2. Without being bound by specific mechanism, we
propose that disruption of this interaction with a peptide
therapeutic or small molecule will reduce the ability of TRPM7 to
function appropriately at the membrane and result in
neuroprotection from ischemic damage.
[0279] INADL is a PDZ protein with multiple PDZ domains. It is
known to organize proteins at the cell membrane photoreceptor
complex, including the Drosophila TRP channel. We disclose herein
that TRPM7 binds the third PDZ domain of INADL. Without being bound
by specific mechanism, we propose that disruption of this
interaction with a peptide therapeutic or small molecule will
reduce the ability of TRPM7 to associate with necessary accessory
proteins and thereby function, resulting in neuroprotection from
ischemic damage.
[0280] G. Measurement of PDZ-Ligand Binding Affinity
[0281] The A and G assays of the invention can be used to determine
the "apparent affinity" of binding of a PDZ ligand peptide to a
PDZ-domain polypeptide. Apparent affinity is determined based on
the concentration of one molecule required to saturate the binding
of a second molecule (e.g., the binding of a ligand to a receptor).
Two particularly useful approaches for quantitation of apparent
affinity of PDZ-ligand binding are provided infra.
[0282] Approach 1:
[0283] (1) A GST/PDZ fusion protein, as well as GST alone as a
negative control, are bound to a surface (e.g., a 96-well plate)
and the surface blocked and washed as described supra for the G
assay.
[0284] (2) 50 .mu.l per well of a solution of biotinylated PL
peptide (e.g. as shown in TABLE 2) is added to the surface in
increasing concentrations in PBS/BSA (e.g. at 0.1 .mu.M, 0.33
.mu.M, 1 .mu.M, 3.3 .mu.M, 10 .mu.M, 33 .mu.M, and 100 .mu.M). In
one embodiment, the PL peptide is allowed to react with the bound
GST/PDZ fusion protein (as well as the GST alone negative control)
for 10 minutes at 4.degree. C. followed by 20 minutes at 25.degree.
C. The plate is washed 3 times with ice cold PBS to remove unbound
labeled peptide.
[0285] (3) The binding of the PL peptide to the immobilized
PDZ-domain polypeptide is detected as described supra for the G
assay.
[0286] (4) For each concentration of peptide, the net binding
signal is determined by subtracting the binding of the peptide to
GST alone from the binding of the peptide to the GST/PDZ fusion
protein. The net binding signal is then plotted as a function of
ligand concentration and the plot is fit (e.g. by using the
Kaleidagraph Synergy Software, Reading, Pa.) software package curve
fitting algorithm) to the following equation, where
"Signal.sub.[ligand]" is the net binding signal at PL peptide
concentration "[ligand]," "K.sub.d" is the apparent affinity of the
binding event, and "Saturation Binding" is a constant determined by
the curve fitting algorithm to optimize the fit to the experimental
data:
Signal.sub.[ligand]=Saturation
Binding.times.([ligand]/([ligand]+K.sub.d))
[0287] For reliable application of the above equation it is
necessary that the highest peptide ligand concentration
successfully tested experimentally be greater than, or at least
similar to, the calculated K.sub.d (equivalently, the maximum
observed binding should be similar to the calculated saturation
binding). In cases where satisfying the above criteria proves
difficult, an alternative approach (infra) can be used.
[0288] Approach 2:
[0289] (1) A fixed concentration of a PDZ-domain polypeptide and
increasing concentrations of a labeled PL peptide (labeled with,
for example, biotin or fluorescein, see TABLE 2 and TABLE 4 for
representative peptide amino acid sequences) are mixed together in
solution and allowed to react. In one embodiment, preferred peptide
concentrations are 0.1 .mu.M, 1 .mu.M, 10 .mu.M, 100 .mu.M, 1 mM.
In various embodiments, appropriate reaction times can range from
10 minutes to 2 days at temperatures ranging from 4.degree. C. to
37.degree. C. In some embodiments, the identical reaction can also
be carried out using a non-PDZ domain-containing protein as a
control (e.g., if the PDZ-domain polypeptide is fusion protein, the
fusion partner can be used).
[0290] (2) PDZ-ligand complexes can be separated from unbound
labeled peptide using a variety of methods known in the art. For
example, the complexes can be separated using high performance
size-exclusion chromatography (HPSEC, gel filtration) (Rabinowitz
et al., 1998, Immunity 9:699), affinity chromatography (e.g. using
glutathione SEPHAROSE beads), and affinity absorption (e.g., by
binding to an anti-GST-coated plate as described supra).
[0291] (3) The PDZ-ligand complex is detected based on presence of
the label on the peptide ligand using a variety of methods and
detectors known to one of skill in the art. For example, if the
label is fluorescein and the separation is achieved using HPSEC, an
in-line fluorescence detector can be used. The binding can also be
detected as described supra for the G assay.
[0292] (4) The PDZ-ligand binding signal is plotted as a function
of ligand concentration and the plot is fit. (e.g., by using the
Kaleidagraph software package curve fitting algorithm) to the
following equation, where "Signal.sub.[ligand]" is the binding
signal at PL peptide concentration "[ligand]," "K.sub.d" is the
apparent affinity of the binding event, and "Saturation Binding" is
a constant determined by the curve fitting algorithm to optimize
the fit to the experimental data:
Signal.sub.[Ligand]=Saturation
Binding.times.([ligand]/([ligand+K.sub.d])
[0293] Measurement of the affinity of a labeled peptide ligand
binding to a PDZ-domain polypeptide is useful because knowledge of
the affinity (or apparent affinity) of this interaction allows
rational design of inhibitors of the interaction with known
potency. The potency of inhibitors in inhibition would be similar
to (i.e. within one-order of magnitude of) the apparent affinity of
the labeled peptide ligand binding to the PDZ-domain.
[0294] Thus, in one aspect, the invention provides a method of
determining the apparent affinity of binding between a PDZ domain
and a ligand by immobilizing a polypeptide comprising the PDZ
domain and a non-PDZ domain on a surface, contacting the
immobilized polypeptide with a plurality of different
concentrations of the ligand, determining the amount of binding of
the ligand to the immobilized polypeptide at each of the
concentrations of ligand, and calculating the apparent affinity of
the binding based on that data. Typically, the polypeptide
comprising the PDZ domain and a non-PDZ domain is a fusion protein.
In one embodiment, the e.g., fusion protein is GST-PDZ fusion
protein, but other polypeptides can also be used (e.g., a fusion
protein including a PDZ domain and any of a variety of epitope
tags, biotinylation signals and the like) so long as the
polypeptide can be immobilized in an orientation that does not
abolish the ligand binding properties of the PDZ domain, e.g, by
tethering the polypeptide to the surface via the non-PDZ domain via
an anti-domain antibody and leaving the PDZ domain as the free end.
It was discovered, for example, reacting a PDZ-GST fusion
polypeptide directly to a plastic plate provided suboptimal
results. The calculation of binding affinity itself can be
determined using any suitable equation (e.g., as shown supra; also
see Cantor and Schimmel (1980) Biophysical Chemistry WH Freeman
& Co., San Francisco) or software.
[0295] Thus, in a preferred embodiment, the polypeptide is
immobilized by binding the polypeptide to an immobilized
immunoglobulin that binds the non-PDZ domain (e.g., an anti-GST
antibody when a GST-PDZ fusion polypeptide is used). In a preferred
embodiment, the step of contacting the ligand and PDZ-domain
polypeptide is carried out under the conditions provided supra in
the description of the G assay. It will be appreciated that binding
assays are conveniently carried out in multiwell plates (e.g.,
24-well, 96-well plates, or 384 well plates).
[0296] The present method has considerable advantages over other
methods for measuring binding affinities PDZ-PL affinities, which
typically involve contacting varying concentrations of a GST-PDZ
fusion protein to a ligand-coated surface. For example, some
previously described methods for determining affinity (e.g., using
immobilized ligand and GST-PDZ protein in solution) did not account
for oligomerization state of the fusion proteins used, resulting in
potential errors of more than an order of magnitude.
[0297] Although not sufficient for quantitative measurement of
PDZ-PL binding affinity, an estimate of the relative strength of
binding of different PDZ-PL pairs can be made based on the absolute
magnitude of the signals observed in the G assay. This estimate
will reflect several factors, including biologically relevant
aspects of the interaction, including the affinity and the
dissociation rate. For comparisons of different ligands binding to
a given PDZ domain-containing protein, differences in absolute
binding signal likely relate primarily to the affinity and/or
dissociation rate of the interactions of interest.
[0298] H. Assays to Identify Novel PDZ Domain Binding Moieties and
to Identify Modulators of PDZ Protein-PL Protein Binding
[0299] Although described supra primarily in terms of identifying
interactions between PDZ-domain polypeptides and PL proteins, the
assays described supra and other assays can also be used to
identify the binding of other molecules (e.g., peptide mimetics,
small molecules, and the like) to PDZ domain sequences. For
example, using the assays disclosed herein, combinatorial and other
libraries of compounds can be screened, e.g., for molecules that
specifically bind to PDZ domains. Screening of libraries can be
accomplished by any of a variety of commonly known methods. See,
e.g., the following references, which disclose screening of peptide
libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol.
251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et
al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc.
Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell
76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al.,
1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad.
Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852;
U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No.
5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science
263:671-673; and PCT Publication No. WO 94/18318.
[0300] In a specific embodiment, screening can be carried out by
contacting the library members with a PDZ-domain polypeptide
immobilized on a solid support (e.g. as described supra in the G
assay) and harvesting those library members that bind to the
protein. Examples of such screening methods, termed "panning"
techniques are described by way of example in Parmley and Smith,
1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques
13:422-427; PCT Publication No. WO 94/18318; and in references
cited hereinabove.
[0301] In another embodiment, the two-hybrid system for selecting
interacting proteins in yeast (Fields and Song, 1989, Nature
340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA
88:9578-9582) can be used to identify molecules that specifically
bind to a PDZ domain-containing protein. Furthermore, the
identified molecules are further tested for their ability to
inhibit transmembrane receptor interactions with a PDZ domain.
[0302] In one aspect of the invention, antagonists of an
interaction between a PDZ protein and a PL protein are identified.
In one embodiment, a modification of the A assay described supra is
used to identify antagonists. In one embodiment, a modification of
the G assay described supra is used to identify antagonists.
[0303] In certain embodiments, screening assays are used to detect
molecules that specifically bind to PDZ domains. Such molecules are
useful as agonists or antagonists of PDZ-protein-mediated cell
function (e.g., cell activation, e.g., T cell activation, vesicle
transport, cytokine release, growth factors, transcriptional
changes, cytoskeleton rearrangement, cell movement, chemotaxis,
intercellular signaling, regulation of synaptic function, neuronal
excitation, cytoskeletal integrity, and neurotransmitter release).
In one embodiment, such assays are performed to screen for
leukocyte activation inhibitors for drug development. The invention
thus provides assays to detect molecules that specifically bind to
PDZ domain-containing proteins. For example, recombinant cells
expressing PDZ domain-encoding nucleic acids can be used to produce
PDZ domains in these assays and to screen for molecules that bind
to the domains. Molecules are contacted with the PDZ domain (or
fragment thereof) under conditions conducive to binding, and then
molecules that specifically bind to such domains are identified.
Methods that can be used to carry out the foregoing are commonly
known in the art.
[0304] It will be appreciated by the ordinarily skilled
practitioner that, in one embodiment, antagonists are identified by
conducting the A or G assays in the presence and absence of a known
or candidate antagonist. When decreased binding is observed in the
presence of a compound, that compound is identified as an
antagonist. Increased binding in the presence of a compound
signifies that the compound is an agonist.
[0305] For example, in one assay, a test compound can be identified
as an inhibitor (antagonist) of binding between a PDZ protein and a
PL protein by contacting a PDZ domain polypeptide and a PL peptide
or protein in the presence and absence of the test compound, under
conditions in which they would (but for the presence of the test
compound) form a complex, and detecting the formation of the
complex in the presence and absence of the test compound. It will
be appreciated that less complex formation in the presence of the
test compound than in the absence of the compound indicates that
the test compound is an inhibitor of a PDZ protein-PL protein
binding.
[0306] In one embodiment, the G assay is used in the presence or
absence of an candidate inhibitor. In one embodiment, the A assay
is used in the presence or absence of a candidate inhibitor.
[0307] In one embodiment (in which a G assay is used), one or more
PDZ domain-containing GST-fusion proteins are bound to the surface
of wells of a 96-well plate as described supra (with appropriate
controls including nonfusion GST protein). All fusion proteins are
bound in multiple wells so that appropriate controls and
statistical analysis can be done. A test compound in BSA/PBS
(typically at multiple different concentrations) is added to wells.
Immediately thereafter, 30 .mu.l of a detectably labeled (e.g.,
biotinylated) PL peptide or protein known to bind to the relevant
PDZ domain (see, e.g., TABLE 5) is added in each of the wells at a
final concentration of, e.g., between about 2 .mu.M and about 40
.mu.M, typically 5 .mu.M, 15 .mu.M, or 25 .mu.M. This mixture is
then allowed to react with the PDZ fusion protein bound to the
surface for 10 minutes at 4.degree. C. followed by 20 minutes at
25.degree. C. The surface is washed free of unbound PL polypeptide
three times with ice cold PBS and the amount of binding of the
polypeptide in the presence and absence of the test compound is
determined. Usually, the level of binding is measured for each set
of replica wells (e.g. duplicates) by subtracting the mean GST
alone background from the mean of the raw measurement of
polypeptide binding in these wells.
[0308] In an alternative embodiment, the A assay is carried out in
the presence or absence of a test candidate to identify inhibitors
of PL-PDZ interactions.
[0309] If assays are conducted in the presence of test compound and
compared against binding in the absence of test compound, then the
assay can be conducted to determine if the difference between
binding in the presence and absence of the test compound is a
statistically significant difference.
[0310] In certain screening assays, assays are conducted to
identify compounds that can inhibit a binding interaction between a
TRP channel or TRP associated protein and a PDZ listed in TABLE 5.
In other screening assays involve screening to identify an
inhibitor that interferes with binding between TRPM7 and a PDZ
listed in TABLE 5.
[0311] In one embodiment, a test compound is determined to be a
specific inhibitor of the binding of the PDZ domain (P) and a PL
(L) sequence when, at a test compound concentration of less than or
equal to 1 mM (e.g., less than or equal to: 500 .mu.M, 100 .mu.M,
10 .mu.M, 1 .mu.M, 100 nM or 1 nM) the binding of P to L in the
presence of the test compound less than about 50% of the binding in
the absence of the test compound (in various embodiments, less than
about 25%, less than about 10%, or less than about 1%). Preferably,
the net signal of binding of P to L in the presence of the test
compound plus six (6) times the standard error of the signal in the
presence of the test compound is less than the binding signal in
the absence of the test compound.
[0312] In one embodiment, assays for an inhibitor are carried out
using a single PDZ protein-PL protein pair (e.g., a PDZ domain
fusion protein and a PL peptide or protein). In a related
embodiment, the assays are carried out using a plurality of pairs,
such as a plurality of different pairs listed in TABLES 2, 4 and
5.
[0313] In some embodiments, it is desirable to identify compounds
that, at a given concentration, inhibit the binding of one PL-PDZ
pair, but do not inhibit (or inhibit to a lesser degree) the
binding of a specified second PL-PDZ pair. These antagonists can be
identified by carrying out a series of assays using a candidate
inhibitor and different PL-PDZ pairs (e.g., as shown in TABLE 6, 7,
and 8) and comparing the results of the assays. All such pairwise
combinations are contemplated by the invention (e.g., test compound
inhibits binding of PL.sub.1 to PDZ.sub.1 to a greater degree than
it inhibits binding of PL.sub.1 to PDZ.sub.2 or PL.sub.2 to
PDZ.sub.2). Importantly, it will be appreciated that, based on the
data provided in TABLE 6, 7, and 8 and disclosed herein (and
additional data that can be generated using the methods described
herein) inhibitors with different specificities can readily be
designed.
[0314] For example, according to the invention, the K.sub.i
("potency") of an inhibitor of a PDZ-PL interaction can be
determined. K.sub.i is a measure of the concentration of an
inhibitor required to have a biological effect. For example,
administration of an inhibitor of a PDZ-PL interaction in an amount
sufficient to result in an intracellular inhibitor concentration of
at least between about 1 and about 100 K.sub.i is expected to
inhibit the biological response mediated by the target PDZ-PL
interaction. In one aspect of the invention, the K.sub.d
measurement of PDZ-PL binding as determined using the methods supra
is used in determining K.sub.i.
[0315] Thus, in one aspect, the invention provides a method of
determining the potency (K.sub.i) of an inhibitor or suspected
inhibitor of binding between a PDZ domain and a ligand by
immobilizing a polypeptide comprising the PDZ domain and a non-PDZ
domain on a surface, contacting the immobilized polypeptide with a
plurality of different mixtures of the ligand and inhibitor,
wherein the different mixtures comprise a fixed amount of ligand
and different concentrations of the inhibitor, determining the
amount of ligand bound at the different concentrations of
inhibitor, and calculating the K.sub.i of the binding based on the
amount of ligand bound in the presence of different concentrations
of the inhibitor. In an embodiment the polypeptide is immobilized
by binding the polypeptide to an immobilized immunoglobulin that
binds the non-PDZ domain. This method, which is based on the G
assay described supra, is particularly suited for high-throughput
analysis of the K.sub.i for inhibitors of PDZ-ligand interactions.
Further, using this method, the inhibition of the PDZ-ligand
interaction itself is measured, without distortion of measurements
by avidity effects.
[0316] Typically, at least a portion of the ligand is detectably
labeled to permit easy quantitation of ligand binding.
[0317] It will be appreciated that the concentration of ligand and
concentrations of inhibitor are selected to allow meaningful
detection of inhibition. Thus, the concentration of the ligand
whose binding is to be blocked is close to or less than its binding
affinity (e.g., preferably less than the 5.times.K.sub.d of the
interaction, more preferably less than 2.times.K.sub.d, most
preferably less than 1.times.K.sub.d). Thus, the ligand is
typically present at a concentration of less than 2 K.sub.d (e.g.,
between about 0.01 K.sub.d and about 2 K.sub.d) and the
concentrations of the test inhibitor typically range from 1 nM to
100 .mu.M (e.g. a 4-fold dilution series with highest concentration
10 .mu.M or 1 mM). In a preferred embodiment, the K.sub.d is
determined using the assay disclosed supra.
[0318] The K.sub.i of the binding can be calculated by any of a
variety of methods routinely used in the art, based on the amount
of ligand bound in the presence of different concentrations of the
inhibitor. In an illustrative embodiment, for example, a plot of
labeled ligand binding versus inhibitor concentration is fit to the
equation:
S.sub.inhibitor=S.sub.0*K.sub.i/([I]+K.sub.i)
where S.sub.inhibitor is the signal of labeled ligand binding to
immobilized PDZ domain in the presence of inhibitor at
concentration [I] and S.sub.0 is the signal in the absence of
inhibitor (i.e., [I]=0). Typically [I] is expressed as a molar
concentration.
[0319] In another aspect of the invention, an enhancer (sometimes
referred to as, augmentor or agonist) of binding between a PDZ
domain and a ligand is identified by immobilizing a polypeptide
comprising the PDZ domain and a non-PDZ domain on a surface,
contacting the immobilized polypeptide with the ligand in the
presence of a test agent and determining the amount of ligand
bound, and comparing the amount of ligand bound in the presence of
the test agent with the amount of ligand bound by the polypeptide
in the absence of the test agent. At least two-fold (often at least
5-fold) greater binding in the presence of the test agent compared
to the absence of the test agent indicates that the test agent is
an agent that enhances the binding of the PDZ domain to the ligand.
As noted supra, agents that enhance PDZ-ligand interactions are
useful for disruption (dysregulation) of biological events
requiring normal PDZ-ligand function (e.g., cancer cell division
and metastasis, and activation and migration of immune cells,
intercellular communication, neurotransmitter release, membrane
receptor turnover, second messenger signaling responsible for cell
homeostasis and function).
[0320] The invention also provides methods for determining the
"potency" or "K.sub.enhancer" of an enhancer of a PDZ-ligand
interaction. For example, according to the invention, the
K.sub.enhancer of an enhancer of a PDZ-PL interaction can be
determined, e.g., using the Kd of PDZ-PL binding as determined
using the methods described supra. K.sub.enhancer is a measure of
the concentration of an enhancer expected to have a biological
effect. For example, administration of an enhancer of a PDZ-PL
interaction in an amount sufficient to result in an intracellular
inhibitor concentration of at least between about 0.1 and about 100
K.sub.enhancer (e.g., between about 0.5 and about 50
K.sub.enhancer) is expected to disrupt the biological response
mediated by the target PDZ-PL interaction.
[0321] Thus, in one aspect the invention provides a method of
determining the potency (K.sub.enhancer) of an enhancer or
suspected enhancer of binding between a PDZ domain and a ligand by
immobilizing a polypeptide comprising the PDZ domain and a non-PDZ
domain on a surface, contacting the immobilized polypeptide with a
plurality of different mixtures of the ligand and enhancer, wherein
the different mixtures comprise a fixed amount of ligand, at least
a portion of which is detectably labeled, and different
concentrations of the enhancer, determining the amount of ligand
bound at the different concentrations of enhancer, and calculating
the potency (K.sub.enhancer) of the enhancer from the binding based
on the amount of ligand bound in the presence of different
concentrations of the enhancer. Typically, at least a portion of
the ligand is detectably labeled to permit easy quantitation of
ligand binding. This method, which is based on the G assay
described supra, is particularly suited for high-throughput
analysis of the K.sub.enhancer for enhancers of PDZ-ligand
interactions.
[0322] It will be appreciated that the concentration of ligand and
concentrations of enhancer are selected to allow meaningful
detection of enhanced binding. Thus, the ligand is typically
present at a concentration of between about 0.01 K.sub.d and about
0.5 K.sub.d and the concentrations of the test agent/enhancer
typically range from 1 nM to 1 mM (e.g. a 4-fold dilution series
with highest concentration 10 .mu.M or 1 mM). In a preferred
embodiment, the K.sub.d is determined using the assay disclosed
supra.
[0323] The potency of the binding can be determined by a variety of
standard methods based on the amount of ligand bound in the
presence of different concentrations of the enhancer or augmentor.
For example, a plot of labeled ligand binding versus enhancer
concentration can be fit to the equation:
S([E])=S(0)+(S(0)*(D.sub.enhancer-1)*[E]/([E]+K.sub.enhancer)
where "K.sub.enhancer" is the potency of the augmenting compound,
and "D.sub.enhancer" is the fold-increase in binding of the labeled
ligand obtained with addition of saturating amounts of the
enhancing compound, [E] is the concentration of the enhancer. It
will be understood that saturating amounts are the amount of
enhancer such that further addition does not significantly increase
the binding signal. Knowledge of "K.sub.enhancer" is useful because
it describes a concentration of the augmenting compound in a target
cell that will result in a biological effect due to dysregulation
of the PDZ-PL interaction. Typical therapeutic concentrations are
between about 0.1 and about 100 K.sub.enhancer.
V. Validation of Binding Assays
[0324] Compounds identified in the foregoing binding assays can be
further analyzed using a variety of biological assays to confirm
that the ability of the compound to inhibit a PDZ:PL protein
interaction actually inhibits a cellular activity correlated with
the PDZ:PL binding interaction. Alternatively, these assays can be
used directly to assay the activity of a potential inhibitory
compound without conducting a binding assay beforehand. These
assays can be conducted using various in vitro assays, or in vivo
assays using various appropriate animal model systems.
[0325] The PDZ:PL binding interactions described herein include
those involved in various biological activities in neurons. As
already noted, one set of cellular activities of interest are those
associated with various types of neurological disorders or injury,
such as cellular responses associated with stroke and ischemia.
Because neurological injury is often associated with cell death,
apoptosis and excitotoxicity responses, assays for each of these
responses can be conducted to validate the inhibitory activity of a
compound identified through a binding assay.
[0326] For example, a variety of different parameters can be
monitored to assess toxicity. Examples of such parameters include,
but are not limited to, cell proliferation, monitoring activation
of cellular pathways for toxicological responses by gene or protein
expression analysis, DNA fragmentation, changes in the composition
of cellular membranes, membrane permeability, activation of
components of death-receptors or downstream signaling pathways
(e.g., caspases), generic stress responses, NF-kappaB activation
and responses to mitogens. Related assays are used to assay for
apoptosis (a programmed process of cell death) and necrosis,
including cGMP formation and NO formation. The following are
illustrative of the type of biological assays that can be conducted
to assess whether a inhibitory agent has a protective effect
against neuronal injury or disease.
[0327] A. Morphological Changes
[0328] Apoptosis in many cell types is correlated with altered
morphological appearances. Examples of such alterations include,
but are not limited to, plasma membrane blebbing, cell shape
change, loss of substrate adhesion properties. Such changes are
readily detectable with a light microscope. Cells undergoing
apoptosis can also be detected by fragmentation and disintegration
of chromosomes. These changes can be detected using light
microscopy and/or DNA or chromatin specific dyes.
[0329] B. Altered Membrane Permeability
[0330] Often the membranes of cells undergoing apoptosis become
increasingly permeable. This change in membrane properties can be
readily detected using vital dyes (e.g., propidium iodide and
trypan blue). Dyes can be used to detect the presence of necrotic
cells. For example, certain methods utilize a green-fluorescent
LIVE/DEAD Cytotoxicity Kit #2, available from Molecular Probes. The
dye specifically reacts with cellular amine groups. In necrotic
cells, the entire free amine content is available to react with the
dye, thus resulting in intense fluorescent staining. In contrast,
only the cell-surface amines of viable cells are available to react
with the dye. Hence, the fluorescence intensity for viable cells is
reduced significantly relative to necrotic cells (see, e.g.,
Haugland, 1996 Handbook of Fluorescent Probes and Research
Chemicals, 6th ed., Molecular Probes, OR).
[0331] C. Dysfunction of Mitochondrial Membrane Potential
[0332] Mitochondria provide direct and indirect biochemical
regulation of diverse cellular processes as the main energy source
in cells of higher organisms. These processes include the electron
transport chain activity, which drives oxidative phosphorylation to
produce metabolic energy in the form of adenosine triphosphate
(i.e., ATP). Altered or defective mitochondrial activity can result
in mitochondrial collapse called the "permeability transition" or
mitochondrial permeability transition. Proper mitochondrial
functioning requires maintenance of the membrane potential
established across the membrane. Dissipation of the membrane
potential prevents ATP synthesis and thus halts or restricts the
production of a vital biochemical energy source.
[0333] Consequently, a variety of assays designed to assess
toxicity and cell death involve monitoring the effect of a test
agent on mitochondrial membrane potentials or on the mitochondrial
permeability transition. One approach is to utilize fluorescent
indicators (see, e.g., Haugland, 1996 Handbook of Fluorescent
Probes and Research Chemicals, 6th ed., Molecular Probes, OR, pp.
266-274 and 589-594). Various non-fluorescent probes can also be
utilized (see, e.g., Kamo et al. (1979) J. Membrane Biol. 49:105).
Mitochondrial membrane potentials can also be determined indirectly
from mitochondrial membrane permeability (see, e.g., Quinn (1976)
The Molecular Biology of Cell Membranes, University Park Press,
Baltimore, Md., pp. 200-217). Further guidance on methods for
conducting such assays is provided in PCT publication WO 00/19200
to Dykens et al.
[0334] D. Caspase Activation
[0335] Apoptosis is the process of programmed cell death and
involves the activation of a genetic program when cells are no
longer needed or have become seriously damaged. Apoptosis involves
a cascade of biochemical events and is under the regulation of a
number of different genes. One group of genes act as effectors of
apoptosis and are referred to as the interleukin-1 beta.converting
enzyme (ICE) family of genes. These genes encode a family of
cysteine proteases whose activity is increased in apoptosis. The
ICE family of proteases is generically referred to as caspase
enzymes. The "c" in the name reflects the fact that the enzymes are
cysteine proteases, while "aspase" refers to the ability of these
enzymes to cleave after aspartic acid residues.
[0336] Consequently, some assays for apoptosis are based upon the
observation that caspases are induced during apoptosis. Induction
of these enzymes can be detected by monitoring the cleavage of
specifically-recognized substrates for these enzymes. A number of
naturally occurring and synthetic protein substrates are known
(see, e.g., Ellerby et al. (1997) J. Neurosci. 17:6165; Kluck, et
al. (1997) Science 275:1132; Nicholson et al. (1995) Nature 376:37;
and Rosen and Casciola-Rosen (1997) J. Cell Biochem. 64:50).
Methods for preparing a number of different substrates that can be
utilized in these assays are described in U.S. Pat. No. 5,976,822.
This patent also describes assays that can be conducted using whole
cells that are amendable to certain of the microfluidic devices
described herein. Other methods using FRET techniques are discussed
in Mahajan, et al. (1999) Chem. Biol. 6:401-9; and Xu, et al.
(1998) Nucl. Acids. Res. 26:2034-5.
[0337] E. Cytochrome c Release
[0338] In healthy cells, the inner mitochondrial membrane is
impermeable to macromolecules. Thus, one indicator of cell
apoptosis is the release or leakage of cytochrome c from the
mitochondria. Detection of cytochrome c can be performed using
spectroscopic methods because of the inherent absorption properties
of the protein. Thus, one detection option with the present devices
is to place the cells within a holding space and monitor absorbance
at a characteristic absorption wavelength for cytochrome c.
Alternatively, the protein can be detected using standard
immunological methods (e.g., ELISA assays) with an antibody that
specifically binds to cytochrome c (see, e.g., Liu et al. (1996)
Cell 86:147).
[0339] F. Assays for Cell Lysis
[0340] The final stage of cell death is typically lysis of the
cell. When cells die they typically release a mixture of chemicals,
including nucleotides, and a variety of other substances (e.g.,
proteins and carbohydrates) into their surroundings. Some of the
substances released include ADP and ATP, as well as the enzyme
adenylate cyclase, which catalyzes the conversion of ADP to ATP in
the presence of excess ADP. Thus, certain assays involve providing
sufficient ADP in the assay medium to drive the equilibrium towards
the generation of ATP which can subsequently be detected via a
number of different means. One such approach is to utilize a
luciferin/luciferase system that is well known to those of ordinary
skill in the art in which the enzyme luciferase utilizes ATP and
the substrate luciferin to generate a photometrically detectable
signal. Further details regarding certain cell lysis assays that
can be performed are set forth in PCT publication WO 00/70082.
[0341] G. Ischemic Model Systems
[0342] Methods for assaying whether a compound can confer
protective neurological effects against ischemia and stroke are
discussed by Aarts, et al. (Science 298:846-850, 2002). In general,
this assay involves subjecting rats to a middle cerebral artery
occlusion (MCAO) for a relatively short period of time (e.g., about
90 minutes). MCAO can be induced using various methods, including
an intraluminal suture method (see, e.g., Longa, E. Z. et al.
(1989) Stroke 20:84; and Belayev, L., et al. (1996) Stroke
27:1616). A composition containing the putative inhibitor is
introduced into the rat using conventional methods (e.g., via
intravenous injection). To evaluate the composition's prophylactic
effect, the composition is administered before performing MCAO. If
the compound is to be evaluated for its ability to mitigate against
an ischemic event that has already occurred, the composition with
the compound is introduced after MCAO has been initiated. The
extent of cerebral infarction is then evaluated using various
measures of neurological function. Examples of such measures
include the postural reflex test (Bederson, J. B. et al. (1986)
Stroke 17:472) and the forelimb placing test (De Ryck, M. et al.
(1989) Stroke 20:1383). Methods are also described in Aarts et al
(supra) assessing the effects of NMDA-induced excitotoxicity using
in vitro assays.
VI. Global Analysis of PDZ-PL Interactions
[0343] As described supra, the present invention provides powerful
methods for analysis of PDZ-ligand interactions, including
high-throughput methods such as the G assay and affinity assays
described supra. In one embodiment of the invention, the affinity
is determined for a particular ligand and a plurality of PDZ
proteins. Typically the plurality is at least 5, and often at least
25, or at least 40 different PDZ proteins. In a preferred
embodiment, the plurality of different PDZ proteins are from a
particular tissue (e.g., central nervous system) or a particular
class or type of cell, (e.g., a neuron) and the like. In a most
preferred embodiment, the plurality of different PDZ proteins
represents a substantial fraction (e.g., typically a majority, more
often at least 80%) of all of the PDZ proteins known to be, or
suspected of being, expressed in the tissue or cell(s), e.g., all
of the PDZ proteins known to be present in neuronal cells. In an
embodiment, the plurality is at least 50%, usually at least 80%, at
least 90% or all of the PDZ proteins disclosed herein as being
expressed in neuronal cells.
[0344] In one embodiment of the invention, the binding of a ligand
to the plurality of PDZ proteins is determined. Using this method,
it is possible to identify a particular PDZ domain bound with
particular specificity by the ligand. The binding may be designated
as "specific" if the affinity of the ligand to the particular PDZ
domain is at least 2-fold that of the binding to other PDZ domains
in the plurality (e.g., present in that cell type). The binding is
deemed "very specific" if the affinity is at least 10-fold higher
than to any other PDZ in the plurality or, alternatively, at least
10-fold higher than to at least 90%, more often 95% of the other
PDZs in a defined plurality. Similarly, the binding is deemed
"exceedingly specific" if it is at least 100-fold higher. For
example, a ligand could bind to 2 different PDZs with an affinity
of 1 .mu.M and to no other PDZs out of a set 40 with an affinity of
less than 100 .mu.M. This would constitute specific binding to
those 2 PDZs. Similar measures of specificity are used to describe
binding of a PDZ to a plurality of PLs.
[0345] It will be recognized that high specificity PDZ-PL
interactions represent potentially more valuable targets for
achieving a desired biological effect. The ability of an inhibitor
or enhancer to act with high specificity is often desirable. In
particular, the most specific PDZ-ligand interactions are also the
best therapeutic targets, allowing specific inhibition of the
interaction.
[0346] Thus, in one embodiment, the invention provides a method of
identifying a high specificity interaction between a particular PDZ
domain and a ligand known or suspected of binding at least one PDZ
domain, by providing a plurality of different immobilized
polypeptides, each of said polypeptides comprising a PDZ domain and
a non-PDZ domain; determining the affinity of the ligand for each
of said polypeptides, and comparing the affinity of binding of the
ligand to each of said polypeptides, wherein an interaction between
the ligand and a particular PDZ domain is deemed to have high
specificity when the ligand binds an immobilized polypeptide
comprising the particular PDZ domain with at least 2-fold higher
affinity than to immobilized polypeptides not comprising the
particular PDZ domain.
[0347] In a related aspect, the affinity of binding of a specific
PDZ domain to a plurality of ligands (or suspected ligands) is
determined. For example, in one embodiment, the invention provides
a method of identifying a high specificity interaction between a
PDZ domain and a particular ligand known or suspected of binding at
least one PDZ domain, by providing an immobilized polypeptide
comprising the PDZ domain and a non-PDZ domain; determining the
affinity of each of a plurality of ligands for the polypeptide, and
comparing the affinity of binding of each of the ligands to the
polypeptide, wherein an interaction between a particular ligand and
the PDZ domain is deemed to have high specificity when the ligand
binds an immobilized polypeptide comprising the PDZ domain with at
least 2-fold higher affinity than other ligands tested. Thus, the
binding may be designated as "specific" if the affinity of the PDZ
to the particular PL is at least 2-fold that of the binding to
other PLs in the plurality (e.g., present in that cell type). The
binding is deemed "very specific" if the affinity is at least
10-fold higher than to any other PL in the plurality or,
alternatively, at least 10-fold higher than to at least 90%, more
often 95% of the other PLs in a defined plurality. Similarly, the
binding is deemed "exceedingly specific" if it is at least 100-fold
higher. Typically the plurality is at least 5 different ligands,
more often at least 10.
[0348] 1. Use of Array for Global Predictions
[0349] One discovery of the present inventors relates to the
important and extensive roles played by interactions between PDZ
proteins and PL proteins, particularly in the biological function
of neuronal cells. Further, it has been discovered that valuable
information can be ascertained by analysis (e.g., simultaneous
analysis) of a large number of PDZ-PL interactions. In a preferred
embodiment, the analysis encompasses all of the PDZ proteins
expressed in a particular tissue (e.g., brain) or type or class of
cell (e.g., neuron). Alternatively, the analysis encompasses at
least about 5, or at least about 10, or at least about 12, or at
least about 15 and often at least 50 different polypeptides, up to
about 60, about 80, about 100, about 150, about 200, or even more
different polypeptides; or a substantial fraction (e.g., typically
a majority, more often at least 80%) of all of the PDZ proteins
known to be, or suspected of being, expressed in the tissue or
cell(s), (e.g., all of the PDZ proteins known to be present in
neurons).
[0350] It will be recognized that the arrays and methods of the
invention are directed to analyze of PDZ and PL interactions, and
involve selection of such proteins for analysis. While the devices
and methods of the invention may include or involve a small number
of control polypeptides, they typically do not include significant
numbers of proteins or fusion proteins that do not include either
PDZ or PL domains (e.g., typically, at least about 90% of the
arrayed or immobilized polypeptides in a method or device of the
invention is a PDZ or PL sequence protein, more often at least
about 95%, or at least about 99%).
[0351] It will be apparent from this disclosure that analysis of
the relatively large number of different interactions preferably
takes place simultaneously. In this context, "simultaneously" means
that the analysis of several different PDZ-PL interactions (or the
effect of a test agent on such interactions) is assessed at the
same time. Typically the analysis is carried out in a high
throughput (e.g., robotic) fashion. One advantage of this method of
simultaneous analysis is that it permits rigorous comparison of
multiple different PDZ-PL interactions. For example, as explained
in detail elsewhere herein, simultaneous analysis (and use of the
arrays described infra) facilitates, for example, the direct
comparison of the effect of an agent (e.g., a potential interaction
inhibitor) on the interactions between a substantial portion of
PDZs and/or PLs in a tissue or cell.
[0352] Accordingly, in one aspect, the invention provides an array
of immobilized polypeptide comprising the PDZ domain and a non-PDZ
domain on a surface. Typically, the array comprises at least about
5, or at least about 10, or at least about 12, or at least about 15
and often at least 50 different polypeptides. In one preferred
embodiment, the different PDZ proteins are from a particular tissue
(e.g., central nervous system) or a particular class or type of
cell, (e.g., a neuron) and the like. In a most preferred
embodiment, the plurality of different PDZ proteins represents a
substantial fraction (e.g., typically a majority, more often at
least 60%, 70% or 80%) of all of the PDZ proteins known to be, or
suspected of being, expressed in the tissue or cell(s), (e.g., all
of the PDZ proteins known to be present in neurons).
[0353] Certain embodiments are arrays which include a plurality,
usually at least 5, 10, 25, or 50 PDZ proteins present in a
particular cell of interest. In this context, "array" refers to an
ordered series of immobilized polypeptides in which the identity of
each polypeptide is associated with its location. In some
embodiments the plurality of polypeptides are arrayed in a "common"
area such that they can be simultaneously exposed to a solution
(e.g., containing a ligand or test agent). For example, the
plurality of polypeptides can be on a slide, plate or similar
surface, which may be plastic, glass, metal, silica, beads or other
surface to which proteins can be immobilized. In a different
embodiment, the different immobilized polypeptides are situated in
separate areas, such as different wells of multi-well plate (e.g.,
a 24-well plate, a 96-well plate, a 384 well plate, and the like).
It will be recognized that a similar advantage can be obtained by
using multiple arrays in tandem.
[0354] 2. Analysis of PDZ-PL Inhibition Profile
[0355] In one aspect, the invention provides a method for
determining if a test compound inhibits any PDZ-ligand interaction
in large set of PDZ-ligand interaction (e.g., a plurality of the
PDZ-ligand interactions described in TABLE 6 and TABLE 7 and TABLE
8; a majority of the PDZ-ligands identified in a particular cell or
tissue as described supra (e.g., neurons) and the like. In one
embodiment, the PDZ domains of interest are expressed as GST-PDZ
fusion proteins and immobilized as described herein. For each PDZ
domain, a labeled ligand that binds to the domain with a known
affinity is identified as described herein.
[0356] For any known or suspected modulator (e.g., inhibitor) of a
PDL-PL interaction(s), it is useful to know which interactions are
inhibited (or augmented). For example, an agent that inhibits all
PDZ-PL interactions in a cell (e.g., a neuron) will have different
uses than an agent that inhibits only one, or a small number, of
specific PDZ-PL interactions. The profile of PDZ interactions
inhibited by a particular agent is referred to as the "inhibition
profile" for the agent, and is described in detail below. The
profile of PDZ interactions enhanced by a particular agent is
referred to as the "enhancement profile" for the agent. It will be
readily apparent to one of skill guided by the description of the
inhibition profile how to determine the enhancement profile for an
agent. The present invention provides methods for determining the
PDZ interaction (inhibition/enhancement) profile of an agent in a
single assay.
[0357] In one aspect, the invention provides a method for
determining the PDZ-PL inhibition profile of a compound by
providing (i) a plurality of different immobilized polypeptides,
each of said polypeptides comprising a PDZ domain and a non-PDZ
domain and (ii) a plurality of corresponding ligands, wherein each
ligand binds at least one PDZ domain in (i), then contacting each
of said immobilized polypeptides in (i) with a corresponding ligand
in (ii) in the presence and absence of a test compound, and
determining for each polypeptide-ligand pair whether the test
compound inhibits binding between the immobilized polypeptide and
the corresponding ligand.
[0358] Typically the plurality is at least 5, and often at least
25, or at least 40 different PDZ proteins. In a preferred
embodiment, the plurality of different ligands and the plurality of
different PDZ proteins are from the same tissue or a particular
class or type of cell, (e.g., a neuron). In a most preferred
embodiment, the plurality of different PDZs represents a
substantial fraction (e.g., at least 80%) of all of the PDZs known
to be, or suspected of being, expressed in the tissue or cell(s),
e.g., all of the PDZs known to be present in neurons (for example,
at least 80%, at least 90% or all of the PDZs disclosed herein as
being expressed in neuronal cells).
[0359] In one embodiment, the inhibition profile is determined as
follows: A plurality (e.g., all known) PDZ domains expressed in a
cell (e.g., neurons) are expressed as GST-fusion proteins and
immobilized without altering their ligand binding properties as
described supra. For each PDZ domain, a labeled ligand that binds
to this domain with a known affinity is identified. If the set of
PDZ domains expressed in neurons is denoted by {P1 . . . Pn}, any
given PDZ domain Pi binds a (labeled) ligand Li with affinity
K.sub.di. To determine the inhibition profile for a test agent
"compound X" the G assay (supra) can be performed as follows in
96-well plates with rows A-H and columns 1-12. Column 1 is coated
with P1 and washed. The corresponding ligand L1 is added to each
washed coated well of column 1 at a concentration 0.5 K.sub.d1 with
(rows B, D, F, H) or without (rows A, C, E, F) between about 1 and
about 1000 .mu.M) of test compound X. Column 2 is coated with P2,
and L2 (at a concentration 0.5 K.sub.d2) is added with or without
inhibitor X. Additional PDZ domains and ligands are similarly
tested.
[0360] Compound X is considered to inhibit the binding of Li to Pi
if the average signal in the wells of column i containing X is less
than half the signal in the equivalent wells of the column lacking
X. Thus, in this single assay one determines the full set of neural
PDZs that are inhibited by compound X.
[0361] In some embodiments, the test compound X is a mixture of
compounds, such as the product of a combinatorial chemistry
synthesis as described supra. In some embodiments, the test
compound is known to have a desired biological effect, and the
assay is used to determine the mechanism of action (i.e., if the
biological effect is due to modulating a PDZ-PL interaction).
[0362] It will be apparent that an agent that modulates only one,
or a few PDZ-PL interactions, in a panel (e.g., a panel of all
known PDZs in neurons, a panel of at least 10, at least 20 or at
least 50 PDZ domains) is a more specific modulator than an agent
that modulate many or most interactions. Typically, an agent that
modulates less than 20% of PDZ domains in a panel (e.g., TABLE 5)
is deemed a "specific" inhibitor, less than 6% a "very specific"
inhibitor, and a single PDZ domain a "maximally specific"
inhibitor.
[0363] It will be recognized that high specificity modulators of
PDZ-PL interactions represent potentially more valuable drug
targets for achieving a desired biological effect. The ability of
an inhibitor or enhancer to act with "maximal specificity" is most
desirable.
[0364] In one embodiment, the assays of the invention can be used
to determine a maximally specific modulator of the interaction
between a TRP channel and a PDZ domain.
[0365] In a preferred embodiment, the assays of the invention are
used to identify a maximally specific modulator of the interaction
between TRPM7 and a PDZ domain.
[0366] It will also be appreciated that "compound X" may be a
composition containing mixture of compounds (e.g., generated using
combinatorial chemistry methods) rather than a single compound.
[0367] Several variations of this assay are contemplated:
[0368] In some alternative embodiments, the assay above is
performed using varying concentrations of the test compound X,
rather than fixed concentration. This allows determination of the
Ki of the X for each PDZ as described above.
[0369] In an alternative embodiment, instead of pairing each PDZ Pi
with a specific labeled ligand Li, a mixture of different labeled
ligands is created that such that for every PDZ at least one of the
ligands in the mixture binds to this PDZ sufficiently to detect the
binding in the G assay. This mixture is then used for every PDZ
domain.
[0370] In one embodiment, compound X is known to have a desired
biological effect, but the chemical mechanism by which it has that
effect is unknown. The assays of the invention can then be used to
determine if compound X has its effect by binding to a PDZ
domain.
[0371] In one embodiment, PDZ-domain containing proteins are
classified in to groups based on their biological function, e.g.
into those that regulate apoptosis versus those that regulate
transcription. An optimal inhibitor of a particular function (e.g.,
including but not limited to an anti-apoptotic agent, an anti-T
cell activation agent, cell-cycle control, vesicle transport, etc.)
will inhibit multiple PDZ-ligand interactions involved in the
function (e.g., apoptosis, activation) but few other interactions.
Thus, the assay is used in one embodiment in screening and design
of a drug that specifically blocks a particular function. For
example, an agent designed to block apoptosis might be identified
because, at a given concentration, the agent inhibits 2 or more
PDZs involved in apoptosis but fewer than 3 other PDZs, or that
inhibits PDZs involved in apoptosis with a K.sub.i>10-fold
better than for other PDZs. Thus, the invention provides a method
for identifying an agent that inhibits a first selected PDZ-PL
interaction or plurality of interactions but does not inhibit a
second selected PDZ-PL interaction or plurality of interactions.
The two (or more) sets of interactions can be selected on the basis
of the known biological function of the PDZ proteins, the tissue
specificity of the PDZ proteins, or any other criteria. Moreover,
the assay can be used to determine effective doses (i.e., drug
concentrations) that result in desired biological effects while
avoiding undesirable effects.
