U.S. patent application number 10/653517 was filed with the patent office on 2004-06-17 for human sel-10 polypeptides and polynucleotides that encode them.
Invention is credited to Gurney, Mark E., Li, Jinhe, Pauley, Adele M..
Application Number | 20040115700 10/653517 |
Document ID | / |
Family ID | 22081324 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040115700 |
Kind Code |
A1 |
Gurney, Mark E. ; et
al. |
June 17, 2004 |
Human sel-10 polypeptides and polynucleotides that encode them
Abstract
The present invention provides isolated nucleic acid molecules
comprising a polynucleotide encoding either of two alternative
splice variants of human sel-10, one of which is expressed in
hippocampal cells, and one of which is expressed in mammary cells.
The invention also provides isolated sel-10 polypeptides and cell
lines which express them in which A.beta. processing is
altered.
Inventors: |
Gurney, Mark E.; (Reykjavik,
IS) ; Pauley, Adele M.; (Plainwell, MI) ; Li,
Jinhe; (Kalamazoo, MI) |
Correspondence
Address: |
PHARMACIA & UPJOHN
301 HENRIETTA ST
0228-32-LAW
KALAMAZOO
MI
49007
US
|
Family ID: |
22081324 |
Appl. No.: |
10/653517 |
Filed: |
September 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10653517 |
Sep 2, 2003 |
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09213888 |
Dec 17, 1998 |
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6638731 |
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60068243 |
Dec 19, 1997 |
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Current U.S.
Class: |
435/6.16 ;
435/226; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 14/4702 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/226; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/64 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide
having a sequence at least 95% identical to a sequence selected
from the group consisting of: (a) a nucleotide sequence encoding a
human sel-10 polypeptide having the complete amino acid sequence
selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or as encoded by the cDNA
clone contained in ATCC Deposit No.98978; (b) a nucleotide sequence
encoding a human sel-10 polypeptide having the complete amino acid
sequence selected from the group consisting of SEQ ID NO:8, SEQ ID
NO:9, and SEQ ID NO:10, or as encoded by the cDNA clone contained
in ATCC Deposit No. 98979; and (c) a nucleotide sequence
complementary to the nucleotide sequence of (a) or (b).
2. An isolated nucleic acid molecule comprising polynucleotide
which hybridizes under stringent conditions to a polynucleotide
having the nucleotide sequence in (a), (b), or (c) of claim 1.
3. The nucleic acid molecule of claim 1, wherein said
polynucleotide of 1(a) encodes a human sel-10 polypeptide having
the complete amino acid sequence of SEQ ID NO:3.
4. The nucleic acid molecule of claim 3, wherein said
polynucleotide molecule of 1(a) comprises the nucleotide sequence
of residues 45-1928 of SEQ ID NO:1.
5. The nucleic acid molecule of claim 1, wherein said
polynucleotide of 1(a) encodes a human sel-10polypeptide having the
complete amino acid sequence of SEQ ID NO:4.
6. The nucleic acid molecule of claim 5, wherein said
polynucleotide molecule of 1(a) comprises the nucleotide sequence
of residues 150-1928 of SEQ ID NO:1.
7. The nucleic acid molecule of claim 1, wherein said
polynucleotide of 1(a) encodes a human sel-10 polypeptide having
the complete amino acid sequence of SEQ ID NO:5.
8. The nucleic acid molecule of claim 7, wherein said
polynucleotide molecule of 1(a) comprises the nucleotide sequence
of residues 267-1928 of SEQ ID NO:1.
9. The nucleic acid molecule of claim 1, wherein said
polynucleotide of 1(a) encodes a human sel-10 polypeptide having
the complete amino acid sequence of SEQ ID NO:6.
10. The nucleic acid molecule of claim 9, wherein said
polynucleotide molecule of 1(a) comprises the nucleotide sequence
of residues 291-1928 of SEQ ID NO:1.
11. The nucleic acid molecule of claim 1, wherein said
polynucleotide of 1(a) encodes a human sel-10 polypeptide having
the complete amino acid sequence of SEQ ID NO:7.
12. The nucleic acid molecule of claim 11, wherein said
polynucleotide molecule of 1(a) comprises the nucleotide sequence
of residues 306-1928 of SEQ ID NO:1.
13. The nucleic acid molecule of claim 1, wherein said
polynucleotide of 1(b) encodes a human sel-10 polypeptide having
the complete amino acid sequence of SEQ ID NO:8.
14. The nucleic acid molecule of claim 13 wherein said
polynucleotide molecule of 1(b) comprises the nucleotide sequence
of residues 180-1949 of SEQ ID NO:2.
15. The nucleic acid molecule of claim 1, wherein said
polynucleotide of 1(b) encodes a human sel-10polypeptide having the
complete amino acid sequence of SEQ ID NO:9.
16. The nucleic acid molecule of claim 15, wherein said
polynucleotide molecule of 1(b) comprises the nucleotide sequence
of residues 270-1949 of SEQ ID NO:2.
17. The nucleic acid molecule of claim 1, wherein said
polynucleotide of 1(b) encodes a human sel-10 polypeptide having
the complete amino acid sequence of SEQ ID NO:10.
18. The nucleic acid molecule of claim 17, wherein said
polynucleotide molecule of 1(b) comprises the nucleotide sequence
of residues 327-1949 of SEQ ID NO:2.
19. A vector comprising the nucleic acid molecule of claim 1.
20. The vector of claim 19, wherein said nucleic acid molecule of
claim 1 is operably linked to a promoter for the expression of a
sel-10 polypeptide.
21. A host cell comprising the vector of claim 19.
22. The host cell of claim 21, wherein said host is a eukaryotic
host.
23. A method of obtaining a sel-10 polypeptide comprising culturing
the host cell of claim 22 and isolating said sel-10
polypeptide.
24. An isolated sel-10 polypeptide comprising (a) an amino acid
sequence selected from the group consisting of SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or as encoded by
the cDNA clone contained in ATCC Deposit No. 98978; (b) an amino
acid sequence selected from the group consisting of SEQ ID NO:8,
SEQ ID NO:9, and SEQ ID NO:10, or as encoded by the cDNA clone
contained in ATCC Deposit No. 98979.
25. The isolated sel-10 polypeptide of claim 24, wherein said
polypeptide comprises the amino acid sequence of SEQ ID NO:3.
26. The isolated sel-10 polypeptide of claim 24, wherein said
polypeptide comprises the amino acid sequence of SEQ ID NO:4.
27. The isolated sel-10 polypeptide of claim 24, wherein said
polypeptide comprises the amino acid sequence of SEQ ID NO:5.
28. The isolated sel-10 polypeptide of claim 24, wherein said
polypeptide comprises the amino acid sequence of SEQ ID NO:6.
29. The isolated sel-10 polypeptide of claim 24, wherein said
polypeptide comprises the amino acid sequence of SEQ ID NO:7.
30. The isolated sel-10 polypeptide of claim 24, wherein said
polypeptide comprises the amino acid sequence of SEQ ID NO:8.
31. The isolated sel-10 polypeptide of claim 24, wherein said
polypeptide comprises the amino acid sequence of SEQ ID NO:9.
32. The isolated sel-10 polypeptide of claim 24, wherein said
polypeptide comprises the amino acid sequence of SEQ ID NO:10.
33. An isolated antibody that binds specifically to the sel-10
polypeptide of claim 24.
34. A cell line having altered A.beta. processing that expresses
any of the sel-10 isolated nucleic acid molecules of claim 1.
35. The cell line of claim 34, wherein said A.beta. processing is
increased.
36. The cell line of claim 34, wherein said A.beta. processing is
decreased.
37. The cell line of claim 34, wherein said cell line is
6myc-N-sel10/2.
38. The cell line of claim 34, wherein said cell line is
6myc-N-sel10/6.
