U.S. patent application number 10/477076 was filed with the patent office on 2005-04-14 for alzheimer's disease secretase, app substrates therefor, and uses therefor.
Invention is credited to Bienkowski, Michael J., Gurney, Mark E., Heinrikson, Robert L., Parodi, Luis A., Yan, Riqiang.
Application Number | 20050080232 10/477076 |
Document ID | / |
Family ID | 23651777 |
Filed Date | 2005-04-14 |
United States Patent
Application |
20050080232 |
Kind Code |
A1 |
Gurney, Mark E. ; et
al. |
April 14, 2005 |
Alzheimer's disease secretase, app substrates therefor, and uses
therefor
Abstract
The present invention provides the enzyme and enzymatic
procedures for cleaving the .beta. secretase cleavage site of the
APP protein and associated nucleic acids, peptides, vectors, cells
and cell isolates and assays. The invention further provides a
modified APP protein and associated nucleic acids, peptides,
vectors, cells, and cell isolates, and assays that are particularly
useful for identifying candidate therapeutics for treatment or
prevention of Alzheimer's disease.
Inventors: |
Gurney, Mark E.; (Grand
Rapids, MI) ; Bienkowski, Michael J.; (Portage,
MI) ; Heinrikson, Robert L.; (Plainwell, MI) ;
Parodi, Luis A.; (Stockholm, SE) ; Yan, Riqiang;
(Kalamazoo, MI) |
Correspondence
Address: |
David A Gass
Marshall O'Toole Murray & Borun
6300 Sears Tower
233 South Wacker Drive
Chicago
IL
60606-6402
US
|
Family ID: |
23651777 |
Appl. No.: |
10/477076 |
Filed: |
November 6, 2003 |
PCT Filed: |
May 9, 2001 |
PCT NO: |
PCT/IB01/00799 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10477076 |
Nov 6, 2003 |
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09416901 |
Oct 13, 1999 |
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6699671 |
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09416901 |
Oct 13, 1999 |
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09404133 |
Sep 23, 1999 |
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09416901 |
Oct 13, 1999 |
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PCT/US99/20881 |
Sep 23, 1999 |
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60101594 |
Sep 24, 1998 |
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60155493 |
Sep 23, 1999 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
G01N 2333/96472
20130101; C12N 9/6478 20130101; C07K 14/4711 20130101; C12N
2799/026 20130101; C07K 2319/00 20130101; C12Q 1/37 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 014/47 |
Claims
1-150. (canceled)
151. A purified and isolated polypeptide comprising an amino acid
sequence that is at least 95% identical to a fragment of the
aspartyl protease amino acid sequence of SEQ ID NO: 4, wherein the
fragment includes the aspartyl protease active site tripeptides DTG
and DSG of SEQ ID NO: 4, wherein said polypeptide lacks a
transmembrane domain, and wherein said polypeptide exhibits
aspartyl protease activity involved in processing APP into amyloid
beta.
152. An isolated or purified polypeptide encoded by a
polynucleotide comprising a nucleotide sequence that hybridizes
under the following stringent hybridization conditions to the
complement of SEQ ID NO: 3: (1) hybridization at 42.degree. C. in a
hybridization buffer comprising 6.times.SSC and 0.1% SDS, and (2)
washing at 65.degree. C. in a wash solution comprising 1.times.SSC
and 0.1% SDS; wherein the polypeptide includes the aspartyl
protease active site tripeptides DTG and DSG of SEQ ID NO: 4,
wherein said polypeptide lacks a transmembrane domain, and wherein
said polypeptide exhibits aspartyl protease activity involved in
processing APP into amyloid beta.
153. An isolated or purified nucleic acid comprising a nucleotide
sequence that encodes a polypeptide, wherein the polypeptide
comprises an amino acid sequence that is at least 95% identical to
a fragment of the aspartyl protease amino acid sequence of SEQ ID
NO: 4, wherein the fragment includes the aspartyl protease active
site tripeptides DTG and DSG of SEQ ID NO: 4, wherein said
polypeptide lacks a transmembrane domain, and wherein said
polypeptide exhibits aspartyl protease activity involved in
processing APP into amyloid beta.
154. A method for identifying an agent that decreases the protease
activity of tie aspartyl protease polypeptide of any one of claims
151-153 comprising steps of: (a) measuring protease activity of
said polypeptide in the presence and absence of a test agent; and
(b) comparing protease activity of the polypeptide in the presence
and absence of the test agent, wherein decreased protease activity
in the presence of the test agent identifies the test agent as an
agent that decreases the protease activity of the aspartyl protease
polypeptide.
Description
[0001] The present application is a continuation of U.S.
application Ser. No. 09/416,901, filed Oct. 13, 1999 which claims
priority benefit of U.S. Provisional Patent Application No.
60/155,493, filed Sep. 23, 1999. The present application also
claims priority benefit as a continuation-in-part of U.S. patent
application Ser. No. 09/404,133 and PCT/US99/20881, both filed Sep.
23, 1999, both of which in turn claim priority benefit of U.S.
Provisional Patent Application No. 60/101,594, filed Sep. 24, 1998.
All of these priority applications are hereby incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to Alzheimer's Disease,
amyloid protein precursor, amyloid beta peptide, and human aspartyl
proteases, as well as a method for the identification of agents
that modulate the activity of these polypeptides and thereby are
candidates to modulate the progression of Alzheimer's disease.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) causes progressive dementia with
consequent formation of amyloid plaques, neurofibrillary tangles,
gliosis and neuronal loss. The disease occurs in both genetic and
sporadic forms whose clinical course and pathological features are
quite similar. Three genes have been discovered to date which, when
mutated, cause an autosomal dominant form of Alzheimer's disease.
These encode the amyloid protein precursor (APP) and two related
proteins, presenilin-1 (PS1) and presenilin-2 (PS2), which, as
their names suggest, are structurally and functionally related.
Mutations in any of the three proteins have been observed to
enhance proteolytic processing of APP via an intracellular pathway
that produces amyloid beta peptide (A.beta. peptide, or sometimes
here as Abeta), a 40-42 amino acid long peptide that is the primary
component of amyloid plaque in AD.
[0004] Dysregulation of intracellular pathways for proteolytic
processing may be central to the pathophysiology of AD. In the case
of plaque formation, mutations in APP, PS1 or PS2 consistently
alter the proteolytic processing of APP so as to enhance formation
of A.beta. 1-42, a form of the A.beta. peptide which seems to be
particularly amyloidogenic, and thus very important in AD.
Different forms of APP range in size from 695-770 amino acids,
localize to the cell surface, and have a single C-terminal
transmembrane domain. Examples of specific isotypes of APP which
are currently known to exist in humans are the 695-amino acid
polypeptide described by Kang et. al. (1987), Nature 325: 733-736
which is designated as the "normal" APP; the 751 amino acid
polypeptide described by Ponte et al. (1988), Nature 331: 525-527
(1988) and Tanzi et al. (1988), Nature 331: 528-530; and the 770
amino acid polypeptide described by Kitaguchi et. al. (1988),
Nature 331: 530-532. The Abeta peptide is derived from a region of
APP adjacent to and containing a portion of the transmembrane
domain. Normally, processing of APP at the .alpha.-secretase site
cleaves the midregion of the A.beta. sequence adjacent to the
membrane and releases the soluble, extracellular domain of APP from
the cell surface. This .alpha.-secretase APP processing creates
soluble APP-.alpha., which is normal and not thought to contribute
to AD. Pathological processing of APP at the .beta.- and
.gamma.-secretase sites, which are located N-terminal and
C-terminal to the .alpha.-secretase site, respectively, produces a
very different result than processing at the a site. Sequential
processing at the .beta.- and .gamma.-secretase sites releases the
A.beta. peptide, a peptide possibly very important in AD
pathogenesis. Processing at the .beta.- and .gamma.-secretase sites
can occur in both the endoplasmic reticulum (in neurons) and in the
endosomal/lysosomal pathway after reinternalization of cell surface
APP (in all cells). Despite intense efforts, for 10 years or more,
to identify the enzymes responsible for processing APP at the
.beta. and .gamma. sites, to produce the A.beta. peptide, those
proteases remained unknown until this disclosure.
SUMMARY OF THE INVENTION
[0005] Here, for the first time, we report the identification and
characterization of the .beta. secretase enzyme, termed Aspartyl
Protease 2 (Asp2). We disclose some known and some novel human
aspartic proteases that can act as .beta.-secretase proteases and,
for the first time, we explain the role these proteases have in AD.
We describe regions in the proteases critical for their unique
function and for the first time characterize their substrate. This
is the first description of expressed isolated purified active
protein of this type, assays that use the protein, in addition to
the identification and creation of useful cell lines and
inhibitors.
[0006] Here we disclose a number of variants of the Asp2 gene and
peptide.
[0007] In one aspect, the invention provides any isolated or
purified nucleic acid polynucleotide that codes for a protease
capable of cleaving the beta (.beta.) secretase cleavage site of
APP that contains two or more sets of special nucleic acids, where
the special nucleic acids are separated by nucleic acids that code
for about 100 to 300 amino acid positions, where the amino acids in
those positions may be any amino acids, where the first set of
special nucleic acids consists of the nucleic acids that code for
the peptide DTG, where the first nucleic acid of the first special
set of nucleic acids is the first special nucleic acid, and where
the second set of nucleic acids code for either the peptide DSG or
DTG, where the last nucleic acid of the second set of nucleic acids
is the last special nucleic acid, with the proviso that the nucleic
acids disclosed in SEQ ID NO. 1 and SEQ ID NO. 3 are not included.
In a preferred embodiment, the two sets of special nucleic acids
are separated by nucleic acids that code for about 125 to 222 amino
acid positions, which may be any amino acids. In a highly preferred
embodiment, the two sets of special nucleic acids are separated by
nucleic acids that code for about 150 to 196, or 150-190, or 150 to
172 amino acid positions, which may be any amino acids. In a
particular preferred embodiment, the two sets are separated by
nucleic acids that code for about 172 amino acid positions, which
may be any amino acids. An exemplary nucleic acid polynucleotide
comprises the acid nucleotide sequence in SEQ ID NO. 5. In another
particular preferred embodiment, the two sets are separated by
nucleic acids that code for about 196 amino acids. An exemplary
polynucleotide comprises the nucleotide sequence in SEQ ID NO. 5.
In another particular embodiment, the two sets of nucleotides are
separated by nucleic acids that code for about 190 amino acids. An
exemplary polynucleotide comprises the nucleotide sequence in SEQ
ID NO. 1. Preferably, the first nucleic acid of the first special
set of amino acids, that is, the first special nucleic acid, is
operably linked to any codon where the nucleic acids of that codon
codes for any peptide comprising from 1 to 10,000 amino acid
(positions). In one variation, the first special nucleic acid is
operably linked to nucleic acid polymers that code for any peptide
selected from the group consisting of: any reporter proteins or
proteins which facilitate purification. For example, the first
special nucleic acid is operably linked to nucleic acid polymers
that code for any peptide selected from the group consisting of:
immunoglobin-heavy chain, maltose binding protein, glutathione S
transferase, Green Fluorescent protein, and ubiquitin. In another
variation, the last nucleic acid of the second set of special amino
acids, that is, the last special nucleic acid, is operably linked
to nucleic acid polymers that code for any peptide comprising any
amino acids from 1 to 10,000 amino acids. In still another
variation, the last special nucleic acid is operably linked to
nucleic acid polymers that code for any peptide selected from the
group consisting of: any reporter proteins or proteins which
facilitate purification. For example, the last special nucleic acid
is operably linked to nucleic acid polymers that code for any
peptide selected from the group consisting of: immunoglobin-heavy
chain, maltose binding protein, glutathione S transferase, Green
Fluorescent protein, and ubiquitin.
[0008] In a related aspect, the invention provides any isolated or
purified nucleic acid polynucleotide that codes for a protease
capable of cleaving the beta secretase cleavage site of APP that
contains two or more sets of special nucleic acids, where the
special nucleic acids are separated by nucleic acids that code for
about 100 to 300 amino acid positions, where the amino acids in
those positions may be any amino acids, where the first set of
special nucleic acids consists of the nucleic acids that code for
DTG, where the first nucleic acid of the first special set of
nucleic acids is the first special nucleic acid, and where the
second set of nucleic acids code for either DSG or DTG, where the
last nucleic acid of the second set of special nucleic acids is the
last special nucleic acid, where the first special nucleic acid is
operably linked to nucleic acids that code for any number of amino
acids from zero to 81 amino acids and where each of those codons
may code for any amino acid. In a preferred embodiment, the first
special nucleic acid is operably linked to nucleic acids that code
for any number of from 64 to 77 amino acids where each codon may
code for any amino acid. In a particular embodiment, the first
special nucleic acid is operably linked to nucleic acids that code
for 71 amino acids. For example, the first special nucleic acid is
operably linked to 71 amino acids and where the first of those 71
amino acids is the amino acid T. In a preferred embodiment, the
polynucleotide comprises a sequence that is at least 95% identical
to a human Asp1 or Asp2 sequence as taught herein. In another
preferred embodiment, the first special nucleic acid is operably
linked to nucleic acids that code for any number of from 30 to 54
amino acids, or 35 to 47 amino acids, or 40 to 54 amino acids where
each codon may code for any amino acid. In a particular embodiment,
the first special nucleic acid is operably linked to nucleic acids
that code for 47 amino acids. For example, the first special
nucleic acid is operably linked to 47 codons where the first those
47 amino acids is the amino acid E.
[0009] In another related aspect, the invention provides for any
isolated or purified nucleic acid polynucleotide that codes for a
protease capable of cleaving the beta (.beta.) secretase cleavage
site of APP and that contains two or more sets of special nucleic
acids, where the special nucleic acids are separated by nucleic
acids that code for about 100 to 300 amino acid positions, where
the amino acids in those positions may be any amino acids, where
the first set of special nucleic acids consists of the nucleic
acids that code for the peptide DTG, where the first nucleic acid
of the first special set of amino acids is, the first special
nucleic acid, and where the second set of special nucleic acids
code for either the peptide DSG or DTG, where the last nucleic acid
of the second set of special nucleic acids, the last special
nucleic acid, is operably linked to nucleic acids that code for any
number of codons from 50 to 170 codons. In a preferred embodiment,
the last special nucleic acid is operably linked to nucleic acids
comprising from 100 to 170 codons. In a highly preferred
embodiment, the last special nucleic acid is operably linked to
nucleic acids comprising from 142 to 163 codons. In a particular
embodiment, the last special nucleic acid is operably linked to
nucleic acids comprising about 142 codons, or about 163 codons, or
about 170 codons. In a highly preferred embodiment, the
polynucleotide comprises a sequence that is at least 95% identical
to aspartyl-protease encoding sequences taught herein. In one
variation, the second set of special nucleic acids code for the
peptide DSG. In another variation, the first set of nucleic acid
polynucleotide is operably linked to a peptide purification tag.
For example, the nucleic acid polynucleotide is operably linked to
a peptide purification tag which is six histidine. In still another
variation, the first set of special nucleic acids are on one
polynucleotide and the second set of special nucleic acids are on a
second polynucleotide, where both first and second polynucleotides
have at lease 50 codons. In one embodiment of this type, both of
the polynucleotides are in the same solution. In a related aspect,
the invention provides a vector which contains a polynucleotide as
described above, or a cell or cell line which is transformed or
transfected with a polynucleotide as described above or with a
vector containing such a polynucleotide.
[0010] In still another aspect, the invention provides an isolated
or purified peptide or protein comprising an amino acid polymer
that is a protease capable of cleaving the beta (.beta.) secretase
cleavage site of APP that contains two or more sets of special
amino acids, where the special amino acids are separated by about
100 to 300 amino acid positions, where each amino acid position can
be any amino acid, where the first set of special amino acids
consists of the peptide DTG, where the first amino acid of the
first special set of amino acids is, the first special amino acid,
where the second set of amino acids is selected from the peptide
comprising either DSG or DTG, where the last amino acid of the
second set of special amino acids is the last special amino acid,
with the proviso that the proteases disclosed in SEQ ID NO. 2 and
SEQ ID NO. 4 are not included. In preferred embodiments, the two
sets of amino acids are separated by about 125 to 222 amino acid
positions or about 150 to 196 amino acids, or about 150-190 amino
acids, or about 150 to 172 amino acids, where in each position it
may be any amino acid. In a particular embodiment, the two sets of
amino acids are separated by about 172 amino acids. For example,
the protease has the amino acid sequence described in SEQ ID NO 6.
In another particular embodiment, the two sets of amino acids are
separated by about 196 amino acids. For example, the two sets of
amino acids are separated by the same amino acid sequences that
separate the same set of special amino acids in SEQ ID NO 4. In
another particular embodiment, the two sets of nucleotides are
separated by about 190 amino acids. For example, the two sets of
nucleotides are separated by the same amino acid sequences that
separate the same set of special amino acids in SEQ ID NO 2. In one
embodiment, the first amino acid of the first special set of amino
acids, that is, the first special amino acid, is operably linked to
any peptide comprising from 1 to 10,000 amino acids. In another
embodiment, the first special amino acid is operably linked to any
peptide selected from the group consisting of: any reporter
proteins or proteins which facilitate purification. In particular
embodiments, the first special amino acid is operably linked to any
peptide selected from the group consisting of: immunoglobin-heavy
chain, maltose binding protein, glutathione S transferase, Green
Fluorescent-protein, and ubiquitin. In still another variation, the
last amino acid of the second set of special amino acids, that is,
the last special amino acid, is operably linked to any peptide
comprising any amino acids from 1 to 10,000 amino acids. By way of
nonlimiting example, the last special amino acid is operably linked
any peptide selected from the group consisting of any reporter
proteins or proteins which facilitate purification. In particular
embodiments, the last special amino acid is operably linked to any
peptide selected from the group consisting of: immunoglobin-heavy
chain, maltose binding protein, glutathione S transferase, Green
Fluorescent protein, and ubiquitin.
[0011] In a related aspect, the invention provides any isolated or
purified peptide or protein comprising an amino acid polypeptide
that codes for a protease capable of cleaving the beta secretase
cleavage site of APP that contains two or more sets of special
amino acids, where the special amino acids are separated by about
100 to 300 amino acid positions, where each amino acid in each
position can be any amino acid, where the first set of special
amino acids consists of the amino acids DTG, where the first amino
acid of the first special set of amino acids is, the first special
amino acid, D, and where the second set of amino acids is either
DSG or DTG, where the last amino acid of the second set of special
amino acids is the last special amino acid, G, where the first
special amino acid is operably linked to amino acids that code for
any number of amino acids from zero to 81 amino acid positions
where in each position it may be any amino acid. In a preferred
embodiment, the first special amino acid is operably linked to a
peptide from about 30-77 or about 64 to 77 amino acids positions
where each amino acid position may be any amino acid. In a
particular embodiment, the first special amino acid is operably
linked to a peptide 35, 47, 71, or 77 amino acids. In a very
particular embodiment, the first special amino acid is operably
linked to 71 amino acids and the first of those 71 amino acids is
the amino acid T. For example, the polypeptide comprises a sequence
that is at least 95% identical to an aspartyl protease sequence as
described herein. In another embodiment, the first special amino
acid is operably linked to any number of from 40 to 54 amino acids
(positions) where each amino acid position may be any amino acid.
In a particular embodiment, the first special amino acid is
operably linked to amino acids that code for a peptide of 47 amino
acids. In a very particular embodiment, the first special amino
acid is operably linked to a 47 amino acid peptide where the first
those 47 amino acids is the amino acid E. In another particular
embodiment, the first special amino acid is operably linked to the
same corresponding peptides from SEQ ID NO. 3 that are 35, 47, 71,
or 77 peptides in length, beginning counting with the amino acids
on the first special sequence, DTG, towards the N-terminal of SEQ
ID NO. 3. In another particular embodiment, the polypeptide
comprises a sequence that is at least 95% identical to the same
corresponding amino acids in SEQ ID NO. 4, that is, identical to
that portion of the sequences in SEQ ID NO. 4, including all the
sequences from both the first and or the second special nucleic
acids, toward the--terminal, through and including 71, 47, 35 amino
acids before the first special amino acids. For example, the
complete polypeptide comprises the peptide of 71 amino acids, where
the first of the amino acid is T and the second is Q.
[0012] In still another related aspect, the invention provides any
isolated or purified amino acid polypeptide that is a protease
capable of cleaving the beta (.beta.) secretase cleavage site of
APP that contains two or more sets of special amino acids, where
the special amino acids are separated by about 100 to 300 amino
acid positions, where each amino acid in each position can be any
amino acid, where the first set of special amino acids consists of
the amino acids that code for DTG, where the first amino acid of
the first special set of amino acids is, the first special amino
acid, D, and where the second set of amino acids are either DSG or
DTG, where the last amino acid of the second set of special amino
acids is the last special amino acid, G, which is operably linked
to any number of amino acids from 50 to 170 amino acids, which may
be any amino acids. In preferred embodiments, the last special
amino acid is operably linked to a peptide of about 100 to 170
amino acids or about 142-163 amino acids. In particular
embodiments, the last special amino acid is operably linked to a
peptide of about 142 amino acids, or about 163 amino acids, or
about 170 amino acids. For example, the polypeptide comprises a
sequence that is at least 95% identical (and preferably 100%
identical) to an aspartyl protease sequence as described herein. In
one particular embodiment, the second set of special amino acids is
comprised of the peptide with the amino acid sequence DSG.
Optionally, the amino acid polypeptide is operably linked to a
peptide purification tag, such as purification tag which is six
histidine. In one variation, the first set of special amino acids
are on one polypeptide and the second set of special amino acids
are on a second polypeptide, where both first and second
polypeptide have at lease 50 amino acids, which maybe any amino
acids. In one embodiment of this type, both of the polypeptides are
in the same vessel. The invention further includes a process of
making any of the polynucleotides, vectors, or cells described
herein; and a process of making any of the polypeptides described
herein.
[0013] In yet another related aspect, the invention provides a
purified polynucleotide comprising a nucleotide sequence that
encodes a polypeptide having aspartyl protease activity, wherein
the polypeptide has an amino acid sequence characterized by: (a) a
first tripeptide sequence DTG; (b) a second tripeptide sequence
selected from the group consisting of DSG and DTG; and (c) about
100 to 300 amino acids separating the first and second tripeptide
sequences, wherein the polypeptide cleaves the beta secretase
cleavage site of amyloid protein precursor. In one embodiment, the
polypeptide comprises an amino acid sequence depicted in SEQ ID NO:
2 or 4, whereas in another embodiment, the polypeptide comprises an
amino acid sequence other than the amino acid sequences set forth
in SEQ ID NOs: 2 and 4. Similarly, the invention provides a
purified polynucleotide comprising a nucleotide sequence that
encodes a polypeptide that cleaves the beta secretase cleavage site
of amyloid protein precursor; wherein the polynucleotide includes a
strand that hybridizes to one or more of SEQ ID NOs: 3, 5, and 7
under the following hybridization conditions: hybridization
overnight at 42.degree. C. for 2.5 hours in 6.times.SSC/0.1% SDS,
followed by washing in 1.0.times.SSC at 65.degree. C., 0.1% SDS. In
one embodiment, the polypeptide comprises an amino acid sequence
depicted in SEQ ID NO: 2 or 4, whereas in another embodiment, the
polypeptide comprises an amino acid sequence other than the amino
acid sequences set forth in SEQ ID NOs: 2 and 4. Likewise, the
invention provides a purified polypeptide having aspartyl protease
activity, wherein the polypeptide is encoded by polynucleotides as
described in the preceding sentences. The invention also provides a
vector or host cell comprising such polynucleotides, and a method
of making the polypeptides using the vectors or host cells to
recombinantly express the polypeptide.
[0014] In yet another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide, said
polynucleotide encoding a Hu-Asp polypeptide and having a
nucleotide sequence at least 95% identical to a sequence selected
from the group consisting of:
[0015] (a) a nucleotide sequence encoding a Hu-Asp polypeptide
selected from the group consisting of Hu-Asp1, Hu-Asp2(a), and
Hu-Asp2(b), wherein said Hu-Asp1, Hu-Asp2(a) and Hu-Asp2(b)
polypeptides have the complete amino acid sequence of SEQ ID NO. 2,
SEQ ID NO. 4, and SEQ ID NO. 6, respectively, and
[0016] (b) a nucleotide sequence complementary to the nucleotide
sequence of (a).
[0017] Several species are particularly contemplated. For example,
the invention provides a nucleic acid and molecule wherein said
Hu-Asp polypeptide is Hu-Asp1, and said polynucleotide molecule of
1 (a) comprises the nucleotide sequence of SEQ ID NO. 1; and a
nucleic acid molecule wherein said Hu-Asp polypeptide is
Hu-Asp2(a), and said polynucleotide molecule of 1(a) comprises the
nucleotide sequence of SEQ ID NO. 4; and a nucleic acid molecule
wherein said Hu-Asp polypeptide is Hu-Asp2(b), and said
polynucleotide molecule of 1(a) comprises the nucleotide sequence
of SEQ ID NO. 5. In addition to the foregoing, the invention
provides an isolated nucleic acid molecule comprising
polynucleotide which hybridizes under stringent conditions to a
polynucleotide having the nucleotide sequence in (a) or (b) as
described above.
[0018] Additionally, the invention provides a vector comprising a
nucleic acid molecule as described in the preceding paragraph. In a
preferred embodiment, the nucleic acid molecule is operably linked
to a promoter for the expression of a Hu-Asp polypeptide.
Individual vectors which encode Hu-Asp1, and Hu-Asp2(a), and
Hu-Asp2(b) are all contemplated. Likewise, the invention
contemplates a host cell comprising any of the foregoing vectors,
as well as a method of obtaining a Hu-Asp polypeptide comprising
culturing such a host cell and isolating the Hu-Asp polypeptide.
Host cells of the invention include bacterial cells, such as E.
coli, and eukaryotic cells. Among the eukaryotic cells that are
contemplated are insect cells, such as sf9 or High 5 cells; and
mammalian cells, such as human, rodent, lagomorph, and primate.
Preferred human cells include HEK293, and IMR-32 cells. Other
preferred mammalian cells include COS-7, CHO-K1, Neuro-2A, and 3T3
cells. Also among the eukaryotic cells that are contemplated are a
yeast cell and an avian cell.
[0019] In a related aspect, the invention provides an isolated
Hu-Asp1 polypeptide comprising an amino acid sequence at least 95%
identical to a sequence comprising the amino acid sequence of SEQ
ID NO. 2. The invention also provides an isolated Hu-Asp2(a)
polypeptide comprising an amino acid sequence at least 95%
identical to a sequence comprising the amino acid sequence of SEQ
ID NO. 4. The invention also provides an isolated Hu-Asp2(a)
polypeptide comprising an amino acid sequence at least 95%
identical to a sequence comprising the amino acid sequence of SEQ
ID NO. 8.
[0020] In still another aspect, the invention provides an isolated
antibody that binds specifically to any Hu-Asp polypeptide
described herein, especially the polypeptide described in the
preceding paragraphs.
[0021] The invention also provides several assays involving
aspartyl protease enzymes of the invention. For example, the
invention provides
[0022] a method to identify a cell that can be used to screen for
inhibitors of .beta. secretase activity comprising:
[0023] (a) identifying a cell that expresses a protease capable of
cleaving APP at the .beta. secretase site, comprising:
[0024] i) collect the cells or the supernatant from the cells to be
identified
[0025] ii) measure the production of a critical peptide, where the
critical peptide is selected from the group consisting of either
the APP C-terminal peptide or soluble APP,
[0026] iii) select the cells which produce the critical
peptide.
[0027] In one variation, the cells are collected and the critical
peptide is the APP C-terminal peptide created as a result of the
.beta. secretase cleavage. In another variation, the supernatant is
collected and the critical peptide is soluble APP, where the
soluble APP has a C-terminus created by .beta. secretase cleavage.
In preferred embodiments, the cells contain any of the nucleic
acids or polypeptides described above and the cells are shown to
cleave the .beta. secretase site of any peptide having the
following peptide structure, P2, P1, P1', P2'; where P2 is K or N,
where P1 is M or L, where P1' is D, where P2' is A. The method of
claim 111 where P2 is K and P1 is M. The method of claim 112 where
P2 is N and P1 is L.
[0028] In still another aspect, the invention provides novel
isoforms of amyloid protein precursor (APP) where the last two
carboxy terminus amino acids of that isoform are both lysine
residues. In this context, the term "isoform" is defined as any APP
polypeptide, including APP variants (including mutations), and APP
fragments that exists in humans, such as those described in U.S.
Pat. No. 5,766,846, col 7, lines 45-67, incorporated into this
document by reference, modified as described herein by the
inclusion of two C-terminal lysine residues. For example, the
invention provides a polypeptide comprising the isoform known as
APP695, modified to include two lysine residues as its last two
carboxy terminus amino acids. An exemplary polypeptide comprises
the amino acid sequence set forth in SEQ ID NO. 16. The invention
further includes APP isoform variants as set forth in SEQ ID NOs.
18 and 20. The invention further includes all polynucleotides that
encode an APP protein that has been modified to include two
C-terminal lysines; as well has any eukaryotic cell line comprising
such nucleic acids or polypeptides. Preferred cell lines include a
mammalian cell line (e.g., HEK293, Neuro2a).
[0029] Thus, in one embodiment, the invention provides a
polypeptide comprising the amino acid sequence of a mammalian
amyloid protein precursor (APP) or fragment thereof containing an
APP cleavage site recognizable by a mammalian .beta.-secretase, and
further comprising two lysine residues at the carboxyl terminus of
the amino acid sequence of the mammalian APP or APP fragment. As
taught herein in detail, the addition of two additional lysine
residues to APP sequences has been found to greatly increase
A.beta. processing of the APP in APP processing assays. Thus, the
di-lysine modified APP reagents of the invention are particularly
useful in assays to identify modulators of A.beta. production, for
use in designing therapeutics for the treatment or prevention of
Alzheimer's disease. In one embodiment, the polypeptide comprises
the complete amino acid sequence of a mammalian amyloid protein
precursor (APP), and further comprises the two lysine residues at
the carboxyl terminus of the amino acid sequence of the mammalian
amyloid protein precursor. In an alternative embodiment, the
polypeptide comprises only a fragment of the APP, the fragment
containing at least that portion of APP that is cleaved by a
mammalian .beta.-secretase in the formation of A.beta.
peptides.
[0030] The practice of assays that monitor cleavage of APP can be
facilitated by attaching a marker to a portion of the APP.
Measurment of retained or liberated marker can be used to
quantitate the amount of APP cleavage that occurs in the assay,
e.g., in the presence or absence of a putative modulator of
cleavage activity. Thus, in one preferred embodiment, the
polypeptide of the invention further includes a marker. For
example, the marker comprises a reporter protein amino acid
sequence attached to the APP amino acid sequence. Exemplary
reporter proteins include a fluorescing protein (e.g., green
fluorescing proteins, luciferase) or an enzyme that is used to
cleave a substrate to produce a colorimetric cleavage product. Also
contemplated are tag sequences which are commonly used as epitopes
for quantitative immunoassays.
[0031] In a preferred embodiment, the di-lysine-modified APP of the
invention is a human APP. For example, human APP isoforms such as
APP695, APP751, and APP770, modified to include the two lysines,
are contemplated. In a preferred embodiment, the APP isoform
comprises at least one variation selected from the group consisting
of a Swedish KM.fwdarw.NL mutation and a London V717.fwdarw.F
mutation, or any other mutation that has been observed in a
subpopulation that is particularly prone to development of
Alzheimer's disease. These mutations are recognized as mutations
that influence APP processing into A.beta.. In a highly preferred
embodiment, the APP protein or fragment thereof comprises the
APP-Sw .beta.-secretase peptide sequence NLDA (SEQ D NO: 66), which
is associated with increased levels of A.beta. processing and
therefore is particularly useful in assays relating to Alzheimer's
research. More particularly, the APP protein or fragment thereof
preferably comprises the APP-Sw .beta.-secretase peptide sequence
SEVNLDAEFR (SEQ ID NO: 63).
[0032] In one preferred embodiment, the APP protein or fragment
thereof further includes an APP transmembrane domain
carboxy-terminal to the APP-Sw .beta.-secretase peptide sequence.
Polypeptides that include the TM domain are particularly useful in
cell-based APP processing assays. In contrast, embodiments lacking
the TM domain are useful in cell-free assays of APP processing.
[0033] In addition to working with APP from humans and various
animal models, researchers in the field of Alzheimer's also have
construct chimeric APP polypeptides which include stretches of
amino acids from APP of one species (e.g., humans) fused to
streches of APP from one or more other species (e.g., rodent).