[0372] 3. Side Effects of PDZ-PL Modulator Interactions
[0373] In a related embodiment, the invention provides a method for
determining likely side effects of a therapeutic that inhibits
PDZ-ligand interactions. The method entails identifying those
target tissues, organs or cell types that express PDZ proteins and
ligands that are disrupted by a specified inhibitor. If, at a
therapeutic dosage, a drug intended to have an effect in one organ
system (e.g., central nervous system) disrupts PDZ-PL interactions
in a different system (e.g., hematopoietic system) it can be
predicted that the drug will have effects ("side effects") on the
second system. It will be apparent that the information obtained
from this assay will be useful in the rational design and selection
of drugs that do not have the side-effect.
[0374] In one embodiment, for example, a comprehensive PDZ protein
set is obtained. A "perfectly comprehensive" PDZ protein set is
defined as the set of all PDZ proteins expressed in the subject
animal (e.g., humans). A comprehensive set may be obtained by
analysis of, for example, the human genome sequence. However, a
"perfectly comprehensive" set is not required and any reasonably
large set of PDZ domain proteins (e.g., the set of all known PDZ
proteins; or the set listed in TABLE 5) will provide valuable
information.
[0375] In one embodiment, the method involves some of all of the
following steps:
[0376] a) For each PDZ protein, determine the tissues in which it
is highly expressed. This can be done experimentally although the
information generally will be available in the scientific
literature;
[0377] b) For each PDZ protein (or as many as possible), identify
the cognate PL(s) bound by the PDZ protein;
[0378] c) Determine the K.sub.i at which the test agent inhibits
each PDZ-PL interaction, using the methods described supra;
[0379] d) From this information it is possible to calculate the
pattern of PDZ-PL interactions disrupted at various concentrations
of the test agent.
[0380] By correlating the set of PDZ-PL interactions disrupted with
the expression pattern of the members of that set, it will be
possible to identify the tissues likely affected by the agent.
[0381] Additional steps can also be carried out, including
determining whether a specified tissue or cell type is exposed to
an agent following a particular route of administration. This can
be determined using basis pharmacokinetic methods and
principles.
[0382] 4. Modulation of Activities
[0383] The PDZ binding moieties and inhibitors described herein
that disrupt PDZ:PL protein interactions can be used to modulate
biological activities or functions of cells (e.g., neurons). These
agents can also be utilized to treat diseases and conditions in
human and nonhuman animals (e.g., experimental models). Exemplary
biological activities are listed supra.
[0384] When administered to patients, the compounds of the
invention (e.g., PL-PDZ interaction inhibitors) are useful for
treating (ameliorating symptoms of) a variety of neurological
disorders, including those associated with some type of injury to
neuronal cells or the death of neurons. Such disorders include, but
are not limited to, stroke, ischemia, brain traumas and chronic
pain. Certain inhibitors can also be used to treat other types of
neurological disorders like Alzheimer's disease, epilepsy,
Parkinson's disease, Huntington's disease, motor neuron diseases
and inherited ataxias.
[0385] Some other inhibitors can be utilized to treat other disease
types, including, for instance, inflammatory and humoral immune
responses, e.g., inflammation, allergy (e.g., systemic anaphylaxis,
hypersensitivity responses, drug allergies, insect sting
allergies); infectious diseases (e.g., viral infection, such as
HIV, measles, parainfluenza, virus-mediated cell fusion,), and
ischemia (e.g., post-myocardial infarction complications, joint
injury, kidney, scleroderma).
VII. Antagonists of PDZ-PL Interactions
[0386] As described herein, interactions between PDZ proteins and
PL proteins in cells (e.g., neurons) may be disrupted or inhibited
by the administration of inhibitors or antagonists. Inhibitors can
be identified using screening assays described herein. In
embodiment, the motifs disclosed herein are used to design
inhibitors. In some embodiments, the antagonists of the invention
have a structure (e.g., peptide sequence) based on the C-terminal
residues of PL-domain proteins listed in TABLE 2 and TABLE 4. In
some embodiments, the antagonists of the invention have a structure
(e.g., peptide sequence) based on a PL motif disclosed herein.
[0387] The PDZ/PL antagonists and antagonists of the invention can
be any of a large variety of compounds, both naturally occurring
and synthetic, organic and inorganic, and including polymers (e.g.,
oligopeptides, polypeptides, oligonucleotides, and
polynucleotides), small molecules, antibodies, sugars, fatty acids,
nucleotides and nucleotide analogs, analogs of naturally occurring
structures (e.g., peptide mimetics, nucleic acid analogs, and the
like), and numerous other compounds. Although, for convenience, the
present discussion primarily refers antagonists of PDZ-PL
interactions, it will be recognized that PDZ-PL interaction
agonists can also be use in the methods disclosed herein.
[0388] In one aspect, the peptides and peptide mimetics or
analogues of the invention contain an amino acid sequence that
binds a PDZ domain in a cell of interest. In one embodiment, the
antagonists comprise a peptide that has a sequence corresponding to
the carboxy-terminal sequence of a PL protein listed in TABLE 2 or
TABLE 4, e.g., a peptide listed TABLE 2. Typically, the peptide
comprises at least the C-terminal two (3), three (3) or four (4)
residues of the PL protein, and often the inhibitory peptide
comprises more than four residues (e.g., at least five, six, seven,
eight, nine, ten, twelve or fifteen residues) from the PL protein
C-terminus.
[0389] In some embodiments, the inhibitor is a peptide, e.g.,
having a sequence of a PL C-terminal protein sequence.
[0390] In some embodiments, the antagonist is a fusion protein
comprising such a sequence. Fusion proteins containing a
transmembrane transporter amino acid sequence can be used to
facilitate transport of the inhibitor into a cell.
[0391] In some embodiments, the inhibitor is conserved variant of
the PL C-terminal protein sequence having inhibitory activity.
[0392] In some embodiments, the antagonist is a peptide mimetic of
a PL C-terminal sequence.
[0393] In some embodiments, the inhibitor is a small molecule
(i.e., having a molecular weight less than 1 kDa).
[0394] A. Polypeptide Antagonists
[0395] 1. Inhibitors with a PL Sequence
[0396] One class of inhibitors or antagonists that are provided
comprise a peptide that has a sequence of a PL protein
carboxy-terminus listed in TABLE 4. The PL protein carboxy-terminus
sequences can be considered as the "core PDZ motif sequence"
because of the ability of the short sequence from the carboxy
terminus to interact with the PDZ domain. For example, in some
inhibitors the "core PDZ motif sequence" or simply the "PL
sequence" contains the last 2, 3 or 4 C-terminus amino acids. In
other instances, however, the core PDZ motif comprises more than
2-4 residues (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 residues) from the PL protein C-terminus. For
some inhibitors, the PDZ motif sequence peptide is from 4-15 amino
acids in length. Other inhibitors have a PDZ motif sequence that is
6-10 amino acids in length, or 3-8 amino acids in length, or 3-7
amino acids in length. Certain inhibitors have a PDZ motif sequence
that is 8 amino acids in length. Although the residues shared by
the inhibitory peptide and the PL protein are often found at the
C-terminus of the peptide, some inhibitors incorporate a PL
sequence that is located in an internal region of a PL protein.
Similarly, in some cases, the inhibitory peptide comprises residues
from a PL sequence that is near, but not at the C-terminus of a PL
protein (see, Gee et al., 1998, J. Biol. Chem. 273:21980-87).
Another set of inhibitors are based upon the identification of
amino acid sequences that specifically disrupt binding between
NMDAR proteins and PSD-95. This particular class of inhibitors are
polypeptides that share the following characteristics: 1) a size
ranging from 3-20 amino acids in length (although somewhat longer
polypeptides can be used), and 2) a C-terminal consensus sequence
of X-L/I/V-X-V/L/A (the slash separates different amino acids that
can appear at a given position).
[0397] Specific examples of peptides which are useful as inhibitors
of the interaction between TRPM7 and the PDZ domains RIM-2 d1,
INADL d3, ZO-1 d2, PAR3 d3, and syntrophin1 alpha d1 are shown in
TABLES 10 and 11 Variants of these peptides are which bind PDZ
domains RIM-2 d1, INADL d3, ZO-1 d2, PAR3 d3, and syntrophin1 alpha
d1 are also useful as inhibitors.
TABLE-US-00012 TABLE 10 titration of peptides with ZO-1 d2 and
INADL d3 Pep- SEQ tide ID PDZ EC50, error of fit, ODmax # Peptide
Sequence NO: domain uM EC50, uM (450 nm) 1791
biotin-YGRKKRRQRRRAVAATSINL 261 ZO-1 d2 <0.1 ND 2.50 1791
biotin-YGRKKRRQRRRAVAATSINL 261 INADL d3 ND ND 0.09 1829
biotin-YGRKKRRQRRRARSDRTIWA 262 ZO-1 d2 0.007 0.0027 2.90 1829
biotin-YGRKKRRQRRRARSDRTIWA 262 INADL d3 ND ND 1.40 1830
biotin-YGRKKRRQRRRARSDRTIIA 263 ZO-1 d2 <0.02 ND 1.80 1830
biotin-YGRKKRRQRRRARSDRTTIA 263 INADL d3 <2 ND 1.20 1832
biotin-YGRKKRRQRRRSRTDRKYWA 264 ZO-1 d2 <0.02 ND 2.10 1832
biotin-YGRKKRRQRRRSRTDRKYWA 264 INADL d3 0.20 0.023 0.60 1836
biotin-YGRKKRRQRRRARGDRKIRV 265 ZO-1 d2 <0.01 ND 2.60 1836
biotin-YGRKKRRQRRRARGDRKIRV 265 INADL d3 ND ND 0.30 1837
biotin-YGRKKRRQRRRARTDRKVEV 266 Z0-1 d2 <0.02 ND 1.80 1837
biotin-YGRKKRRQRRRARTDRKVEV 266 INADL d3 ND ND 0.37 1838
biotin-YGRKKRRQRRRARGDRKYIV 267 ZO-1 d2 <0.1 ND 2.60 1838
biotin-YGRKKRRQRRRARGDRKYIV 267 INADL d3 <0.1 ND 2.60 1839
biotin-YGRKKRRQRRRSRTDRKYQI 268 ZO-1 d2 <0.02 ND 2.50 1839
biotin-YGRKKRRQRRRSRTDRKYQI 268 INADL d3 0.13 0.007 1.60 1841
biotin-YGRKKRRQRRRARGDRKVPV 269 ZO-1 d2 <0.02 ND 2.10 1841
biotin-YGRKKRRQRRRARGDRKVPV 269 INADL d3 ND ND 0.20 1842
biotin-YGRKKRRQRRRQDERRLIVL 270 ZO-1 d2 0.035 0.008 2.30 1842
biotin-YGRKKRRQRRRQDERRLIVL 270 INADL d3 <1 ND 1.90 1843
biotin-YGRKKRRQRRRARGDRLVSL 271 ZO-1 d2 0.017 0.003 2.20 1843
biotin-YGRKKRRQRRRARGDRLVSL 271 INADL d3 <2 uM ND 1.20 1844
biotin-YGRKKRRQRRRARGTRLVWV 272 ZO-1 d2 <0.02 ND 2.60 1844
biotin-YGRKKRRQRRRARGTRLVWV 272 INADL d3 <0.02 ND 2.40 1845
biotin-YGRKKRRQRRRARGDRYRIV 273 ZO-1 d2 <0.1 ND 2.30 1845
biotin-YGRKKRRQRRRARGDRYRIV 273 INADL d3 <0.1 ND 2.20 1846
biotin-YGRKKRRQRRRSRTDRLEYV 274 INADL d3 ND ND 1.50 1848
biotin-YGRKKRRQRRRARGDRTIIY 275 ZO-1 d2 <0.02 ND 2.20 1848
biotin-YGRKKRRQRRRARGDRTIIY 275 INADL d3 ~<2 ND 1.20 1852
biotin-YGRKKRRQRRRKNKDKEYYV 276 ZO-1 d2 <0.02 ND 2.60 1852
biotin-YGRKKRRQRRRKNKDKEYYV 276 INADL d3 <0.02 ND 2.40 1854
biotin-YGRKKRRQRRRARGRRETWV 277 ZO-1 d2 ND ND 2.80 1854
biotin-YGRKKRRQRRRARGRRETWV 277 INADL d3 <0.02 ND 2.10 AA353
biotin-YGRKKRRQRRREKHFRETEV 278 TIP1 <0.002 ND 3.00
TABLE-US-00013 TABLE 11 titrations or binding assays of peptides
with RIM-2 d1, INADL d3, ZO-1 d2, PAR3 d3, and syntrophin1 alpha d1
PDZ SEQ domain ID bound Peptide Sequence NO: INADL D3
ATDYLVQPFMDQLAFHQFYI 280 INADL D3 DFRPSFKHILFRRARRGFRQ 282 INADL D3
ELLQFCRTPNPALKNGQYWV 285 INADL D3 ENLELPVNPSSVVSERISSV 286 INADL D3
FHSKTAGANTTDKELEVLSL 287 INADL D3 GRWTGRAMSAWKPTRRETEV 289 INADL D3
HAMNAAPRAMENAPALRTSH 290 INADL D3 HDFRRAFKKILARGDRKRIV 291 INADL D3
HHLVAQRDIRQFQLQHWLAI 292 INADL D3 HSCCNRARQERLQRRRETQV 293 INADL D3
ILNSIQVMRAQMNQIQSVEV 294 INADL D3 KHSRKSSSYSSSSTTVKTSY 296 INADL D3
KKKKQPGNSTKESESTNSVRLML 297 INADL D3 LASKSAEEGKQIPDSLSTDL 302 INADL
D3 LAVLAYSITLVMLWSIWQYA 303 INADL D3 LNSCSNRRVYKKMPSIESDV 305 INADL
D3 LQFHRGSRAQSFLQTETSVI 306 INADL D3 PGQPPKVKSEFNSYSLTGYV 308 INADL
D3 PIPAGGCTFSGIFPTLTSPL 309 INADL D3 QDFRRAFRRILARPWTQTAW 312 INADL
D3 RELVDRGEVRQFTLRHWLKV 315 INADL D3 RSGATIPLVGQDIIDLQTEV 318 INADL
D3 SLIGPVQKEYQRELGKLSSP 319 INADL D3 SSKSKSSEESQTFFGLYKL 320 INADL
D3 STDNLVRPFMDTLASHQLYI 323 INADL D3 TQGFPGPATWRRISSLESEV 327 INADL
D3 TTNNNPNSAVNIKKIFTDV 328 INADL D3 VDPNSPAAKKKYVSYNNLVI 329 INADL
D3 VHKVRNKFKAKCSLCRLYII 331 INADL D3 VPSDNIDSQGRNASTNDSLL 333 INADL
D3 YGRKKRRQRRRARGDRKYIV 339 INADL D3 YGRKKRRQRRRARSDRTIIA 350 INADL
D3 YGRKKRRQRRRARSDRTIWA 351 INADL D3 YGRKKRRQRRRAVAATSANL 355 INADL
D3 YGRKKRRQRRREYLGLDVPV 360 INADL D3 YGRKKRRQRRRSRTDRKYQI 373 INADL
D3 YGRKKRRQRRRSRTDRKYWA 374 INADL D3 YSATYSELEDPGEMSPPIDL 378 PAR3
D3 ATDYLVQPFMDQLAFHQFYI 280 PAR3 D3 DFRPSFKHILFRRARRGFRQ 282 PAR3
D3 DGGARTEDEVQSYPSKHDYV 283 PAR3 D3 DTLLLTENEGDKTEEQVSYV 284 PAR3
D3 ELLQFCRTPNPALKNGQYWV 285 PAR3 D3 FHSKTAGANTTDKELEVLSL 287 PAR3
D3 HDFRRAFKKILARGDRKRIV 291 PAR3 D3 HHLVAQRDIRQFQLQHWLAI 292 PAR3
D3 HSCCNRARQERLQRRRETQV 293 PAR3 D3 ILNSIQVMRAQMNQIQSVEV 294 PAR3
D3 KAGYRAPRSYPKSNSSKEYV 295 PAR3 D3 KHSRKSSSYSSSSTTVKTSY 296 PAR3
D3 KKKKQPGNSTKESESTNSVRLML 297 PAR3 D3 KTMPAAMFRLLTGQETPLYI 298
PAR3 D3 KTMPAAMYRLLTAQEQPVYI 299 PAR3 D3 KTMPAATYRLLTGQEQPVYL 300
PAR3 D3 KYSAPRRPTATGDYDKKNYV 301 PAR3 D3 LASKSAEEGKQIPDSLSTDL 302
PAR3 D3 LAVLAYSITLVMLWSIWQYA 303 PAR3 D3 LERTSSVSPSTAEPELSIVF 304
PAR3 D3 LQFHRGSRAQSFLQTETSVI 306 PAR3 D3 PGQPPKVKSEFNSYSLTGYV 308
PAR3 D3 PIPAGGCTFSGIFPTLTSPL 309 PAR3 D3 QDFRRAFRRILARPWTQTAW 312
PAR3 D3 QGDPALQDAGDSSRKEYFI 313 PAR3 D3 RELVDRGEVRQFTLRHWLKV 315
PAR3 D3 SSKSKSSEESQTFFGLYKL 320 PAR3 D3 SSPDSSYQGKGFVMSRAMYV 321
PAR3 D3 STDNLVRPFMDTLASHQLYI 323 PAR3 D3 VDPNSPAAKKKYVSYNNLVI 329
PAR3 D3 VHKVRNKFKAKCSLCRLYII 331 PAR3 D3 YGRKKRRQRRRAVAATSANL 355
PAR3 D3 YGRKKRRQRRRAVMTSINL 356 PAR3 D3 YGRKKRRQRRREYLGLDVPV 360
PAR3 D3 YGRKKRRQRRRGASADSTQA 361 PAR3 D3 YGRKKRRQRRRKLSSIESDV 363
PAR3 D3 YGRKKRRQRRRNDNIALLVQ 366 PAR3 D3 YGRKKRRQRRRSEGVPDLLV 372
RIM2 D1 DFRPSFKHILFRRARRGFRQ 282 RIM2 D1 FHSKTAGANTTDKELEVLSL 287
RIM2 D1 HDFRRAFKKILARGDRKRIV 291 RIM2 D1 HHLVAQRDIRQFQLQHWLAI 292
RIM2 D1 KHSRKSSSYSSSSTTVKTSY 296 RIM2 D1 KKKKQPGNSTKESESTNSVRLML
297 RIM2 D1 LAVLAYSITLVMLWSIWQYA 303 RIM2 D1 NYKLNTDHAGSNDNIALLVQ
307 RIM2 D1 QDFRRAFRRILARPWTQTAW 312 RIM2 D1 RELVDRGEVRQFTLRHWLKV
315 RIM2 D1 SSKSKSSEESQTFFGLYKL 320 RIM2 D1 SSSRRDSSWSETSEASYSGL
322 RIM2 D1 VDPNSPAAKKKYVSYNNLVI 329 RIM2 D1 VHKVRNKFKAKCSLCRLYII
331 RIM2 D1 YGRKKRRQRRRARGDRKIRV 334 RIM2 D1 YGRKKRRQRRRARGDRKKIV
335 RIM2 D1 YGRKKRRQRRRARGDRKRIV 335 RIM2 D1 YGRKKRRQRRRARGDRKRWA
336 RIM2 D1 YGRKKRRQRRRARGDRKRWL 337 RIM2 D1 YGRKKRRQRRRARGDRKVPV
338 RIM2 D1 YGRKKRRQRRRARGDRKYIV 339 RIM2 D1 YGRKKRRQRRRARGDRLEIV
340 RIM2 D1 YGRKKRRQRRRARGDRLVSL 341 RIM2 D1 YGRKKRRQRRRARGDRRRIV
342 RIM2 D1 YGRKKRRQRRRARGDRTIIY 343 RIM2 D1 YGRKKRRQRRRARGDRYRIV
344 RIM2 D1 YGRKKRRQRRRARGDVRLML 345 RIM2 D1 YGRKKRRQRRRARGRRETWV
346 RIM2 D1 YGRKKRRQRRRARGTRLVWV 346 RIM2 D1 YGRKKRRQRRRARSDRGIWA
347 RIM2 D1 YGRKKRRQRRRARSDRKRIA 348 RIM2 D1 YGRKKRRQRRRARSDRKRIV
349 RIM2 D1 YGRKKRRQRRRARSDRTIIA 350 RIM2 D1 YGRKKRRQRRRARSDRTIWA
351 RIM2 D1 YGRKKRRQRRRARTDRKVEV 352 RIM2 D1 YGRKKRRQRRRAVAAASANL
353 RIM2 D1 YGRKKRRQRRRAVAATGIWA 354 RIM2 D1 YGRKKRRQRRRAVAATSANL
355 RIM2 D1 YGRKKRRQRRRAVAATSINL 356 RIM2 D1 YGRKKRRQRRRAVAATYSNL
357 RIM2 D1 YGRKKRRQRRREKHFRETEV 359 RIM2 D1 YGRKKRRQRRREYLGLDVPV
360 RIM2 D1 YGRKKRRQRRRGASADSTQA 361 RIM2 D1 YGRKKRRQRRRGMTSSSSVV
362 RIM2 D1 YGRKKRRQRRRKNKDKEYYV 364
RIM2 D1 YGRKKRRQRRRLQRRRETQV 365 RIM2 D1 YGRKKRRQRRRNDNIALLVQ 366
RIM2 D1 YGRKKRRQRRRQDEEEGIWA 367 RIM2 D1 YGRKKRRQRRRQDEEEGIWS 368
RIM2 D1 YGRKKRRQRRRQDEEETIWA 369 RIM2 D1 YGRKKRRQRRRQDERRLIVL 370
RIM2 D1 YGRKKRRQRRRQDERVETRV 371 RIM2 D1 YGRKKRRQRRRSRTDRKYQI 373
RIM2 D1 YGRKKRRQRRRSRTDRKYWA 374 RIM2 D1 YGRKKRRQRRRSRTDRLEYV 375
RIM2 D1 YGRKKRRQRRRSRTVREIWA 376 RIM2 D1 YGRKKRRQRRRSVTSTSINL 377
Syntrophin 1 AAGGRSARGGRLQGRRETAL 279 alpha D1 Syntrophin 1
ATDYLVQPFMDQLAFHQFYI 280 alpha D1 Syntrophin 1 AVGGRPARGGRLQGRRQTQV
281 alpha D1 Syntrophin 1 DFRPSFKHILFRRARRGFRQ 282 alpha D1
Syntrophin 1 DTLLLTENEGDKTEEQVSYV 284 alpha D1 Syntrophin 1
FNGSSNGHVYEKLSSIESDV 288 alpha D1 Syntrophin 1 GRWTGRAMSAWKPTRRETEV
289 alpha D1 Syntrophin 1 HDFRRAFKKILARGDRKRIV 291 alpha D1
Syntrophin 1 HHLVAQRDIRQFQLQHWLAI 292 alpha D1 Syntrophin 1
HSCCNRARQERLQRRRETQV 293 alpha D1 Syntrophin 1 ILNSIQVMRAQMNQIQSVEV
294 alpha D1 Syntrophin 1 KKKKQPGNSTKESESTNSVRLML 297 alpha D1
Syntrophin 1 LAVLAYSITLVMLWSIWQYA 303 alpha D1 Syntrophin 1
PIPAGGCTFSGIFPTLTSPL 309 alpha D1 Syntrophin 1 PYSELNYETSHYPASPDSWV
311 alpha D1 Syntrophin 1 QDFRRAFRRILARPWTQTAW 312 alpha D1
Syntrophin 1 QISPGGLEPPSEKHFRETEV 314 alpha D1 Syntrophin 1
RELVDRGEVRQFTLRHWLKV 315 alpha D1 Syntrophin 1 SSKSKSSEESQTFFGLYKL
320 alpha D1 Syntrophin 1 TFKGTPTAENPEYLGLDVPV 325 alpha D1
Syntrophin 1 TGSALQAWRHTSRQATESTV 326 alpha D1 Syntrophin 1
TQGFPGPATWRRISSLESEV 327 alpha D1 Syntrophin 1 VHDAESSDEDGYDWGPATDL
330 alpha D1 Syntrophin 1 VHKVRNKFKAKCSLCRLYII 331 alpha D1
Syntrophin 1 VPGALDYAAFSSALYGESDL 332 alpha D1 Syntrophin 1
YGRKKRRQRRRKLSSIESDV 363 alpha D1 Syntrophin 1 YGRKKRRQRRRNDNIALLVQ
366 alpha D1 ZO-1 D2 DFRPSFKHILFRRARRGFRQ 282 ZO-1 D2
ELLQFCRTPNPALKNGQYWV 285 ZO-1 D2 FHSKTAGANTTDKELEVLSL 287 ZO-1 D2
HDFRRAFKKILARGDRKRIV 291 ZO-1 D2 HHLVAQRDIRQFQLQHWLAI 292 ZO-1 D2
HSCCNRARQERLQRRRETQV 293 ZO-1 D2 ILNSIQVMRAQMNQIQSVEV 294 ZO-1 D2
KHSRKSSSYSSSSTTVKTSY 296 ZO-1 D2 KKKKQPGNSTKESESTNSVRLML 297 ZO-1
D2 LAVLAYSITLVMLWSIWQYA 303 ZO-I D2 LNSCSNRRVYKKMPSIESDV 305 ZO-1
D2 NYKLNTDHAGSNDNIALLVQ 307 ZO-1 D2 PSSRASSRASSRPRPDDLEI 310 ZO-1
D2 QDFRRAFRRILARPWTQTAW 312 ZO-I D2 RELVDRGEVRQFTLRHWLKV 315 ZO-1
D2 RRRRRRRGNTTDKELEVLSL 316 ZO-1 D2 RRRRRRRGTNPAVAATSANL 317 ZO-1
D2 SSKSKSSEESQTFFGLYKL 320 ZO-1 D2 SSSRRDSSWSETSEASYSGL 322 ZO-1 D2
TEGNESSEATSPVNAIYSLA 324 ZO-1 D2 TTNNNPNSAVNIKKIFTDV 328 ZO-1 D2
VDPNSPAAKKKYVSYNNLVI 329 ZO-1 D2 VHKVRNKFKAKCSLCRLYII 331 ZO-1 D2
YGRKKRRQRRRARGDRKRIV 335 ZO-1 D2 YGRKKRRQRRRARGDRKRWA 336 ZO-1 D2
YGRKKRRQRRRARGDRKRWL 337 ZO-l D2 YGRKKRRQRRRARGDRKVPV 338 ZO-1 D2
YGRKKRRQRRRARGDRLVSL 341 ZO-l D2 YGRKKRRQRRRARGDRTIIY 343 ZO-1 D2
YGRKKRRQRRRARGDRYRIV 344 ZO-1 D2 YGRKKRRQRRRARGRRETWV 346 ZO-1 D2
YGRKKRRQRRRARGTRLVWV 346 ZO-1 D2 YGRKKRRQRRRARSDRGIWA 347 ZO-1 D2
YGRKKRRQRRRARSDRTIIA 350 ZO-1 D2 YGRKKRRQRRRARSDRTIWA 351 ZO-1 D2
YGRKKRRQRRRAVAAASANL 353 ZO-1 D2 YGRKKRRQRRRAVAATSANL 355 ZO-1 D2
YGRKKRRQRRRAVAATSINL 356 ZO-1 D2 YGRKKRRQRRRAVAATYSNL 357 ZO-1 D2
YGRKKRRQRRRDKELEVLSL 358 ZO-1 D2 YGRKKRRQRRREKHFRETEV 359 ZO-1 D2
YGRKKRRQRRREYLGLDVPV 360 ZO-1 D2 YGRKKRRQRRRGASADSTQA 361 ZO-1 D2
YGRKKRRQRRRKLSSIESDV 363 ZO-1 D2 YGRKKRRQRRRKNKDKEYYV 364 ZO-1 D2
YGRKKRRQRRRNDNIALLVQ 366 ZO-1 D2 YGRKKRRQRRRQDEEEGIWA 367 ZO-1 D2
YGRKKRRQRRRQDERRLIVL 370 ZO-1 D2 YGRKKRRQRRRSEGVPDLLV 372 ZO-1 D2
YGRKKRRQRRRSRTDRKYWA 374 ZO-1 D2 YVYSRVKNLNSSEGVPDLLV 379
[0398] These specific examples should not be considered as limiting
but simply illustrative of inhibitors having the general
characteristics listed above.
[0399] As described in greater detail below, short PL peptides,
such as just described can be used in the rational design of other
small molecules with similar properties according to established
techniques.
[0400] Core PDZ motif sequences/PL sequences such as those just
listed can optionally be joined to additional amino acids at their
amino terminus to further increase binding affinity and/or
stability and/or transportability into cells. These additional
sequences located at the amino terminus can be from the natural
sequence of a neuronal cell surface receptor or from other sources.
The PDZ motif sequence and additional N-terminal sequences can
optionally be joined by a linker. The additional amino acids can
also be conservatively substituted. The total peptide length (i.e.,
core PDZ motif sequence plus optional N-terminal segment) can be of
a variety of lengths (e.g., at least 2, 3, 4, 5, 6, 8, 10, 15, 20,
25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids).
Typically, the overall length is in the range of 30-40 amino acids)
For those inhibitors in which additional sequences are attached at
the N-terminus of the core PDZ motif sequence (PL sequence), the
overall structure is thus: N-terminal segment--core PDZ motif
sequence (PL sequence), or N-terminal segment--linker--core PDZ
motif sequence (PL sequence). As discussed further below, one
useful class of proteins that can be fused to the core PDZ motifs
or PL sequences are transmembrane transporter peptides. These
peptides can be fused to the inhibitory sequences to facilitate
transport into a target cell (e.g., neuron). Further details are
provided below. Purification tags that are known in the art can
also optionally be fused to the N-terminus of the PL sequence.
[0401] 2. Inhibitors with a PDZ-Domain Polypeptide
[0402] Some of the inhibitors that are provided contain all or a
portion of a PDZ binding domain rather than containing a PL
sequence. The PDZ-domain sequence included in these inhibitors is
selected to mimic (i.e., have similar binding characteristics) of
the PDZ domain in the PDZ protein of interest (i.e., the PDZ
protein whose binding interaction with a PL protein one seeks to
disrupt). The PDZ-domain sequence is long enough to include at
least enough of the PDZ domain such that the resulting polypeptide
inhibitor can effectively bind to the cognate PL protein. This
typically means that the PDZ-domain sequence is at least 50, 55,
60, 65, 70, 75, 80, 85, 90 or more amino acids long. But certain
inhibitors can include the entire PDZ-domain, or even additional
amino acids from the PDZ protein that extend beyond the
PDZ-domain.
[0403] 3. Optional Features of Inhibitors
[0404] Polypeptide inhibitors such as those just described can
optionally be derivatized (e.g., acetylated, phosphorylated and/or
glycoslylated) to improve the binding affinity of the inhibitor, to
improve the ability of the inhibitor to be transported across a
cell membrane or to improve stability. As a specific example, for
inhibitors in which the third residue from the C-terminus is S, T
or Y, this residue can be phosphorylated prior to the use of the
peptide.
[0405] The polypeptide inhibitors can also optionally be linked
directly or via a linker to a transmembrane transporter peptide.
Specific examples of these sequences are described in the section
on formulation and administration of the polypeptides of the
invention. But certain polypeptide inhibitors do not include a
transporter peptide.
[0406] B. Peptide Variants
[0407] Having identified PDZ binding peptides and PDZ-PL
interaction inhibitory sequences, variations of these sequences can
be made and the resulting peptide variants can be tested for PDZ
domain binding or PDZ-PL inhibitory activity. In embodiments, the
variants have the same or a different ability to bind a PDZ domain
as the parent peptide. Typically, such amino acid substitutions are
conservative, i.e., the amino acid residues are replaced with other
amino acid residues having physical and/or chemical properties
similar to the residues they are replacing. Preferably,
conservative amino acid substitutions are those wherein an amino
acid is replaced with another amino acid encompassed within the
same designated class.
[0408] C. Peptide Mimetics
[0409] Having identified PDZ binding peptides and PDZ-PL
interaction inhibitory sequences, peptide mimetics can be prepared
using routine methods, and the inhibitory activity of the mimetics
can be confirmed using the assays of the invention. Thus, in some
embodiments, the antagonist is a peptide mimetic of a PL C-terminal
sequence. The skilled artisan will recognize that individual
synthetic residues and polypeptides incorporating mimetics can be
synthesized using a variety of procedures and methodologies, which
are well described in the scientific and patent literature, e.g.,
Organic Syntheses Collective Volumes, Gilman et al. (Eds) John
Wiley & Sons, Inc., NY. Polypeptides incorporating mimetics can
also be made using solid phase synthetic procedures, as described,
e.g., by Di Marchi, et al., U.S. Pat. No. 5,422,426. Mimetics of
the invention can also be synthesized using combinatorial
methodologies. Various techniques for generation of peptide and
peptidomimetic libraries are well known, and include, e.g.,
multipin, tea bag, and split-couple-mix techniques; see, e.g.,
al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr.
Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.
3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.
[0410] D. Small Molecules
[0411] In some embodiments, the inhibitor is a small molecule
(i.e., having a molecular weight less than 1 kDa). Methods for
screening small molecules are well known in the art and include
those described supra.
[0412] E. Binding Affinity
[0413] Regardless of type, the inhibitors generally have an
EC.sub.50 of less than 50 .mu.m. Some inhibitors have an EC.sub.50
of less than 10 .mu.M, others have an EC.sub.50 of 1 .mu.M, and
still others an EC.sub.50 of less than 100 nM. The inhibitors
typically have an EC.sub.50 value of 20-100 nM.
VIII. Uses of PDZ Domain Binding and Antagonist Compounds
[0414] Because the inhibitors that are described herein are useful
in interfering with binding between certain PDZ and PL proteins in
neurons (e.g., the TRPM7/PDZ interaction), the inhibitors can be
utilized in the treatment of a variety of biological processes in
neuron cells. For instance, the inhibitors can be utilized to treat
problems associated with excitotoxicity and apoptosis occasioned by
neuronal damage. The inhibitors can also be utilized to treat
various neurological diseases, including those associated with
stroke and ischemia. Specific examples of neurological diseases
that can be treated with certain inhibitors include, Alzheimer's
disease, epilepsy, Parkinson's disease, Huntington's disease, motor
neuron diseases and inherited ataxias.
[0415] Because PDZ proteins are involved in a number of biological
functions besides involvement in excitotoxicity responses, some of
the inhibitors that are provided can be used in the treatment of
other conditions and activities correlated with the PDZ:PL protein
interactions described herein. Examples of such activities include,
but are not limited to, organization and regulation of multiprotein
complexes, vesicular trafficking, tumor suppression, protein
sorting, establishment of membrane polarity, apoptosis, regulation
of immune response and organization of synapse formation. In
general, PDZ proteins have a common function of facilitating the
assembly of multi-protein complexes, often serving as a bridge
between several proteins, or regulating the function of other
proteins. Additionally, as also noted supra, these proteins are
found in essentially all cell types.
[0416] Consequently, modulation of these interactions can be
utilized to control a wide variety of biological conditions and
physiological conditions. In particular, modulation of interactions
such as those disclosed herein can be utilized to control movement
of vesicles within a cell, inhibition of tumor formation, as well
as in the treatment of immune disorders, neurological disorders,
muscular disorders, and intestinal disorders.
[0417] Certain compounds which modulate binding of the PDZ proteins
and PL proteins can be used to inhibit leukocyte activation, which
is manifested in measurable events including but not limited to,
cytokine production, cell adhesion, expansion of cell numbers,
apoptosis and cytotoxicity. Thus, some compounds of the invention
can be used to treat diverse conditions associated with undesirable
leukocyte activation, including but not limited to, acute and
chronic inflammation, graft-versus-host disease, transplantation
rejection, hypersensitivities and autoimmunity such as multiple
sclerosis, rheumatoid arthritis, peridontal disease, systemic lupus
erythematosis, juvenile diabetes mellitus, non-insulin-dependent
diabetes, and allergies, and other conditions listed herein.
[0418] Thus, the invention also relates to methods of using such
compositions in modulating leukocyte activation as measured by, for
example, cytotoxicity, cytokine production, cell proliferation, and
apoptosis.
IX. Formulation and Route of Administration
[0419] A. Introduction of Antagonists (e.g., Peptides and Fusion
Proteins) into Cells
[0420] The inhibitors disclosed herein or identified using the
screening methods that are provided can be used in the manufacture
of a medicament or pharmaceutical composition. These can then be
administered according to a number of different methods.
[0421] In one aspect, the PDZ-PL antagonists of the invention are
introduced into a cell to modulate (i.e., increase or decrease) a
biological function or activity of the cell. Many small organic
molecules readily cross the cell membranes (or can be modified by
one of skill using routine methods to increase the ability of
compounds to enter cells, e.g., by reducing or eliminating charge,
increasing lipophilicity, conjugating the molecule to a moiety
targeting a cell surface receptor such that after interacting with
the receptor). Methods for introducing larger molecules, e.g.,
peptides and fusion proteins are also well known, including, e.g.,
injection, liposome-mediated fusion, application of a hydrogel,
conjugation to a targeting moiety conjugate endocytozed by the
cell, electroporation, and the like).
[0422] In one embodiment, the antagonist or agent is a fusion
polypeptide or derivatized polypeptide. A fusion or derivatized
protein may include a targeting moiety that increases the ability
of the polypeptide to traverse a cell membrane or causes the
polypeptide to be delivered to a specified cell type (e.g., a
neuron) preferentially or cell compartment (e.g., nuclear
compartment) preferentially. Examples of targeting moieties include
lipid tails, amino acid sequences such as antennapoedia peptide or
a nuclear localization signal (NLS; e.g., Xenopus nucleoplasmin
Robbins et al., 1991, Cell 64:615).
[0423] In one embodiment of the invention, a peptide sequence or
peptide analog determined to inhibit a PDZ domain-PL protein
binding interaction as described herein is introduced into a cell
by linking the sequence to an amino acid sequence that facilitates
its transport through the plasma membrane (a "transmembrane
transporter sequence"). The peptides of the invention may be used
directly or fused to a transmembrane transporter sequence to
facilitate their entry into cells. In the case of such a fusion
peptide, each peptide may be fused with a heterologous peptide at
its amino terminus directly or by using a flexible polylinker such
as the pentamer G-G-G-G-S (SEQ ID NO:254) repeated 1 to 3 times.
Such linker has been used in constructing single chain antibodies
(scFv) by being inserted between V.sub.H and V.sub.L (Bird et al.,
1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad.
Sci. U.S.A. 85:5979-5883). The linker is designed to enable the
correct interaction between two beta-sheets forming the variable
region of the single chain antibody. Other linkers which may be
used include
Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID
NO:255) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A.
87:1066-1070) and
Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp
(SEQ ID NO:256) (Bird et al., 1988, Science 242:423-426).
[0424] A number of peptide sequences have been described in the art
as capable of facilitating the entry of a peptide linked to these
sequences into a cell through the plasma membrane (Derossi et al.,
1998, Trends in Cell Biol. 8:84). For the purpose of this
invention, such peptides are collectively referred to as
transmembrane transporter peptides. Examples of these peptides
include, but are not limited to, tat derived from mV (Vives et al.,
1997, J. Biol. Chem. 272:16010; Nagahara et al., 1998, Nat. Med.
4:1449), antennapedia from Drosophila (Derossi et al., 1994, J.
Biol. Chem. 261:10444), VP22 from herpes simplex virus (Elliot and
D'Hare, 1997, Cell 88:223-233), complementarity-determining regions
(CDR) 2 and 3 of anti-DNA antibodies (Avrameas et al., 1998, Proc.
Natl. Acad. Sci. U.S.A., 95:5601-5606), 70 kDa heat shock protein
(Fujihara, 1999, EMBO J. 18:411-419), transportan (Pooga et al.,
1998, FASEB J. 12:67-77), penetratin, SynB1, SynB3, amphipathic
model peptide, signal sequence-based peptides, and Arg, as
described in Temsamani et al. (2004) Drug Discovery Today
9:1012-1019. In a preferred embodiment of the invention, a
truncated HIV tat peptide having the sequence of YGRKKRRQRRR (SEQ
ID NO:257) is used.
[0425] In some instances, a transmembrane transporter sequence is
fused to a neuronal cell surface receptor carboxyl terminal
sequence at its amino-terminus with or without a linker. Generally,
the C-terminus of a PDZ motif sequence (PL sequence) is free to
interact with a PDZ domain. The transmembrane transporter sequence
can be used in whole or in part as long as it is capable of
facilitating entry of the peptide into a cell.
[0426] In an alternate embodiment of the invention, a neuronal cell
surface receptor C-terminal sequence can be used alone when it is
delivered in a manner that allows its entry into cells in the
absence of a transmembrane transporter sequence. For example, the
peptide may be delivered in a liposome formulation or using a gene
therapy approach by delivering a coding sequence for the PDZ motif
alone or as a fusion molecule into a target cell.
[0427] The compounds of the invention can also be administered via
liposomes, which serve to target the conjugates to a particular
tissue, such as neural tissue, or targeted selectively to infected
cells, as well as increase the half-life of the peptide
composition. Liposomes include emulsions, foams, micelles,
insoluble monolayers, liquid crystals, phospholipid dispersions,
lamellar layers and the like. In these preparations, the peptide to
be delivered is incorporated as part of a liposome, alone or in
conjunction with a molecule which binds to, e.g., a receptor
prevalent among neural cells, such as monoclonal antibodies which
bind to the NMDA Receptor. Thus, liposomes filled with a desired
peptide or conjugate of the invention can be directed to the site
of neural cells, where the liposomes then deliver the selected
inhibitor compositions. Liposomes for use in the invention are
formed from standard vesicle-forming lipids, which generally
include neutral and negatively charged phospholipids and a sterol,
such as cholesterol. The selection of lipids is generally guided by
consideration of, e.g., liposome size, acid lability and stability
of the liposomes in the blood stream. A variety of methods are
available for preparing liposomes, as described in, e.g., Szoka et
al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos.
4,235,871, 4,501,728 and 4,837,028.
[0428] The targeting of liposomes using a variety of targeting
agents is well known in the art (see, e.g., U.S. Pat. Nos.
4,957,773 and 4,603,044). For targeting to the neural cells, a
ligand to be incorporated into the liposome can include, e.g.,
antibodies or fragments thereof specific for cell surface
determinants of the desired nervous system cells. A liposome
suspension containing a peptide or conjugate may be administered
intravenously, locally, topically, etc. in a dose which varies
according to, inter alia, the manner of administration, the
conjugate being delivered, and the stage of the disease being
treated.
[0429] In order to specifically deliver a PDZ motif sequence or PL
sequence peptide into a specific cell type, the peptide can be
linked to a cell-specific targeting moiety, which include but are
not limited to, ligands for diverse neuron surface molecules such
as growth factors, hormones and cytokines, neuronal receptors, ion
transporters, as well as antibodies or antigen-binding fragments
thereof. Since a large number of cell surface receptors have been
identified in neurons, ligands or antibodies specific for these
receptors may be used as cell-specific targeting moieties.
[0430] Antibodies are the most versatile cell-specific targeting
moieties because they can be generated against any cell surface
antigen. Monoclonal antibodies have been generated against
neuron-specific markers. Antibody variable region genes can be
readily isolated from hybridoma cells by methods well known in the
art. However, since antibodies are assembled between two heavy
chains and two light chains, it is preferred that a scFv be used as
a cell-specific targeting moiety in the present invention. Such
scFv are comprised of V.sub.H and V.sub.L domains linked into a
single polypeptide chain by a flexible linker peptide.
[0431] The PDZ motif sequence (PL sequence) may be linked to a
transmembrane transporter sequence and a cell-specific targeting
moiety to produce a tri-fusion molecule. This molecule can bind to
a neuron surface molecule, pass through the membrane and target PDZ
domains. Alternatively, a PDZ motif sequence (PL sequence) may be
linked to a cell-specific targeting moiety that binds to a surface
molecule that internalizes the fusion peptide.
[0432] In another approach, microspheres of artificial polymers of
mixed amino acids (proteinoids) have been used to deliver
pharmaceuticals. For example, U.S. Pat. No. 4,925,673 describes
drug-containing proteinoid microsphere carriers as well as methods
for their preparation and use. These proteinoid microspheres are
useful for the delivery of a number of active agents. Also see,
U.S. Pat. Nos. 5,907,030 and 6,033,884, which are incorporated
herein by reference.
[0433] B. Introduction of Polynucleotides into Cells
[0434] By introducing gene sequences into cells, gene therapy can
be used to treat diseased cells (e.g., neuron cells that are
associated with apoptosis or an excitotoxic response due to a
neuronal insult). In one embodiment, a polynucleotide that encodes
a PL sequence peptide of the invention is introduced into a cell
where it is expressed. The expressed peptide then inhibits the
interaction of PDZ proteins and PL proteins in the cell. In another
embodiment, the expression of a given protein would be suppressed,
thus inhibiting its interactions with other proteins. In a specific
embodiment, a polynucleotide that encoders an interfering RNA
duplex is introduced into the cell. This results in RNA
interference (RNAi), a process of post transcriptional gene
silencing that inhibits, with high specificity, the expression of
native genes in mammalian cells (Elbashir et al., 2001; Cullen,
2002). In a specific embodiment, the polynucleotides of the
invention would suppress the expression of a given TRP channel such
as TRPM7, or the expression of a protein with which the TRP channel
interacts.
[0435] Examples of specific siRNAs against TRPM7 corresponded to
coding regions 5152-5172, 5023-5043 and 1318-1338
(siRNA.sub.TRPM7-1 to SiRNA.sub.TRPM7-3, respectively) relative to
the first nucleotide of the start codon of murine TRPM7 (GenBank
accession #AY032951). These siRNAs can reduce the expression levels
of TRPM7 in cells (FIG. 8) and have functional consequences (FIG.
3). Thus, these sequences or related sequences could be used
directly or with an appropriate delivery system to treat cells
damaged by OGD or other cytodestructive disorders.
[0436] In one embodiment, the polypeptides of the invention are
expressed in a cell by introducing a nucleic acid (e.g., a DNA
expression vector or mRNA) encoding the desired protein or peptide
into the cell. Expression can be either constitutive or inducible
depending on the vector and choice of promoter. Methods for
introduction and expression of nucleic acids into a cell are well
known in the art and described herein.
[0437] In a specific embodiment, nucleic acids comprising a
sequence encoding a peptide disclosed herein, are administered to a
human subject. In this embodiment of the invention, the nucleic
acid produces its encoded product that mediates a therapeutic
effect. Any of the methods for gene therapy available in the art
can be used according to the present invention. Exemplary methods
are described below.