39. A method for the identification of an agent capable of altering
the ratio of A.beta..sub.1-40/A.beta..sub.1-40+A.beta..sub.1-42
produced in any of the cell lines of claims 34, 37, and 38,
comprising the steps of: (a) obtaining a test culture and a control
culture of said cell line; (b) contacting said test culture with a
test agent; (c) measuring the levels of A.beta..sub.1-40 and
A.beta..sub.1-42 produced by said test culture of step (b) and said
control culture; (d) calculating the ratio of
A.beta..sub.1-40/A.beta..sub.1-40+A.beta..sub.1-42 for said test
culture and said control culture from the levels of
A.beta..sub.1-40 and A.beta..sub.1-42 measured in step (c); and (e)
comparing the ratio of
A.beta..sub.1-40/A.beta..sub.1-40+A.beta..sub.1-42 measured for
said test culture and said control culture in step (d); whereby a
determination that the ratio of
A.beta..sub.1-40/A.beta..sub.1-40+A.beta..sub.1-42 for said test
culture is higher or lower than ratio of
A.beta..sub.1-40/A.beta..sub.1-40+A.beta..sub.1-42 for said control
culture indicates that said test agent has altered the ratio of
A.beta..sub.1-40/A.beta..sub.1-40+A.beta..sub.1-42.
40. The method of claim 39, wherein said ratio of
A.beta..sub.1-40/A.beta.- .sub.1-40+A.beta..sub.1-42 is increased
by said test agent.
41. The method of claim 39, wherein said ratio of
A.beta..sub.1-40/A.beta.- .sub.1-40+A.beta..sub.1-42 is decreased
by said test agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/213,888, filed Dec. 17, 1998, which claims the benefit of the
following provisional application: U.S. Ser. No. 60/068,243, filed
19 Dec. 1997, under 35 USC 119(e)(1).
FIELD OF THE INVENTION
[0002] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding either of two
alternative splice variants of human sel-10, one of which is
expressed in hippocampal cells, and one of which is expressed in
mammary cells. The invention also provides isolated sel-10
polypeptides.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) is a degenerative disorder of the
central nervous system which causes progressive memory and
cognitive decline during mid to late adult life. The disease is
accompanied by a wide range of neuropathologic features including
extracellular amyloid plaques and intra-neuronal neurofibrillary
tangles. (Sherrington, R., et al.; Nature 375: 754-60 (1995)).
Although the pathogenic pathway leading to AD is not well
understood, several genetic loci are known to be involved in the
development of the disease.
[0004] Genes associated with early onset Alzheimer's disease (AD)
have been identified by the use of mapping studies in families with
early-onset AD. These studies have shown that genetic loci on
chromosomes 1 and 14 were likely to be involved in AD. Positional
cloning of the chromosome 14 locus identified a novel mutant gene
encoding an eight-transmembrane domain protein which subsequently
was named presenilin-1 (PS-1). (Sherrington, R., et al.; Nature
375: 754-60 (1995)). Blast search of the human EST database
revealed a single EST exhibiting homology to PS-1, designated
presenilin-2 (PS-2) which was shown to be the gene associated with
AD on chromosome 1. (Levy-Lahad, E. et al., Science 269:973-977
(1995); Rogaev, E. I., et al., Nature 376: 775-8 (1995); Li, J. et
al., Proc. Natl. Acad. Sci. U.S.A. 92: 12180-12184 (1995)). altered
by the point mutations found in familial Alzheimer's disease
[Perez-Tur, J. et al., Neuroreport 7: 297-301 (1995); Mercken, M.
et al., FEBS Lett. 389: 297-303 (1996)]. PS-1 gene expression is
widely distributed across tissues, while the highest levels of PS-2
mRNA are found in pancreas and skeletal muscle. (Li, J. et al.,
Proc. Natl. Acad. Sci. U.S.A. 92: 12180-12184 (1995); Jinhe Li,
personal communication). The highest levels of PS-2 protein,
however, are found in brain (Jinhe Li, personal communication).
Both PS-1 and PS-2 proteins have been localized to the endoplasmic
reticulum, the Golgi apparatus, and the nuclear envelope. (Jinhe
Li, personal communication; Kovacs, D. M. et al., Nat. Med.
2:224-229 (1996); Doan, A. et al., Neuron 17: 1023-1030 (1996)).
Mutations in either the PS-1 gene or the PS-2 gene alter the
processing of the amyloid protein precursor (APP) such that the
ratio of A-beta.sub.1-42 is increased relative to A-beta.sub.1-40
(Scheuner, D. et al., Nat. Med. 2: 864-870 (1996)). When
coexpressed in transgenic mice with human APP, a similar increase
in the ratio of A-beta.sub.1-42 as compared to A-beta.sub.1-40 is
observed (Borchelt, D. R. et al., Neuron 17: 1005-1013 (1996);
Citron, M. et al., Nat. Med. 3: 67-72 (1997); Duff, K. et al.,
Nature 383: 710-713 (1996)), together with an acceleration of the
deposition of A-beta in amyloid plaques (Borchelt et al., Neuron
19: 939 (1997).
[0005] Despite the above-described observations made with respect
to the role of PS-1 and PS-2 in AD, their biological function
remains unknown, placing them alongside a large number of human
disease genes having an unknown biological function. Where the
function of a gene or its product is unknown, genetic analysis in
model organisms can be useful in placing such genes in known
biochemical or genetic pathways. This is done by screening for
extragenic mutations that either suppress or enhance the effect of
mutations in the gene under analysis. For example, extragenic
suppressors of loss-of-function mutations in a disease gene may
turn on the affected genetic or biochemical pathway downstream of
the mutant gene, while suppressers of gain-of-function mutations
will probably turn the pathway off.
[0006] One model organism that can be used in the elucidation of
the function of the presenilin genes is C. elegans, which contains
three genes having homology to PS-1 and PS-2, with sel-12 having
the highest degree of homology to the genes encoding the human
presenilins. Sel-12 was discovered in a screen for genetic
suppressers of an activated notch receptor, lin-12(d) (Levitan, D.
et al., Nature 377: 351-354 (1995)). Lin-12 functions in
development to pattern cell lineages. Hypermorphic mutations such
as lin-12(d), which increase lin-12 activity, cause a
"multi-vulval" phenotype, while hypomorphic mutations which
decrease activity cause eversion of the vulva, as well as homeotic
changes in several other cell lineages (Greenwald, I., et al.,
Nature 346: 197-199 (1990); Sundaram, M. et al., Genetics 135:
755-763 (1993)). Sel-12 mutations suppress hypermorphic lin-12(d)
mutations, but only if the lin-12(d) mutations activate signaling
by the intact lin-12(d) receptor (Levitan, D. et al., Nature 377:
351-354 (1995)). Lin-12 mutations that truncate the cytoplasmic
domain of the receptor also activate signaling (Greenwald, I., et
al., Nature 346: 197-199 (1990)), but are not suppressed by
mutations of sel-12 (Levitan, D. et al., Nature 377: 351-354
(1995)). This implies that sel-12 mutations act upstream of the
lin-12 signaling pathway, perhaps by decreasing the amount of
functional lin-12 receptor present in the plasma membrane. In
addition to suppressing certain lin-12 hypermorphic mutations,
mutations to sel-12 cause a loss-of-function for egg laying, and
thus internal accumulation of eggs, although the mutants otherwise
appear anatomically normal (Levitan, D. et al., Nature 377: 351-354
(1995)). Sel-12 mutants can be rescued by either human PS-1 or
PS-2, indicating that sel-12, PS-1 and PS-2 are functional
homologues (Levitan, D., et al., Proc. Natl. Acad. Sci. U.S.A 93:
14940-14944 (1996)).
[0007] A second gene, sel-10, has been identified in a separate
genetic screen for suppressors of lin-12 hypomorphic mutations.
Loss-of-function mutations in sel-10 restore signaling by lin-12
hypomorphic mutants. As the lowering of sel-10 activity elevates
lin-12 activity, it can be concluded that sel-10 acts as a negative
regulator of lin-12 signaling. Sel-10 also acts as a negative
regulator of sel-12, the C. elegans presenilin homologue
(Levy-Lahad, E. et al., Science 269:973-977 (1995)). Loss of sel-10
activity suppresses the egg laying defect associated with
hypomorphic mutations in sel-12 (Iva Greenwald, personal
communication). The effect of loss-of-function mutations to sel-10
on lin-12 and sel-12 mutations indicates that sel-10 acts as a
negative regulator of both lin-12/notch and presenilin activity.