Thus, in another embodiment of the polypeptide of the invention,
the APP protein or fragment thereof comprises a chimeric APP, the
chimeric APP including partial APP amino acid sequences from at
least two species. A chimeric APP that includes amino acid sequence
of a human APP and a rodent APP is particularly contemplated.
[0034] In a related aspect, the invention provides a polynucleotide
comprising a nucleotide sequence that encodes a polypeptide as
described in the preceding paragraphs. Such a polynucleotide is
useful for recominant expression of the polypeptide of the
invention for use in APP processing assays. In addition, the
polynucleotide is useful for transforming into cells to produce
recombinant cells that express the polypeptide of the invention,
which cells are useful in cell-based assays to identify modulators
of APP processing. Thus, in addition to polynucleotides, the
invention provides a vector comprising such polynucleotides,
especially expression vectors where the polynucleotide is operably
linked to a promoter to promote expression of the polypeptide
encoded by the polynucleotide in a host cell. The invention further
provides a host cell transformed or transfected with a
polynucleotide according to claim 14 or a vector according to claim
15 or 16. Among the preferred host cells are mammalian cells,
especially human cells.
[0035] In another, related embodiment, the invention provides a
polypeptide useful for assaying for modulators of .beta.-secretase
activity, said polypeptide comprising an amino acid sequence of the
formula NH.sub.2--X--Y-Z-KK--COOH; wherein X, Y, and Z each
comprise an amino acid sequence of at least one amino acid;
wherein-NH.sub.2--X comprises an amino-terminal amino acid sequence
having at least one amino acid residue; wherein Y comprises an
amino acid sequence of a .beta.-secretase recognition site of a
mammalian amyloid protein precursor (APP); and wherein Z-KK--COOH
comprises a carboxy-terminal amino acid sequence ending in two
lysine (K) residues. In one preferred variation, the
carboxyl-terminal amino acid sequence Z includes a hyrdrophobic
domain that is a transmembrane domain in host cells that express
the polypeptide. Host cells that express such a polypeptide are
particularly useful in assays described herein for identifying
modulators of APP processing. In another preferred variation, the
amino-terminal amino acid sequence X includes an amino acid
sequence of a reporter or marker protein, as described above. In
still another preferred variation, the .beta.-secretase recognition
site Y comprises the human APP-Sw .beta.-secretase peptide sequence
NLDA (SEQ DI NO: 66). It will be apparent that these preferred
variations are not mutually exclusive of each other--they may be
combined in a single polypeptide. The invention further provides a
polynucleotide comprising a nucleotide sequence that encodes such
polypeptides, vectors which comprise such polynucleotides, and host
cells which comprises such vectors, polynucleotides, and/or
polypeptides.
[0036] In yet another aspect, the invention provides a method for
identifying inhibitors of an enzyme that cleaves the beta secretase
cleavable site of APP comprising:
[0037] a) culturing cells in a culture medium under conditions in
which the enzyme causes processing of APP and release of amyloid
beta-peptide into the medium and causes the accumulation of CTF99
fragments of APP in cell lysates,
[0038] b) exposing the cultured cells to a test compound; and
specifically determining whether the test compound inhibits the
function of the enzyme by measuring the amount of amyloid
beta-peptide released into the medium and/or the amount of CTF99
fragments of APP in cell lysates;
[0039] c) identifying test compounds diminishing the amount of
soluble amyloid beta peptide present in the culture medium and
diminution of CTF99 fragments of APP in cell lysates as Asp2
inhibitors. In preferred embodiments, the cultured cells are a
human, rodent or insect cell line. It is also preferred that the
human or rodent cell line exhibits .beta. secretase activity in
which processing of APP occurs with release of amyloid beta-peptide
into the culture medium and accumulation of CTF99 in cell lysates.
Among the contemplated test compounds are antisense oligomers
directed against the enzyme that exhibits .beta. secretase
activity, which oligomers reduce release of soluble amyloid
beta-peptide into the culture medium and accumulation of CTF99 in
cell lysates.
[0040] In yet another aspect, the invention provides a method for
the identification of an agent that decreases the activity of a
Hu-Asp polypeptide selected from the group consisting of Hu-Asp1,
Hu-Asp2(a), and Hu-Asp2(b), the method comprising:
[0041] a) determining the activity of said Hu-Asp polypeptide in
the presence of a test agent and in the absence of a test agent;
and
[0042] b) comparing the activity of said Hu-Asp polypeptide
determined in the presence of said test agent to the activity of
said Hu-Asp polypeptide determined in the absence of said test
agent; whereby a lower level of activity in the presence of said
test agent than in the absence of said test agent indicates that
said test agent has decreased the activity of said Hu-Asp
polypeptide.
[0043] In a related aspect, the invention provides a method for
assaying for modulators of .beta.-secretase activity, comprising
the steps of:
[0044] (a) contacting a first composition with a second composition
both in the presence and in the absence of a putative modulator
compound, wherein the first composition comprises a mammalian
.beta.-secretase polypeptide or biologically active fragment
thereof, and wherein the second composition comprises a substrate
polypeptide having an amino acid sequence comprising a
.beta.-secretase cleavage site;
[0045] (b) measuring cleavage of the substrate polypeptide in the
presence and in the absence of the putative modulator compound; and
(c) identifying modulators of .beta.-secretase activity from a
difference in cleavage in the presence versus in the absence of the
putative modulator compound. A modulator that is a .beta.-secretase
antagonist (inhibitor) reduces such cleavage, whereas a modulator
that is a .beta.-secretase agonist increases such cleavage. Since
such assays are relevant to development of Alzheimer's disease
therapeutics for humans, it will be readily apparent that, in one
preferred embodiment, the first composition comprises a purified
human Asp2 polypeptide. In one variation, the first composition
comprises a soluble fragment of a human Asp2 polypeptide that
retains Asp2 .beta.-secretase activity. Several such fragments
(including .DELTA.TM fragments) are described herein in detail.
Thus, in a particular embodiment, the soluble fragment is a
fragment lacking an Asp2 transmembrane domain.
[0046] The .beta.-secretase cleavage site in APP is known, and it
will be appreciated that the oassays of the invention can be
performed with either intact APP or fragments or analogs of APP
that retain the .beta.-secretase recognition and cleavage site.
Thus, in one variation, the substrate polypeptide of the second
composition comprises the amino acid sequence SEVNLDAEFR (SEQ ID
NO: 63), which includes the .beta.-secretase recognition site of
human APP that contains the "Swiss" mutation. In another variation,
the substrate polypeptide of the second composition comprises the
amino acid sequence EVKMDAEF (SEQ ID NO: 67). In another variation,
the second composition comprises a polypeptide having an amino acid
sequence of a human amyloid precursor protein (APP). For example,
the human amyloid precursor protein is selected from the group
consisting of: APP695, APP75 1, and APP770. Preferably, the human
amyloid precursor protein (irrespective of isoform selected)
includes at least on mutation selected from a KM.fwdarw.NL Swiss
mutation and a V.fwdarw.F London mutation. As explained elsewhere,
one preferred embodiment involves a variation wherein the
polypeptide having an amino acid sequence of a human APP further
comprises an amino acid sequence comprising a marker sequence
attached amino-terminal to the amino acid sequence of the human
amyloid precursor protein. Preferably, the polypeptide having an
amino acid sequence of a human APP further comprises two lysine
residues attached to the carboxyl terminus of the amino acid
sequence of the human APP. The assays can be performed in a cell
free setting, using cell-free enzyme and cell-free substrate, or
can be performed in a cell-based assay wherein the second
composition comprises a eukaryotic cell that expresses amyloid
precursor protein (APP) or a fragment thereof containing a
.beta.-secretase cleavage site. Preferably, the APP expressed by
the host cell is an APP variant that includes two carboxyl-terminal
lysine residues. It will also be appreciated that the
.beta.-secretase enzyme can be an enzyme that is expressed on the
surface of the same cells.
[0047] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide that codes for a polypeptide
selected from the group consisting of human aspartyl proteases. In
particular, human aspartyl protease 1 (Hu-Asp1) and two alternative
splice variants of human aspartyl protease-2 (Hu-Asp2), a "long"
(L) form designated herein as Hu-Asp2(a) and a "short" (S) form
designated Hu-Asp2(b). As used herein, all references to "Hu-Asp"
should be understood to refer to all of Hu-Asp1, Hu-Asp2(a), and
Hu-Asp2(b). In addition, as used herein, all references to
"Hu-Asp2" should be understood to refer to both Hu-Asp2(a) and
Hu-Asp2(b). Hu-Asp1 is expressed most abundantly in pancreas and
prostate tissues, while Hu-Asp2(a) and Hu-Asp2(b) are expressed
most abundantly in pancreas and brain tissues. The invention also
provides isolated Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b) polypeptides,
as well as fragments thereof which exhibit aspartyl protease
activity.
[0048] In a preferred embodiment, the nucleic acid molecules
comprise a polynucleotide having a nucleotide sequence selected
from the group consisting of residues 1-1554 of SEQ D NO. 1,
encoding Hu-Asp1, residues 1-1503 of SEQ ID NO. 3, encoding
Hu-Asp2(a), and residues 1-1428 of SEQ ID NO.5, encoding
Hu-Asp2(b). In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent conditions to a polynucleotide encoding Hu-Asp 1,
Hu-Asp2(a), Hu-Asp-2(b), or fragments thereof European patent
application EP 0 848 062 discloses a polypeptide referred to as
"Asp 1," that bears substantial homology to Hu-Asp1, while
international application WO 98/22597 discloses a polypeptide
referred to as "Asp 2," that bears substantial homology to
Hu-Asp2(a).
[0049] The present invention also provides vectors comprising the
isolated nucleic acid molecules of the invention, host cells into
which such vectors have been introduced, and recombinant methods of
obtaining a Hu-Asp 1, Hu-Asp2(a), or Hu-Asp2(b) polypeptide
comprising culturing the above-described host cell and isolating
the relevant polypeptide.
[0050] In another aspect, the invention provides isolated Hu-Asp1,
Hu-Asp2(a), and Hu-Asp2(b) polypeptides, as well as fragments
thereof. In a preferred embodiment, the Hu-Asp 1, Hu-Asp2(a), and
Hu-Asp2(b) polypeptides have the amino acid sequence given in SEQ
ID NO. 2, SEQ ID NO. 4, or SEQ ID NO.6, respectively. The present
invention also describes active forms of Hu-Asp2, methods for
preparing such active forms, methods for preparing soluble forms,
methods for measuring Hu-Asp2 activity, and substrates for Hu-Asp2
cleavage. The invention also describes antisense oligomers
targeting the Hu-Asp1, Hu-Asp2(a) and Hu-Asp2(b) mRNA transcripts
and the use of such antisense reagents to decrease such mRNA and
consequently the production of the corresponding polypeptide.
Isolated antibodies, both polyclonal and monoclonal, that binds
specifically to any of the Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b)
polypeptides of the invention are also provided.
[0051] The invention also provides a method for the identification
of an agent that modulates the activity of any of Hu-Asp-1,
Hu-Asp2(a), and Hu-Asp2(b). The inventions describes methods to
test such agents in cell-free assays to which Hu-Asp2 polypeptide
is added, as well as methods to test such agents in human or other
mammalian cells in which Hu-Asp2 is present.
[0052] Additional features and variations of the invention will be
apparent to those skilled in the art from the entirety of this
application, including the drawing and detailed description, and
all such features are intended as aspects of the invention.
Likewise, features of the invention described herein can be
re-combined into additional embodiments that are also intended as
aspects of the invention, irrespective of whether the combination
of features is specifically mentioned above as an aspect or
embodiment of the invention. Also, only such limitations which are
described herein as critical to the invention should be viewed as
such; variations of the invention lacking limitations which have
not been described herein as critical are intended as aspects of
the invention.
[0053] In addition to the foregoing, the invention includes, as an
additional aspect, all embodiments of the invention narrower in
scope in any way than the variations specifically mentioned above.
Although the applicant(s) invented the full scope of the claims
appended hereto, the claims appended hereto are not intended to
encompass within their scope the prior art work of others.
Therefore, in the event that statutory prior art within the scope
of a claim is brought to the attention of the applicants by a
Patent Office or other entity or individual, the applicant(s)
reserve the right to exercise amendment rights under applicable
patent laws to redefine the subject matter of such a claim to
specifically exclude such statutory prior art or obvious variations
of statutory prior art from the scope of such a claim. Variations
of the invention defined by such amended claims also are intended
as aspects of the invention.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0054] Sequence ID No. 1: Human Asp-1, nucleotide sequence.
[0055] Sequence ID No. 2: Human Asp-1, predicted amino acid
sequence.
[0056] Sequence ID No. 3: Human Asp-2(a), nucleotide sequence.
[0057] Sequence ID No. 4: Human Asp-2(a), predicted amino acid
sequence. The Asp2(a) amino acid sequence includes a putative
signal peptide comprising residues 1 to 21; and a putative
pre-propeptide after the signal peptide that extends through
residue 45 (as assessed by processing observed of recombinant
Asp2(a) in CHO cells), and a putative propeptide that may extend to
at least about residue 57, based on the observation of an observed
GRR.dwnarw.GS (SEQ ID NO: 68) sequence which has characteristics of
a protease recognition sequence. The Asp2(a) further includes a
transmembrane domain comprising residues 455-477, a cytoplasmic
domain comprising residues 478-501, and a putative alpha-helical
spacer region, comprising residues 420-454, believed to be
unnecessary for proteolytic activity, between the protease
catalytic domain and the transmembrane domain.
[0058] Sequence ID No. 5: Human Asp-2(b), nucleotide sequence.
[0059] Sequence ID No. 6: Human Asp-2(b), predicted amino acid
sequence. The Asp2(b) amino acid sequence includes a putative
signal peptide, pre-propeptide, and propeptide as described above
for Asp2(a). The Asp2(b) further includes a transmembrane domain
comprising residues 430-452, a cytoplasmic domain comprising
residues 453-476, and a putative alpha-helical spacer region,
comprising residues 395-429, believed to be unnecessary for
proteolytic activity, between the protease catalytic domain and the
transmembrane domain.
[0060] Sequence ID No. 7: Murine Asp-2(a), nucleotide sequence.
[0061] Sequence ID No. 8: Murine Asp-2(a), predicted amino acid
sequence. The proteolytic processing of murine Asp2(a) is believed
to be analogous to the processing described above for human
Asp2(a). In addition, a variant lacking amino acid residues 190-214
of SEQ ID NO: 8 is specifically contemplated as a murine Asp2(b)
polypeptide.
[0062] Sequence ID No. 9: Human APP695, nucleotide sequence.
[0063] Sequence ID No.10: Human APP695, predicted amino acid
sequence.
[0064] Sequence ID No.11: Human APP695-Sw, nucleotide sequence.
[0065] Sequence ID No.12: Human APP695-Sw. predicted amino acid
sequence. In the APP695 isoform, the Sw mutation is characterized
by a KM.fwdarw.NL alteration at positions 595-596 (compared to
normal APP695).
[0066] Sequence ID No.13: Human APP695-VF, nucleotide sequence.
[0067] Sequence ID No.14: Human APP695-VF, predicted amino acid
sequence. In the APP 695 isoform, the VF mutation is characterized
by a V.fwdarw.F alteration at position 642 (compared to normal APP
695).
[0068] Sequence ID No.15: Human APP695-KK, nucleotide sequence.
[0069] Sequence ID No.16: Human APP695-KK, predicted amino acid
sequence. (APP695 with two carboxy-terminal lysine residues.)
[0070] Sequence ID No.17: Human APP695-Sw-KK, nucleotide
sequence.
[0071] Sequence ID No.18: Human APP695-Sw-KK, predicted amino acid
sequence
[0072] Sequence ID No.19: Human APP695-VF-KK, nucleotide
sequence
[0073] Sequence ID No.20: Human APP695-VF-KK, predicted amino acid
sequence
[0074] Sequence ID No.21: T7-Human-pro-Asp-2(a).DELTA.TM,
nucleotide sequence
[0075] Sequence ID No.22: T7-Human-pro-Asp-2(a).DELTA.TM, amino
acid sequence
[0076] Sequence ID No.23: T7-Caspase-Human-pro-Asp-2(a).DELTA.TM,
nucleotide sequence
[0077] Sequence ID No.24: T7-Caspase-Human-pro-Asp-2(a).DELTA.TM,
amino acid sequence
[0078] Sequence ID No.25: Human-pro-Asp-2(a).DELTA.TM (low GC),
nucleotide sequence
[0079] Sequence ID No.26: Human-pro-Asp-2(a).DELTA.TM, (low GC),
amino acid sequence
[0080] Sequence ID No.27: T7-Caspase-Caspase 8
cleavage-Human-pro-Asp-2(a)- .DELTA.TM, nucleotide sequence
[0081] Sequence ID No.28: T7-Caspase-Caspase 8
cleavage-Human-pro-Asp-2(a)- .DELTA.TM, amino acid sequence
[0082] Sequence ID No.29: Human Asp-2(a).DELTA.TM, nucleotide
sequence
[0083] Sequence ID No.30: Human Asp-2(a).DELTA.TM, amino acid
sequence
[0084] Sequence ID No.31: Human Asp-2(a).DELTA.TM(His).sub.6,
nucleotide sequence
[0085] Sequence ID No. 32: Human Asp-2(a).DELTA.TM(His).sub.6,
amino acid sequence
[0086] Sequence ID Nos. 33-49 are short synthetic peptide and
oligonucleotide sequences that are described below in the Detailed
Description of the Invention.
[0087] Sequence ID No. 50: Human Asp2(b).DELTA.TM polynucleotide
sequence.
[0088] Sequence ID No. 51: Human Asp2(b).DELTA.TM polypeptide
sequence (exemplary variant of Human Asp2(b) lacking transmembrane
and intracellular domains of Hu-Asp2(b) set forth in SEQ ID NO:
6.
[0089] Sequence ID No. 52: Human Asp2(b).DELTA.TM(His).sub.6
polynucleotide sequence.
[0090] Sequence ID No. 53: Human Asp2(b).DELTA.TM(His).sub.6
polypeptide sequence (Human Asp2(b).DELTA.TM with six histidine tag
attached to C-terminus),
[0091] Sequence ID No. 54: Human APP770-encoding polynucleotide
sequence.
[0092] Sequence ID No. 55: Human APP770 polypeptide sequence. To
introduce the KM.fwdarw.NL Swedish mutation, residues KM at
positions 670-71 are changed to NL. To introduce the V.fwdarw.F
London mutation, the V residue at position 717 is changed to F.
[0093] Sequence ID No. 56: Human APP751 encoding polynucleotide
sequence.
[0094] Sequence ID No. 57: Human APP751 polypeptide sequence (Human
APP751 isoform).
[0095] Sequence ID No. 58: Human APP770-KK encoding polynucleotide
sequence.
[0096] Sequence ID No. 59: Human APP770-KK polypeptide sequence.
(Human APP770 isoform to which two C-terminal lysines have been
added).
[0097] Sequence ID No. 60: Human APP751-KK encoding polynucleotide
sequence.
[0098] Sequence ID No. 61: Human APP751-KK polypeptide sequence
(Human APP751 isoform to which two C-terminal lysines have been
added).
[0099] Sequence ID No. 62-65: Various short peptide sequences
described in detail in detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0100] FIG. 1: FIG. 1 shows the nucleotide (SEQ ID NO: 1) and
predicted amino acid sequence (SEQ ID NO:2) of human Asp1.
[0101] FIG. 2: FIG. 2 shows the nucleotide (SEQ ID NO:3) and
predicted amino acid sequence (SEQ ID NO:4) of human Asp2(a).
[0102] FIG. 3: FIG. 3 shows the nucleotide (SEQ ID NO:5) and
predicted amino acid sequence (SEQ ID NO:6) of human Asp2(b). The
predicted transmembrane domain of Hu-Asp2(b) is enclosed in
brackets.
[0103] FIG. 4: FIG. 4 shows the nucleotide (SEQ ID No. 7) and
predicted amino acid sequence (SEQ ID No. 8) of murine Asp2(a)
[0104] FIG. 5: FIG. 5 shows the BestFit alignment of the predicted
amino acid sequences of Hu-Asp2(a) (SEQ ID NO: 4) and murine
Asp2(a) (SEQ ID NO: 8).
[0105] FIG. 6: FIG. 6 shows the nucleotide (SEQ ID No. 21) and
predicted amino acid sequence (SEQ ID No. 22) of
T7-Human-pro-Asp-2(a).DELTA.TM
[0106] FIG. 7: FIG. 7 shows the nucleotide (SEQ ID No. 23) and
predicted amino acid sequence (SEQ ID No. 24) of
T7-caspase-Human-pro-Asp-2(a).DELT- A.TM
[0107] FIG. 8: FIG. 8 shows the nucleotide (SEQ ID No. 25) and
predicted amino acid sequence (SEQ ID No. 26) of
Human-pro-Asp-2(a).DELTA.TM (low GC)
[0108] FIG. 9: Western blot showing reduction of CTF99 production
by HEK125.3 cells transfected with antisense oligomers targeting
the Hu-Asp2 mRNA.
[0109] FIG. 10: Western blot showing increase in CTF99 production
in mouse Neuro-2a cells cotransfected with APP-KK with and without
Hu-Asp2 only in those cells cotransfected with Hu-Asp2. A further
increase in CTF99 production is seen in cells cotransfected with
APP-Sw-KK with and without Hu-Asp2 only in those cells
cotransfected with Hu-Asp2
[0110] FIG. 11: FIG. 11 shows the predicted amino acid sequence
(SEQ ID No. 30) of Human-Asp2(a).DELTA.TM
[0111] FIG. 12: FIG. 11 shows the predicted amino acid sequence
(SEQ ID No. 30) of Human-Asp2(a).DELTA.TM(His).sub.6
DETAILED DESCRIPTION OF THE INVENTION
[0112] A few definitions used in this invention follow, most
definitions to be used are those that would be used by one
ordinarily skilled in the art.
[0113] The term ".beta. amyloid peptide" means any peptide
resulting from beta secretase cleavage of APP. This includes
peptides of 39, 40,41, 42 and 43 amino acids, extending from the
.beta.-secretase cleavage site to 39, 40, 41, 42 and 43 amino acids
C-terminal to the .beta.-secretase cleavage site. .beta. amyloid
peptide also includes sequences 1-6, SEQ ID NOs. 1-6 of U.S. Pat.
No. 5,750,349, issued 12 May 1998 (incorporated into this document
by reference). A .beta.-secretase cleavage fragment disclosed here
is called CTF-99, which extends from .beta.-secretase cleavage site
to the carboxy terminus of APP.
[0114] When an isoform of APP is discussed then what is meant is
any APP polypeptide, including APP variants (including mutations),
and APP fragments that exists in humans such as those described in
U.S. Pat. No. 5,766,846, col 7, lines 45-67, incorporated into this
document by reference.
[0115] The term ".beta.-amyloid precursor protein" (APP) as used
herein is defined as a polypeptide that is encoded by a gene of the
same name localized in humans on the long arm of chromosome 21 and
that includes ".beta.AP--here ".beta.-amyloid protein" see above,
within its carboxyl third. APP is a glycosylated, single-membrane
spanning protein expressed in a wide variety of cells in many
mammalian tissues. Examples of specific isotypes of APP which are
currently known to exist in humans are the 695 amino acid
polypeptide described by Kang et. al. (1987) Nature 325:733-736
which is designated as the "normal" APP (SEQ ID NOs: 9-10); the 751
amino acid polypeptide described by Ponte et al. (1988) Nature
331:525-527 (1988) and Tanzi et al. (1988) Nature 331:528-530 (SEQ
ID NOs: 56-57); and the 770-amino acid polypeptide described by
Kitaguchi et. al. (1988) Nature 331:530-532 (SEQ ID NOs: 54-55).
Examples of specific variants of APP include point mutation which
can differ in both position and phenotype (for review of known
variant mutation see Hardy (1992) Nature Genet. 1:233-234). All
references cited here incorporated by reference. The term "APP
fragments" as used herein refers to fragments of APP other than
those which consist solely of .beta.AP or .beta.AP fragments. That
is, APP fragments will include amino acid sequences of APP in
addition to those which form intact .beta.AP or a fragment of
.beta.AP.
[0116] When the term "any amino acid" is used, the amino acids
referred to are to be selected from the following, three letter and
single letter abbreviations--which may also be used, are provided
as follows:
[0117] Alanine, Ala, A; Arginine, Arg, R; Asparagine, Asn, N;
Aspartic acid, Asp, D; Cysteine, Cys, C; Glutamine, Gln, Q;
Glutamic Acid, Glu, E; Glycine, Gly, G; Histidine, His, H;
Isoleucine, Ile, I; Leucine, Leu, L; Lysine, Lys, K; Methionine,
Met, M; Phenylalanine, Phe, F; Proline, Pro, P; Serine, Ser, S;
Threonine, Thr, T; Tryptophan, Trp, W; Tyrosine, Tyr, Y; Valine,
Val, V; Aspartic acid or Asparagine, Asx, B; Glutamic acid or
Glutamine, Glx, Z; Any amino acid, Xaa, X.
[0118] The present invention describes a method to scan gene
databases for the simple active site motif characteristic of
aspartyl proteases. Eukaryotic aspartyl proteases such as pepsin
and renin possess a two-domain structure which folds to bring two
aspartyl residues into proximity within the active site. These are
embedded in the short tripeptide motif DTG, or more rarely, DSG.
Most aspartyl proteases occur as proenzyme whose N-terminus must be
cleaved for activation. The DTG or DSG active site motif appears at
about residue 65-70 in the proenzyme (prorenin, pepsinogen), but at
about residue 25-30 in the active enzyme after cleavage of the
N-terminal prodomain. The limited length of the active site motif
makes it difficult to search collections of short, expressed
sequence tags (EST) for novel aspartyl proteases. EST sequences
typically average 250 nucleotides or less, and so would encode
80-90 amino acid residues or less. That would be too short a
sequence to span the two active site motifs. The preferred method
is to scan databases of hypothetical or assembled protein coding
sequences. The present invention describes a computer method to
identify candidate aspartyl proteases in protein sequence
databases. The method was used to identify seven candidate aspartyl
protease sequences in the Caenorhabditis elegans genome. These
sequences were then used to identify by homology search Hu-Asp1 and
two alternative splice variants of Hu-Asp2, designated herein as
Hu-Asp2(a) and Hu-Asp2(b).
[0119] In a major aspect of the invention disclosed here we provide
new information about APP processing. Pathogeneic processing of the
amyloid precursor protein (APP) via the A.beta. pathway requires
the sequential action of two proteases referred to as
.beta.secretase and .gamma.-secretase. Cleavage of APP by the
.beta.-secretase and .gamma.-secretase generates the N-terminus and
C-terminus of the A.beta. peptide, respectively. Because over
production of the A.beta. peptide, particularly the
A.beta..sub.41-2, has been implicated in the initiation of
Alzheimer's disease, inhibitors of either the .beta.-secretase
and/or the .gamma.-secretase have potential in the treatment of
Alzheimer's disease. Despite the importance of the .beta.-secretase
and .gamma.-secretase in the pathogenic processing of APP,
molecular definition of these enzymes has not been accomplished to
date. That is, it was not known what enzymes were required for
cleavage at either the .beta.-secretase or the .gamma.-secretase
cleavage site. The sites themselves were known because APP was
known and the A.beta..sub.1-42, peptide was known, see U.S. Pat.
No. 5,766,846 and U.S. Pat. No. 5,837,672, (incorporated by
reference, with the exception to reference to "soluble" peptides).
But what enzyme was involved in producing the A.beta..sub.1-42,
peptide was unknown.
[0120] Alignment of the amino acid sequences of Hu-Asp2 with other
known aspartyl proteases reveals a similar domain organization. All
of the sequences contain a signal sequence followed by a
pro-segment and the catalytic domain containing 2 copies of the
aspartyl protease active site motif (DTG/DSG) separated by
approximately 180 amino acid residues. Comparison of the processing
site for proteolytic removal of the pro-segment in the mature forms
of pepsin A, pepsin C, cathepsin D, cathepsin E and renin reveals
that the mature forms of these enzymes contain between 31-35 amino
acid residues upstream of the first DTG motif. Inspection of this
region in the Hu-Asp-2 amino acid sequence indicates a preferred
processing site within the sequence GRR.dwnarw.GS (SEQ ID NO: 68)
as proteolytic processing of pro-protein precursors commonly occurs
at site following dibasic amino acid pairs (eg. RR). Also,
processing at this site would yield a mature enzyme with 35 amino
acid residues upstream of the first DTG, consistent with the
processing sites for other aspartyl proteases. In the absence of
self-activation of Hu-Asp2 or a knowledge of the endogenous
protease that processes Hu-Asp2 at this site, a recombinant form
was engineered by introducing a recognition site for the PreSission
protease (LEVLFQ.dwnarw.GP; SEQ ID NO: 62) into the expression
plasmids for bacterial, insect cell, and mammalian cell expression
of pro-Hu-Asp2. In each case, the Gly residue in P1' position
corresponds to the Gly residue 35 amino acids upstream of the first
DTG motif in Hu-Asp2.
[0121] The present invention involves the molecular definition of
several novel human aspartyl proteases and one of these, referred
to as Hu-Asp-2(a) and Hu-Asp2(b), has been characterized in detail.
Previous forms of asp1 and asp 2 have been disclosed, see EP
0848062 A2 and EP 0855444A2, inventors David Powel et al., assigned
to Smith Kline Beecham Corp. (incorporated by reference). Herein
are disclosed old and new forms of Hu-Asp 2. For the first time
they are expressed in active form, their substrates are disclosed,
and their specificity is disclosed. Prior to this disclosure cell
or cell extracts were required to cleave the .beta.-secretase site,
now purified protein can be used in assays, also described here.
Based on the results of (1) antisense knock out experiments, (2)
transient transfection knock in experiments, and (3) biochemical
experiments using purified recombinant Hu-Asp-2, we demonstrate
that Hu-Asp-2 is the .beta.-secretase involved in the processing of
APP. Although the nucleotide and predicted amino acid sequence of
Hu-Asp-2(a) has been reported, see above, see EP 0848062 A2 and EP
0855444A2, no functional characterization of the enzyme was
disclosed. Here the authors characterize the Hu-Asp-2 enzyme and
are able to explain why it is a critical and essential enzyme
required in the formation of A.beta..sub.1-42, peptide and possible
a critical step in the development of AD.
[0122] In another embodiment the present invention also describes a
novel splice variant of Hu-Asp2, referred to as Hu-Asp-2(b), that
has never before been disclosed.
[0123] In another embodiment, the invention provides isolated
nucleic acid molecules comprising a polynucleotide encoding a
polypeptide selected from the group consisting of human aspartyl
protease 1 (Hu-Asp1) and two alternative splice variants of human
aspartyl protease-2 (Hu-Asp2), designated herein as Hu-Asp2(a) and
Hu-Asp2(b). As used herein, all references to "Hu-Asp2" should be
understood to refer to both Hu-Asp2(a) and Hu-Asp2(b). Hu-Asp1 is
expressed most abundantly in pancreas and prostate tissues, while
Hu-Asp2(a) and Hu-Asp2(b) are expressed most abundantly in pancreas
and brain tissues. The invention also provides isolated Hu-Asp1,
Hu-Asp2(a), and Hu-Asp2(b) polypeptides, as well as fragments
thereof which exhibit aspartyl protease activity.
[0124] The predicted amino acid sequences of Hu-Asp1, Hu-Asp2(a)
and Hu-Asp2(b) share significant homology with previously
identified mammalian aspartyl proteases such as pepsinogen A,
pepsinogen B, cathepsin D, cathepsin E, and renin. P. B. Szecs,
Scand. J. Clin. Lab. Invest. 52:(Suppl. 210 5-22 (1992)). These
enzymes are characterized by the presence of a duplicated DTG/DSG
sequence motif. The Hu-Asp1 and HuAsp2 polypeptides disclosed
herein also exhibit extremely high homology with the ProSite
consensus motif for aspartyl proteases extracted from the SwissProt
database.
[0125] The nucleotide sequence given as residues 1-1554 of SEQ ID
NO:1 corresponds to the nucleotide sequence encoding Hu-Asp1, the
nucleotide sequence given as residues 1-1503 of SEQ ID NO:3
corresponds to the nucleotide sequence encoding Hu-Asp2(a), and the
nucleotide sequence given as residues 1-1428 of SEQ ID NO:5
corresponds to the nucleotide sequence encoding Hu-Asp2(b). The
isolation and sequencing of DNA encoding Hu-Asp1, Hu-Asp2(a), and
Hu-Asp2(b) is described below in Examples 1 and 2.