[0438] For general reviews of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIBTECH 11(5):155-215. Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular
Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
[0439] In a preferred embodiment of the invention, the therapeutic
composition comprises a coding sequence that is part of an
expression vector. In particular, such a nucleic acid has a
promoter operably linked to the coding sequence, said promoter
being inducible or constitutive, and, optionally, tissue-specific.
In another specific embodiment, a nucleic acid molecule is used in
which the coding sequence and any other desired sequences are
flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the nucleic acid (Koller and Smithies, 1989, Proc.
Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature
342:435-438).
[0440] Delivery of the nucleic acid into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vector, or indirect, in which
case, cells are first transformed with the nucleic acid in vitro,
then transplanted into the patient. These two approaches are known,
respectively, as in vivo or ex vivo gene therapy.
[0441] In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any methods known in the art,
e.g., by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by infection using a defective or attenuated
retroviral or other viral vector (see U.S. Pat. No. 4,980,286), by
direct injection of naked DNA, by use of microparticle bombardment
(e.g., a gene gun; Biolistic, DuPont), by coating with lipids or
cell-surface receptors or transfecting agents, by encapsulation in
liposomes, microparticles, or microcapsules, by administering it in
linkage to a peptide which is known to enter the nucleus, or by
administering it in linkage to a ligand subject to
receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol.
Chem. 262:4429-4432) which can be used to target cell types
specifically expressing the receptors. In another embodiment, a
nucleic acid-ligand complex can be formed in which the ligand
comprises a fusogenic viral peptide to disrupt endosomes, allowing
the nucleic acid to avoid lysosomal degradation. In yet another
embodiment, the nucleic acid can be targeted in vivo for cell
specific uptake and expression, by targeting a specific receptor
(see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992; WO
92/22635 dated Dec. 23, 1992; WO92/20316 dated Nov. 26, 1992;
WO93/14188 dated Jul. 22, 1993; WO 93/20221 dated Oct. 14, 1993).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
[0442] In a preferred embodiment of the invention, adenoviruses as
viral vectors can be used in gene therapy. Adenoviruses have the
advantage of being capable of infecting non-dividing cells
(Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503). Other instances of the use of adenoviruses
in gene therapy can be found in Rosenfeld et al., 1991, Science
252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; and
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234. Furthermore,
adenoviral vectors with modified tropism may be used for cell
specific targeting (WO98/40508). Adeno-associated virus (AAV) has
also been proposed for use in gene therapy (Walsh et al., 1993,
Proc. Soc. Exp. Biol. Med. 204:289-300).
[0443] In addition, retroviral vectors (see Miller et al., 1993,
Meth. Enzymol. 217:581-599) have been modified to delete retroviral
sequences that are not necessary for packaging of the viral genome
and integration into host cell DNA. The coding sequence to be used
in gene therapy is cloned into the vector, which facilitates
delivery of the gene into a patient. More detail about retroviral
vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302,
which describes the use of a retroviral vector to deliver the mdr1
gene to hematopoietic stem cells in order to make the stem cells
more resistant to chemotherapy. Other references illustrating the
use of retroviral vectors in gene therapy are: Clowes et al., 1994,
J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473;
Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and
Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.
3:110-114.
[0444] Another approach to gene therapy involves transferring a
gene to cells in tissue culture. Usually, the method of transfer
includes the transfer of a selectable marker to the cells. The
cells are then placed under selection to isolate those cells that
have taken up and are expressing the transferred gene. Those cells
are then delivered to a patient.
[0445] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, lipofection, microinjection, infection with a
viral or bacteriophage vector containing the nucleic acid
sequences, cell fusion, chromosome-mediated gene transfer,
microcell-mediated gene transfer, spheroplast fusion, etc. Numerous
techniques are known in the art for the introduction of foreign
genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol.
217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline,
1985, Pharmac. Ther. 29:69-92) and may be used in accordance with
the present invention, provided that the necessary developmental
and physiological functions of the recipient cells are not
disrupted. The technique should provide for the stable transfer of
the nucleic acid to the cell, so that the nucleic acid is
expressible by the cell and preferably heritable and expressible by
its cell progeny. In a preferred embodiment, the cell used for gene
therapy is autologous to the patient.
[0446] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding sequence, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0447] Oligonucleotides such as anti-sense RNA and DNA molecules,
and ribozymes that function to inhibit the translation of a
targeted mRNA, especially its C-terminus are also within the scope
of the invention. Anti-sense RNA and DNA molecules act to directly
block the translation of mRNA by binding to targeted mRNA and
preventing protein translation. In regard to antisense DNA,
oligodeoxyribonucleotides derived from the translation initiation
site, e.g., between -10 and +10 regions of a nucleotide sequence,
are preferred.
[0448] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0449] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. The mechanism of ribozyme action
involves sequence specific hybridization of the ribozyme molecule
to complementary target RNA, followed by endonucleolytic cleavage.
Within the scope of the invention are engineered hammerhead motif
ribozyme molecules that specifically and efficiently catalyze
endonucleolytic cleavage of target RNA sequences.
[0450] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences, GUA,
GUU and GUC. Once identified, short RNA sequences of between 15 and
20 ribonucleotides corresponding to the region of the target gene
containing the cleavage site may be evaluated for predicted
structural features such as secondary structure that may render the
oligonucleotide sequence unsuitable. The suitability of candidate
targets may also be evaluated by testing their accessibility to
hybridization with complementary oligonucleotides, using
ribonuclease protection assays.
[0451] The anti-sense RNA and DNA molecules and ribozymes of the
invention may be prepared by any method known in the art for the
synthesis of nucleic acid molecules. These include techniques for
chemically synthesizing oligodeoxyribonucleotides well known in the
art such as for example solid phase phosphoramidite chemical
synthesis. Alternatively, RNA molecules may be generated by in
vitro and in vivo transcription of DNA sequences encoding the RNA
molecule. Such DNA sequences may be incorporated into a wide
variety of vectors which contain suitable RNA polymerase promoters
such as the T7 or SP6 polymerase promoters. Alternatively,
antisense cDNA constructs that synthesize antisense RNA
constitutively or inducibly, depending on the promoter used, can be
introduced stably into cell lines.
[0452] Various modifications to the DNA molecules may be introduced
as a means of increasing intracellular stability and half-life.
Possible modifications include, but are not limited to, the
addition of flanking sequences of ribo- or deoxy-nucleotides to the
5' and/or 3' ends of the molecule or the use of phosphorothioate or
2' O-methyl rather than phosphodiesterase linkages within the
oligodeoxyribonucleotide backbone.
[0453] C. Other Pharmaceutical Compositions
[0454] The compounds of the invention, may be administered to a
subject per se or in the form of a sterile composition or a
pharmaceutical composition. Pharmaceutical compositions comprising
the compounds of the invention may be manufactured by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Pharmaceutical compositions may be formulated in
conventional manner using one or more physiologically acceptable
carriers, diluents, excipients or auxiliaries that facilitate
processing of the active peptides or peptide analogues into
preparations which can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen.
[0455] For topical administration the compounds of the invention
can be formulated as solutions, gels, ointments, creams,
suspensions, etc. as are well-known in the art.
[0456] Systemic formulations include those designed for
administration by injection, e.g. subcutaneous, intravenous,
intramuscular, intrathecal or intraperitoneal injection, as well as
those designed for transdermal, transmucosal, oral or pulmonary
administration.
[0457] For injection, the compounds of the invention can be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. The solution can contain formulatory
agents such as suspending, stabilizing and/or dispersing
agents.
[0458] Alternatively, the compounds can be in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use.
[0459] For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art. This route of
administration may be used to deliver the compounds to the nasal
cavity.
[0460] For oral administration, the compounds can be readily
formulated by combining the active peptides or peptide analogues
with pharmaceutically acceptable carriers well known in the art.
Such carriers enable the compounds of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated. For oral solid formulations such as, for
example, powders, capsules and tablets, suitable excipients include
fillers such as sugars, such as lactose, sucrose, mannitol and
sorbitol; cellulose preparations such as maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP);
granulating agents; and binding agents. If desired, disintegrating
agents may be added, such as the cross-linked polyvinylpyrrolidone,
agar, or alginic acid or a salt thereof such as sodium
alginate.
[0461] If desired, solid dosage forms may be sugar-coated or
enteric-coated using standard techniques.
[0462] For oral liquid preparations such as, for example,
suspensions, elixirs and solutions, suitable carriers, excipients
or diluents include water, glycols, oils, alcohols, etc.
Additionally, flavoring agents, preservatives, coloring agents and
the like may be added.
[0463] For buccal administration, the compounds may take the form
of tablets, lozenges, etc. formulated in conventional manner.
[0464] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray from pressurized packs or a nebulizer,
with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0465] The compounds may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0466] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0467] Alternatively, other pharmaceutical delivery systems may be
employed. Liposomes and emulsions are well known examples of
delivery vehicles that may be used to deliver peptides and peptide
analogues of the invention. Certain organic solvents such as
dimethylsulfoxide also may be employed, although usually at the
cost of greater toxicity. Additionally, the compounds may be
delivered using a sustained-release system, such as semipermeable
matrices of solid polymers containing the therapeutic agent.
Various of sustained-release materials have been established and
are well known by those skilled in the art. Sustained-release
capsules may, depending on their chemical nature, release the
compounds for a few weeks up to over 100 days. Depending on the
chemical nature and the biological stability of the therapeutic
reagent, additional strategies for protein stabilization may be
employed.
[0468] As the compounds of the invention may contain charged side
chains or termini, they may be included in any of the
above-described formulations as the free acids or bases or as
pharmaceutically acceptable salts. Pharmaceutically acceptable
salts are those salts which substantially retain the biologic
activity of the free bases and which are prepared by reaction with
inorganic acids. Pharmaceutical salts tend to be more soluble in
aqueous and other protic solvents than are the corresponding free
base forms.
[0469] D. Effective Dosages
[0470] The compounds of the invention will generally be used in an
amount effective to achieve the intended purpose (e.g., treatment
of a neuronal injury). The compounds of the invention or
pharmaceutical compositions thereof, are administered or applied in
a therapeutically effective amount. By therapeutically effective
amount is meant an amount effective ameliorate or prevent the
symptoms, or prolong the survival of, the patient being treated.
Determination of a therapeutically effective amount is well within
the capabilities of those skilled in the art, especially in light
of the detailed disclosure provided herein. An "inhibitory amount"
or "inhibitory concentration" of a PL-PDZ binding inhibitor is an
amount that reduces binding by at least about 40%, preferably at
least about 50%, often at least about 70%, and even as much as at
least about 90%. Binding can be measured in vitro (e.g., in an A
assay or G assay) or in situ.
[0471] For systemic administration, a therapeutically effective
dose can be estimated initially from in vitro assays. For example,
a dose can be formulated in animal models to achieve a circulating
concentration range that includes the IC.sub.50 as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans.
[0472] Initial dosages can also be estimated from in vivo data,
e.g., animal models, using techniques that are well known in the
art. One having ordinary skill in the art could readily optimize
administration to humans based on animal data.
[0473] Dosage amount and interval may be adjusted individually to
provide plasma levels of the compounds that are sufficient to
maintain therapeutic effect. Usual patient dosages for
administration by injection range from about 0.1 to 5 mg/kg/day,
preferably from about 0.5 to 1 mg/kg/day. Therapeutically effective
serum levels may be achieved by administering multiple doses each
day. For usual peptide therapeutic treatment of stroke, acute
administration of 0.03 mmol/g to 30 nmol/g within 6 hours of stroke
or brain ischemia is typical. In other instances, 0.1 nmol/g to 20
nmol/g within 6 hours are administered. And in still other
instances 1 nmol/g to 10 nmol/g is administered with in 6
hours.
[0474] In cases of local administration or selective uptake, the
effective local concentration of the compounds may not be related
to plasma concentration. One having skill in the art will be able
to optimize therapeutically effective local dosages without undue
experimentation.
[0475] The amount of compound administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
[0476] The therapy may be repeated intermittently while symptoms
detectable or even when they are not detectable. The therapy may be
provided alone or in combination with other drugs.
[0477] E. Toxicity
[0478] Preferably, a therapeutically effective dose of the
compounds described herein will provide therapeutic benefit without
causing substantial toxicity.
[0479] Toxicity of the compounds described herein can be determined
by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., by determining the LD.sub.50 (the dose
lethal to 50% of the population) or the LD.sub.100 (the dose lethal
to 100% of the population). The dose ratio between toxic and
therapeutic effect is the therapeutic index. Compounds which
exhibit high therapeutic indices are preferred. The data obtained
from these cell culture assays and animal studies can be used in
formulating a dosage range that is not toxic for use in human. The
dosage of the compounds described herein lies preferably within a
range of circulating concentrations that include the effective dose
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See, e.g., Fingl et al., 1975,
In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1).
EXAMPLES
Example 1
Identification of TRPM7 Interactions with PDZ Domains
[0480] This example describes assays to identify binding between
TRPM7 and PDZ domains. GST-PDZ fusions are produced that each
contain an entire PDZ domain (or multiple PDZ domains), the
collection encompassing approximately 90% of all PDZ domains
identified in the human genome. (See Table 5 and Section V (PDZ
Proteins and PL Protein Interactions Disclosure). Biotinylated
peptide corresponding to 11 residues of Tat coupled to the
C-terminal 9 amino acids of TRPM7 (YGRKKRRQRRRSTNSVRLML; SEQ ID
NO:258) is synthesized and purified by HPLC.
[0481] Binding between these entities is detected through the G or
the G3 Assays, calorimetric assays using avidin-HRP to bind the
biotin and a peroxidase substrate.
[0482] Another type of assay is a fluorescence-based binding assay
using the rhodamine derivative TAMRA (Molecular Probes) conjugated
to the C-terminal 9 amino acids of TRPM7 (TAMRA-RRSTNSVRLML; SEQ ID
NO:259).
[0483] A. G Assay for Identification of Interactions Between
Peptides and Fusion Proteins
Reagents and Materials:
[0484] Nunc Maxisorp 96 well immuno-plate [0485] PBS pH 7.4 (Gibco
BRL Cat #16777-148) or (AVC phosphate buffered saline, 8 g NaCl,
0.29 g KCl, 1.44 g Na.sub.2HPO.sub.4, 0.24 g KH.sub.2PO.sub.4, add
H2O to 1 liter and pH 7.4; 0.2 .mu.m filter) [0486] 2% BSA/PBS (10
g of bovine serum albumin, fraction V (ICN Biomedicals Cat.
#IC15142983) into 500 ml PBS [0487] Goat anti-GST mAb stock @ 5
mg/ml, store at 4.degree. C., (Amersham Pharmacia Cat.
#27-4577-01), dilute 1:1000 in PBS, final concentration 5 .mu.g/ml
[0488] HRP-Streptavidin, 2.5 mg/2 ml stock stored at 4.degree. C.
(Zymed Cat. #43-4323), dilute 1:2000 into 2% BSA, final
concentration at 0.5 .mu.g/ml [0489] Wash Buffer, PBS [0490] TMB
ready to use (Dako Cat #S1600) [0491] 1 MH.sub.2SO.sub.4 [0492] 12
w multichannel pipettor, [0493] 50 ml reagent reservoirs, [0494] 15
ml polypropylene conical tubes
Protocol
[0494] [0495] 1) Coat plate with 100 .mu.l of 5 .mu.g/ml goat anti
GST, overnight at 4.degree. C. [0496] 2) Dump coating antibodies
out and tap dry [0497] 3) Blocking--Add 200 .mu.l per well 2% BSA,
2 hrs at room temperature [0498] 4) Prepare proteins at 5 .mu.g/ml
in 2% BSA (2 ml per row or per two columns) [0499] 5) 3 washes with
cold PBS (must be cold through entire experiment) (at last wash
leave PBS in wells until immediately adding next step) [0500] 6)
Add proteins at 50 .mu.l per well on ice (1 to 2 hrs at 4.degree.
C.) [0501] 7) Prepare Peptides at desired concentration in 2% BSA
(2 ml/row or/columns) [0502] 8) 3.times. wash with cold PBS [0503]
9) Add peptides at 50 .mu.l per well on ice (time on/time off)
[0504] a. keep on ice after last peptide has been added for 10
minutes exactly [0505] b. place at room temp for 20 minutes exactly
[0506] 10) Prepare 12 ml/plate of HRP-Streptavidin (1:2000 dilution
in 2% BSA) [0507] 11) 3.times. wash with cold PBS, (must be cold,
critical) [0508] 12) Add HRP-Streptavidin at 100 .mu.l per well on
ice, 20 minutes at 4.degree. C. [0509] 13) Turn on plate reader
[0510] 14) 5.times. washes with PBS at room temperature, avoid
bubbles [0511] 15) Add TMB substrate at 100 .mu.l per well [0512]
a. incubate in dark at room temperature [0513] b. check plate
periodically (5, 10, & 20 minutes) [0514] c. take early
readings, if necessary, at 650 nm (blue) [0515] d. at 30 minutes,
stop reaction with 100 .mu.l of 1M H.sub.2SO.sub.4 [0516] e. take
last reading at 450 nm (yellow
[0517] B. G3 Assay For Identification of Interactions Between
Peptides and Fusion Proteins
Reagents and Supplies
[0518] Nunc MaxiSorp 96 well Immuno-plate, Nunc [0519] PBS pH 7.4
(phosphate buffered saline, 8 g NaCl, 0.29 g KCl, 1.44 g
Na.sub.2HPO.sub.4, 0.24 g KH.sub.2PO.sub.4, add H.sub.2O to 1 liter
and pH 7.4; 0.2 .mu.m filter) [0520] Assay Buffer: 2% BSA in PBS
(20 g of bovine serum albumin per liter PBS, fraction V, ICN
Biomedicals, cat#IC15142983 [0521] Goat anti-GST polyclonal Ab,
stock 5 mg/ml, stored at 4.degree. C., Amersham Pharmacia
cat#27-4577-01 [0522] Dilute 1:1000 in PBS, final concentration 5
.mu.g/ml. [0523] HRP-Streptavidin, 2.5 mg/2 ml stock stored at
4.degree. C., Zymed cat#43-4323, dilute 1:2000 into Assay buffer,
final concentration of 0.5 .mu.g/ml [0524] Wash Buffer, PBS [0525]
Biotinylated peptides (HPLC purified, stock solution stored in
-20.degree. C. freezer) [0526] GST-PRISM proteins (stock stored at
-80.degree. C., after 1.sup.st thaw store in -10.degree. C.
freezer) [0527] TMB (3,3',5,5', teramethylbensidine), tablets,
Sigma cat.#T5525 [0528] Per plate, dissolve 1 tablet in 1 ml DMSO,
add 9 ml Citrate/Phosphate buffer pH 5.4 and 2 .mu.L H.sub.2O.sub.2
[0529] 0.1 M H.sub.2SO.sub.4, Sigma cat.#S1526 [0530] 12-w
multichannel pipettor & tips [0531] 50 ml reagent reservoirs,
Costar#4870 [0532] 50, 15 ml polypropylene conical tubes [0533]
Costar Transtar 96 Costar #7605 [0534] Transtar 96 Cartridge Costar
#7610 [0535] Molecular Devices microplate reader (450 & 650 nm
filters) with SoftMax Pro software *When using reagents stored at
or 4.degree. C. or -20.degree. C., remove & keep on ice
Protocol
[0535] [0536] 1. Coat plate with 100 .mu.l of 5 .mu.g/ml anti-GST,
overnight at 4.degree. C. [0537] 2. Dump contents of plate &
out tap dry on paper towels [0538] 3. Block with 200 .mu.l Assay
Buffer for 2 hrs at room temperature [0539] 4. Prepare proteins at
5 .mu.g/ml in Assay Buffer [0540] 5. Wash 3.times. with cold PBS
(4.degree. C.), avoiding letting the plates dry out [0541] 6. Add
proteins at 50 .mu.l per well, incubate 1 to 2 hrs at 4.degree. C.
[0542] 7. Prepare peptides to desired concentration in Assay Buffer
[0543] 8. Wash 3.times. with cold PBS (4.degree. C.), avoiding
letting the plates dry out [0544] 9. Add HRP-Streptavidin/peptide
complex [prepared by incubating for 20 minutes at room temperature
HRP-Streptavidin (1:2000) with the desired biotin-peptide
concentration for 20 minutes at room temperature] at 50 .mu.l per
well on ice [0545] 10. Place at room temperature for exactly 30
minutes [0546] 11. Promptly wash 5.times. with PBS at room
temperature [0547] 12. Add 100 .mu.l/well TMB substrate (write time
on plate) [0548] 13. Incubate in dark at room temperature for a
maximum of 30 minutes [0549] 14. Read plate at 25 minutes (650 nm),
optional [0550] 15. Stop reaction with 100 .mu.l of 0.18 M
H.sub.2SO.sub.4, 30 minutes after adding TMB [0551] Take last
reading at 450 nm soon after stopping reaction
[0552] C. Results of Binding Experiments
[0553] The G3 assay was performed using 0.1 .mu.M peptide
representing 11 amino acids of Tat fused to the final 9 amino acids
of TRPM7 (YGRKKRRQRRRSTNSVRLML; SEQ ID NO:258). Table 8 shows the
PDZ domains which bound the peptide with an OD at least 2-fold over
GST-only OD in the G3 assay as described above and/or had an
ED.sub.50 of less than 0.17 .mu.M in the titration experiments
described in Example 2 below.
Example 2
Titration Experiments
[0554] The G3 assay was performed on a subset of the PDZ domains as
in Example II except that the concentration of peptide was 0.002
.mu.M, 002 .mu.M, 0.1 .mu.M, 0.2 .mu.M, 1 .mu.M, and 2 .mu.M. Data
was fitted to the equation:
y=100.0/(1+10.sup.((log(m1)-log(m0))*m2)), where m1=EC.sub.50,
m2=Hill coefficient, and m0=peptide concentration to calculate the
EC.sub.50 for the binding of the peptides to the PDZ domains.
[0555] Results:
[0556] EC.sub.50 data are presented in Table 9. Graphs of
titrations for the biotinylated peptide representing 11 amino acids
of Tat fused to the final 9 amino acids of TRPM7
(YGRKKRRQRRRSTNSVRLML; SEQ ID NO:258) peptide with RIM-2 d1, INADL
d3, ZO-1 d2, and Par3 d3 are shown in FIGS. 9A, 9B, 9C, and 9D.
Example 3
Peptide Binding
[0557] The G3 assay was performed to determine the binding of PDZ
domains ZO-1 d2 and INADL d3 with the peptides listed in Table 10.
EC.sub.50 results are shown in Table 10. FIGS. 10A and 10B show
graphs of the titrations for the Peptide #1829 with ZO-1 d2 and for
the Peptide #1839 with INADL d3.
[0558] The peptides shown in Table 11 have also been demonstrated
to bind ZO-1 d2, INADL d3, PAR3 d3, syntrophin 1 alpha d1, and RIM2
d1 in G assays.
Example 4
Treatment of Ischemic Brain Damage by Modulating TRPM7
Expression
Generation of a DNA Vector-Based siRNA System.
[0559] In brief, a small DNA insert (49 bp) encoding a short
hairpin RNA targeting TRPM7 (the siRNA against TRPM7 corresponded
to coding regions 5152-5172 (siRNA.sub.TRPM7-1)) was cloned into a
commercially available pAdTrack vector (see FIG. 11). The sequence
was placed under the control of the H1 promoter and GFP was placed
under a CMV promoter. The TRPM7 siRNA-pAdTrack insert-containing
vector as well as adeno recombination sequences were co-transfected
with pAdEasy (containing viral sequences) into HEK cells and the
cells were selected with kanamycin. Recombined virus containing the
TRPM7 siRNA and GFP sequences was produced from these cells. The
hairpin RNA was rapidly processed by the cellular machinery into 45
nt double stranded RNA (siRNA). In this way, the siRNA was
delivered using a viral vector. Two additional effective RNAi
sequences for TRPM7 include siRNAs against TRPM7 corresponding to
coding regions, 5023-5043 and 1318-1338 (siRNA.sub.TRPM7-2 and
siRNATRPM.sub.7-3, respectively) relative to the first nucleotide
of the start codon of murine TRPM7 (GeneBank accession
#AY032951).
[0560] FIG. 12 shows the effects of infecting primary cultured
neurons on TRPM7 mRNA by RT-PCR. Cultured cortical neurons plated
in 12 well plates (106 cells/well) were infected at the time of
plating with the corresponding adenoviral constructs and the RNA
was harvested 5 days later. RT-PCR was performed on equivalent
amounts of RNA using primers for TRPM7 or .beta.-actin.
[0561] FIG. 13 shows the effect of oxygen glucose deprivation (OGD)
for the indicated duration on neuronal cell death in the presence
(MCN(+)) or absence (MCN(-)) of the combination of MK-801 (10
.mu.M), CNQX (10 .mu.M) and nimodipine (2 .mu.M), denoted as MCN,
antagonists of NMDA and AMPA/kainate glutamate receptors and L-type
Ca.sup.2+ channels, respectively. The fraction of dead cells was
determining by dividing the number of neurons expressing GFP which
became stained with propidium iodide 20 h after OGD by the number
of neurons expressing GFP at the beginning of the experiment. Data
for each culture was obtained from counting cells in 3 high power
microscope fields per experiment.
[0562] The siRNA sequences for TRPM7 may also be placed under the
control of the human U6 promoter in the shuttle vector pTrack
CMV-EGFP, whereby the siRNA sequences and the EGFP sequence will be
driven off separate promoters. A control adenovirus expressing EGFP
alone, using the same shuttle vector as above, will also be
produced.
Regional Infection of Rat Cortex and Hippocampus with RNAi
Adenoviruses by Microinjection.
[0563] 3 .mu.l of either TRPM7 vector or control vector are infused
to the cortex (from bregma: AP=2 mm, ML=2.8 mm, DV=1.7 mm) or in
the CA1 sector of the hippocampus. We infuse in the order of
10.sup.4 particles. Animals are sacrificed 2, 5 and 10 days later
and brain sections prepared and analyzed for GFP fluorescence in
frozen brain sections and for GFP staining with anti-GFP antibodies
in fixed sections. The technique can be titrated (amount of
infectious particles, duration of treatment, number of treatments)
to optimize TRPM7 suppression. All animals undergo bilateral
injections, using the active vector on one side, and the control on
the other, to control for effects of the adenovirus.
Effect of In-Vivo TRPM7 Suppression on Cerebral Ischemic
Damage.
[0564] Experiments are performed using a permanent distal middle
cerebral artery occlusion (MCAO) model as described by Brint et al.
(1988). This results primarily in the death of cortical neurons. In
brief, Wistar rats (250-300 g) are anesthetized with 1-2%
halothane. Snares are placed around the CCAs and a 2 mm burr hole
made at the junction of the zygomatic arch and squamous bone. The
distal MCA is exposed and divided above the rhinal fissure. The CCA
snares is tightened to occlude the CCAs, and released after 4
h.
[0565] Experiments are performed in animals in which TRPM7 was
suppressed as in 5.2 bilaterally in the cortex. Animals are
sacrificed 48 hr later and brain sections prepared and analyzed.
Neuronal cell loss will be assessed in transfected areas as gauged
by GFP expression, which is co-expressed with siRNA in the same
vector and for infarct volumes. Experimental groups will include
animals receiving injections of saline (10), Scrambled (inactive)
RNAi vector (10), and TRPM7 RNAi vector (10). The hemisphere
contralateral to the MCAO will serve as a control in each animal.
If TRPM7 suppression is protective, then there will be improved
survival of neurons in transfected (GFP positive) areas.
Example 5
Treatment of Ischemic Brain Damage by Modulating TRPM7-PDZ Protein
Interactions
[0566] Small peptides or fusion proteins containing C- and
N-terminus TRPM7 subunit sequences, or encompassing intrinsic TRPM
channel enzymatic domains will be synthesized or grown in bacteria.
Some will be grown as Flag- or HA-tagged proteins fused to a
peptide corresponding to the cell-membrane transduction domain of
the HIV-1-Tat protein (YGRKKRRQRRR; Tat; SEQ ID NO:257).
Tat-peptides and proteins permeate into cells in a rapid,
dose-dependent manner independent of receptors and
transporters.
[0567] FIG. 14 shows the effect of treating the cultures with
Tat-9cTRPM7. The sequence of Tat-9cTRPM7 is:
[YGRKKRRQRRR-STNSVRLML; SEQ ID NO:258], whereby the first 11
residues correspond to the cell-membrane transduction domain of the
human immunodeficiency virus type 1 (HIV-1) Tat protein and the
last 9 residues correspond to the last 9 amino acids of the
C-terminus of human TRPM7 (accession Q96QT4). (A) Neuronal survival