Thus, a human homologue of C. elegans sel-10 would be expected to
interact genetically and/or physiologically with human presenilin
genes in ways relevant to the pathogenesis of Alzheimer's
Disease.
[0008] In view of the foregoing, it will be clear that there is a
continuing need for the identification of genes related to AD, and
for the development of assays for the identification of agents
capable of interfering with the biological pathways that lead to
AD.
Information Disclosure
[0009] Hubbard E J A, Wu G, Kitajewski J, and Greenwald I (1997)
Sel-10, a negative regulator of lin-12 activity in Caenorhabditis
elegans, encodes a member of the CDC4 family of proteins. Genes
& Dev 11:3182-3193.
[0010] Greenwald-I; Seydoux-G (1990) Analysis of gain-of-function
mutations of the lin-12 gene of Caenorhabditis elegans. Nature.
346: 197-9
[0011] Kim T-W, Pettingell W H, Hallmark O G, Moir R D, Wasco W,
Tanzi R (1997) Endoproteolytic cleavage and proteasomal degradation
of presenilin 2 in transfected cells. J Biol Chem
272:11006-11010.
[0012] Levitan-D; Greenwald-I (1995) Facilitation of
lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182
Alzheimer's disease gene. Nature. 377: 351-4.
[0013] Levitan-D; Doyle-T G; Brousseau-D; Lee-M K; Thinakaran-G;
Slunt-H H; Sisodia-S S; Greenwald-I (1996) Assessment of normal and
mutant human presenilin function in Caenorhabditis elegans. Proc.
Natl. Acad. Sci. U.S.A. 93: 14940-4.
[0014] Sundaram-M; Greenwald-I (1993) Suppressors of a lin-12
hypomorph define genes that interact with both lin-12 and glp-1 in
Caenorhabditis elegans. Genetics. 135: 765-83.
[0015] Sundaram-M; Greenwald-I (1993) Genetic and phenotypic
studies of hypomorphic lin-12 mutants in Caenorhabditis elegans.
Genetics. 135: 755-63.
[0016] F55B12.3 GenPep Report (WMBL locus CEF55B12, accession
z79757).
SUMMARY OF THE INVENTION
[0017] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding human sel-10, which
is expressed in hippocampal cells and in mammary cells. Unless
otherwise noted, any reference herein to sel-10 will be understood
to refer to human sel-10, and to encompass both hippocampal and
mammary sel-10. Fragments of hippocampal sel-10 and mammary sel-10
are also provided.
[0018] In a preferred embodiment, the invention provides an
isolated nucleic acid molecule comprising a polynucleotide having a
sequence at least 95% identical to a sequence selected from the
group consisting of:
[0019] (a) a nucleotide sequence encoding a human sel-10
polypeptide having the complete amino acid sequence selected from
the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ
ID NO:6, and SEQ ID NO:7, or as encoded by the cDNA clone contained
in ATCC Deposit No.98978;
[0020] (b) a nucleotide sequence encoding a human sel-10
polypeptide having the complete amino acid sequence selected from
the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10,
or as encoded by the cDNA clone contained in ATCC Deposit No.
98979; and
[0021] (c) a nucleotide sequence complementary to the nucleotide
sequence of (a) or (b).
[0022] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent conditions to a polynucleotide encoding sel-10, or
fragments thereof.
[0023] 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 sel-10 polypeptide comprising culturing the
above-described host cell and isolating the sel-10 polypeptide.
[0024] In another aspect, the invention provides isolated sel-10
polypeptides, as well as fragments thereof. In a preferred
embodiment, the sel-10 polypeptides have an amino acid sequence
selected from the group consisting of SEQ ID NO:3, 4, 5, 6, 7, 8,
9, and 10. Isolated antibodies, both polyclonal and monoclonal,
that bind specifically to sel-10 polypeptides are also
provided.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIGS. 1A and 1B: FIGS. 1A and 1B are western blots showing
protein expression in HEK293 cells transfected with PS1-C-FLAG,
6-myc-N-sel-10, and APP695NL-KK cDNAs.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding human sel-10. The
nucleotide sequence of human hippocampal sel-10 (hhsel-10), which
sequence is given in SEQ ID NO:1, encodes five hhsel-10
polypeptides (hhsel-10-(1), hhsel-10-(2), hhsel-10-(3),
hhsel-10-(4), and hhsel-10-(5), referred to collectively herein as
hhsel-10). The nucleotide sequence of human mammary sel-10
(hmsel-10), which sequence is given in SEQ ID NO:2, encodes three
hmsel-10 polypeptides (hmSel-10-(1), hmSel-10-(2), and
hmsel-10-(3), referred to collectively herein as hmsel-10). The
nucleotide sequences of the hhsel-10 polynucleotides are given in
SEQ ID NO. 1, where nucleotide residues 45-1928 of SEQ ID NO. 1
correspond to hhsel-10-(1), nucleotide residues 150-1928 of SEQ ID
NO. 1 correspond to hhSel-10-(2), nucleotide residues 267-1928 of
SEQ ID NO. 1 correspond to hhSel-10-(3), nucleotide residues
291-1928 of SEQ ID NO. 1 correspond to hhSel-10-(4), and nucleotide
residues 306-1928 of SEQ ID NO. 1 correspond to hhSel-10-(5). The
nucleotide sequences of the hmSel-10 polynucleotides are given in
SEQ ID NO. 2, where nucleotide residues 180-1949 of SEQ ID NO. 2
correspond to hmSel-10-(1), nucleotide residues 270-1949 of SEQ ID
NO. 2 correspond to hmSel-10-(2), and nucleotide residues 327-1949
of SEQ ID NO. 2 correspond to hmSel-10-(3). The amino acid
sequences of the polypeptides encoded by the hhSel-10 and hm-Sel-10
nucleic acid molecules are given as follows: SEQ ID NOS: 3, 4, 5,
6, and 7 correspond to the hhSel-10-(1), hhSel-10-(2),
hhSel-10-(3). hhSel-10-(4), and hhSel-10-(5) polypeptides,
respectively, and SEQ ID NOS: 8, 9, and 10 correspond to the
hmSel-10-(1), hmSel-10-(2), and hmSel-10-(3) polypeptides,
respectively. Unless otherwise noted, any reference herein to
sel-10 will be understood to refer to human sel-10, and to
encompass all of the hippocampal and mammary sel-10 nucleic acid
molecules (in the case of reference to sel-10 nucleic acid,
polynucleotide, DNA, RNA, or gene) or polypeptides (in the case of
reference to sel-10 protein, polypeptide, amino acid sequnce).
Fragments of hippocampal sel-10 and mammary sel-10 nucleic acid
molecules and polypeptides are also provided.
[0027] The nucleotide sequence of SEQ ID NO:1 was obtained as
described in Example 1, and is contained in cDNA clone PNV 102-1,
which was deposited on Nov. 9, 1998, at the American Type Culture
Collection, 10801 University Blvd., Manassas, Va. 20110, and given
accession number 98978. The nucleotide sequence of SEQ ID NO:2 was
obtained as described in Example 1, and is contained in cDNA clone
PNV 108-2, which was deposited on Nov. 9, 1998, at the American
Type Culture Collection, 10801 University Blvd., Manassas, Va.
20110, and given accession number 98979.
[0028] The human sel-10 polypeptides of the invention share
homology with C. elegans sel-10, as well as with members of the
.beta.-transducin protein family, including yeast CDC4, and human
LIS-1. This family is characterized by the presence of an F-box and
multiple WD-40 repeats (Li, J., et al., Proc. Natl. Acad. Sci.