[0126] As is described in Examples 1 and 2, automated sequencing
methods were used to obtain the nucleotide sequence of Hu-Asp1,
Hu-Asp2(a), and Hu-Asp-2(b). The Hu-Asp nucleotide sequences of the
present invention were obtained for both DNA strands, and are
believed to be 100% accurate. However, as is known in the art,
nucleotide sequence obtained by such automated methods may contain
some errors. Nucleotide sequences determined by automation are
typically at least about 90%, more typically at least about 95% to
at least about 99.9% identical to the actual nucleotide sequence of
a given nucleic acid molecule. The actual sequence may be more
precisely determined using manual sequencing methods, which are
well known in the art. An error in sequence which results in an
insertion or deletion of one or more nucleotides may result in a
frame shift in translation such that the predicted amino acid
sequence will differ from that which would be predicted from the
actual nucleotide sequence of the nucleic acid molecule, starting
at the point of the mutation. The Hu-Asp DNA of the present
invention includes cDNA, chemically synthesized DNA, DNA isolated
by PCR, genomic DNA, and combinations thereof. Genomic Hu-Asp DNA
may be obtained by screening a genomic library with the Hu-Asp2
cDNA described herein, using methods that are well known in the
art, or with oligonucleotides chosen from the Hu-Asp2 sequence that
will prime the polymerase chain reaction (PCR). RNA transcribed
from Hu-Asp DNA is also encompassed by the present invention.
[0127] Due to the degeneracy of the genetic code, two DNA sequences
may differ and yet encode identical amino acid sequences. The
present invention thus provides isolated nucleic acid molecules
having a polynucleotide sequence encoding any of the Hu-Asp
polypeptides of the invention, wherein said polynucleotide sequence
encodes a Hu-Asp polypeptide having the complete amino acid
sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or fragments
thereof.
[0128] Also provided herein are purified Hu-Asp polypeptides, both
recombinant and non-recombinant. Most importantly, methods to
produce Hu-Asp2 polypeptides in active form are provided. These
include production of Hu-Asp2 polypeptides and variants thereof in
bacterial cells, insect cells, and mammalian cells, also in forms
that allow secretion of the Hu-Asp2 polypeptide from bacterial,
insect or mammalian cells into the culture medium, also methods to
produce variants of Hu-Asp2 polypeptide incorporating amino acid
tags that facilitate subsequent purification. In a preferred
embodiment of the invention the Hu-Asp2 polypeptide is converted to
a proteolytically active form either in transformed cells or after
purification and cleavage by a second protease in a cell-free
system, such active forms of the Hu-Asp2 polypeptide beginning with
the N-terminal sequence TQHGIR (SEQ ID NO: 69) or ETDEEP (SEQ ID
NO: 70). The sequence TQHGIR (SEQ ID NO: 69) represents the
amino-terminus of Asp2(a) or Asp2(b) beginning with residue 22 of
SEQ ID NO: 4 or 6, after cleavage of a putative 21 residue signal
peptide. Recombinant Asp2(a) expressed in and purified from insect
cells was observed to have this amino terminus, presumably as a
result of cleavage by a signal peptidase. The sequence ETDEEP (SEQ
ID NO: 70) represents the amino-terminus of Asp2(a) or Asp2(b)
beginning with residue 46 of SEQ ID NO: 4 or 6, as observed when
Asp2(a) has been recombinantly produced in CHO cells (presumably
after cleavage by both a rodent signal peptidase and another rodent
peptidase that removes a propeptide sequence). The Asp2(a) produced
in the CHO cells possesses .beta.-secretase activity, as described
in greater detail in Examples 11 and 12. Variants and derivatives,
including fragments, of Hu-Asp proteins having the native amino
acid sequences given in SEQ D Nos: 2, 4, and 6 that retain any of
the biological activities of Hu-Asp are also within the scope of
the present invention. Of course, one of ordinary skill in the art
will readily be able to determine whether a variant, derivative, or
fragment of a Hu-Asp protein displays Hu-Asp activity by subjecting
the variant, derivative, or fragment to a standard aspartyl
protease assay. Fragments of Hu-Asp within the scope of this
invention include those that contain the active site domain
containing the amino acid sequence DTG, fragments that contain the
active site domain amino acid sequence DSG, fragments containing
both the DTG and DSG active site sequences, fragments in which the
spacing of the DTG and DSG active site sequences has been
lengthened, fragments in which the spacing has been shortened. Also
within the scope of the invention are fragments of Hu-Asp in which
the transmembrane domain has been removed to allow production of
Hu-Asp2 in a soluble form. In another embodiment of the invention,
the two halves of Hu-Asp2, each containing a single active site DTG
or DSG sequence can be produced independently as recombinant
polypeptides, then combined in solution where they reconstitute an
active protease.
[0129] Thus, the invention provides a purified polypeptide
comprising a fragment of a mammalian Asp2 protein, wherein said
fragment lacks the Asp2 transmembrane domain of said Asp2 protein,
and wherein the polypeptide and the fragment retain the
.beta.-secretase activity of said mammalian Asp2 protein. In a
preferred embodiment, the purified polypeptide comprises a fragment
of a human Asp2 protein that retains the .beta.-secretase activity
of the human Asp2 protein from which it was derived. Examples
include:
[0130] a purified polypeptide that comprises a fragment of Asp2(a)
having the amino acid sequence set forth in SEQ ID NO: 4, wherein
the polypeptide lacks transmembrane domain amino acids 455 to 477
of SEQ ID NO: 4;
[0131] a purified polypeptide as described in the preceding
paragraph that further lacks cytoplasmic domain amino acids 478 to
501 of SEQ ID NO: 4;
[0132] a purified polypeptide as described in either of the
preceding paragraphs that further lacks amino acids 420-454 of SEQ
ID NO: 4, which constitute a putative alpha helical region between
the catalytic domain and the transmembrane domain that is believed
to be unnecessary for .beta.-secretase activity;
[0133] a purified polypeptide that comprises an amino acid sequence
that includes amino acids 58 to 419 of SEQ ID NO: 4, and that lacks
amino acids 22 to 57 of SEQ ID NO:4;
[0134] a purified polypeptide that comprises an amino acid sequence
that includes amino acids 46 to 419 of SEQ ID NO: 4, and that lacks
amino acids 22 to 45 of SEQ ID NO: 4;
[0135] a purified polypeptide that comprises an amino acid sequence
that includes amino acids 22 to 454 of SEQ ID NO: 4.
[0136] a purified polypeptide that comprises a fragment of Asp2(b)
having the amino acid sequence set forth in SEQ ID NO: 6, and
wherein said polypeptide lacks transmembrane domain amino acids 430
to 452 of SEQ ID NO: 6;
[0137] a purified polypeptide as described in the preceding
paragraph that further lacks cytoplasmic domain amino acids 453 to
476 of SEQ ID NO: 6;
[0138] a purified polypeptide as described in either of the
preceding two paragraphs that further lacks amino acids 395-429 of
SEQ ID NO: 4, which constitute a putative alpha helical region
between the catalytic domain and the transmembrane domain that is
believed to be unnecessary for .beta.-secretase activity;
[0139] a purified polypeptide comprising an amino acid sequence
that includes amino acids 58 to 394 of SEQ ID NO: 4, and that lacks
amino acids 22 to 57 of SEQ ID NO: 4;
[0140] a purified polypeptide comprising an amino acid sequence
that includes amino acids 46 to 394 of SEQ ID NO: 4, and that lacks
amino acids 22 to 45 of SEQ ID NO: 4; and
[0141] a purified polypeptide comprising an amino acid sequence
that includes amino acids 22 to 429 of SEQ ID NO: 4.
[0142] Also included as part of the invention is a purified
polynucleotide comprising a nucleotide sequence that encodes such
polypeptides; a vector comprising a polynucleotide that encodes
such polypeptides; and a host cell transformed or transfected with
such a polynucleotide or vector.
[0143] Hu-Asp variants may be obtained by mutation of native
Hu-Asp-encoding nucleotide sequences, for example. A Hu-Asp
variant, as referred to herein, is a polypeptide substantially
homologous to a native Hu-Asp polypeptide but which has an amino
acid sequence different from that of native Hu-Asp because of one
or more deletions, insertions, or substitutions in the amino acid
sequence. The variant amino acid or nucleotide sequence is
preferably at least about 80% identical, more preferably at least
about 90% identical, and most preferably at least about 95%
identical, to a native Hu-Asp sequence. Thus, a variant nucleotide
sequence which contains, for example, 5 point mutations for every
one hundred nucleotides, as compared to a native Hu-Asp gene, will
be 95% identical to the native protein. The percentage of sequence
identity, also termed homology, between a native and a variant
Hu-Asp sequence may also be determined, for example, by comparing
the two sequences using any of the computer programs commonly
employed for this purpose, such as the Gap program (Wisconsin
Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, Madison Wis.), which uses the
algorithm of Smith and Waterman (Adv. Appl. Math. 2: 482-489
(1981)).
[0144] Alterations of the native amino acid sequence may be
accomplished by any of a number of known techniques. For example,
mutations may be introduced at particular locations by procedures
well known to the skilled artisan, such as oligonucleotide-directed
mutagenesis, which is described by Walder et al. (Gene 42:133
(1986)); Bauer et al. (Gene 37:73 (1985)); Craik (BioTechniques,
January 1985, pp. 12-19); Smith et al. (Genetic Engineering:
Principles and Methods, Plenum Press (1981)); and U.S. Pat. Nos.
4,518,584 and 4,737,462.
[0145] Hu-Asp variants within the scope of the invention may
comprise conservatively substituted sequences, meaning that one or
more amino acid residues of a Hu-Asp polypeptide are replaced by
different residues that do not alter the secondary and/or tertiary
structure of the Hu-Asp polypeptide. Such substitutions may include
the replacement of an amino acid by a residue having similar
physicochemical properties, such as substituting one aliphatic
residue (Ile, Val, Leu or Ala) for another, or substitution between
basic residues Lys and Arg, acidic residues Glu and Asp, amide
residues Gln and Asn, hydroxyl residues Ser and Tyr, or aromatic
residues Phe and Tyr. Further information regarding making
phenotypically silent amino acid exchanges maybe found in Bowie et
al., Science 247:1306-1310 (1990). Other Hu-Asp variants which
might retain substantially the biological activities of Hu-Asp are
those where amino acid substitutions have been made in areas
outside functional regions of the protein.
[0146] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent conditions to a portion of the nucleic acid
molecules described above, e.g., to at least about 15 nucleotides,
preferably to at least about 20 nucleotides, more preferably to at
least about 30 nucleotides, and still more preferably to at least
about from 30 to at least about 100 nucleotides, of one of the
previously described nucleic acid molecules. Such portions of
nucleic acid molecules having the described lengths refer to, e.g.,
at least about 15 contiguous nucleotides of the reference nucleic
acid molecule. By stringent hybridization conditions is intended
overnight incubation at about 42.degree. C. for about 2.5 hours in
6.times.SSC/0.1% SDS, followed by washing of the filters four times
for 15 minutes in 1.0.times.SSC at 65.degree. C., 0.1% SDS.
[0147] Fragments of the Hu-Asp encoding nucleic acid molecules
described herein, as well as polynucleotides capable of hybridizing
to such nucleic acid molecules may be used as a probe or as primers
in a polymerase chain reaction (PCR). Such probes may be used,
e.g., to detect the presence of Hu-Asp nucleic acids in in vitro
assays, as well as in Southern and northern blots. Cell types
expressing Hu-Asp may also be identified by the use of such probes.
Such procedures are well known, and the skilled artisan will be
able to choose a probe of a length suitable to the particular
application. For PCR, 5' and 3' primers corresponding to the
termini of a desired Hu-Asp nucleic acid molecule are employed to
isolate and amplify that sequence using conventional
techniques.
[0148] Other useful fragments of the Hu-Asp nucleic acid molecules
are antisense or sense oligonucleotides comprising a single
stranded nucleic acid sequence capable of binding to a target
Hu-Asp mRNA (using a sense strand), or Hu-Asp DNA (using an
antisense strand) sequence. In a preferred embodiment of the
invention these Hu-Asp antisense oligonucleotides reduce Hu-Asp
mRNA and consequent production of Hu-Asp polypeptides.
[0149] In another aspect, the invention includes Hu-Asp
polypeptides with or without associated native pattern
glycosylation. Both Hu-Asp1 and Hu-Asp2 have canonical acceptor
sites for Asn-linked sugars, with Hu-Asp1 having two of such sites,
and Hu-Asp2 having four. Hu-Asp expressed in yeast or mammalian
expression systems (discussed below) may be similar to or
significantly different from a native Hu-Asp polypeptide in
molecular weight and glycosylation pattern. Expression of Hu-Asp in
bacterial expression systems will provide non-glycosylated
Hu-Asp.
[0150] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. Hu-Asp polypeptides may be recovered and purified from
tissues, cultured cells, or recombinant cell cultures by well-known
methods, including ammonium sulfate or ethanol precipitation, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, and high
performance liquid chromatography (HPLC). In a preferred
embodiment, an amino acid tag is added to the Hu-Asp polypeptide
using genetic engineering techniques that are well known to
practitioners of the art which include addition of six histidine
amino acid residues to allow purification by binding to nickel
immobilized on a suitable support, epitopes for polyclonal or
monoclonal antibodies including but not limited to the T7 epitope,
the myc epitope, and the V5a epitope, and fusion of Hu-Asp2 to
suitable protein partners including but not limited to
glutathione-S-transferase or maltose binding protein. In a
preferred embodiment these additional amino acid sequences are
added to the C-terminus of Hu-Asp but may be added to the
N-terminus or at intervening positions within the Hu-Asp2
polypeptide.
[0151] The present invention also relates to vectors comprising the
polynucleotide molecules of the invention, as well as host cell
transformed with such vectors. Any of the polynucleotide molecules
of the invention may be joined to a vector, which generally
includes a selectable marker and an origin of replication, for
propagation in a host. Because the invention also provides Hu-Asp
polypeptides expressed from the polynucleotide molecules described
above, vectors for the expression of Hu-Asp are preferred. The
vectors include DNA encoding any of the Hu-Asp polypeptides
described above or below, operably linked to suitable
transcriptional or translational regulatory sequences, such as
those derived from a mammalian, microbial, viral, or insect gene.
Examples of regulatory sequences include transcriptional promoters,
operators, or enhancers, mRNA ribosomal binding sites, and
appropriate sequences which control transcription and translation.
Nucleotide sequences are operably linked when the regulatory
sequence functionally relates to the DNA encoding Hu-Asp. Thus, a
promoter nucleotide sequence is operably linked to a Hu-Asp DNA
sequence if the promoter nucleotide sequence directs the
transcription of the Hu-Asp sequence.
[0152] Selection of suitable vectors to be used for the cloning of
polynucleotide molecules encoding Hu-Asp, or for the expression of
Hu-Asp polypeptides, will of course depend upon the host cell in
which the vector will be transformed, and, where applicable, the
host cell from which the Hu-Asp polypeptide is to be expressed.
Suitable host cells for expression of Hu-Asp polypeptides include
prokaryotes, yeast, and higher eukaryotic cells, each of which is
discussed below.
[0153] The Hu-Asp polypeptides to be expressed in such host cells
may also be fusion proteins which include regions from heterologous
proteins. Such regions may be included to allow, e.g., secretion,
improved stability, or facilitated purification of the polypeptide.
For example, a sequence encoding an appropriate signal peptide can
be incorporated into expression vectors. A DNA sequence for a
signal peptide (secretory leader) may be fused inframe to the
Hu-Asp sequence so that Hu-Asp is translated as a fusion protein
comprising the signal peptide. A signal peptide that is functional
in the intended host cell promotes extracellular secretion of the
Hu-Asp polypeptide. Preferably, the signal sequence will be cleaved
from the Hu-Asp polypeptide upon secretion of Hu-Asp from the cell.
Nonlimiting examples of signal sequences that can be used in
practicing the invention include the yeast Ifactor and the honeybee
melatin leader in sf9 insect cells.
[0154] In a preferred embodiment, the Hu-Asp polypeptide will be a
fusion protein which includes a heterologous region used to
facilitate purification of the polypeptide. Many of the available
peptides used for such a function allow selective binding of the
fusion protein to a binding partner. For example, the Hu-Asp
polypeptide may be modified to comprise a peptide to form a fusion
protein which specifically binds to a binding partner, or peptide
tag. Nonlimiting examples of such peptide tags include the 6-His
tag, thioredoxin tag, hemaglutinin tag, GST tag, and OmpA signal
sequence tag. As will be understood by one of skill in the art, the
binding partner which recognizes and binds to the peptide may be
any molecule or compound including metal ions (e.g., metal affinity
columns), antibodies, or fragments thereof, and any protein or
peptide which binds the peptide, such as the FLAG tag.
[0155] Suitable host cells for expression of Hu-Asp polypeptides
includes prokaryotes, yeast, and higher eukaryotic cells. Suitable
prokaryotic hosts to be used for the expression of Hu-Asp include
bacteria of the genera Escherichia, Bacillus, and Salmonella, as
well as members of the genera Pseudomonas, Streptomyces, and
Staphylococcus. For expression in, e.g., E. coli, a Hu-Asp
polypeptide may include an N-terminal methionine residue to
facilitate expression of the recombinant polypeptide in a
prokaryotic host. The N-terminal Met may optionally then be cleaved
from the expressed Hu-Asp polypeptide. Other N-terminal amino acid
residues can be added to the Hu-Asp polypeptide to facilitate
expression in Escherichia coli including but not limited to the T7
leader sequence, the T7-caspase 8 leader sequence, as well as
others leaders including tags for purification such as the 6-His
tag (Example 9). Hu-Asp polypeptides expressed in E. coli may be
shortened by removal of the cytoplasmic tail, the transmembrane
domain, or the membrane proximal region. Hu-Asp polypeptides
expressed in E. coli may be obtained in either a soluble form or as
an insoluble form which may or may not be present as an inclusion
body. The insoluble polypeptide may be rendered soluble by
guanidine HCl, urea or other protein denaturants, then refolded
into a soluble form before or after purification by dilution or
dialysis into a suitable aqueous buffer. If the inactive proform of
the Hu-Asp was produced using recombinant methods, it may be
rendered active by cleaving off the prosegment with a second
suitable protease such as human immunodeficiency virus
protease.
[0156] Expression vectors for use in prokaryotic hosts generally
comprises one or more phenotypic selectable marker genes. Such
genes generally encode, e.g., a protein that confers antibiotic
resistance or that supplies an auxotrophic requirement. A wide
variety of such vectors are readily available from commercial
sources. Examples include pSPORT vectors, pGEM vectors (Promega),
pPROEX vectors (LTI, Bethesda, MD), Bluescript vectors
(Stratagene), pET vectors (Novagen) and pQE vectors (Qiagen).
[0157] Hu-Asp may also be expressed in yeast host cells from genera
including Saccharomyces, Pichia, and Kluveromyces. Preferred yeast
hosts are S. cerevisiae and P. pastoris. Yeast vectors will often
contain an origin of replication sequence from a 2T yeast plasmid,
an autonomously replicating sequence (ARS), a promoter region,
sequences for polyadenylation, sequences for transcription
termination, and a selectable marker gene. Vectors replicable in
both yeast and E. coli (termed shuttle vectors) may also be used.
In addition to the above-mentioned features of yeast vectors, a
shuttle vector will also include sequences for replication and
selection in E. coli. Direct secretion of Hu-Asp polypeptides
expressed in yeast hosts may be accomplished by the inclusion of
nucleotide sequence encoding the yeast I-factor leader sequence at
the 5' end of the Hu-Asp-encoding nucleotide sequence.
[0158] Insect host cell culture systems may also be used for the
expression of Hu-Asp polypeptides. In a preferred embodiment, the
Hu-Asp polypeptides of the invention are expressed using an insect
cell expression system (see Example 10). Additionally, a
baculovirus expression system can be used for expression in insect
cells as reviewed by Luckow and Summers, Bio/Technology 6:47
(1988).
[0159] In another preferred embodiment, the Hu-Asp polypeptide is
expressed in mammalian host cells. Nonlimiting examples of suitable
mammalian cell lines include the COS7 line of monkey kidney cells
(Gluzman et al., Cell 23:175 (1981)), human embyonic kidney cell
line 293, and Chinese hamster ovary (CHO) cells. Preferably,
Chinese hamster ovary (CHO) cells are used for expression of Hu-Asp
proteins (Example 11).
[0160] The choice of a suitable expression vector for expression of
the Hu-Asp polypeptides of the invention will of course depend upon
the specific mammalian host cell to be used, and is within the
skill of the ordinary artisan. Examples of suitable expression
vectors include pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). A
preferred vector for expression of Hu-Asp polypeptides is
pcDNA3.1-Hygro (Invitrogen). Expression vectors for use in
mammalian host cells may include transcriptional and translational
control sequences derived from viral genomes. Commonly used
promoter sequences and enhancer sequences which may be used in the
present invention include, but are not limited to, those derived
from human cytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and
Simian virus 40 (SV40). Methods for the construction of mammalian
expression vectors are disclosed, for example, in Okayama and Berg
(Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol. Immunol.
23:935 (1986)); Cosman et al. (Nature 312:768 (1984));
EP-A-0367566; and WO 91/18982.
[0161] The polypeptides of the present invention may also be used
to raise polyclonal and monoclonal antibodies, which are useful in
diagnostic assays for detecting Hu-Asp polypeptide expression. Such
antibodies may be prepared by conventional techniques. See, for
example, Antibodies: A Laboratory Manual, Harlow and Land (eds.),
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
(1988); Monoclonal Antibodies, Hybridomas: A New Dimension in
Biological Analyses, Kennet et al. (eds.), Plenum Press, New York
(1980). Synthetic peptides comprising portions of Hu-Asp containing
5 to 20 amino acids may also be used for the production of
polyclonal or monoclonal antibodies after linkage to a suitable
carrier protein including but not limited to keyhole limpet
hemacyanin (KLH), chicken ovalbumin, or bovine serum albumin using
various cross-linking reagents including carbodimides,
glutaraldehyde, or if the peptide contains a cysteine,
N-methylmaleimide. A preferred peptide for immunization when
conjugated to KLH contains the C-terminus of Hu-Asp1 or Hu-Asp2
comprising QRRPRDPEVVNDESSLVRHRWK (SEQ ID NO: 2, residues 497-518)
or LRQQHDDFADDISLLK (SEQ ID NO:4, residues 486-501), respectively.
See SEQ ID Nos. 33-34.
[0162] The Hu-Asp nucleic acid molecules of the present invention
are also valuable for chromosome identification, as they can
hybridize with a specific location on a human chromosome. Hu-Asp1
has been localized to chromosome 21, while Hu-Asp2 has been
localized to chromosome 11q23.3-24.1. There is a current need for
identifying particular sites on the chromosome, as few chromosome
marking reagents based on actual sequence data (repeat
polymorphisms) are presently available for marking chromosomal
location. Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. The relationship between
genes and diseases that have been mapped to the same chromosomal
region can then be identified through linkage analysis, wherein the
coinheritance of physically adjacent genes is determined. Whether a
gene appearing to be related to a particular disease is in fact the
cause of the disease can then be determined by comparing the
nucleic acid sequence between affected and unaffected
individuals.
[0163] In another embodiment, the invention relates to a method of
assaying Hu-Asp function, specifically Hu-Asp2 function which
involves incubating in solution the Hu-Asp polypeptide with a
suitable substrate including but not limited to a synthetic peptide
containing the .beta.-secretase cleavage site of APP, preferably
one containing the mutation found in a Swedish kindred with
inherited AD in which KM is changed to NL, such peptide comprising
the sequence SEVNLDAEFR (SEQ ID NO: 63) in an acidic buffering
solution, preferably an acidic buffering solution of pH5.5 (see
Example 12) using cleavage of the peptide monitored by high
performance liquid chromatography as a measure of Hu-Asp
proteolytic activity. Preferred assays for proteolytic activity
utilize internally quenched peptide assay substrates. Such suitable
substrates include peptides which have attached a paired flurophore
and quencher including but not limited to 7-amino4-methyl coumarin
and dinitrophenol, respectively, such that cleavage of the peptide
by the Hu-Asp results in increased fluorescence due to physical
separation of the flurophore and quencher. Other paired flurophores
and quenchers include bodipy-tetramethylrhodamine and QSY-5
(Molecular Probes, Inc.). In a variant of this assay, biotin or
another suitable tag may be placed on one end of the peptide to
anchor the peptide to a substrate assay plate and a flurophore may
be placed at the other end of the peptide. Useful flurophores
include those listed above as well as Europium labels such as W8044
(EG&g Wallac, Inc.). Cleavage of the peptide by Asp2 will
release the flurophore or other tag from the plate, allowing
compounds to be assayed for inhibition of Asp2 proteolytic cleavage
as shown by an increase in retained fluorescence. Preferred
colorimetric assays of Hu-Asp proteolytic activity utilize other
suitable substrates that include the P2 and P1 amino acids
comprising the recognition site for cleavage linked to
o-nitrophenol through an amide linkage, such that cleavage by the
Hu-Asp results in an increase in optical density after altering the
assay buffer to alkaline pH.
[0164] In another embodiment, the invention relates to a method for
the identification of an agent that increases the activity of a
Hu-Asp polypeptide selected from the group consisting of Hu-Asp1,
Hu-Asp2(a), and Hu-Asp2(b), the method comprising
[0165] (a) determining the activity of said Hu-Asp polypeptide in
the presence of a test agent and in the absence of a test agent;
and
[0166] (b) comparing the activity of said Hu-Asp polypeptide
determined in the presence of said test agent to the activity of
said Hu-Asp polypeptide determined in the absence of said test
agent;
[0167] whereby a higher level of activity in the presence of said
test agent than in the absence of said test agent indicates that
said test agent has increased the activity of said Hu-Asp
polypeptide. Such tests can be performed with Hu-Asp polypeptide in
a cell free system and with cultured cells that express Hu-Asp as
well as variants or isoforms thereof.
[0168] In another embodiment, the invention relates to a method for
the identification of an agent that decreases the activity of a
Hu-Asp polypeptide selected from the group consisting of Hu-Asp1,
Hu-Asp2(a), and Hu-Asp2(b), the method comprising
[0169] (a) determining the activity of said Hu-Asp polypeptide in
the presence of a test agent and in the absence of a test agent;
and
[0170] (b) comparing the activity of said Hu-Asp polypeptide
determined in the presence of said test agent to the activity of
said Hu-Asp polypeptide determined in the absence of said test
agent;
[0171] whereby a lower level of activity in the presence of said
test agent than in the absence of said test agent indicates that
said test agent has decreased the activity of said Hu-Asp
polypeptide. Such tests can be performed with Hu-Asp polypeptide in
a cell free system and with cultured cells that express Hu-Asp as
well as variants or isoforms thereof.
[0172] In another embodiment, the invention relates to a novel cell
line (HEK125.3 cells) for measuring processing of amyloid .beta.
peptide (A.beta.) from the amyloid protein precursor (APP). The
cells are stable transformants of human embryonic kidney 293 cells
(HEK293) with a bicistronic vector derived from pIRES-EGFP
(Clontech) containing a modified human APP cDNA, an internal
ribosome entry site and an enhanced green fluorescent protein
(EGFP) cDNA in the second cistron. The APP cDNA was modified by
adding two lysine codons to the carboxyl terminus of the APP coding
sequence. This increases processing of A.beta. peptide from human
APP by 24 fold. This level of A.beta. peptide processing is 60 fold
higher than is seen in nontransformed HEK293 cells. HEK125.3 cells
will be useful for assays of compounds that inhibit .beta. peptide
processing. This invention also includes addition of two lysine
residues to the C-terminus of other APP isoforms including the 751
and 770 amino acid isoforms, to isoforms of APP having mutations
found in human AD including the Swedish KM.fwdarw.NL and V7
7.fwdarw.F mutations, to C-terminal fragments of APP, such as those
beginning with the .beta.-secretase cleavage site, to C-terminal
fragments of APP containing the .beta.-secretase cleavage site
which have been operably linked to an N-terminal signal peptide for
membrane insertion and secretion, and to C-terminal fragments of
APP which have been operably linked to an N-terminal signal peptide
for membrane insertion and secretion and a reporter sequence
including but not limited to green fluorescent protein or alkaline
phosphatase, such that .beta.-secretase cleavage releases the
reporter protein from the surface of cells expressing the
polypeptide.
[0173] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLE 1
Development of a Search Algorithm Useful for the Identification of
Aspartyl Proteases, and Identification of C. elegans Aspartyl
Protease Genes in Wormpep 12
[0174] Materials and Methods:
[0175] Classical aspartyl proteases such as pepsin and renin
possess a two-domain structure which folds to bring two aspartyl
residues into proximity within the active site. These are embedded
in the short tripeptide motif DTG, or more rarely, DSG. The DTG or
DSG active site motif appears at about residue 25-30 in the enzyme,
but at about 65-70 in the proenzyme (prorenin, pepsinogen). This
motif appears again about 150-200 residues downstream. The
proenzyme is activated by cleavage of the N-terminal prodomain.
This pattern exemplifies the double domain structure of the modern
day aspartyl enzymes which apparently arose by gene duplication and
divergence. Thus;
NH.sub.2--X-D.sup.25TG-Y-D.sup.Y+25TG-C
[0176] where X denotes the beginning of the enzyme, following the
N-terminal prodomain, and Y denotes the center of the molecule
where the gene repeat begins again.
[0177] In the case of the retroviral enzymes such as the HI
protease, they represent only a half of the two-domain structures
of well-known enzymes like pepsin, cathepsin D, renin, etc. They
have no prosegment, but are carved out of a polyprotein precursor
containing the gag and pol proteins of the virus. They can be
represented by:
NH.sub.2-D.sup.25TG-C100
[0178] This "monomer" only has about 100 aa, so is extremely
parsimonious as compared to the other aspartyl protease "dimers"
which have of the order of 330 or so aa, not counting the
N-terminal prodomain.
[0179] The limited length of the eukaryotic aspartyl protease
active site motif makes it difficult to search EST collections for
novel sequences. EST sequences typically average 250 nucleotides,
and so in this case would be unlikely to span both aspartyl
protease active site motifs. Instead, we turned to the C. elegans
genome. The C. elegans genome is estimated to contain around 13,000
genes. Of these, roughly 12,000 have been sequenced and the
corresponding hypothetical open reading frame (ORF) has been placed
in the database Wormpep12. We used this database as the basis for a
whole genome scan of a higher eukaryote for novel aspartyl
proteases, using an algorithm that we developed specifically for
this purpose. The following AWK script for locating proteins
containing two DTG or DSG motifs was used for the search, which was
repeated four times to recover all pairwise combinations of the
aspartyl motif.
1 BEGIN {RS=">"} /* defines ">" as record separator for FASTA
format */ { pos = index($0,"DTG") /*finds "DTG" in record*/ if
(pos>0) { rest = substr($0,pos+3) /*get rest of record after
first DTG*/ pos2 = index(rest,"DTG") /*find second DTG*/ if
(pos2>0) printf ("%s%s.backslash.n",">",$0)} /*report hits*/
} }
[0180] The AWK script shown above was used to search Wormpep12,
which was downloaded from ftp.sanger.ac.uk/pub/databases/wormpep,
for sequence entries containing at least two DTG or DSG motifs.
Using AWK limited each record to 3000 characters or less. Thus, 35
or so larger records were eliminated manually from Wormpep12 as in
any case these were unlikely to encode aspartyl proteases.
[0181] Results and Discussion:
[0182] The Wormpep 12 database contains 12,178 entries, although
some of these (<10%) represent alternatively spliced transcripts
from the same gene. Estimates of the number of genes encoded in the
C. elegans genome is on the order of 13,000 genes, so Wormpep 12
may be estimated to cover greater than 90% of the C. elegans
genome.
[0183] Eukaryotic aspartyl proteases contain a two-domain
structure, probably arising from ancestral gene duplication. Each
domain contains the active site motif D(S/T)G located from 20-25
amino acid residues into each domain. The retroviral (e.g., HIV
protease) or retrotransposon proteases are homodimers of subunits
which are homologous to a single eukaryotic aspartyl protease
domain. An AWK script was used to search the Wormpep12 database for
proteins in which the D(S/T)G motif occurred at least twice. This
identified >60 proteins with two DTG or DSG motifs. Visual
inspection was used to select proteins in which the position of the
aspartyl domains was suggestive of a two-domain structure meeting
the criteria described above.
[0184] In addition, the PROSITE eukaryotic and viral aspartyl
protease active site pattern PS00141 was used to search Wormpep12
for candidate aspartyl proteases. (Bairoch A., Bucher P., Hofmann
K., The PROSITE database: its status in 1997, Nucleic Acids Res.