at 20 h in the indicated concentrations of Tat-9cTRPM7 in the
absence of excitotoxic challenge. (B) Neuronal survival 20 h after
challenging the cultures for 1 h with the indicated concentration
of NMDA. Tat-9cTRPM7 was applied immediately after the NMDA
challenge. Therefore, neurons treated with 9C-TRPM7 exhibited
enhanced survival when challenged with NMDA.
[0568] We predict similar results with a Tat-conjugated peptide
encoding the last 9 residues of the mouse TRPM7 C-terminus
(YGRKKRRQRRR-ATNSVRLML; SEQ ID NO:380; accession Q923J1).
[0569] To examine whether treatment with these Tat-conjugated TRPM7
C-terminal peptides would reduce stroke damage, experiments are
carried out in adult male Sprague-Dawley rats subjected to
transient middle cerebral artery occlusion (MCAO) for 90 minutes by
the intraluminal suture method. This produces an extensive
infarction encompassing the cerebral cortex and basal ganglia.
Animals are treated with either saline, the Tat-TRPM7 C-terminus,
or with Tat-conjugated to a scrambled C-terminal peptide by a
single intravenous bolus injection 1 hour after the onset of MCAO
(3 nMoles/g). Physiological parameters (body temperature, blood
pressure, blood gases) are monitored and maintained throughout the
experiment. All experimental manipulations and analyses of data are
performed by individuals blinded to the treatment groups. The
extent of cerebral infarction is measured 24 h after MCAO onset
(FIG. 3C inset). The postural reflex test, and the forelimb placing
test is used to grade neurological function on a scale of 0 to 12
(normal=0; worst=12) during MCAO (at 50 minutes) and 24 h
thereafter.
[0570] The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention and any sequences which are
functionally equivalent are within the scope of the invention.
Indeed, various modifications of the invention in addition to those
shown and described herein will become apparent to those skilled in
the art from the foregoing description and accompanying drawings.
Such modifications are intended to fall within the scope of the
appended claims.
[0571] All publications, patents and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes to the same extent as if each individual publication,
patent or patent application were specifically and individually
indicated to be so incorporated by reference.
Sequence CWU 1
1
38414PRTArtificial SequenceSynthetic peptide 1Xaa Leu Met
Leu126PRTArtificial SequenceSynthetic peptide 2Glu Trp Lys Phe Ala
Arg1 5393PRTArtificial SequenceSynthetic peptide 3Leu Arg Lys Glu
Pro Glu Ile Ile Thr Val Thr Leu Lys Lys Gln Asn1 5 10 15Gly Met Gly
Leu Ser Ile Val Ala Ala Lys Gly Ala Gly Gln Asp Lys 20 25 30Leu Gly
Ile Tyr Val Lys Ser Val Val Lys Gly Gly Ala Ala Asp Val 35 40 45Asp
Gly Arg Leu Ala Ala Gly Asp Gln Leu Leu Ser Val Asp Gly Arg 50 55
60Ser Leu Val Gly Leu Ser Gln Glu Arg Ala Ala Glu Leu Met Thr Arg65
70 75 80Thr Ser Ser Val Val Thr Leu Glu Val Ala Lys Gln Gly 85
904105PRTArtificial SequenceSynthetic peptide 4Leu Ile Arg Pro Ser
Val Ile Ser Ile Ile Gly Leu Tyr Lys Glu Lys1 5 10 15Gly Lys Gly Leu
Gly Phe Ser Ile Ala Gly Gly Arg Asp Cys Ile Arg 20 25 30Gly Gln Met
Gly Ile Phe Val Lys Thr Ile Phe Pro Asn Gly Ser Ala 35 40 45Ala Glu
Asp Gly Arg Leu Lys Glu Gly Asp Glu Ile Leu Asp Val Asn 50 55 60Gly
Ile Pro Ile Lys Gly Leu Thr Phe Gln Glu Ala Ile His Thr Phe65 70 75
80Lys Gln Ile Arg Ser Gly Leu Phe Val Leu Thr Val Arg Thr Lys Leu
85 90 95Val Ser Pro Ser Leu Thr Asn Ser Ser 100
1055105PRTArtificial SequenceSynthetic peptide 5Gln Ser Glu Asn Glu
Glu Asp Val Cys Phe Ile Val Leu Asn Arg Lys1 5 10 15Glu Gly Ser Gly
Leu Gly Phe Ser Val Ala Gly Gly Thr Asp Val Glu 20 25 30Pro Lys Ser
Ile Thr Val His Arg Val Phe Ser Gln Gly Ala Ala Ser 35 40 45Gln Glu
Gly Thr Met Asn Arg Gly Asp Phe Leu Leu Ser Val Asn Gly 50 55 60Ala
Ser Leu Ala Gly Leu Ala His Gly Asn Val Leu Lys Val Leu His65 70 75
80Gln Ala Gln Leu His Lys Asp Ala Leu Val Val Ile Lys Lys Gly Met
85 90 95Asp Gln Pro Arg Pro Ser Asn Ser Ser 100
1056132PRTArtificial SequenceSynthetic peptide 6Gly Ile Ser Ser Leu
Gly Arg Lys Thr Pro Gly Pro Lys Asp Arg Ile1 5 10 15Val Met Glu Val
Thr Leu Asn Lys Glu Pro Arg Val Gly Leu Gly Ile 20 25 30Gly Ala Cys
Cys Leu Ala Leu Glu Asn Ser Pro Pro Gly Ile Tyr Ile 35 40 45His Ser
Leu Ala Pro Gly Ser Val Ala Lys Met Glu Ser Asn Leu Ser 50 55 60Arg
Gly Asp Gln Ile Leu Glu Val Asn Ser Val Asn Val Arg His Ala65 70 75
80Ala Leu Ser Lys Val His Ala Ile Leu Ser Lys Cys Pro Pro Gly Pro
85 90 95Val Arg Leu Val Ile Gly Arg His Pro Asn Pro Lys Val Ser Glu
Gln 100 105 110Glu Met Asp Glu Val Ile Ala Arg Ser Thr Tyr Gln Glu
Ser Lys Glu 115 120 125Ala Asn Ser Ser 1307101PRTArtificial
SequenceSynthetic peptide 7Leu Gly Arg Ser Val Ala Val His Asp Ala
Leu Cys Val Glu Val Leu1 5 10 15Lys Thr Ser Ala Gly Leu Gly Leu Ser
Leu Asp Gly Gly Lys Ser Ser 20 25 30Val Thr Gly Asp Gly Pro Leu Val
Ile Lys Arg Val Tyr Lys Gly Gly 35 40 45Ala Ala Glu Gln Ala Gly Ile
Ile Glu Ala Gly Asp Glu Ile Leu Ala 50 55 60Ile Asn Gly Lys Pro Leu
Val Gly Leu Met His Phe Asp Ala Trp Asn65 70 75 80Ile Met Lys Ser
Val Pro Glu Gly Pro Val Gln Leu Leu Ile Arg Lys 85 90 95His Arg Asn
Ser Ser 100898PRTArtificial SequenceSynthetic peptide 8Arg Glu Glu
Gly Gly Met Pro Gln Thr Val Ile Leu Pro Gly Pro Ala1 5 10 15Pro Trp
Gly Phe Arg Leu Ser Gly Gly Ile Asp Phe Asn Gln Pro Leu 20 25 30Val
Ile Thr Arg Ile Thr Pro Gly Ser Lys Ala Ala Ala Ala Asn Leu 35 40
45Cys Pro Gly Asp Val Ile Leu Ala Ile Asp Gly Phe Gly Thr Glu Ser
50 55 60Met Thr His Ala Asp Ala Gln Asp Arg Ile Lys Ala Ala Ala His
Gln65 70 75 80Leu Cys Leu Lys Ile Asp Arg Gly Glu Thr His Leu Trp
Ser Pro Asn 85 90 95Ser Ser 985PRTArtificial SequenceSynthetic
peptide 9Ile Leu Val Glu Val Gln Leu Ser Gly Gly Ala Pro Trp Gly
Phe Thr1 5 10 15Leu Lys Gly Gly Arg Glu His Gly Glu Pro Leu Val Ile
Thr Lys Ile 20 25 30Glu Glu Gly Ser Lys Ala Ala Ala Val Asp Lys Leu
Leu Ala Gly Asp 35 40 45Glu Ile Val Gly Ile Asn Asp Ile Gly Leu Ser
Gly Phe Arg Gln Glu 50 55 60Ala Ile Cys Leu Val Lys Gly Ser His Lys
Thr Leu Lys Leu Val Val65 70 75 80Lys Arg Asn Ser Ser
8510106PRTArtificial SequenceSynthetic peptide 10Ser Val Gly His
Val Arg Gly Pro Gly Pro Ser Val Gln His Thr Thr1 5 10 15Leu Asn Gly
Asp Ser Leu Thr Ser Gln Leu Thr Leu Leu Gly Gly Asn 20 25 30Ala Arg
Gly Ser Phe Val His Ser Val Lys Pro Gly Ser Leu Ala Glu 35 40 45Lys
Ala Gly Leu Arg Glu Gly His Gln Leu Leu Leu Leu Glu Gly Cys 50 55
60Ile Arg Gly Glu Arg Gln Ser Val Pro Leu Asp Thr Cys Thr Lys Glu65
70 75 80Glu Ala His Trp Thr Ile Gln Arg Cys Ser Gly Pro Val Thr Leu
His 85 90 95Tyr Lys Val Asn His Glu Gly Tyr Arg Lys 100
10511105PRTArtificial SequenceSynthetic peptide 11Arg Arg Pro Ala
Arg Arg Ile Leu Ser Gln Val Thr Met Leu Ala Phe1 5 10 15Gln Gly Asp
Ala Leu Leu Glu Gln Ile Ser Val Ile Gly Gly Asn Leu 20 25 30Thr Gly
Ile Phe Ile His Arg Val Thr Pro Gly Ser Ala Ala Asp Gln 35 40 45Met
Ala Leu Arg Pro Gly Thr Gln Ile Val Met Val Asp Tyr Glu Ala 50 55
60Ser Glu Pro Leu Phe Lys Ala Val Leu Glu Asp Thr Thr Leu Glu Glu65
70 75 80Ala Val Gly Leu Leu Arg Arg Val Asp Gly Phe Cys Cys Leu Ser
Val 85 90 95Lys Val Asn Thr Asp Gly Tyr Lys Arg 100
10512100PRTArtificial SequenceSynthetic peptide 12Ile Leu Ser Gln
Val Thr Met Leu Ala Phe Gln Gly Asp Ala Leu Leu1 5 10 15Glu Gln Ile
Ser Val Ile Gly Gly Asn Leu Thr Gly Ile Phe Ile His 20 25 30Arg Val
Thr Pro Gly Ser Ala Ala Asp Gln Met Ala Leu Arg Pro Gly 35 40 45Thr
Gln Ile Val Met Val Asp Tyr Glu Ala Ser Glu Pro Leu Phe Lys 50 55
60Ala Val Leu Glu Asp Thr Thr Leu Glu Glu Ala Val Gly Leu Leu Arg65
70 75 80Arg Val Asp Gly Phe Cys Cys Leu Ser Val Lys Val Asn Thr Asp
Gly 85 90 95Tyr Lys Arg Leu 1001390PRTArtificial SequenceSynthetic
peptide 13Thr Arg Val Arg Leu Val Gln Phe Gln Lys Asn Thr Asp Glu
Pro Met1 5 10 15Gly Ile Thr Leu Lys Met Asn Glu Leu Asn His Cys Ile
Val Ala Arg 20 25 30Ile Met His Gly Gly Met Ile His Arg Gln Gly Thr
Leu His Val Gly 35 40 45Asp Glu Ile Arg Glu Ile Asn Gly Ile Ser Val
Ala Asn Gln Thr Val 50 55 60Glu Gln Leu Gln Lys Met Leu Arg Glu Met
Arg Gly Ser Ile Thr Phe65 70 75 80Lys Ile Val Pro Ser Tyr Arg Thr
Gln Ser 85 901488PRTArtificial SequenceSynthetic peptide 14Leu Glu
Gln Lys Ala Val Leu Glu Gln Val Gln Leu Asp Ser Pro Leu1 5 10 15Gly
Leu Glu Ile His Thr Thr Ser Asn Cys Gln His Phe Val Ser Gln 20 25
30Val Asp Thr Gln Val Pro Thr Asp Ser Arg Leu Gln Ile Gln Pro Gly
35 40 45Asp Glu Val Val Gln Ile Asn Glu Gln Val Val Val Gly Trp Pro
Arg 50 55 60Lys Asn Met Val Arg Glu Leu Leu Arg Glu Pro Ala Gly Leu
Ser Leu65 70 75 80Val Leu Lys Lys Ile Pro Ile Pro
851592PRTArtificial SequenceSynthetic peptide 15Gln Arg Lys Leu Val
Thr Val Glu Lys Gln Asp Asn Glu Thr Phe Gly1 5 10 15Phe Glu Ile Gln
Ser Tyr Arg Pro Gln Asn Gln Asn Ala Cys Ser Ser 20 25 30Glu Met Phe
Thr Leu Ile Cys Lys Ile Gln Glu Asp Ser Pro Ala His 35 40 45Cys Ala
Gly Leu Gln Ala Gly Asp Val Leu Ala Asn Ile Asn Gly Val 50 55 60Ser
Thr Glu Gly Phe Thr Tyr Lys Gln Val Val Asp Leu Ile Arg Ser65 70 75
80Ser Gly Asn Leu Leu Thr Ile Glu Thr Leu Asn Gly 85
9016109PRTArtificial SequenceSynthetic peptide 16Arg Cys Leu Ile
Gln Thr Lys Gly Gln Arg Ser Met Asp Gly Tyr Pro1 5 10 15Glu Gln Phe
Cys Val Arg Ile Glu Lys Asn Pro Gly Leu Gly Phe Ser 20 25 30Ile Ser
Gly Gly Ile Ser Gly Gln Gly Asn Pro Phe Lys Pro Ser Asp 35 40 45Lys
Gly Ile Phe Val Thr Arg Val Gln Pro Asp Gly Pro Ala Ser Asn 50 55
60Leu Leu Gln Pro Gly Asp Lys Ile Leu Gln Ala Asn Gly His Ser Phe65
70 75 80Val His Met Glu His Glu Lys Ala Val Leu Leu Leu Lys Ser Phe
Gln 85 90 95Asn Thr Val Asp Leu Val Ile Gln Arg Glu Leu Thr Val 100
1051797PRTArtificial SequenceSynthetic peptide 17Pro Thr Ser Pro
Glu Ile Gln Glu Leu Arg Gln Met Leu Gln Ala Pro1 5 10 15His Phe Lys
Gly Ala Thr Ile Lys Arg His Glu Met Thr Gly Asp Ile 20 25 30Leu Val
Ala Arg Ile Ile His Gly Gly Leu Ala Glu Arg Ser Gly Leu 35 40 45Leu
Tyr Ala Gly Asp Lys Leu Val Glu Val Asn Gly Val Ser Val Glu 50 55
60Gly Leu Asp Pro Glu Gln Val Ile His Ile Leu Ala Met Ser Arg Gly65
70 75 80Thr Ile Met Phe Lys Val Val Pro Val Ser Asp Pro Pro Val Asn
Ser 85 90 95Ser 18141PRTArtificial SequenceSynthetic peptide 18Pro
Thr Ser Pro Glu Ile Gln Glu Leu Arg Gln Met Leu Gln Ala Pro1 5 10
15His Phe Lys Ala Leu Leu Ser Ala His Asp Thr Ile Ala Gln Lys Asp
20 25 30Phe Glu Pro Leu Leu Pro Pro Leu Pro Asp Asn Ile Pro Glu Ser
Glu 35 40 45Glu Ala Met Arg Ile Val Cys Leu Val Lys Asn Gln Gln Pro
Leu Gly 50 55 60Ala Thr Ile Lys Arg His Glu Met Thr Gly Asp Ile Leu
Val Ala Arg65 70 75 80Ile Ile His Gly Gly Leu Ala Glu Arg Ser Gly
Leu Leu Tyr Ala Gly 85 90 95Asp Lys Leu Val Glu Val Asn Gly Val Ser
Val Glu Gly Leu Asp Pro 100 105 110Glu Gln Val Ile His Ile Leu Ala
Met Ser Arg Gly Thr Ile Met Phe 115 120 125Lys Val Val Pro Val Ser
Asp Pro Pro Val Asn Ser Ser 130 135 14019101PRTArtificial
SequenceSynthetic peptide 19Ile Gln Val Asn Gly Thr Asp Ala Asp Tyr
Glu Tyr Glu Glu Ile Thr1 5 10 15Leu Glu Arg Gly Asn Ser Gly Leu Gly
Phe Ser Ile Ala Gly Gly Thr 20 25 30Asp Asn Pro His Ile Gly Asp Asp
Ser Ser Ile Phe Ile Thr Lys Ile 35 40 45Ile Thr Gly Gly Ala Ala Ala
Gln Asp Gly Arg Leu Arg Val Asn Asp 50 55 60Cys Ile Leu Gln Val Asn
Glu Val Asp Val Arg Asp Val Thr His Ser65 70 75 80Lys Ala Val Glu
Ala Leu Lys Glu Ala Gly Ser Ile Val Arg Leu Tyr 85 90 95Val Lys Arg
Arg Asn 1002095PRTArtificial SequenceSynthetic peptide 20Ile Gln
Leu Ile Lys Gly Pro Lys Gly Leu Gly Phe Ser Ile Ala Gly1 5 10 15Gly
Val Gly Asn Gln His Ile Pro Gly Asp Asn Ser Ile Tyr Val Thr 20 25
30Lys Ile Ile Glu Gly Gly Ala Ala His Lys Asp Gly Lys Leu Gln Ile
35 40 45Gly Asp Lys Leu Leu Ala Val Asn Asn Val Cys Leu Glu Glu Val
Thr 50 55 60His Glu Glu Ala Val Thr Ala Leu Lys Asn Thr Ser Asp Phe
Val Tyr65 70 75 80Leu Lys Val Ala Lys Pro Thr Ser Met Tyr Met Asn
Asp Gly Asn 85 90 9521203PRTArtificial SequenceSynthetic peptide
21Val Asn Gly Thr Asp Ala Asp Tyr Glu Tyr Glu Glu Ile Thr Leu Glu1
5 10 15Arg Gly Asn Ser Gly Leu Gly Phe Ser Ile Ala Gly Gly Thr Asp
Asn 20 25 30Pro His Ile Gly Asp Asp Ser Ser Ile Phe Ile Thr Lys Ile
Ile Thr 35 40 45Gly Gly Ala Ala Ala Gln Asp Gly Arg Leu Arg Val Asn
Asp Cys Ile 50 55 60Leu Gln Val Asn Glu Val Asp Val Arg Asp Val Thr
His Ser Lys Ala65 70 75 80Val Glu Ala Leu Lys Glu Ala Gly Ser Ile
Val Arg Leu Tyr Val Lys 85 90 95Arg Arg Lys Pro Val Ser Glu Lys Ile
Met Glu Ile Lys Leu Ile Lys 100 105 110Gly Pro Lys Gly Leu Gly Phe
Ser Ile Ala Gly Gly Val Gly Asn Gln 115 120 125His Ile Pro Gly Asp
Asn Ser Ile Tyr Val Thr Lys Ile Ile Glu Gly 130 135 140Gly Ala Ala
His Lys Asp Gly Lys Leu Gln Ile Gly Asp Lys Leu Leu145 150 155
160Ala Val Asn Asn Val Cys Leu Glu Glu Val Thr His Glu Glu Ala Val
165 170 175Thr Ala Leu Lys Asn Thr Ser Asp Phe Val Tyr Leu Lys Val
Ala Lys 180 185 190Pro Thr Ser Met Tyr Met Asn Asp Gly Tyr Ala 195
2002285PRTArtificial SequenceSynthetic peptide 22Ile Leu His Arg
Gly Ser Thr Gly Leu Gly Phe Asn Ile Val Gly Gly1 5 10 15Glu Asp Gly
Glu Gly Ile Phe Ile Ser Phe Ile Leu Ala Gly Gly Pro 20 25 30Ala Asp
Leu Ser Gly Glu Leu Arg Lys Gly Asp Arg Ile Ile Ser Val 35 40 45Asn
Ser Val Asp Leu Arg Ala Ala Ser His Glu Gln Ala Ala Ala Ala 50 55
60Leu Lys Asn Ala Gly Gln Ala Val Thr Ile Val Ala Gln Tyr Arg Pro65
70 75 80Glu Glu Tyr Ser Arg 8523110PRTArtificial SequenceSynthetic
peptide 23Ile Glu Gly Arg Gly Ile Leu Glu Gly Glu Pro Arg Lys Val
Val Leu1 5 10 15His Lys Gly Ser Thr Gly Leu Gly Phe Asn Ile Val Gly
Gly Glu Asp 20 25 30Gly Glu Gly Ile Phe Val Ser Phe Ile Leu Ala Gly
Gly Pro Ala Asp 35 40 45Leu Ser Gly Glu Leu Gln Arg Gly Asp Gln Ile
Leu Ser Val Asn Gly 50 55 60Ile Asp Leu Arg Gly Ala Ser His Glu Gln
Ala Ala Ala Ala Leu Lys65 70 75 80Gly Ala Gly Gln Thr Val Thr Ile
Ile Ala Gln His Gln Pro Glu Asp 85 90 95Tyr Ala Arg Phe Glu Ala Lys
Ile His Asp Leu Asn Ser Ser 100 105 11024101PRTArtificial
SequenceSynthetic peptide 24Ile Ser Tyr Val Asn Gly Thr Glu Ile Glu
Tyr Glu Phe Glu Glu Ile1 5 10 15Thr Leu Glu Arg Gly Asn Ser Gly Leu
Gly Phe Ser Ile Ala Gly Gly 20 25 30Thr Asp Asn Pro His Ile Gly Asp
Asp Pro Gly Ile Phe Ile Thr Lys 35 40 45Ile Ile Pro Gly Gly Ala Ala
Ala Glu Asp Gly Arg Leu Arg Val Asn 50 55 60Asp Cys Ile Leu Arg Val
Asn Glu Val Asp Val Ser Glu Val Ser His65 70 75 80Ser Lys Ala Val
Glu Ala Leu Lys Glu Ala Gly
Ser Ile Val Arg Leu 85 90 95Tyr Val Arg Arg Arg
10025113PRTArtificial SequenceSynthetic peptide 25Ile Pro Ile Leu
Glu Thr Val Val Glu Ile Lys Leu Phe Lys Gly Pro1 5 10 15Lys Gly Leu
Gly Phe Ser Ile Ala Gly Gly Val Gly Asn Gln His Ile 20 25 30Pro Gly
Asp Asn Ser Ile Tyr Val Thr Lys Ile Ile Asp Gly Gly Ala 35 40 45Ala
Gln Lys Asp Gly Arg Leu Gln Val Gly Asp Arg Leu Leu Met Val 50 55
60Asn Asn Tyr Ser Leu Glu Glu Val Thr His Glu Glu Ala Val Ala Ile65
70 75 80Leu Lys Asn Thr Ser Glu Val Val Tyr Leu Lys Val Gly Lys Pro
Thr 85 90 95Thr Ile Tyr Met Thr Asp Pro Tyr Gly Pro Pro Asn Ser Ser
Leu Thr 100 105 110Asp26101PRTArtificial SequenceSynthetic peptide
26Gly Ile Pro Tyr Val Glu Glu Pro Arg His Val Lys Val Gln Lys Gly1
5 10 15Ser Glu Pro Leu Gly Ile Ser Ile Val Ser Gly Glu Lys Gly Gly
Ile 20 25 30Tyr Val Ser Lys Val Thr Val Gly Ser Ile Ala His Gln Ala
Gly Leu 35 40 45Glu Tyr Gly Asp Gln Leu Leu Glu Phe Asn Gly Ile Asn
Leu Arg Ser 50 55 60Ala Thr Glu Gln Gln Ala Arg Leu Ile Ile Gly Gln
Gln Cys Asp Thr65 70 75 80Ile Thr Ile Leu Ala Gln Tyr Asn Pro His
Val His Gln Leu Arg Asn 85 90 95Ser Ser Leu Thr Asp
10027103PRTArtificial SequenceSynthetic peptide 27Gly Ile Leu Ala
Gly Asp Ala Asn Lys Lys Thr Leu Glu Pro Arg Val1 5 10 15Val Phe Ile
Lys Lys Ser Gln Leu Glu Leu Gly Val His Leu Cys Gly 20 25 30Gly Asn
Leu His Gly Val Phe Val Ala Glu Val Glu Asp Asp Ser Pro 35 40 45Ala
Lys Gly Pro Asp Gly Leu Val Pro Gly Asp Leu Ile Leu Glu Tyr 50 55
60Gly Ser Leu Asp Val Arg Asn Lys Thr Val Glu Glu Val Tyr Val Glu65
70 75 80Met Leu Lys Pro Arg Asp Gly Val Arg Leu Lys Val Gln Tyr Arg
Pro 85 90 95Glu Glu Phe Ile Val Thr Asp 1002893PRTArtificial
SequenceSynthetic peptide 28Leu Asn Ile Val Thr Val Thr Leu Asn Met
Glu Arg His His Phe Leu1 5 10 15Gly Ile Ser Ile Val Gly Gln Ser Asn
Asp Arg Gly Asp Gly Gly Ile 20 25 30Tyr Ile Gly Ser Ile Met Lys Gly
Gly Ala Val Ala Ala Asp Gly Arg 35 40 45Ile Glu Pro Gly Asp Met Leu
Leu Gln Val Asn Asp Val Asn Phe Glu 50 55 60Asn Met Ser Asn Asp Asp
Ala Val Arg Val Leu Arg Glu Ile Val Ser65 70 75 80Gln Thr Gly Pro
Ile Ser Leu Thr Val Ala Lys Cys Trp 85 9029100PRTArtificial
SequenceSynthetic peptide 29Leu Asn Ile Ile Thr Val Thr Leu Asn Met
Glu Lys Tyr Asn Phe Leu1 5 10 15Gly Ile Ser Ile Val Gly Gln Ser Asn
Glu Arg Gly Asp Gly Gly Ile 20 25 30Tyr Ile Gly Ser Ile Met Lys Gly
Gly Ala Val Ala Ala Asp Gly Arg 35 40 45Ile Glu Pro Gly Asp Met Leu
Leu Gln Val Asn Asp Met Asn Phe Glu 50 55 60Asn Met Ser Asn Asp Asp
Ala Val Arg Val Leu Arg Asp Ile Val His65 70 75 80Lys Pro Gly Pro
Ile Val Leu Thr Val Ala Lys Cys Trp Asp Pro Ser 85 90 95Pro Gln Asn
Ser 1003095PRTArtificial SequenceSynthetic peptide 30Ile Ile Thr
Val Thr Leu Asn Met Glu Lys Tyr Asn Phe Leu Gly Ile1 5 10 15Ser Ile
Val Gly Gln Ser Asn Glu Arg Gly Asp Gly Gly Ile Tyr Ile 20 25 30Gly
Ser Ile Met Lys Gly Gly Ala Val Ala Ala Asp Gly Arg Ile Glu 35 40
45Pro Gly Asp Met Leu Leu Gln Val Asn Glu Ile Asn Phe Glu Asn Met
50 55 60Ser Asn Asp Asp Ala Val Arg Val Leu Arg Glu Ile Val His Lys
Pro65 70 75 80Gly Pro Ile Thr Leu Thr Val Ala Lys Cys Trp Asp Pro
Ser Pro 85 90 953198PRTArtificial SequenceSynthetic peptide 31Gln
Gln Arg Glu Leu Arg Pro Arg Leu Cys Thr Met Lys Lys Gly Pro1 5 10
15Ser Gly Tyr Gly Phe Asn Leu His Ser Asp Lys Ser Lys Pro Gly Gln
20 25 30Phe Ile Arg Ser Val Asp Pro Asp Ser Pro Ala Glu Ala Ser Gly
Leu 35 40 45Arg Ala Gln Asp Arg Ile Val Glu Val Asn Gly Val Cys Met
Glu Gly 50 55 60Lys Gln His Gly Asp Val Val Ser Ala Ile Arg Ala Gly
Gly Asp Glu65 70 75 80Thr Lys Leu Leu Val Val Asp Arg Glu Thr Asp
Glu Phe Phe Lys Asn 85 90 95Ser Ser32155PRTArtificial
SequenceSynthetic peptide 32Gly Ile Gln Met Ser Ala Asp Ala Ala Ala
Gly Ala Pro Leu Pro Arg1 5 10 15Leu Cys Cys Leu Glu Lys Gly Pro Asn
Gly Tyr Gly Phe His Leu His 20 25 30Gly Glu Lys Gly Lys Leu Gly Gln
Tyr Ile Arg Leu Val Glu Pro Gly 35 40 45Ser Pro Ala Glu Lys Ala Gly
Leu Leu Ala Gly Asp Arg Leu Val Glu 50 55 60Val Asn Gly Glu Asn Val
Glu Lys Glu Thr His Gln Gln Val Val Ser65 70 75 80Arg Ile Arg Ala
Ala Leu Asn Ala Val Arg Leu Leu Val Val Asp Pro 85 90 95Glu Thr Asp
Glu Gln Leu Gln Lys Leu Gly Val Gln Val Arg Glu Glu 100 105 110Leu
Leu Arg Ala Gln Glu Ala Pro Gly Gln Ala Glu Pro Pro Ala Ala 115 120
125Ala Glu Val Gln Gly Ala Gly Asn Glu Asn Glu Pro Arg Glu Ala Asp
130 135 140Lys Ser His Pro Glu Gln Arg Glu Leu Arg Asn145 150
15533243PRTArtificial SequenceSynthetic peptide 33Gly Ile Gln Met
Ser Ala Asp Ala Ala Ala Gly Ala Pro Leu Pro Arg1 5 10 15Leu Cys Cys
Leu Glu Lys Gly Pro Asn Gly Tyr Gly Phe His Leu His 20 25 30Gly Glu
Lys Gly Lys Leu Gly Gln Tyr Ile Arg Leu Val Glu Pro Gly 35 40 45Ser
Pro Ala Glu Lys Ala Gly Leu Leu Ala Gly Asp Arg Leu Val Glu 50 55
60Val Asn Gly Glu Asn Val Glu Lys Glu Thr His Gln Gln Val Val Ser65
70 75 80Arg Ile Arg Ala Ala Leu Asn Ala Val Arg Leu Leu Val Val Asp
Pro 85 90 95Glu Thr Asp Glu Gln Leu Gln Lys Leu Gly Val Gln Val Arg
Glu Glu 100 105 110Leu Leu Arg Ala Gln Glu Ala Pro Gly Gln Ala Glu
Pro Pro Ala Ala 115 120 125Ala Glu Val Gln Gly Ala Gly Asn Glu Asn
Glu Pro Arg Glu Ala Asp 130 135 140Lys Ser His Pro Glu Gln Arg Glu
Leu Arg Pro Arg Leu Cys Thr Met145 150 155 160Lys Lys Gly Pro Ser
Gly Tyr Gly Phe Asn Leu His Ser Asp Lys Ser 165 170 175Lys Pro Gly
Gln Phe Ile Arg Ser Val Asp Pro Asp Ser Pro Ala Glu 180 185 190Ala
Ser Gly Leu Arg Ala Gln Asp Arg Ile Val Glu Val Asn Gly Val 195 200
205Cys Met Glu Gly Lys Gln His Gly Asp Val Val Ser Ala Ile Arg Ala
210 215 220Gly Gly Asp Glu Thr Lys Leu Leu Val Val Asp Arg Glu Thr
Asp Glu225 230 235 240Phe Phe Lys34155PRTArtificial
SequenceSynthetic peptide 34Gln Met Ser Ala Asp Ala Ala Ala Gly Ala
Pro Leu Pro Arg Leu Cys1 5 10 15Cys Leu Glu Lys Gly Pro Asn Gly Tyr
Gly Phe His Leu His Gly Glu 20 25 30Lys Gly Lys Leu Gly Gln Tyr Ile
Arg Leu Val Glu Pro Gly Ser Pro 35 40 45Ala Glu Lys Ala Gly Leu Leu
Ala Gly Asp Arg Leu Val Glu Val Asn 50 55 60Gly Glu Asn Val Glu Lys
Glu Thr His Gln Gln Val Val Ser Arg Ile65 70 75 80Arg Ala Ala Leu
Asn Ala Val Arg Leu Leu Val Val Asp Pro Glu Thr 85 90 95Asp Glu Gln
Leu Gln Lys Leu Gly Val Gln Val Arg Glu Glu Leu Leu 100 105 110Arg
Ala Gln Glu Ala Pro Gly Gln Ala Glu Pro Pro Ala Ala Ala Glu 115 120
125Val Gln Gly Ala Gly Asn Glu Asn Glu Pro Arg Glu Ala Asp Lys Ser
130 135 140His Pro Glu Gln Arg Glu Leu Arg Asn Ser Ser145 150
1553598PRTArtificial SequenceSynthetic peptide 35Leu Thr Thr Gln
Gln Ile Asp Leu Gln Gly Pro Gly Pro Trp Gly Phe1 5 10 15Arg Leu Val
Gly Gly Lys Asp Phe Glu Gln Pro Leu Ala Ile Ser Arg 20 25 30Val Thr
Pro Gly Ser Lys Ala Ala Leu Ala Asn Leu Cys Ile Gly Asp 35 40 45Val
Ile Thr Ala Ile Asp Gly Glu Asn Thr Ser Asn Met Thr His Leu 50 55
60Glu Ala Gln Asn Arg Ile Lys Gly Cys Thr Asp Asn Leu Thr Leu Thr65
70 75 80Val Ala Arg Ser Glu His Lys Val Trp Ser Pro Leu Val Thr Asn
Ser 85 90 95Ser Trp3689PRTArtificial SequenceSynthetic peptide
36Ile Phe Met Asp Ser Phe Lys Val Val Leu Glu Gly Pro Ala Pro Trp1
5 10 15Gly Phe Arg Leu Gln Gly Gly Lys Asp Phe Asn Val Pro Leu Ser
Ile 20 25 30Ser Arg Leu Thr Pro Gly Gly Lys Ala Ala Gln Ala Gly Val
Ala Val 35 40 45Gly Asp Trp Val Leu Ser Ile Asp Gly Glu Asn Ala Gly
Ser Leu Thr 50 55 60His Ile Glu Ala Gln Asn Lys Ile Arg Ala Cys Gly
Glu Arg Leu Ser65 70 75 80Leu Gly Leu Ser Arg Ala Gln Pro Val
8537100PRTArtificial SequenceSynthetic peptide 37Gln Gly His Glu
Leu Ala Lys Gln Glu Ile Arg Val Arg Val Glu Lys1 5 10 15Asp Pro Glu
Leu Gly Phe Ser Ile Ser Gly Gly Val Gly Gly Arg Gly 20 25 30Asn Pro
Phe Arg Pro Asp Asp Asp Gly Ile Phe Val Thr Arg Val Gln 35 40 45Pro
Glu Gly Pro Ala Ser Lys Leu Leu Gln Pro Gly Asp Lys Ile Ile 50 55
60Gln Ala Asn Gly Tyr Ser Phe Ile Asn Ile Glu His Gly Gln Ala Val65
70 75 80Ser Leu Leu Lys Thr Phe Gln Asn Thr Val Glu Leu Ile Ile Val
Arg 85 90 95Glu Val Ser Ser 10038107PRTArtificial SequenceSynthetic
peptide 38Lys Asn Pro Ser Gly Glu Leu Lys Thr Val Thr Leu Ser Lys
Met Lys1 5 10 15Gln Ser Leu Gly Ile Ser Ile Ser Gly Gly Ile Glu Ser
Lys Val Gln 20 25 30Pro Met Val Lys Ile Glu Lys Ile Phe Pro Gly Gly
Ala Ala Phe Leu 35 40 45Ser Gly Ala Leu Gln Ala Gly Phe Glu Leu Val
Ala Val Asp Gly Glu 50 55 60Asn Leu Glu Gln Val Thr His Gln Arg Ala
Val Asp Thr Ile Arg Arg65 70 75 80Ala Tyr Arg Asn Lys Ala Arg Glu
Pro Met Glu Leu Val Val Arg Val 85 90 95Pro Gly Pro Ser Pro Arg Pro
Ser Pro Ser Asp 100 1053997PRTArtificial SequenceSynthetic peptide
39Glu Gly His Ser His Pro Arg Val Val Glu Leu Pro Lys Thr Glu Glu1
5 10 15Gly Leu Gly Phe Asn Ile Met Gly Gly Lys Glu Gln Asn Ser Pro
Ile 20 25 30Tyr Ile Ser Arg Ile Ile Pro Gly Gly Ile Ala Asp Arg His
Gly Gly 35 40 45Leu Lys Arg Gly Asp Gln Leu Leu Ser Val Asn Gly Val
Ser Val Glu 50 55 60Gly Glu His His Glu Lys Ala Val Glu Leu Leu Lys
Ala Ala Gln Gly65 70 75 80Lys Val Lys Leu Val Val Arg Tyr Thr Pro
Lys Val Leu Glu Glu Met 85 90 95Glu 4088PRTArtificial
SequenceSynthetic peptide 40Pro Gly Ala Pro Tyr Ala Arg Lys Thr Phe
Thr Ile Val Gly Asp Ala1 5 10 15Val Gly Trp Gly Phe Val Val Arg Gly
Ser Lys Pro Cys His Ile Gln 20 25 30Ala Val Asp Pro Ser Gly Pro Ala
Ala Ala Ala Gly Met Lys Val Cys 35 40 45Gln Phe Val Val Ser Val Asn
Gly Leu Asn Val Leu His Val Asp Tyr 50 55 60Arg Thr Val Ser Asn Leu
Ile Leu Thr Gly Pro Arg Thr Ile Val Met65 70 75 80Glu Val Met Glu
Glu Leu Glu Cys 854197PRTArtificial SequenceSynthetic peptide 41Gly
Gln Tyr Gly Gly Glu Thr Val Lys Ile Val Arg Ile Glu Lys Ala1 5 10
15Arg Asp Ile Pro Leu Gly Ala Thr Val Arg Asn Glu Met Asp Ser Val
20 25 30Ile Ile Ser Arg Ile Val Lys Gly Gly Ala Ala Glu Lys Ser Gly
Leu 35 40 45Leu His Glu Gly Asp Glu Val Leu Glu Ile Asn Gly Ile Glu
Ile Arg 50 55 60Gly Lys Asp Val Asn Glu Val Phe Asp Leu Leu Ser Asp
Met His Gly65 70 75 80Thr Leu Thr Phe Val Leu Ile Pro Ser Gln Gln
Ile Lys Pro Pro Pro 85 90 95Ala42104PRTArtificial SequenceSynthetic
peptide 42Lys Pro Ser Gln Ala Ser Gly His Phe Ser Val Glu Leu Val
Arg Gly1 5 10 15Tyr Ala Gly Phe Gly Leu Thr Leu Gly Gly Gly Arg Asp
Val Ala Gly 20 25 30Asp Thr Pro Leu Ala Val Arg Gly Leu Leu Lys Asp
Gly Pro Ala Gln 35 40 45Arg Cys Gly Arg Leu Glu Val Gly Asp Leu Val
Leu His Ile Asn Gly 50 55 60Glu Ser Thr Gln Gly Leu Thr His Ala Gln
Ala Val Glu Arg Ile Arg65 70 75 80Ala Gly Gly Pro Gln Leu His Leu
Val Ile Arg Arg Pro Leu Glu Thr 85 90 95His Pro Gly Lys Pro Arg Gly
Val 10043101PRTArtificial SequenceSynthetic peptide 43Pro Val Met
Ser Gln Cys Ala Cys Leu Glu Glu Val His Leu Pro Asn1 5 10 15Ile Lys
Pro Gly Glu Gly Leu Gly Met Tyr Ile Lys Ser Thr Tyr Asp 20 25 30Gly
Leu His Val Ile Thr Gly Thr Thr Glu Asn Ser Pro Ala Asp Arg 35 40
45Ser Gln Lys Ile His Ala Gly Asp Glu Val Thr Gln Val Asn Gln Gln
50 55 60Thr Val Val Gly Trp Gln Leu Lys Asn Leu Val Lys Lys Leu Arg
Glu65 70 75 80Asn Pro Thr Gly Val Val Leu Leu Leu Lys Lys Arg Pro
Thr Gly Ser 85 90 95Phe Asn Phe Thr Pro 1004497PRTArtificial
SequenceSynthetic peptide 44Ile Asp Asp Glu Glu Asp Ser Val Lys Ile
Ile Arg Leu Val Lys Asn1 5 10 15Arg Glu Pro Leu Gly Ala Thr Ile Lys
Lys Asp Glu Gln Thr Gly Ala 20 25 30Ile Ile Val Ala Arg Ile Met Arg
Gly Gly Ala Ala Asp Arg Ser Gly 35 40 45Leu Ile His Val Gly Asp Glu
Leu Arg Glu Val Asn Gly Ile Pro Val 50 55 60Glu Asp Lys Arg Pro Glu
Glu Ile Ile Gln Ile Leu Ala Gln Ser Gln65 70 75 80Gly Ala Ile Thr
Phe Lys Ile Ile Pro Gly Ser Lys Glu Glu Thr Pro 85 90
95Ser45452PRTArtificial SequenceSynthetic peptide 45Met Gly Ser Ser
Gln Ser Val Glu Ile Pro Gly Gly Gly Thr Glu Gly1 5 10 15Tyr His Val
Leu Arg Val Gln Glu Asn Ser Pro Gly His Arg Ala Gly 20 25 30Leu Glu
Pro Phe Phe Asp Phe Ile Val Ser Ile Asn Gly Ser Arg Leu 35 40 45Asn
Lys Asp Asn Asp Thr Leu Lys Asp Leu Leu Lys Ala Asn Val Glu 50 55
60Lys Pro Val Lys Met Leu Ile Tyr Ser Ser Lys Thr Leu Glu Leu Arg65
70 75 80Glu Thr Ser Val Thr Pro Ser Asn Leu Trp Gly Gly Gln Gly Leu
Leu 85 90 95Gly Val Ser Ile Arg Phe Cys Ser Phe Asp Gly Ala Asn Glu
Asn Val 100 105
110Trp His Val Leu Glu Val Glu Ser Asn Ser Pro Ala Ala Leu Ala Gly
115 120 125Leu Arg Pro His Ser Asp Tyr Ile Ile Gly Ala Asp Thr Val
Met Asn 130 135 140Glu Ser Glu Asp Leu Phe Ser Leu Ile Glu Thr His
Glu Ala Lys Pro145 150 155 160Leu Lys Leu Tyr Val Tyr Asn Thr Asp
Thr Asp Asn Cys Arg Glu Val 165 170 175Ile Ile Thr Pro Asn Ser Ala
Trp Gly Gly Glu Gly Ser Leu Gly Cys 180 185 190Gly Ile Gly Tyr Gly
Tyr Leu His Arg Ile Pro Thr Arg Pro Phe Glu 195 200 205Glu Gly Lys
Lys Ile Ser Leu Pro Gly Gln Met Ala Gly Thr Pro Ile 210 215 220Thr
Pro Leu Lys Asp Gly Phe Thr Glu Val Gln Leu Ser Ser Val Asn225 230
235 240Pro Pro Ser Leu Ser Pro Pro Gly Thr Thr Gly Ile Glu Gln Ser
Leu 245 250 255Thr Gly Leu Ser Ile Ser Ser Thr Pro Pro Ala Val Ser
Ser Val Leu 260 265 270Ser Thr Gly Val Pro Thr Val Pro Leu Leu Pro
Pro Gln Val Asn Gln 275 280 285Ser Leu Thr Ser Val Pro Pro Met Asn
Pro Ala Thr Thr Leu Pro Gly 290 295 300Leu Met Pro Leu Pro Ala Gly
Leu Pro Asn Leu Pro Asn Leu Asn Leu305 310 315 320Asn Leu Pro Ala
Pro His Ile Met Pro Gly Val Gly Leu Pro Glu Leu 325 330 335Val Asn
Pro Gly Leu Pro Pro Leu Pro Ser Met Pro Pro Arg Asn Leu 340 345
350Pro Gly Ile Ala Pro Leu Pro Leu Pro Ser Glu Phe Leu Pro Ser Phe
355 360 365Pro Leu Val Pro Glu Ser Ser Ser Ala Ala Ser Ser Gly Glu
Leu Leu 370 375 380Ser Ser Leu Pro Pro Thr Ser Asn Ala Pro Ser Asp
Pro Ala Thr Thr385 390 395 400Thr Ala Lys Ala Asp Ala Ala Ser Ser
Leu Thr Val Asp Val Thr Pro 405 410 415Pro Thr Ala Lys Ala Pro Thr
Thr Val Glu Asp Arg Val Gly Asp Ser 420 425 430Thr Pro Val Ser Glu
Lys Pro Val Ser Ala Ala Val Asp Ala Asn Ala 435 440 445Ser Glu Ser
Pro 4504697PRTArtificial SequenceSynthetic peptide 46Asn Glu Asn
Val Trp His Val Leu Glu Val Glu Ser Asn Ser Pro Ala1 5 10 15Ala Leu
Ala Gly Leu Arg Pro His Ser Asp Tyr Ile Ile Gly Ala Asp 20 25 30Thr
Val Met Asn Glu Ser Glu Asp Leu Phe Ser Leu Ile Glu Thr His 35 40
45Glu Ala Lys Pro Leu Lys Leu Tyr Val Tyr Asn Thr Asp Thr Asp Asn
50 55 60Cys Arg Glu Val Ile Ile Thr Pro Asn Ser Ala Trp Gly Gly Glu
Gly65 70 75 80Ser Leu Gly Cys Gly Ile Gly Tyr Gly Tyr Leu His Arg
Ile Pro Thr 85 90 95Arg47110PRTArtificial SequenceSynthetic peptide
47Met Gly Ser Ser Gln Ser Val Glu Ile Pro Gly Gly Gly Thr Glu Gly1
5 10 15Tyr His Val Leu Arg Val Gln Glu Asn Ser Pro Gly His Arg Ala
Gly 20 25 30Leu Glu Pro Phe Phe Asp Phe Ile Val Ser Ile Asn Gly Ser
Arg Leu 35 40 45Asn Lys Asp Asn Asp Thr Leu Lys Asp Leu Leu Lys Ala
Asn Val Glu 50 55 60Lys Pro Val Lys Met Leu Ile Tyr Ser Ser Lys Thr
Leu Glu Leu Arg65 70 75 80Glu Thr Ser Val Thr Pro Ser Asn Leu Trp
Gly Gly Gln Gly Leu Leu 85 90 95Gly Val Ser Ile Arg Phe Cys Ser Phe
Asp Gly Ala Asn Glu 100 105 1104899PRTArtificial SequenceSynthetic
peptide 48Arg Ala Ser Glu Gln Val Trp His Val Leu Asp Val Glu Pro
Ser Ser1 5 10 15Pro Ala Ala Leu Ala Gly Leu Arg Pro Tyr Thr Asp Tyr
Val Val Gly 20 25 30Ser Asp Gln Ile Leu Gln Glu Ser Glu Asp Phe Phe
Thr Leu Ile Glu 35 40 45Ser His Glu Gly Lys Pro Leu Lys Leu Met Val
Tyr Asn Ser Lys Ser 50 55 60Asp Ser Cys Arg Glu Val Thr Val Thr Pro
Asn Ala Ala Trp Gly Gly65 70 75 80Glu Gly Ser Leu Gly Cys Gly Ile
Gly Tyr Gly Tyr Leu His Arg Ile 85 90 95Pro Thr
Gln49110PRTArtificial SequenceSynthetic peptide 49Met Gly Leu Gly
Val Ser Ala Glu Gln Pro Ala Gly Gly Ala Glu Gly1 5 10 15Phe His Leu
His Gly Val Gln Glu Asn Ser Pro Ala Gln Gln Ala Gly 20 25 30Leu Glu
Pro Tyr Phe Asp Phe Ile Ile Thr Ile Gly His Ser Arg Leu 35 40 45Asn
Lys Glu Asn Asp Thr Leu Lys Ala Leu Leu Lys Ala Asn Val Glu 50 55
60Lys Pro Val Lys Leu Glu Val Phe Asn Met Lys Thr Met Arg Val Arg65
70 75 80Glu Val Glu Val Val Pro Ser Asn Met Trp Gly Gly Gln Gly Leu
Leu 85 90 95Gly Ala Ser Val Arg Phe Cys Ser Phe Arg Arg Ala Ser Glu
100 105 11050440PRTArtificial SequenceSynthetic peptide 50Met Gly
Leu Gly Val Ser Ala Glu Gln Pro Ala Gly Gly Ala Glu Gly1 5 10 15Phe
His Leu His Gly Val Gln Glu Asn Ser Pro Ala Gln Gln Ala Gly 20 25
30Leu Glu Pro Tyr Phe Asp Phe Ile Ile Thr Ile Gly His Ser Arg Leu
35 40 45Asn Lys Glu Asn Asp Thr Leu Lys Ala Leu Leu Lys Ala Asn Val
Glu 50 55 60Lys Pro Val Lys Leu Glu Val Phe Asn Met Lys Thr Met Arg
Val Arg65 70 75 80Glu Val Glu Val Val Pro Ser Asn Met Trp Gly Gly
Gln Gly Leu Leu 85 90 95Gly Ala Ser Val Arg Phe Cys Ser Phe Arg Arg
Ala Ser Glu Gln Val 100 105 110Trp His Val Leu Asp Val Glu Pro Ser
Ser Pro Ala Ala Leu Ala Gly 115 120 125Leu Arg Pro Tyr Thr Asp Tyr
Val Val Gly Ser Asp Gln Ile Leu Gln 130 135 140Glu Ser Glu Asp Phe
Phe Thr Leu Ile Glu Ser His Glu Gly Lys Pro145 150 155 160Leu Lys
Leu Met Val Tyr Asn Ser Lys Ser Asp Ser Cys Arg Glu Val 165 170
175Thr Val Thr Pro Asn Ala Ala Trp Gly Gly Glu Gly Ser Leu Gly Cys
180 185 190Gly Ile Gly Tyr Gly Tyr Leu His Arg Ile Pro Thr Gln Pro
Pro Ser 195 200 205Tyr His Lys Lys Pro Pro Gly Thr Pro Pro Pro Ser
Ala Leu Pro Leu 210 215 220Gly Ala Pro Pro Pro Asp Ala Leu Pro Pro
Gly Pro Thr Pro Glu Asp225 230 235 240Ser Pro Ser Leu Glu Thr Gly