U.S.A. 92:12180-12184 (1995)). The repeats are 20-40 amino acids
long and are bounded by gly-his (GH) and trp-asp (WD) residues. The
three dimensional structure of .beta.-transducin indicates that the
WD40 repeats form the arms of a seven-bladed propeller like
structure (Sondek, J., et al., Nature 379:369-374 (1996)). Each
blade is formed by four alternating pleats of beta-sheet with a
pair of the conserved aspartic acid residues in the protein motif
forming the limits of one internal beta strand. WD40 repeats are
found in over 27 different proteins which represent diverse
functional classes (Neer, E. J., et al., Nature 371:297-300
(1994)). These regulate cellular functions including cell division,
cell fate determination, gene transcription, signal transduction,
protein degradation, mRNA modification and vesicle fusion. This
diversity in function has led to the hypothesis that
.beta.-transducin family members provide a common scaffolding upon
which multiprotein complexes can be assembled.
[0029] The nucleotide sequence given in SEQ ID NO:1 corresponds to
the nucleotide sequence encoding hhsel-10, while the nucleotide
sequence given in SEQ ID NO:2 corresponds to the nucleotide
sequence encoding hmsel-10. The isolation and sequencing of DNA
encoding sel-10 is described below in Examples 1 and 2.
[0030] As is described in Examples 1 and 2, automated sequencing
methods were used to obtain the nucleotide sequence of sel-10. The
sel-10 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
sel-10 DNA of the present invention includes cDNA, chemically
synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations
thereof. Genomic sel-10 DNA may be obtained by screening a genomic
library with the sel-10 cDNA described herein, using methods that
are well known in the art. RNA transcribed from sel-10 DNA is also
encompassed by the present invention.
[0031] 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 sel-10
polypeptides of the invention, wherein said polynucleotide sequence
encodes a sel-10 polypeptide having the complete amino acid
sequence of SEQ ID NOs:3-10, or fragments thereof.
[0032] Also provided herein are purified sel-10 polypeptides, both
recombinant and non-recombinant. Variants and derivatives of native
sel-10 proteins that retain any of the biological activities of
sel-10 are also within the scope of the present invention. As is
described above, the sel-10 polypeptides of the present invention
share homology with yeast CDC4. As CDC4 is known to catalyze
ubiquitination of specific cellular proteins (Feldman et al., Cell
91:221 (1997)), it may be inferred that sel-10 will also have this
activity. Assay procedures for demonstrating such activity are well
known, and involve reconstitution of the ubiquitinating system
using purified human sel-10 protein together with the yeast
proteins Cdc4p, Cdc53p and Skp1p, or their human orthologs, and an
E1 enzyme, the E2 enzyme Cdc34p or its human ortholog, ubiquitin, a
target protein and an ATP regenerating system (Feldman et al.,
1997). Skp1p associates with Cdc4p through a protein domain called
an F-box (Bai et al., Cell 86:263 (1996)). The F-box protein motif
is found in yeast CDC4, C. elegans sel-10, mouse sel-10 and human
sel-10. The sel-10 ubiquitination system may be reconstituted with
the C. elegans counterparts of the yeast components, e.g., cu1-1
(also known as lin-19) protein substituting for Cdc53p (Kipreos et
al., Cell 85:829 (1996)) and the protein F46A9 substituting for
Skp1p, or with their mammalian counterparts, e.g., Cu1-2 protein
substituting for Cdc53p (Kipreos et al., 1996) and mammalian Skp1p
substituting for yeast Skp1p. A phosphorylation system provided by
a protein kinase is also included in the assay system as per
Feldman et al., 1997.
[0033] Sel-10 variants may be obtained by mutation of native
sel-10-encoding nucleotide sequences, for example. A sel-10
variant, as referred to herein, is a polypeptide substantially
homologous to a native sel-10 but which has an amino acid sequence
different from that of native sel-10 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 sel-10 sequence. Thus, a variant nucleotide sequence which
contains, for example, 5 point mutations for every one hundred
nucleotides, as compared to a native sel-10 gene, will be 95%
identical to the native protein. The percentage of sequence
identity, also termed homology, between a native and a variant
sel-10 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)).
[0034] 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.
[0035] Sel-10 variants within the scope of the invention may
comprise conservatively substituted sequences, meaning that one or
more amino acid residues of a sel-10 polypeptide are replaced by
different residues that do not alter the secondary and/or tertiary
structure of the sel-10 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 may be found in Bowie et
al., Science 247:1306-1310 (1990). Other sel-10 variants which
might retain substantially the biological activities of sel-10 are
those where amino acid substitutions have been made in areas
outside functional regions of the protein.
[0036] 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/C for about 2.5 hours in
6.times.SSC/0.1% SDS, followed by washing of the filters in
1.0.times.SSC at 65/C, 0.1% SDS.
[0037] Fragments of the sel-10-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 sel-10 nucleic acids in in vitro
assays, as well as in Southern and northern blots. Cell types
expressing sel-10 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 sel-10 nucleic acid molecule are employed to
isolate and amplify that sequence using conventional
techniques.
[0038] Other useful fragments of the sel-10 nucleic acid molecules
are antisense or sense oligonucleotides comprising a
single-stranded nucleic acid sequence capable of binding to a
target sel-10 mRNA (using a sense strand), or sel-10 DNA (using an
antisense strand) sequence.
[0039] In another aspect, the invention includes sel-10
polypeptides with or without associated native pattern
glycosylation. Sel-10 expressed in yeast or mammalian expression
systems (discussed below) may be similar to or significantly
different from a native sel-10 polypeptide in molecular weight and
glycosylation pattern. Expression of sel-10 in bacterial expression
systems will provide non-glycosylated sel-10.
[0040] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. Sel-10 polypeptides may be recovered and purified from
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 and lectin chromatography. In a
preferred embodiment, high performance liquid chromatography (HPLC)
is employed for purification.
[0041] 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 sel-10
polypeptides expressed from the polynucleotide molecules described
above, vectors for the expression of sel-10 are preferred. The
vectors include DNA encoding any of the sel-10 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 sel-10. Thus, a
promoter nucleotide sequence is operably linked to a sel-10 DNA
sequence if the promoter nucleotide sequence directs the
transcription of the sel-10 sequence.
[0042] Selection of suitable vectors to be used for the cloning of
polynucleotide molecules encoding sel-10, or for the expression of
sel-10 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 sel-10 polypeptide is to be expressed.
Suitable host cells for expression of sel-10 polypeptides include
prokaryotes, yeast, and higher eukaryotic cells, each of which is
discussed below.
[0043] The sel-10 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 in-frame to the
sel-10 sequence so that sel-10 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
sel-10 polypeptide. Preferably, the signal sequence will be cleaved
from the sel-10 polypeptide upon secretion of sel-10 from the cell.
Non-limiting examples of signal sequences that can be used in
practicing the invention include the yeast I-factor and the
honeybee melatin leader in sf9 insect cells.
[0044] In a preferred embodiment, the sel-10 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 sel-10
polypeptide may be modified to comprise a peptide to form a fusion
protein which specifically binds to a binding partner, or peptide
tag. Non-limiting examples of such peptide tags include the 6-His
tag, thioredoxin tag, FLAG 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. These tags may be
recognized by fluorescein or rhodamine labeled antibodies that
react specifically with each type of tag
[0045] Suitable host cells for expression of sel-10 polypeptides
include prokaryotes, yeast, and higher eukaryotic cells. Suitable
prokaryotic hosts to be used for the expression of sel-10 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 sel-10
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 sel-10 polypeptide.
[0046] Expression vectors for use in prokaryotic hosts generally
comprise 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), and pQE vectors (Qiagen).
[0047] Sel-10 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.
[0048] 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 sel-10 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 sel-10-encoding
nucleotide sequence.
[0049] Insect host cell culture systems may also be used for the
expression of Sel-10 polypeptides. In a preferred embodiment, the
sel-10 polypeptides of the invention are expressed using a
baculovirus expression system. Further information regarding the
use of baculovirus systems for the expression of heterologous
proteins in insect cells are reviewed by Luckow and Summers,
Bio/Technology 6:47 (1988).
[0050] In another preferred embodiment, the sel-10 polypeptide is
expressed in mammalian host cells. Non-limiting examples of
suitable mammalian cell lines include the COS-7 line of monkey
kidney cells (Gluzman et al., Cell 23:175 (1981)) and Chinese
hamster ovary (CHO) cells.
[0051] The choice of a suitable expression vector for expression of
the sel-10 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).