24:217-221(1997)). This generated an overlapping set of Wormpep12
sequences. Of these, seven sequences contained two DTG or DSG
motifs and the PROSITE aspartyl protease active site pattern. Of
these seven, three were found in the same cosmid clone (F21F8.3,
F21F8.4, and F21F8.7) suggesting that they represent a family of
proteins that arose by ancestral gene duplication. Two other ORFs
with extensive homology to F21F8.3, F21F8.4 and F21F8.7 are present
in the same gene cluster (F21F8.2 and F21F8.6), however, these
contain only a single DTG motif. Exhaustive BLAST searches with
these seven sequences against Wormpep12 failed to reveal additional
candidate aspartyl proteases in the C. elegans genome containing
two repeats of the DTG or DSG motif
[0185] BLASTX search with each C. elegans sequence against
SWISS-PROT, GenPep and TREMBL revealed that R12H7.2 was the closest
worm homologue to the known mammalian aspartyl proteases, and that
T18H9.2 was somewhat more distantly related, while CEASP1, F21F8.3,
F21F8.4, and F21F8.7 formed a subcluster which had the least
sequence homology to the mammalian sequences.
[0186] Discussion:
[0187] APP, the presenilins, and p35, the activator of cdk5, all
undergo intracellular proteolytic processing at sites which conform
to the substrate specificity of the HIV protease. Dysregulation of
a cellular aspartyl protease with the same substrate specificity,
might therefore provide a unifying mechanism for causation of the
plaque and tangle pathologies in AD. Therefore, we sought to
identify novel human aspartyl proteases. A whole genome scan in C.
elegans identified seven open reading frames that adhere to the
aspartyl protease profile that we had identified. These seven
aspartyl proteases probably comprise the complete complement of
such proteases in a simple, multicellular eukaryote. These include
four closely related aspartyl proteases unique to C. elegans which
probably arose by duplication of an ancestral gene. The other three
candidate aspartyl proteases (T18H9.2, R12H7.2 and C11D2.2) were
found to have homology to mammalian gene sequences.
EXAMPLE 2
Identification of Novel Human Aspartyl Proteases Using Database
Mining by Genome Bridging
[0188] Materials and Methods:
[0189] Computer-assisted analysis of EST databases, cDNA, and
predicted polypeptide sequences:
[0190] Exhaustive homology searches of EST databases with the
CEASP1, F21F8.3, F21F8.4, and F21F8.7 sequences failed to reveal
any novel mammalian homologues. TBLASTN searches with R12H7.2
showed homology to cathepsin D, cathepsin E, pepsinogen A,
pepsinogen C and renin, particularly around the DTG motif within
the active site, but also failed to identify any additional novel
mammalian aspartyl proteases. This indicates that the C. elegans
genome probably contains only a single lysosomal aspartyl protease
which in mammals is represented by a gene family that arose through
duplication and consequent modification of an ancestral gene.
[0191] TBLASTN searches with T18H9.2, the remaining C. elegans
sequence, identified several ESTs which assembled into a contig
encoding a novel human aspartyl protease (Hu-ASP1). As is described
above in Example 1, BLASTX search with the Hu-ASP1 contig against
SWISS-PROT revealed that the active site motifs in the sequence
aligned with the active sites of other aspartyl proteases.
Exhaustive, repetitive rounds of BLASTN searches against LifeSeq,
LifeSeqFL, and the public EST collections identified 102 EST from
multiple cDNA libraries that assembled into a single contig. The 51
sequences in this contig found in public EST collections also have
been assembled into a single contig (THC213329) by The Institute
for Genome Research (TIGR). The TIGR annotation indicates that they
failed to find any hits in the database for the contig. Note that
the TIGR contig is the reverse complement of the LifeSeq contig
that we assembled. BLASTN search of Hu-ASP1 against the rat and
mouse EST sequences in ZooSeq revealed one homologous EST in each
database (Incyte clone 700311523 and IMAGE clone 313341, GenBank
accession number W10530, respectively).
[0192] TBLASTN searches with the assembled DNA sequence for Hu-ASP1
against both LifeSeqFL and the public EST databases identified a
second, related human sequence (Hu-Asp2) represented by a single
EST (2696295). Translation of this partial cDNA sequence reveals a
single DTG motif which has homology to the active site motif of a
bovine aspartyl protease, NM1.
[0193] BLAST searches, contig assemblies and multiple sequence
alignments were performed using the bioinformatics tools provided
with the LifeSeq, LifeSeqFL and LifeSeq Assembled databases from
Incyte. Predicted protein motifs were identified using either the
ProSite dictionary (Motifs in GCG 9) or the Pfam database.
[0194] Full-length cDNA Cloning of Hu-Asp1
[0195] The open reading frame of C. elegans gene T18H9.2CE was used
to query Incyte LifeSeq and LifeSeq-FL databases and a single
electronic assembly referred to as 1863920CE1 was detected. The 5'
most cDNA clone in this contig, 1863920, was obtained from Incyte
and completely sequenced on both strands. Translation of the open
reading frame contained within clone 1863920 revealed the presence
of the duplicated aspartyl protease active site motif (DTG/DSG) but
the 5' end was incomplete. The remainder of the Hu-Asp1 coding
sequence was determined by 5' Marathon RACE analysis using a human
placenta Marathon ready cDNA template (Clontech). A 3'-antisense
oligonucleotide primer specific for the 5' end of clone 1863920 was
paired with the 5'-sense primer specific for the Marathon ready
cDNA synthetic adaptor in the PCR. Specific PCR products were
directly sequenced by cycle sequencing and the resulting sequence
assembled with the sequence of clone 1863920 to yield the complete
coding sequence of Hu-Asp-1 (SEQ ID No. 1).
[0196] Several interesting features are present in the primary
amino acid sequence of Hu-Asp1 (FIG. 1, SEQ ID No. 2). The sequence
contains a signal peptide (residues 1-20 in SEQ ID No. 2), a
pro-segment, and a catalytic domain containing two copies of the
aspartyl protease active site motif (DTG/DSG). The spacing between
the first and second active site motifs is about 200 residues which
should correspond to the expected size of a single, eukaryotic
aspartyl protease domain. More interestingly, the sequence contains
a predicted transmembrane domain (residues 469-492 in SEQ ID No.2)
near its C-terminus which suggests that the protease is anchored in
the membrane. This feature is not found in any other aspartyl
protease.
[0197] Cloning of a Full-Length Hu-Asp-2 cDNAs:
[0198] As is described above in Example 1, genome wide scan of the
Caenorhabditis elegans database WormPep12 for putative aspartyl
proteases and subsequent mining of human EST databases revealed a
human ortholog to the C. elegans gene T18H9.2 referred to as
Hu-Asp1. The assembled contig for Hu-Asp1 was used to query for
human paralogs using the BLAST search tool in human EST databases
and a single significant match (2696295CE1) with approximately 60%
shared identity was found in the LifeSeq FL database. Similar
queries of either gb105PubEST or the family of human databases
available from TIGR did not identify similar EST clones. cDNA clone
2696295, identified by single pass sequence analysis from a human
uterus cDNA library, was obtained from Incyte and completely
sequence on both strands. This clone contained an incomplete 1266
bp open-reading frame that encoded a 422 amino acid polypeptide but
lacked an initiator ATG on the 5' end. inspection of the predicted
sequence revealed the presence of the duplicated aspartyl protease
active site motif DTG/DSG, separated by 194 amino acid residues.
Subsequent queries of later releases of the LifeSeq EST database
identified an additional ESTs, sequenced from a human astrocyte
cDNA library (4386993), that appeared to contain additional 5'
sequence relative to clone 2696295. Clone 4386993 was obtained from
Incyte and completely sequenced on both strands. Comparative
analysis of clone 4386993 and clone 2696295 confirmed that clone
4386993 extended the open-reading frame by 31 amino acid residues
including two in-frame translation initiation codons. Despite the
presence of the two in-frame ATGs, no in-frame stop codon was
observed upstream of the ATG indicating that the 4386993 may not be
full-length. Furthermore, alignment of the sequences of clones
2696295 and 4386993 revealed a 75 base pair insertion in clone
2696295 relative to clone 4386993 that results in the insertion of
25 additional amino acid residues in 2696295. The remainder of the
Hu-Asp2 coding sequence was determined by 5' Marathon RACE analysis
using a human hippocampus Marathon ready cDNA template (Clontech).
A 3'-antisense oligonucleotide primer specific for the shared
5'-region of clones 2696295 and 4386993 was paired with the
5'-sense primer specific for the Marathon ready cDNA synthetic
adaptor in the PCR. Specific PCR products were directly sequenced
by cycle sequencing and the resulting sequence assembled with the
sequence of clones 2696295 and 4386993 to yield the complete coding
sequence of Hu-Asp2(a) (SEQ ID No. 3) and Hu-Asp2(b) (SEQ ID No.
5), respectively.
[0199] Several interesting features are present in the primary
amino acid sequence of Hu-Asp2(a) (FIG. 2 and SEQ ID No. 4) and
Hu-Asp-2(b) (FIG. 3, SEQ ID No. 6). Both sequences contain a signal
peptide (residues 1-21 in SEQ ID No. 4 and SEQ ID No. 6), a
pro-segment, and a catalytic domain containing two copies of the
aspartyl protease active site motif (DTG/DSG). The spacing between
the first and second active site motifs is variable due to the 25
amino acid residue deletion in Hu-Asp-2(b) and consists of
168-versus-194 amino acid residues, for Hu-Asp2(b) and Hu-Asp-2(a),
respectively. More interestingly, both sequences contains a
predicted transmembrane domain (residues 455-477 in SEQ ID No.4 and
430-452 in SEQ ID No. 6) near their C-termini which indicates that
the protease is anchored in the membrane. This feature is not found
in any other aspartyl protease except Hu-Asp1.
EXAMPLE 3
Molecular Cloning of Mouse Asp2 cDNA and Genomic DNA
[0200] Cloning and Characterization of Murine Asp2 cDNA.
[0201] The murine ortholog of Hu-Asp2 was cloned using a
combination of cDNA library screening, PCR, and genomic cloning.
Approximately 500,000 independent clones from a mouse brain cDNA
library were screened using a .sup.32P-labeled coding sequence
probe prepared from Hu-Asp2. Replicate positives were subjected to
DNA sequence analysis and the longest cDNA contained the entire 3'
untranslated region and 47 amino acids in the coding region. PCR
amplification of the same mouse brain cDNA library with an
antisense oligonucleotide primer specific for the 5'-most cDNA
sequence determined above and a sense primer specific for the 5'
region of human Asp2 sequence followed by DNA sequence analysis
gave an additional 980 bp of the coding sequence. The remainder of
the 5' sequence of murine Asp-2 was derived from genomic sequence
(see below).
[0202] Isolation and sequence Analysis of the Murine Asp-2 Gene
[0203] A murine EST sequence encoding a portion of the murine Asp2
cDNA was identified in the GenBank EST database using the BLAST
search tool and the Hu-Asp2 coding sequence as the query. Clone
g3160898 displayed 88% shared identity to the human sequence over
352 bp. Oligonucleotide primer pairs specific for this region of
murine Asp2 were then synthesized and used to amplify regions of
the murine gene. Murine genomic DNA, derived from strain 129/SvJ,
was amplified in the PCR (25 cycles) using various primer sets
specific for murine Asp2 and the products analyzed by agarose gel
electrophoresis. The primer set Zoo-1 and Zoo-4 amplified a 750 bp
fragment that contained approximately 600 bp of intron sequence
based on comparison to the known cDNA sequence. This primer set was
then used to screen a murine BAC library by PCR, a single genomic
clone was isolated and this cloned was confirmed contain the murine
Asp2 gene by DNA sequence analysis. Shotgun DNA sequencing of this
Asp2 genomic clone and comparison to the cDNA sequences of both
Hu-Asp2 and the partial murine cDNA sequences defined the
full-length sequence of murine Asp2 (SEQ ID No. 7). The predicted
amino acid sequence of murine Asp2 (SEQ ID No. 8) showed 96.4%
shared identity (GCG BestFit algorithm) with 18/501 amino acid
residue substitutions compared to the human sequence (FIG. 4). The
proteolytic processing of murine Asp2(a) is believed to be
analogous to the processing described above for human Asp2(a). In
addition, a variant lacking amino acid residues 190-214 of SEQ D
NO: 8 is specifically contemplated as a murine Asp2(b) polypeptide.
All forms of murine Asp2(b) gene and protein are intended as
aspects of the invention.
EXAMPLE 4
Tissue Distribution of Expression of Hu-Asp2 Transcripts
[0204] Materials and Methods:
[0205] The tissue distribution of expression of Hu-Asp-2 was
determined using multiple tissue Northern blots obtained from
Clontech (Palo Alto, Calif.). Incyte clone 2696295 in the vector
pINCY was digested to completion with EcoRI/NotI and the 1.8 kb
cDNA insert purified by preparative agarose gel electrophoresis.
This fragment was radiolabeled to a specific activity
>1.times.10.sup.9 dpm/.mu.g by random priming in the presence of
[.alpha.-.sup.32P-dATP] (>3000 Ci/mmol, Amersham, Arlington
Heights, Ill.) and Klenow fragment of DNA polymerase I. Nylon
filters containing denatured, size fractionated poly A.sup.+ RNAs
isolated from different human tissues were hybridized with
2.times.10.sup.6 dpm/ml probe in ExpressHyb buffer (Clontech, Palo
Alto, Calif.) for 1 hour at 68.degree. C. and washed as recommended
by the manufacture. Hybridization signals were visualized by
autoradiography using BioMax XR film (Kodak, Rochester, N.Y.) with
intensifying screens at -80.degree. C.
[0206] Results and Discussion:
[0207] Limited information on the tissue distribution of expression
of Hu-Asp-2 transcripts was obtained from database analysis due to
the relatively small number of ESTs detected using the methods
described above (<5). In an effort to gain further information
on the expression of the Hu-Asp2 gene, Northern analysis was
employed to determine both the size(s) and abundance of Hu-Asp2
transcripts. PolyA.sup.+ RNAs isolated from a series of peripheral
tissues and brain regions were displayed on a solid support
following separation under denaturing conditions and Hu-Asp2
transcripts were visualized by high stringency hybridization to
radiolabeled insert from clone 2696295. The 2696295 cDNA probe
visualized a constellation of transcripts that migrated with
apparent sizes of 3.0 kb, 4.4 kb and 8.0 kb with the latter two
transcript being the most abundant.
[0208] Across the tissues surveyed, Hu-Asp2 transcripts were most
abundant in pancreas and brain with lower but detectable levels
observed in all other tissues examined except thymus and PBLs.
Given the relative abundance of Hu-Asp2 transcripts in brain, the
regional expression in brain regions was also established. A
similar constellation of transcript sizes were detected in all
brain regions examined [cerebellum, cerebral cortex, occipital
pole, frontal lobe, temporal lobe and putamen] with the highest
abundance in the medulla and spinal cord.
EXAMPLE 5
Northern Blot Detection of HuAsp-1 and HuAsp-2 Transcripts in Human
Cell Lines
[0209] A variety of human cell lines were tested for their ability
to produce Hu-Asp1 and Asp2 mRNA. Human embryonic kidney (HEK-293)
cells, African green monkey (Cos-7) cells, Chinese hamster ovary
(CHO) cells, HELA cells, and the neuroblastoma cell line IMR-32
were all obtained from the ATCC. Cells were cultured in DME
containing 10% FCS except CHO cells which were maintained in
.alpha.-MEM/10% FCS at 37.degree. C. in 5% CO.sub.2 until they were
near confluence. Washed monolayers of cells (3.times.10.sup.7) were
lysed on the dishes and poly A.sup.+ RNA extracted using the Qiagen
Oligotex Direct mRNA kit. Samples containing 2 .mu.g of poly
A.sup.+ RNA from each cell line were fractionated under denaturing
conditions (glyoxal-treated), transferred to a solid nylon membrane
support by capillary action, and transcripts visualized by
hybridization with random-primed labeled (.sup.32P) coding sequence
probes derived from either Hu-Asp1 or Hu-Asp2. Radioactive signals
were detected by exposure to X-ray film and by image analysis with
a Phosphorimager.
[0210] The Hu-Asp1 cDNA probe visualized a similar constellation of
transcripts (2.6 kb and 3.5 kb) that were previously detected is
human tissues. The relative abundance determined by quantification
of the radioactive signal was Cos-7>HEK 292=HELA>IMR32.
[0211] The Hu-Asp2 cDNA probe also visualized a similar
constellation of transcripts compared to tissue (3.0 kb, 4.4 kb,
and 8.0 kb) with the following relative abundance; HEK 293>Cos
7>IMR32>HELA.
EXAMPLE 6
[0212] Modification of APP to Increase A.beta. Processing for In
Vitro Screening
[0213] Human cell lines that process A.beta. peptide from APP
provide a means to screen in cellular assays for inhibitors of
.beta.- and .gamma.-secretase. Production and release of A.beta.
peptide into the culture supernatant is monitored by an
enzyme-linked immunosorbent assay (EIA). Although expression of APP
is widespread and both neural and non-neuronal cell lines process
and release A.beta. peptide, levels of endogenous APP processing
are low and difficult to detect by EIA. A.beta. processing can be
increased by expressing in transformed cell lines mutations of APP
that enhance A.beta. processing. We made the serendipitous
observation that addition of two lysine residues to the carboxyl
terminus of APP695 increases A.beta. processing still further. This
allowed us to create a transformed cell line that releases A.beta.
peptide into the culture medium at the remarkable level of 20,000
pg/ml.
[0214] Materials and Methods
[0215] Materials:
[0216] Human embryonic kidney cell line 293 (HEK293 cells) were
obtained internally. The vector pIRES-EGFP was purchased from
Clontech. Oligonucleotides for mutation using the polymerase chain
reaction (PCR) were purchased from Genosys. A plasmid containing
human APP695 (SEQ ID No. 9 [nucleotide] and SEQ ID No. 10 [amino
acid]) was obtained from Northwestern University Medical School.
This was subcloned into pSK (Stratagene) at the Not1 site creating
the plasmid pAPP695.
[0217] Mutagenesis Protocol:
[0218] The Swedish mutation (K670N, M671L) was introduced into
pAPP695 using the Stratagene Quick Change Mutagenesis Kit to create
the plasmid pAPP695NL (SEQ ID No. 11 [nucleotide] and SEQ ID No. 12
[amino acid]). To introduce a di-lysine motif at the C-terminus of
APP695, the forward primer #276 5' GACTGACCACTCGACCAGGTTC (SEQ ID
No. 47) was used with the "patch" primer #274 5'
CGAATTAAATTCCAGCACACTGGCTACTTCTTGTTCTGCATCTCAAAGAA- C (SEQ ID No.
48) and the flanking primer #275 CGAATTAAATTCCAGCACACTGGCTA (SEQ ID
No. 49) to modify the 3' end of the APP695 cDNA (SEQ ID No. 15
[nucleotide] and SEQ ID No. 16 [amino acid]). This also added a
BstX1 restriction site that will be compatible with the BstX1 site
in the multiple cloning site of pIRES-EGFP. PCR amplification was
performed with a Clontech HF Advantage cDNA PCR kit using the
polymerase mix and buffers supplied by the manufacturer. For
"patch" PCR, the patch primer was used at {fraction (1/20)}th the
molar concentration of the flanking primers. PCR amplification
products were purified using a QIAquick PCR purification kit
(Qiagen). After digestion with restriction enzymes, products were
separated on 0.8% agarose gels and then excised DNA fragments were
purified using a QIAquick gel extraction kit (Qiagen).
[0219] To reassemble a modified APP695-Sw cDNA, the 5' Not1-Bg12
fragment of the APP695-Sw cDNA and the 3' Bg12-BstX1 APP695 cDNA
fragment obtained by PCR were ligated into pIRES-EGFP plasmid DNA
opened at the Not1 and BstX1 sites. Ligations were performed for 5
minutes at room temperature using a Rapid DNA Ligation kit
(Boehringer Mannheim) and transformed into Library Efficiency DH5a
Competent Cells (GibcoBRL Life Technologies). Bacterial colonies
were screened for inserts by PCR amplification using primers #276
and #275. Plasmid DNA was purified for mammalian cell transfection
using a QIAprep Spin Miniprep kit (Qiagen). The construct obtained
was designated pMG125.3 (APPSW-KK, SEQ ID No. 17 [nucleotide] and
SEQ ID No. 18 [amino acid]).
[0220] Mammalian Cell Transfection:
[0221] HEK293 cells for transfection were grown to 80% confluence
in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine
serum. Cotransfections were performed using LipofectAmine
(Gibco-BRL) with 3 .mu.g pMG125.3 DNA and 9 [g pcDNA3.1 DNA per
10.times.10.sup.6 cells. Three days posttransfection, cells were
passaged into medium containing G418 at a concentration of 400
.mu.g/ml. After three days growth in selective medium, cells were
sorted by their fluorescence.
[0222] Clonal Selection of 125.3 Cells by FACS:
[0223] Cell samples were analyzed on an EPICS Elite ESP flow
cytometer (Coulter, Hialeah, Fla.) equipped with a 488 nm
excitation line supplied by an air-cooled argon laser. EGFP
emission was measured through a 525 nm band-pass filter and
fluorescence intensity was displayed on a 4-decade log scale after
gating on viable cells as determined by forward and right angle
light scatter. Single green cells were separated into each well of
one 96 well plate containing growth medium without G418. After a
four day recovery period, G418 was added to the medium to a final
concentration of 400 .mu.g/ml. After selection, 32% of the wells
contained expanding clones. Wells with clones were expanded from
the 96 well plate to a 24 well plate and then a 6 well plate with
the fastest growing colonies chosen for expansion at each passage.
The final cell line selected was the fastest growing of the final
six passaged. This clone, designated 125.3, has been maintained in
G418 at 400 ug/ml with passage every four days into fresh medium.
No loss of A.beta. production of EGFP fluorescence has been seen
over 23 passages.
[0224] A.beta.EM4 Analysis (Double Antibody Sandwich ELISA for
hA.beta. 1-40/42):
[0225] Cell culture supernatants harvested 48 hours after
transfection were analyzed in a standard A.beta. EIA as follows.
Human A.beta. 1-40 or 1-42 was measured using monoclonal antibody
(mAb) 6E10 (Senetek, St. Louis, Mo.) and biotinylated rabbit
antiserum 162 or 164 (New York State Institute for Basic Research,
Staten Island, N.Y.) in a double antibody sandwich ELISA. The
capture antibody 6E10 is specific to an epitope present on the
N-terminal amino acid residues 1-16 of hA.beta.. The conjugated
detecting antibodies 162 and 164 are specific for hA.beta. 1-40 and
1-42, respectively. Briefly, a Nunc Maxisorp 96 well immunoplate
was coated with 100 .mu.l/well of mAb 6E10 (5 .mu.g/ml diluted in
0.1M carbonate-bicarbonate buffer, pH 9.6 and incubated at
4.degree. C. overnight. After washing the plate 3.times. with 0.01M
DPBS (Modified Dulbecco's Phosphate Buffered Saline (0.008M sodium
phosphate, 0.002M potassium phosphate, 0.14M sodium chloride, 0.01
M potassium chloride, pH 7.4) from Pierce, Rockford, Ill.)
containing 0.05% of Tween-20 (DPBST), the plate was blocked for 60
minutes with 200 .mu.l of 10% normal sheep serum (Sigma) in 0.01M
DPBS to avoid non-specific binding. Human A.beta. 1-40 or 1-42
standards 100 .mu.l/well (Bachem, Torrance, Calif.) diluted, from a
1 mg/ml stock solution in DMSO, in culture medium was added after
washing the plate, as well as 100 .mu.l/well of sample, e.g.,
conditioned medium of transfected cells.
[0226] The plate was incubated for 2 hours at room temperature and
4.degree. C. overnight. The next day, after washing the plate, 100
.mu.l/well biotinylated rabbit antiserum 162 1:400 or 164 1:50
diluted in DPBST +0.5% BSA was added and incubated at room
temperature for 1 hour, 15 minutes. Following washes, 100
.mu.l/well neutravidin-horseradish peroxidase (Pierce, Rockford,
Ill.) diluted 1:10,000 in DPBST was applied and incubated for 1
hour at room temperature. After the last washes 100 .mu.l/well of
o-phenylnediamine dihydrochloride (Sigma Chemicals, St. Louis, Mo.)
in 50 mM citric acid/100 mM sodium phosphate buffer (Sigma
Chemicals, St. Louis, Mo.), pH 5.0, was added as substrate and the
color development was monitored at 450 nm in a kinetic microplate
reader for 20 minutes using Soft max Pro software. All standards
and samples were run in triplicates. The samples with absorbance
values falling within the standard curve were extrapolated from the
standard curves using Soft max Pro software and expressed in pg/ml
culture medium.
[0227] Results:
[0228] Addition of two lysine residues to the carboxyl terminus of
APP695 greatly increases A.beta. processing in HEK293 cells as
shown by transient expression (Table 1). Addition of the di-lysine
motif to APP695 increases A.beta. processing to that seen with the
APP695 containing the Swedish mutation. Combining the di-lysine
motif with the Swedish mutation further increases processing by an
additional 2.8 fold.
[0229] Cotransformation of HEK293 cells with pMG125.3 and pcDNA3.1
allowed dual selection of transformed cells for G418 resistance and
high level expression of EGFP. After clonal selection by FACS, the
cell line obtained, produces a remarkable 20,000 pg A.beta. peptide
per ml of culture medium after growth for 36 hours in 24 well
plates. Production of A.beta. peptide under various growth
conditions is summarized in Table 2.
2TABLE 1 Release of A.beta. peptide into the culture medium 48
hours after transient transfection of HEK293 cells with the
indicated vectors containing wildtype or modified APP. Values
tabulated are mean + SD and P-value for pairwise comparison using
Student's t-test assuming unequal variances. A.beta. 1-40 peptide
Fold APP Construct (pg/ml) Increase P-value pIRES-EGFP vector 147 +
28 1.0 wt APP695 (142.3) 194 + 15 1.3 0.051 wt APP695-KK (124.1)
424 + 34 2.8 3 .times. 10-5 APP695-Sw (143.3) 457 + 65 3.1 2
.times. 10-3 APP695-SwKK (125.3) 1308 + 98 8.9 3 .times. 10-4
[0230]
3TABLE 2 Release of A.beta. peptide from HEK125.3 cells under
various growth conditions. Type of Culture Volume of Duration of
A.beta. 1-40 A.beta. 1-42 Plate Medium Culture (pg/ml) (pg/ml) 24
well plate 400 ul 36 hr 28,036 1,439
EXAMPLE 7
[0231] Antisense Oligomer Inhibition of Abeta Processing in
HEK125.3 Cells
[0232] The sequences of Hu-Asp1 and Hu-Asp2 were provided to
Sequitur, Inc (Natick, Mass.) for selection of targeted sequences
and design of 2nd generation chimeric antisense oligomers using
prorietary technology (Sequitur Ver. D Pat pending #3002).
Antisense oligomers Lot #S644, S645, S646 and S647 were targeted
against Asp1. Antisense oligomers Lot #S648, S649, S650 and S651
were targeted against Asp2. Control antisense oligomers Lot #S652,
S653, S655, and S674 were targeted against an irrelevant gene and
antisense oligomers Lot #S656, S657, S658, and S659 were targeted
against a second irrelevant gene.
[0233] For transfection with the antisense oligomers, HEK125.3
cells were grown to about 50% confluence in 6 well plates in
Minimal Essential Medium (MEM) supplemented with 10% fetal calf
serum. A stock solution of oligofectin G (Sequitur Inc., Natick,
Mass.) at 2 mg/ml was diluted to 50 .mu.g/ml in serum free MEM.
Separately, the antisense oligomer stock solution at 100 .mu.M was
diluted to 800 nM in Opti-MEM (GIBCO-BRL, Grand Island, N.Y.). The
diluted stocks of oligofectin G and antisense oligomer were then
mixed at a ratio of 1:1 and incubated at room temperature. After 15
minutes incubation, the reagent was diluted 10 fold into MEM
containing 10% fetal calf serum and 2 ml was added to each well of
the 6 well plate after first removing the old medium. After
transfection, cells were grown in the continual presence of the
oligofectin G/antisense oligomer. To monitor A.beta. peptide
release, 400 .mu.l of conditioned medium was removed periodically
from the culture well and replaced with fresh medium beginning 24
hours after transfection. A.beta. peptides in the conditioned
medium were assayed via immunoprecipitation and Western blotting.
Data reported are from culture supernatants harvested 48 hours
after transfection.
[0234] The 16 different antisense oligomers obtained from Sequitur
Inc. were transfected separately into HEK125.3 cells to determine
their affect on A.beta. peptide processing. Only antisense
oligomers targeted against Asp2 significantly reduced Abeta
processing by HEK125.3 cells. Both A.beta. (1-40) and A.beta.
(1-42) were inhibited by the same degree. In Table 3, percent
inhibition is calculated with respect to untransfected cells.
Antisense oligomer reagents giving greater than 50% inhibition are
marked with an asterisk. For ASP2, 4 of 4 antisense oligomers gave
greater than 50% inhibition with an average inhibition of 62% for
A.beta. 1-40 processing and 60% for A.beta. 1-42 processing.
4TABLE 3 Inhibition of A.beta. peptide release from HEK125.3 cells
treated with antisense oligomers. Gene Antisense Abeta Abeta
Targeted Oligomer (1-40) (1-42) Asp2-1 S648 71%* 67%* Asp2-2 S649
83%* 76%* Asp2-3 S650 46%* 50%* Asp2-4 S651 47%* 46%* Con1-1 S652
13% 18% Con1-2 S653 35% 30% Con1-3 S655 9% 18% Con1-4 S674 29% 18%
Con2-1 S656 12% 18% Con2-2 S657 16% 19% Con2-3 S658 8% 35% Con2-4
S659 3% 18%
[0235] Since HEK293 cells derive from kidney, the experiment was
extended to human IMR-32 neuroblastoma cells which express all
three APP isoforms and which release A.beta. peptides into
conditioned medium at measurable levels. [See Neill et al., J.
NeuroSci. Res., (1994) 39: 482-93; and Asami-Odaka et al.,
Biochem., (1995) 34:10272-8.] Essentially identical results were
obtained in the neuroblastoma cells as the HEK293 cells. As shown
in Table 3B, the pair of Asp2 antisense oligomers reduced Asp2 mRNA
by roughly one-half, while the pair of reverse control oligomers
lacked this effect (Table 3B).
5TABLE 3B Reduction of A.beta.40 and A.beta.42 in human
neuroblastoma IMR-32 cells and mouse neuroblastoma Neuro-2A cells
treated with Asp2 antisense and control oligomers as indicated.
Oligomers were transfected in quadruplicate cultures. Values
tabulated are normalized against cultures treated with
oligofectin-G .TM. only (mean + SD, **p < 0.001 compared to
reverse control oligomer). Asp2 IMR-32 cells Neuro-2A cells mRNA
A.beta.40 A.beta.42 A.beta.40 A.beta.42 Asp2-1A -75% -49 + 2%** -42
+ 14%** -70 + 7%** -67 + 2%** Asp2-1R 0.16 -0 + 3% 21.26 -9 + 15%
1.05 Asp2-2A -39% -43 + 3%** -44 + 18%** -61 + 12%** -61 + 12%**
Asp2-2R 0.47 12.2 19.22 6.15 -8 + 10%
[0236] Together with the reduction in Asp2 mRNA there was a
concomitant reduction in the release of A.beta.40 and A.beta.42
peptides into the conditioned medium. Thus, Asp2 functions directly
or indirectly in a human kidney and a human neuroblastoma cell line
to facilitate the processing of APP into A.beta. peptides.
Molecular cloning of the mouse Asp2 cDNA revealed a high degree of
homology to human (>96% amino acid identity, see Example 3), and
indeed, complete nucleotide identity at the sites targeted by the
Asp2-1A and Asp2-2A antisense oligomers. Similar results were
obtained in mouse Neuro-2a cells engineered to express APP-Sw-KK.
The Asp2 antisense oligomers reduced release of A.beta. peptides
into the medium while the reverse control oligomers did not (Table
3B). Thus, the three antisense experiments with HEK293, IMR-32 and
Neuro-2a cells indicate that Asp2 acts directly or indirectly to
facilitate A.beta. processing in both somatic and neural cell
lines.
EXAMPLE 8
[0237] Demonstration of Hu-Asp2 .beta.-Secretase Activity in
Cultured Cells
[0238] Several mutations in APP associated with early onset
Alzheimer's disease have been shown to alter A.beta. peptide
processing. These flank the--and C-terminal cleavage sites that
release A.beta. from APP. These cleavage sites are referred to as
the .beta.-secretase and .gamma.-secretase cleavage sites,
respectively. Cleavage of APP at the .beta.-secretase site creates
a C-terminal fragment of APP containing 99 amino acids of 11,145
daltons molecular weight. The Swedish KM.fwdarw.NL mutation
immediately upstream of the .beta.-secretase cleavage site causes a
general increase in production of both the 1-40 and 1-42 amino acid
forms of A.beta. peptide. The London VF mutation (V717.fwdarw.F in
the APP770 isoform) has little effect on total A.beta. peptide
production, but appears to preferentially increase the percentage
of the longer 1-42 amino acid form of A.beta. peptide by affecting
the choice of .beta.-secretase cleavage site used during APP
processing. Thus, we sought to determine if these mutations altered
the amount and type of A.beta. peptide produced by cultured cells
cotransfected with a construct directing expression of Hu-Asp2.