Ser Arg Gln Ser Asp Tyr Met Glu Ala 245 250 255Leu Leu Gln Ala Pro
Gly Ser Ser Met Glu Asp Pro Leu Pro Gly Pro 260 265 270Gly Ser Pro
Ser His Ser Ala Pro Asp Pro Asp Gly Leu Pro His Phe 275 280 285Met
Glu Thr Pro Leu Gln Pro Pro Pro Pro Val Gln Arg Val Met Asp 290 295
300Pro Gly Phe Leu Asp Val Ser Gly Ile Ser Leu Leu Asp Asn Ser
Asn305 310 315 320Ala Ser Val Trp Pro Ser Leu Pro Ser Ser Thr Glu
Leu Thr Thr Thr 325 330 335Ala Val Ser Thr Ser Gly Pro Glu Asp Ile
Cys Ser Ser Ser Ser Ser 340 345 350His Glu Arg Gly Gly Glu Ala Thr
Trp Ser Gly Ser Glu Phe Glu Val 355 360 365Ser Phe Leu Asp Ser Pro
Gly Ala Gln Ala Gln Ala Asp His Leu Pro 370 375 380Gln Leu Thr Leu
Pro Asp Ser Leu Thr Ser Ala Ala Ser Pro Glu Asp385 390 395 400Gly
Leu Ser Ala Glu Leu Leu Glu Ala Gln Ala Glu Glu Glu Pro Ala 405 410
415Ser Thr Glu Gly Leu Asp Thr Gly Thr Glu Ala Glu Gly Leu Asp Ser
420 425 430Gln Ala Gln Ile Ser Thr Thr Glu 435 4405190PRTArtificial
SequenceSynthetic peptide 51Ile Tyr Thr Val Glu Leu Lys Arg Tyr Gly
Gly Pro Leu Gly Ile Thr1 5 10 15Ile Ser Gly Thr Glu Glu Pro Phe Asp
Pro Ile Ile Ile Ser Ser Leu 20 25 30Thr Lys Gly Gly Leu Ala Glu Arg
Thr Gly Ala Ile His Ile Gly Asp 35 40 45Arg Ile Leu Ala Ile Asn Ser
Ser Ser Leu Lys Gly Lys Pro Leu Ser 50 55 60Glu Ala Ile His Leu Leu
Gln Met Ala Gly Glu Thr Val Thr Leu Lys65 70 75 80Ile Lys Lys Gln
Thr Asp Ala Gln Ser Ala 85 905283PRTArtificial SequenceSynthetic
peptide 52Val Val Glu Leu Met Lys Lys Glu Gly Thr Thr Leu Gly Leu
Thr Val1 5 10 15Ser Gly Gly Ile Asp Lys Asp Gly Lys Pro Arg Val Ser
Asn Leu Arg 20 25 30Gln Gly Gly Ile Ala Ala Arg Ser Asp Gln Leu Asp
Val Gly Asp Tyr 35 40 45Ile Lys Ala Val Asn Gly Ile Asn Leu Ala Lys
Phe Arg His Asp Glu 50 55 60Ile Ile Ser Leu Leu Lys Asn Val Gly Glu
Arg Val Val Leu Glu Val65 70 75 80Glu Tyr Glu53106PRTArtificial
SequenceSynthetic peptide 53His Val Ala Thr Ala Ser Gly Pro Leu Leu
Val Glu Val Ala Lys Thr1 5 10 15Pro Gly Ala Ser Leu Gly Val Ala Leu
Thr Thr Ser Met Cys Cys Asn 20 25 30Lys Gln Val Ile Val Ile Asp Lys
Ile Lys Ser Ala Ser Ile Ala Asp 35 40 45Arg Cys Gly Ala Leu His Val
Gly Asp His Ile Leu Ser Ile Asp Gly 50 55 60Thr Ser Met Glu Tyr Cys
Thr Leu Ala Glu Ala Thr Gln Phe Leu Ala65 70 75 80Asn Thr Thr Asp
Gln Val Lys Leu Glu Ile Leu Pro His His Gln Thr 85 90 95Arg Leu Ala
Leu Lys Gly Pro Asn Ser Ser 100 1055495PRTArtificial
SequenceSynthetic peptide 54Ile Met Ser Pro Thr Pro Val Glu Leu His
Lys Val Thr Leu Tyr Lys1 5 10 15Asp Ser Asp Met Glu Asp Phe Gly Phe
Ser Val Ala Asp Gly Leu Leu 20 25 30Glu Lys Gly Val Tyr Val Lys Asn
Ile Arg Pro Ala Gly Pro Gly Asp 35 40 45Leu Gly Gly Leu Lys Pro Tyr
Asp Arg Leu Leu Gln Val Asn His Val 50 55 60Arg Thr Arg Asp Phe Asp
Cys Cys Leu Val Val Pro Leu Ile Ala Glu65 70 75 80Ser Gly Asn Lys
Leu Asp Leu Val Ile Ser Arg Asn Pro Leu Ala 85 90
955590PRTArtificial SequenceSynthetic peptide 55Ile Tyr Thr Val Glu
Leu Lys Arg Tyr Gly Gly Pro Leu Gly Ile Thr1 5 10 15Ile Ser Gly Thr
Glu Glu Pro Phe Asp Pro Ile Ile Ile Ser Ser Leu 20 25 30Thr Lys Gly
Gly Leu Ala Glu Arg Thr Gly Ala Ile His Ile Gly Asp 35 40 45Arg Ile
Leu Ala Ile Asn Ser Ser Ser Leu Lys Gly Lys Pro Leu Ser 50 55 60Glu
Ala Ile His Leu Leu Gln Met Ala Gly Glu Thr Val Thr Leu Lys65 70 75
80Ile Lys Lys Gln Thr Asp Ala Gln Ser Ala 85 905695PRTArtificial
SequenceSynthetic peptide 56Ile Met Ser Pro Thr Pro Val Glu Leu His
Lys Val Thr Leu Tyr Lys1 5 10 15Asp Ser Asp Met Glu Asp Phe Gly Phe
Ser Val Ala Asp Gly Leu Leu 20 25 30Glu Lys Gly Val Tyr Val Lys Asn
Ile Arg Pro Ala Gly Pro Gly Asp 35 40 45Leu Gly Gly Leu Lys Pro Tyr
Asp Arg Leu Leu Gln Val Asn His Val 50 55 60Arg Thr Arg Asp Phe Asp
Cys Cys Leu Val Val Pro Leu Ile Ala Glu65 70 75 80Ser Gly Asn Lys
Leu Asp Leu Val Ile Ser Arg Asn Pro Leu Ala 85 90
955788PRTArtificial SequenceSynthetic peptide 57Ser Arg Gly Cys Glu
Thr Arg Glu Leu Ala Leu Pro Arg Asp Gly Gln1 5 10 15Gly Arg Leu Gly
Phe Glu Val Asp Ala Glu Gly Phe Val Thr His Val 20 25 30Glu Arg Phe
Thr Phe Ala Glu Thr Ala Gly Leu Arg Pro Gly Ala Arg 35 40 45Leu Leu
Arg Val Cys Gly Gln Thr Leu Pro Ser Leu Arg Pro Glu Ala 50 55 60Ala
Ala Gln Leu Leu Arg Ser Ala Pro Lys Val Cys Val Thr Val Leu65 70 75
80Pro Pro Asp Glu Ser Gly Arg Pro 8558108PRTArtificial
SequenceSynthetic peptide 58Cys Ser Val Met Ile Phe Glu Val Val Glu
Gln Ala Gly Ala Ile Ile1 5 10 15Leu Glu Asp Gly Gln Glu Leu Asp Ser
Trp Tyr Val Ile Leu Asn Gly 20 25 30Thr Val Glu Ile Ser His Pro Asp
Gly Lys Val Glu Asn Leu Phe Met 35 40 45Gly Asn Ser Phe Gly Ile Thr
Pro Thr Leu Asp Lys Gln Tyr Met His 50 55 60Gly Ile Val Arg Thr Lys
Val Asp Asp Cys Gln Phe Val Cys Ile Ala65 70 75 80Gln Gln Asp Tyr
Trp Arg Ile Leu Asn His Val Glu Lys Asn Thr His 85 90 95Lys Val Glu
Glu Glu Gly Glu Ile Val Met Val His 100 1055982PRTArtificial
SequenceSynthetic peptide 59Pro Arg Glu Thr Val Lys Ile Pro Asp Ser
Ala Asp Gly Leu Gly Phe1 5 10 15Gln Ile Arg Gly Phe Gly Pro Ser Val
Val His Ala Val Gly Arg Gly 20 25 30Thr Val Ala Ala Ala Ala Gly Leu
His Pro Gly Gln Cys Ile Ile Lys 35 40 45Val Asn Gly Ile Asn Val Ser
Lys Glu Thr His Ala Ser Val Ile Ala 50 55 60His Val Thr Ala Cys Arg
Lys Tyr Arg Arg Pro Thr Lys Gln Asp Ser65 70 75 80Ile
Gln6089PRTArtificial SequenceSynthetic peptide 60Leu Glu Asn Val
Ile Ala Lys Ser Leu Leu Ile Lys Ser Asn Glu Gly1 5 10 15Ser Tyr Gly
Phe Gly Leu Glu Asp Lys Asn Lys Val Pro Ile Ile Lys 20 25 30Leu Val
Glu Lys Gly Ser Asn Ala Glu Met Ala Gly Met Glu Val Gly 35 40 45Lys
Lys Ile Phe Ala Ile Asn Gly Asp Leu Val Phe Met Arg Pro Phe 50 55
60Asn Glu Val Asp Cys Phe Leu Lys Ser Cys Leu Asn Ser Arg Lys Pro65
70 75 80Leu Arg Val Leu Val Ser Thr Lys Pro 8561100PRTArtificial
SequenceSynthetic peptide 61Glu Asp Phe Cys Tyr Val Phe Thr Val Glu
Leu Glu Arg Gly Pro Ser1 5 10 15Gly Leu Gly Met Gly Leu Ile Asp Gly
Met His Thr His Leu Gly Ala 20 25 30Pro Gly Leu Tyr Ile Gln Thr Leu
Leu Pro Gly Ser Pro Ala Ala Ala 35 40 45Asp Gly Arg Leu Ser Leu Gly
Asp Arg Ile Leu Glu Val Asn Gly Ser 50 55 60Ser Leu Leu Gly Leu Gly
Tyr Leu Arg Ala Val Asp Leu Ile Arg His65 70 75 80Gly Gly Lys Lys
Met Arg Phe Leu Val Ala Lys Ser Asp Val Glu Thr 85 90 95Ala Lys Lys
Ile 1006291PRTArtificial SequenceSynthetic peptide 62Ile Tyr Leu
Glu Ala Phe Leu Glu Gly Gly Ala Pro Trp Gly Phe Thr1 5 10 15Leu Lys
Gly Gly Leu Glu His Gly Glu Pro Leu Ile Ile Ser Lys Val 20 25 30Glu
Glu Gly Gly Lys Ala Asp Thr Leu Ser Ser Lys Leu Gln Ala Gly 35 40
45Asp Glu Val Val His Ile Asn Glu Val Thr Leu Ser Ser Ser Arg Lys
50 55 60Glu Ala Val Ser Leu Val Lys Gly Ser Tyr Lys Thr Leu Arg Leu
Val65 70 75 80Val Arg Arg Asp Val Cys Thr Asp Pro Gly His 85
9063100PRTArtificial SequenceSynthetic peptide 63Asn Asn Glu Leu
Thr Gln Phe Leu Pro Arg Thr Ile Thr Leu Lys Lys1 5 10 15Pro Pro Gly
Ala Gln Leu Gly Phe Asn Ile Arg Gly Gly Lys Ala Ser 20 25 30Gln Leu
Gly Ile Phe Ile Ser Lys Val Ile Pro Asp Ser Asp Ala His 35 40 45Arg
Ala Gly Leu Gln Glu Gly Asp Gln Val Leu Ala Val Asn Asp Val 50
55
60Asp Phe Gln Asp Ile Glu His Ser Lys Ala Val Glu Ile Leu Lys Thr65
70 75 80Ala Arg Glu Ile Ser Met Arg Val Arg Phe Phe Pro Tyr Asn Tyr
His 85 90 95Arg Gln Lys Glu 10064107PRTArtificial SequenceSynthetic
peptide 64Phe Leu Thr Glu Phe Gln Asp Lys Gln Ile Lys Asp Trp Lys
Lys Arg1 5 10 15Phe Ile Gly Ile Arg Met Arg Thr Ile Thr Pro Ser Leu
Val Asp Glu 20 25 30Leu Lys Ala Ser Asn Pro Asp Phe Pro Glu Val Ser
Ser Gly Ile Tyr 35 40 45Val Gln Glu Val Ala Pro Asn Ser Pro Ser Gln
Arg Gly Gly Ile Gln 50 55 60Asp Gly Asp Ile Ile Val Lys Val Asn Gly
Arg Pro Leu Val Asp Ser65 70 75 80Ser Glu Leu Gln Glu Ala Val Leu
Thr Glu Ser Pro Leu Leu Leu Glu 85 90 95Val Arg Arg Gly Asn Asp Asp
Leu Leu Phe Ser 100 1056594PRTArtificial SequenceSynthetic peptide
65Asn Lys Lys Tyr Leu Gly Leu Gln Met Leu Ser Leu Thr Val Pro Leu1
5 10 15Ser Glu Glu Leu Lys Met His Tyr Pro Asp Phe Pro Asp Val Ser
Ser 20 25 30Gly Val Tyr Val Cys Lys Val Val Glu Gly Thr Ala Ala Gln
Ser Ser 35 40 45Gly Leu Arg Asp His Asp Val Ile Val Asn Ile Asn Gly
Lys Pro Ile 50 55 60Thr Thr Thr Thr Asp Val Val Lys Ala Leu Asp Ser
Asp Ser Leu Ser65 70 75 80Met Ala Val Leu Arg Gly Lys Asp Asn Leu
Leu Leu Thr Val 85 9066111PRTArtificial SequenceSynthetic peptide
66Pro Gly Ser Asp Ser Ser Leu Phe Glu Thr Tyr Asn Val Glu Leu Val1
5 10 15Arg Lys Asp Gly Gln Ser Leu Gly Ile Arg Ile Val Gly Tyr Val
Gly 20 25 30Thr Ser His Thr Gly Glu Ala Ser Gly Ile Tyr Val Lys Ser
Ile Ile 35 40 45Pro Gly Ser Ala Ala Tyr His Asn Gly His Ile Gln Val
Asn Asp Lys 50 55 60Ile Val Ala Val Asp Gly Val Asn Ile Gln Gly Phe
Ala Asn His Asp65 70 75 80Val Val Glu Val Leu Arg Asn Ala Gly Gln
Val Val His Leu Thr Leu 85 90 95Val Arg Arg Lys Thr Ser Ser Ser Thr
Ser Arg Ile His Arg Asp 100 105 1106793PRTArtificial
SequenceSynthetic peptide 67Pro Ala Thr Cys Pro Ile Val Pro Gly Gln
Glu Met Ile Ile Glu Ile1 5 10 15Ser Lys Gly Arg Ser Gly Leu Gly Leu
Ser Ile Val Gly Gly Lys Asp 20 25 30Thr Pro Leu Asn Ala Ile Val Ile
His Glu Val Tyr Glu Glu Gly Ala 35 40 45Ala Ala Arg Asp Gly Arg Leu
Trp Ala Gly Asp Gln Ile Leu Glu Val 50 55 60Asn Gly Val Asp Leu Arg
Asn Ser Ser His Glu Glu Ala Ile Thr Ala65 70 75 80Leu Arg Gln Thr
Pro Gln Lys Val Arg Leu Val Val Tyr 85 9068100PRTArtificial
SequenceSynthetic peptide 68Leu Pro Glu Thr Val Cys Trp Gly His Val
Glu Glu Val Glu Leu Ile1 5 10 15Asn Asp Gly Ser Gly Leu Gly Phe Gly
Ile Val Gly Gly Lys Thr Ser 20 25 30Gly Val Val Val Arg Thr Ile Val
Pro Gly Gly Leu Ala Asp Arg Asp 35 40 45Gly Arg Leu Gln Thr Gly Asp
His Ile Leu Lys Ile Gly Gly Thr Asn 50 55 60Val Gln Gly Met Thr Ser
Glu Gln Val Ala Gln Val Leu Arg Asn Cys65 70 75 80Gly Asn Ser Val
Arg Met Leu Val Ala Arg Asp Pro Ala Gly Asp Ile 85 90 95Gln Ser Pro
Ile 10069119PRTArtificial SequenceSynthetic peptide 69Pro Asn Phe
Ser His Trp Gly Pro Pro Arg Ile Val Glu Ile Phe Arg1 5 10 15Glu Pro
Asn Val Ser Leu Gly Ile Ser Ile Val Val Gly Gln Thr Val 20 25 30Ile
Lys Arg Leu Lys Asn Gly Glu Glu Leu Lys Gly Ile Phe Ile Lys 35 40
45Gln Val Leu Glu Asp Ser Pro Ala Gly Lys Thr Asn Ala Leu Lys Thr
50 55 60Gly Asp Lys Ile Leu Glu Val Ser Gly Val Asp Leu Gln Asn Ala
Ser65 70 75 80His Ser Glu Ala Val Glu Ala Ile Lys Asn Ala Gly Asn
Pro Val Val 85 90 95Phe Ile Val Gln Ser Leu Ser Ser Thr Pro Arg Val
Ile Pro Asn Val 100 105 110His Asn Lys Ala Asn Ser Ser
1157099PRTArtificial SequenceSynthetic peptide 70Pro Gly Glu Leu
His Ile Ile Glu Leu Glu Lys Asp Lys Asn Gly Leu1 5 10 15Gly Leu Ser
Leu Ala Gly Asn Lys Asp Arg Ser Arg Met Ser Ile Phe 20 25 30Val Val
Gly Ile Asn Pro Glu Gly Pro Ala Ala Ala Asp Gly Arg Met 35 40 45Arg
Ile Gly Asp Glu Leu Leu Glu Ile Asn Asn Gln Ile Leu Tyr Gly 50 55
60Arg Ser His Gln Asn Ala Ser Ala Ile Ile Lys Thr Ala Pro Ser Lys65
70 75 80Val Lys Leu Val Phe Ile Arg Asn Glu Asp Ala Val Asn Gln Met
Ala 85 90 95Asn Ser Ser71102PRTArtificial SequenceSynthetic peptide
71Leu Ser Ser Pro Glu Val Lys Ile Val Glu Leu Val Lys Asp Cys Lys1
5 10 15Gly Leu Gly Phe Ser Ile Leu Asp Tyr Gln Asp Pro Leu Asp Pro
Thr 20 25 30Arg Ser Val Ile Val Ile Arg Ser Leu Val Ala Asp Gly Val
Ala Glu 35 40 45Arg Ser Gly Gly Leu Leu Pro Gly Asp Arg Leu Val Ser
Val Asn Glu 50 55 60Tyr Cys Leu Asp Asn Thr Ser Leu Ala Glu Ala Val
Glu Ile Leu Lys65 70 75 80Ala Val Pro Pro Gly Leu Val His Leu Gly
Ile Cys Lys Pro Leu Val 85 90 95Glu Phe Ile Val Thr Asp
10072104PRTArtificial SequenceSynthetic peptide 72Ile Trp Gln Ile
Glu Tyr Ile Asp Ile Glu Arg Pro Ser Thr Gly Gly1 5 10 15Leu Gly Phe
Ser Val Val Ala Leu Arg Ser Gln Asn Leu Gly Lys Val 20 25 30Asp Ile
Phe Val Lys Asp Val Gln Pro Gly Ser Val Ala Asp Arg Asp 35 40 45Gln
Arg Leu Lys Glu Asn Asp Gln Ile Leu Ala Ile Asn His Thr Pro 50 55
60Leu Asp Gln Asn Ile Ser His Gln Gln Ala Ile Ala Leu Leu Gln Gln65
70 75 80Thr Thr Gly Ser Leu Arg Leu Ile Val Ala Arg Glu Pro Val His
Thr 85 90 95Lys Ser Ser Thr Ser Ser Ser Glu 1007396PRTArtificial
SequenceSynthetic peptide 73Asn Ser Asp Asp Ala Glu Leu Gln Lys Tyr
Ser Lys Leu Leu Pro Ile1 5 10 15His Thr Leu Arg Leu Gly Val Glu Val
Asp Ser Phe Asp Gly His His 20 25 30Tyr Ile Ser Ser Ile Val Ser Gly
Gly Pro Val Asp Thr Leu Gly Leu 35 40 45Leu Gln Pro Glu Asp Glu Leu
Leu Glu Val Asn Gly Met Gln Leu Tyr 50 55 60Gly Lys Ser Arg Arg Glu
Ala Val Ser Phe Leu Lys Glu Val Pro Pro65 70 75 80Pro Phe Thr Leu
Val Cys Cys Arg Arg Leu Phe Asp Asp Glu Ala Ser 85 90
9574118PRTArtificial SequenceSynthetic peptide 74His Leu Arg Leu
Leu Asn Ile Ala Cys Ala Ala Lys Ala Lys Arg Arg1 5 10 15Leu Met Thr
Leu Thr Lys Pro Ser Arg Glu Ala Pro Leu Pro Phe Ile 20 25 30Leu Leu
Gly Gly Ser Glu Lys Gly Phe Gly Ile Phe Val Asp Ser Val 35 40 45Asp
Ser Gly Ser Lys Ala Thr Glu Ala Gly Leu Lys Arg Gly Asp Gln 50 55
60Ile Leu Glu Val Asn Gly Gln Asn Phe Glu Asn Ile Gln Leu Ser Lys65
70 75 80Ala Met Glu Ile Leu Arg Asn Asn Thr His Leu Ser Ile Thr Val
Lys 85 90 95Thr Asn Leu Phe Val Phe Lys Glu Leu Leu Thr Arg Leu Ser
Glu Glu 100 105 110Lys Arg Asn Gly Ala Pro 1157588PRTArtificial
SequenceSynthetic peptide 75Ile Pro Pro Ala Pro Arg Lys Val Glu Met
Arg Arg Asp Pro Val Leu1 5 10 15Gly Phe Gly Phe Val Ala Gly Ser Glu
Lys Pro Val Val Val Arg Ser 20 25 30Val Thr Pro Gly Gly Pro Ser Glu
Gly Lys Leu Ile Pro Gly Asp Gln 35 40 45Ile Val Met Ile Asn Asp Glu
Pro Val Ser Ala Ala Pro Arg Glu Arg 50 55 60Val Ile Asp Leu Val Arg
Ser Cys Lys Glu Ser Ile Leu Leu Thr Val65 70 75 80Ile Gln Pro Tyr
Pro Ser Pro Lys 8576101PRTArtificial SequenceSynthetic peptide
76Leu Asn Lys Arg Thr Thr Met Pro Lys Asp Ser Gly Ala Leu Leu Gly1
5 10 15Leu Lys Val Val Gly Gly Lys Met Thr Asp Leu Gly Arg Leu Gly
Ala 20 25 30Phe Ile Thr Lys Val Lys Lys Gly Ser Leu Ala Asp Val Val
Gly His 35 40 45Leu Arg Ala Gly Asp Glu Val Leu Glu Trp Asn Gly Lys
Pro Leu Pro 50 55 60Gly Ala Thr Asn Glu Glu Val Tyr Asn Ile Ile Leu
Glu Ser Lys Ser65 70 75 80Glu Pro Gln Val Glu Ile Ile Val Ser Arg
Pro Ile Gly Asp Ile Pro 85 90 95Arg Ile His Arg Asp
1007777PRTArtificial SequenceSynthetic peptide 77Arg Cys Val Ile
Ile Gln Lys Asp Gln His Gly Phe Gly Phe Thr Val1 5 10 15Ser Gly Asp
Arg Ile Val Leu Val Gln Ser Val Arg Pro Gly Gly Ala 20 25 30Ala Met
Lys Ala Gly Val Lys Glu Gly Asp Arg Ile Ile Lys Val Asn 35 40 45Gly
Thr Met Val Thr Asn Ser Ser His Leu Glu Val Val Lys Leu Ile 50 55
60Lys Ser Gly Ala Tyr Val Ala Leu Thr Leu Leu Gly Ser65 70
757887PRTArtificial SequenceSynthetic peptide 78Ile Leu Val Gln Arg
Cys Val Ile Ile Gln Lys Asp Asp Asn Gly Phe1 5 10 15Gly Leu Thr Val
Ser Gly Asp Asn Pro Val Phe Val Gln Ser Val Lys 20 25 30Glu Asp Gly
Ala Ala Met Arg Ala Gly Val Gln Thr Gly Asp Arg Ile 35 40 45Ile Lys
Val Asn Gly Thr Leu Val Thr His Ser Asn His Leu Glu Val 50 55 60Val
Lys Leu Ile Lys Ser Gly Ser Tyr Val Ala Leu Thr Val Gln Gly65 70 75
80Arg Pro Pro Gly Asn Ser Ser 857978PRTArtificial SequenceSynthetic
peptide 79Ser Val Glu Met Thr Leu Arg Arg Asn Gly Leu Gly Gln Leu
Gly Phe1 5 10 15His Val Asn Tyr Glu Gly Ile Val Ala Asp Val Glu Pro
Tyr Gly Tyr 20 25 30Ala Trp Gln Ala Gly Leu Arg Gln Gly Ser Arg Leu
Val Glu Ile Cys 35 40 45Lys Val Ala Val Ala Thr Leu Ser His Glu Gln
Met Ile Asp Leu Leu 50 55 60Arg Thr Ser Val Thr Val Lys Val Val Ile
Ile Pro Pro His65 70 758096PRTArtificial SequenceSynthetic peptide
80Leu Lys Val Met Thr Ser Gly Trp Glu Thr Val Asp Met Thr Leu Arg1
5 10 15Arg Asn Gly Leu Gly Gln Leu Gly Phe His Val Lys Tyr Asp Gly
Thr 20 25 30Val Ala Glu Val Glu Asp Tyr Gly Phe Ala Trp Gln Ala Gly
Leu Arg 35 40 45Gln Gly Ser Arg Leu Val Glu Ile Cys Lys Val Ala Val
Val Thr Leu 50 55 60Thr His Asp Gln Met Ile Asp Leu Leu Arg Thr Ser
Val Thr Val Lys65 70 75 80Val Val Ile Ile Pro Pro Phe Glu Asp Gly
Thr Pro Arg Arg Gly Trp 85 90 9581105PRTArtificial
SequenceSynthetic peptide 81His Tyr Ile Phe Pro His Ala Arg Ile Lys
Ile Thr Arg Asp Ser Lys1 5 10 15Asp His Thr Val Ser Gly Asn Gly Leu
Gly Ile Arg Ile Val Gly Gly 20 25 30Lys Glu Ile Pro Gly His Ser Gly
Glu Ile Gly Ala Tyr Ile Ala Lys 35 40 45Ile Leu Pro Gly Gly Ser Ala
Glu Gln Thr Gly Lys Leu Met Glu Gly 50 55 60Met Gln Val Leu Glu Trp
Asn Gly Ile Pro Leu Thr Ser Lys Thr Tyr65 70 75 80Glu Glu Val Gln
Ser Ile Ile Ser Gln Gln Ser Gly Glu Ala Glu Ile 85 90 95Cys Val Arg
Leu Asp Leu Asn Met Leu 100 1058286PRTArtificial SequenceSynthetic
peptide 82Ser Tyr Ser Val Thr Leu Thr Gly Pro Gly Pro Trp Gly Phe
Arg Leu1 5 10 15Gln Gly Gly Lys Asp Phe Asn Met Pro Leu Thr Ile Ser
Arg Ile Thr 20 25 30Pro Gly Ser Lys Ala Ala Gln Ser Gln Leu Ser Gln
Gly Asp Leu Val 35 40 45Val Ala Ile Asp Gly Val Asn Thr Asp Thr Met
Thr His Leu Glu Ala 50 55 60Gln Asn Lys Ile Lys Ser Ala Ser Tyr Asn
Leu Ser Leu Thr Leu Gln65 70 75 80Lys Ser Lys Asn Ser Ser
858394PRTArtificial SequenceSynthetic peptide 83Phe Ser Asp Met Arg
Ile Ser Ile Asn Gln Thr Pro Gly Lys Ser Leu1 5 10 15Asp Phe Gly Phe
Thr Ile Lys Trp Asp Ile Pro Gly Ile Phe Val Ala 20 25 30Ser Val Glu
Ala Gly Ser Pro Ala Glu Phe Ser Gln Leu Gln Val Asp 35 40 45Asp Glu
Ile Ile Ala Ile Asn Asn Thr Lys Phe Ser Tyr Asn Asp Ser 50 55 60Lys
Glu Trp Glu Glu Ala Met Ala Lys Ala Gln Glu Thr Gly His Leu65 70 75
80Val Met Asp Val Arg Arg Tyr Gly Lys Ala Gly Ser Pro Glu 85
908498PRTArtificial SequenceSynthetic peptide 84Gln Ser Ala His Leu
Glu Val Ile Gln Leu Ala Asn Ile Lys Pro Ser1 5 10 15Glu Gly Leu Gly
Met Tyr Ile Lys Ser Thr Tyr Asp Gly Leu His Val 20 25 30Ile Thr Gly
Thr Thr Glu Asn Ser Pro Ala Asp Arg Cys Lys Lys Ile 35 40 45His Ala
Gly Asp Glu Val Ile Gln Val Asn His Gln Thr Val Val Gly 50 55 60Trp
Gln Leu Lys Asn Leu Val Asn Ala Leu Arg Glu Asp Pro Ser Gly65 70 75
80Val Ile Leu Thr Leu Lys Lys Arg Pro Gln Ser Met Leu Thr Ser Ala
85 90 95Pro Ala85100PRTArtificial SequenceSynthetic peptide 85Ile
Leu Thr Gln Thr Leu Ile Pro Val Arg His Thr Val Lys Ile Asp1 5 10
15Lys Asp Thr Leu Leu Gln Asp Tyr Gly Phe His Ile Ser Glu Ser Leu
20 25 30Pro Leu Thr Val Val Ala Val Thr Ala Gly Gly Ser Ala His Gly
Lys 35 40 45Leu Phe Pro Gly Asp Gln Ile Leu Gln Met Asn Asn Glu Pro
Ala Glu 50 55 60Asp Leu Ser Trp Glu Arg Ala Val Asp Ile Leu Arg Glu
Ala Glu Asp65 70 75 80Ser Leu Ser Ile Thr Val Val Arg Cys Thr Ser
Gly Val Pro Lys Ser 85 90 95Ser Asn Ser Ser 1008691PRTArtificial
SequenceSynthetic peptide 86Arg Ser Phe Gln Tyr Val Pro Val Gln Leu
Gln Gly Gly Ala Pro Trp1 5 10 15Gly Phe Thr Leu Lys Gly Gly Leu Glu
His Cys Glu Pro Leu Thr Val 20 25 30Ser Lys Ile Glu Asp Gly Gly Lys
Ala Ala Leu Ser Gln Lys Met Arg 35 40 45Thr Gly Asp Glu Leu Val Asn
Ile Asn Gly Thr Pro Leu Tyr Gly Ser 50 55 60Arg Gln Glu Ala Leu Ile
Leu Ile Lys Gly Ser Phe Arg Ile Leu Lys65 70 75 80Leu Ile Val Arg
Arg Arg Asn Ala Pro Val Ser 85 9087103PRTArtificial
SequenceSynthetic peptide 87Ile Leu Glu Lys Leu Glu Leu Phe Pro Val
Glu Leu Glu Lys Asp Glu1 5 10 15Asp Gly Leu Gly Ile Ser Ile Ile Gly
Met Gly Val Gly Ala Asp Ala 20 25 30Gly Leu Glu Lys Leu Gly Ile Phe
Val Lys Thr Val Thr Glu Gly Gly 35 40 45Ala Ala Gln Arg Asp Gly Arg
Ile
Gln Val Asn Asp Gln Ile Val Glu 50 55 60Val Asp Gly Ile Ser Leu Val
Gly Val Thr Gln Asn Phe Ala Ala Thr65 70 75 80Val Leu Arg Asn Thr
Lys Gly Asn Val Arg Phe Val Ile Gly Arg Glu 85 90 95Lys Pro Gly Gln
Val Ser Glu 10088113PRTArtificial SequenceSynthetic peptide 88Lys
Asp Val Asn Val Tyr Val Asn Pro Lys Lys Leu Thr Val Ile Lys1 5 10
15Ala Lys Glu Gln Leu Lys Leu Leu Glu Val Leu Val Gly Ile Ile His
20 25 30Gln Thr Lys Trp Ser Trp Arg Arg Thr Gly Lys Gln Gly Asp Gly
Glu 35 40 45Arg Leu Val Val His Gly Leu Leu Pro Gly Gly Ser Ala Met
Lys Ser 50 55 60Gly Gln Val Leu Ile Gly Asp Val Leu Val Ala Val Asn
Asp Val Asp65 70 75 80Val Thr Thr Glu Asn Ile Glu Arg Val Leu Ser
Cys Ile Pro Gly Pro 85 90 95Met Gln Val Lys Leu Thr Phe Glu Asn Ala
Tyr Asp Val Lys Arg Glu 100 105 110Thr8990PRTArtificial
SequenceSynthetic peptide 89Thr Arg Gly Cys Glu Thr Val Glu Met Thr
Leu Arg Arg Asn Gly Leu1 5 10 15Gly Gln Leu Gly Phe His Val Asn Phe
Glu Gly Ile Val Ala Asp Val 20 25 30Glu Pro Phe Gly Phe Ala Trp Lys
Ala Gly Leu Arg Gln Gly Ser Arg 35 40 45Leu Val Glu Ile Cys Lys Val
Ala Val Ala Thr Leu Thr His Glu Gln 50 55 60Met Ile Asp Leu Leu Arg
Thr Ser Val Thr Val Lys Val Val Ile Ile65 70 75 80Gln Pro His Asp
Asp Gly Ser Pro Arg Arg 85 909096PRTArtificial SequenceSynthetic
peptide 90Val Glu Asn Ile Leu Ala Lys Arg Leu Leu Ile Leu Pro Gln
Glu Glu1 5 10 15Asp Tyr Gly Phe Asp Ile Glu Glu Lys Asn Lys Ala Val
Val Val Lys 20 25 30Ser Val Gln Arg Gly Ser Leu Ala Glu Val Ala Gly
Leu Gln Val Gly 35 40 45Arg Lys Ile Tyr Ser Ile Asn Glu Asp Leu Val
Phe Leu Arg Pro Phe 50 55 60Ser Glu Val Glu Ser Ile Leu Asn Gln Ser
Phe Cys Ser Arg Arg Pro65 70 75 80Leu Arg Leu Leu Val Ala Thr Lys
Ala Lys Glu Ile Ile Lys Ile Pro 85 90 9591103PRTArtificial
SequenceSynthetic peptide 91Pro Asp Ser Ala Gly Pro Gly Glu Val Arg
Leu Val Ser Leu Arg Arg1 5 10 15Ala Lys Ala His Glu Gly Leu Gly Phe
Ser Ile Arg Gly Gly Ser Glu 20 25 30His Gly Val Gly Ile Tyr Val Ser
Leu Val Glu Pro Gly Ser Leu Ala 35 40 45Glu Lys Glu Gly Leu Arg Val
Gly Asp Gln Ile Leu Arg Val Asn Asp 50 55 60Lys Ser Leu Ala Arg Val
Thr His Ala Glu Ala Val Lys Ala Leu Lys65 70 75 80Gly Ser Lys Lys
Leu Val Leu Ser Val Tyr Ser Ala Gly Arg Ile Pro 85 90 95Gly Gly Tyr
Val Thr Asn His 10092100PRTArtificial SequenceSynthetic peptide
92Leu Gln Gly Gly Asp Glu Lys Lys Val Asn Leu Val Leu Gly Asp Gly1
5 10 15Arg Ser Leu Gly Leu Thr Ile Arg Gly Gly Ala Glu Tyr Gly Leu
Gly 20 25 30Ile Tyr Ile Thr Gly Val Asp Pro Gly Ser Glu Ala Glu Gly
Ser Gly 35 40 45Leu Lys Val Gly Asp Gln Ile Leu Glu Val Asn Gly Arg
Ser Phe Leu 50 55 60Asn Ile Leu His Asp Glu Ala Val Arg Leu Leu Lys
Ser Ser Arg His65 70 75 80Leu Ile Leu Thr Val Lys Asp Val Gly Arg
Leu Pro His Ala Arg Thr 85 90 95Thr Val Asp Glu
1009398PRTArtificial SequenceSynthetic peptide 93Leu Arg Arg Ala
Glu Leu Val Glu Ile Ile Val Glu Thr Glu Ala Gln1 5 10 15Thr Gly Val
Ser Gly Ile Asn Val Ala Gly Gly Gly Lys Glu Gly Ile 20 25 30Phe Val
Arg Glu Leu Arg Glu Asp Ser Pro Ala Ala Arg Ser Leu Ser 35 40 45Leu
Gln Glu Gly Asp Gln Leu Leu Ser Ala Arg Val Phe Phe Glu Asn 50 55
60Phe Lys Tyr Glu Asp Ala Leu Arg Leu Leu Gln Cys Ala Glu Pro Tyr65
70 75 80Lys Val Ser Phe Cys Leu Lys Arg Thr Val Pro Thr Gly Asp Leu
Ala 85 90 95Leu Arg9494PRTArtificial SequenceSynthetic peptide
94Ile Gln Thr Thr Gly Ala Val Ser Tyr Thr Val Glu Leu Lys Arg Tyr1
5 10 15Gly Gly Pro Leu Gly Ile Thr Ile Ser Gly Thr Glu Glu Pro Phe
Asp 20 25 30Pro Ile Val Ile Ser Gly Leu Thr Lys Arg Gly Leu Ala Glu
Arg Thr 35 40 45Gly Ala Ile His Val Gly Asp Arg Ile Leu Ala Ile Asn
Asn Val Ser 50 55 60Leu Lys Gly Arg Pro Leu Ser Glu Ala Ile His Leu
Leu Gln Val Ala65 70 75 80Gly Glu Thr Val Thr Leu Lys Ile Lys Lys
Gln Leu Asp Arg 85 9095105PRTArtificial SequenceSynthetic peptide
95Ile Leu Glu Met Glu Glu Leu Leu Leu Pro Thr Pro Leu Glu Met His1
5 10 15Lys Val Thr Leu His Lys Asp Pro Met Arg His Asp Phe Gly Phe
Ser 20 25 30Val Ser Asp Gly Leu Leu Glu Lys Gly Val Tyr Val His Thr
Val Arg 35 40 45Pro Asp Gly Pro Ala His Arg Gly Gly Leu Gln Pro Phe
Asp Arg Val 50 55 60Leu Gln Val Asn His Val Arg Thr Arg Asp Phe Asp
Cys Cys Leu Ala65 70 75 80Val Pro Leu Leu Ala Glu Ala Gly Asp Val
Leu Glu Leu Ile Ile Ser 85 90 95Arg Lys Pro His Thr Ala His Ser Ser
100 10596102PRTArtificial SequenceSynthetic peptide 96Ile His Thr
Val Ala Asn Ala Ser Gly Pro Leu Met Val Glu Ile Val1 5 10 15Lys Thr
Pro Gly Ser Ala Leu Gly Ile Ser Leu Thr Thr Thr Ser Leu 20 25 30Arg
Asn Lys Ser Val Ile Thr Ile Asp Arg Ile Lys Pro Ala Ser Val 35 40
45Val Asp Arg Ser Gly Ala Leu His Pro Gly Asp His Ile Leu Ser Ile
50 55 60Asp Gly Thr Ser Met Glu His Cys Ser Leu Leu Glu Ala Thr Lys
Leu65 70 75 80Leu Ala Ser Ile Ser Glu Lys Val Arg Leu Glu Ile Leu
Pro Val Pro 85 90 95Gln Ser Gln Arg Pro Leu 1009784PRTArtificial
SequenceSynthetic peptide 97Ile Thr Val Val Glu Leu Ile Lys Lys Glu
Gly Ser Thr Leu Gly Leu1 5 10 15Thr Ile Ser Gly Gly Thr Asp Lys Asp
Gly Lys Pro Arg Val Ser Asn 20 25 30Leu Arg Pro Gly Gly Leu Ala Ala
Arg Ser Asp Leu Leu Asn Ile Gly 35 40 45Asp Tyr Ile Arg Ser Val Asn
Gly Ile His Leu Thr Arg Leu Arg His 50 55 60Asp Glu Ile Ile Thr Leu
Leu Lys Asn Val Gly Glu Arg Val Val Leu65 70 75 80Glu Val Glu
Tyr98103PRTArtificial SequenceSynthetic peptide 98Ile Gln Ile Val
His Thr Glu Thr Thr Glu Val Val Leu Cys Gly Asp1 5 10 15Pro Leu Ser
Gly Phe Gly Leu Gln Leu Gln Gly Gly Ile Phe Ala Thr 20 25 30Glu Thr
Leu Ser Ser Pro Pro Leu Val Cys Phe Ile Glu Pro Asp Ser 35 40 45Pro
Ala Glu Arg Cys Gly Leu Leu Gln Val Gly Asp Arg Val Leu Ser 50 55
60Ile Asn Gly Ile Ala Thr Glu Asp Gly Thr Met Glu Glu Ala Asn Gln65
70 75 80Leu Leu Arg Asp Ala Ala Leu Ala His Lys Val Val Leu Glu Val
Glu 85 90 95Phe Asp Val Ala Glu Ser Val 1009992PRTArtificial
SequenceSynthetic peptide 99Ile Leu Asp Val Ser Leu Tyr Lys Glu Gly
Asn Ser Phe Gly Phe Val1 5 10 15Leu Arg Gly Gly Ala His Glu Asp Gly
His Lys Ser Arg Pro Leu Val 20 25 30Leu Thr Tyr Val Arg Pro Gly Gly
Pro Ala Asp Arg Glu Gly Ser Leu 35 40 45Lys Val Gly Asp Arg Leu Leu
Ser Val Asp Gly Ile Pro Leu His Gly 50 55 60Ala Ser His Ala Thr Ala
Leu Ala Thr Leu Arg Gln Cys Ser His Glu65 70 75 80Ala Leu Phe Gln
Val Glu Tyr Asp Val Ala Thr Pro 85 90100102PRTArtificial
SequenceSynthetic peptide 100Gln Phe Asp Val Ala Glu Ser Val Ile
Pro Ser Ser Gly Thr Phe His1 5 10 15Val Lys Leu Pro Lys Lys Arg Ser
Val Glu Leu Gly Ile Thr Ile Ser 20 25 30Ser Ala Ser Arg Lys Arg Gly
Glu Pro Leu Ile Ile Ser Asp Ile Lys 35 40 45Lys Gly Ser Val Ala His
Arg Thr Gly Thr Leu Glu Pro Gly Asp Lys 50 55 60Leu Leu Ala Ile Asp
Asn Ile Arg Leu Asp Asn Cys Pro Met Glu Asp65 70 75 80Ala Val Gln
Ile Leu Arg Gln Cys Glu Asp Leu Val Lys Leu Lys Ile 85 90 95Arg Lys
Asp Glu Asp Asn 10010191PRTArtificial SequenceSynthetic peptide
101Met Ala Leu Thr Val Asp Val Ala Gly Pro Ala Pro Trp Gly Phe Arg1
5 10 15Ile Thr Gly Gly Arg Asp Phe His Thr Pro Ile Met Val Thr Lys
Val 20 25 30Ala Glu Arg Gly Lys Ala Lys Asp Ala Asp Leu Arg Pro Gly
Asp Ile 35 40 45Ile Val Ala Ile Asn Gly Glu Ser Ala Glu Gly Met Leu
His Ala Glu 50 55 60Ala Gln Ser Lys Ile Arg Gln Ser Pro Ser Pro Leu
Arg Leu Gln Leu65 70 75 80Asp Arg Ser Gln Ala Thr Ser Pro Gly Gln
Thr 85 9010284PRTArtificial SequenceSynthetic peptide 102Ser Asn
Tyr Ser Val Ser Leu Val Gly Pro Ala Pro Trp Gly Phe Arg1 5 10 15Leu
Gln Gly Gly Lys Asp Phe Asn Met Pro Leu Thr Ile Ser Ser Leu 20 25
30Lys Asp Gly Gly Lys Ala Ala Gln Ala Asn Val Arg Ile Gly Asp Val
35 40 45Val Leu Ser Ile Asp Gly Ile Asn Ala Gln Gly Met Thr His Leu
Glu 50 55 60Ala Gln Asn Lys Ile Lys Gly Cys Thr Gly Ser Leu Asn Met
Thr Leu65 70 75 80Gln Arg Ala Ser103133PRTArtificial
SequenceSynthetic peptide 103Thr Leu Val Glu His Ser Lys Leu Tyr
Cys Gly His Cys Tyr Tyr Gln1 5 10 15Thr Val Val Thr Pro Val Ile Glu
Gln Ile Leu Pro Asp Ser Pro Gly 20 25 30Ser His Leu Pro His Thr Val
Thr Leu Val Ser Ile Pro Ala Ser Ser 35 40 45His Gly Lys Arg Gly Leu
Ser Val Ser Ile Asp Pro Pro His Gly Pro 50 55 60Pro Gly Cys Gly Thr
Glu His Ser His Thr Val Arg Val Gln Gly Val65 70 75 80Asp Pro Gly
Cys Met Ser Pro Asp Val Lys Asn Ser Ile His Val Gly 85 90 95Asp Arg
Ile Leu Glu Ile Asn Gly Thr Pro Ile Arg Asn Val Pro Leu 100 105
110Asp Glu Ile Asp Leu Leu Ile Gln Glu Thr Ser Arg Leu Leu Gln Leu
115 120 125Thr Leu Glu His Asp 13010492PRTArtificial
SequenceSynthetic peptide 104Pro Tyr Ser Val Thr Leu Ile Ser Met
Pro Ala Thr Thr Glu Gly Arg1 5 10 15Arg Gly Phe Ser Val Ser Val Glu
Ser Ala Cys Ser Asn Tyr Ala Thr 20 25 30Thr Val Gln Val Lys Glu Val
Asn Arg Met His Ile Ser Pro Asn Asn 35 40 45Arg Asn Ala Ile His Pro
Gly Asp Arg Ile Leu Glu Ile Asn Gly Thr 50 55 60Pro Val Arg Thr Leu
Arg Val Glu Glu Val Glu Asp Ala Ile Ser Gln65 70 75 80Thr Ser Gln
Thr Leu Gln Leu Leu Ile Glu His Asp 85 9010582PRTArtificial
SequenceSynthetic peptide 105Ile His Ser Val Thr Leu Arg Gly Pro
Ser Pro Trp Gly Phe Arg Leu1 5 10 15Val Gly Arg Asp Phe Ser Ala Pro
Leu Thr Ile Ser Arg Val His Ala 20 25 30Gly Ser Lys Ala Ser Leu Ala
Ala Leu Cys Pro Gly Asp Leu Ile Gln 35 40 45Ala Ile Asn Gly Glu Ser
Thr Glu Leu Met Thr His Leu Glu Ala Gln 50 55 60Asn Arg Ile Lys Gly
Cys His Asp His Leu Thr Leu Ser Val Ser Arg65 70 75 80Pro
Glu10674PRTArtificial SequenceSynthetic peptide 106Val Cys Tyr Arg
Thr Asp Asp Glu Glu Asp Leu Gly Ile Tyr Val Gly1 5 10 15Glu Val Asn
Pro Asn Ser Ile Ala Ala Lys Asp Gly Arg Ile Arg Glu 20 25 30Gly Asp
Arg Ile Ile Gln Ile Asn Gly Val Asp Val Gln Asn Arg Glu 35 40 45Glu
Ala Val Ala Ile Leu Ser Gln Glu Glu Asn Thr Asn Ile Ser Leu 50 55
60Leu Val Ala Arg Pro Glu Ser Gln Leu Ala65 7010793PRTArtificial
SequenceSynthetic peptide 107Ile Pro Ala Thr Gln Pro Glu Leu Ile
Thr Val His Ile Val Lys Gly1 5 10 15Pro Met Gly Phe Gly Phe Thr Ile
Ala Asp Ser Pro Gly Gly Gly Gly 20 25 30Gln Arg Val Lys Gln Ile Val
Asp Ser Pro Arg Cys Arg Gly Leu Lys 35 40 45Glu Gly Asp Leu Ile Val
Glu Val Asn Lys Lys Asn Val Gln Ala Leu 50 55 60Thr His Asn Gln Val
Val Asp Met Leu Val Glu Cys Pro Lys Gly Ser65 70 75 80Glu Val Thr
Leu Leu Val Gln Arg Gly Gly Asn Ser Ser 85 90108103PRTArtificial
SequenceSynthetic peptide 108Ile Pro Asp Tyr Gln Glu Gln Asp Ile
Phe Leu Trp Arg Lys Glu Thr1 5 10 15Gly Phe Gly Phe Arg Ile Leu Gly
Gly Asn Glu Pro Gly Glu Pro Ile 20 25 30Tyr Ile Gly His Ile Val Pro
Leu Gly Ala Ala Asp Thr Asp Gly Arg 35 40 45Leu Arg Ser Gly Asp Glu
Leu Ile Cys Val Asp Gly Thr Pro Val Ile 50 55 60Gly Lys Ser His Gln
Leu Val Val Gln Leu Met Gln Gln Ala Ala Lys65 70 75 80Gln Gly His
Val Asn Leu Thr Val Arg Arg Lys Val Val Phe Ala Val 85 90 95Pro Lys
Thr Glu Asn Ser Ser 100109120PRTArtificial SequenceSynthetic
peptide 109Ile Pro Gly Val Val Ser Thr Val Val Gln Pro Tyr Asp Val
Glu Ile1 5 10 15Arg Arg Gly Glu Asn Glu Gly Phe Gly Phe Val Ile Val
Ser Ser Val 20 25 30Ser Arg Pro Glu Ala Gly Thr Thr Phe Ala Gly Asn
Ala Cys Val Ala 35 40 45Met Pro His Lys Ile Gly Arg Ile Ile Glu Gly
Ser Pro Ala Asp Arg 50 55 60Cys Gly Lys Leu Lys Val Gly Asp Arg Ile
Leu Ala Val Asn Gly Cys65 70 75 80Ser Ile Thr Asn Lys Ser His Ser
Asp Ile Val Asn Leu Ile Lys Glu 85 90 95Ala Gly Asn Thr Val Thr Leu
Arg Ile Ile Pro Gly Asp Glu Ser Ser 100 105 110Asn Ala Glu Phe Ile
Val Thr Asp 115 120110107PRTArtificial SequenceSynthetic peptide
110Ile Pro Ser Glu Leu Lys Gly Lys Phe Ile His Thr Lys Leu Arg Lys1
5 10 15Ser Ser Arg Gly Phe Gly Phe Thr Val Val Gly Gly Asp Glu Pro
Asp 20 25 30Glu Phe Leu Gln Ile Lys Ser Leu Val Leu Asp Gly Pro Ala
Ala Leu 35 40 45Asp Gly Lys Met Glu Thr Gly Asp Val Ile Val Ser Val
Asn Asp Thr 50 55 60Cys Val Leu Gly His Thr His Ala Gln Val Val Lys
Ile Phe Gln Ser65 70 75 80Ile Pro Ile Gly Ala Ser Val Asp Leu Glu
Leu Cys Arg Gly Tyr Pro 85 90 95Leu Pro Phe Asp Pro Asp Gly Ile His
Arg Asp 100 10511191PRTArtificial SequenceSynthetic peptide 111Gln
Ala Thr Gln Glu Gln Asp Phe Tyr Thr Val Glu Leu Glu Arg Gly1 5 10
15Ala Lys Gly
Phe Gly Phe Ser Leu Arg Gly Gly Arg Glu Tyr Asn Met 20 25 30Asp Leu
Tyr Val Leu Arg Leu Ala Glu Asp Gly Pro Ala Glu Arg Cys 35 40 45Gly
Lys Met Arg Ile Gly Asp Glu Ile Leu Glu Ile Asn Gly Glu Thr 50 55
60Thr Lys Asn Met Lys His Ser Arg Ala Ile Glu Leu Ile Lys Asn Gly65
70 75 80Gly Arg Arg Val Arg Leu Phe Leu Lys Arg Gly 85
90112104PRTArtificial SequenceSynthetic peptide 112Arg Glu Lys Pro
Leu Phe Thr Arg Asp Ala Ser Gln Leu Lys Gly Thr1 5 10 15Phe Leu Ser
Thr Thr Leu Lys Lys Ser Asn Met Gly Phe Gly Phe Thr 20 25 30Ile Ile