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.
[0052] The polypeptides of the present invention may also be used
to raise polyclonal and monoclonal antibodies, which are useful in
diagnostic assays for detecting sel-10 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, N.Y.
(1980).
[0053] The sel-10 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. 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.
[0054] The sel-10 polypeptides of the invention, and the DNA
encoding them, may also be used to further elucidate the biological
mechanism of AD, and may ultimately lead to the identification of
compounds that can be used to alter such mechanisms. The sel-10
polypeptides of the invention are 47.6% identical and 56.7% similar
to C. elegans sel-10. As is described above, mutations to C.
elegans sel-10 are known to suppress mutations to sel-12 that
result in a loss-of-function for egg laying, and also to suppress
certain hypomorphic mutations to lin-12. Mutations to C. elegans
sel-12 can also be rescued by either of the human AD-linked genes
PS-1 (42.7% identical to sel-12) or PS-2 (43.4% identical to
sel-12). However, human PS-1 with a familial AD-linked mutant has a
reduced ability to rescue sel-12 mutants (Levitan, D. et al., Proc.
Natl. Acad. Sci. USA 93: 14940-14944 (1996)).
[0055] This demonstrated interchangeability of human and C. elegans
genes in the notch signaling pathway makes it reasonable to predict
that mutations of human sel-10 will suppress mutations to PS-1 or
PS-2 that lead to AD, especially in light of the predicted
structure of sel-10. As described above, PS-1 and PS-2 mutations
that lead to AD are those which interfere with the proteolytic
processing of PS-1 or PS-2. The sel-10 polypeptides of the
invention are members of the .beta.-transducin protein family,
which includes yeast CDC4, a component of an enzyme which functions
in the ubiquitin-dependent protein degradation pathway. Thus, human
sel-10 may regulate presenilin degradation via the
ubiquitin-proteasome pathway. Alternatively, or in addition, human
sel-10 may alter presenilin function by targeting for degradation
through ubiquitination a modulator of presenilin activity, e.g., a
negative regulator. Therefore, mutations to sel-10 may reverse the
faulty proteolytic processing of PS-1 or PS-2 which occurs as a
result of mutation to PS-1 or PS-2 or otherwise increase presenilin
function. For the same reason, inhibition of sel-10 activity may
also act to reverse PS-1 or PS-2 mutations. Thus, it may be
hypothesized that compounds which inhibit either the expression or
the activity of the human sel-10 polypeptides of the invention may
reverse the effects of mutations to PS-1 or PS-2, and thus be
useful for the prevention or treatment of AD.
[0056] Thus, C. elegans may be used as a genetic system for the
identification of agents capable of inhibiting the activity or
expression of the human sel-10 polypeptides of the invention. A
suitable C. elegans strain for use in such assays lacks a gene
encoding active C. elegans sel-10, and exhibits a loss-of-function
for egg-laying resulting from an inactivated sel-12 gene.
Construction of C. elegans strains having a loss-of-function for
egg-laying due to mutation of sel-12 may be accomplished using
routine methods, as both the sequence of sel-12 (Genebank accession
number U35660) and mutations to sel-12 resulting in a
loss-of-function for egg laying are known (see Levitan et al.,
Nature 377: 351-354 (1995), which describes construction of C.
elegans sel-12(ar171)). An example of how to make such a strain is
also given in Levitan et al. (Nature 377: 351-354 (1995)).
Wild-type C. elegans sel-10 in the C. elegans sel-12(ar171)) , is
also mutagenized using routine methods, such as the technique used
for sel-12 mutagenesis in Levitan et al., supra.
[0057] In order to identify compounds inhibiting human sel-10
activity, a DNA vector containing a human sel-10 gene encoding any
of the wild-type human sel-10 proteins of the invention is
introduced into the above-described C. elegans strain. In a
preferred embodiment, the heterologous human sel-10 gene is
integrated into the C. elegans genome. The gene is then expressed,
using techniques described in Levitan et al. (Proc. Natl. Acad.
Sci. USA 93: 14940-14944 (1996)). Test compounds are then
administered to this strain in order to determine whether a given
agent is capable of inhibiting sel-10 activity so as to suppress
mutations to sel-12 or lin-12 that result in egg-laying defects.
Egg-laying in this strain is then determined, e.g. by the assay
described in Levitan et al. (Proc. Natl. Acad. Sci. USA 93:
14940-14944 (1996)). To confirm that the compound's effect on
egg-laying is due to inhibition of sel-10 activity, the action of
the compound can be tested in a second biochemical or genetic
pathway that is known to be affected by loss-of-function mutations
in sel-10 (e.g., further elevation of lin-12 activity in lin-12(d)
hypomorphic strains). Such assays may be performed as described in
Sundarem and Greenwald (Genetics 135: 765-783 (1993)).
[0058] Alternatively, compounds are tested for their ability to
inhibit the E3 Ubiquitin Ligating Enzyme. Assays procedures for
demonstrating such activity are well known, and involve
reconstitution of the ubiquitinating system using purified human
sel-10 protein together with the yeast proteins Cdc4p, Cdc53p and
Skp1p and an E1 enzyme, the E2 enzyme Cdc34p, ubiquitin, a target
protein and an ATP regenerating system (Feldman et al., 1997). The
sel-10 ubiquitination system may also be reconstituted with the C.
elegans counterparts of the yeast components, e.g., cu1-1 (also
known as lin-19) protein substituting for Cdc53p (Kipreos et al.,
Cell 85:829 (1996)) and the protein F46A9 substituting for Skp1p,
or with their mammalian counterparts, e.g., Cu1-2 protein
substituting for Cdc53p (Kipreos et al., ibid.) and mammalian Skp1p
substituting for yeast Skp1p. A phosphorylation system provided by
a protein kinase is also to be included in the assay system as per
Feldman et al., 1997.
[0059] Alternatively, cell lines which express human sel-10 due to
transformation with a human sel-10 cDNA and which as a consequence
have elevated APP processing and formation of A.beta..sub.1-40 or
A.beta..sub.1-42 may also be used for such assays as in Example 3.
Compounds may be tested for their ability to reduce the elevated
A.beta. processing seen in the sel-10 transformed cell line.
[0060] Compounds that rescue the egg-laying defect or that inhibit
E3 Ubiquitin Ligating Enzyme are then screened for their ability to
cause a reduction in the production of A-beta.sub.1-40 or
A-beta.sub.1-42 in a human cell line. Test compounds are used to
expose IMR-32 or other human cell lines known to produce
A-beta.sub.1-40 or A-beta.sub.1-42 (Asami-Okada et al.,
Biochemistry 34: 10272-10278 (1995)), or in human cell lines
engineered to express human APP at high levels. In these assays,
A-beta.sub.1-40 or A-beta.sub.1-42 is measured in cell extracts or
after release into the medium by ELISA or other assays which are
known in the art (Borchelt et al., Neuron 17: 1005-1013 (1996);
Citron et al., Nat. Med. 3: 67-72 (1997)).
[0061] 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.
EXAMPLES
Example 1
Identification of a Human Homologue to C. elegans sel-10
[0062] Results
[0063] Identification of sel-10 in ACEDB: Sel-10 maps between the
cloned polymorphisms arP3 and TCPARI just to the left of him-5
[ACEDB entry wm95p536]. Three phage lambda clones have been
sequenced across the interval, F53C11, F09F3, and F55B12. Sel-10 is
reported to have homology to yeast cdc4 [ACEDB entry wm97ab259].
Blast search revealed a single ORF with homology to yeast cdc4
(CC4_YST) within the interval defined by arP3 and TCPARI
corresponding to the GenPep entry F55B12.3. F55B12.3, like yeast
cdc4, is a member of the .beta.-transducin protein family. This
family is characterized by the presence of multiple WD40 repeats
[Neer, E. J. et al., Nature 371: 297-300 (1994)].