[0239] Two experiments were performed which demonstrate Hu-Asp2
.beta.-secretase activity in cultured cells. In the first
experiment, treatment of HEK125.3 cells with antisense oligomers
directed against Hu-Asp2 transcripts as described in Example 7 was
found to decrease the amount of the C-terminal fragment of APP
created by .beta.-secretase cleavage (CTF99) (FIG. 9). This shows
that Hu-Asp2 acts directly or indirectly to facilitate
.beta.-secretase cleavage. In the second experiment, increased
expression of Hu-Asp2 in transfected mouse Neuro2A cells is shown
to increase accumulation of the CTF99 .beta.-secretase cleavage
fragment (FIG. 10). This increase is seen most easily when a mutant
APP-KK clone containing a C-terminal di-lysine motif is used for
transfection. A further increase is seen when Hu-Asp2 is
cotransfected with APP-Sw-KK containing the Swedish mutation
KM.fwdarw.NL. The Swedish mutation is known to increase cleavage of
APP by the .beta.-secretase.
[0240] A second set of experiments demonstrate Hu-Asp2 facilitates
.gamma.-secretase activity in cotransfection experiments with human
embryonic kidney HEK293 cells. Cotransfection of Hu-Asp2 with an
APP-KK clone greatly increases production and release of soluble
A.beta.1-40 and A.beta.1-42 peptides from HEK293 cells. There is a
proportionately greater increase in the release of A.beta.1-42. A
further increase in production of A.beta.1-42 is seen when Hu-Asp2
is cotransfected with APP-VF (SEQ ID No. 13 [nucleotide] and SEQ ID
No. 14 [amino acid]) or APP-VF-KK SEQ ID No. 19 [nucleotide] and
SEQ ID No. 20 [amino acid]) clones containing the London mutation
V717.fwdarw.F. The V717.fwdarw.F mutation is known to alter
cleavage specificity of the APP .gamma.-secretase such that the
preference for cleavage at the A.beta.42 site is increased. Thus,
Asp2 acts directly or indirectly to facilitate .gamma.-secretase
processing of APP at the .beta.42 cleavage site.
[0241] Materials
[0242] Antibodies 6E10 and 4G8 were purchased from Senetek (St.
Louis, Mo.). Antibody 369 was obtained from the laboratory of Paul
Greengard at the Rockefeller University. Antibody C8 was obtained
from the laboratory of Dennis Selkoe at the Harvard Medical School
and Brigham and Women's Hospital.
[0243] APP Constructs Used
[0244] The APP constructs used for transfection experiments
comprised the following
[0245] APP: wild-type APP695 (SEQ ID No. 9 and No. 10)
[0246] APP-Sw: APP695 containing the Swedish KM.fwdarw.NL mutation
(SEQ ID No. 11 and No. 12, wherein the lysine (K) at residue 595 of
APP695 is changed to asparagine (N) and the methionine (M) at
residue 596 of APP695 is changed to leucine (L).),
[0247] APP-VF: APP695 containing the London V.fwdarw.F mutation
(SEQ ID Nos. 13 & 14) (Affected residue 717 of the APP770
isoform corresponds with residue 642 of the APP695 isoform. Thus,
APP-VF as set in SEQ ID NO: 14 comprises the APP695 sequence,
wherein the valine (V) at residue 642 is changed to phenylalanine
(F).)
[0248] APP-KK: APP695 containing a C-terminal KK motif (SEQ ID Nos.
15 & 16),
[0249] APP-Sw-KK: APP695-Sw containing a C-terminal KK motif (SEQ
ID No. 17 & 18),
[0250] APP-VF-KK: APP695-VF containing a C-terminal KK motif (SEQ D
Nos. 19& 20).
[0251] These were inserted into the vector pIRES-EGFP (Clontech,
Palo Alto Calif.) between the Not1 and BstX1 sites using
appropriate linker sequences introduced by PCR.
[0252] Transfection of Antisense Oligomers Or Plasmid DNA
Constructs in HEK293 Cells, HEK125.3 Cells and Neuro-2A Cells,
[0253] Human embryonic kidney HEK293 cells and mouse Neuro-2a cells
were transfected with expression constructs using the Lipofectamine
Plus reagent from Gibco/BRL. Cells were seeded in 24 well tissue
culture plates to a density of 70-80% confluence. Four wells per
plate were transfected with 2 .mu.g DNA (3:1, APP:cotransfectant),
8 .mu.l Plus reagent, and 4 .mu.l Lipofectamine in OptiMEM. OptiMEM
was added to a total volume of 1 ml, distributed 200 .mu.l per well
and incubated 3 hours. Care was taken to hold constant the ratios
of the two plasmids used for cotransfection as well as the total
amount of DNA used in the transfection. The transfection media was
replaced with DMEM, 10% FBS, NaPyruvate, with
antibiotic/antimycotic and the cells were incubated under normal
conditions (37.degree. C., 5% CO.sub.2) for 48 hours. The
conditioned media were removed to polypropylene tubes and stored at
-80.degree. C. until assayed for the content of A.beta.1-40 and
A.beta.1-42 by EIA as described in the preceding examples.
Transfection of antisense oligomers into HEK125.3 cells was as
described in Example 7.
[0254] Preparation of Cell Extracts, Western Blot Protocol
[0255] Cells were harvested after being transfected with plasmid
DNA for about 60 hours. First, cells were transferred to 15-ml
conical tube from the plate and centrifuged at 1,500 rpm for 5
minutes to remove the medium. The cell pellets were washed once
with PBS. We then lysed the cells with lysis buffer (10 mM HEPES,
pH 7.9, 150 mM NaCl, 10% glycerol, 1 mM EGTA, 1 mM EDTA, 0.1 mM
sodium vanadate and 1% NP-40). The lysed cell mixtures were
centrifuged at 5000 rpm and the supernatant was stored at
-20.degree. C. as the cell extracts. Equal amounts of extracts from
HEK125.3 cells transfected with the Asp2 antisense oligomers and
controls were precipitated with antibody 369 that recognizes the
C-terminus of APP and then CTF99 was detected in the
immunoprecipitate with antibody 6E10. The experiment was repeated
using C8, a second precipitating antibody that also recognizes the
C-terminus of APP. For Western blot of extracts from mouse Neuro-2a
cells cotransfected with Hu-Asp2 and APP-KK, APP-Sw-KK, APP-VF-KK
or APP-VF, equal amounts of cell extracts were electrophoresed
through 4-10% or 10-20% Tricine gradient gels (NOVEX, San Diego,
Calif.). Full length APP and the CTF99 .beta.-secretase product
were detected with antibody 6E10.
[0256] Results
[0257] Transfection of HEK125.3 cells with Asp2-1 or Asp2-2
antisense oligomers reduces production of the CTF .beta.-secretase
product in comparison to cells similarly transfected with control
oligomers having the reverse sequence (Asp2-1 reverse & Asp2-2
reverse), see FIG. 9. Correspondingly, cotransfection of Hu-Asp2
into mouse Neuro-2a cells with the APP-KK construct increased the
formation of CTF99. (See FIG. 10.) This was further increased if
Hu-Asp2 was coexpressed with APP-Sw-KK, a mutant form of APP
containing the Swedish KM.fwdarw.NL mutation that increases
.beta.-secretase processing.
[0258] Effects of Asp2 on the production of Ab peptides from
endogenously expressed APP isoforms were assessed in HEK293 cells
transfected with a construct expressing Asp2 or with the empty
vector after selection of transformants with the antibiotic G418.
A.beta.40 production was increased in cells transformed with the
Asp2 construct in comparison to those transformed with the empty
vector DNA. A.beta.40 levels in conditioned medium collected from
the Asp2 transformed and control cultures was 424.+-.45 pg/ml and
113.+-.58 pg/ml, respectively (p<0.001). A.beta.42 release was
below the limit of detection by the EIA, while the release of
sAPP.alpha. was unaffected, 112.+-.8 ng/ml versus 111.+-.40 ng/ml.
This further indicates that Asp2 acts directly or indirectly to
facilitate the processing and release of A.beta. from endogenously
expressed APP.
[0259] Co-transfection of Hu-Asp2 with APP has little effect on
A.beta.40 production but increases A.beta.42 production above
background (Table 4). Addition of the di-lysine motif to the
C-terminus of APP increases A.beta. peptide processing about two
fold, although A.beta.40 and A.beta.42 production remain quite low
(352 pg/ml and 21 pg/ml, respectively). Cotransfection of Asp2 with
APP-KK further increases both A.beta.40 and A.beta.42
production.
[0260] The APP V717.fwdarw.F mutation has been shown to increase
.gamma.-secretase processing at the A.beta.42 cleavage site.
Cotransfection of Hu-Asp2 with the APP-VF or APP-VF-KK constructs
increased A.beta.42 production (a two fold increase with APP-VF and
a four-fold increase with APP-VF-KK, Table 4), but had mixed
effects on A.beta.40 production (a slight decrease with APP-VF, and
a two fold increase with APP-VF-KK in comparison to the pcDNA
cotransfection control. Thus, the effect of Asp2 on A.beta.42
production was proportionately greater leading to an increase in
the ratio of A.beta.42/total Ab. Indeed, the ratio of
A.beta.42/total A.beta. reaches a very high value of 42% in HEK293
cells cotransfected with Hu-Asp2 and APP-VF-KK.
6TABLE 4 Results of cotransfecting Hu-Asp2 or pcDNA plasmid DNA
with various APP constructs containing the V717.fwdarw.F mutation
that modifies .gamma.- secretase processing. Cotransfection with
Asp2 consistently increases the ratio of A.beta.42/total A.beta..
Values tabulated are A.beta. peptide pg/ml. pcDNA Asp2
Cotransfection Cotransfection A.beta.40 A.beta.42 A.beta.42/Total
A.beta.40 A.beta.42 A.beta.42/Total APP 192 .+-. 18 <4 <2%
188 .+-. 40 8 .+-. 10 3.9% APP-VF 118 .+-. 15 15 .+-. 19 11.5% 85
.+-. 7 24 .+-. 12 22.4% APP-KK 352 .+-. 24 21 .+-. 6 5.5% 1062 .+-.
101 226 .+-. 49 17.5% APP-VF-KK 230 .+-. 31 88 .+-. 24 27.7% 491
.+-. 35 355 .+-. 36 42%
EXAMPLE 9
Bacterial Expression of Human Asp2(a)
[0261] Expression of Recombinant Hu-Asp2(a) in E. coli.
[0262] Hu-Asp2(a) can be expressed in E. coli after addition of
N-terminal sequences such as a T7 tag (SEQ ID No. 21 and No. 22) or
a T7 tag followed by a caspase 8 leader sequence (SEQ ID No. 23 and
No. 24). Alternatively, reduction of the GC content of the 5'
sequence by site directed mutagenesis can be used to increase the
yield of Hu-Asp2 (SEQ ID No. 25 and No. 26). In addition, Asp2(a)
can be engineered with a proteolytic cleavage site (SEQ ID No. 27
and No. 28). To produce a soluble protein after expression and
refolding, deletion of the transmembrane domain and cytoplasmic
tail, or deletion of the membrane proximal region, transmembrane
domain, and cytoplasmic tail is preferred. Any materials (vectors,
host cells, etc.) and methods described herein to express
Hu-Asp2(a) should in principle be equally effective for expression
of Hu-Asp2(b).
[0263] Methods
[0264] PCR with primers containing appropriate linker sequences was
used to assemble fusions of Asp2(a) coding sequence with N-terminal
sequence modifications including a T7 tag (SEQ ID Nos. 21 and 22)
or a T7-caspase 8 leader (SEQ ID Nos. 23 and 24). These constructs
were cloned into the expression vector pet23a(+) [Novagen] in which
a T7 promoter directs expression of a T7 tag preceding a sequence
of multiple cloning sites. To clone Hu-Asp2 sequences behind the T7
leader of pet23a+, the following oligonucleotides were used for
amplification of the selected Hu-Asp2(a) sequence:
#553=GTGGATCCACCCAGCACGGCATCCGGCTG (SEQ ID No. 35),
#554=GAAAGCTTTCATGACTCATCTGTCTGTGGAATGTTG (SEQ ID No. 36) which
placed BamHI and HindIII sites flanking the 5' and 3' ends of the
insert, respectively. The Asp2(a) sequence was amplified from the
full length Asp2(a) cDNA cloned into pcDNA3.1 using the
Advantage-GC cDNA PCR [Clontech] following the manufacturer's
supplied protocol using annealing & extension at 68.degree. C.
in a two-step PCR cycle for 25 cycles. The insert and vector were
cut with BamHI and HindIII, purified by electrophoresis through an
agarose gel, then ligated using the Rapid DNA Ligation kit
[Boerhinger Mannheim]. The ligation reaction was used to transform
the E. coli strain JM109 (Promega) and colonies were picked for the
purification of plasmid (Qiagen,Qiaprep minispin) and DNA sequence
analysis. For inducible expression using induction with isopropyl
b-D-thiogalactopyranoside (IPTG), the expression vector was
transferred into E. coli strain BL21 (Statagene). Bacterial
cultures were grown in LB broth in the presence of ampicillin at
100 ug/ml, and induced in log phase growth at an OD600 of 0.6-1.0
with 1 mM IPTG for 4 hour at 37.degree. C. The cell pellet was
harvested by centrifugation.
[0265] To clone Hu-Asp2 sequences behind the T7 tag and caspase
leader (SEQ ID Nos. 23 and 24), the construct created above
containing the T7-Hu-Asp2 sequence (SEQ ID Nos. 21 and 22) was
opened at the BamH1 site, and then the phosphorylated caspase 8
leader oligonucleotides
#559=GATCGATGACTATCTCTGACTCTCCGCGTGAACAGGACG (SEQ ID No. 37),
#560=GATCCGTCCTGTTCACGCGGAGAGTCAGAGATAGTCATC (SEQ ID No. 38) were
annealed and ligated to the vector DNA. The 5' overhang for each
set of oligonucleotides was designed such that it allowed ligation
into the BamHI site but not subsequent digestion with BamHI. The
ligation reaction was transformed into JM109 as above for analysis
of protein expression after transfer to E. coli strain BL21.
[0266] In order to reduce the GC content of the 5' terminus of
asp2(a), a pair of antiparallel oligos were designed to change
degenerate codon bases in 15 amino acid positions from G/C to A/T
(SEQ ID Nos. 25 and 26). The new nucleotide sequence at the 5' end
of asp2 did not change the encoded amino acid and was chosen to
optimize E. Coli expression. The sequence of the sense linker is
5.dbd. CGGCATCCGGCTGCCCCTGCGTAGCGGTCTGGGT- GGTGCTCCACTGGGTCT
GCGTCTGCCCCGGGAGACCGACGAA G 3' (SEQ ID No. 39). The sequence of the
antisense linker is: 5' CTTCGTCGGTCTCCCGGGGCAGACGCAGACCCA-
GTGGAGCACCACCCAGA CCGCTACGCAGGGGCAGCCGGATGCCG 3' (SEQ ID No. 40).
After annealing the phosphorylated linkers together in 0.1 M
NaCl-10 mM Tris, pH 7.4 they were ligated into unique Cla I and Sma
I sites in Hu-Asp2 in the vector pTAC. For inducible expression
using induction with isopropyl b-D-thiogalactopyranoside (IPTG),
bacterial cultures were grown in LB broth in the presence of
ampicillin at 100 ug/ml, and induced in log phase growth at an
OD600 of 0.6-1.0 with 1 mM IPTG for 4 hour at 37.degree. C. The
cell pellet was harvested by centrifugation.
[0267] To create a vector in which the leader sequences can be
removed by limited proteolysis with caspase 8 such that this
liberates a Hu-Asp2 polypeptide beginning with the N-terminal
sequence GSFV (SEQ ID Nos. 27 and 28), the following procedure was
followed. Two phosphorylated oligonucleotides containing the
caspase 8 cleavage site IETD, #571=5'
GATCGATGACTATCTCTGACTCTCCGCTGGACTCTGGTATCGAAACCGACG (SEQ ID No. 41)
and #572=GATCCGTCGGTTTCGATACCAGAGTCCAGCGGAGAGTCAGAGATAGTCAT C (SEQ
ID No. 42) were annealed and ligated into pET23a+ that had been
opened with BamHI. After transformation into JM109, the purified
vector DNA was recovered and orientation of the insert was
confirmed by DNA sequence analysis.
[0268] The following oligonucleotides were used for amplification
of the selected Hu-Asp2(a) sequence: #573=5'
AAGGATCCTTTGTGGAGATGGTGGACAACCTG, (SEQ ID No. 43)
#554=GAAAGCTTTCATGACTCATCTGTCTGTGGAATGTTG (SEQ ID No. 44) which
placed BamHI and HindIII sites flanking the 5' and 3' ends of the
insert, respectively. The Hu-Asp2(a) sequence was amplified from
the full length Hu-Asp2(a) cDNA cloned into pcDNA3.1 using the
Advantage-GC cDNA PCR [Clontech] following the manufacturer's
supplied protocol using annealing & extension at 68.degree. C.
in a two-step PCR cycle for 25 cycles. The insert and vector were
cut with BamHI and HindIII, purified by electrophoresis through an
agarose gel, then ligated using the Rapid DNA Ligation kit
[Boerhinger Mannheim]. The ligation reaction was used to transform
the E. coli strain JM109 [Promega] and colonies were picked for the
purification of plasmid (Qiagen,Qiaprep minispin) and DNA sequence
analysis. For inducible expression using induction with isopropyl
b-D-thiogalactopyranoside (IPTG), the expression vector was
transferred into E. coli strain BL21 (Statagene). Bacterial
cultures were grown in LB broth in the presence of ampicillin at
100 ug/ml, and induced in log phase growth at an OD600 of 0.6-1.0
with 1 mM IPTG for 4 hour at 37.degree. C. The cell pellet was
harvested by centrifugation.
[0269] To assist purification, a 6-His tag can be introduced into
any of the above constructs following the T7 leader by opening the
construct at the BamHI site and then ligating in the annealed,
phosphorylated oligonucleotides containing the six histidine
sequence #565=GATCGCATCATCACCATCACCATG (SEQ ID No. 45),
#566=GATCCATGGTGATGGTGATGA- TGC (SEQ ID No. 46). The 5' overhang
for each set of oligonucleotides was designed such that it allowed
ligation into the BamHI site but not subsequent digestion with
BamHI.
[0270] Preparation of Bacterial Pellet:
[0271] 36.34 g of bacterial pellet representing 10.8 L of growth
was dispersed into a total volume of 200 ml using a 20 mm tissue
homogenizer probe at 3000 to 5000 rpm in 2M KCl, 0.1M Tris, 0.05M
EDTA, 1 mM DTT. The conductivity adjusted to about 193 mMhos with
water. After the pellet was dispersed, an additional amount of the
KCl solution was added, bringing the total volume to 500 ml. This
suspension was homogenized further for about 3 minutes at 5000 rpm
using the same probe. The mixture was then passed through a Rannie
high-pressure homogenizer at 10,000 psi.
[0272] In all cases, the pellet material was carried forward, while
the soluble fraction was discarded. The resultant solution was
centrifuged in a GSA rotor for 1 hour at 12,500 rpm. The pellet was
resuspended in the same solution (without the DTT) using the same
tissue homogenizer probe at 2,000 rpm. After homogenizing for 5
minutes at 3000 rpm, the volume was adjusted to 500 ml with the
same solution, and spun for 1 hour at 12,500 rpm. The pellet was
then resuspended as before, but this time the final volume was
adjusted to 1.5 L with the same solution prior to homogenizing for
5 minutes. After centrifuging at the same speed for 30 minutes,
this procedure was repeated. The pellet was then resuspended into
about 150 ml of cold water, pooling the pellets from the six
centrifuge tubes used in the GSA rotor. The pellet has homogenized
for 5 minutes at 3,000 rpm, volume adjusted to 250 ml with cold
water, then spun for 30 minutes. Weight of the resultant pellet was
17.75 g.
[0273] Summary: Lysis of bacterial pellet in KCl solution, followed
by centrifugation in a GSA rotor was used to initially prepare the
pellet. The same solution was then used an additional three times
for resuspension/homogenization. A final water wash/homogenization
was then performed to remove excess KCl and EDTA.
[0274] Solublization of Recombinant Hu-Asp2(a):
[0275] A ratio of 9-10 ml/gram of pellet was utilized for
solubilizing the rHuAsp2L from the pellet previously described.
17.75 g of pellet was thawed, and 150 ml of 8M guanidine HCl, 5 mM
.beta.ME, 0.1% DEA, was added. 3M Tris was used to titrate the pH
to 8.6. The pellet was initially resuspended into the guanidine
solution using a 20 mm tissue homogenizer probe at 1000 rpm. The
mixture was then stirred at 4.degree. C. for 1 hour prior to
centrifugation at 12,500 rpm for 1 hour in GSA rotor. The resultant
supernatant was then centrifuged for 30 minutes at 40,000.times.g
in an SS-34 rotor. The final supernatant was then stored at
-20.degree. C., except for 50 ml.
[0276] Immobilized Nickel Affinity Chromatography of Solubilized
Recombinant Hu-Asp2(a):
[0277] The following solutions were utilized:
[0278] A) 6M Guanidine HCl, 00.1M NaP, pH 8.0, 0.01M Tris, 5 mM
.beta.ME, 0.5 mM Imidazole
[0279] A') 6M Urea, 20 mM NaP, pH 6.80, 50 mM NaCl
[0280] B') 6M Urea, 20 mM NaP, pH 6.20, 50 mM NaCl, 12 mM
Imidazole
[0281] C') 6M Urea, 20 mM NaP, pH 6.80, 50 mM NaCl, 300 nM
Imidazole
[0282] Note: Buffers A' and C' were mixed at the appropriate ratios
to give intermediate concentrations of Imidazole.
[0283] The 50 ml of solubilized material was combined with 50 ml of
buffer A prior to adding to 100-125 ml Qiagen Ni-NTA SuperFlow
(re-equilibrated with buffer A) in a 5.times.10 cm Bio-Rad econo
column. This was shaken gently overnight at 4.degree. C. in the
cold room.
[0284] Chromatography Steps:
[0285] Drained the resultant flow through.
[0286] Washed with 50 ml buffer A (collecting into flow through
fraction)
[0287] Washed with 250 ml buffer A (wash 1)
[0288] Washed with 250 ml buffer A (wash 2)
[0289] Washed with 250 ml buffer A'
[0290] Washed with 250 ml buffer B'
[0291] Washed with 250 ml buffer A'
[0292] Eluted with 250 ml 75 mM Imidazole
[0293] Eluted with 250 ml 150 mM Imidazole (150-1)
[0294] Eluted with 250 ml 150 mM Imidazole (150-2)
[0295] Eluted with 250 ml 300 mM Imidazole (300-1)
[0296] Eluted with 250 ml 300 mM Imidazole (300-2)
[0297] Eluted with 250 ml 300 mM Imidazole (300-3)
[0298] Chromatography Results:
[0299] The Hu-Asp(a) eluted at 75 mM Imidazole through 300 mM
Imidazole. The 75 mM fraction, as well as the first 150 mM
Imidazole (150-1) fraction contained contaminating proteins as
visualized on Coomassie Blue stained gels. Therefore, fractions
150-2 and 300-1 will be utilized for refolding experiments since
they contained the greatest amount of protein as visualized on a
Coomassie Blue stained gel.
[0300] Refolding Experiments of Recombinant Hu-Asp2(a):
[0301] Experiment 1:
[0302] Forty ml of 150-2 was spiked with 1M DTT, 3M Tris, pH 7.4
and DEA to a final concentration of 6 mM, 50 mM, and 0.1%
respectively. This was diluted suddenly (while stirring) with 200
ml of (4.degree. C.) cold 20 mM NaP, pH 6.8, 150 mM NaCl. This
dilution gave a final Urea concentration of 1M. This solution
remained clear, even if allowed to set open to the air at room
temperature (RT) or at 4.degree. C.
[0303] After setting open to the air for 4-5 hours at 4.degree. C.,
this solution was then dialyzed overnight against 20 mM NaP, pH
7.4, 150 mM NaCl, 20% glycerol. This method effectively removes the
urea in the solution without precipitation of the protein.
[0304] Experiment 2:
[0305] Some of the 150-2 eluate was concentrated 2.times. on an
Amicon Centriprep, 10,000 MWCO, then treated as in Experiment 1.
This material also stayed in solution, with no visible
precipitation.
[0306] Experiment 3:
[0307] 89 ml of the 150-2 eluate was spiked with 1M DTT, 3M Tris,
pH 7.4 and DEA to a final concentration of 6 mM, 50 mM, and 0.1%
respectively. This was diluted suddenly (while stirring) with 445
ml of (4.degree. C.) cold 20 mM NaP, pH 6.8, 150 mM NaCl. This
solution appeared clear, with no apparent precipitation. The
solution was removed to RT and stirred for 10 minutes prior to
adding MEA to a final concentration of 0.1 mM. This was stirred
slowly at RT for 1 hour. Cystamine and CuSO.sub.4 were then added
to final concentrations of 1 mM and 10 .mu.M respectively. The
solution was stirred slowly at RT for 10 minutes prior to being
moved to the 4.degree. C. cold room and shaken slowly overnight,
open to the air.
[0308] The following day, the solution (still clear, with no
apparent precipitation) was centrifuged at 100,000.times.g for 1
hour. Supernatants from multiple runs were pooled, and the bulk of
the stabilized protein was dialyzed against 20 mM NaP, pH 7.4, 150
mM NaCl, 20% glycerol. After dialysis, the material was stored at
-20.degree. C.-
[0309] Some (about 10 ml) of the protein solution (still in 1M
Urea) was saved back for biochemical analyses, and frozen at
-20.degree. C. for storage.
EXAMPLE 10
Expression of Hu-Asp2 and Derivatives in Insect Cells
[0310] Any materials (vectors, host cells, etc.) and methods that
are useful to express Hu-Asp2(a) should in principle be equally
effective for expression of Hu-Asp2(b).
[0311] Expression by Baculovirus Infection.
[0312] The coding sequence of Hu-Asp2(a) and Hu-ASp2(b) and several
derivatives were engineered for expression in insect cells using
the PCR. For the full-length sequence, a 5'-sense oligonucleotide
primer that modified the translation initiation site to fit the
Kozak consensus sequence was paired with a 3'-antisense primer that
contains the natural translation termination codon in the Hu-Asp2
sequence. PCR amplification of the pcDNA3.1(hygro)/Hu-Asp2(a)
template was used to prepare two derivatives of Hu-Asp2(a) or
Hu-Asp(b) that delete the C-terminal transmembrane domain (SEQ ID
Nos. 29-30 and 50-51, respectively) or delete the transmembrane
domain and introduce a hexa-histidine tag at the C-terminus (SEQ ID
Nos. 31-32 and 52-53) respectively, were also engineered using PCR.
The same 5'-sense oligonucleotide primer described above was paired
with either a 3'-antisense primer that (1) introduced a translation
termination codon after codon 453 (SEQ ID No.3) or (2) incorporated
a hexa-histidine tag followed by a translation termination codon in
the PCR using pcDNA3.1 (hygro)/Hu-Asp-2(a) as the template. In all
cases, the PCR reactions were performed amplified for 15 cycles
using PwoI DNA polymerase (Boehringer-Mannheim) as outlined by the
supplier. The reaction products were digested to completion with
BamHI and NotI and ligated to BamHI and NotI digested baculovirus
transfer vector pVL1393 (Invitrogen). A portion of the ligations
was used to transform competent E. coli DH5 cells followed by
antibiotic selection on LB-Amp. Plasmid DNA was prepared by
standard alkaline lysis and banding in CsCl to yield the
baculovirus transfer vectors pVL1393/Asp2(a),
pVL1393/Asp2(a).DELTA.TM and pVL1393/Asp2(a).DELTA.TM(His).sub.6.
Creation of recombinant baculoviruses and infection of sf9 insect
cells was performed using standard methods.
[0313] Expression by Transfection
[0314] Transient and stable expression of Hu-Asp2(a).DELTA.TM and
Hu-Asp2(a).DELTA.TM(His).sub.6 in High 5 insect cells was performed
using the insect expression vector pIZ/V5-His. The DNA inserts from
the expression plasmids vectors pVL1393/Asp2(a),
pVL1393/Asp2(a).DELTA.TM and pVL1393/Asp2(a).DELTA.TM(His).sub.6
were excised by double digestion with BamHI and NotI and subcloned
into BamHI and NotI digested pIZ/V5-His using standard methods. The
resulting expression plasmids, referred to as pIZ/Hu-Asp2.DELTA.TM
and pIZ/Hu-Asp2.DELTA.TM(His).sub.6, were prepared as described
above.
[0315] For transfection, High 5 insect cells were cultured in High
Five serum free medium supplemented with 10 .mu.g/ml gentamycin at
27.degree. C. in sealed flasks. Transfections were performed using
High five cells, High five serum free media supplemented with 10
.mu.g/ml gentamycin, and InsectinPlus liposomes (Invitrogen,
Carlsbad, Calif.) using standard methods.
[0316] For large scale transient transfections, 1.2.times.10.sup.7
high five cells were plated in a 150 mm tissue culture dish and
allowed to attach at room temperature for 15-30 minutes. During the
attachment time the DNA/liposome mixture was prepared by mixing 6
ml of serum free media, 60 .mu.g Hu-Asp2(a).DELTA.TM/pIZ (.+-.His)
DNA and 120 .mu.l of Insectin Plus and incubating at room
temperature for 15 minutes. The plating media was removed from the
dish of cells and replaced with the DNA/liposome mixture for 4
hours at room temperature with constant rocking at 2 rpm. An
additional 6 ml of media was added to the dish prior to incubation
for 4 days at 27.degree. C. in a humid incubator. Four days post
transfection the media was harvested, clarified by centrifugation
at 500.times.g, assayed for Hu-Asp2(a) expression by Western
blotting. For stable expression, the cells were treated with 50
.mu.g/ml Zeocin and the surviving pool used to prepared clonal
cells by limiting dilution followed by analysis of the expression
level as noted above.
[0317] Purification of Hu-Asp2(a).DELTA.TM and
Hu-Asp2(a).DELTA.TM(His).su- b.6
[0318] Removal of the transmembrane segment from Hu-Asp2(a)
resulted in the secretion of the polypeptide into the culture
medium. Following protein production by either baculovirus
infection or transfection, the conditioned medium was harvested,
clarified by centrifugation, and dialyzed against Tris-HCl (pH
8.0). This material was then purified by successive chromatography
by anion exchange (Tris-HCl, pH 8.0) followed by cation exchange
chromatography (Acetate buffer at pH 4.5) using NaCl gradients. The
elution profile was monitored by (1) Western blot analysis and (2)
by activity assay using the peptide substrate described in Example
12. For the Hu-Asp2(a).DELTA.TM(His).sub.6, the conditioned medium
was dialyzed against Tris buffer (pH 8.0) and purified by
sequential chromatography on IMAC resin followed by anion exchange
chromatography.
[0319] Amino-terminal sequence analysis of the purified
Hu-Asp2(a).DELTA.TM(His).sub.6 protein revealed that the signal
peptide had been cleaved [TQHGIRLPLR, corresponding to SEQ ID NO:
32, residues 22-3].
EXAMPLE 11
Expression of Hu-Asp2(a) and Hu-Asp(b) in CHO Cells
[0320] The materials (vectors, host cells, etc.) and methods
described herein for expression of Hu-Asp2(a) are intended to be
equally applicable for expression of Hu-Asp2(b).
[0321] Heterologous Expression of Hu-Asp-2(a) in CHO-K1 Cells
[0322] The entire coding sequence of Hu-Asp2(a) was cloned into the
mammalian expression vector pcDNA3. 1 (+)Hygro (invitrogen,
Carlsbad, Calif.) which contains the CMV immediate early promoter
and bGH polyadenylation signal to drive over expression. The
expression plasmid, pcDNA3.1(+)Hygro/Hu-Asp2(a), was prepared by
alkaline lysis and banding in CsCl and completely sequenced on both
strands to verify the integrity of the coding sequence.