Gly Gly Asp Glu Pro Asp Glu Phe Leu Gln Val Lys Ser Val 35 40 45Ile
Pro Asp Gly Pro Ala Ala Gln Asp Gly Lys Met Glu Thr Gly Asp 50 55
60Val Ile Val Tyr Ile Asn Glu Val Cys Val Leu Gly His Thr His Ala65
70 75 80Asp Val Val Lys Leu Phe Gln Ser Val Pro Ile Gly Gln Ser Val
Asn 85 90 95Leu Val Leu Cys Arg Gly Tyr Pro 10011393PRTArtificial
SequenceSynthetic peptide 113His Tyr Lys Glu Leu Asp Val His Leu
Arg Arg Met Glu Ser Gly Phe1 5 10 15Gly Phe Arg Ile Leu Gly Gly Asp
Glu Pro Gly Gln Pro Ile Leu Ile 20 25 30Gly Ala Val Ile Ala Met Gly
Ser Ala Asp Arg Asp Gly Arg Leu His 35 40 45Pro Gly Asp Glu Leu Val
Tyr Val Asp Gly Ile Pro Val Ala Gly Lys 50 55 60Thr His Arg Tyr Val
Ile Asp Leu Met His His Ala Ala Arg Asn Gly65 70 75 80Gln Val Asn
Leu Thr Val Arg Arg Lys Val Leu Cys Gly 85 90114106PRTArtificial
SequenceSynthetic peptide 114Glu Gly Arg Gly Ile Ser Ser His Ser
Leu Gln Thr Ser Asp Ala Val1 5 10 15Ile His Arg Lys Glu Asn Glu Gly
Phe Gly Phe Val Ile Ile Ser Ser 20 25 30Leu Asn Arg Pro Glu Ser Gly
Ser Thr Ile Thr Val Pro His Lys Ile 35 40 45Gly Arg Ile Ile Asp Gly
Ser Pro Ala Asp Arg Cys Ala Lys Leu Lys 50 55 60Val Gly Asp Arg Ile
Leu Ala Val Asn Gly Gln Ser Ile Ile Asn Met65 70 75 80Pro His Ala
Asp Ile Val Lys Leu Ile Lys Asp Ala Gly Leu Ser Val 85 90 95Thr Leu
Arg Ile Ile Pro Gln Glu Glu Leu 100 10511591PRTArtificial
SequenceSynthetic peptide 115Leu Ser Gly Ala Thr Gln Ala Glu Leu
Met Thr Leu Thr Ile Val Lys1 5 10 15Gly Ala Gln Gly Phe Gly Phe Thr
Ile Ala Asp Ser Pro Thr Gly Gln 20 25 30Arg Val Lys Gln Ile Leu Asp
Ile Gln Gly Cys Pro Gly Leu Cys Glu 35 40 45Gly Asp Leu Ile Val Glu
Ile Asn Gln Gln Asn Val Gln Asn Leu Ser 50 55 60His Thr Glu Val Val
Asp Ile Leu Lys Asp Cys Pro Ile Gly Ser Glu65 70 75 80Thr Ser Leu
Ile Ile His Arg Gly Gly Phe Phe 85 9011698PRTArtificial
SequenceSynthetic peptide 116Leu Ser Asp Tyr Arg Gln Pro Gln Asp
Phe Asp Tyr Phe Thr Val Asp1 5 10 15Met Glu Lys Gly Ala Lys Gly Phe
Gly Phe Ser Ile Arg Gly Gly Arg 20 25 30Glu Tyr Lys Met Asp Leu Tyr
Val Leu Arg Leu Ala Glu Asp Gly Pro 35 40 45Ala Ile Arg Asn Gly Arg
Met Arg Val Gly Asp Gln Ile Ile Glu Ile 50 55 60Asn Gly Glu Ser Thr
Arg Asp Met Thr His Ala Arg Ala Ile Glu Leu65 70 75 80Ile Lys Ser
Gly Gly Arg Arg Val Arg Leu Leu Leu Lys Arg Gly Thr 85 90 95Gly
Gln11790PRTArtificial SequenceSynthetic peptide 117His Glu Ser Val
Ile Gly Arg Asn Pro Glu Gly Gln Leu Gly Phe Glu1 5 10 15Leu Lys Gly
Gly Ala Glu Asn Gly Gln Phe Pro Tyr Leu Gly Glu Val 20 25 30Lys Pro
Gly Lys Val Ala Tyr Glu Ser Gly Ser Lys Leu Val Ser Glu 35 40 45Glu
Leu Leu Leu Glu Val Asn Glu Thr Pro Val Ala Gly Leu Thr Ile 50 55
60Arg Asp Val Leu Ala Val Ile Lys His Cys Lys Asp Pro Leu Arg Leu65
70 75 80Lys Cys Val Lys Gln Gly Gly Ile His Arg 85
90118100PRTArtificial SequenceSynthetic peptide 118Ala Ser Ser Gly
Ser Ser Gln Pro Glu Leu Val Thr Ile Pro Leu Ile1 5 10 15Lys Gly Pro
Lys Gly Phe Gly Phe Ala Ile Ala Asp Ser Pro Thr Gly 20 25 30Gln Lys
Val Lys Met Ile Leu Asp Ser Gln Trp Cys Gln Gly Leu Gln 35 40 45Lys
Gly Asp Ile Ile Lys Glu Ile Tyr His Gln Asn Val Gln Asn Leu 50 55
60Thr His Leu Gln Val Val Glu Val Leu Lys Gln Phe Pro Val Gly Ala65
70 75 80Asp Val Pro Leu Leu Ile Leu Arg Gly Gly Pro Pro Ser Pro Thr
Lys 85 90 95Thr Ala Lys Met 10011998PRTArtificial SequenceSynthetic
peptide 119Gln Asn Leu Gly Cys Tyr Pro Val Glu Leu Glu Arg Gly Pro
Arg Gly1 5 10 15Phe Gly Phe Ser Leu Arg Gly Gly Lys Glu Tyr Asn Met
Gly Leu Phe 20 25 30Ile Leu Arg Leu Ala Glu Asp Gly Pro Ala Ile Lys
Asp Gly Arg Ile 35 40 45His Val Gly Asp Gln Ile Val Glu Ile Asn Gly
Glu Pro Thr Gln Gly 50 55 60Ile Thr His Thr Arg Ala Ile Glu Leu Ile
Gln Ala Gly Gly Asn Lys65 70 75 80Val Leu Leu Leu Leu Arg Pro Gly
Thr Gly Leu Ile Pro Asp His Gly 85 90 95Leu Ala120108PRTArtificial
SequenceSynthetic peptide 120Leu Tyr Glu Asp Lys Pro Pro Asn Thr
Lys Asp Leu Asp Val Phe Leu1 5 10 15Arg Lys Gln Glu Ser Gly Phe Gly
Phe Arg Val Leu Gly Gly Asp Gly 20 25 30Pro Asp Gln Ser Ile Tyr Ile
Gly Ala Ile Ile Pro Leu Gly Ala Ala 35 40 45Glu Lys Asp Gly Arg Leu
Arg Ala Ala Asp Glu Leu Met Cys Ile Asp 50 55 60Gly Ile Pro Val Lys
Gly Lys Ser His Lys Gln Val Leu Asp Leu Met65 70 75 80Thr Thr Ala
Ala Arg Asn Gly His Val Leu Leu Thr Val Arg Arg Lys 85 90 95Ile Phe
Tyr Gly Glu Lys Gln Pro Glu Asp Asp Ser 100 105121102PRTArtificial
SequenceSynthetic peptide 121Pro Ser Gln Leu Lys Gly Val Leu Val
Arg Ala Ser Leu Lys Lys Ser1 5 10 15Thr Met Gly Phe Gly Phe Thr Ile
Ile Gly Gly Asp Arg Pro Asp Glu 20 25 30Phe Leu Gln Val Lys Asn Val
Leu Lys Asp Gly Pro Ala Ala Gln Asp 35 40 45Gly Lys Ile Ala Pro Gly
Asp Val Ile Val Asp Ile Asn Gly Asn Cys 50 55 60Val Leu Gly His Thr
His Ala Asp Val Val Gln Met Phe Gln Leu Val65 70 75 80Pro Val Asn
Gln Tyr Val Asn Leu Thr Leu Cys Arg Gly Tyr Pro Leu 85 90 95Pro Asp
Asp Ser Glu Asp 100122102PRTArtificial SequenceSynthetic peptide
122Pro Ala Pro Gln Glu Pro Tyr Asp Val Val Leu Gln Arg Lys Glu Asn1
5 10 15Glu Gly Phe Gly Phe Val Ile Leu Thr Ser Lys Asn Lys Pro Pro
Pro 20 25 30Gly Val Ile Pro His Lys Ile Gly Arg Val Ile Glu Gly Ser
Pro Ala 35 40 45Asp Arg Cys Gly Lys Leu Lys Val Gly Asp His Ile Ser
Ala Val Asn 50 55 60Gly Gln Ser Ile Val Glu Leu Ser His Asp Asn Ile
Val Gln Leu Ile65 70 75 80Lys Asp Ala Gly Val Thr Val Thr Leu Thr
Val Ile Ala Glu Glu Glu 85 90 95His His Gly Pro Pro Ser
10012394PRTArtificial SequenceSynthetic peptide 123Gly Leu Arg Ser
Pro Ile Thr Ile Gln Arg Ser Gly Lys Lys Tyr Gly1 5 10 15Phe Thr Leu
Arg Ala Ile Arg Val Tyr Met Gly Asp Thr Asp Val Tyr 20 25 30Ser Val
His His Ile Val Trp His Val Glu Glu Gly Gly Pro Ala Gln 35 40 45Glu
Ala Gly Leu Cys Ala Gly Asp Leu Ile Thr His Val Asn Gly Glu 50 55
60Pro Val His Gly Met Val His Pro Glu Val Val Glu Leu Ile Leu Lys65
70 75 80Ser Gly Asn Lys Val Ala Val Thr Thr Thr Pro Phe Glu Asn 85
90124101PRTArtificial SequenceSynthetic peptide 124Ile Ser Ala Leu
Gly Ser Met Arg Pro Pro Ile Ile Ile His Arg Ala1 5 10 15Gly Lys Lys
Tyr Gly Phe Thr Leu Arg Ala Ile Arg Val Tyr Met Gly 20 25 30Asp Ser
Asp Val Tyr Thr Val His His Met Val Trp His Val Glu Asp 35 40 45Gly
Gly Pro Ala Ser Glu Ala Gly Leu Arg Gln Gly Asp Leu Ile Thr 50 55
60His Val Asn Gly Glu Pro Val His Gly Leu Val His Thr Glu Val Val65
70 75 80Glu Leu Ile Leu Lys Ser Gly Asn Lys Val Ala Ile Ser Thr Thr
Pro 85 90 95Leu Glu Asn Ser Ser 100125103PRTArtificial
SequenceSynthetic peptide 125Leu Cys Gly Ser Leu Arg Pro Pro Ile
Val Ile His Ser Ser Gly Lys1 5 10 15Lys Tyr Gly Phe Ser Leu Arg Ala
Ile Arg Val Tyr Met Gly Asp Ser 20 25 30Asp Val Tyr Thr Val His His
Val Val Trp Ser Val Glu Asp Gly Ser 35 40 45Pro Ala Gln Glu Ala Gly
Leu Arg Ala Gly Asp Leu Ile Thr His Ile 50 55 60Asn Gly Glu Ser Val
Leu Gly Leu Val His Met Asp Val Val Glu Leu65 70 75 80Leu Leu Lys
Ser Gly Asn Lys Ile Ser Leu Arg Thr Thr Ala Leu Glu 85 90 95Asn Thr
Ser Ile Lys Val Gly 10012691PRTArtificial SequenceSynthetic peptide
126Pro His Gln Pro Ile Val Ile His Ser Ser Gly Lys Asn Tyr Gly Phe1
5 10 15Thr Ile Arg Ala Ile Arg Val Tyr Val Gly Asp Ser Asp Ile Tyr
Thr 20 25 30Val His His Ile Val Trp Asn Val Glu Glu Gly Ser Pro Ala
Cys Gln 35 40 45Ala Gly Leu Lys Ala Gly Asp Leu Ile Thr His Ile Asn
Gly Glu Pro 50 55 60Val His Gly Leu Val His Thr Glu Val Ile Glu Leu
Leu Leu Lys Ser65 70 75 80Gly Asn Lys Val Ser Ile Thr Thr Thr Pro
Phe 85 90127100PRTArtificial SequenceSynthetic peptide 127Pro Ala
Lys Met Glu Lys Glu Glu Thr Thr Arg Glu Leu Leu Leu Pro1 5 10 15Asn
Trp Gln Gly Ser Gly Ser His Gly Leu Thr Ile Ala Gln Arg Asp 20 25
30Asp Gly Val Phe Val Gln Glu Val Thr Gln Asn Ser Pro Ala Ala Arg
35 40 45Thr Gly Val Val Lys Glu Gly Asp Gln Ile Val Gly Ala Thr Ile
Tyr 50 55 60Phe Asp Asn Leu Gln Ser Gly Glu Val Thr Gln Leu Leu Asn
Thr Met65 70 75 80Gly His His Thr Val Gly Leu Lys Leu His Arg Lys
Gly Asp Arg Ser 85 90 95Pro Asn Ser Ser 10012898PRTArtificial
SequenceSynthetic peptide 128Ser Glu Asn Cys Lys Asp Val Phe Ile
Glu Lys Gln Lys Gly Glu Ile1 5 10 15Leu Gly Val Val Ile Val Glu Ser
Gly Trp Gly Ser Ile Leu Pro Thr 20 25 30Val Ile Ile Ala Asn Met Met
His Gly Gly Pro Ala Glu Lys Ser Gly 35 40 45Lys Leu Asn Ile Gly Asp
Gln Ile Met Ser Ile Asn Gly Thr Ser Leu 50 55 60Val Gly Leu Pro Leu
Ser Thr Cys Gln Ser Ile Ile Lys Gly Leu Lys65 70 75 80Asn Gln Ser
Arg Val Lys Leu Asn Ile Val Arg Cys Pro Pro Val Asn 85 90 95Ser
Ser129178PRTArtificial SequenceSynthetic peptide 129Ser Glu Asn Cys
Lys Asp Val Phe Ile Glu Lys Gln Lys Gly Glu Ile1 5 10 15Leu Gly Val
Val Ile Val Glu Ser Gly Trp Gly Ser Ile Leu Pro Thr 20 25 30Val Ile
Ile Ala Asn Met Met His Gly Gly Pro Ala Glu Lys Ser Gly 35 40 45Lys
Leu Asn Ile Gly Asp Gln Ile Met Ser Ile Asn Gly Thr Ser Leu 50 55
60Val Gly Leu Pro Leu Ser Thr Cys Gln Ser Ile Ile Lys Gly Leu Glu65
70 75 80Asn Gln Ser Arg Val Lys Leu Asn Ile Val Arg Cys Pro Pro Val
Thr 85 90 95Thr Val Leu Ile Arg Arg Pro Asp Leu Arg Tyr Gln Leu Gly
Phe Ser 100 105 110Val Gln Asn Gly Ile Ile Cys Ser Leu Met Arg Gly
Gly Ile Ala Glu 115 120 125Arg Gly Gly Val Arg Val Gly His Arg Ile
Ile Glu Ile Asn Gly Gln 130 135 140Ser Val Val Ala Thr Pro His Glu
Lys Ile Val His Ile Leu Ser Asn145 150 155 160Ala Val Gly Glu Ile
His Met Lys Thr Met Pro Ala Ala Met Tyr Arg 165 170 175Leu
Leu13092PRTArtificial SequenceSynthetic peptide 130Leu Arg Cys Pro
Pro Val Thr Thr Val Leu Ile Arg Arg Pro Asp Leu1 5 10 15Arg Tyr Gln
Leu Gly Phe Ser Val Gln Asn Gly Ile Ile Cys Ser Leu 20 25 30Met Arg
Gly Gly Ile Ala Glu Arg Gly Gly Val Arg Val Gly His Arg 35 40 45Ile
Ile Glu Ile Asn Gly Gln Ser Val Val Ala Thr Pro His Glu Lys 50 55
60Ile Val His Ile Leu Ser Asn Ala Val Gly Glu Ile His Met Lys Thr65
70 75 80Met Pro Ala Ala Met Tyr Arg Leu Leu Asn Ser Ser 85
90131106PRTArtificial SequenceSynthetic peptide 131His Asn Gly Asp
Leu Asp His Phe Ser Asn Ser Asp Asn Cys Arg Glu1 5 10 15Val His Leu
Glu Lys Arg Arg Gly Glu Gly Leu Gly Val Ala Leu Val 20 25 30Glu Ser
Gly Trp Gly Ser Leu Leu Pro Thr Ala Val Ile Ala Asn Leu 35 40 45Leu
His Gly Gly Pro Ala Glu Arg Ser Gly Ala Leu Ser Ile Gly Asp 50 55
60Arg Leu Thr Ala Ile Asn Gly Thr Ser Leu Val Gly Leu Pro Leu Ala65
70 75 80Ala Cys Gln Ala Ala Val Arg Glu Thr Lys Ser Gln Thr Ser Val
Thr 85 90 95Leu Ser Ile Val His Cys Pro Pro Val Thr 100
10513290PRTArtificial SequenceSynthetic peptide 132Pro Val Thr Thr
Ala Ile Ile His Arg Pro His Ala Arg Glu Gln Leu1 5 10 15Gly Phe Cys
Val Glu Asp Gly Ile Ile Cys Ser Leu Leu Arg Gly Gly 20 25 30Ile Ala
Glu Arg Gly Gly Ile Arg Val Gly His Arg Ile Ile Glu Ile 35 40 45Asn
Gly Gln Ser Val Val Ala Thr Pro His Ala Arg Ile Ile Glu Leu 50 55
60Leu Thr Glu Ala Tyr Gly Glu Val His Ile Lys Thr Met Pro Ala Ala65
70 75 80Thr Tyr Arg Leu Leu Thr Gly Asn Ser Ser 85
90133103PRTArtificial SequenceSynthetic peptide 133Leu Ser Asn Ser
Asp Asn Cys Arg Glu Val His Leu Glu Lys Arg Arg1 5 10 15Gly Glu Gly
Leu Gly Val Ala Leu Val Glu Ser Gly Trp Gly Ser Leu 20 25 30Leu Pro
Thr Ala Val Ile Ala Asn Leu Leu His Gly Gly Pro Ala Glu 35 40 45Arg
Ser Gly Ala Leu Ser Ile Gly Asp Arg Leu Thr Ala Ile Asn Gly 50 55
60Thr Ser Leu Val Gly Leu Pro Leu Ala Ala Cys Gln Ala Ala Val Arg65
70 75 80Glu Thr Lys Ser Gln Thr Ser Val Thr Leu Ser Ile Val His Cys
Pro 85 90 95Pro Val Thr Thr Ala Ile Met 10013486PRTArtificial
SequenceSynthetic peptide 134Arg Lys Val Arg Leu Ile Gln Phe Glu
Lys Val Thr Glu Glu Pro Met1 5
10 15Gly Ile Thr Leu Lys Leu Asn Glu Lys Gln Ser Cys Thr Val Ala
Arg 20 25 30Ile Leu His Gly Gly Met Ile His Arg Gln Gly Ser Leu His
Val Gly 35 40 45Asp Glu Ile Leu Glu Ile Asn Gly Thr Asn Val Thr Asn
His Ser Val 50 55 60Asp Gln Leu Gln Lys Ala Met Lys Glu Thr Lys Gly
Met Ile Ser Leu65 70 75 80Lys Val Ile Pro Asn Gln
8513589PRTArtificial SequenceSynthetic peptide 135Pro Val Pro Pro
Asp Ala Val Arg Met Val Gly Ile Arg Lys Thr Ala1 5 10 15Gly Glu His
Leu Gly Val Thr Phe Arg Val Glu Gly Gly Glu Leu Val 20 25 30Ile Ala
Arg Ile Leu His Gly Gly Met Val Ala Gln Gln Gly Leu Leu 35 40 45His
Val Gly Asp Ile Ile Lys Glu Val Asn Gly Gln Pro Val Gly Ser 50 55
60Asp Pro Arg Ala Leu Gln Glu Leu Leu Arg Asn Ala Ser Gly Ser Val65
70 75 80Ile Leu Lys Ile Leu Pro Asn Tyr Gln 8513699PRTArtificial
SequenceSynthetic peptide 136Asn Ile Asp Glu Asp Phe Asp Glu Glu
Ser Val Lys Ile Val Arg Leu1 5 10 15Val Lys Asn Lys Glu Pro Leu Gly
Ala Thr Ile Arg Arg Asp Glu His 20 25 30Ser Gly Ala Val Val Val Ala
Arg Ile Met Arg Gly Gly Ala Ala Asp 35 40 45Arg Ser Gly Leu Val His
Val Gly Asp Glu Leu Arg Glu Val Asn Gly 50 55 60Ile Ala Val Leu His
Lys Arg Pro Asp Glu Ile Ser Gln Ile Leu Ala65 70 75 80Gln Ser Gln
Gly Ser Ile Thr Leu Lys Ile Ile Pro Ala Thr Gln Glu 85 90 95Glu Asp
Arg137100PRTArtificial SequenceSynthetic peptide 137Trp Glu Ala Gly
Ile Gln His Ile Glu Leu Glu Lys Gly Ser Lys Gly1 5 10 15Leu Gly Phe
Ser Ile Leu Asp Tyr Gln Asp Pro Ile Asp Pro Ala Ser 20 25 30Thr Val
Ile Ile Ile Arg Ser Leu Val Pro Gly Gly Ile Ala Glu Lys 35 40 45Asp
Gly Arg Leu Leu Pro Gly Asp Arg Leu Met Phe Val Asn Asp Val 50 55
60Asn Leu Glu Asn Ser Ser Leu Glu Glu Ala Val Glu Ala Leu Lys Gly65
70 75 80Ala Pro Ser Gly Thr Val Arg Ile Gly Val Ala Lys Pro Leu Pro
Leu 85 90 95Ser Pro Glu Glu 10013896PRTArtificial SequenceSynthetic
peptide 138Leu Gln Gly Leu Arg Thr Val Glu Met Lys Lys Gly Pro Thr
Asp Ser1 5 10 15Leu Gly Ile Ser Ile Ala Gly Gly Val Gly Ser Pro Leu
Gly Asp Val 20 25 30Pro Ile Phe Ile Ala Met Met His Pro Thr Gly Val
Ala Ala Gln Thr 35 40 45Gln Lys Leu Arg Val Gly Asp Arg Ile Val Thr
Ile Cys Gly Thr Ser 50 55 60Thr Glu Gly Met Thr His Thr Gln Ala Val
Asn Leu Leu Lys Asn Ala65 70 75 80Ser Gly Ser Ile Glu Met Gln Val
Val Ala Gly Gly Asp Val Ser Val 85 90 9513997PRTArtificial
SequenceSynthetic peptide 139Pro Val His Trp Gln His Met Glu Thr
Ile Glu Leu Val Asn Asp Gly1 5 10 15Ser Gly Leu Gly Phe Gly Ile Ile
Gly Gly Lys Ala Thr Gly Val Ile 20 25 30Val Lys Thr Ile Leu Pro Gly
Gly Val Ala Asp Gln His Gly Arg Leu 35 40 45Cys Ser Gly Asp His Ile
Leu Lys Ile Gly Asp Thr Asp Leu Ala Gly 50 55 60Met Ser Ser Glu Gln
Val Ala Gln Val Leu Arg Gln Cys Gly Asn Arg65 70 75 80Val Lys Leu
Met Ile Ala Arg Gly Ala Ile Glu Glu Arg Thr Ala Pro 85 90
95Thr14098PRTArtificial SequenceSynthetic peptide 140Gln Glu Ser
Glu Thr Phe Asp Val Glu Leu Thr Lys Asn Val Gln Gly1 5 10 15Leu Gly
Ile Thr Ile Ala Gly Tyr Ile Gly Asp Lys Lys Leu Glu Pro 20 25 30Ser
Gly Ile Phe Val Lys Ser Ile Thr Lys Ser Ser Ala Val Glu His 35 40
45Asp Gly Arg Ile Gln Ile Gly Asp Gln Ile Ile Ala Val Asp Gly Thr
50 55 60Asn Leu Gln Gly Phe Thr Asn Gln Gln Ala Val Glu Val Leu Arg
His65 70 75 80Thr Gly Gln Thr Val Leu Leu Thr Leu Met Arg Arg Gly
Met Lys Gln 85 90 95Glu Ala14198PRTArtificial SequenceSynthetic
peptide 141Lys Glu Glu Glu Val Cys Asp Thr Leu Thr Ile Glu Leu Gln
Lys Lys1 5 10 15Pro Gly Lys Gly Leu Gly Leu Ser Ile Val Gly Lys Arg
Asn Asp Thr 20 25 30Gly Val Phe Val Ser Asp Ile Val Lys Gly Gly Ile
Ala Asp Ala Asp 35 40 45Gly Arg Leu Met Gln Gly Asp Gln Ile Leu Met
Val Asn Gly Glu Asp 50 55 60Val Arg Asn Ala Thr Gln Glu Ala Val Ala
Ala Leu Leu Lys Cys Ser65 70 75 80Leu Gly Thr Val Thr Leu Glu Val
Gly Arg Ile Lys Ala Gly Pro Phe 85 90 95His Ser 14295PRTArtificial
SequenceSynthetic peptide 142Leu Thr Gly Glu Leu His Met Ile Glu
Leu Glu Lys Gly His Ser Gly1 5 10 15Leu Gly Leu Ser Leu Ala Gly Asn
Lys Asp Arg Ser Arg Met Ser Val 20 25 30Phe Ile Val Gly Ile Asp Pro
Asn Gly Ala Ala Gly Lys Asp Gly Arg 35 40 45Leu Gln Ile Ala Asp Glu
Leu Leu Glu Ile Asn Gly Gln Ile Leu Tyr 50 55 60Gly Arg Ser His Gln
Asn Ala Ser Ser Ile Ile Lys Cys Ala Pro Ser65 70 75 80Lys Val Lys
Ile Ile Phe Ile Arg Asn Lys Asp Ala Val Asn Gln 85 90
9514391PRTArtificial SequenceSynthetic peptide 143Leu Gly Pro Pro
Gln Cys Lys Ser Ile Thr Leu Glu Arg Gly Pro Asp1 5 10 15Gly Leu Gly
Phe Ser Ile Val Gly Gly Tyr Gly Ser Pro His Gly Asp 20 25 30Leu Pro
Ile Tyr Val Lys Thr Val Phe Ala Lys Gly Ala Ala Ser Glu 35 40 45Asp
Gly Arg Leu Lys Arg Gly Asp Gln Ile Ile Ala Val Asn Gly Gln 50 55
60Ser Leu Glu Gly Val Thr His Glu Glu Ala Val Ala Ile Leu Lys Arg65
70 75 80Thr Lys Gly Thr Val Thr Leu Met Val Leu Ser 85
9014499PRTArtificial SequenceSynthetic peptide 144Arg Asn Val Ser
Lys Glu Ser Phe Glu Arg Thr Ile Asn Ile Ala Lys1 5 10 15Gly Asn Ser
Ser Leu Gly Met Thr Val Ser Ala Asn Lys Asp Gly Leu 20 25 30Gly Met
Ile Val Arg Ser Ile Ile His Gly Gly Ala Ile Ser Arg Asp 35 40 45Gly
Arg Ile Ala Ile Gly Asp Cys Ile Leu Ser Ile Asn Glu Glu Ser 50 55
60Thr Ile Ser Val Thr Asn Ala Gln Ala Arg Ala Met Leu Arg Arg His65
70 75 80Ser Leu Ile Gly Pro Asp Ile Lys Ile Thr Tyr Val Pro Ala Glu
His 85 90 95Leu Glu Glu14595PRTArtificial SequenceSynthetic peptide
145Leu Pro Gly Cys Glu Thr Thr Ile Glu Ile Ser Lys Gly Arg Thr Gly1
5 10 15Leu Gly Leu Ser Ile Val Gly Gly Ser Asp Thr Leu Leu Gly Ala
Ile 20 25 30Ile Ile His Glu Val Tyr Glu Glu Gly Ala Ala Cys Lys Asp
Gly Arg 35 40 45Leu Trp Ala Gly Asp Gln Ile Leu Glu Val Asn Gly Ile
Asp Leu Arg 50 55 60Lys Ala Thr His Asp Glu Ala Ile Asn Val Leu Arg
Gln Thr Pro Gln65 70 75 80Arg Val Arg Leu Thr Leu Tyr Arg Asp Glu
Ala Pro Tyr Lys Glu 85 90 95146112PRTArtificial SequenceSynthetic
peptide 146Leu Asn Trp Asn Gln Pro Arg Arg Val Glu Leu Trp Arg Glu
Pro Ser1 5 10 15Lys Ser Leu Gly Ile Ser Ile Val Gly Gly Arg Gly Met
Gly Ser Arg 20 25 30Leu Ser Asn Gly Glu Val Met Arg Gly Ile Phe Ile
Lys His Val Leu 35 40 45Glu Asp Ser Pro Ala Gly Lys Asn Gly Thr Leu
Lys Pro Gly Asp Arg 50 55 60Ile Val Glu Val Asp Gly Met Asp Leu Arg
Asp Ala Ser His Glu Gln65 70 75 80Ala Val Glu Ala Ile Arg Lys Ala
Gly Asn Pro Val Val Phe Met Val 85 90 95Gln Ser Ile Ile Asn Arg Pro
Arg Lys Ser Pro Leu Pro Ser Leu Leu 100 105 11014794PRTArtificial
SequenceSynthetic peptide 147Leu Ser Ser Phe Lys Asn Val Gln His
Leu Glu Leu Pro Lys Asp Gln1 5 10 15Gly Gly Leu Gly Ile Ala Ile Ser
Glu Glu Asp Thr Leu Ser Gly Val 20 25 30Ile Ile Lys Ser Leu Thr Glu
His Gly Val Ala Ala Thr Asp Gly Arg 35 40 45Leu Lys Val Gly Asp Gln
Ile Leu Ala Val Asp Asp Glu Ile Val Val 50 55 60Gly Tyr Pro Ile Glu
Lys Phe Ile Ser Leu Leu Lys Thr Ala Lys Met65 70 75 80Thr Val Lys
Leu Thr Ile His Ala Glu Asn Pro Asp Ser Gln 85 9014899PRTArtificial
SequenceSynthetic peptide 148Gln Gly Arg His Val Glu Val Phe Glu
Leu Leu Lys Pro Pro Ser Gly1 5 10 15Gly Leu Gly Phe Ser Val Val Gly
Leu Arg Ser Glu Asn Arg Gly Glu 20 25 30Leu Gly Ile Phe Val Gln Glu
Ile Gln Glu Gly Ser Val Ala His Arg 35 40 45Asp Gly Arg Leu Lys Glu
Thr Asp Gln Ile Leu Ala Ile Asn Gly Gln 50 55 60Ala Leu Asp Gln Thr
Ile Thr His Gln Gln Ala Ile Ser Ile Leu Gln65 70 75 80Lys Ala Lys
Asp Thr Val Gln Leu Val Ile Ala Arg Gly Ser Leu Pro 85 90 95Gln Leu
Val14992PRTArtificial SequenceSynthetic peptide 149Leu Asn Tyr Glu
Ile Val Val Ala His Val Ser Lys Phe Ser Glu Asn1 5 10 15Ser Gly Leu
Gly Ile Ser Leu Glu Ala Thr Val Gly His His Phe Ile 20 25 30Arg Ser
Val Leu Pro Glu Gly Pro Val Gly His Ser Gly Lys Leu Phe 35 40 45Ser
Gly Asp Glu Leu Leu Glu Val Asn Gly Ile Thr Leu Leu Gly Glu 50 55
60Asn His Gln Asp Val Val Asn Ile Leu Lys Glu Leu Pro Ile Glu Val65
70 75 80Thr Met Val Cys Cys Arg Arg Thr Val Pro Pro Thr 85
9015090PRTArtificial SequenceSynthetic peptide 150Ile Thr Leu Leu
Lys Gly Pro Lys Gly Leu Gly Phe Ser Ile Ala Gly1 5 10 15Gly Ile Gly
Asn Gln His Ile Pro Gly Asp Asn Ser Ile Tyr Ile Thr 20 25 30Lys Ile
Ile Glu Gly Gly Ala Ala Gln Lys Asp Gly Arg Leu Gln Ile 35 40 45Gly
Asp Arg Leu Leu Ala Val Asn Asn Thr Asn Leu Gln Asp Val Arg 50 55
60His Glu Glu Ala Val Ala Ser Leu Lys Asn Thr Ser Asp Met Val Tyr65
70 75 80Leu Lys Val Ala Lys Pro Gly Ser Leu Glu 85
9015193PRTArtificial SequenceSynthetic peptide 151Ile Gln Tyr Glu
Glu Ile Val Leu Glu Arg Gly Asn Ser Gly Leu Gly1 5 10 15Phe Ser Ile
Ala Gly Gly Ile Asp Asn Pro His Val Pro Asp Asp Pro 20 25 30Gly Ile
Phe Ile Thr Lys Ile Ile Pro Gly Gly Ala Ala Ala Met Asp 35 40 45Gly
Arg Leu Gly Val Asn Asp Cys Val Leu Arg Val Asn Glu Val Glu 50 55
60Val Ser Glu Val Val His Ser Arg Ala Val Glu Ala Leu Lys Glu Ala65
70 75 80Gly Pro Val Val Arg Leu Val Val Arg Arg Arg Gln Asn 85
90152119PRTArtificial SequenceSynthetic peptide 152Ile Leu Leu His
Lys Gly Ser Thr Gly Leu Gly Phe Asn Ile Val Gly1 5 10 15Gly Glu Asp
Gly Glu Gly Ile Phe Val Ser Phe Ile Leu Ala Gly Gly 20 25 30Pro Ala
Asp Leu Ser Gly Glu Leu Arg Arg Gly Asp Arg Ile Leu Ser 35 40 45Val
Asn Gly Val Asn Leu Arg Asn Ala Thr His Glu Gln Ala Ala Ala 50 55
60Ala Leu Lys Arg Ala Gly Gln Ser Val Thr Ile Val Ala Gln Tyr Arg65
70 75 80Pro Glu Glu Tyr Ser Arg Phe Glu Ser Lys Ile His Asp Leu Arg
Glu 85 90 95Gln Met Met Asn Ser Ser Met Ser Ser Gly Ser Gly Ser Leu
Arg Thr 100 105 110Ser Glu Lys Arg Ser Leu Glu
115153187PRTArtificial SequenceSynthetic peptide 153Tyr Glu Glu Ile
Val Leu Glu Arg Gly Asn Ser Gly Leu Gly Phe Ser1 5 10 15Ile Ala Gly
Gly Ile Asp Asn Pro His Val Pro Asp Asp Pro Gly Ile 20 25 30Phe Ile
Thr Lys Ile Ile Pro Gly Gly Ala Ala Ala Met Asp Gly Arg 35 40 45Leu
Gly Val Asn Asp Cys Val Leu Arg Val Asn Glu Val Glu Val Ser 50 55
60Glu Val Val His Ser Arg Ala Val Glu Ala Leu Lys Glu Ala Gly Pro65
70 75 80Val Val Arg Leu Val Val Arg Arg Arg Gln Pro Pro Pro Glu Thr
Ile 85 90 95Met Glu Val Asn Leu Leu Lys Gly Pro Lys Gly Leu Gly Phe
Ser Ile 100 105 110Ala Gly Gly Ile Gly Asn Gln His Ile Pro Gly Asp
Asn Ser Ile Tyr 115 120 125Ile Thr Lys Ile Ile Glu Gly Gly Ala Ala
Gln Lys Asp Gly Arg Leu 130 135 140Gln Ile Gly Asp Arg Leu Leu Ala
Val Asn Asn Thr Asn Leu Gln Asp145 150 155 160Val Arg His Glu Glu
Ala Val Ala Ser Leu Lys Asn Thr Ser Asp Met 165 170 175Val Tyr Leu
Lys Val Ala Lys Pro Gly Ser Leu 180 185154106PRTArtificial
SequenceSynthetic peptide 154Arg Val Glu Arg Leu Glu Leu Phe Pro
Val Glu Leu Glu Lys Asp Ser1 5 10 15Glu Gly Leu Gly Ile Ser Ile Ile
Gly Met Gly Ala Gly Ala Asp Met 20 25 30Gly Leu Glu Lys Leu Gly Ile
Phe Val Lys Thr Val Thr Glu Gly Gly 35 40 45Ala Ala His Arg Asp Gly
Arg Ile Gln Val Asn Asp Leu Leu Val Glu 50 55 60Val Asp Gly Thr Ser
Leu Val Gly Val Thr Gln Ser Phe Ala Ala Ser65 70 75 80Val Leu Arg
Asn Thr Lys Gly Arg Val Arg Cys Arg Phe Met Ile Gly 85 90 95Arg Glu
Arg Pro Gly Glu Gln Ser Glu Val 100 10515590PRTArtificial
SequenceSynthetic peptide 155Gln Pro Asn Val Ile Ser Val Arg Leu
Phe Lys Arg Lys Val Gly Gly1 5 10 15Leu Gly Phe Leu Val Lys Glu Arg
Val Ser Lys Pro Pro Val Ile Ile 20 25 30Ser Asp Leu Ile Arg Gly Gly
Ala Ala Glu Gln Ser Gly Leu Ile Gln 35 40 45Ala Gly Asp Ile Ile Leu
Ala Val Asn Gly Arg Pro Leu Val Asp Leu 50 55 60Ser Tyr Asp Ser Ala
Leu Glu Val Leu Arg Gly Ile Ala Ser Glu Thr65 70 75 80His Val Val
Leu Ile Leu Arg Gly Pro Glu 85 90156103PRTArtificial
SequenceSynthetic peptide 156Pro Ser Asp Thr Ser Ser Glu Asp Gly
Val Arg Arg Ile Val His Leu1 5 10 15Tyr Thr Thr Ser Asp Asp Phe Cys
Leu Gly Phe Asn Ile Arg Gly Gly 20 25 30Lys Glu Phe Gly Leu Gly Ile
Tyr Val Ser Lys Val Asp His Gly Gly 35 40 45Leu Ala Glu Glu Asn Gly
Ile Lys Val Gly Asp Gln Val Leu Ala Ala 50 55 60Asn Gly Val Arg Phe
Asp Asp Ile Ser His Ser Gln Ala Val Glu Val65 70 75 80Leu Lys Gly
Gln Thr His Ile Met Leu Thr Ile Lys Glu Thr Gly Arg 85 90 95Tyr Pro
Ala Tyr Lys Glu Met 100157104PRTArtificial SequenceSynthetic
peptide 157Glu Ala Asn Ser Asp Glu Ser Asp Ile Ile His Ser Val Arg
Val Glu1 5 10 15Lys Ser Pro Ala Gly Arg Leu Gly Phe Ser Val
Arg Gly Gly Ser Glu 20 25 30His Gly Leu Gly Ile Phe Val Ser Lys Val
Glu Glu Gly Ser Ser Ala 35 40 45Glu Arg Ala Gly Leu Cys Val Gly Asp
Lys Ile Thr Glu Val Asn Gly 50 55 60Leu Ser Leu Glu Ser Thr Thr Met
Gly Ser Ala Val Lys Val Leu Thr65 70 75 80Ser Ser Ser Arg Leu His
Met Met Val Arg Arg Met Gly Arg Val Pro 85 90 95Gly Ile Lys Phe Ser
Lys Glu Lys 100158116PRTArtificial SequenceSynthetic peptide 158Asp
Lys Ile Lys Lys Phe Leu Thr Glu Ser His Asp Arg Gln Ala Lys1 5 10
15Gly Lys Ala Ile Thr Lys Lys Lys Tyr Ile Gly Ile Arg Met Met Ser
20 25 30Leu Thr Ser Ser Lys Ala Lys Glu Leu Lys Asp Arg His Arg Asp
Phe 35 40 45Pro Asp Val Ile Ser Gly Ala Tyr Ile Ile Glu Val Ile Pro
Asp Thr 50 55 60Pro Ala Glu Ala Gly Gly Leu Lys Glu Asn Asp Val Ile
Ile Ser Ile65 70 75 80Asn Gly Gln Ser Val Val Ser Ala Asn Asp Val
Ser Asp Val Ile Lys 85 90 95Arg Glu Ser Thr Leu Asn Met Val Val Arg
Arg Gly Asn Glu Asp Ile 100 105 110Met Ile Thr Val
11515998PRTArtificial SequenceSynthetic peptide 159Tyr Arg Pro Arg
Asp Asp Ser Phe His Val Ile Leu Asn Lys Ser Ser1 5 10 15Pro Glu Glu
Gln Leu Gly Ile Lys Leu Val Arg Lys Val Asp Glu Pro 20 25 30Gly Val
Phe Ile Phe Asn Ala Leu Asp Gly Gly Val Ala Tyr Arg His 35 40 45Gly
Gln Leu Glu Glu Asn Asp Arg Val Leu Ala Ile Asn Gly His Asp 50 55
60Leu Arg Tyr Gly Ser Pro Glu Ser Ala Ala His Leu Ile Gln Ala Ser65
70 75 80Glu Arg Arg Val His Leu Val Val Ser Arg Gln Val Arg Gln Arg
Ser 85 90 95Pro Asp160100PRTArtificial SequenceSynthetic peptide
160Pro Thr Ile Thr Cys His Glu Lys Val Val Asn Ile Gln Lys Asp Pro1
5 10 15Gly Glu Ser Leu Gly Met Thr Val Ala Gly Gly Ala Ser His Arg
Glu 20 25 30Trp Asp Leu Pro Ile Tyr Val Ile Ser Val Glu Pro Gly Gly
Val Ile 35 40 45Ser Arg Asp Gly Arg Ile Lys Thr Gly Asp Ile Leu Leu
Asn Val Asp 50 55 60Gly Val Glu Leu Thr Glu Val Ser Arg Ser Glu Ala
Val Ala Leu Leu65 70 75 80Lys Arg Thr Ser Ser Ser Ile Val Leu Lys
Ala Leu Glu Val Lys Glu 85 90 95Tyr Glu Pro Gln
10016197PRTArtificial SequenceSynthetic peptide 161Pro Asp Gly Glu
Ile Thr Ser Ile Lys Ile Asn Arg Val Asp Pro Ser1 5 10 15Glu Ser Leu
Ser Ile Arg Leu Val Gly Gly Ser Glu Thr Pro Leu Val 20 25 30His Ile
Ile Ile Gln His Ile Tyr Arg Asp Gly Val Ile Ala Arg Asp 35 40 45Gly
Arg Leu Leu Pro Arg Asp Ile Ile Leu Lys Val Asn Gly Met Asp 50 55
60Ile Ser Asn Val Pro His Asn Tyr Ala Val Arg Leu Leu Arg Gln Pro65
70 75 80Cys Gln Val Leu Trp Leu Thr Val Met Arg Glu Gln Lys Phe Arg
Ser 85 90 95Arg16299PRTArtificial SequenceSynthetic peptide 162Pro
Arg Cys Leu Tyr Asn Cys Lys Asp Ile Val Leu Arg Arg Asn Thr1 5 10
15Ala Gly Ser Leu Gly Phe Cys Ile Val Gly Gly Tyr Glu Glu Tyr Asn
20 25 30Gly Asn Lys Pro Phe Phe Ile Lys Ser Ile Val Glu Gly Thr Pro
Ala 35 40 45Tyr Asn Asp Gly Arg Ile Arg Cys Gly Asp Ile Leu Leu Ala
Val Asn 50 55 60Gly Arg Ser Thr Ser Gly Met Ile His Ala Cys Leu Ala
Arg Leu Leu65 70 75 80Lys Glu Leu Lys Gly Arg Ile Thr Leu Thr Ile
Val Ser Trp Pro Gly 85 90 95Thr Phe Leu 163101PRTArtificial
SequenceSynthetic peptide 163Leu Leu Thr Glu Glu Glu Ile Asn Leu
Thr Arg Gly Pro Ser Gly Leu1 5 10 15Gly Phe Asn Ile Val Gly Gly Thr
Asp Gln Gln Tyr Val Ser Asn Asp 20 25 30Ser Gly Ile Tyr Val Ser Arg
Ile Lys Glu Asn Gly Ala Ala Ala Leu 35 40 45Asp Gly Arg Leu Gln Glu
Gly Asp Lys Ile Leu Ser Val Asn Gly Gln 50 55 60Asp Leu Lys Asn Leu
Leu His Gln Asp Ala Val Asp Leu Phe Arg Asn65 70 75 80Ala Gly Tyr
Ala Val Ser Leu Arg Val Gln His Arg Leu Gln Val Gln 85 90 95Asn Gly
Ile His Ser 10016494PRTArtificial SequenceSynthetic peptide 164Pro
Val Asp Ala Ile Arg Ile Leu Gly Ile His Lys Arg Ala Gly Glu1 5 10
15Pro Leu Gly Val Thr Phe Arg Val Glu Asn Asn Asp Leu Val Ile Ala
20 25 30Arg Ile Leu His Gly Gly Met Ile Asp Arg Gln Gly Leu Leu His
Val 35 40 45Gly Asp Ile Ile Lys Glu Val Asn Gly His Glu Val Gly Asn
Asn Pro 50 55 60Lys Glu Leu Gln Glu Leu Leu Lys Asn Ile Ser Gly Ser
Val Thr Leu65 70 75 80Lys Ile Leu Pro Ser Tyr Arg Asp Thr Ile Thr
Pro Gln Gln 85 9016594PRTArtificial SequenceSynthetic peptide
165Gly Lys Arg Leu Asn Ile Gln Leu Lys Lys Gly Thr Glu Gly Leu Gly1
5 10 15Phe Ser Ile Thr Ser Arg Asp Val Thr Ile Gly Gly Ser Ala Pro
Ile 20 25 30Tyr Val Lys Asn Ile Leu Pro Arg Gly Ala Ala Ile Gln Asp
Gly Arg 35 40 45Leu Lys Ala Gly Asp Arg Leu Ile Glu Val Asn Gly Val
Asp Leu Val 50 55 60Gly Lys Ser Gln Glu Glu Val Val Ser Leu Leu Arg
Ser Thr Lys Met65 70 75 80Glu Gly Thr Val Ser Leu Leu Val Phe Arg
Gln Glu Asp Ala 85 90166106PRTArtificial SequenceSynthetic peptide
166Ile Pro Asn Phe Ser Leu Asp Asp Met Val Lys Leu Val Glu Val Pro1
5 10 15Asn Asp Gly Gly Pro Leu Gly Ile His Val Val Pro Phe Ser Ala
Arg 20 25 30Gly Gly Arg Thr Leu Gly Leu Leu Val Lys Arg Leu Glu Lys
Gly Gly 35 40 45Lys Ala Glu His Glu Asn Leu Phe Arg Glu Asn Asp Cys
Ile Val Arg 50 55 60Ile Asn Asp Gly Asp Leu Arg Asn Arg Arg Phe Glu
Gln Ala Gln His65 70 75 80Met Phe Arg Gln Ala Met Arg Thr Pro Ile
Ile Trp Phe His Val Val 85 90 95Pro Ala Ala Asn Lys Glu Gln Tyr Glu
Gln 100 105167113PRTArtificial SequenceSynthetic peptide 167Pro Arg
Glu Phe Leu Thr Phe Glu Val Pro Leu Asn Asp Ser Gly Ser1 5 10 15Ala
Gly Leu Gly Val Ser Val Lys Gly Asn Arg Ser Lys Glu Asn His 20 25
30Ala Asp Leu Gly Ile Phe Val Lys Ser Ile Ile Asn Gly Gly Ala Ala
35 40 45Ser Lys Asp Gly Arg Leu Arg Val Asn Asp Gln Leu Ile Ala Val
Asn 50 55 60Gly Glu Ser Leu Leu Gly Lys Thr Asn Gln Asp Ala Met Glu
Thr Leu65 70 75 80Arg Arg Ser Met Ser Thr Glu Gly Asn Lys Arg Gly
Met Ile Gln Leu 85 90 95Ile Val Ala Ser Arg Ile Ser Lys Cys Asn Glu
Leu Lys Ser Asn Ser 100 105 110Ser16899PRTArtificial
SequenceSynthetic peptide 168Ile Ser Asn Lys Asn Ala Lys Lys Ile
Lys Ile Asp Leu Lys Lys Gly1 5 10 15Pro Glu Gly Leu Gly Phe Thr Val
Val Thr Arg Asp Ser Ser Ile His 20 25 30Gly Pro Gly Pro Ile Phe Val
Lys Asn Ile Leu Pro Lys Gly Ala Ala 35 40 45Ile Lys Asp Gly Arg Leu
Gln Ser Gly Asp Arg Ile Leu Glu Val Asn 50 55 60Gly Arg Asp Val Thr
Gly Arg Thr Gln Glu Glu Leu Val Ala Met Leu65 70 75 80Arg Ser Thr
Lys Gln Gly Glu Thr Ala Ser Leu Val Ile Ala Arg Gln 85 90 95Glu Gly
His169106PRTArtificial SequenceSynthetic peptide 169Ile Thr Ser Glu
Gln Leu Thr Phe Glu Ile Pro Leu Asn Asp Ser Gly1 5 10 15Ser Ala Gly
Leu Gly Val Ser Leu Lys Gly Asn Lys Ser Arg Glu Thr 20 25 30Gly Thr
Asp Leu Gly Ile Phe Ile Lys Ser Ile Ile His Gly Gly Ala 35 40 45Ala
Phe Lys Asp Gly Arg Leu Arg Met Asn Asp Gln Leu Ile Ala Val 50 55
60Asn Gly Glu Ser Leu Leu Gly Lys Ser Asn His Glu Ala Met Glu Thr65
70 75 80Leu Arg Arg Ser Met Ser Met Glu Gly Asn Ile Arg Gly Met Ile
Gln 85 90 95Leu Val Ile Leu Arg Arg Pro Glu Arg Pro 100
105170104PRTArtificial SequenceSynthetic peptide 170Ile Pro Arg Thr
Lys Asp Thr Leu Ser Asp Met Thr Arg Thr Val Glu1 5 10 15Ile Ser Gly
Glu Gly Gly Pro Leu Gly Ile His Val Val Pro Phe Phe 20 25 30Ser Ser
Leu Ser Gly Arg Ile Leu Gly Leu Phe Ile Arg Gly Ile Glu 35 40 45Asp
Asn Ser Arg Ser Lys Arg Glu Gly Leu Phe His Glu Asn Glu Cys 50 55
60Ile Val Lys Ile Asn Asn Val Asp Leu Val Asp Lys Thr Phe Ala Gln65
70 75 80Ala Gln Asp Val Phe Arg Gln Ala Met Lys Ser Pro Ser Val Leu
Leu 85 90 95His Val Leu Pro Pro Gln Asn Arg 100171104PRTArtificial
SequenceSynthetic peptide 171Pro Glu Thr His Arg Arg Val Arg Leu
His Lys His Gly Ser Asp Arg1 5 10 15Pro Leu Gly Phe Tyr Ile Arg Asp
Gly Met Ser Val Arg Val Ala Pro 20 25 30Gln Gly Leu Glu Arg Val Pro
Gly Ile Phe Ile Ser Arg Leu Val Arg 35 40 45Gly Gly Leu Ala Glu Ser
Thr Gly Leu Leu Ala Val Ser Asp Glu Ile 50 55 60Leu Glu Val Asn Gly
Ile Glu Val Ala Gly Lys Thr Leu Asp Gln Val65 70 75 80Thr Asp Met
Met Val Ala Asn Ser His Asn Leu Ile Val Thr Val Lys 85 90 95Pro Ala
Asn Gln Arg Asn Asn Val 100172120PRTArtificial SequenceSynthetic
peptide 172Ile Pro Val Ser Ser Ile Ile Asp Val Asp Ile Leu Pro Glu
Thr His1 5 10 15Arg Arg Val Arg Leu Tyr Lys Tyr Gly Thr Glu Lys Pro
Leu Gly Phe 20 25 30Tyr Ile Arg Asp Gly Ser Ser Val Arg Val Thr Pro
His Gly Leu Glu 35 40 45Lys Val Pro Gly Ile Phe Ile Ser Arg Leu Val