[0064] Identification of a human sel-10 homologue, Incyte 028971:
The GenPep entry F55B12.3 was used to search the LifeSeq, LifeSeq
FL and EMBL data bases using tblastn. The search revealed multiple
homologies to .beta.-transducin family members including LIS-1
(S36113 and P43035), a gene implicated in Miller-Dieker
lissencephaly, a Xenopus laevis gene, TRCPXEN (U63921), and a human
contig in LifeSeq FL, 028971. Since there also are multiple
.beta.-transducin family members within the C. elegans genome,
these were collected using multiple blast searches and then
clustered with the sel-10 candidate genes. Multiple alignments were
performed with the DNAStar program Megalign using the Clustal
method. This revealed that LIS-1 clustered with T03F6.F, a
different .beta.-transducin family member and thus excluded it as a
candidate sel-10 homologue. TRCPXEN clustered with K10B2.1, a gene
which also clusters with F55B12.3 and CC4YST, while Incyte 028971
clustered with sel-10. Thus, Incyte 028971 appears to encode the
human homologue of C. elegans sel-10. Sequence homology between
sel-10 and 028971 is strongest in the region of the protein
containing 7 repeats of the WD40 motif. The Incyte 028971 contig
contains 44 ESTs from multiple libraries including pancreas, lung,
T-lymphocytes, fibroblasts, breast, hippocampus, cardiac muscle,
colon, and others.
[0065] Public EST: Blastx searches with the DNA sequence 028971
against the TREMBLP dataset identified a single homologous mouse
EST (W85144) from the IMAGE Library, Soares mouse embryo
NbME13.5-14.5. The blastx alignment of 028971 with W85144 and then
with F55B12.3 revealed a change in reading frame in 028971 which
probably is due to a sequencing error.
[0066] Blastn searches of the EMBL EST database with the 028971 DNA
sequence revealed in addition to W85144, three human EST that align
with the coding sequence of 028971 and six EST that align with the
3' untranslated region of the 028971 sequence.
[0067] Protein Motifs: Two protein motifs were identified in
F55B12.3 which are shared with yeast cdc4, mouse w85144 and human
028971. These are an F-box in the N-terminal domain and seven
.beta.-transducin repeats in the C-terminal domain.
[0068] Discussion
[0069] The sel-10 gene encodes a member of the .beta.-transducin
protein family. These are characterized by the presence of multiple
WD40 repeats [Neer, E. J. et al., Nature 371: 297-300 (1994)]. The
repeats are 20-40 amino acids long and are bounded by gly-his (GH)
and trp-asp (WD) residues. Solution of the three dimensional
structure of .beta.-transducin indicates that the WD40 repeats form
the arms of a seven-bladed propeller like structure [Sondek, J. et
al., Nature 379: 369-74 (1996)]. Each blade is formed by four
alternating pleats of beta-sheet with a pair of the conserved
aspartic acid residues in the protein motif forming the limits of
one internal beta strand. WD40 repeats are found in over 27
different proteins which represent diverse functional classes
[Neer, E. J. et al., Nature 371: 297-300 (1994)]. These regulate
cellular functions including cell division, cell fate
determination, gene transcription, signal transduction, protein
degradation, mRNA modification and vesicle fusion. This diversity
in function has led to the hypothesis that .beta.-transducin family
members provide a common scaffolding upon which multiprotein
complexes can be assembled.
[0070] The homology of sel-10, 28971 and W85144 to the yeast cdc4
gene suggests a functional role in the ubiquitin-proteasome pathway
for intracellular degradation of protein. Mutations of the yeast
cdc4 gene cause cell cycle arrest by blocking degradation of Sic1,
an inhibitor of S-phase cyclin/cdk complexes [King, R. W. et al.,
Science 274: 1652-9 (1996)]. Phosphorylation of Sic1 targets it for
destruction through the ubiquitin-proteasome pathway. This pathway
consists of three linked enzyme reactions that are catalyzed by
multiprotein complexes [Ciechanover, A., Cell 79: 13-21 (1994);
Ciechanover, A. and A. L. Schwartz, FASEB J. 8: 182-91 (1994)].
Initially, the C-terminal glycine of ubiquitin is activated by ATP
to form a high energy thiol ester intermediate in a reaction
catalyzed by the ubiquitin-activating enzyme, E1. Following
activation, an E2 enzyme (ubiquitin conjugating enzyme) transfers
ubiquitin from E1 to the protein target. In some cases, E2 acts
alone. In others, it acts in concert with an E3 ubiquitin-ligating
enzyme which binds the protein substrate and recruits an E2 to
catalyze ubiquitination. E2 ubiquitin-conjugating enzymes comprise
a fairly conserved gene family, while E3 enzymes are divergent in
sequence [Ciechanover, A., Cell 79: 13-21 (1994); Ciechanover, A.
and A. L. Schwartz, FASEB J. 8: 182-91 (1994)].
[0071] In yeast, mutation of the E2 ubiquitin-conjugating enzyme,
cdc34, causes cell cycle arrest through failure to degrade the Sic1
inhibitor of the S-phase cyclin/cdk complex [King, R. W. et al.,
Science 274: 1652-9 (1996)]. Sic1 normally is degraded as cells
enter the G1-S phase transition, but in the absence of cdc34, Sic1
escapes degradation and its accumulation causes cell cycle arrest.
Besides cdc34, cdc4 is one of three other proteins required for the
G1-S phase transition. The other two are cdc53 and Skp1. As
discussed above, cdc4 contains two structural motifs, seven WD40
repeats (which suggests that the protein forms a beta-propeller)
and a structural motif shared with cyclin F which is an interaction
domain for Skp1 [Bai, C. et al., Cell 86: 263-74 (1996)]. Insect
cell lysates containing cdc53, cdc4 and skp1 (and also ubiquitin,
cdc34 and E1) can transfer ubiquitin to Sic1 suggesting that one or
more of these components functions as an E3 ubiquitin-ligating
enzyme [King, R. W. et al., Science 274: 1652-9 (1996)]. Increased
expression of either cdc4 or Skp1 partially rescues loss of the
other.
[0072] In C. elegans, mutation of sel-10 has no visible phenotype
indicating that sel-10 does not play a role in regulation of the
cell-cycle. A closely related, C. elegans .beta.-transducin family
member, K10B2.6 may play that role as it clusters with the gene
TRCP_XEN from Xenopus laevis which rescues yeast cell cycle mutants
arrested in late anaphase due to a failure to degrade cyclin B
[Spevak,W. et al., Mol. Cell. Biol. 13: 4953-66 (1993)]. If sel-10
does encode a component of an E3-ubiquitin ligating enzyme, how
might it suppress sel-12 and enhance lin-12 mutations? The simplest
hypothesis is that sel-10 regulates degradation of both proteins
via the ubiquitin-proteasome pathway. Both sel-12 and lin-12 are
transmembrane proteins. Sel-12 crosses the membrane 8 times such
that its N- and C-termini face the cytosol [Kim, T. W. et al., J
Biol. Chem. 272: 11006-10 (1997)], while lin-12 is a type 1
transmembrane protein (Greenwald, I. and G. Seydoux, Nature 346:
197-9 (1990)). Both are ubiquitinated, and in the case of human
PS2, steady state levels increase in cells treated with an
inhibitor of the proteasome, N-acetyl-L-leucinal-L-norleucinal and
lactacystin (Li, X. and I. Greenwald, Neuron. 17: 1015-21 (1996)).
Alternatively, sel-10 may target for degradation of a negative
regulator of presenilin function.
[0073] The genetic analysis and protein function suggested by
homology to cdc4 implies that drug inhibitors of human sel-10 may
increase steady state levels of human presenilins. This could
potentiate activity of the presenilin pathway and provide a means
for therapeutic intervention in Alzheimer's disease.
Example 2
5' RACE Cloning of a Human cDNA Encoding Sel-10, an Extragenic
Suppressor of Presenilin Mutations in C. elegans
[0074] Materials and Methods
[0075] Oligonucleotide primers for the amplification of the sel-10
coding sequence from C. elegans cDNA were prepared based on the
sequence of F55B12.3, identified in Example 1 as the coding
sequence for C. elegans sel-10. The primers prepared were:
5'-CGGGATCCACCATGGATGATGGATCGATGACACC-- 3' (SEQ ID NO:11) and
5'-GGAATTCCTTAAGGGTATACAGCATCAAAGTCG-3' (SEQ ID NO:12). C. elegans
mRNA was converted to cDNA using a BRL Superscript II
Preamplification kit. The PCR product was digested with restriction
enzymes BamHI and EcoRI (LTI, Gaithersberg, Md.) and cloned into
pcDNA3.1 (Invitrogen). Two isolates were sequenced (ABI,
Perkin-Elmer Corp).