[0323] Wild-type Chinese hamster ovary cells (CHO-K1) were obtained
from the ATCC. The cells were maintained in monolayer cultures in
.alpha.-MEM containing 10% FCS at 37.degree. C. in 5% CO.sub.2. Two
100 mm dishes of CHO-K1 cells (60% confluent) were transfected with
pcDNA3.1(+)/Hygro alone (mock) or pcDNA3.1(+)Hygro/Hu-Asp2(a) or
pcDNA3.1(+)Hygro/Hu-Asp2(b- ) using the cationic liposome DOTAP as
recommended by the supplier (Roche, Indianapolis, Ind.). The cells
were treated with the plasmid DNA/liposome mixtures for 15 hours
and then the medium replaced with growth medium containing 500
Units/mil hygromycin B. In the case of pcDNA3.1(+)Hygro/Hu-Asp2(a)
or (b) transfected CHO-K1 cells, individual hygromycin B-resistant
cells were cloned by limiting dilution. Following clonal expansion
of the individual cell lines, expression of Hu-Asp2(a) or
Hu-Asp2(b) protein was assessed by Western blot analysis using a
polyclonal rabbit antiserum raised against recombinant Hu-Asp2
prepared by expression in E. coli. Near confluent dishes of each
cell line were harvested by scraping into PBS and the cells
recovered by centrifugation. The cell pellets were resuspended in
cold lysis buffer (25 mM Tris-HCl (pH 8.0)/5 mM EDTA) containing
protease inhibitors and the cells lysed by sonication. The soluble
and membrane fractions were separated by centrifugation
(105,000.times.g, 60 min) and normalized amounts of protein from
each fraction were then separated by SDS-PAGE. Following
electrotransfer of the separated polypeptides to PVDF membranes,
Hu-Asp-2(a) or Hu-Asp2(b) protein was detected using rabbit
anti-Hu-Asp2 antiserum ({fraction (1/1000)} dilution) and the
antibody-antigen complexes were visualized using alkaline
phosphatase conjugated goat anti-rabbit antibodies ({fraction
(1/2500)}). A specific immunoreactive protein with an apparent Mr
value of 65 kDa was detected in pcDNA3.1(+)Hygro/Hu-Asp2
transfected cells and not mock-transfected cells. Also, the Hu-Asp2
polypeptide was only detected in the membrane fraction, consistent
with the presence of a signal peptide and single transmembrane
domain in the predicted sequence. Based on this analysis, clone #5
had the highest expression level of Hu-Asp2(a) protein and this
production cell lines was scaled up to provide material for
purification.
[0324] Purification of Recombinant Hu-Asp-2(a) from CHO-K1/Hu-Asp2
Clone #5
[0325] In a typical purification, clone #5 cell pellets derived
from 20 150 mm dishes of confluent cells, were used as the starting
material. The cell pellets were resuspended in 50 ml cold lysis
buffer as described above. The cells were lysed by polytron
homogenization (2.times.20 sec) and the lysate centrifuged at
338,000.times.g for 20 minutes. The membrane pellet was then
resuspended in 20 ml of cold lysis buffer containing 50 mM
.beta.-octylglucoside followed by rocking at 4.degree. C. for 1
hour. The detergent extract was clarified by centrifugation at
338,000.times.g for 20 minutes and the supernatant taken for
further analysis.
[0326] The .beta.-octylglucoside extract was applied to a Mono Q
anion exchange column that was previously equilibrated with 25 mM
Tris-HCl (pH 8.0)/50 mM .beta.-octylglucoside. Following sample
application, the column was eluted with a linear gradient of
increasing NaCl concentration (0-1.0 M over 30 minutes) and
individual fractions assayed by Western blot analysis and for
.beta.-secretase activity (see below). Fractions containing both
Hu-Asp-2(a) immunoreactivity and .beta.-secretase activity were
pooled and dialyzed against 25 mM NaOAc (pH 4.5)/50 mM
.beta.-octylglucoside. Following dialysis, precipitated material
was removed by centrifugation and the soluble material
chromatographed on a MonoS cation exchange column that was
previously equilibrated in 25 mM NaOAc (pH 4.5)/50 mM
.beta.-octylglucoside. The column was eluted using a linear
gradient of increasing NaCl concentration (0-1.0 M over 30 minutes)
and individual fractions assayed by Western blot analysis and for
.beta.-secretase activity. Fractions containing both Hu-Asp2
immunoreactivity and .beta.-secretase activity were combined and
determined to be >95% pure by SDS-PAGE/Coomassie Blue
staining.
[0327] The same methods were used to express and purify
Hu-Asp2(b).
EXAMPLE 12
Assay of Hu-Asp2 .beta.-Secretase Activity Using Peptide
Substrates
[0328] .beta.-Secretase Assay
[0329] Recombinant human Asp2(a) prepared in CHO cells and purified
as described in Example 11 was used to assay Asp2(a) proteolytic
activity directly. Activity assays for Asp2(a) were performed using
synthetic peptide substrates containing either the wild-type APP
.beta.-secretase site (SEVKM.dwnarw.DAEFR; SEQ ID NO: 64), the
Swedish KM.fwdarw.NL mutation (SEVNL.dwnarw.DAEFR; SEQ ID NO: 63),
or the A.beta.40 and 42 .gamma.-secretase sites
(RRGGVV.dwnarw.IA.dwnarw.TVIVGER; SEQ ID NO: 65). Reactions were
performed in 50 mM 2-[N-morpholino]ethane-sulfonate ("Na-MES," pH
5.5) containing 1% .beta.-octylglucoside, 70 mM peptide substrate,
and recombinant Asp2(a) (1-5 .mu.g protein) for various times at
37.degree. C. The reaction products were quantified by RP-HPLC
using a linear gradient from 0-70 B over 30 minutes (A=0.1% TFA in
water, B=0.1%TFA/10%water/90%AcCN). The elution profile was
monitored by absorbance at 214 nm. In preliminary experiments, the
two product peaks which eluted before the intact peptide substrate,
were confirned to have the sequence DAEFR (SEQ ID. NO: 72)and SEVNL
(SEQ ID NO: 73) using both Edman sequencing and MADLI-TOF mass
spectrometry. Percent hydrolysis of the peptide substrate was
calculated by comparing the integrated peak areas for the two
product peptides and the starting material derived from the
absorbance at 214 nm. The sequence of cleavage/hydrolysis products
was confirmed using Edman sequencing and MADLI-TOF mass
spectrometry.
[0330] The behavior of purified Asp2(a) in the proteolysis assays
was consistent with the prior anti-sense studies which indicated
that Asp2(a) possesses .beta.-secretase activity. Maximal
proteolysis was seen with the Swedigh .beta.-secretase peptide,
which, after 6 hours, was about 10-fold higher than wild type
APP.
[0331] The specificity of the protease cleavage reaction was
determined by performing the .beta.-secretase assay in the presence
of 8 .mu.M pepstatin A and the presence of a cocktail of protease
inhibitors (10 .mu.M leupeptin, 10 .mu.M E64, and 5 mM EDTA).
Proteolytic activity was insensitive to both the pepstatin and the
cocktail, which are inhibitors of cathepsin D (and other aspartyl
proteases), serine proteases, cysteinyl proteases, and
metalloproteases, respectively.
[0332] Hu-Asp2(b) when similarly expressed in CHO cells and
purified using identical conditions for extraction with
.beta.-octylglucoside and sequential chromatography over Mono Q and
Mono S also cleaves the Swedish .beta.-secretase peptide in
proteolysis assays using identical assay conditions.
[0333] Collectively, this data establishes that both forms of Asp2
(Hu-Asp2(a) and Hu-Asp2(b)) act directly in cell-free assays to
cleave synthetic APP-peptides at the .beta.-secretase site, and
that the rate of cleavage is greatly increased by the Swedish
KM.fwdarw.NL mutation that is associated with Alzheimer's
disease.
[0334] An alternative .beta.-secretase assay utilizes internally
quenched fluorescent substrates to monitor enzyme activity using
fluorescence spectroscopy in a single sample or multiwell format.
Each reaction contained 50 mM Na-MES (pH 5.5), peptide substrate
MCA-EVKMDAEF[K-DNP] (SEQ ID NO: 71; BioSource International) (50
.mu.M) and purified Hu-Asp-2 enzyme. These components were
equilibrated to 37.degree. C. for various times and the reaction
initiated by addition of substrate. Excitation was performed at 330
nm and the reaction kinetics were monitored by measuring the
fluorescence emission at 390 nm. To detect compounds that modulate
Hu-Asp-2 activity, the test compounds were added during the
preincubation phase of the reaction and the kinetics of the
reaction monitored as described above. Activators are scored as
compounds that increase the rate of appearance of fluorescence
while inhibitors decrease the rate of appearance of
fluorescence.
[0335] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0336] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the invention. The entire
disclosure of all publications cited herein are hereby incorporated
by reference.
Sequence CWU 1
1
73 1 1804 DNA Homo sapiens 1 atgggcgcac tggcccgggc gctgctgctg
cctctgctgg cccagtggct cctgcgcgcc 60 gccccggagc tggcccccgc
gcccttcacg ctgcccctcc gggtggccgc ggccacgaac 120 cgcgtagttg
cgcccacccc gggacccggg acccctgccg agcgccacgc cgacggcttg 180
gcgctcgccc tggagcctgc cctggcgtcc cccgcgggcg ccgccaactt cttggccatg
240 gtagacaacc tgcaggggga ctctggccgc ggctactacc tggagatgct
gatcgggacc 300 cccccgcaga agctacagat tctcgttgac actggaagca
gtaactttgc cgtggcagga 360 accccgcact cctacataga cacgtacttt
gacacagaga ggtctagcac ataccgctcc 420 aagggctttg acgtcacagt
gaagtacaca caaggaagct ggacgggctt cgttggggaa 480 gacctcgtca
ccatccccaa aggcttcaat acttcttttc ttgtcaacat tgccactatt 540
tttgaatcag agaatttctt tttgcctggg attaaatgga atggaatact tggcctagct
600 tatgccacac ttgccaagcc atcaagttct ctggagacct tcttcgactc
cctggtgaca 660 caagcaaaca tccccaacgt tttctccatg cagatgtgtg
gagccggctt gcccgttgct 720 ggatctggga ccaacggagg tagtcttgtc
ttgggtggaa ttgaaccaag tttgtataaa 780 ggagacatct ggtatacccc
tattaaggaa gagtggtact accagataga aattctgaaa 840 ttggaaattg
gaggccaaag ccttaatctg gactgcagag agtataacgc agacaaggcc 900
atcgtggaca gtggcaccac gctgctgcgc ctgccccaga aggtgtttga tgcggtggtg
960 gaagctgtgg cccgcgcatc tctgattcca gaattctctg atggtttctg
gactgggtcc 1020 cagctggcgt gctggacgaa ttcggaaaca ccttggtctt
acttccctaa aatctccatc 1080 tacctgagag atgagaactc cagcaggtca
ttccgtatca caatcctgcc tcagctttac 1140 attcagccca tgatgggggc
cggcctgaat tatgaatgtt accgattcgg catttcccca 1200 tccacaaatg
cgctggtgat cggtgccacg gtgatggagg gcttctacgt catcttcgac 1260
agagcccaga agagggtggg cttcgcagcg agcccctgtg cagaaattgc aggtgctgca
1320 gtgtctgaaa tttccgggcc tttctcaaca gaggatgtag ccagcaactg
tgtccccgct 1380 cagtctttga gcgagcccat tttgtggatt gtgtcctatg
cgctcatgag cgtctgtgga 1440 gccatcctcc ttgtcttaat cgtcctgctg
ctgctgccgt tccggtgtca gcgtcgcccc 1500 cgtgaccctg aggtcgtcaa
tgatgagtcc tctctggtca gacatcgctg gaaatgaata 1560 gccaggcctg
acctcaagca accatgaact cagctattaa gaaaatcaca tttccagggc 1620
agcagccggg atcgatggtg gcgctttctc ctgtgcccac ccgtcttcaa tctctgttct
1680 gctcccagat gccttctaga ttcactgtct tttgattctt gattttcaag
ctttcaaatc 1740 ctccctactt ccaagaaaaa taattaaaaa aaaaacttca
ttctaaacca aaaaaaaaaa 1800 aaaa 1804 2 518 PRT Homo sapiens 2 Met
Gly Ala Leu Ala Arg Ala Leu Leu Leu Pro Leu Leu Ala Gln Trp 1 5 10
15 3 2070 DNA Homo sapiens 3 atggcccaag ccctgccctg gctcctgctg
tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccg
gctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120 ctgcggctgc
cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 180
gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc
240 gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag
taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat cgctactacc
agaggcagct gtccagcaca 360 taccgggacc tccggaaggg tgtgtatgtg
ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg acctggtaag
catcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca
ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540
gggctggcct atgctgagat tgccaggcct gacgactccc tggagccttt ctttgactct
600 ctggtaaagc agacccacgt tcccaacctc ttctccctgc acctttgtgg
tgctggcttc 660 cccctcaacc agtctgaagt gctggcctct gtcggaggga
gcatgatcat tggaggtatc 720 gaccactcgc tgtacacagg cagtctctgg
tatacaccca tccggcggga gtggtattat 780 gaggtcatca ttgtgcgggt
ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag 840 tacaactatg
acaagagcat tgtggacagt ggcaccacca accttcgttt gcccaagaaa 900
gtgtttgaag ctgcagtcaa atccatcaag gcagcctcct ccacggagaa gttccctgat
960 ggtttctggc taggagagca gctggtgtgc tggcaagcag gcaccacccc
ttggaacatt 1020 ttcccagtca tctcactcta cctaatgggt gaggttacca
accagtcctt ccgcatcacc 1080 atccttccgc agcaatacct gcggccagtg
gaagatgtgg ccacgtccca agacgactgt 1140 tacaagtttg ccatctcaca
gtcatccacg ggcactgtta tgggagctgt tatcatggag 1200 ggcttctacg
ttgtctttga tcgggcccga aaacgaattg gctttgctgt cagcgcttgc 1260
catgtgcacg atgagttcag gacggcagcg gtggaaggcc cttttgtcac cttggacatg
1320 gaagactgtg gctacaacat tccacagaca gatgagtcaa ccctcatgac
catagcctat 1380 gtcatggctg ccatctgcgc cctcttcatg ctgccactct
gcctcatggt gtgtcagtgg 1440 cgctgcctcc gctgcctgcg ccagcagcat
gatgactttg ctgatgacat ctccctgctg 1500 aagtgaggag gcccatgggc
agaagataga gattcccctg gaccacacct ccgtggttca 1560 ctttggtcac
aagtaggaga cacagatggc acctgtggcc agagcacctc aggaccctcc 1620
ccacccacca aatgcctctg ccttgatgga gaaggaaaag gctggcaagg tgggttccag
1680 ggactgtacc tgtaggaaac agaaaagaga agaaagaagc actctgctgg
cgggaatact 1740 cttggtcacc tcaaatttaa gtcgggaaat tctgctgctt
gaaacttcag ccctgaacct 1800 ttgtccacca ttcctttaaa ttctccaacc
caaagtattc ttcttttctt agtttcagaa 1860 gtactggcat cacacgcagg
ttaccttggc gtgtgtccct gtggtaccct ggcagagaag 1920 agaccaagct
tgtttccctg ctggccaaag tcagtaggag aggatgcaca gtttgctatt 1980
tgctttagag acagggactg tataaacaag cctaacattg gtgcaaagat tgcctcttga
2040 attaaaaaaa aaaaaaaaaa aaaaaaaaaa 2070 4 501 PRT Homo sapiens 4
Met Ala Gln Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5
10 15 Leu Pro Ala His Gly Thr Gln His Gly Ile Arg Leu Pro Leu Arg
Ser 20 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg
Glu Thr Asp 35 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser
Phe Val Glu Met Val 50 55 60 Asp Asn Leu Arg Gly Lys Ser Gly Gln
Gly Tyr Tyr Val Glu Met Thr 65 70 75 80 Val Gly Ser Pro Pro Gln Thr
Leu Asn Ile Leu Val Asp Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val
Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg
Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 115 120 125 Tyr
Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135
140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile
145 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly
Ser Asn Trp 165 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile
Ala Arg Pro Asp Asp 180 185 190 Ser Leu Glu Pro Phe Phe Asp Ser Leu
Val Lys Gln Thr His Val Pro 195 200 205 Asn Leu Phe Ser Leu His Leu
Cys Gly Ala Gly Phe Pro Leu Asn Gln 210 215 220 Ser Glu Val Leu Ala
Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile 225 230 235 240 Asp His
Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg 245 250 255
Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly Gln 260
265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile
Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val
Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr
Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp Leu Gly Glu Gln Leu
Val Cys Trp Gln Ala Gly Thr Thr 325 330 335 Pro Trp Asn Ile Phe Pro
Val Ile Ser Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr Asn Gln Ser
Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu Arg 355 360 365 Pro Val
Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr Lys Phe Ala 370 375 380
Ile Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu 385
390 395 400 Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly
Phe Ala 405 410 415 Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr
Ala Ala Val Glu 420 425 430 Gly Pro Phe Val Thr Leu Asp Met Glu Asp
Cys Gly Tyr Asn Ile Pro 435 440 445 Gln Thr Asp Glu Ser Thr Leu Met
Thr Ile Ala Tyr Val Met Ala Ala 450 455 460 Ile Cys Ala Leu Phe Met
Leu Pro Leu Cys Leu Met Val Cys Gln Trp 465 470 475 480 Arg Cys Leu
Arg Cys Leu Arg Gln Gln His Asp Asp Phe Ala Asp Asp 485 490 495 Ile
Ser Leu Leu Lys 500 5 1977 DNA Homo sapiens 5 atggcccaag ccctgccctg
gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc
acggcatccg gctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120
ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt
180 gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt
ggagatgacc 240 gtgggcagcc ccccgcagac gctcaacatc ctggtggata
caggcagcag taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat
cgctactacc agaggcagct gtccagcaca 360 taccgggacc tccggaaggg
tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg
acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480
gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg
540 gggctggcct atgctgagat tgccaggctt tgtggtgctg gcttccccct
caaccagtct 600 gaagtgctgg cctctgtcgg agggagcatg atcattggag
gtatcgacca ctcgctgtac 660 acaggcagtc tctggtatac acccatccgg
cgggagtggt attatgaggt gatcattgtg 720 cgggtggaga tcaatggaca
ggatctgaaa atggactgca aggagtacaa ctatgacaag 780 agcattgtgg
acagtggcac caccaacctt cgtttgccca agaaagtgtt tgaagctgca 840
gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc ctgatggttt ctggctagga
900 gagcagctgg tgtgctggca agcaggcacc accccttgga acattttccc
agtcatctca 960 ctctacctaa tgggtgaggt taccaaccag tccttccgca
tcaccatcct tccgcagcaa 1020 tacctgcggc cagtggaaga tgtggccacg
tcccaagacg actgttacaa gtttgccatc 1080 tcacagtcat ccacgggcac
tgttatggga gctgttatca tggagggctt ctacgttgtc 1140 tttgatcggg
cccgaaaacg aattggcttt gctgtcagcg cttgccatgt gcacgatgag 1200
ttcaggacgg cagcggtgga aggccctttt gtcaccttgg acatggaaga ctgtggctac
1260 aacattccac agacagatga gtcaaccctc atgaccatag cctatgtcat
ggctgccatc 1320 tgcgccctct tcatgctgcc actctgcctc atggtgtgtc
agtggcgctg cctccgctgc 1380 ctgcgccagc agcatgatga ctttgctgat
gacatctccc tgctgaagtg aggaggccca 1440 tgggcagaag atagagattc
ccctggacca cacctccgtg gttcactttg gtcacaagta 1500 ggagacacag
atggcacctg tggccagagc acctcaggac cctccccacc caccaaatgc 1560
ctctgccttg atggagaagg aaaaggctgg caaggtgggt tccagggact gtacctgtag
1620 gaaacagaaa agagaagaaa gaagcactct gctggcggga atactcttgg
tcacctcaaa 1680 tttaagtcgg gaaattctgc tgcttgaaac ttcagccctg
aacctttgtc caccattcct 1740 ttaaattctc caacccaaag tattcttctt
ttcttagttt cagaagtact ggcatcacac 1800 gcaggttacc ttggcgtgtg
tccctgtggt accctggcag agaagagacc aagcttgttt 1860 ccctgctggc
caaagtcagt aggagaggat gcacagtttg ctatttgctt tagagacagg 1920
gactgtataa acaagcctaa cattggtgca aagattgcct cttgaaaaaa aaaaaaa 1977
6 476 PRT Homo sapiens 6 Met Ala Gln Ala Leu Pro Trp Leu Leu Leu
Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr Gln His
Gly Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu Gly Gly Ala Pro
Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40 45 Glu Glu Pro Glu
Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 50 55 60 Asp Asn
Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr 65 70 75 80
Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 85
90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg
Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg
Lys Gly Val 115 120 125 Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly
Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile Pro His Gly Pro Asn
Val Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala Ile Thr Glu Ser
Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175 Glu Gly Ile Leu
Gly Leu Ala Tyr Ala Glu Ile Ala Arg Leu Cys Gly 180 185 190 Ala Gly
Phe Pro Leu Asn Gln Ser Glu Val Leu Ala Ser Val Gly Gly 195 200 205
Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu 210
215 220 Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile
Val 225 230 235 240 Arg Val Glu Ile Asn Gly Gln Asp Leu Lys Met Asp
Cys Lys Glu Tyr 245 250 255 Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly
Thr Thr Asn Leu Arg Leu 260 265 270 Pro Lys Lys Val Phe Glu Ala Ala
Val Lys Ser Ile Lys Ala Ala Ser 275 280 285 Ser Thr Glu Lys Phe Pro
Asp Gly Phe Trp Leu Gly Glu Gln Leu Val 290 295 300 Cys Trp Gln Ala
Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser 305 310 315 320 Leu
Tyr Leu Met Gly Glu Val Thr Asn Gln Ser Phe Arg Ile Thr Ile 325 330
335 Leu Pro Gln Gln Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser Gln
340 345 350 Asp Asp Cys Tyr Lys Phe Ala Ile Ser Gln Ser Ser Thr Gly
Thr Val 355 360 365 Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val
Phe Asp Arg Ala 370 375 380 Arg Lys Arg Ile Gly Phe Ala Val Ser Ala
Cys His Val His Asp Glu 385 390 395 400 Phe Arg Thr Ala Ala Val Glu
Gly Pro Phe Val Thr Leu Asp Met Glu 405 410 415 Asp Cys Gly Tyr Asn
Ile Pro Gln Thr Asp Glu Ser Thr Leu Met Thr 420 425 430 Ile Ala Tyr
Val Met Ala Ala Ile Cys Ala Leu Phe Met Leu Pro Leu 435 440 445 Cys
Leu Met Val Cys Gln Trp Arg Cys Leu Arg Cys Leu Arg Gln Gln 450 455
460 His Asp Asp Phe Ala Asp Asp Ile Ser Leu Leu Lys 465 470 475 7
2043 DNA Mus musculus 7 atggccccag cgctgcactg gctcctgcta tgggtgggct
cgggaatgct gcctgcccag 60 ggaacccatc tcggcatccg gctgcccctt
cgcagcggcc tggcagggcc acccctgggc 120 ctgaggctgc cccgggagac
tgacgaggaa tcggaggagc ctggccggag aggcagcttt 180 gtggagatgg
tggacaacct gaggggaaag tccggccagg gctactatgt ggagatgacc 240
gtaggcagcc ccccacagac gctcaacatc ctggtggaca cgggcagtag taactttgca
300 gtgggggctg ccccacaccc tttcctgcat cgctactacc agaggcagct
gtccagcaca 360 tatcgagacc tccgaaaggg tgtgtatgtg ccctacaccc
agggcaagtg ggagggggaa 420 ctgggcaccg acctggtgag catccctcat
ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcgga
caagttcttc atcaatggtt ccaactggga gggcatccta 540 gggctggcct
atgctgagat tgccaggccc gacgactctt tggagccctt ctttgactcc 600
ctggtgaagc agacccacat tcccaacatc ttttccctgc agctctgtgg cgctggcttc
660 cccctcaacc agaccgaggc actggcctcg gtgggaggga gcatgatcat
tggtggtatc 720 gaccactcgc tatacacggg cagtctctgg tacacaccca
tccggcggga gtggtattat 780 gaagtgatca ttgtacgtgt ggaaatcaat
ggtcaagatc tcaagatgga ctgcaaggag 840 tacaactacg acaagagcat
tgtggacagt gggaccacca accttcgctt gcccaagaaa 900 gtatttgaag
ctgccgtcaa gtccatcaag gcagcctcct cgacggagaa gttcccggat 960
ggcttttggc taggggagca gctggtgtgc tggcaagcag gcacgacccc ttggaacatt
1020 ttcccagtca tttcacttta cctcatgggt gaagtcacca atcagtcctt
ccgcatcacc 1080 atccttcctc agcaatacct acggccggtg gaggacgtgg
ccacgtccca agacgactgt 1140 tacaagttcg ctgtctcaca gtcatccacg
ggcactgtta tgggagccgt catcatggaa 1200 ggtttctatg tcgtcttcga
tcgagcccga aagcgaattg gctttgctgt cagcgcttgc 1260 catgtgcacg
atgagttcag gacggcggca gtggaaggtc cgtttgttac ggcagacatg 1320
gaagactgtg gctacaacat tccccagaca gatgagtcaa cacttatgac catagcctat
1380 gtcatggcgg ccatctgcgc cctcttcatg ttgccactct gcctcatggt
atgtcagtgg 1440 cgctgcctgc gttgcctgcg ccaccagcac gatgactttg
ctgatgacat ctccctgctc 1500 aagtaaggag gctcgtgggc agatgatgga
gacgcccctg gaccacatct gggtggttcc 1560 ctttggtcac atgagttgga
gctatggatg gtacctgtgg ccagagcacc tcaggaccct 1620 caccaacctg
ccaatgcttc tggcgtgaca gaacagagaa atcaggcaag ctggattaca 1680
gggcttgcac ctgtaggaca caggagaggg aaggaagcag cgttctggtg gcaggaatat
1740 ccttaggcac cacaaacttg agttggaaat tttgctgctt gaagcttcag
ccctgaccct 1800 ctgcccagca tcctttagag tctccaacct aaagtattct
ttatgtcctt ccagaagtac 1860 tggcgtcata ctcaggctac ccggcatgtg
tccctgtggt accctggcag agaaagggcc 1920 aatctcattc cctgctggcc
aaagtcagca gaagaaggtg aagtttgcca gttgctttag 1980 tgatagggac
tgcagactca agcctacact ggtacaaaga ctgcgtcttg agataaacaa 2040 gaa
2043 8 501 PRT Mus musculus 8 Met Ala Pro Ala Leu His Trp Leu Leu
Leu Trp Val Gly Ser Gly Met 1 5 10 15 Leu Pro Ala Gln Gly Thr His
Leu Gly Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu Ala Gly Pro
Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40 45 Glu Glu Ser
Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 50 55 60 Asp
Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr 65 70
75 80 Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly
Ser 85 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu
His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp
Leu Arg Lys
Gly Val 115 120 125 Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu
Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val
Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala Ile Thr Glu Ser Asp
Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175 Glu Gly Ile Leu Gly
Leu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp 180 185 190 Ser Leu Glu
Pro Phe Phe Asp Ser Leu Val Lys Gln Thr His Ile Pro 195 200 205 Asn
Ile Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn Gln 210 215
220 Thr Glu Ala Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile
225 230 235 240 Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro
Ile Arg Arg 245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val
Glu Ile Asn Gly Gln 260 265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr
Asn Tyr Asp Lys Ser Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu
Arg Leu Pro Lys Lys Val Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile
Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe
Trp Leu Gly Glu Gln Leu Val Cys Trp Gln Ala Gly Thr Thr 325 330 335
Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val 340
345 350 Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu
Arg 355 360 365 Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr
Lys Phe Ala 370 375 380 Val Ser Gln Ser Ser Thr Gly Thr Val Met Gly
Ala Val Ile Met Glu 385 390 395 400 Gly Phe Tyr Val Val Phe Asp Arg
Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 Val Ser Ala Cys His Val
His Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425 430 Gly Pro Phe Val
Thr Ala Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440 445 Gln Thr
Asp Glu Ser Thr Leu Met Thr Ile Ala Tyr Val Met Ala Ala 450 455 460
Ile Cys Ala Leu Phe Met Leu Pro Leu Cys Leu Met Val Cys Gln Trp 465
470 475 480 Arg Cys Leu Arg Cys Leu Arg His Gln His Asp Asp Phe Ala
Asp Asp 485 490 495 Ile Ser Leu Leu Lys 500 9 2088 DNA Homo sapiens
9 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta
60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt
ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt
cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg
cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga
agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc
agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360
gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg
420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac
atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct
gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct
gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga
tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca
aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720
gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa
780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac
caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag
ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag
aatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca
ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc
aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080
caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag
1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc
cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc
acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga
cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa
agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg
agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440
gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac
1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc
tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg
tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg
gctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc
ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga
caaatatcaa gacggaggag atctctgaag tgaagatgga tgcagaattc 1800
cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg
1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat
agcgacagtg 1920 atcgtcatca ccttggtgat gctgaagaag aaacagtaca
catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag
gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta
caagttcttt gagcagatgc agaactag 2088 10 695 PRT Homo sapiens 10 Met
Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10
15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro
20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn
Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys
Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln
Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu Ala
Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg
Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110 Ile Pro Tyr Arg
Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 Val Pro
Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140
Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145
150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys
Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro
Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu
Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp
Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu
Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp
Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu
Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265
270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg
275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys
Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe
Gln Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu
Arg Met Ser Gln Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg
Gln Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile
Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu 355 360 365 Gln Glu Ala
Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala 370 375 380 Arg
Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390
395 400 Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val
Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp
Arg Gln His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val
Asp Pro Lys Lys Ala 435 440 445 Ala Gln Ile Arg Ser Gln Val Met Thr
His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met Asn Gln Ser Leu Ser
Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile Gln
Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn 485 490 495 Tyr Ser
Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510
Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515
520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu
Gln 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn
Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala
Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr
Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala
Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His His Gln Lys
Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala
Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635
640 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile
645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu
Glu Arg 660 665 670 His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn
Pro Thr Tyr Lys 675 680 685 Phe Phe Glu Gln Met Gln Asn 690 695 11
2088 DNA Homo sapiens 11 atgctgcccg gtttggcact gctcctgctg
gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg
cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc
acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180
acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg
240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg
gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc
cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct
gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca
tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca
acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540
ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat
600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta
tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag
tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat
ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga
gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg
aagaggtggt tcgagttcct acaacagcag ccagtacccc tgatgccgtt 900
gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa
960 gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga
atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga
aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa
gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga
agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg
ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260
aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg
1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac
acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct
acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg
cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag
tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg
aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620
gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaa cacagaaaac
1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac
tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag
tgaatctgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat
caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat
cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920 atcgtcatca
ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980
gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac
2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaactag 2088 12
695 PRT Homo sapiens 12 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala
Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn
Ala Gly Leu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly
Arg Leu Asn Met His Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp
Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu
Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln
Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90
95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val
100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala
Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg
Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp His Thr Val Ala
Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp
Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly
Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn
Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp
Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215
220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu
225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu
Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu
Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu
Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser
Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp
Glu Asn Glu His Ala His Phe Gln Lys Ala Lys 305 310 315 320 Glu Arg
Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg 325 330 335
Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp 340
345 350 Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu
Glu 355 360 365 Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr
His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg
Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu Gln Ala Val
Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr
Val Arg Ala Glu Gln Lys Asp Arg Gln His 420 425 430 Thr Leu Lys His
Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala Gln
Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460
Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465
470 475 480 Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu
Gln Asn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu
Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu
Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly
Glu Phe Ser Leu Asp Asp Leu Gln 530 535 540 Pro Trp His Ser Phe Gly
Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu
Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr
Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585
590 Glu Val Asn Leu Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val
595 600 605 His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val
Ile Ala Thr Val 625 630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys
Lys Lys Gln Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val
Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys
Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe
Glu Gln Met Gln Asn 690 695 13 2088 DNA Homo sapiens 13 atgctgcccg
gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60
cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga
120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc
agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc
aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa
ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac
ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa
gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420
atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag
480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga
caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg
acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg
ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga
agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg
atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780
ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca
840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccc
tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg
cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga
atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa
cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag
tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140
acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac
1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa
tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc
taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag
atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa
tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc
aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500
gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca
1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga
gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg
tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct
gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa
gacggaggag atctctgaag tgaagatgga tgcagaattc 1800 cgacatgact
caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860
ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg
1920 atcttcatca ccttggtgat gctgaagaag aaacagtaca catccattca
tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacc
tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt
gagcagatgc agaactag 2088 14 695 PRT Homo sapiens 14 Met Leu Pro Gly
Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu
Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30
Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35
40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile
Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr
Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro
Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys
Lys Thr His Pro His Phe Val 100 105 110 Ile Pro Tyr Arg Cys Leu Val
Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys
Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140 Glu Thr His
Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160
Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165
170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu
Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp
Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp
Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu
Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu
Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu
Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr
Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285
Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290
295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala
Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser
Gln Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys
Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile Gln His Phe
Gln Glu Lys Val Glu Ser Leu Glu 355 360 365 Gln Glu Ala Ala Asn Glu
Arg Gln Gln Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala
Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr
Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410
415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His
420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys
Lys Ala 435 440 445 Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg
Val Ile Tyr Glu 450 455 460 Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr
Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile Gln Asp Glu Val
Asp Glu Leu Leu Gln Lys Glu Gln Asn 485 490 495 Tyr Ser Asp Asp Val
Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 Tyr Gly Asn
Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val
Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln 530 535
540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn
545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg
Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys
Thr Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala Glu Phe Arg
His Asp Ser Gly Tyr Glu Val 595 600 605 His His Gln Lys Leu Val Phe
Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly
Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Phe
Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile 645 650 655
His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660
665 670 His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr
Lys 675 680 685 Phe Phe Glu Gln Met Gln Asn 690 695 15 2094 DNA
Homo sapiens 15 atgctgcccg gtttggcact gctcctgctg gccgcctgga
cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct
gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt
ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg
ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240
cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg
300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg
cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca
aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg
cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga
ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt
ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600
gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg
660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt
ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg
tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc
agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt
tcgagttcct acaacagcag ccagtacccc tgatgccgtt 900 gacaagtatc
tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa 960
gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag
1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat
ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg
agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc
aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc
tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc
gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320
cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt
1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcc
tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag
agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg
atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac
caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc
agccgtggca ttcttttggg gctgactctg tgccagccaa cacagaaaac 1680
gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac tcgaccaggt
1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaagatgga
tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg
tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc
atggtgggcg gtgttgtcat agcgacagtg 1920 atcgtcatca ccttggtgat
gctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg
acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040
ggctacgaaa atccaaccta caagttcttt gagcagatgc agaacaagaa gtag 2094 16
697 PRT Homo sapiens 16 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala
Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn
Ala Gly Leu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly
Arg Leu Asn Met His Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp
Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu
Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln
Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90
95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val
100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala
Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg
Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp His Thr Val Ala
Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp
Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly
Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn
Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp
Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215
220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu
225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu
Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu
Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu
Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser
Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp
Glu Asn Glu His Ala His Phe Gln Lys Ala Lys 305 310 315 320 Glu Arg
Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg 325 330 335
Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp 340
345 350 Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu
Glu 355 360 365 Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr
His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg
Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu Gln Ala Val
Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr
Val Arg Ala Glu Gln Lys Asp Arg Gln His 420 425 430 Thr Leu Lys His
Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala Gln
Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460
Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465
470 475 480 Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu
Gln Asn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu
Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu
Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly
Glu Phe Ser Leu Asp Asp Leu Gln 530 535 540 Pro Trp His Ser Phe Gly
Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu
Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr
Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585
590 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val
595 600 605 His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val
Ile Ala Thr Val 625 630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys
Lys Lys Gln Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val
Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met
Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu
Gln Met Gln Asn Lys Lys 690 695 17 2094 DNA Homo sapiens 17
atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta
60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt
ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt
cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg
cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga
agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc
agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360
gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg
420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac
atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct
gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct
gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga
tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca
aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720
gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa
780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac
caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag
ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag
aatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca
ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc
aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080
caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag
1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc
cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc
acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga
cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa
agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg
agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440
gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac
1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc
tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg
tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg
gctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc
ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga
caaatatcaa gacggaggag atctctgaag tgaatctgga tgcagaattc 1800
cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg
1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat
agcgacagtg 1920 atcgtcatca ccttggtgat gctgaagaag aaacagtaca
catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag
gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta
caagttcttt gagcagatgc agaacaagaa gtag 2094 18 697 PRT Homo sapiens
18 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg
1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala
Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His
Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly
Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr
Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val
Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg
Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110 Ile Pro
Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125
Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130
135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser
Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu
Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys
Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp
Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp
Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val
Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu
Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250
255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile
260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val
Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val
Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala
His Phe Gln Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His
Arg Glu Arg Met Ser Gln Val Met Arg 325 330 335 Glu Trp Glu Glu Ala
Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala
Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu 355 360 365 Gln
Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala 370 375
380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn
385 390 395 400 Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg
His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln
Lys Asp Arg Gln His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg
Met Val Asp Pro Lys Lys Ala 435 440 445 Ala Gln Ile Arg Ser Gln Val
Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met Asn Gln Ser
Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu
Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn 485 490 495
Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500
505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr
Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp
Asp Leu Gln 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro
Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg
Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly
Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val Asn Leu
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His His
Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620
Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625
630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr
Ser Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr
Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met Gln Gln Asn Gly Tyr
Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu Gln Met Gln Asn Lys
Lys 690 695 19 2094 DNA Homo sapiens 19 atgctgcccg gtttggcact
gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg
gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120
ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa
180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta
ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca
tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac
tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct
tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt
gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480
aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga
540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga
ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag
acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag
gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga
cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag
aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840
gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccc tgatgccgtt
900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca
gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcccagg
tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa
gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt
ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg
ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200
tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag
1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt
cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc
aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc
tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt
tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca
acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560
tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg
1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaa
cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag
gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag
atctctgaag tgaagatgga tgcagaattc 1800 cgacatgact caggatatga
agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca
aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920
atcttcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg
1980 gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat
gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc
agaacaagaa gtag 2094 20 697 PRT Homo sapiens 20 Met Leu Pro Gly Leu
Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu
Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 Gln
Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40
45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp
50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro
Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val
Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys
Thr His Pro His Phe Val 100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly
Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys
Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140 Glu Thr His Leu
His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys
Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170
175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu
180 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser
Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly
Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val
Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp
Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro
Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr
Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val
Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295
300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys
305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln
Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn
Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile Gln His Phe Gln
Glu Lys Val Glu Ser Leu Glu 355 360 365 Gln Glu Ala Ala Asn Glu Arg
Gln Gln Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met
Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile
Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415
Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His 420
425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys
Ala 435 440 445 Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val
Ile Tyr Glu 450 455 460 Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn
Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile Gln Asp Glu Val Asp
Glu Leu Leu Gln Lys Glu Gln Asn 485 490 495 Tyr Ser Asp Asp Val Leu
Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp
Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu
Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln 530 535 540
Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545
550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly
Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr
Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala Glu Phe Arg His
Asp Ser Gly Tyr Glu Val 595 600 605 His His Gln Lys Leu Val Phe Phe
Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu
Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Phe Ile
Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile 645 650 655 His
His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665
670 His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys
675 680 685 Phe Phe Glu Gln Met Gln Asn Lys Lys 690 695 21 1341 DNA
Homo sapiens 21 atggctagca tgactggtgg acagcaaatg ggtcgcggat
ccacccagca cggcatccgg 60 ctgcccctgc gcagcggcct ggggggcgcc
cccctggggc tgcggctgcc ccgggagacc 120 gacgaagagc ccgaggagcc
cggccggagg ggcagctttg tggagatggt ggacaacctg 180 aggggcaagt
cggggcaggg ctactacgtg gagatgaccg tgggcagccc cccgcagacg 240
ctcaacatcc tggtggatac aggcagcagt aactttgcag tgggtgctgc cccccacccc
300 ttcctgcatc gctactacca gaggcagctg tccagcacat accgggacct
ccggaagggt 360 gtgtatgtgc cctacaccca gggcaagtgg gaaggggagc
tgggcaccga cctggtaagc 420 atcccccatg gccccaacgt cactgtgcgt
gccaacattg ctgccatcac tgaatcagac 480 aagttcttca tcaacggctc
caactgggaa ggcatcctgg ggctggccta tgctgagatt 540 gccaggcctg
acgactccct ggagcctttc tttgactctc tggtaaagca gacccacgtt 600
cccaacctct tctccctgca cctttgtggt gctggcttcc ccctcaacca gtctgaagtg
660 ctggcctctg tcggagggag catgatcatt ggaggtatcg accactcgct
gtacacaggc 720 agtctctggt atacacccat ccggcgggag tggtattatg
aggtcatcat tgtgcgggtg 780 gagatcaatg gacaggatct gaaaatggac
tgcaaggagt acaactatga caagagcatt 840 gtggacagtg gcaccaccaa
ccttcgtttg cccaagaaag tgtttgaagc tgcagtcaaa 900 tccatcaagg
cagcctcctc cacggagaag ttccctgatg gtttctggct aggagagcag 960
ctggtgtgct ggcaagcagg caccacccct tggaacattt tcccagtcat ctcactctac
1020 ctaatgggtg aggttaccaa ccagtccttc cgcatcacca tccttccgca
gcaatacctg 1080 cggccagtgg aagatgtggc cacgtcccaa gacgactgtt
acaagtttgc catctcacag 1140 tcatccacgg gcactgttat gggagctgtt
atcatggagg gcttctacgt tgtctttgat 1200 cgggcccgaa aacgaattgg
ctttgctgtc agcgcttgcc atgtgcacga tgagttcagg 1260 acggcagcgg
tggaaggccc ttttgtcacc ttggacatgg aagactgtgg ctacaacatt 1320
ccacagacag atgagtcatg a 1341 22 446 PRT Homo sapiens 22 Met Ala Ser
Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Thr Gln 1 5 10 15 His
Gly Ile Arg Leu Pro Leu Arg Ser Gly Leu Gly Gly Ala Pro Leu 20 25
30 Gly Leu Arg Leu Pro Arg Glu Thr Asp Glu Glu Pro Glu Glu Pro Gly
35 40 45 Arg Arg Gly Ser Phe Val Glu Met Val Asp Asn Leu Arg Gly
Lys Ser 50 55 60 Gly Gln Gly Tyr Tyr Val Glu Met Thr Val Gly Ser
Pro Pro Gln Thr 65 70 75 80 Leu Asn Ile Leu Val Asp Thr Gly Ser Ser
Asn Phe Ala Val Gly Ala 85 90 95 Ala Pro His Pro Phe Leu His Arg
Tyr Tyr Gln Arg Gln Leu Ser Ser 100 105 110 Thr Tyr Arg Asp Leu Arg
Lys Gly Val Tyr Val Pro Tyr Thr Gln Gly 115 120 125 Lys Trp Glu Gly
Glu Leu Gly Thr Asp Leu Val Ser Ile Pro His Gly 130 135 140 Pro Asn
Val Thr Val Arg Ala Asn Ile Ala Ala Ile Thr Glu Ser Asp 145 150 155
160 Lys Phe Phe Ile Asn Gly Ser Asn Trp Glu Gly Ile Leu Gly Leu Ala
165 170 175 Tyr Ala Glu Ile Ala Arg Pro Asp Asp Ser Leu Glu Pro Phe
Phe Asp 180 185 190 Ser Leu Val Lys Gln Thr His Val Pro Asn Leu Phe
Ser Leu His Leu 195 200 205 Cys Gly Ala Gly Phe Pro Leu Asn Gln Ser
Glu Val Leu Ala Ser Val 210 215 220 Gly Gly Ser Met Ile Ile Gly Gly
Ile Asp His Ser Leu Tyr Thr Gly 225 230 235 240 Ser Leu Trp Tyr Thr
Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile 245 250 255 Ile Val Arg
Val Glu Ile Asn Gly Gln Asp Leu Lys Met Asp Cys Lys 260 265 270 Glu
Tyr Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu 275 280
285 Arg Leu Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala
290 295 300 Ala Ser Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly
Glu Gln 305 310 315 320 Leu Val Cys Trp Gln Ala Gly Thr Thr Pro Trp
Asn Ile Phe Pro Val 325 330 335 Ile Ser Leu Tyr Leu Met Gly Glu Val
Thr Asn Gln Ser Phe Arg Ile 340 345 350 Thr Ile Leu Pro Gln Gln Tyr
Leu Arg Pro Val Glu Asp Val Ala Thr 355 360 365 Ser Gln Asp Asp Cys
Tyr Lys Phe Ala Ile Ser Gln Ser Ser Thr Gly 370 375 380 Thr Val Met
Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp 385 390 395 400
Arg Ala Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val His 405
410 415 Asp Glu Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu
Asp 420 425 430 Met Glu Asp Cys Gly Tyr Asn Ile Pro Gln Thr Asp Glu
Ser 435 440 445 23 1380 DNA Homo sapiens 23 atggctagca tgactggtgg
acagcaaatg ggtcgcggat cgatgactat ctctgactct 60 ccgcgtgaac
aggacggatc cacccagcac ggcatccggc tgcccctgcg cagcggcctg 120
gggggcgccc ccctggggct gcggctgccc cgggagaccg acgaagagcc cgaggagccc
180 ggccggaggg gcagctttgt ggagatggtg gacaacctga ggggcaagtc
ggggcagggc 240 tactacgtgg agatgaccgt gggcagcccc ccgcagacgc
tcaacatcct ggtggataca 300 ggcagcagta actttgcagt gggtgctgcc
ccccacccct tcctgcatcg ctactaccag 360 aggcagctgt ccagcacata
ccgggacctc cggaagggtg tgtatgtgcc ctacacccag 420 ggcaagtggg
aaggggagct gggcaccgac ctggtaagca tcccccatgg ccccaacgtc 480
actgtgcgtg ccaacattgc tgccatcact gaatcagaca agttcttcat caacggctcc
540 aactgggaag gcatcctggg gctggcctat gctgagattg ccaggcctga
cgactccctg 600 gagcctttct ttgactctct ggtaaagcag acccacgttc
ccaacctctt ctccctgcac 660 ctttgtggtg ctggcttccc cctcaaccag
tctgaagtgc tggcctctgt cggagggagc 720 atgatcattg gaggtatcga
ccactcgctg tacacaggca gtctctggta tacacccatc 780 cggcgggagt
ggtattatga ggtcatcatt gtgcgggtgg agatcaatgg acaggatctg 840
aaaatggact gcaaggagta caactatgac aagagcattg tggacagtgg caccaccaac
900 cttcgtttgc ccaagaaagt gtttgaagct gcagtcaaat ccatcaaggc
agcctcctcc 960 acggagaagt tccctgatgg tttctggcta ggagagcagc
tggtgtgctg gcaagcaggc 1020 accacccctt ggaacatttt cccagtcatc
tcactctacc taatgggtga ggttaccaac 1080 cagtccttcc gcatcaccat
ccttccgcag caatacctgc ggccagtgga agatgtggcc 1140 acgtcccaag
acgactgtta caagtttgcc atctcacagt catccacggg cactgttatg 1200
ggagctgtta tcatggaggg cttctacgtt gtctttgatc gggcccgaaa acgaattggc
1260 tttgctgtca gcgcttgcca tgtgcacgat gagttcagga cggcagcggt
ggaaggccct 1320 tttgtcacct tggacatgga agactgtggc tacaacattc
cacagacaga tgagtcatga 1380 24 459 PRT Homo sapiens 24 Met Ala Ser
Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Thr 1 5 10 15 Ile
Ser Asp Ser Pro Arg Glu Gln Asp Gly Ser Thr Gln His Gly Ile 20 25
30 Arg Leu Pro Leu Arg Ser Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg
35 40 45 Leu Pro Arg Glu Thr Asp Glu Glu Pro Glu Glu Pro Gly Arg
Arg Gly 50 55 60 Ser Phe Val Glu Met Val Asp Asn Leu Arg Gly Lys
Ser Gly Gln Gly 65 70 75 80 Tyr Tyr Val Glu Met Thr Val Gly Ser Pro
Pro Gln Thr Leu Asn Ile 85 90 95 Leu Val Asp Thr Gly Ser Ser Asn
Phe Ala Val Gly Ala Ala Pro His 100 105 110 Pro Phe Leu His Arg Tyr
Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg 115 120 125 Asp Leu Arg Lys
Gly Val Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu 130 135 140 Gly Glu
Leu Gly Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn Val 145 150 155
160 Thr Val Arg Ala Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe
165 170 175 Ile Asn Gly Ser Asn Trp Glu Gly Ile Leu Gly Leu Ala Tyr
Ala Glu 180 185 190 Ile Ala Arg Pro Asp Asp Ser Leu Glu Pro Phe Phe
Asp Ser Leu Val 195 200 205 Lys Gln Thr His Val Pro Asn Leu Phe Ser
Leu His Leu Cys Gly Ala 210 215 220 Gly Phe Pro Leu Asn Gln Ser Glu
Val Leu Ala Ser Val Gly Gly Ser 225 230 235 240 Met Ile Ile Gly Gly
Ile Asp His Ser Leu Tyr Thr Gly Ser Leu Trp 245 250 255 Tyr Thr Pro
Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg 260 265 270 Val
Glu Ile Asn Gly Gln Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn 275 280
285 Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro
290 295 300 Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala
Ser Ser 305 310 315 320 Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly
Glu Gln Leu Val Cys 325 330 335 Trp Gln Ala Gly Thr Thr Pro Trp Asn
Ile Phe Pro Val Ile Ser Leu 340 345 350 Tyr Leu Met Gly Glu Val Thr
Asn Gln Ser Phe Arg Ile Thr Ile Leu 355 360 365 Pro Gln Gln Tyr Leu
Arg Pro Val Glu Asp Val Ala Thr Ser Gln Asp 370 375 380 Asp Cys Tyr
Lys Phe Ala Ile Ser Gln Ser Ser Thr Gly Thr Val Met 385 390 395 400
Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala Arg 405
410 415 Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu
Phe 420 425 430 Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp
Met Glu Asp 435 440 445 Cys Gly Tyr Asn Ile Pro Gln Thr Asp Glu Ser
450 455 25 1302 DNA Homo sapiens 25 atgactcagc atggtattcg
tctgccactg cgtagcggtc tgggtggtgc tccactgggt 60 ctgcgtctgc
cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 120
gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc
180 gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag
taactttgca 240 gtgggtgctg ccccccaccc cttcctgcat cgctactacc
agaggcagct gtccagcaca 300 taccgggacc tccggaaggg tgtgtatgtg
ccctacaccc agggcaagtg ggaaggggag 360 ctgggcaccg acctggtaag
catcccccat ggccccaacg tcactgtgcg tgccaacatt 420 gctgccatca
ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 480
gggctggcct atgctgagat tgccaggcct gacgactccc tggagccttt ctttgactct
540 ctggtaaagc agacccacgt tcccaacctc ttctccctgc acctttgtgg
tgctggcttc 600 cccctcaacc agtctgaagt gctggcctct gtcggaggga
gcatgatcat tggaggtatc 660 gaccactcgc tgtacacagg cagtctctgg
tatacaccca tccggcggga gtggtattat 720 gaggtcatca ttgtgcgggt
ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag 780 tacaactatg
acaagagcat tgtggacagt ggcaccacca accttcgttt gcccaagaaa 840
gtgtttgaag ctgcagtcaa atccatcaag gcagcctcct ccacggagaa gttccctgat
900 ggtttctggc taggagagca gctggtgtgc tggcaagcag gcaccacccc
ttggaacatt 960 ttcccagtca tctcactcta cctaatgggt gaggttacca
accagtcctt ccgcatcacc 1020 atccttccgc agcaatacct gcggccagtg
gaagatgtgg ccacgtccca agacgactgt 1080 tacaagtttg ccatctcaca
gtcatccacg ggcactgtta tgggagctgt tatcatggag 1140 ggcttctacg
ttgtctttga tcgggcccga aaacgaattg gctttgctgt cagcgcttgc 1200
catgtgcacg atgagttcag gacggcagcg gtggaaggcc cttttgtcac cttggacatg
1260 gaagactgtg gctacaacat tccacagaca gatgagtcat ga 1302 26 433 PRT
Homo sapiens 26 Met Thr Gln His Gly Ile Arg Leu Pro Leu Arg Ser Gly
Leu Gly Gly 1 5 10 15 Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr
Asp Glu Glu Pro Glu 20 25 30 Glu Pro Gly Arg Arg Gly Ser Phe Val
Glu Met Val Asp Asn Leu Arg 35 40 45 Gly Lys Ser Gly Gln Gly Tyr
Tyr Val Glu Met Thr Val Gly Ser Pro 50 55 60 Pro Gln Thr Leu Asn
Ile Leu Val Asp Thr Gly Ser Ser Asn Phe Ala 65 70 75 80 Val Gly Ala
Ala Pro His Pro Phe Leu His Arg Tyr Tyr Gln Arg Gln 85 90 95 Leu
Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val Tyr Val Pro Tyr 100 105
110 Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp Leu Val Ser Ile
115 120 125 Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile Ala Ala
Ile Thr 130 135 140 Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp
Glu Gly Ile Leu 145 150 155 160 Gly Leu Ala Tyr Ala Glu Ile Ala Arg
Pro Asp Asp Ser Leu Glu Pro 165 170 175 Phe Phe Asp Ser Leu Val Lys
Gln Thr His Val Pro Asn Leu Phe Ser 180 185 190 Leu His Leu Cys Gly
Ala Gly Phe Pro Leu Asn Gln Ser Glu Val Leu 195 200 205 Ala Ser Val
Gly Gly Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu 210 215 220 Tyr
Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr 225 230
235 240 Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly Gln Asp Leu Lys
Met 245 250 255 Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val Asp
Ser Gly Thr 260 265 270 Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu
Ala Ala Val Lys Ser 275 280 285 Ile Lys Ala Ala Ser Ser Thr Glu Lys
Phe Pro Asp Gly Phe Trp Leu 290 295 300 Gly Glu Gln Leu Val Cys Trp
Gln Ala Gly Thr Thr Pro Trp Asn Ile 305 310 315 320 Phe Pro Val Ile
Ser Leu Tyr Leu Met Gly Glu Val Thr Asn Gln Ser 325 330 335 Phe Arg
Ile Thr Ile Leu Pro Gln Gln Tyr Leu Arg Pro Val Glu Asp 340 345 350
Val Ala Thr Ser Gln Asp Asp Cys Tyr Lys Phe Ala Ile Ser Gln Ser 355
360 365 Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu Gly Phe Tyr
Val 370 375 380 Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala Val
Ser Ala Cys 385 390 395 400 His Val His Asp Glu Phe Arg Thr Ala Ala
Val Glu Gly Pro Phe Val 405 410 415 Thr Leu Asp Met Glu Asp Cys Gly
Tyr Asn Ile Pro Gln Thr Asp Glu 420 425 430 Ser 27 1278 DNA Homo
sapiens 27 atggctagca tgactggtgg acagcaaatg ggtcgcggat cgatgactat
ctctgactct 60 ccgctggact ctggtatcga aaccgacgga tcctttgtgg
agatggtgga caacctgagg 120 ggcaagtcgg ggcagggcta ctacgtggag
atgaccgtgg gcagcccccc gcagacgctc 180 aacatcctgg tggatacagg
cagcagtaac tttgcagtgg gtgctgcccc ccaccccttc 240 ctgcatcgct
actaccagag gcagctgtcc agcacatacc gggacctccg gaagggtgtg 300
tatgtgccct acacccaggg caagtgggaa ggggagctgg gcaccgacct ggtaagcatc
360 ccccatggcc ccaacgtcac tgtgcgtgcc aacattgctg ccatcactga
atcagacaag 420 ttcttcatca acggctccaa ctgggaaggc atcctggggc
tggcctatgc tgagattgcc 480 aggcctgacg actccctgga gcctttcttt
gactctctgg taaagcagac ccacgttccc 540 aacctcttct ccctgcacct
ttgtggtgct ggcttccccc tcaaccagtc tgaagtgctg 600 gcctctgtcg
gagggagcat gatcattgga ggtatcgacc actcgctgta cacaggcagt 660
ctctggtata cacccatccg gcgggagtgg tattatgagg tcatcattgt gcgggtggag
720 atcaatggac aggatctgaa aatggactgc aaggagtaca actatgacaa
gagcattgtg 780 gacagtggca ccaccaacct tcgtttgccc aagaaagtgt
ttgaagctgc agtcaaatcc 840 atcaaggcag cctcctccac ggagaagttc
cctgatggtt tctggctagg agagcagctg 900 gtgtgctggc aagcaggcac
caccccttgg aacattttcc cagtcatctc actctaccta 960 atgggtgagg
ttaccaacca gtccttccgc atcaccatcc ttccgcagca atacctgcgg 1020
ccagtggaag atgtggccac gtcccaagac gactgttaca agtttgccat ctcacagtca
1080 tccacgggca ctgttatggg agctgttatc atggagggct tctacgttgt
ctttgatcgg 1140 gcccgaaaac gaattggctt tgctgtcagc gcttgccatg
tgcacgatga gttcaggacg 1200 gcagcggtgg aaggcccttt tgtcaccttg
gacatggaag actgtggcta caacattcca 1260 cagacagatg agtcatga 1278 28
425 PRT Homo sapiens 28 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly
Arg Gly Ser Met Thr 1 5 10 15 Ile Ser Asp Ser Pro Leu Asp Ser Gly
Ile Glu Thr Asp Gly Ser Phe 20 25 30 Val Glu Met Val Asp Asn Leu
Arg Gly Lys Ser Gly Gln Gly Tyr Tyr 35 40 45 Val Glu Met Thr Val
Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val 50 55 60 Asp Thr Gly
Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe 65 70 75 80 Leu
His Arg Tyr Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu 85 90
95 Arg Lys Gly Val Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu
100 105 110 Leu Gly Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn Val
Thr Val 115 120 125 Arg Ala Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys
Phe Phe Ile Asn 130 135 140 Gly Ser Asn Trp Glu Gly Ile Leu Gly Leu
Ala Tyr Ala Glu Ile Ala 145 150 155 160 Arg Pro Asp Asp Ser Leu Glu
Pro Phe Phe Asp Ser Leu Val Lys Gln 165 170 175 Thr His Val Pro Asn
Leu Phe Ser Leu His Leu Cys Gly Ala Gly Phe 180 185 190 Pro Leu Asn
Gln Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile 195 200 205 Ile
Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr 210 215
220 Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu
225 230 235 240 Ile Asn Gly Gln Asp Leu Lys Met Asp Cys Lys Glu Tyr
Asn Tyr Asp 245 250 255 Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu
Arg Leu Pro Lys Lys 260 265 270 Val Phe Glu Ala Ala Val Lys Ser Ile
Lys Ala Ala Ser Ser Thr Glu 275 280 285 Lys Phe Pro Asp Gly Phe Trp
Leu Gly Glu Gln Leu Val Cys Trp Gln 290 295 300 Ala Gly Thr Thr Pro
Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu 305 310 315 320 Met Gly
Glu Val Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln 325 330 335
Gln Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys 340
345 350 Tyr Lys Phe Ala Ile Ser Gln Ser Ser Thr Gly Thr Val Met Gly
Ala 355 360 365 Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala
Arg Lys Arg 370 375 380 Ile Gly Phe Ala Val Ser Ala Cys His Val His
Asp Glu Phe Arg Thr 385 390 395 400 Ala Ala Val Glu Gly Pro Phe Val
Thr Leu Asp Met Glu Asp Cys Gly 405 410 415 Tyr Asn Ile Pro Gln Thr
Asp Glu Ser 420 425 29 1362 DNA Homo sapiens 29 atggcccaag
ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60
ggcacccagc acggcatccg gctgcccctg cgcagcggcc tggggggcgc ccccctgggg
120 ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag
gggcagcttt 180 gtggagatgg tggacaacct gaggggcaag tcggggcagg
gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac gctcaacatc
ctggtggata caggcagcag taactttgca 300 gtgggtgctg ccccccaccc
cttcctgcat cgctactacc agaggcagct gtccagcaca 360 taccgggacc
tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420
ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt
480 gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga
aggcatcctg 540 gggctggcct atgctgagat tgccaggcct gacgactccc
tggagccttt ctttgactct 600 ctggtaaagc agacccacgt tcccaacctc
ttctccctgc acctttgtgg tgctggcttc 660 cccctcaacc agtctgaagt
gctggcctct gtcggaggga gcatgatcat tggaggtatc 720 gaccactcgc
tgtacacagg cagtctctgg tatacaccca tccggcggga gtggtattat 780
gaggtcatca ttgtgcgggt ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag
840 tacaactatg acaagagcat tgtggacagt ggcaccacca accttcgttt
gcccaagaaa 900 gtgtttgaag ctgcagtcaa atccatcaag gcagcctcct
ccacggagaa gttccctgat 960 ggtttctggc taggagagca gctggtgtgc
tggcaagcag gcaccacccc ttggaacatt 1020 ttcccagtca tctcactcta
cctaatgggt gaggttacca accagtcctt ccgcatcacc 1080 atccttccgc
agcaatacct gcggccagtg gaagatgtgg ccacgtccca agacgactgt 1140
tacaagtttg ccatctcaca gtcatccacg ggcactgtta tgggagctgt tatcatggag
1200 ggcttctacg ttgtctttga tcgggcccga aaacgaattg gctttgctgt
cagcgcttgc 1260 catgtgcacg atgagttcag gacggcagcg gtggaaggcc
cttttgtcac cttggacatg 1320 gaagactgtg gctacaacat tccacagaca
gatgagtcat ga 1362 30 453 PRT Homo sapiens 30 Met Ala Gln Ala Leu
Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala
His Gly Thr Gln His Gly Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly
Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40
45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val
50 55 60 Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu
Met Thr 65 70 75 80 Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val
Asp Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His
Pro Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser Ser Thr
Tyr Arg Asp Leu Arg Lys Gly Val 115 120 125 Tyr Val Pro Tyr Thr Gln
Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile
Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 145 150 155 160 Ala
Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170
175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp
180 185 190 Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr His
Val Pro 195 200 205 Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe
Pro Leu Asn Gln 210 215 220 Ser Glu Val Leu Ala Ser Val Gly Gly Ser
Met Ile Ile Gly Gly Ile 225 230 235
240 Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg
245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn
Gly Gln 260 265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp
Lys Ser Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro
Lys Lys Val Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile Lys Ala Ala
Ser Ser Thr Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp Leu Gly
Glu Gln Leu Val Cys Trp Gln Ala Gly Thr Thr 325 330 335 Pro Trp Asn
Ile Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr
Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu Arg 355 360
365 Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr Lys Phe Ala
370 375 380 Ile Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile
Met Glu 385 390 395 400 Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys
Arg Ile Gly Phe Ala 405 410 415 Val Ser Ala Cys His Val His Asp Glu
Phe Arg Thr Ala Ala Val Glu 420 425 430 Gly Pro Phe Val Thr Leu Asp
Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440 445 Gln Thr Asp Glu Ser
450 31 1380 DNA Homo sapiens 31 atggcccaag ccctgccctg gctcctgctg
tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccg
gctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120 ctgcggctgc
cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 180
gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc
240 gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag
taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat cgctactacc
agaggcagct gtccagcaca 360 taccgggacc tccggaaggg tgtgtatgtg
ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg acctggtaag
catcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca
ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540
gggctggcct atgctgagat tgccaggcct gacgactccc tggagccttt ctttgactct
600 ctggtaaagc agacccacgt tcccaacctc ttctccctgc acctttgtgg