Pro Gly Gly Leu Ala 50 55 60Gln Ser Thr Gly Leu Leu Ala Val Asn Asp
Glu Val Leu Glu Val Asn65 70 75 80Gly Ile Glu Val Ser Gly Lys Ser
Leu Asp Gln Val Thr Asp Met Met 85 90 95Ile Ala Asn Ser Arg Asn Leu
Ile Ile Thr Val Arg Pro Ala Asn Gln 100 105 110Arg Asn Asn Arg Ile
His Arg Asp 115 120173111PRTArtificial SequenceSynthetic peptide
173Ile Asp Val Asp Leu Val Pro Glu Thr His Arg Arg Val Arg Leu His1
5 10 15Arg His Gly Cys Glu Lys Pro Leu Gly Phe Tyr Ile Arg Asp Gly
Ala 20 25 30Ser Val Arg Val Thr Pro His Gly Leu Glu Lys Val Pro Gly
Ile Phe 35 40 45Ile Ser Arg Met Val Pro Gly Gly Leu Ala Glu Ser Thr
Gly Leu Leu 50 55 60Ala Val Asn Asp Glu Val Leu Glu Val Asn Gly Ile
Glu Val Ala Gly65 70 75 80Lys Thr Leu Asp Gln Val Thr Asp Met Met
Ile Ala Asn Ser His Asn 85 90 95Leu Ile Val Thr Val Lys Pro Ala Asn
Gln Arg Asn Asn Val Val 100 105 110174103PRTArtificial
SequenceSynthetic peptide 174Pro Glu Gln Ile Met Gly Lys Asp Val
Arg Leu Leu Arg Ile Lys Lys1 5 10 15Glu Gly Ser Leu Asp Leu Ala Leu
Glu Gly Gly Val Asp Ser Pro Ile 20 25 30Gly Lys Val Val Val Ser Ala
Val Tyr Glu Arg Gly Ala Ala Glu Arg 35 40 45His Gly Gly Ile Val Lys
Gly Asp Glu Ile Met Ala Ile Asn Gly Lys 50 55 60Ile Val Thr Asp Tyr
Thr Leu Ala Glu Ala Asp Ala Ala Leu Gln Lys65 70 75 80Ala Trp Asn
Gln Gly Gly Asp Trp Ile Asp Leu Val Val Ala Val Cys 85 90 95Pro Pro
Lys Glu Tyr Asp Asp 100175102PRTArtificial SequenceSynthetic
peptide 175Ile Pro Gly Asn Arg Glu Asn Lys Glu Lys Lys Val Phe Ile
Ser Leu1 5 10 15Val Gly Ser Arg Gly Leu Gly Cys Ser Ile Ser Ser Gly
Pro Ile Gln 20 25 30Lys Pro Gly Ile Phe Ile Ser His Val Lys Pro Gly
Ser Leu Ser Ala 35 40 45Glu Val Gly Leu Glu Ile Gly Asp Gln Ile Val
Glu Val Asn Gly Val 50 55 60Asp Phe Ser Asn Leu Asp His Lys Glu Ala
Val Asn Val Leu Lys Ser65 70 75 80Ser Arg Ser Leu Thr Ile Ser Ile
Val Ala Ala Ala Gly Arg Glu Leu 85 90 95Phe Met Thr Asp Glu Phe
100176100PRTArtificial SequenceSynthetic peptide 176Arg Ser Arg Lys
Leu Lys Glu Val Arg Leu Asp Arg Leu His Pro Glu1 5 10 15Gly Leu Gly
Leu Ser Val Arg Gly Gly Leu Glu Phe Gly Cys Gly Leu 20 25 30Phe Ile
Ser His Leu Ile Lys Gly Gly Gln Ala Asp Ser Val Gly Leu 35 40 45Gln
Val Gly Asp Glu Ile Val Arg Ile Asn Gly Tyr Ser Ile Ser Ser 50 55
60Cys Thr His Glu Glu Val Ile Asn Leu Ile Arg Thr Lys Lys Thr Val65
70 75 80Ser Ile Lys Val Arg His Ile Gly Leu Ile Pro Val Lys Ser Ser
Pro 85 90 95Asp Glu Phe His 10017792PRTArtificial SequenceSynthetic
peptide 177Arg Leu Cys Tyr Leu Val Lys Glu Gly Gly Ser Tyr Gly Phe
Ser Leu1 5 10 15Lys Thr Val Gln Gly Lys Lys Gly Val Tyr Met Thr Asp
Ile Thr Pro 20 25 30Gln Gly Val Ala Met Arg Ala Gly Val Leu Ala Asp
Asp His Leu Ile 35 40 45Glu Val Asn Gly Glu Asn Val Glu Asp Ala Ser
His Glu Glu Val Val 50 55 60Glu Lys Val Lys Lys Ser Gly Ser Arg Val
Met Phe Leu Leu Val Asp65 70 75 80Lys Glu Thr Asp Lys Arg Glu Phe
Ile Val Thr Asp 85 90178112PRTArtificial SequenceSynthetic peptide
178Gln Phe Lys Arg Glu Thr Ala Ser Leu Lys Leu Leu Pro His Gln Pro1
5 10 15Arg Ile Val Glu Met Lys Lys Gly Ser Asn Gly Tyr Gly Phe Tyr
Leu 20 25 30Arg Ala Gly Ser Glu Gln Lys Gly Gln Ile Ile Lys Asp Ile
Asp Ser 35 40 45Gly Ser Pro Ala Glu Glu Ala Gly Leu Lys Asn Asn Asp
Leu Val Val 50 55 60Ala Val Asn Gly Glu Ser Val Glu Thr Leu Asp His
Asp Ser Val Val65 70 75 80Glu Met Ile Arg Lys Gly Gly Asp Gln Thr
Ser Leu Leu Val Val Asp 85 90 95Lys Glu Thr Asp Asn Met Tyr Arg Leu
Ala Glu Phe Ile Val Thr Asp 100 105 110179324PRTArtificial
SequenceSynthetic peptide 179Arg Leu Cys Tyr Leu Val Lys Glu Gly
Gly Ser Tyr Gly Phe Ser Leu1 5 10 15Lys Thr Val Gln Gly Lys Lys Gly
Val Tyr Met Thr Asp Ile Thr Pro 20 25 30Gln Gly Val Ala Met Arg Ala
Gly Val Leu Ala Asp Asp His Leu Ile 35 40 45Glu Val Asn Gly Glu Asn
Val Glu Asp Ala Ser His Glu Lys Val Val 50 55 60Glu Lys Val Lys Lys
Ser Gly Ser Arg Val Met Phe Leu Leu Val Asp65 70 75 80Lys Glu Thr
Asp Lys Arg His Val Glu Gln Lys Ile Gln Phe Lys Arg 85
90 95Glu Thr Ala Ser Leu Lys Leu Leu Pro His Gln Pro Arg Ile Val
Glu 100 105 110Met Lys Lys Gly Ser Asn Gly Tyr Gly Phe Tyr Leu Arg
Ala Gly Ser 115 120 125Glu Gln Lys Gly Gln Ile Ile Lys Asp Ile Asp
Ser Gly Ser Pro Ala 130 135 140Glu Glu Ala Gly Leu Lys Asn Asn Asp
Leu Val Val Ala Val Asn Gly145 150 155 160Glu Ser Val Glu Thr Leu
Asp His Asp Ser Val Val Glu Met Ile Arg 165 170 175Lys Gly Gly Asp
Gln Thr Ser Leu Leu Val Val Asp Lys Glu Thr Asp 180 185 190Asn Met
Tyr Arg Leu Ala His Phe Ser Pro Phe Leu Tyr Tyr Gln Ser 195 200
205Gln Glu Leu Pro Asn Gly Ser Val Lys Glu Ala Pro Ala Pro Thr Pro
210 215 220Thr Ser Leu Glu Val Ser Ser Pro Pro Asp Thr Thr Glu Glu
Val Asp225 230 235 240His Lys Pro Lys Leu Cys Arg Leu Ala Lys Gly
Glu Asn Gly Tyr Gly 245 250 255Phe His Leu Asn Ala Ile Arg Gly Leu
Pro Gly Ser Phe Ile Lys Glu 260 265 270Val Gln Lys Gly Gly Pro Ala
Asp Leu Ala Gly Leu Glu Asp Glu Asp 275 280 285Val Ile Ile Glu Val
Asn Gly Val Asn Val Leu Asp Glu Pro Tyr Glu 290 295 300Lys Val Val
Asp Arg Ile Gln Ser Ser Gly Lys Asn Val Thr Leu Leu305 310 315
320Val Cys Gly Lys18094PRTArtificial SequenceSynthetic peptide
180Pro Asp Thr Thr Glu Glu Val Asp His Lys Pro Lys Leu Cys Arg Leu1
5 10 15Ala Lys Gly Glu Asn Gly Tyr Gly Phe His Leu Asn Ala Ile Arg
Gly 20 25 30Leu Pro Gly Ser Phe Ile Lys Glu Val Gln Lys Gly Gly Pro
Ala Asp 35 40 45Leu Ala Gly Leu Glu Asp Glu Asp Val Ile Ile Glu Val
Asn Gly Val 50 55 60Asn Val Leu Asp Glu Pro Tyr Glu Lys Val Val Asp
Arg Ile Gln Ser65 70 75 80Ser Gly Lys Asn Val Thr Leu Leu Val Gly
Lys Asn Ser Ser 85 90181101PRTArtificial SequenceSynthetic peptide
181Leu Thr Ser Thr Phe Asn Pro Arg Glu Cys Lys Leu Ser Lys Gln Glu1
5 10 15Gly Gln Asn Tyr Gly Phe Phe Leu Arg Ile Glu Lys Asp Thr Glu
Gly 20 25 30His Leu Val Arg Val Val Glu Lys Cys Ser Pro Ala Glu Lys
Ala Gly 35 40 45Leu Gln Asp Gly Asp Arg Val Leu Arg Ile Asn Gly Val
Phe Val Asp 50 55 60Lys Glu Glu His Met Gln Val Val Asp Leu Val Arg
Lys Ser Gly Asn65 70 75 80Ser Val Thr Leu Leu Val Leu Asp Gly Asp
Ser Tyr Glu Lys Ala Gly 85 90 95Ser His Glu Pro Ser
10018299PRTArtificial SequenceSynthetic peptide 182Leu Gly Ile Pro
Thr Val Pro Gly Lys Val Thr Leu Gln Lys Asp Ala1 5 10 15Gln Asn Leu
Ile Gly Ile Ser Ile Gly Gly Gly Ala Gln Tyr Cys Pro 20 25 30Cys Leu
Tyr Ile Val Gln Val Phe Asp Asn Thr Pro Ala Ala Leu Asp 35 40 45Gly
Thr Val Ala Ala Gly Asp Glu Ile Thr Gly Val Asn Gly Arg Ser 50 55
60Ile Lys Gly Lys Thr Lys Val Glu Val Ala Lys Met Ile Gln Glu Val65
70 75 80Lys Gly Glu Val Thr Ile His Tyr Asn Lys Leu Gln Ala Asp Pro
Lys 85 90 95Gln Gly Met18398PRTArtificial SequenceSynthetic peptide
183Ser Gln Gly Val Gly Pro Ile Arg Lys Val Leu Leu Leu Lys Glu Asp1
5 10 15His Glu Gly Leu Gly Ile Ser Ile Thr Gly Gly Lys Glu His Gly
Val 20 25 30Pro Ile Leu Ile Ser Glu Ile His Pro Gly Gln Pro Ala Asp
Arg Cys 35 40 45Gly Gly Leu His Val Gly Asp Ala Ile Leu Ala Val Asn
Gly Val Asn 50 55 60Leu Arg Asp Thr Lys His Lys Glu Ala Val Thr Ile
Leu Ser Gln Gln65 70 75 80Arg Gly Glu Ile Glu Phe Glu Val Val Tyr
Val Ala Pro Glu Val Asp 85 90 95Ser Asp18498PRTArtificial
SequenceSynthetic peptide 184Thr Ala Glu Ala Thr Val Cys Thr Val
Thr Leu Glu Lys Met Ser Ala1 5 10 15Gly Leu Gly Phe Ser Leu Glu Gly
Gly Lys Gly Ser Leu His Gly Asp 20 25 30Lys Pro Leu Thr Ile Asn Arg
Ile Phe Lys Gly Ala Ala Ser Glu Gln 35 40 45Ser Glu Thr Val Gln Pro
Gly Asp Glu Ile Leu Gln Leu Gly Gly Thr 50 55 60Ala Met Gln Gly Leu
Thr Arg Phe Glu Ala Trp Asn Ile Ile Lys Ala65 70 75 80Leu Pro Asp
Gly Pro Val Thr Ile Val Ile Arg Arg Lys Ser Leu Gln 85 90 95Ser
Lys18597PRTArtificial SequenceSynthetic peptide 185Ile His Val Thr
Ile Leu His Lys Glu Glu Gly Ala Gly Leu Gly Phe1 5 10 15Ser Leu Ala
Gly Gly Ala Asp Leu Glu Asn Lys Val Ile Thr Val His 20 25 30Arg Val
Phe Pro Asn Gly Leu Ala Ser Gln Glu Gly Thr Ile Gln Lys 35 40 45Gly
Asn Glu Val Leu Ser Ile Asn Gly Lys Ser Leu Lys Gly Thr Thr 50 55
60His His Asp Ala Leu Ala Ile Leu Arg Gln Ala Arg Glu Pro Arg Gln65
70 75 80Ala Val Ile Val Thr Arg Lys Leu Thr Pro Glu Glu Phe Ile Val
Thr 85 90 95Asp186214PRTArtificial SequenceSynthetic peptide 186Ile
His Val Thr Ile Leu His Lys Glu Glu Gly Ala Gly Leu Gly Phe1 5 10
15Ser Leu Ala Gly Gly Ala Asp Leu Glu Asn Lys Val Ile Thr Val His
20 25 30Arg Val Phe Pro Asn Gly Leu Ala Ser Gln Glu Gly Thr Ile Gln
Lys 35 40 45Gly Asn Glu Val Leu Ser Ile Asn Gly Lys Ser Leu Lys Gly
Thr Thr 50 55 60His His Asp Ala Leu Ala Ile Leu Arg Gln Ala Arg Glu
Pro Arg Gln65 70 75 80Ala Val Ile Val Thr Arg Lys Leu Thr Pro Glu
Ala Met Pro Asp Leu 85 90 95Asn Ser Ser Thr Asp Ser Ala Ala Ser Ala
Ser Ala Ala Ser Asp Val 100 105 110Ser Val Glu Ser Thr Ala Glu Ala
Thr Val Cys Thr Val Thr Leu Glu 115 120 125Lys Met Ser Ala Gly Leu
Gly Phe Ser Leu Glu Gly Gly Lys Gly Ser 130 135 140Leu His Gly Asp
Lys Pro Leu Thr Ile Asn Arg Ile Phe Lys Gly Ala145 150 155 160Ala
Ser Glu Gln Ser Glu Thr Val Gln Pro Gly Asp Glu Ile Leu Gln 165 170
175Leu Gly Gly Thr Ala Met Gln Gly Leu Thr Arg Phe Glu Ala Trp Asn
180 185 190Ile Ile Lys Ala Leu Pro Asp Gly Pro Val Thr Ile Val Ile
Arg Arg 195 200 205Lys Ser Leu Gln Ser Lys 210187162PRTArtificial
SequenceSynthetic peptide 187Ile Arg Glu Ala Lys Tyr Ser Gly Val
Leu Ser Ser Ile Gly Lys Ile1 5 10 15Phe Lys Glu Glu Gly Leu Leu Gly
Phe Phe Val Gly Leu Ile Pro His 20 25 30Leu Leu Gly Asp Val Val Phe
Leu Trp Gly Cys Asn Leu Leu Ala His 35 40 45Phe Ile Asn Ala Tyr Leu
Val Asp Asp Ser Val Ser Asp Thr Pro Gly 50 55 60Gly Leu Gly Asn Asp
Gln Asn Pro Gly Ser Gln Phe Ser Gln Ala Leu65 70 75 80Ala Ile Arg
Ser Tyr Thr Lys Phe Val Met Gly Ile Ala Val Ser Met 85 90 95Leu Thr
Tyr Pro Phe Leu Leu Val Gly Asp Leu Met Ala Val Asn Asn 100 105
110Cys Gly Leu Gln Ala Gly Leu Pro Pro Tyr Ser Pro Val Phe Lys Ser
115 120 125Trp Ile His Cys Trp Lys Tyr Leu Ser Val Gln Gly Gln Leu
Phe Arg 130 135 140Gly Ser Ser Leu Leu Phe Arg Arg Val Ser Ser Gly
Ser Cys Phe Ala145 150 155 160Leu Glu 188338PRTArtificial
SequenceSynthetic peptide 188Glu Gly Glu Met Glu Tyr Glu Glu Ile
Thr Leu Glu Arg Gly Asn Ser1 5 10 15Gly Leu Gly Phe Ser Ile Ala Gly
Gly Thr Asp Asn Pro His Ile Gly 20 25 30Asp Asp Pro Ser Ile Phe Ile
Thr Lys Ile Ile Pro Gly Gly Ala Ala 35 40 45Ala Gln Asp Gly Arg Leu
Arg Val Asn Asp Ser Ile Leu Phe Val Asn 50 55 60Glu Val Asp Val Arg
Glu Val Thr His Ser Ala Ala Val Glu Ala Leu65 70 75 80Lys Glu Ala
Gly Ser Ile Val Arg Leu Tyr Val Met Arg Arg Lys Pro 85 90 95Pro Ala
Glu Lys Val Met Glu Ile Lys Leu Ile Lys Gly Pro Lys Gly 100 105
110Leu Gly Phe Ser Ile Ala Gly Gly Val Gly Asn Gln His Ile Pro Gly
115 120 125Asp Asn Ser Ile Tyr Val Thr Lys Ile Ile Glu Gly Gly Ala
Ala His 130 135 140Lys Asp Gly Arg Leu Gln Ile Gly Asp Lys Ile Leu
Ala Val Asn Ser145 150 155 160Val Gly Leu Glu Asp Val Met His Glu
Asp Ala Val Ala Ala Leu Lys 165 170 175Asn Thr Tyr Asp Val Val Tyr
Leu Lys Val Ala Lys Pro Ser Asn Ala 180 185 190Tyr Leu Ser Asp Ser
Tyr Ala Pro Pro Asp Ile Thr Thr Ser Tyr Ser 195 200 205Gln His Leu
Asp Asn Glu Ile Ser His Ser Ser Tyr Leu Gly Thr Asp 210 215 220Tyr
Pro Thr Ala Met Thr Pro Thr Ser Pro Arg Arg Tyr Ser Pro Val225 230
235 240Ala Lys Asp Leu Leu Gly Glu Glu Asp Ile Pro Arg Glu Pro Arg
Arg 245 250 255Ile Val Ile His Arg Gly Ser Thr Gly Leu Gly Phe Asn
Ile Val Gly 260 265 270Gly Glu Asp Gly Glu Gly Ile Phe Ile Ser Phe
Ile Leu Ala Gly Gly 275 280 285Pro Ala Asp Leu Ser Gly Glu Leu Arg
Lys Gly Asp Gln Ile Leu Ser 290 295 300Val Asn Gly Val Asp Leu Arg
Asn Ala Ser His Glu Gln Ala Ala Ile305 310 315 320Ala Leu Lys Asn
Ala Gly Gln Thr Val Thr Ile Ile Ala Gln Tyr Lys 325 330 335Pro
Glu189105PRTArtificial SequenceSynthetic peptide 189His Val Met Arg
Arg Lys Pro Pro Ala Glu Lys Val Met Glu Ile Lys1 5 10 15Leu Ile Lys
Gly Pro Lys Gly Leu Gly Phe Ser Ile Ala Gly Gly Val 20 25 30Gly Asn
Gln His Ile Pro Gly Asp Asn Ser Ile Tyr Val Thr Lys Ile 35 40 45Ile
Glu Gly Gly Ala Ala His Lys Asp Gly Arg Leu Gln Ile Gly Asp 50 55
60Lys Ile Leu Ala Val Asn Ser Val Gly Leu Glu Asp Val Met His Glu65
70 75 80Asp Ala Val Ala Ala Leu Lys Asn Thr Tyr Asp Val Val Tyr Leu
Lys 85 90 95Val Ala Lys Pro Ser Asn Ala Tyr Leu 100
10519097PRTArtificial SequenceSynthetic peptide 190Arg Glu Asp Ile
Pro Arg Glu Pro Arg Arg Ile Val Ile His Arg Gly1 5 10 15Ser Thr Gly
Leu Gly Phe Asn Ile Val Gly Gly Glu Asp Gly Glu Gly 20 25 30Ile Phe
Ile Ser Phe Ile Leu Ala Gly Gly Pro Ala Asp Leu Ser Gly 35 40 45Glu
Leu Arg Lys Gly Asp Gln Ile Leu Ser Val Asn Gly Val Asp Leu 50 55
60Arg Asn Ala Ser His Glu Gln Ala Ala Ile Ala Leu Lys Asn Ala Gly65
70 75 80Gln Thr Val Thr Ile Ile Ala Gln Tyr Lys Pro Glu Phe Ile Val
Thr 85 90 95Asp19199PRTArtificial SequenceSynthetic peptide 191Leu
Glu Tyr Glu Glu Ile Thr Leu Glu Arg Gly Asn Ser Gly Leu Gly1 5 10
15Phe Ser Ile Ala Gly Gly Thr Asp Asn Pro His Ile Gly Asp Asp Pro
20 25 30Ser Ile Phe Ile Thr Lys Ile Ile Pro Gly Gly Ala Ala Ala Gln
Asp 35 40 45Gly Arg Leu Arg Val Asn Asp Ser Ile Leu Phe Val Asn Glu
Val Asp 50 55 60Val Arg Glu Val Thr His Ser Ala Ala Val Glu Ala Leu
Lys Glu Ala65 70 75 80Gly Ser Ile Val Arg Leu Tyr Val Met Arg Arg
Lys Pro Pro Ala Glu 85 90 95Asn Ser Ser 19288PRTArtificial
SequenceSynthetic peptide 192Arg Asp Met Ala Glu Ala His Lys Glu
Ala Met Ser Arg Lys Leu Gly1 5 10 15Gln Ser Glu Ser Gln Gly Pro Pro
Arg Ala Phe Ala Lys Val Asn Ser 20 25 30Ile Ser Pro Gly Ser Pro Ala
Ser Ile Ala Gly Leu Gln Val Asp Asp 35 40 45Glu Ile Val Glu Phe Gly
Ser Val Asn Thr Gln Asn Phe Gln Ser Leu 50 55 60His Asn Ile Gly Ser
Val Val Gln His Ser Glu Gly Ala Leu Ala Pro65 70 75 80Thr Ile Leu
Leu Ser Val Ser Met 85193102PRTArtificial SequenceSynthetic peptide
193Gln Asn Asp Asn Gly Asp Ser Tyr Leu Val Leu Ile Arg Ile Thr Pro1
5 10 15Asp Glu Asp Gly Lys Phe Gly Phe Asn Leu Lys Gly Gly Val Asp
Gln 20 25 30Lys Met Pro Leu Val Val Ser Arg Ile Asn Pro Glu Ser Pro
Ala Asp 35 40 45Thr Cys Ile Pro Lys Leu Asn Glu Gly Asp Gln Ile Val
Leu Ile Asn 50 55 60Gly Arg Asp Ile Ser Glu His Thr His Asp Gln Val
Val Met Phe Ile65 70 75 80Lys Ala Ser Arg Glu Ser His Ser Arg Glu
Leu Ala Leu Val Ile Arg 85 90 95Arg Arg Ala Val Arg Ser
10019488PRTArtificial SequenceSynthetic peptide 194Ile Arg Met Lys
Pro Asp Glu Asn Gly Arg Phe Gly Phe Asn Val Lys1 5 10 15Gly Gly Tyr
Asp Gln Lys Met Pro Val Ile Val Ser Arg Val Ala Pro 20 25 30Gly Thr
Pro Ala Asp Leu Cys Val Pro Arg Leu Asn Glu Gly Asp Gln 35 40 45Val
Val Leu Ile Asn Gly Arg Asp Ile Ala Glu His Thr His Asp Gln 50 55
60Val Val Leu Phe Ile Lys Ala Ser Cys Glu Arg His Ser Gly Glu Leu65
70 75 80Met Leu Leu Val Arg Pro Asn Ala 8519595PRTArtificial
SequenceSynthetic peptide 195Gly Asp Ile Phe Glu Val Glu Leu Ala
Lys Asn Asp Asn Ser Leu Gly1 5 10 15Ile Ser Val Thr Gly Gly Val Asn
Thr Ser Val Arg His Gly Gly Ile 20 25 30Tyr Val Lys Ala Val Ile Pro
Gln Gly Ala Ala Glu Ser Asp Gly Arg 35 40 45Ile His Lys Gly Asp Arg
Val Leu Ala Val Asn Gly Val Ser Leu Glu 50 55 60Gly Ala Thr His Lys
Gln Ala Val Glu Thr Leu Arg Asn Thr Gly Gln65 70 75 80Val Val His
Leu Leu Leu Glu Lys Gly Gln Ser Pro Thr Ser Lys 85 90
95196106PRTArtificial SequenceSynthetic peptide 196Pro Glu Arg Glu
Ile Thr Leu Val Asn Leu Lys Lys Asp Ala Lys Tyr1 5 10 15Gly Leu Gly
Phe Gln Ile Ile Gly Gly Glu Lys Met Gly Arg Leu Asp 20 25 30Leu Gly
Ile Phe Ile Ser Ser Val Ala Pro Gly Gly Pro Ala Asp Phe 35 40 45His
Gly Cys Leu Lys Pro Gly Asp Arg Leu Ile Ser Val Asn Ser Val 50 55
60Ser Leu Glu Gly Val Ser His His Ala Ala Ile Glu Ile Leu Gln Asn65
70 75 80Ala Pro Glu Asp Val Thr Leu Val Ile Ser Gln Pro Lys Glu Lys
Ile 85 90 95Ser Lys Val Pro Ser Thr Pro Val His Leu 100
10519798PRTArtificial SequenceSynthetic peptide 197Glu Leu Glu Val
Glu Leu Leu Ile Thr Leu Ile Lys Ser Glu Lys Ala1 5 10 15Ser Leu Gly
Phe Thr Val Thr Lys Gly Asn Gln Arg Ile Gly Cys Tyr 20 25 30Val His
Asp Val Ile Gln Asp Pro Ala Lys Ser Asp Gly Arg Leu Lys 35 40 45Pro
Gly Asp Arg Leu Ile Lys Val Asn Asp Thr Asp Val Thr Asn Met 50 55
60Thr His Thr Asp Ala Val
Asn Leu Leu Arg Ala Ala Ser Lys Thr Val65 70 75 80Arg Leu Val Ile
Gly Arg Val Leu Glu Leu Pro Arg Ile Pro Met Leu 85 90 95Pro
His198104PRTArtificial SequenceSynthetic peptide 198Thr Glu Glu Asn
Thr Phe Glu Val Lys Leu Phe Lys Asn Ser Ser Gly1 5 10 15Leu Gly Phe
Ser Phe Ser Arg Glu Asp Asn Leu Ile Pro Glu Gln Ile 20 25 30Asn Ala
Ser Ile Val Arg Val Lys Lys Leu Phe Ala Gly Gln Pro Ala 35 40 45Ala
Glu Ser Gly Lys Ile Asp Val Gly Asp Val Ile Leu Lys Val Asn 50 55
60Gly Ala Ser Leu Lys Gly Leu Ser Gln Gln Glu Val Ile Ser Ala Leu65
70 75 80Arg Gly Thr Ala Pro Glu Val Phe Leu Leu Leu Cys Arg Pro Pro
Pro 85 90 95Gly Val Leu Pro Glu Ile Asp Thr 10019994PRTArtificial
SequenceSynthetic peptide 199Met Leu Pro His Leu Leu Pro Asp Ile
Thr Leu Thr Cys Asn Lys Glu1 5 10 15Glu Leu Gly Phe Ser Leu Cys Gly
Gly His Asp Ser Leu Tyr Gln Val 20 25 30Val Tyr Ile Ser Asp Ile Asn
Pro Arg Ser Val Ala Ala Ile Glu Gly 35 40 45Asn Leu Gln Leu Leu Asp
Val Ile His Tyr Val Asn Gly Val Ser Thr 50 55 60Gln Gly Met Thr Leu
Glu Glu Val Asn Arg Ala Leu Asp Met Ser Leu65 70 75 80Pro Ser Leu
Val Leu Lys Ala Thr Arg Asn Asp Leu Pro Val 85 9020092PRTArtificial
SequenceSynthetic peptide 200Val Cys Ser Glu Arg Arg Tyr Arg Gln
Ile Thr Ile Pro Arg Gly Lys1 5 10 15Asp Gly Phe Gly Phe Thr Ile Cys
Cys Asp Ser Pro Val Arg Val Gln 20 25 30Ala Val Asp Ser Gly Gly Pro
Ala Glu Arg Ala Gly Leu Gln Gln Leu 35 40 45Asp Thr Val Leu Gln Leu
Asn Glu Arg Pro Val Glu His Trp Lys Cys 50 55 60Val Glu Leu Ala His
Glu Ile Arg Ser Cys Pro Ser Glu Ile Ile Leu65 70 75 80Leu Val Trp
Arg Met Val Pro Gln Val Lys Pro Gly 85 9020193PRTArtificial
SequenceSynthetic peptide 201Arg Pro Ser Pro Pro Arg Val Arg Ser
Val Glu Val Ala Arg Gly Arg1 5 10 15Ala Gly Tyr Gly Phe Thr Leu Ser
Gly Gln Ala Pro Cys Val Leu Ser 20 25 30Cys Val Met Arg Gly Ser Pro
Ala Asp Phe Val Gly Leu Arg Ala Gly 35 40 45Asp Gln Ile Leu Ala Val
Asn Glu Ile Asn Val Lys Lys Ala Ser His 50 55 60Glu Asp Val Val Lys
Leu Ile Gly Lys Cys Ser Gly Val Leu His Met65 70 75 80Val Ile Ala
Glu Gly Val Gly Arg Phe Glu Ser Cys Ser 85 90202124PRTArtificial
SequenceSynthetic peptide 202Ser Glu Asp Glu Thr Phe Ser Trp Pro
Gly Pro Lys Thr Val Thr Leu1 5 10 15Lys Arg Thr Ser Gln Gly Phe Gly
Phe Thr Leu Arg His Phe Ile Val 20 25 30Tyr Pro Pro Glu Ser Ala Ile
Gln Phe Ser Tyr Lys Asp Glu Glu Asn 35 40 45Gly Asn Arg Gly Gly Lys
Gln Arg Asn Arg Leu Glu Pro Met Asp Thr 50 55 60Ile Phe Val Lys Gln
Val Lys Glu Gly Gly Pro Ala Phe Glu Ala Gly65 70 75 80Leu Cys Thr
Gly Asp Arg Ile Ile Lys Val Asn Gly Glu Ser Val Ile 85 90 95Gly Lys
Thr Tyr Ser Gln Val Ile Ala Leu Ile Gln Asn Ser Asp Thr 100 105
110Thr Leu Glu Leu Ser Val Met Pro Lys Asp Glu Asp 115
12020396PRTArtificial SequenceSynthetic peptide 203Ser Ala Lys Asn
Arg Trp Arg Leu Val Gly Pro Val His Leu Thr Arg1 5 10 15Gly Glu Gly
Gly Phe Gly Leu Thr Leu Arg Gly Asp Ser Pro Val Leu 20 25 30Ile Ala
Ala Val Ile Pro Gly Ser Gln Ala Ala Ala Ala Gly Leu Lys 35 40 45Glu
Gly Asp Tyr Ile Val Ser Val Asn Gly Gln Pro Cys Arg Trp Trp 50 55
60Arg His Ala Glu Val Val Thr Glu Leu Lys Ala Ala Gly Glu Ala Gly65
70 75 80Ala Ser Leu Gln Val Val Ser Leu Leu Pro Ser Ser Arg Leu Pro
Ser 85 90 95204104PRTArtificial SequenceSynthetic peptide 204Ile
Ser Phe Ser Ala Asn Lys Arg Trp Thr Pro Pro Arg Ser Ile Arg1 5 10
15Phe Thr Ala Glu Glu Gly Asp Leu Gly Phe Thr Leu Arg Gly Asn Ala
20 25 30Pro Val Gln Val His Phe Leu Asp Pro Tyr Cys Ser Ala Ser Val
Ala 35 40 45Gly Ala Arg Glu Gly Asp Tyr Ile Val Ser Ile Gln Leu Val
Asp Cys 50 55 60Lys Trp Leu Thr Leu Ser Glu Val Met Lys Leu Leu Lys
Ser Phe Gly65 70 75 80Glu Asp Glu Ile Glu Met Lys Val Val Ser Leu
Leu Asp Ser Thr Ser 85 90 95Ser Met His Asn Lys Ser Ala Thr
100205126PRTArtificial SequenceSynthetic peptide 205Thr Leu Asn Glu
Glu His Ser His Ser Asp Lys His Pro Val Thr Trp1 5 10 15Gln Pro Ser
Lys Asp Gly Asp Arg Leu Ile Gly Arg Ile Leu Leu Asn 20 25 30Lys Arg
Leu Lys Asp Gly Ser Val Pro Arg Asp Ser Gly Ala Met Leu 35 40 45Gly
Leu Lys Val Val Gly Gly Lys Met Thr Glu Ser Gly Arg Leu Cys 50 55
60Ala Phe Ile Thr Lys Val Lys Lys Gly Ser Leu Ala Asp Thr Val Gly65
70 75 80His Leu Arg Pro Gly Asp Glu Val Leu Glu Trp Asn Gly Arg Leu
Leu 85 90 95Gln Gly Ala Thr Phe Glu Glu Val Tyr Asn Ile Ile Leu Glu
Ser Lys 100 105 110Pro Glu Pro Gln Val Glu Leu Val Val Ser Arg Pro
Ile Gly 115 120 125206101PRTArtificial SequenceSynthetic peptide
206Gln Glu Met Asp Arg Glu Glu Leu Glu Leu Glu Glu Val Asp Leu Tyr1
5 10 15Arg Met Asn Ser Gln Asp Lys Leu Gly Leu Thr Val Cys Tyr Arg
Thr 20 25 30Asp Asp Glu Asp Asp Ile Gly Ile Tyr Ile Ser Glu Ile Asp
Pro Asn 35 40 45Ser Ile Ala Ala Lys Asp Gly Arg Ile Arg Glu Gly Asp
Arg Ile Ile 50 55 60Gln Ile Asn Gly Ile Glu Val Gln Asn Arg Glu Glu
Ala Val Ala Leu65 70 75 80Leu Thr Ser Glu Glu Asn Lys Asn Phe Ser
Leu Leu Ile Ala Arg Pro 85 90 95Glu Leu Gln Leu Asp
100207107PRTArtificial SequenceSynthetic peptide 207Gln Gly Glu Glu
Thr Lys Ser Leu Thr Leu Val Leu His Arg Asp Ser1 5 10 15Gly Ser Leu
Gly Phe Asn Ile Ile Gly Gly Arg Pro Ser Val Asp Asn 20 25 30His Asp
Gly Ser Ser Ser Glu Gly Ile Phe Val Ser Lys Ile Val Asp 35 40 45Ser
Gly Pro Ala Ala Lys Glu Gly Gly Leu Gln Ile His Asp Arg Ile 50 55
60Ile Glu Val Asn Gly Arg Asp Leu Ser Arg Ala Thr His Asp Gln Ala65
70 75 80Val Glu Ala Phe Lys Thr Ala Lys Glu Pro Ile Val Val Gln Val
Leu 85 90 95Arg Arg Thr Pro Arg Thr Lys Met Phe Thr Pro 100
10520898PRTArtificial SequenceSynthetic peptide 208Ile Leu Ala His
Val Lys Gly Ile Glu Lys Glu Val Asn Val Tyr Lys1 5 10 15Ser Glu Asp
Ser Leu Gly Leu Thr Ile Thr Asp Asn Gly Val Gly Tyr 20 25 30Ala Phe
Ile Lys Arg Ile Lys Asp Gly Gly Val Ile Asp Ser Val Lys 35 40 45Thr
Ile Cys Val Gly Asp His Ile Glu Ser Ile Asn Gly Glu Asn Ile 50 55
60Val Gly Trp Arg His Tyr Asp Val Ala Lys Lys Leu Lys Glu Leu Lys65
70 75 80Lys Glu Glu Leu Phe Thr Met Lys Leu Ile Glu Pro Lys Lys Ala
Phe 85 90 95Glu Ile 209109PRTArtificial SequenceSynthetic peptide
209Arg Gly Glu Lys Lys Asn Ser Ser Ser Gly Ile Ser Gly Ser Gln Arg1
5 10 15Arg Tyr Ile Gly Val Met Met Leu Thr Leu Ser Pro Ser Ile Leu
Ala 20 25 30Glu Leu Gln Leu Arg Glu Pro Ser Phe Pro Asp Val Gln His
Gly Val 35 40 45Leu Ile His Lys Val Ile Leu Gly Ser Pro Ala His Arg
Ala Gly Leu 50 55 60Arg Pro Gly Asp Val Ile Leu Ala Ile Gly Glu Gln
Met Val Gln Asn65 70 75 80Ala Glu Asp Val Tyr Glu Ala Val Arg Thr
Gln Ser Gln Leu Ala Val 85 90 95Gln Ile Arg Arg Gly Arg Glu Thr Leu
Thr Leu Tyr Val 100 105210110PRTArtificial SequenceSynthetic
peptide 210Ile Leu Glu Glu Lys Thr Val Val Leu Gln Lys Lys Asp Asn
Glu Gly1 5 10 15Phe Gly Phe Val Leu Arg Gly Ala Lys Ala Asp Thr Pro
Ile Glu Glu 20 25 30Phe Thr Pro Thr Pro Ala Phe Pro Ala Leu Gln Tyr
Leu Glu Ser Val 35 40 45Asp Glu Gly Gly Val Ala Trp Gln Ala Gly Leu
Arg Thr Gly Asp Phe 50 55 60Leu Ile Glu Val Asn Asn Glu Asn Val Val
Lys Val Gly His Arg Gln65 70 75 80Val Val Asn Met Ile Arg Gln Gly
Gly Asn His Leu Val Leu Lys Val 85 90 95Val Thr Val Thr Arg Asn Leu
Asp Pro Asp Asp Asn Ser Ser 100 105 110211113PRTArtificial
SequenceSynthetic peptide 211Ile Leu Lys Glu Lys Thr Val Leu Leu
Gln Lys Lys Asp Ser Glu Gly1 5 10 15Phe Gly Phe Val Leu Arg Gly Ala
Lys Ala Gln Thr Pro Ile Glu Glu 20 25 30Phe Thr Pro Thr Pro Ala Phe
Pro Ala Leu Gln Tyr Leu Glu Ser Val 35 40 45Asp Glu Gly Gly Val Ala
Trp Arg Ala Gly Leu Arg Met Gly Asp Phe 50 55 60Leu Ile Glu Val Asn
Gly Gln Asn Val Val Lys Val Gly His Arg Gln65 70 75 80Val Val Asn
Met Ile Arg Gln Gly Gly Asn Thr Leu Met Val Lys Val 85 90 95Val Met
Val Thr Arg His Pro Asp Met Asp Glu Ala Val Gln Asn Ser 100 105
110Ser212110PRTArtificial SequenceSynthetic peptide 212Ser Asp Tyr
Val Ile Asp Asp Lys Val Ala Val Leu Gln Lys Arg Asp1 5 10 15His Glu
Gly Phe Gly Phe Val Leu Arg Gly Ala Lys Ala Glu Thr Pro 20 25 30Ile
Glu Glu Phe Thr Pro Thr Pro Ala Phe Pro Ala Leu Gln Tyr Leu 35 40
45Glu Ser Val Asp Val Glu Gly Val Ala Trp Arg Ala Gly Leu Arg Thr
50 55 60Gly Asp Phe Leu Ile Glu Val Asn Gly Val Asn Val Val Lys Val
Gly65 70 75 80His Lys Gln Val Val Ala Leu Ile Arg Gln Gly Gly Asn
Arg Leu Val 85 90 95Met Lys Val Val Ser Val Thr Arg Lys Pro Glu Glu
Asp Gly 100 105 11021398PRTArtificial SequenceSynthetic peptide
213Ser Asn Ser Pro Arg Glu Glu Ile Phe Gln Val Ala Leu His Lys Arg1
5 10 15Asp Ser Gly Glu Gln Leu Gly Ile Lys Leu Val Arg Arg Thr Asp
Glu 20 25 30Pro Gly Val Phe Ile Leu Asp Leu Leu Glu Gly Gly Leu Ala
Ala Gln 35 40 45Asp Gly Arg Leu Ser Ser Asn Asp Arg Val Leu Ala Ile
Asn Gly His 50 55 60Asp Leu Lys Tyr Gly Thr Pro Glu Leu Ala Ala Gln
Ile Ile Gln Ala65 70 75 80Ser Gly Glu Arg Val Asn Leu Thr Ile Ala
Arg Pro Gly Lys Pro Gln 85 90 95Pro Gly214104PRTArtificial
SequenceSynthetic peptide 214Ile Gln Cys Val Thr Cys Gln Glu Lys
His Ile Thr Val Lys Lys Glu1 5 10 15Pro His Glu Ser Leu Gly Met Thr
Val Ala Gly Gly Arg Gly Ser Lys 20 25 30Ser Gly Glu Leu Pro Ile Phe
Val Thr Ser Val Pro Pro His Gly Cys 35 40 45Leu Ala Arg Asp Gly Arg
Ile Lys Arg Gly Asp Val Leu Leu Asn Ile 50 55 60Asn Gly Ile Asp Leu
Thr Asn Leu Ser His Ser Glu Ala Val Ala Met65 70 75 80Leu Lys Ala
Ser Ala Ala Ser Pro Ala Val Ala Leu Lys Ala Leu Glu 85 90 95Val Gln
Ile Val Glu Glu Ala Thr 100215110PRTArtificial SequenceSynthetic
peptide 215Met Gly Leu Gly Val Ser Ala Glu Gln Pro Ala Gly Gly Ala
Glu Gly1 5 10 15Phe His Leu His Gly Val Gln Glu Asn Ser Pro Ala Gln
Gln Ala Gly 20 25 30Leu Glu Pro Tyr Phe Asp Phe Ile Ile Thr Ile Gly
His Ser Arg Leu 35 40 45Asn Lys Glu Asn Asp Thr Leu Lys Ala Leu Leu
Lys Ala Asn Val Glu 50 55 60Lys Pro Val Lys Leu Glu Val Phe Asn Met
Lys Thr Met Arg Val Arg65 70 75 80Glu Val Glu Val Val Pro Ser Asn
Met Trp Gly Gly Gln Gly Leu Leu 85 90 95Gly Ala Ser Val Arg Phe Cys
Ser Phe Arg Arg Ala Ser Glu 100 105 110216109PRTArtificial
SequenceSynthetic peptide 216Arg Ala Ser Glu Gln Val Trp His Val
Leu Asp Val Glu Pro Ser Ser1 5 10 15Pro Ala Ala Leu Ala Gly Leu Arg
Pro Tyr Thr Asp Tyr Val Val Gly 20 25 30Ser Asp Gln Ile Leu Gln Glu
Ser Glu Asp Phe Phe Thr Leu Ile Glu 35 40 45Ser His Glu Gly Lys Pro
Leu Lys Leu Met Val Tyr Asn Ser Lys Ser 50 55 60Asp Ser Cys Arg Glu
Ser Gly Met Trp His Trp Leu Trp Val Ser Thr65 70 75 80Pro Asp Pro
Asn Ser Ala Pro Gln Leu Pro Gln Glu Ala Thr Trp His 85 90 95Pro Thr
Thr Phe Cys Ser Thr Thr Trp Cys Pro Thr Thr 100
105217101PRTArtificial SequenceSynthetic peptide 217Ile Ser Val Thr
Asp Gly Pro Lys Phe Glu Val Lys Leu Lys Lys Asn1 5 10 15Ala Asn Gly
Leu Gly Phe Ser Phe Val Gln Met Glu Lys Glu Ser Cys 20 25 30Ser His
Leu Lys Ser Asp Leu Val Arg Ile Lys Arg Leu Phe Pro Gly 35 40 45Gln
Pro Ala Glu Glu Asn Gly Ala Ile Ala Ala Gly Asp Ile Ile Leu 50 55
60Ala Val Asn Gly Arg Ser Thr Glu Gly Leu Ile Phe Gln Glu Val Leu65
70 75 80His Leu Leu Arg Gly Ala Pro Gln Glu Val Thr Leu Leu Leu Cys
Arg 85 90 95Pro Pro Pro Gly Ala 100218100PRTArtificial
SequenceSynthetic peptide 218Gln Pro Glu Pro Leu Arg Pro Arg Leu
Cys Arg Leu Val Arg Gly Glu1 5 10 15Gln Gly Tyr Gly Phe His Leu His
Gly Glu Lys Gly Arg Arg Gly Gln 20 25 30Phe Ile Arg Arg Val Glu Pro
Gly Ser Pro Ala Glu Ala Ala Ala Leu 35 40 45Arg Ala Gly Asp Arg Leu
Val Glu Val Asn Gly Val Asn Val Glu Gly 50 55 60Glu Thr His His Gln
Val Val Gln Arg Ile Lys Ala Val Glu Gly Gln65 70 75 80Thr Arg Leu
Leu Val Val Asp Gln Glu Thr Asp Glu Glu Leu Arg Arg 85 90 95Arg Asn
Ser Ser 10021999PRTArtificial SequenceSynthetic peptide 219Pro Leu
Arg Glu Leu Arg Pro Arg Leu Cys His Leu Arg Lys Gly Pro1 5 10 15Gln
Gly Tyr Gly Phe Asn Leu His Ser Asp Lys Ser Arg Pro Gly Gln 20 25
30Tyr Ile Arg Ser Val Asp Pro Gly Ser Pro Ala Ala Arg Ser Gly Leu
35 40 45Arg Ala Gln Asp Arg Leu Ile Glu Val Asn Gly Gln Asn Val Glu
Gly 50 55 60Leu Arg His Ala Glu Val Val Ala Ser Ile Lys Ala Arg Glu
Asp Glu65 70 75 80Ala Arg Leu Leu Val Val Asp Pro Glu Thr Asp Glu
His Phe Lys Arg 85 90 95Asn Ser Ser22092PRTArtificial
SequenceSynthetic peptide 220Pro Gly Val Arg Glu
Ile His Leu Cys Lys Asp Glu Arg Gly Lys Thr1 5 10 15Gly Leu Arg Leu
Arg Lys Val Asp Gln Gly Leu Phe Val Gln Leu Val 20 25 30Gln Ala Asn
Thr Pro Ala Ser Leu Val Gly Leu Arg Phe Gly Asp Gln 35 40 45Leu Leu
Gln Ile Asp Gly Arg Asp Cys Ala Gly Trp Ser Ser His Lys 50 55 60Ala
His Gln Val Val Lys Lys Ala Ser Gly Asp Lys Ile Val Val Val65 70 75
80Val Arg Asp Arg Pro Phe Gln Arg Thr Val Thr Met 85
9022190PRTArtificial SequenceSynthetic peptide 221Pro Phe Gln Arg
Thr Val Thr Met His Lys Asp Ser Met Gly His Val1 5 10 15Gly Phe Val
Ile Lys Lys Gly Lys Ile Val Ser Leu Val Lys Gly Ser 20 25 30Ser Ala
Ala Arg Asn Gly Leu Leu Thr Asn His Tyr Val Cys Glu Val 35 40 45Asp
Gly Gln Asn Val Ile Gly Leu Lys Asp Lys Lys Ile Met Glu Ile 50 55
60Leu Ala Thr Ala Gly Asn Val Val Thr Leu Thr Ile Ile Pro Ser Val65
70 75 80Ile Tyr Glu His Ile Val Glu Phe Ile Val 85
9022296PRTArtificial SequenceSynthetic peptide 222Ser Leu Glu Arg
Pro Arg Phe Cys Leu Leu Ser Lys Glu Glu Gly Lys1 5 10 15Ser Phe Gly
Phe His Leu Gln Gln Glu Leu Gly Arg Ala Gly His Val 20 25 30Val Cys
Arg Val Asp Pro Gly Thr Ser Ala Gln Arg Gln Gly Leu Gln 35 40 45Glu
Gly Asp Arg Ile Leu Ala Val Asn Asn Asp Val Val Glu His Glu 50 55
60Asp Tyr Ala Val Val Val Arg Arg Ile Arg Ala Ser Ser Pro Arg Val65
70 75 80Leu Leu Thr Val Leu Ala Arg His Ala His Asp Val Ala Arg Ala
Gln 85 90 9522392PRTArtificial SequenceSynthetic peptide 223Leu Arg
Asp Arg Pro Phe Glu Arg Thr Ile Thr Met His Lys Asp Ser1 5 10 15Thr
Gly His Val Gly Phe Ile Phe Lys Asn Gly Lys Ile Thr Ser Ile 20 25
30Val Lys Asp Ser Ser Ala Ala Arg Asn Gly Leu Leu Thr Glu His Asn
35 40 45Ile Cys Glu Ile Asn Gly Gln Asn Val Ile Gly Leu Lys Asp Ser
Gln 50 55 60Ile Ala Asp Ile Leu Ser Thr Ser Gly Thr Val Val Thr Ile
Thr Ile65 70 75 80Met Pro Ala Phe Ile Phe Glu His Met Asn Ser Ser
85 9022488PRTArtificial SequenceSynthetic peptide 224Leu Glu Ile
Lys Gln Gly Ile Arg Glu Val Ile Leu Cys Lys Asp Gln1 5 10 15Asp Gly
Lys Ile Gly Leu Arg Leu Lys Ser Ile Asp Asn Gly Ile Phe 20 25 30Val
Gln Leu Val Gln Ala Asn Ser Pro Ala Ser Leu Val Gly Leu Arg 35 40
45Phe Gly Asp Gln Val Leu Gln Ile Asn Gly Glu Asn Cys Ala Gly Trp
50 55 60Ser Ser Asp Lys Ala His Lys Val Leu Lys Gln Ala Phe Gly Glu
Lys65 70 75 80Ile Thr Met Arg Ile His Arg Asp 8522594PRTArtificial
SequenceSynthetic peptide 225Gln Arg Arg Arg Val Thr Val Arg Lys
Ala Asp Ala Gly Gly Leu Gly1 5 10 15Ile Ser Ile Lys Gly Gly Arg Glu
Asn Lys Met Pro Ile Leu Ile Ser 20 25 30Lys Ile Phe Lys Gly Leu Ala
Ala Asp Gln Thr Glu Ala Leu Phe Val 35 40 45Gly Asp Ala Ile Leu Ser
Val Asn Gly Glu Asp Leu Ser Ser Ala Thr 50 55 60His Asp Glu Ala Val
Gln Val Leu Lys Lys Thr Gly Lys Glu Val Val65 70 75 80Leu Glu Val
Lys Tyr Met Lys Asp Val Ser Pro Tyr Phe Lys 85 9022688PRTArtificial
SequenceSynthetic peptide 226Pro Val Arg Arg Val Arg Val Val Lys
Gln Glu Ala Gly Gly Leu Gly1 5 10 15Ile Ser Ile Lys Gly Gly Arg Glu
Asn Arg Met Pro Ile Leu Ile Ser 20 25 30Lys Ile Phe Pro Gly Leu Ala
Ala Asp Gln Ser Arg Ala Leu Arg Leu 35 40 45Gly Asp Ala Ile Leu Ser
Val Asn Gly Thr Asp Leu Arg Gln Ala Thr 50 55 60His Asp Gln Ala Val
Gln Ala Leu Lys Arg Ala Gly Lys Glu Val Leu65 70 75 80Leu Glu Val
Lys Phe Ile Arg Glu 85227100PRTArtificial SequenceSynthetic peptide
227Glu Pro Phe Tyr Ser Gly Glu Arg Thr Val Thr Ile Arg Arg Gln Thr1
5 10 15Val Gly Gly Phe Gly Leu Ser Ile Lys Gly Gly Ala Glu His Asn