[0076] The sequence of Incyte clone 028971 (encoding a portion of
the human homologue of C. elegans sel-10), was used to design four
antisense oligonucleotide primers: 5'-TCACTTCATGTCCACATCAAAGTCC-3'
(SEQ ID NO:13), 5'-GGTAATTACAAGTTCTTGTTGAACTG (SEQ ID NO:14),
5'-CCCTGCAACGTGTGTAGACAGG-3- ' (SEQ ID NO:15), and
5'-CCAGTCTCTGCATTCCACACTTTG-3' (SEQ ID NO:16) to amplify the
missing 5' end of human sel-10. The Incyte LifeSeq "Electronic
Northern" analysis was used to identify tissues in which sel-10 was
expressed. Two of these, hippocampus and mammary gland, were chosen
for 5' RACE cloning using a CloneTech Marathon kit and prepared
Marathon-ready cDNA from hippocampus and mammary gland. PCR
products were cloned into the TA vector pCR3.1 (Invitrogen), and
isolates were sequenced. An alternate 5' oligonucleotide primer was
also designed based on Incyte clones which have 5' ends that differ
from the hippocampal sel-10 sequence: 5'-CTCAGACAGGTCAGGACATTTGG-3'
(SEQ ID NO:17).
[0077] Blastn was used to search Incyte databases LifeSeq and
LifeSeqFL. Gap alignments and translations were performed with GCG
programs (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, Madison
Wis.).
[0078] Results
[0079] The coding sequence of the C. elegans sel-10: The predicted
coding sequence of the C. elegans sel-10, F55B12.3, had originally
been determined at the Genome Sequencing Center, Washington
University, St. Louis, by using the computer program GeneFinder to
predict introns and exons in the genomic cosmid F55B12. The
hypothetical cDNA sequence was confirmed by amplifying this region
from C. elegans cDNA, cloning, and sequencing it.
[0080] The coding sequence of the human sel-10 gene homologue: All
of the 028971 antisense oligonucleotides amplified a 5' RACE
product from human hippocampal and mammary cDNA. The longest PCR
product from the hippocampal reactions was cloned and sequenced.
This PCR reaction was designed to generate products which end at
the predicted stop codon. Two isolates contained identical sequence
which begins 880 bases before the beginning of the 028971 sequence.
This sequence was confirmed by comparison with spanning Incyte cDNA
clones. The Incyte clones that spanned the 5' end of the human
sel-10 homologue were not annotated as F55B 12.3, as the homology
in this region between the human and C. elegans genes is low, and
as the overlap between these clones and the annotated clones
happened to be too small for them to be clustered in the Incyte
database or uncovered by our blasting the Incyte database with the
028971 sequence.
[0081] The predicted protein sequences of human sel-10 have 47.6%
identity and 56.7% similarity to C. elegans sel-10. The N-terminus
of the human sel-10 sequence begins with 4 in-frame methionines. In
addition to the WD40 repeats described above, the human sequence
also contains a region homologous to the CDC4 F-box for binding
Skp1, as expected for a sel-10 homologue.
[0082] Different Human sel-10 mRNAs Expressed in Mammary and
Hippocampal Tissues:
[0083] Several additional human sel-10 ESTs which differ from the
hippocampal sequence were identified. These are an exact match,
which indicates that the alternative transcript is probably real.
Comparison of these sequences with the human hippocampal sel-10
sequence shows divergence prior to the 4th in-frame methionine and
then exact sequence match thereafter. An oligonucleotide primer
specific for the 5' end of this alternative transcript was found to
amplify a product from mammary but not hippocampal cDNA. This
indicates either that the human sel-10 transcript undergoes
differential splicing in a tissue-specific fashion or that the gene
contains multiple, tissue specific promoters.
[0084] Discussion
[0085] 5'RACE and PCR amplification were used to clone a
full-length cDNA encoding the human homologue of the C. elegans
gene, sel-10. Sequence analysis confirms the earlier prediction
that sel-10 is a member of the CDC4 family of proteins containing
F-Box and WD40 Repeat domains. Two variants of the human sel-10
cDNA were cloned from hippocampus and mammary gland which differed
in 5' sequence preceding the apparent site of translation
initiation. This implies that the gene may have two or more start
sites for transcription initiation which are tissue-specific or
that the pattern of exon splicing is tissue-specific.
Example 3
Expression Of Epitope-Tagged Sel-10 In Human Cells, and
Perturbation Of Amyloid .beta. Peptide Processing By Human
Sel-10
[0086] Materials And Methods
[0087] Construction of Epitope-Tagged Sel-10: Subcloning, Cell
Growth and Transfection:
[0088] An EcoR1 site was introduced in-frame into the human sel-10
cDNA using a polymerase chain reaction (PCR) primed with the
oligonucleotides 237 (5'-GGAATTCCATGAAAAGATTGGACCATGGTTCTG-3') (SEQ
ID NO:18) and 206 (5'-GGAATTCCTCACTTCATGT-CACATCAAAGTCCAG-3') (SEQ
ID NO:19). The resulting PCR product was cloned into the EcoR1 site
of the vector pCS2+MT. This fused a 5' 6-myc epitope tag in-frame
to the fifth methionine of the hippocampal sel-10 cDNA, i.e.,
upstream of nucleotide 306 of the sequence given in SEQ ID NO:1.
The nucleotide sequence of this construct, designated 6
myc-N-sel-10, is given in SEQ ID NO: 20, while the amino acid
sequence of the polypeptide encoded thereby is given in SEQ ID NO:
21. The hippocampal and mammary sel-10 cDNA diverge upstream of
this methionine. A PS1 cDNA with a 3'-FLAG tag (PS1-C-FLAG) was
subcloned into the pcDNA3.1 vector. An APP cDNA containing the
Swedish NL mutation and an attenuated ER retention sequence
consisting of the addition of a di-lysyl motif to the C-terminus of
APP695 (APP695NL-KK) was cloned into vector pIRES-EGFP (Mullan et
al., Nat Genet 1992 August;1(5):345-7). HEK293 and IMR32 cells were
grown to 80% confluence in DMEM with 10% FBS and transfected with
the above cDNA. A total of 10 mg total DNA/6.times.10.sup.6 cells
was used for transfection with a single plasmid. For
cotransfections of multiple plasmids, an equal amount of each
plasmid was used for a total of 10 mg DNA using LipofectAmine
(BRL).
[0089] In order to construct C-term V5 his tagged sel-10 and the
C-term mychis tagged sel-10, the coding sequence of human
hippocampal sel-10 was amplified using oligonucleotides primers
containing a KpnI restriction site on the 5' primer:
5'-GGGTACCCCTCATTATTCCCTCGAGTTCTTC-3' (SEQ ID NO:22) and an EcoRI
site on the 3' primer: 5'-GGAATTCCTTCATGTCCACATCAAAGT- CC-3' (SEQ
ID NO:23), using the original human sel-10 RACE pcr product as
template. The product was digested with both KpnI and EcoRI and
cloned into either the vector pcDNA6/V5-His A or
pcDNA3.1/Myc-His(+) A (Invitrogen). The nucleotide sequence of
independent isolates was confirmed by dideoxy sequencing. The
nucleotide sequence of the C-term V5 his tagged sel-10 is given in
SEQ ID NO: 24, while the amino acid sequence of the polypeptide
encoded thereby is given in SEQ ID NO: 25. The nucleotide sequence
of independent isolates was confirmed by dideoxy sequencing. The
nucleotide sequence of the C-term mychis tagged sel-10 is given in
SEQ ID NO: 26, while the amino acid sequence of the polypeptide
encoded thereby is given in SEQ ID NO: 27.