tgctggcttc 660 cccctcaacc agtctgaagt gctggcctct gtcggaggga
gcatgatcat tggaggtatc 720 gaccactcgc tgtacacagg cagtctctgg
tatacaccca tccggcggga gtggtattat 780 gaggtcatca ttgtgcgggt
ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag 840 tacaactatg
acaagagcat tgtggacagt ggcaccacca accttcgttt gcccaagaaa 900
gtgtttgaag ctgcagtcaa atccatcaag gcagcctcct ccacggagaa gttccctgat
960 ggtttctggc taggagagca gctggtgtgc tggcaagcag gcaccacccc
ttggaacatt 1020 ttcccagtca tctcactcta cctaatgggt gaggttacca
accagtcctt ccgcatcacc 1080 atccttccgc agcaatacct gcggccagtg
gaagatgtgg ccacgtccca agacgactgt 1140 tacaagtttg ccatctcaca
gtcatccacg ggcactgtta tgggagctgt tatcatggag 1200 ggcttctacg
ttgtctttga tcgggcccga aaacgaattg gctttgctgt cagcgcttgc 1260
catgtgcacg atgagttcag gacggcagcg gtggaaggcc cttttgtcac cttggacatg
1320 gaagactgtg gctacaacat tccacagaca gatgagtcac agcagcagca
gcagcagtga 1380 32 459 PRT Homo sapiens 32 Met Ala Gln Ala Leu Pro
Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His
Gly Thr Gln His Gly Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu
Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40 45
Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 50
55 60 Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met
Thr 65 70 75 80 Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp
Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro
Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser Ser Thr Tyr
Arg Asp Leu Arg Lys Gly Val 115 120 125 Tyr Val Pro Tyr Thr Gln Gly
Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile Pro
His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala
Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175
Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp 180
185 190 Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr His Val
Pro 195 200 205 Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro
Leu Asn Gln 210 215 220 Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met
Ile Ile Gly Gly Ile 225 230 235 240 Asp His Ser Leu Tyr Thr Gly Ser
Leu Trp Tyr Thr Pro Ile Arg Arg 245 250 255 Glu Trp Tyr Tyr Glu Val
Ile Ile Val Arg Val Glu Ile Asn Gly Gln 260 265 270 Asp Leu Lys Met
Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val 275 280 285 Asp Ser
Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala 290 295 300
Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp 305
310 315 320 Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Gln Ala Gly
Thr Thr 325 330 335 Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu
Met Gly Glu Val 340 345 350 Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu
Pro Gln Gln Tyr Leu Arg 355 360 365 Pro Val Glu Asp Val Ala Thr Ser
Gln Asp Asp Cys Tyr Lys Phe Ala 370 375 380 Ile Ser Gln Ser Ser Thr
Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390 395 400 Gly Phe Tyr
Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 Val
Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425
430 Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro
435 440 445 Gln Thr Asp Glu Ser His His His His His His 450 455 33
25 PRT Homo sapiens 33 Ser Glu Gln Gln Arg Arg Pro Arg Asp Pro Glu
Val Val Asn Asp Glu 1 5 10 15 Ser Ser Leu Val Arg His Arg Trp Lys
20 25 34 19 PRT Homo sapiens 34 Ser Glu Gln Leu Arg Gln Gln His Asp
Asp Phe Ala Asp Asp Ile Ser 1 5 10 15 Leu Leu Lys 35 29 DNA Homo
sapiens 35 gtggatccac ccagcacggc atccggctg 29 36 36 DNA Homo
sapiens 36 gaaagctttc atgactcatc tgtctgtgga atgttg 36 37 39 DNA
Homo sapiens 37 gatcgatgac tatctctgac tctccgcgtg aacaggacg 39 38 39
DNA Homo sapiens 38 gatccgtcct gttcacgcgg agagtcagag atagtcatc 39
39 77 DNA Artificial Sequence Description of Artificial Sequence
Hu-Asp2 39 cggcatccgg ctgcccctgc gtagcggtct gggtggtgct ccactgggtc
tgcgtctgcc 60 ccgggagacc gacgaag 77 40 77 DNA Artificial Sequence
Description of Artificial Sequence Hu-Asp2 40 cttcgtcggt ctcccggggc
agacgcagac ccagtggagc accacccaga ccgctacgca 60 ggggcagccg gatgccg
77 41 51 DNA Artificial Sequence Description of Artificial Sequence
Caspase 8 Cleavage Site 41 gatcgatgac tatctctgac tctccgctgg
actctggtat cgaaaccgac g 51 42 51 DNA Artificial Sequence
Description of Artificial Sequence Caspase 8 Cleavage Site 42
gatccgtcgg tttcgatacc agagtccagc ggagagtcag agatagtcat c 51 43 32
DNA Homo sapiens 43 aaggatcctt tgtggagatg gtggacaacc tg 32 44 36
DNA Homo sapiens 44 gaaagctttc atgactcatc tgtctgtgga atgttg 36 45
24 DNA Artificial Sequence Description of Artificial Sequence 6-His
tag 45 gatcgcatca tcaccatcac catg 24 46 24 DNA Artificial Sequence
Description of Artificial Sequence 6-His tag 46 gatccatggt
gatggtgatg atgc 24 47 22 DNA Artificial Sequence Description of
Artificial Sequence primer 47 gactgaccac tcgaccaggt tc 22 48 51 DNA
Artificial Sequence Description of Artificial Sequence primer 48
cgaattaaat tccagcacac tggctacttc ttgttctgca tctcaaagaa c 51 49 26
DNA Artificial Sequence Description of Artificial Sequence primer
49 cgaattaaat tccagcacac tggcta 26 50 1287 DNA Artificial Sequence
Description of Artificial Sequence Hu-Asp2(b) delta TM 50
atggcccaag ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac
60 ggcacccagc acggcatccg gctgcccctg cgcagcggcc tggggggcgc
ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagag cccgaggagc
ccggccggag gggcagcttt 180 gtggagatgg tggacaacct gaggggcaag
tcggggcagg gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac
gctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctg
ccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 360
taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag
420 ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg
tgccaacatt 480 gctgccatca ctgaatcaga caagttcttc atcaacggct
ccaactggga aggcatcctg 540 gggctggcct atgctgagat tgccaggctt
tgtggtgctg gcttccccct caaccagtct 600 gaagtgctgg cctctgtcgg
agggagcatg atcattggag gtatcgacca ctcgctgtac 660 acaggcagtc
tctggtatac acccatccgg cgggagtggt attatgaggt catcattgtg 720
cgggtggaga tcaatggaca ggatctgaaa atggactgca aggagtacaa ctatgacaag
780 agcattgtgg acagtggcac caccaacctt cgtttgccca agaaagtgtt
tgaagctgca 840 gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc
ctgatggttt ctggctagga 900 gagcagctgg tgtgctggca agcaggcacc
accccttgga acattttccc agtcatctca 960 ctctacctaa tgggtgaggt
taccaaccag tccttccgca tcaccatcct tccgcagcaa 1020 tacctgcggc
cagtggaaga tgtggccacg tcccaagacg actgttacaa gtttgccatc 1080
tcacagtcat ccacgggcac tgttatggga gctgttatca tggagggctt ctacgttgtc
1140 tttgatcggg cccgaaaacg aattggcttt gctgtcagcg cttgccatgt
gcacgatgag 1200 ttcaggacgg cagcggtgga aggccctttt gtcaccttgg
acatggaaga ctgtggctac 1260 aacattccac agacagatga gtcatga 1287 51
428 PRT Artificial Sequence Description of Artificial Sequence
Hu-Asp2(b) delta TM 51 Met Ala Gln Ala Leu Pro Trp Leu Leu Leu Trp
Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr Gln His Gly
Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu Gly Gly Ala Pro Leu
Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40 45 Glu Glu Pro Glu Glu
Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 50 55 60 Asp Asn Leu
Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr 65 70 75 80 Val
Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 85 90
95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr
100 105 110 Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys
Gly Val 115 120 125 Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu
Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val
Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala Ile Thr Glu Ser Asp
Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175 Glu Gly Ile Leu Gly
Leu Ala Tyr Ala Glu Ile Ala Arg Leu Cys Gly 180 185 190 Ala Gly Phe
Pro Leu Asn Gln Ser Glu Val Leu Ala Ser Val Gly Gly 195 200 205 Ser
Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu 210 215
220 Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val
225 230 235 240 Arg Val Glu Ile Asn Gly Gln Asp Leu Lys Met Asp Cys
Lys Glu Tyr 245 250 255 Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr
Thr Asn Leu Arg Leu 260 265 270 Pro Lys Lys Val Phe Glu Ala Ala Val
Lys Ser Ile Lys Ala Ala Ser 275 280 285 Ser Thr Glu Lys Phe Pro Asp
Gly Phe Trp Leu Gly Glu Gln Leu Val 290 295 300 Cys Trp Gln Ala Gly
Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser 305 310 315 320 Leu Tyr
Leu Met Gly Glu Val Thr Asn Gln Ser Phe Arg Ile Thr Ile 325 330 335
Leu Pro Gln Gln Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser Gln 340
345 350 Asp Asp Cys Tyr Lys Phe Ala Ile Ser Gln Ser Ser Thr Gly Thr
Val 355 360 365 Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe
Asp Arg Ala 370 375 380 Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys
His Val His Asp Glu 385 390 395 400 Phe Arg Thr Ala Ala Val Glu Gly
Pro Phe Val Thr Leu Asp Met Glu 405 410 415 Asp Cys Gly Tyr Asn Ile
Pro Gln Thr Asp Glu Ser 420 425 52 1305 DNA Artificial Sequence
Description of Artificial Sequence Hu-Asp2(b) delta TM 52
atggcccaag ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac
60 ggcacccagc acggcatccg gctgcccctg cgcagcggcc tggggggcgc
ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagag cccgaggagc
ccggccggag gggcagcttt 180 gtggagatgg tggacaacct gaggggcaag
tcggggcagg gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac
gctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctg
ccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 360
taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag
420 ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg
tgccaacatt 480 gctgccatca ctgaatcaga caagttcttc atcaacggct
ccaactggga aggcatcctg 540 gggctggcct atgctgagat tgccaggctt
tgtggtgctg gcttccccct caaccagtct 600 gaagtgctgg cctctgtcgg
agggagcatg atcattggag gtatcgacca ctcgctgtac 660 acaggcagtc
tctggtatac acccatccgg cgggagtggt attatgaggt catcattgtg 720
cgggtggaga tcaatggaca ggatctgaaa atggactgca aggagtacaa ctatgacaag
780 agcattgtgg acagtggcac caccaacctt cgtttgccca agaaagtgtt
tgaagctgca 840 gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc
ctgatggttt ctggctagga 900 gagcagctgg tgtgctggca agcaggcacc
accccttgga acattttccc agtcatctca 960 ctctacctaa tgggtgaggt
taccaaccag tccttccgca tcaccatcct tccgcagcaa 1020 tacctgcggc
cagtggaaga tgtggccacg tcccaagacg actgttacaa gtttgccatc 1080
tcacagtcat ccacgggcac tgttatggga gctgttatca tggagggctt ctacgttgtc
1140 tttgatcggg cccgaaaacg aattggcttt gctgtcagcg cttgccatgt
gcacgatgag 1200 ttcaggacgg cagcggtgga aggccctttt gtcaccttgg
acatggaaga ctgtggctac 1260 aacattccac agacagatga gtcacagcag
cagcagcagc agtga 1305 53 434 PRT Artificial Sequence Description of
Artificial Sequence Hu-Asp2(b) delta TM 53 Met Ala Gln Ala Leu Pro
Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His
Gly Thr Gln His Gly Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu
Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40 45
Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 50
55 60 Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met
Thr 65 70 75 80 Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp
Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro
Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser Ser Thr Tyr
Arg Asp Leu Arg Lys Gly Val 115 120 125 Tyr Val Pro Tyr Thr Gln Gly
Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile Pro
His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala
Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175
Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Leu Cys Gly 180
185 190 Ala Gly Phe Pro Leu Asn Gln Ser Glu Val Leu Ala Ser Val Gly
Gly 195 200 205 Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr Thr
Gly Ser Leu 210 215 220 Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr
Glu Val Ile Ile Val 225 230 235 240 Arg Val Glu Ile Asn Gly Gln Asp
Leu Lys Met Asp Cys Lys Glu Tyr 245 250 255 Asn Tyr Asp Lys Ser Ile
Val Asp Ser Gly Thr Thr Asn Leu Arg Leu 260 265 270 Pro Lys Lys Val
Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser 275 280 285
Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu Gln Leu Val 290
295 300 Cys Trp Gln Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile
Ser 305 310 315 320 Leu Tyr Leu Met Gly Glu Val Thr Asn Gln Ser Phe
Arg Ile Thr Ile 325 330 335 Leu Pro Gln Gln Tyr Leu Arg Pro Val Glu
Asp Val Ala Thr Ser Gln 340 345 350 Asp Asp Cys Tyr Lys Phe Ala Ile
Ser Gln Ser Ser Thr Gly Thr Val 355 360 365 Met Gly Ala Val Ile Met
Glu Gly Phe Tyr Val Val Phe Asp Arg Ala 370 375 380 Arg Lys Arg Ile
Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu 385 390 395 400 Phe
Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu 405 410
415 Asp Cys Gly Tyr Asn Ile Pro Gln Thr Asp Glu Ser His His His His
420 425 430 His His 54 2310 DNA Homo sapiens 54 atgctgcccg
gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60
cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga
120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc
agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc
aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa
ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac
ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa
gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420
atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag
480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga
caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg
acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg
ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga
agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg
atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780
ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca
840 gagtctgtgg aagaggtggt tcgagaggtg tgctctgaac aagccgagac
ggggccgtgc 900 cgagcaatga tctcccgctg gtactttgat gtgactgaag
ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaa ccggaacaac
tttgacacag aagagtactg catggccgtg 1020 tgtggcagcg ccatgtccca
aagtttactc aagactaccc aggaacctct tggccgagat 1080 cctgttaaac
ttcctacaac agcagccagt acccctgatg ccgttgacaa gtatctcgag 1140
acacctgggg atgagaatga acatgcccat ttccagaaag ccaaagagag gcttgaggcc
1200 aagcaccgag agagaatgtc ccaggtcatg agagaatggg aagaggcaga
acgtcaagca 1260 aagaacttgc ctaaagctga taagaaggca gttatccagc
atttccagga gaaagtggaa 1320 tctttggaac aggaagcagc caacgagaga
cagcagctgg tggagacaca catggccaga 1380 gtggaagcca tgctcaatga
ccgccgccgc ctggccctgg agaactacat caccgctctg 1440 caggctgttc
ctcctcggcc tcgtcacgtg ttcaatatgc taaagaagta tgtccgcgca 1500
gaacagaagg acagacagca caccctaaag catttcgagc atgtgcgcat ggtggatccc
1560 aagaaagccg ctcagatccg gtcccaggtt atgacacacc tccgtgtgat
ttatgagcgc 1620 atgaatcagt ctctctccct gctctacaac gtgcctgcag
tggccgagga gattcaggat 1680 gaagttgatg agctgcttca gaaagagcaa
aactattcag atgacgtctt ggccaacatg 1740 attagtgaac caaggatcag
ttacggaaac gatgctctca tgccatcttt gaccgaaacg 1800 aaaaccaccg
tggagctcct tcccgtgaat ggagagttca gcctggacga tctccagccg 1860
tggcattctt ttggggctga ctctgtgcca gccaacacag aaaacgaagt tgagcctgtt
1920 gatgcccgcc ctgctgccga ccgaggactg accactcgac caggttctgg
gttgacaaat 1980 atcaagacgg aggagatctc tgaagtgaag atggatgcag
aattccgaca tgactcagga 2040 tatgaagttc atcatcaaaa attggtgttc
tttgcagaag atgtgggttc aaacaaaggt 2100 gcaatcattg gactcatggt
gggcggtgtt gtcatagcga cagtgatcgt catcaccttg 2160 gtgatgctga
agaagaaaca gtacacatcc attcatcatg gtgtggtgga ggttgacgcc 2220
gctgtcaccc cagaggagcg ccacctgtcc aagatgcagc agaacggcta cgaaaatcca
2280 acctacaagt tctttgagca gatgcagaac 2310 55 770 PRT Homo sapiens
55 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg
1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala
Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His
Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly
Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr
Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val
Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg
Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110 Ile Pro
Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125
Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130
135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser
Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu
Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys
Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp
Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp
Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val
Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu
Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250
255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile
260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val
Val Arg 275 280 285 Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys
Arg Ala Met Ile 290 295 300 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly
Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys Gly Gly Asn
Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met Ala Val Cys
Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr 340 345 350 Thr Gln Glu
Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365 Ala
Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375
380 Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala
385 390 395 400 Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp
Glu Glu Ala 405 410 415 Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp
Lys Lys Ala Val Ile 420 425 430 Gln His Phe Gln Glu Lys Val Glu Ser
Leu Glu Gln Glu Ala Ala Asn 435 440 445 Glu Arg Gln Gln Leu Val Glu
Thr His Met Ala Arg Val Glu Ala Met 450 455 460 Leu Asn Asp Arg Arg
Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu 465 470 475 480 Gln Ala
Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495
Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe 500
505 510 Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg
Ser 515 520 525 Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met
Asn Gln Ser 530 535 540 Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala
Glu Glu Ile Gln Asp 545 550 555 560 Glu Val Asp Glu Leu Leu Gln Lys
Glu Gln Asn Tyr Ser Asp Asp Val 565 570 575 Leu Ala Asn Met Ile Ser
Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585 590 Leu Met Pro Ser
Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605 Val Asn
Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe 610 615 620
Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val 625
630 635 640 Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro
Gly Ser 645 650 655 Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu
Val Lys Met Asp 660 665 670 Ala Glu Phe Arg His Asp Ser Gly Tyr Glu
Val His His Gln Lys Leu 675 680 685 Val Phe Phe Ala Glu Asp Val Gly
Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700 Leu Met Val Gly Gly Val
Val Ile Ala Thr Val Ile Val Ile Thr Leu 705 710 715 720 Val Met Leu
Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val 725 730 735 Glu
Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745
750 Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met
755 760 765 Gln Asn 770 56 2253 DNA Homo sapiens 56 atgctgcccg
gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60
cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga
120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc
agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc
aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa
ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac
ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa
gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420
atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag
480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga
caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg
acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg
ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga
agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg
atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780
ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca
840 gagtctgtgg aagaggtggt tcgagaggtg tgctctgaac aagccgagac
ggggccgtgc 900 cgagcaatga tctcccgctg gtactttgat gtgactgaag
ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaa ccggaacaac
tttgacacag aagagtactg catggccgtg 1020 tgtggcagcg ccattcctac
aacagcagcc agtacccctg atgccgttga caagtatctc 1080 gagacacctg
gggatgagaa tgaacatgcc catttccaga aagccaaaga gaggcttgag 1140
gccaagcacc gagagagaat gtcccaggtc atgagagaat gggaagaggc agaacgtcaa
1200 gcaaagaact tgcctaaagc tgataagaag gcagttatcc agcatttcca
ggagaaagtg 1260 gaatctttgg aacaggaagc agccaacgag agacagcagc
tggtggagac acacatggcc 1320 agagtggaag ccatgctcaa tgaccgccgc
cgcctggccc tggagaacta catcaccgct 1380 ctgcaggctg ttcctcctcg
gcctcgtcac gtgttcaata tgctaaagaa gtatgtccgc 1440 gcagaacaga
aggacagaca gcacacccta aagcatttcg agcatgtgcg catggtggat 1500
cccaagaaag ccgctcagat ccggtcccag gttatgacac acctccgtgt gatttatgag
1560 cgcatgaatc agtctctctc cctgctctac aacgtgcctg cagtggccga
ggagattcag 1620 gatgaagttg atgagctgct tcagaaagag caaaactatt
cagatgacgt cttggccaac 1680 atgattagtg aaccaaggat cagttacgga
aacgatgctc tcatgccatc tttgaccgaa 1740 acgaaaacca ccgtggagct
ccttcccgtg aatggagagt tcagcctgga cgatctccag 1800 ccgtggcatt
cttttggggc tgactctgtg ccagccaaca cagaaaacga agttgagcct 1860
gttgatgccc gccctgctgc cgaccgagga ctgaccactc gaccaggttc tgggttgaca
1920 aatatcaaga cggaggagat ctctgaagtg aagatggatg cagaattccg
acatgactca 1980 ggatatgaag ttcatcatca aaaattggtg ttctttgcag
aagatgtggg ttcaaacaaa 2040 ggtgcaatca ttggactcat ggtgggcggt
gttgtcatag cgacagtgat cgtcatcacc 2100 ttggtgatgc tgaagaagaa
acagtacaca tccattcatc atggtgtggt ggaggttgac 2160 gccgctgtca
ccccagagga gcgccacctg tccaagatgc agcagaacgg ctacgaaaat 2220
ccaacctaca agttctttga gcagatgcag aac 2253 57 751 PRT Homo sapiens
57 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg
1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala
Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His
Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly
Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr
Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val
Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg
Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110 Ile Pro
Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125
Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130
135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser
Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu
Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys
Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp
Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp
Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val
Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu
Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250
255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile
260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val
Val Arg 275 280 285 Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys
Arg Ala Met Ile 290 295 300 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly
Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys Gly Gly Asn
Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met Ala Val Cys
Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr 340 345 350 Pro Asp Ala
Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu 355 360 365 His
Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg 370 375
380 Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln
385 390 395 400 Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile
Gln His Phe 405 410 415 Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala
Ala Asn Glu Arg Gln 420 425 430 Gln Leu Val Glu Thr His Met Ala Arg
Val Glu Ala Met Leu Asn Asp 435 440 445 Arg Arg Arg Leu Ala Leu Glu
Asn Tyr Ile Thr Ala Leu Gln Ala Val 450 455 460 Pro Pro Arg Pro Arg
His Val Phe Asn Met Leu Lys Lys Tyr Val Arg 465 470 475 480 Ala Glu
Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val 485 490 495
Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met 500
505 510 Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser
Leu 515 520 525 Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp
Glu Val Asp 530 535 540 Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp
Asp Val Leu Ala Asn 545 550 555 560 Met Ile Ser Glu Pro Arg Ile Ser
Tyr Gly Asn Asp Ala Leu Met Pro 565 570 575 Ser Leu Thr Glu Thr Lys
Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580 585 590 Glu Phe Ser Leu
Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp 595 600 605 Ser Val
Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg 610 615 620
Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr 625
630 635 640 Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala
Glu Phe 645 650 655 Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys
Leu Val Phe Phe 660 665 670 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala
Ile Ile Gly Leu Met Val 675 680 685 Gly Gly Val Val Ile Ala Thr Val
Ile Val Ile Thr Leu Val Met Leu 690 695 700 Lys Lys Lys Gln Tyr Thr
Ser Ile His His Gly Val Val Glu Val Asp 705 710 715 720 Ala Ala Val
Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn 725 730 735 Gly
Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn 740 745 750
58 2316 DNA Homo sapiens 58 atgctgcccg gtttggcact gctcctgctg
gccgcctgga cggctcgggc gctggaggta 60
cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga
120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc
agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc
aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa
ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac
ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa
gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420
atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag
480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga
caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg
acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg
ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga
agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg
atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780
ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca
840 gagtctgtgg aagaggtggt tcgagaggtg tgctctgaac aagccgagac
ggggccgtgc 900 cgagcaatga tctcccgctg gtactttgat gtgactgaag
ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaa ccggaacaac
tttgacacag aagagtactg catggccgtg 1020 tgtggcagcg ccatgtccca
aagtttactc aagactaccc aggaacctct tggccgagat 1080 cctgttaaac
ttcctacaac agcagccagt acccctgatg ccgttgacaa gtatctcgag 1140
acacctgggg atgagaatga acatgcccat ttccagaaag ccaaagagag gcttgaggcc
1200 aagcaccgag agagaatgtc ccaggtcatg agagaatggg aagaggcaga
acgtcaagca 1260 aagaacttgc ctaaagctga taagaaggca gttatccagc
atttccagga gaaagtggaa 1320 tctttggaac aggaagcagc caacgagaga
cagcagctgg tggagacaca catggccaga 1380 gtggaagcca tgctcaatga
ccgccgccgc ctggccctgg agaactacat caccgctctg 1440 caggctgttc
ctcctcggcc tcgtcacgtg ttcaatatgc taaagaagta tgtccgcgca 1500
gaacagaagg acagacagca caccctaaag catttcgagc atgtgcgcat ggtggatccc
1560 aagaaagccg ctcagatccg gtcccaggtt atgacacacc tccgtgtgat
ttatgagcgc 1620 atgaatcagt ctctctccct gctctacaac gtgcctgcag
tggccgagga gattcaggat 1680 gaagttgatg agctgcttca gaaagagcaa
aactattcag atgacgtctt ggccaacatg 1740 attagtgaac caaggatcag
ttacggaaac gatgctctca tgccatcttt gaccgaaacg 1800 aaaaccaccg
tggagctcct tcccgtgaat ggagagttca gcctggacga tctccagccg 1860
tggcattctt ttggggctga ctctgtgcca gccaacacag aaaacgaagt tgagcctgtt
1920 gatgcccgcc ctgctgccga ccgaggactg accactcgac caggttctgg
gttgacaaat 1980 atcaagacgg aggagatctc tgaagtgaag atggatgcag
aattccgaca tgactcagga 2040 tatgaagttc atcatcaaaa attggtgttc
tttgcagaag atgtgggttc aaacaaaggt 2100 gcaatcattg gactcatggt
gggcggtgtt gtcatagcga cagtgatcgt catcaccttg 2160 gtgatgctga
agaagaaaca gtacacatcc attcatcatg gtgtggtgga ggttgacgcc 2220
gctgtcaccc cagaggagcg ccacctgtcc aagatgcagc agaacggcta cgaaaatcca
2280 acctacaagt tctttgagca gatgcagaac aagaag 2316 59 772 PRT Homo
sapiens 59 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr
Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu
Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn
Met His Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro
Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu
Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn
Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys
Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110
Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115
120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val
Cys 130 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr
Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met
Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe
Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser
Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly
Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val
Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235
240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu
245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr
Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu
Glu Val Val Arg 275 280 285 Glu Val Cys Ser Glu Gln Ala Glu Thr Gly
Pro Cys Arg Ala Met Ile 290 295 300 Ser Arg Trp Tyr Phe Asp Val Thr
Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys Gly
Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met Ala
Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr 340 345 350 Thr
Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360
365 Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp
370 375 380 Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu
Glu Ala 385 390 395 400 Lys His Arg Glu Arg Met Ser Gln Val Met Arg
Glu Trp Glu Glu Ala 405 410 415 Glu Arg Gln Ala Lys Asn Leu Pro Lys
Ala Asp Lys Lys Ala Val Ile 420 425 430 Gln His Phe Gln Glu Lys Val
Glu Ser Leu Glu Gln Glu Ala Ala Asn 435 440 445 Glu Arg Gln Gln Leu
Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460 Leu Asn Asp
Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu 465 470 475 480
Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485
490 495 Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His
Phe 500 505 510 Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln
Ile Arg Ser 515 520 525 Gln Val Met Thr His Leu Arg Val Ile Tyr Glu
Arg Met Asn Gln Ser 530 535 540 Leu Ser Leu Leu Tyr Asn Val Pro Ala
Val Ala Glu Glu Ile Gln Asp 545 550 555 560 Glu Val Asp Glu Leu Leu
Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val 565 570 575 Leu Ala Asn Met
Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585 590 Leu Met
Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605
Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe 610
615 620 Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro
Val 625 630 635 640 Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr
Arg Pro Gly Ser 645 650 655 Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile
Ser Glu Val Lys Met Asp 660 665 670 Ala Glu Phe Arg His Asp Ser Gly
Tyr Glu Val His His Gln Lys Leu 675 680 685 Val Phe Phe Ala Glu Asp
Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700 Leu Met Val Gly
Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu 705 710 715 720 Val
Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val 725 730
735 Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met
740 745 750 Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu
Gln Met 755 760 765 Gln Asn Lys Lys 770 60 2259 DNA Homo sapiens 60
atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta
60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt
ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt
cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg
cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga
agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc
agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360
gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg
420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac
atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct
gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct
gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga
tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca
aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720
gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa
780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac
caccaccaca 840 gagtctgtgg aagaggtggt tcgagaggtg tgctctgaac
aagccgagac ggggccgtgc 900 cgagcaatga tctcccgctg gtactttgat
gtgactgaag ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaa
ccggaacaac tttgacacag aagagtactg catggccgtg 1020 tgtggcagcg
ccattcctac aacagcagcc agtacccctg atgccgttga caagtatctc 1080
gagacacctg gggatgagaa tgaacatgcc catttccaga aagccaaaga gaggcttgag
1140 gccaagcacc gagagagaat gtcccaggtc atgagagaat gggaagaggc
agaacgtcaa 1200 gcaaagaact tgcctaaagc tgataagaag gcagttatcc
agcatttcca ggagaaagtg 1260 gaatctttgg aacaggaagc agccaacgag
agacagcagc tggtggagac acacatggcc 1320 agagtggaag ccatgctcaa
tgaccgccgc cgcctggccc tggagaacta catcaccgct 1380 ctgcaggctg
ttcctcctcg gcctcgtcac gtgttcaata tgctaaagaa gtatgtccgc 1440
gcagaacaga aggacagaca gcacacccta aagcatttcg agcatgtgcg catggtggat
1500 cccaagaaag ccgctcagat ccggtcccag gttatgacac acctccgtgt
gatttatgag 1560 cgcatgaatc agtctctctc cctgctctac aacgtgcctg
cagtggccga ggagattcag 1620 gatgaagttg atgagctgct tcagaaagag
caaaactatt cagatgacgt cttggccaac 1680 atgattagtg aaccaaggat
cagttacgga aacgatgctc tcatgccatc tttgaccgaa 1740 acgaaaacca
ccgtggagct ccttcccgtg aatggagagt tcagcctgga cgatctccag 1800
ccgtggcatt cttttggggc tgactctgtg ccagccaaca cagaaaacga agttgagcct
1860 gttgatgccc gccctgctgc cgaccgagga ctgaccactc gaccaggttc
tgggttgaca 1920 aatatcaaga cggaggagat ctctgaagtg aagatggatg
cagaattccg acatgactca 1980 ggatatgaag ttcatcatca aaaattggtg
ttctttgcag aagatgtggg ttcaaacaaa 2040 ggtgcaatca ttggactcat
ggtgggcggt gttgtcatag cgacagtgat cgtcatcacc 2100 ttggtgatgc
tgaagaagaa acagtacaca tccattcatc atggtgtggt ggaggttgac 2160
gccgctgtca ccccagagga gcgccacctg tccaagatgc agcagaacgg ctacgaaaat
2220 ccaacctaca agttctttga gcagatgcag aacaagaag 2259 61 753 PRT
Homo sapiens 61 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp
Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly
Leu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu
Asn Met His Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp
Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile
Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr
Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp
Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105
110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu
115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp
Val Cys 130 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu
Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly
Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu
Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp
Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly
Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val
Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230
235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu
Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr
Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val
Glu Glu Val Val Arg 275 280 285 Glu Val Cys Ser Glu Gln Ala Glu Thr
Gly Pro Cys Arg Ala Met Ile 290 295 300 Ser Arg Trp Tyr Phe Asp Val
Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys
Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met
Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr 340 345 350
Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu 355
360 365 His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His
Arg 370 375 380 Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala
Glu Arg Gln 385 390 395 400 Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys
Ala Val Ile Gln His Phe 405 410 415 Gln Glu Lys Val Glu Ser Leu Glu
Gln Glu Ala Ala Asn Glu Arg Gln 420 425 430 Gln Leu Val Glu Thr His
Met Ala Arg Val Glu Ala Met Leu Asn Asp 435 440 445 Arg Arg Arg Leu
Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val 450 455 460 Pro Pro
Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg 465 470 475
480 Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val
485 490 495 Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln
Val Met 500 505 510 Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln
Ser Leu Ser Leu 515 520 525 Leu Tyr Asn Val Pro Ala Val Ala Glu Glu
Ile Gln Asp Glu Val Asp 530 535 540 Glu Leu Leu Gln Lys Glu Gln Asn
Tyr Ser Asp Asp Val Leu Ala Asn 545 550 555 560 Met Ile Ser Glu Pro
Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro 565 570 575 Ser Leu Thr
Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580 585 590 Glu
Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp 595 600
605 Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg
610 615 620 Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly
Leu Thr 625 630 635 640 Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys
Met Asp Ala Glu Phe 645 650 655 Arg His Asp Ser Gly Tyr Glu Val His
His Gln Lys Leu Val Phe Phe 660 665 670 Ala Glu Asp Val Gly Ser Asn
Lys Gly Ala Ile Ile Gly Leu Met Val 675 680 685 Gly Gly Val Val Ile
Ala Thr Val Ile Val Ile Thr Leu Val Met Leu 690 695 700 Lys Lys Lys
Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp 705 710 715 720
Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn 725
730 735 Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn
Lys 740 745 750 Lys 62 8 PRT Artificial Sequence Description of
Artificial Sequence synthetic 62 Leu Glu Val Leu Phe Gln Gly Pro 1
5 63 10 PRT Artificial Sequence Description of Artificial Sequence
synthetic 63 Ser Glu Val Asn Leu Asp Ala Glu Phe Arg 1 5 10 64 10
PRT Artificial Sequence Description of Artificial Sequence
synthetic 64 Ser Glu Val Lys Met Asp Ala Glu Phe Arg 1 5 10 65 15
PRT Artificial Sequence Description of Artificial Sequence
synthetic 65 Arg Arg Gly Gly Val Val Ile Ala Thr Val Ile Val Gly
Glu Arg 1 5 10 15 66 4 PRT Artificial Sequence Description of
Artificial Sequence Peptide 66 Asn Leu Asp Ala 1 67 8 PRT
Artificial Sequence Description of Artificial Sequence Peptide 67
Glu Val Lys Met Asp Ala Glu Phe 1 5 68 5 PRT Artificial Sequence
Description of Artificial Sequence Peptide 68 Gly Arg Arg Gly Ser 1
5 69 6 PRT Artificial Sequence Description of Artificial Sequence
Peptide 69 Thr Gln His Gly Ile Arg 1 5 70 6 PRT Artificial Sequence
Description of Artificial Sequence Peptide 70 Glu Thr Asp Glu Glu
Pro 1 5 71 15
PRT Artificial Sequence Description of Artificial Sequence Peptide
71 Met Cys Ala Glu Val Lys Met Asp Ala Glu Phe Lys Asp Asn Pro 1 5
10 15 72 5 PRT Artificial Sequence Description of Artificial
Sequence synthetic 72 Asp Ala Glu Phe Arg 1 5 73 5 PRT Artificial
Sequence Description of Artificial Sequence synthetic 73 Ser Glu
Val Asn Leu 1 5
* * * * *