Ile 20 25 30Pro Val Val Val Ser Lys Ile Ser Lys Glu Gln Arg Ala Glu
Leu Ser 35 40 45Gly Leu Leu Phe Ile Gly Asp Ala Ile Leu Gln Ile Asn
Gly Ile Asn 50 55 60Val Arg Lys Cys Arg His Glu Glu Val Val Gln Val
Leu Arg Asn Ala65 70 75 80Gly Glu Glu Val Thr Leu Thr Val Ser Phe
Leu Lys Arg Ala Pro Ala 85 90 95Phe Leu Lys Leu
10022899PRTArtificial SequenceSynthetic peptide 228Ser His Gln Gly
Arg Asn Arg Arg Thr Val Thr Leu Arg Arg Gln Pro1 5 10 15Val Gly Gly
Leu Gly Leu Ser Ile Lys Gly Gly Ser Glu His Asn Val 20 25 30Pro Val
Val Ile Ser Lys Ile Phe Glu Asp Gln Ala Ala Asp Gln Thr 35 40 45Gly
Met Leu Phe Val Gly Asp Ala Val Leu Gln Val Asn Gly Ile His 50 55
60Val Glu Asn Ala Thr His Glu Glu Val Val His Leu Leu Arg Asn Ala65
70 75 80Gly Asp Glu Val Thr Ile Thr Val Glu Tyr Leu Arg Glu Ala Pro
Ala 85 90 95Phe Leu Lys22991PRTArtificial SequenceSynthetic peptide
229Arg Gly Glu Thr Lys Glu Val Glu Val Thr Lys Thr Glu Asp Ala Leu1
5 10 15Gly Leu Thr Ile Thr Asp Asn Gly Ala Gly Tyr Ala Phe Ile Lys
Arg 20 25 30Ile Lys Glu Gly Ser Ile Ile Asn Arg Ile Glu Ala Val Cys
Val Gly 35 40 45Asp Ser Ile Glu Ala Ile Asn Asp His Ser Ile Val Gly
Cys Arg His 50 55 60Tyr Glu Val Ala Lys Met Leu Arg Glu Leu Pro Lys
Ser Gln Pro Phe65 70 75 80Thr Leu Arg Leu Val Gln Pro Lys Arg Ala
Phe 85 9023088PRTArtificial SequenceSynthetic peptide 230His Ser
Ile His Ile Glu Lys Ser Asp Thr Ala Ala Asp Thr Tyr Gly1 5 10 15Phe
Ser Leu Ser Ser Val Glu Glu Asp Gly Ile Arg Arg Leu Tyr Val 20 25
30Asn Ser Val Lys Glu Thr Gly Leu Ala Ser Lys Lys Gly Leu Lys Ala
35 40 45Gly Asp Glu Ile Leu Glu Ile Asn Asn Arg Ala Ala Asp Ala Leu
Asn 50 55 60Ser Ser Met Leu Lys Asp Phe Leu Ser Gln Pro Ser Leu Gly
Leu Leu65 70 75 80Val Arg Thr Tyr Pro Glu Leu Glu
8523197PRTArtificial SequenceSynthetic peptide 231Pro Leu Asn Val
Tyr Asp Val Gln Leu Thr Lys Thr Gly Ser Val Cys1 5 10 15Asp Phe Gly
Phe Ala Val Thr Ala Gln Val Asp Glu Arg Gln His Leu 20 25 30Ser Arg
Ile Phe Ile Ser Asp Val Leu Pro Asp Gly Leu Ala Tyr Gly 35 40 45Glu
Gly Leu Arg Lys Gly Asn Glu Ile Met Thr Leu Asn Gly Glu Ala 50 55
60Val Ser Asp Leu Asp Leu Lys Gln Met Glu Ala Leu Phe Ser Glu Lys65
70 75 80Ser Val Gly Leu Thr Leu Ile Ala Arg Pro Pro Asp Thr Lys Ala
Thr 85 90 95Leu 232103PRTArtificial SequenceSynthetic peptide
232Gln Arg Val Glu Ile His Lys Leu Arg Gln Gly Glu Asn Leu Ile Leu1
5 10 15Gly Phe Ser Ile Gly Gly Gly Ile Asp Gln Asp Pro Ser Gln Asn
Pro 20 25 30Phe Ser Glu Asp Lys Thr Asp Lys Gly Ile Tyr Val Thr Arg
Val Ser 35 40 45Glu Gly Gly Pro Ala Glu Ile Ala Gly Leu Gln Ile Gly
Asp Lys Ile 50 55 60Met Gln Val Asn Gly Trp Asp Met Thr Met Val Thr
His Asp Gln Ala65 70 75 80Arg Lys Arg Leu Thr Lys Arg Ser Glu Glu
Val Val Arg Leu Leu Val 85 90 95Thr Arg Gln Ser Leu Gln Lys
10023386PRTArtificial SequenceSynthetic peptide 233Arg Lys Glu Val
Glu Val Phe Lys Ser Glu Asp Ala Leu Gly Leu Thr1 5 10 15Ile Thr Asp
Asn Gly Ala Gly Tyr Ala Phe Ile Lys Arg Ile Lys Glu 20 25 30Gly Ser
Val Ile Asp His Ile His Leu Ile Ser Val Gly Asp Met Ile 35 40 45Glu
Ala Ile Asn Gly Gln Ser Leu Leu Gly Cys Arg His Tyr Glu Val 50 55
60Ala Arg Leu Leu Lys Glu Leu Pro Arg Gly Arg Thr Phe Thr Leu Lys65
70 75 80Leu Thr Glu Pro Arg Lys 8523491PRTArtificial
SequenceSynthetic peptide 234His Ser His Pro Arg Val Val Glu Leu
Pro Lys Thr Asp Glu Gly Leu1 5 10 15Gly Phe Asn Val Met Gly Gly Lys
Glu Gln Asn Ser Pro Ile Tyr Ile 20 25 30Ser Arg Ile Ile Pro Gly Gly
Val Ala Glu Arg His Gly Gly Leu Lys 35 40 45Arg Gly Asp Gln Leu Leu
Ser Val Asn Gly Val Ser Val Glu Gly Glu 50 55 60His His Glu Lys Ala
Val Glu Leu Leu Lys Ala Ala Lys Asp Ser Val65 70 75 80Lys Leu Val
Val Arg Tyr Thr Pro Lys Val Leu 85 9023597PRTArtificial
SequenceSynthetic peptide 235Leu Ser Asn Gln Lys Arg Gly Val Lys
Val Leu Lys Gln Glu Leu Gly1 5 10 15Gly Leu Gly Ile Ser Ile Lys Gly
Gly Lys Glu Asn Lys Met Pro Ile 20 25 30Leu Ile Ser Lys Ile Phe Lys
Gly Leu Ala Ala Asp Gln Thr Gln Ala 35 40 45Leu Tyr Val Gly Asp Ala
Ile Leu Ser Val Asn Gly Ala Asp Leu Arg 50 55 60Asp Ala Thr His Asp
Glu Ala Val Gln Ala Leu Lys Arg Ala Gly Lys65 70 75 80Glu Val Leu
Leu Glu Val Lys Tyr Met Arg Glu Ala Thr Pro Tyr Val 85 90
95Lys23698PRTArtificial SequenceSynthetic peptide 236Ile Gln Arg
Ser Ser Ile Lys Thr Val Glu Leu Ile Lys Gly Asn Leu1 5 10 15Gln Ser
Val Gly Leu Thr Leu Arg Leu Val Gln Ser Thr Asp Gly Tyr 20 25 30Ala
Gly His Val Ile Ile Glu Thr Val Ala Pro Asn Ser Pro Ala Ala 35 40
45Ile Ala Asp Leu Gln Arg Gly Asp Arg Leu Ile Ala Ile Gly Gly Val
50 55 60Lys Ile Thr Ser Thr Leu Gln Val Leu Lys Leu Ile Lys Gln Ala
Gly65 70 75 80Asp Arg Val Leu Val Tyr Tyr Glu Arg Pro Val Gly Gln
Ser Asn Gln 85 90 95Gly Ala237103PRTArtificial SequenceSynthetic
peptide 237Ile Leu Thr Leu Thr Ile Leu Arg Gln Thr Gly Gly Leu Gly
Ile Ser1 5 10 15Ile Ala Gly Gly Lys Gly Ser Thr Pro Tyr Lys Gly Asp
Asp Glu Gly 20 25 30Ile Phe Ile Ser Arg Val Ser Glu Glu Gly Pro Ala
Ala Arg Ala Gly 35 40 45Val Arg Val Gly Asp Lys Leu Leu Glu Val Asn
Gly Val Ala Leu Gln 50 55 60Gly Ala Glu His His Glu Ala Val Glu Ala
Leu Arg Gly Ala Gly Thr65 70 75 80Ala Val Gln Met Arg Val Trp Arg
Glu Arg Met Val Glu Pro Glu Asn 85 90 95Ala Glu Phe Ile Val Thr Asp
100238105PRTArtificial SequenceSynthetic peptide 238Arg Glu Leu Cys
Ile Gln Lys Ala Pro Gly Glu Arg Leu Gly Ile Ser1 5 10 15Ile Arg Gly
Gly Ala Arg Gly His Ala Gly Asn Pro Arg Asp Pro Thr 20 25 30Asp Glu
Gly Ile Phe Ile Ser Lys Val Ser Pro Thr Gly Ala Ala Gly 35 40 45Arg
Asp Gly Arg Leu Arg Val Gly Leu Arg Leu Leu Glu Val Asn Gln 50 55
60Gln Ser Leu Leu Gly Leu Thr His Gly Glu Ala Val Gln Leu Leu Arg65
70 75 80Ser Val Gly Asp Thr Leu Thr Val Leu Val Cys Asp Gly Phe Glu
Ala 85 90 95Ser Thr Asp Ala Ala Leu Glu Val Ser 100
105239105PRTArtificial SequenceSynthetic peptide 239Leu Glu Gly Pro
Tyr Pro Val Glu Glu Ile Arg Leu Pro Arg Ala Gly1 5 10 15Gly Pro Leu
Gly Leu Ser Ile Val Gly Gly Ser Asp His Ser Ser His 20 25 30Pro Phe
Gly Val Gln Glu Pro Gly Val Phe Ile Ser Lys Val Leu Pro 35 40 45Arg
Gly Leu Ala Ala Arg Ser Gly Leu Arg Val Gly Asp Arg Ile Leu 50 55
60Ala Val Asn Gly Gln Asp Val Arg Asp Ala Thr His Gln Glu Ala Val65
70 75 80Ser Ala Leu Leu Arg Pro Cys Leu Glu Leu Ser Leu Leu Val Arg
Arg 85 90 95Asp Pro Ala Glu Phe Ile Val Thr Asp 100
10524097PRTArtificial SequenceSynthetic peptide 240Pro Leu Arg Gln
Arg His Val Ala Cys Leu Ala Arg Ser Glu Arg Gly1 5 10 15Leu Gly Phe
Ser Ile Ala Gly Gly Lys Gly Ser Thr Pro Tyr Arg Ala 20 25 30Gly Asp
Ala Gly Ile Phe Val Ser Arg Ile Ala Glu Gly Gly Ala Ala 35 40 45His
Arg Ala Gly Thr Leu Gln Val Gly Asp Arg Val Leu Ser Ile Asn 50 55
60Gly Val Asp Val Thr Glu Ala Arg His Asp His Ala Val Ser Leu Leu65
70 75 80Thr Ala Ala Ser Pro Thr Ile Ala Leu Leu Leu Glu Arg Glu Ala
Gly 85 90 95Gly241114PRTArtificial SequenceSynthetic peptide 241Thr
Leu Thr Ile Leu Arg Gln Thr Gly Gly Leu Gly Ile Ser Ile Ala1 5 10
15Gly Gly Lys Gly Ser Thr Pro Tyr Lys Gly Asp Asp Glu Gly Ile Phe
20 25 30Ile Ser Arg Val Ser Glu Glu Gly Pro Ala Ala Arg Ala Gly Val
Arg 35 40 45Val Gly Asp Lys Leu Leu Glu Gly Ile Phe Val Ser Arg Ile
Ala Glu 50 55 60Gly Gly Ala Ala His Arg Ala Gly Thr Leu Gln Val Gly
Asp Arg Val65 70 75 80Leu Ser Ile Asn Gly Val Asp Val Thr Glu Ala
Arg His Asp His Ala 85 90 95Val Ser Leu Leu Thr Ala Ala Ser Pro Thr
Ile Ala Leu Leu Leu Glu 100 105 110Arg Glu24295PRTArtificial
SequenceSynthetic peptide 242Ile Pro Pro Val Thr Thr Val Leu Ile
Lys Arg Pro Asp Leu Lys Tyr1 5 10 15Gln Leu Gly Phe Ser Val Gln Asn
Gly Ile Ile Cys Ser Leu Met Arg 20 25 30Gly Gly Ile Ala Glu Arg Gly
Gly Val Arg Val Gly His Arg Ile Ile 35 40 45Glu Ile Asn Gly Gln Ser
Val Val Ala Thr Ala His Glu Lys Ile Val 50 55 60Gln Ala Leu Ser Asn
Ser Val Gly Glu Ile His Met Lys Thr Met Pro65 70 75 80Ala Ala Met
Phe Arg Leu Leu Thr Gly Gln Glu Asn Ser Ser Leu 85 90
95243110PRTArtificial SequenceSynthetic peptide 243Ile His Phe Ser
Asn Ser Glu Asn Cys Lys Glu Leu Gln Leu Glu Lys1 5 10 15His Lys Gly
Glu Ile Leu Gly Val Val Val Val Glu Ser Gly Trp Gly 20 25 30Ser Ile
Leu Pro Thr Val Ile Leu Ala Asn Met Met Asn Gly Gly Pro 35 40 45Ala
Ala Arg Ser Gly Lys Leu Ser Ile Gly Asp Gln Ile Met Ser Ile 50 55
60Asn Gly Thr Ser Leu Val Gly Leu Pro Leu Ala Thr Cys Gln Gly Ile65
70 75 80Ile Lys Gly Leu Lys Asn Gln Thr Gln Val Lys Leu Asn Ile Val
Ser 85 90 95Cys Pro Pro Val Thr Thr Val Leu Ile Lys Arg Asn Ser Ser
100 105 110244101PRTArtificial SequenceSynthetic peptide 244Ile Trp
Glu Gln His Thr Val Thr Leu His Arg Ala Pro Gly Phe Gly1 5
10 15Phe Gly Ile Ala Ile Ser Gly Gly Arg Asp Asn Pro His Phe Gln
Ser 20 25 30Gly Glu Thr Ser Ile Val Ile Ser Asp Val Leu Lys Gly Gly
Pro Ala 35 40 45Glu Gly Gln Leu Gln Glu Asn Asp Arg Val Ala Met Val
Asn Gly Val 50 55 60Ser Met Asp Asn Val Glu His Ala Phe Ala Val Gln
Gln Leu Arg Lys65 70 75 80Ser Gly Lys Asn Ala Lys Ile Thr Ile Arg
Arg Lys Lys Lys Val Gln 85 90 95Ile Pro Asn Ser Ser
10024595PRTArtificial SequenceSynthetic peptide 245Ile Ser Ser Gln
Pro Ala Lys Pro Thr Lys Val Thr Leu Val Lys Ser1 5 10 15Arg Lys Asn
Glu Glu Tyr Gly Leu Arg Leu Ala Ser His Ile Phe Val 20 25 30Lys Glu
Ile Ser Gln Asp Ser Leu Ala Ala Arg Asp Gly Asn Ile Gln 35 40 45Glu
Gly Asp Val Val Leu Lys Ile Asn Gly Thr Val Thr Glu Asn Met 50 55
60Ser Leu Thr Asp Ala Lys Thr Leu Ile Glu Arg Ser Lys Gly Lys Leu65
70 75 80Lys Met Val Val Gln Arg Asp Arg Ala Thr Leu Leu Asn Ser Ser
85 90 9524690PRTArtificial SequenceSynthetic peptide 246Ile Arg Met
Lys Leu Val Lys Phe Arg Lys Gly Asp Ser Val Gly Leu1 5 10 15Arg Leu
Ala Gly Gly Asn Asp Val Gly Ile Phe Val Ala Gly Val Leu 20 25 30Glu
Asp Ser Pro Ala Ala Lys Glu Gly Leu Glu Glu Gly Asp Gln Ile 35 40
45Leu Arg Val Asn Asn Val Asp Phe Thr Asn Ile Ile Arg Glu Glu Ala
50 55 60Val Leu Phe Leu Leu Asp Leu Pro Lys Gly Glu Glu Val Thr Ile
Leu65 70 75 80Ala Gln Lys Lys Lys Asp Val Phe Ser Asn 85
9024799PRTArtificial SequenceSynthetic peptide 247Ile Gln His Thr
Val Thr Leu His Arg Ala Pro Gly Phe Gly Phe Gly1 5 10 15Ile Ala Ile
Ser Gly Gly Arg Asp Asn Pro His Phe Gln Ser Gly Glu 20 25 30Thr Ser
Ile Val Ile Ser Asp Val Leu Lys Gly Gly Pro Ala Glu Gly 35 40 45Gln
Leu Gln Glu Asn Asp Arg Val Ala Met Val Asn Gly Val Ser Met 50 55
60Asp Asn Val Glu His Ala Phe Ala Val Gln Gln Leu Arg Lys Ser Gly65
70 75 80Lys Asn Ala Lys Ile Thr Ile Arg Arg Lys Lys Lys Val Gln Ile
Pro 85 90 95Asn Ser Ser 24890PRTArtificial SequenceSynthetic
peptide 248His Ala Pro Asn Thr Lys Met Val Arg Phe Lys Lys Gly Asp
Ser Val1 5 10 15Gly Leu Arg Leu Ala Gly Gly Asn Asp Val Gly Ile Phe
Val Ala Gly 20 25 30Ile Gln Glu Gly Thr Ser Ala Glu Gln Glu Gly Leu
Gln Glu Gly Asp 35 40 45Gln Ile Leu Lys Val Asn Thr Gln Asp Phe Arg
Gly Leu Val Arg Glu 50 55 60Asp Ala Val Leu Tyr Leu Leu Glu Ile Pro
Lys Gly Glu Met Val Thr65 70 75 80Ile Leu Ala Gln Ser Arg Ala Asp
Val Tyr 85 9024979PRTArtificial SequenceSynthetic peptide 249Arg
Val Leu Leu Met Lys Ser Arg Ala Asn Glu Glu Tyr Gly Leu Arg1 5 10
15Leu Gly Ser Gln Ile Phe Val Lys Glu Met Thr Arg Thr Gly Leu Ala
20 25 30Thr Lys Asp Gly Asn Leu His Glu Gly Asp Ile Ile Leu Lys Ile
Asn 35 40 45Gly Thr Val Thr Glu Asn Met Ser Leu Thr Asp Ala Arg Lys
Leu Ile 50 55 60Glu Lys Ser Arg Gly Lys Leu Gln Leu Val Val Leu Arg
Asp Ser65 70 75250104PRTArtificial SequenceSynthetic peptide 250Arg
Gly Tyr Ser Pro Asp Thr Arg Val Val Arg Phe Leu Lys Gly Lys1 5 10
15Ser Ile Gly Leu Arg Leu Ala Gly Gly Asn Asp Val Gly Ile Phe Val
20 25 30Ser Gly Val Gln Ala Gly Ser Pro Ala Asp Gly Gln Gly Ile Gln
Glu 35 40 45Gly Asp Gln Ile Leu Gln Val Asn Asp Val Pro Phe Gln Asn
Leu Thr 50 55 60Arg Glu Glu Ala Val Gln Phe Leu Leu Gly Leu Pro Pro
Gly Glu Glu65 70 75 80Met Glu Leu Val Thr Gln Arg Lys Gln Asp Ile
Phe Trp Lys Met Val 85 90 95Gln Ser Glu Phe Ile Val Thr Asp
100251106PRTArtificial SequenceSynthetic peptide 251Ile Pro Gly Asn
Ser Thr Ile Trp Glu Gln His Thr Ala Thr Leu Ser1 5 10 15Lys Asp Pro
Arg Arg Gly Phe Gly Ile Ala Ile Ser Gly Gly Arg Asp 20 25 30Arg Pro
Gly Gly Ser Met Val Val Ser Asp Val Val Pro Gly Gly Pro 35 40 45Ala
Glu Gly Arg Leu Gln Thr Gly Asp His Ile Val Met Val Asn Gly 50 55
60Val Ser Met Glu Asn Ala Thr Ser Ala Phe Ala Ile Gln Ile Leu Lys65
70 75 80Thr Cys Thr Lys Met Ala Asn Ile Thr Val Lys Arg Pro Arg Arg
Ile 85 90 95His Leu Pro Ala Glu Phe Ile Val Thr Asp 100
10525298PRTArtificial SequenceSynthetic peptide 252Gln Asp Val Gln
Met Lys Pro Val Lys Ser Val Leu Val Lys Arg Arg1 5 10 15Asp Ser Glu
Glu Phe Gly Val Lys Leu Gly Ser Gln Ile Phe Ile Lys 20 25 30His Ile
Thr Asp Ser Gly Leu Ala Ala Arg His Arg Gly Leu Gln Glu 35 40 45Gly
Asp Leu Ile Leu Gln Ile Asn Gly Val Ser Ser Gln Asn Leu Ser 50 55
60Leu Asn Asp Thr Arg Arg Leu Ile Glu Lys Ser Glu Gly Lys Leu Ser65
70 75 80Leu Leu Val Leu Arg Asp Arg Gly Gln Phe Leu Val Asn Ile Pro
Asn 85 90 95Ser Ser 2535PRTArtificial SequenceSynthetic peptide
253Gly Ile Pro Gly Asn1 52545PRTArtificial SequenceSynthetic
peptide 254Gly Gly Gly Gly Ser1 525514PRTArtificial
SequenceSynthetic peptide 255Glu Gly Lys Ser Ser Gly Ser Gly Ser
Glu Ser Lys Val Asp1 5 1025618PRTArtificial SequenceSynthetic
peptide 256Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe
Arg Ser1 5 10 15Leu Asp25711PRTArtificial SequenceSynthetic peptide
257Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5
1025820PRTArtificial SequenceSynthetic peptide 258Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Ser Thr Asn Ser Val1 5 10 15Arg Leu Met
Leu 2025911PRTArtificial SequenceSynthetic peptide 259Arg Arg Ser
Thr Asn Ser Val Arg Leu Met Leu1 5 102609PRTArtificial
SequenceSynthetic peptide 260Ser Thr Asn Ser Val Arg Leu Met Leu1
526120PRTArtificial SequenceSynthetic peptide 261Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Ala Val Ala Ala Thr1 5 10 15Ser Ile Asn
Leu 2026220PRTArtificial SequenceSynthetic peptide 262Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Ser Asp Arg1 5 10 15Thr Ile
Trp Ala 2026320PRTArtificial SequenceSynthetic peptide 263Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Ser Asp Arg1 5 10 15Thr
Ile Ile Ala 2026420PRTArtificial SequenceSynthetic peptide 264Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Arg Thr Asp Arg1 5 10
15Lys Tyr Trp Ala 2026520PRTArtificial SequenceSynthetic peptide
265Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1
5 10 15Lys Ile Arg Val 2026620PRTArtificial SequenceSynthetic
peptide 266Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Thr
Asp Arg1 5 10 15Lys Val Glu Val 2026720PRTArtificial
SequenceSynthetic peptide 267Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ala Arg Gly Asp Arg1 5 10 15Lys Tyr Ile Val
2026820PRTArtificial SequenceSynthetic peptide 268Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Ser Arg Thr Asp Arg1 5 10 15Lys Tyr Gln
Ile 2026920PRTArtificial SequenceSynthetic peptide 269Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1 5 10 15Lys Val
Pro Val 2027020PRTArtificial SequenceSynthetic peptide 270Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Gln Asp Glu Arg Arg1 5 10 15Leu
Ile Val Leu 2027120PRTArtificial SequenceSynthetic peptide 271Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1 5 10
15Leu Val Ser Leu 2027220PRTArtificial SequenceSynthetic peptide
272Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Thr Arg1
5 10 15Leu Val Trp Val 2027320PRTArtificial SequenceSynthetic
peptide 273Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly
Asp Arg1 5 10 15Tyr Arg Ile Val 2027420PRTArtificial
SequenceSynthetic peptide 274Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ser Arg Thr Asp Arg1 5 10 15Leu Glu Tyr Val
2027520PRTArtificial SequenceSynthetic peptide 275Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1 5 10 15Thr Ile Ile
Tyr 2027620PRTArtificial SequenceSynthetic peptide 276Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Lys Asn Lys Asp Lys1 5 10 15Glu Tyr
Tyr Val 2027720PRTArtificial SequenceSynthetic peptide 277Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Arg Arg1 5 10 15Glu
Thr Trp Val 2027820PRTArtificial SequenceSynthetic peptide 278Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Lys His Phe Arg1 5 10
15Glu Thr Glu Val 2027920PRTArtificial SequenceSynthetic peptide
279Ala Ala Gly Gly Arg Ser Ala Arg Gly Gly Arg Leu Gln Gly Arg Arg1
5 10 15Glu Thr Ala Leu 2028020PRTArtificial SequenceSynthetic
peptide 280Ala Thr Asp Tyr Leu Val Gln Pro Phe Met Asp Gln Leu Ala
Phe His1 5 10 15Gln Phe Tyr Ile 2028120PRTArtificial
SequenceSynthetic peptide 281Ala Val Gly Gly Arg Pro Ala Arg Gly
Gly Arg Leu Gln Gly Arg Arg1 5 10 15Gln Thr Gln Val
2028220PRTArtificial SequenceSynthetic peptide 282Asp Phe Arg Pro
Ser Phe Lys His Ile Leu Phe Arg Arg Ala Arg Arg1 5 10 15Gly Phe Arg
Gln 2028320PRTArtificial SequenceSynthetic peptide 283Asp Gly Gly
Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser Lys1 5 10 15His Asp
Tyr Val 2028420PRTArtificial SequenceSynthetic peptide 284Asp Thr
Leu Leu Leu Thr Glu Asn Glu Gly Asp Lys Thr Glu Glu Gln1 5 10 15Val
Ser Tyr Val 2028520PRTArtificial SequenceSynthetic peptide 285Glu
Leu Leu Gln Phe Cys Arg Thr Pro Asn Pro Ala Leu Lys Asn Gly1 5 10
15Gln Tyr Trp Val 2028620PRTArtificial SequenceSynthetic peptide
286Glu Asn Leu Glu Leu Pro Val Asn Pro Ser Ser Val Val Ser Glu Arg1
5 10 15Ile Ser Ser Val 2028720PRTArtificial SequenceSynthetic
peptide 287Phe His Ser Lys Thr Ala Gly Ala Asn Thr Thr Asp Lys Glu
Leu Glu1 5 10 15Val Leu Ser Leu 2028820PRTArtificial
SequenceSynthetic peptide 288Phe Asn Gly Ser Ser Asn Gly His Val
Tyr Glu Lys Leu Ser Ser Ile1 5 10 15Glu Ser Asp Val
2028920PRTArtificial SequenceSynthetic peptide 289Gly Arg Trp Thr
Gly Arg Ala Met Ser Ala Trp Lys Pro Thr Arg Arg1 5 10 15Glu Thr Glu
Val 2029020PRTArtificial SequenceSynthetic peptide 290His Ala Met
Asn Ala Ala Pro Arg Ala Met Glu Asn Ala Pro Ala Leu1 5 10 15Arg Thr
Ser His 2029120PRTArtificial SequenceSynthetic peptide 291His Asp
Phe Arg Arg Ala Phe Lys Lys Ile Leu Ala Arg Gly Asp Arg1 5 10 15Lys
Arg Ile Val 2029220PRTArtificial SequenceSynthetic peptide 292His
His Leu Val Ala Gln Arg Asp Ile Arg Gln Phe Gln Leu Gln His1 5 10
15Trp Leu Ala Ile 2029320PRTArtificial SequenceSynthetic peptide
293His Ser Cys Cys Asn Arg Ala Arg Gln Glu Arg Leu Gln Arg Arg Arg1
5 10 15Glu Thr Gln Val 2029420PRTArtificial SequenceSynthetic
peptide 294Ile Leu Asn Ser Ile Gln Val Met Arg Ala Gln Met Asn Gln
Ile Gln1 5 10 15Ser Val Glu Val 2029520PRTArtificial
SequenceSynthetic peptide 295Lys Ala Gly Tyr Arg Ala Pro Arg Ser
Tyr Pro Lys Ser Asn Ser Ser1 5 10 15Lys Glu Tyr Val
2029620PRTArtificial SequenceSynthetic peptide 296Lys His Ser Arg
Lys Ser Ser Ser Tyr Ser Ser Ser Ser Thr Thr Val1 5 10 15Lys Thr Ser
Tyr 2029723PRTArtificial SequenceSynthetic peptide 297Lys Lys Lys
Lys Gln Pro Gly Asn Ser Thr Lys Glu Ser Glu Ser Thr1 5 10 15Asn Ser
Val Arg Leu Met Leu 2029820PRTArtificial SequenceSynthetic peptide
298Lys Thr Met Pro Ala Ala Met Phe Arg Leu Leu Thr Gly Gln Glu Thr1
5 10 15Pro Leu Tyr Ile 2029920PRTArtificial SequenceSynthetic
peptide 299Lys Thr Met Pro Ala Ala Met Tyr Arg Leu Leu Thr Ala Gln
Glu Gln1 5 10 15Pro Val Tyr Ile 2030020PRTArtificial
SequenceSynthetic peptide 300Lys Thr Met Pro Ala Ala Thr Tyr Arg
Leu Leu Thr Gly Gln Glu Gln1 5 10 15Pro Val Tyr Leu
2030120PRTArtificial SequenceSynthetic peptide 301Lys Tyr Ser Ala
Pro Arg Arg Pro Thr Ala Thr Gly Asp Tyr Asp Lys1 5 10 15Lys Asn Tyr
Val 2030220PRTArtificial SequenceSynthetic peptide 302Leu Ala Ser
Lys Ser Ala Glu Glu Gly Lys Gln Ile Pro Asp Ser Leu1 5 10 15Ser Thr
Asp Leu 2030320PRTArtificial SequenceSynthetic peptide 303Leu Ala
Val Leu Ala Tyr Ser Ile Thr Leu Val Met Leu Trp Ser Ile1 5 10 15Trp
Gln Tyr Ala 2030420PRTArtificial SequenceSynthetic peptide 304Leu
Glu Arg Thr Ser Ser Val Ser Pro Ser Thr Ala Glu Pro Glu Leu1 5 10
15Ser Ile Val Phe 2030520PRTArtificial SequenceSynthetic peptide
305Leu Asn Ser Cys Ser Asn Arg Arg Val Tyr Lys Lys Met Pro Ser Ile1
5 10 15Glu Ser Asp Val 2030620PRTArtificial SequenceSynthetic
peptide 306Leu Gln Phe His Arg Gly Ser Arg Ala Gln Ser Phe Leu Gln
Thr Glu1 5 10 15Thr Ser Val Ile 2030720PRTArtificial
SequenceSynthetic peptide 307Asn Tyr Lys Leu Asn Thr Asp His Ala
Gly Ser Asn Asp Asn Ile Ala1 5 10 15Leu Leu Val Gln
2030820PRTArtificial SequenceSynthetic peptide 308Pro Gly Gln Pro
Pro Lys Val Lys Ser Glu Phe Asn Ser Tyr Ser Leu1 5 10 15Thr Gly Tyr
Val 2030920PRTArtificial SequenceSynthetic peptide 309Pro Ile Pro
Ala Gly Gly Cys Thr Phe Ser Gly Ile Phe Pro Thr Leu1 5 10 15Thr Ser
Pro Leu 2031020PRTArtificial SequenceSynthetic peptide 310Pro Ser
Ser Arg Ala Ser Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp1 5 10 15Asp
Leu Glu Ile 2031120PRTArtificial SequenceSynthetic peptide 311Pro
Tyr Ser Glu Leu Asn Tyr Glu Thr Ser His Tyr Pro Ala Ser Pro1 5 10
15Asp Ser Trp Val 2031220PRTArtificial SequenceSynthetic peptide
312Gln Asp Phe Arg Arg Ala Phe Arg Arg Ile Leu Ala Arg Pro Trp Thr1
5 10 15Gln Thr Ala Trp 2031319PRTArtificial SequenceSynthetic
peptide 313Gln Gly Asp Pro Ala Leu Gln Asp Ala Gly Asp Ser Ser Arg
Lys Glu1 5 10
15Tyr Phe Ile31420PRTArtificial SequenceSynthetic peptide 314Gln
Ile Ser Pro Gly Gly Leu Glu Pro Pro Ser Glu Lys His Phe Arg1 5 10
15Glu Thr Glu Val 2031520PRTArtificial SequenceSynthetic peptide
315Arg Glu Leu Val Asp Arg Gly Glu Val Arg Gln Phe Thr Leu Arg His1
5 10 15Trp Leu Lys Val 2031620PRTArtificial SequenceSynthetic
peptide 316Arg Arg Arg Arg Arg Arg Arg Gly Asn Thr Thr Asp Lys Glu
Leu Glu1 5 10 15Val Leu Ser Leu 2031720PRTArtificial
SequenceSynthetic peptide 317Arg Arg Arg Arg Arg Arg Arg Gly Thr
Asn Pro Ala Val Ala Ala Thr1 5 10 15Ser Ala Asn Leu
2031820PRTArtificial SequenceSynthetic peptide 318Arg Ser Gly Ala
Thr Ile Pro Leu Val Gly Gln Asp Ile Ile Asp Leu1 5 10 15Gln Thr Glu
Val 2031920PRTArtificial SequenceSynthetic peptide 319Ser Leu Ile
Gly Pro Val Gln Lys Glu Tyr Gln Arg Glu Leu Gly Lys1 5 10 15Leu Ser
Ser Pro 2032019PRTArtificial SequenceSynthetic peptide 320Ser Ser
Lys Ser Lys Ser Ser Glu Glu Ser Gln Thr Phe Phe Gly Leu1 5 10 15Tyr
Lys Leu32120PRTArtificial SequenceSynthetic peptide 321Ser Ser Pro
Asp Ser Ser Tyr Gln Gly Lys Gly Phe Val Met Ser Arg1 5 10 15Ala Met
Tyr Val 2032220PRTArtificial SequenceSynthetic peptide 322Ser Ser
Ser Arg Arg Asp Ser Ser Trp Ser Glu Thr Ser Glu Ala Ser1 5 10 15Tyr
Ser Gly Leu 2032320PRTArtificial SequenceSynthetic peptide 323Ser
Thr Asp Asn Leu Val Arg Pro Phe Met Asp Thr Leu Ala Ser His1 5 10
15Gln Leu Tyr Ile 2032420PRTArtificial SequenceSynthetic peptide
324Thr Glu Gly Asn Glu Ser Ser Glu Ala Thr Ser Pro Val Asn Ala Ile1
5 10 15Tyr Ser Leu Ala 2032520PRTArtificial SequenceSynthetic
peptide 325Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr Leu
Gly Leu1 5 10 15Asp Val Pro Val 2032620PRTArtificial
SequenceSynthetic peptide 326Thr Gly Ser Ala Leu Gln Ala Trp Arg
His Thr Ser Arg Gln Ala Thr1 5 10 15Glu Ser Thr Val
2032720PRTArtificial SequenceSynthetic peptide 327Thr Gln Gly Phe
Pro Gly Pro Ala Thr Trp Arg Arg Ile Ser Ser Leu1 5 10 15Glu Ser Glu
Val 2032819PRTArtificial SequenceSynthetic peptide 328Thr Thr Asn
Asn Asn Pro Asn Ser Ala Val Asn Ile Lys Lys Ile Phe1 5 10 15Thr Asp
Val32920PRTArtificial SequenceSynthetic peptide 329Val Asp Pro Asn
Ser Pro Ala Ala Lys Lys Lys Tyr Val Ser Tyr Asn1 5 10 15Asn Leu Val
Ile 2033020PRTArtificial SequenceSynthetic peptide 330Val His Asp
Ala Glu Ser Ser Asp Glu Asp Gly Tyr Asp Trp Gly Pro1 5 10 15Ala Thr
Asp Leu 2033120PRTArtificial SequenceSynthetic peptide 331Val His
Lys Val Arg Asn Lys Phe Lys Ala Lys Cys Ser Leu Cys Arg1 5 10 15Leu
Tyr Ile Ile 2033220PRTArtificial SequenceSynthetic peptide 332Val
Pro Gly Ala Leu Asp Tyr Ala Ala Phe Ser Ser Ala Leu Tyr Gly1 5 10
15Glu Ser Asp Leu 2033320PRTArtificial SequenceSynthetic peptide
333Val Pro Ser Asp Asn Ile Asp Ser Gln Gly Arg Asn Ala Ser Thr Asn1
5 10 15Asp Ser Leu Leu 2033420PRTArtificial SequenceSynthetic
peptide 334Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly
Asp Arg1 5 10 15Lys Ile Arg Val 2033520PRTArtificial
SequenceSynthetic peptide 335Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ala Arg Gly Asp Arg1 5 10 15Lys Lys Ile Val
2033620PRTArtificial SequenceSynthetic peptide 336Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1 5 10 15Lys Arg Trp
Ala 2033720PRTArtificial SequenceSynthetic peptide 337Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1 5 10 15Lys Arg
Trp Leu 2033820PRTArtificial SequenceSynthetic peptide 338Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1 5 10 15Lys
Val Pro Val 2033920PRTArtificial SequenceSynthetic peptide 339Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1 5 10
15Lys Tyr Ile Val 2034020PRTArtificial SequenceSynthetic peptide
340Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1
5 10 15Leu Glu Ile Val 2034120PRTArtificial SequenceSynthetic
peptide 341Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly
Asp Arg1 5 10 15Leu Val Ser Leu 2034220PRTArtificial
SequenceSynthetic peptide 342Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ala Arg Gly Asp Arg1 5 10 15Arg Arg Ile Val
2034320PRTArtificial SequenceSynthetic peptide 343Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1 5 10 15Thr Ile Ile
Tyr 2034420PRTArtificial SequenceSynthetic peptide 344Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Arg1 5 10 15Tyr Arg
Ile Val 2034520PRTArtificial SequenceSynthetic peptide 345Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Asp Val1 5 10 15Arg
Leu Met Leu 2034620PRTArtificial SequenceSynthetic peptide 346Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Gly Arg Arg1 5 10
15Glu Thr Trp Val 2034720PRTArtificial SequenceSynthetic peptide
347Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Ser Asp Arg1
5 10 15Gly Ile Trp Ala 2034820PRTArtificial SequenceSynthetic
peptide 348Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Ser
Asp Arg1 5 10 15Lys Arg Ile Ala 2034920PRTArtificial
SequenceSynthetic peptide 349Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ala Arg Ser Asp Arg1 5 10 15Lys Arg Ile Val
2035020PRTArtificial SequenceSynthetic peptide 350Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Ala Arg Ser Asp Arg1 5 10 15Thr Ile Ile
Ala 2035120PRTArtificial SequenceSynthetic peptide 351Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Ser Asp Arg1 5 10 15Thr Ile
Trp Ala 2035220PRTArtificial SequenceSynthetic peptide 352Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Arg Thr Asp Arg1 5 10 15Lys
Val Glu Val 2035320PRTArtificial SequenceSynthetic peptide 353Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Val Ala Ala Ala1 5 10
15Ser Ala Asn Leu 2035420PRTArtificial SequenceSynthetic peptide
354Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Val Ala Ala Thr1
5 10 15Gly Ile Trp Ala 2035520PRTArtificial SequenceSynthetic
peptide 355Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Val Ala
Ala Thr1 5 10 15Ser Ala Asn Leu 2035620PRTArtificial
SequenceSynthetic peptide 356Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ala Val Ala Ala Thr1 5 10 15Ser Ile Asn Leu
2035720PRTArtificial SequenceSynthetic peptide 357Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Ala Val Ala Ala Thr1 5 10 15Tyr Ser Asn
Leu 2035820PRTArtificial SequenceSynthetic peptide 358Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Asp Lys Glu Leu Glu1 5 10 15Val Leu
Ser Leu 2035920PRTArtificial SequenceSynthetic peptide 359Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Lys His Phe Arg1 5 10 15Glu
Thr Glu Val 2036020PRTArtificial SequenceSynthetic peptide 360Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Tyr Leu Gly Leu1 5 10
15Asp Val Pro Val 2036120PRTArtificial SequenceSynthetic peptide
361Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ala Ser Ala Asp1
5 10 15Ser Thr Gln Ala 2036220PRTArtificial SequenceSynthetic
peptide 362Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Met Thr
Ser Ser1 5 10 15Ser Ser Val Val 2036320PRTArtificial
SequenceSynthetic peptide 363Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Lys Leu Ser Ser Ile1 5 10 15Glu Ser Asp Val
2036420PRTArtificial SequenceSynthetic peptide 364Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Lys Asn Lys Asp Lys1 5 10 15Glu Tyr Tyr
Val 2036520PRTArtificial SequenceSynthetic peptide 365Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Leu Gln Arg Arg Arg1 5 10 15Glu Thr
Gln Val 2036620PRTArtificial SequenceSynthetic peptide 366Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Asn Asp Asn Ile Ala1 5 10 15Leu
Leu Val Gln 2036720PRTArtificial SequenceSynthetic peptide 367Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gln Asp Glu Glu Glu1 5 10
15Gly Ile Trp Ala 2036820PRTArtificial SequenceSynthetic peptide
368Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gln Asp Glu Glu Glu1
5 10 15Gly Ile Trp Ser 2036920PRTArtificial SequenceSynthetic
peptide 369Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gln Asp Glu
Glu Glu1 5 10 15Thr Ile Trp Ala 2037020PRTArtificial
SequenceSynthetic peptide 370Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Gln Asp Glu Arg Arg1 5 10 15Leu Ile Val Leu
2037120PRTArtificial SequenceSynthetic peptide 371Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Gln Asp Glu Arg Val1 5 10 15Glu Thr Arg
Val 2037220PRTArtificial SequenceSynthetic peptide 372Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Ser Glu Gly Val Pro1 5 10 15Asp Leu
Leu Val 2037320PRTArtificial SequenceSynthetic peptide 373Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Arg Thr Asp Arg1 5 10 15Lys
Tyr Gln Ile 2037420PRTArtificial SequenceSynthetic peptide 374Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Arg Thr Asp Arg1 5 10
15Lys Tyr Trp Ala 2037520PRTArtificial SequenceSynthetic peptide
375Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Arg Thr Asp Arg1
5 10 15Leu Glu Tyr Val 2037620PRTArtificial SequenceSynthetic
peptide 376Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Arg Thr
Val Arg1 5 10 15Glu Ile Trp Ala 2037720PRTArtificial
SequenceSynthetic peptide 377Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ser Val Thr Ser Thr1 5 10 15Ser Ile Asn Leu
2037820PRTArtificial SequenceSynthetic peptide 378Tyr Ser Ala Thr
Tyr Ser Glu Leu Glu Asp Pro Gly Glu Met Ser Pro1 5 10 15Pro Ile Asp
Leu 2037920PRTArtificial SequenceSynthetic peptide 379Tyr Val Tyr
Ser Arg Val Lys Asn Leu Asn Ser Ser Glu Gly Val Pro1 5 10 15Asp Leu
Leu Val 2038020PRTArtificial SequenceSynthetic peptide 380Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Thr Asn Ser Val1 5 10 15Arg
Leu Met Leu 20381167DNAArtificial SequenceViral construct
381gaattcatat ttgcatgtcg ctatgtgttc tgggaaatca ccataaacgt
gaaatgtctt 60tggatttggg aatcttataa gttctgtatg agaccactcg gatccgagtg
catgactggt 120gaatttcaag agaattcacc agtcatgcac tctttttgga aaagctt
16738249RNAArtifical SequencesiRNA sequence 382gagugcauga
cuggugaauu ucaagagaau ucaccaguca ugcacucuu 4938345RNAArtifical
SequencesiRNA sequence 383gagugcauga cuggugauuc aagagaucac
cagucaugca cucuu 453849PRTArtificial SequenceSynthetic peptide
384Ala Thr Asn Ser Val Arg Leu Met Leu1 5
* * * * *
References