[0090] Clonal Selection of transformed cells by FACS: 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 mg/ml. Wells
with clones were expanded from the 96 well plate to a 24 well plate
and then to a 6 well plate with the fastest growing colonies chosen
for expansion at each passage.
[0091] Immunofluorescence: Cells grown on slides were fixed 48 hrs
after transfection with 4% formaldehyde and 0.1% Triton X-100 in
PBS for 30 min on ice and blocked with 10% Goat serum in PBS
(blocking solution) 1 hr RT (i.e., 25.degree. C.), followed by
incubation with mouse anti-myc (10 mg/ml) or rabbit anti-FLAG (0.5
mg/ml) antibody 4.degree. C. O/N and then fluorescein-labeled goat
anti-mouse or anti-rabbit antibody (5 mg/ml) in blocking solution 1
hr at 25.degree. C.
[0092] Western blotting: Cell lysates were made 48 hrs after
transfection by incubating 10.sup.5 cells with 100 ml TENT (50 mM
Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1.times.
protease inhibitor cocktail) 10 min on ice followed by
centrifugation at 14,000 g. The supernatant was loaded on 4-12%
NuPage gels (50 mg protein/lane) and electrophoresis and transfer
were conducted using an Xcell II Mini-Cell system (Novex). The blot
was blocked with 5% milk in PBS 1 hr RT and incubated with anti-myc
or anti-FLAG antibody (described in "Immunofluorescence" above)
4.degree. C. O/N, then sheep anti-mouse or anti-rabbit antibody-HRP
(0.1 mg/ml) 1 hr RT, followed by Supersignal (Pierce)
detection.
[0093] ELISA: Cell culture supernatant or cell lysates (100 ml
formic acid/10.sup.6 cells) were assayed in the following double
antibody sandwich ELISA, which is capable of detecting levels of
A.beta..sub.1-40 and A.beta..sub.1-42 peptide in culture
supernatant.
[0094] 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 (NYS 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. The sandwich ELISA was performed according to
the method of Pirttila et al. (Neurobiology of Aging 18: 121-7
(1997)). 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
min 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 non transfected conditioned cell medium
was added after washing the plate, as well as 100 .mu.l/well of
sample i.e. filtered conditioned medium of transfected cells. 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
hr 15 min. Following washes, 100 .mu.l/well neutravidin-horseradish
peroxidase (Pierce, Rockford, Ill.) diluted 1:10,000 in DPBST was
applied and incubated for 1 hr 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 min. using Soft max Pro
software.
[0095] Results
[0096] Transfection of HEK293 cells: Transfection efficiency was
monitored through the use of vectors that express green fluorescent
protein (GFP) or by immunofluorescent detection of epitope-tagged
sel-10 or PS1. An N-terminal 6-myc epitope was used to tag human
sel-10 (6myc-N-sel-10), while PS1 was tagged with a C-terminal FLAG
epitope (PS1-C-FLAG). APP695 was modified by inclusion of the
Swedish NL mutation to increase A.beta. processing and an
attenuated endoplasmic reticulum (ER) retention signal consisting
of a C-terminal di-lysine motif (APP695NL-KK). The di-lysine motif
increases A.beta. processing about two fold. The APP695NL-KK
construct was inserted into the first cistron of a bicistronic
vector containing GFP (pIRES-EGFP, Invitrogen) to allow us to
monitor transfection efficiency. Transfection efficiency in HEK293
cells was about 50% for transfections with a single plasmid DNA.
For cotransfections with two plasmids, about 30-40% of the cells
expressed both proteins as detected by double
immunofluorescence.
[0097] Expression of recombinant protein in transfected HEK293
cells was confirmed by Western blot as illustrated for PS1-C-FLAG
and 6myc-N-sel-10 (FIG. 1A). In the case of cotransfections with
three plasmids (PS1-C-FLAG+6myc-N-sel-10+APP), all three proteins
were detected in the same cell lysate by Western blot (FIG. 1B)
using appropriate antibodies.
[0098] Effect of 6myc-N-sel-10 and PS1-C-FLAG on A.beta.
processing: Cotransfection of APP695NL-KK with 6myc-N-sel-10 or
PS1-C-FLAG into HEK293 cells increased the release of Ab1-40 and
Ab1-42 peptide into the culture supernatant by 2- to 3-fold over
transfections with just APP695NL-KK (Table 1). Cotransfection of
APP695NL-KK with both 6myc-N-sel-10 and PS1-C-FLAG increased Ab
release still further (i.e., 4- to 6-fold increase). In contrast,
the ratio of Ab1-42/(Ab1-40+Ab1-42) released into the supernatant
decreased about 50%. The subtle decrease in the ratio of
Ab1-42/(Ab1-40+Ab1-42) reflects the larger increase in Ab 1-40
relative to Ab 1-42. Neither 6myc-N-sel-10 nor PS1-C-FLAG affected
endogenous Ab production in HEK293 cells. Similar observations were
also obtained in IMR32 cells (Table 2). However, IMR32 cells
transfected less well than HEK293 cells, so the stimulation of
APP695NL-KK processing by cotransfection with 6myc-N-sel-10 or
PS1-C-FLAG was lower.
[0099] Levels of Ab 1-40 expressed in HEK293 cells transfected with
APP695NL-KK were sufficient to measure Ab peptide in both the
culture supernatant and cell pellet. Considerably more Ab 1-40 is
detected in the HEK293 cell pellet than in the supernatant in cells
transfected with just APP695NL-KK. Cotransfection with
6myc-N-sel-10 or PS1-C-FLAG proportionately decreased Ab 1-40 in
the cell pellet and increased Ab in the culture supernatant. This
implies that 6myc-N-sel-10 and PS1-C-FLAG alter processing or
trafficking of APP such that proportionately more Ab is released
from the cell.
[0100] Effect of 6myc-N-sel-10 and PS1 -C-FLAG expression on
endogenous A.beta. processing: The effect of 6myc-N-sel-10 on the
processing of endogenous APP by human cells was assessed by
creating stably transformed HEK293 cell lines expressing these
proteins. Two cell lines expressing 6myc-N-sel-10 were derived
(sel-10/2 & sel-10/6) as well as a control cell line
transformed with pcDNA3.1 vector DNA. Both 6myc-N-sel-10 cell lines
expressed the protein as shown by Western blot analysis. Endogenous
production of Ab 1-40 was increased in both 6myc-N-sel-10 cell
lines in contrast to vector DNA transformed cells Table 3). In
addition, stable expression of 6myc-N-sel-10 significantly
increased Ab production after transfection with APP695NL-KK plasmid
DNA (Table 3). Similar results were obtained with 6 stable cell
lines expressing PS1-C-FLAG. All 6 cell lines showed significant
elevation of endogenous A.beta. processing and all also showed
enhanced processing of Ab after transfection with APP695NL-KK
(Table 3). In addition, the increase of A.beta. processing seen
with 6myc-N-sel-10 was also seen with sel-10 tagged at the
C-terminus with either mychis or v5his (See Table 4). Both
C-terminal and N-terminal tags resulted in an increase in A.beta.
processing.
[0101] Discussion
[0102] These data suggest that, when over expressed, 6myc-N-sel-10
as well as PS1-C-FLAG alter A.beta. processing in both transient
and stable expression systems. A 6-myc epitope tag was used in
these experiments to allow detection of sel-10 protein expression
by Western blot analysis. If as its sequence homology to yeast CDC4
suggests, sel-10 is an E2-E3 ubiquitin ligase, it should be
possible to identify the proteins it targets for ubiquitination.
Since the presenilins are degraded via the ubiquitin-proteasome
pathway, PS1 & PS2 are logical targets of sel-10 catalyzed
ubiquitination [Kim et al., J. Biol. Chem. 272:11006-11010 (1997)].
How sel-10 affects A.beta. processing is not understood at this
point. In the future, it will be necessary to determine if sel-10
& PS1 increase A.beta. processing by altering production,
processing, transport, or turn-over of APP, and whether the effect
of PS1 is mediated or regulated by sel-10.
[0103] These experiments suggest that sel-10 is a potential drug
target for decreasing Ab levels in the treatment of AD. They also
show that C. elegans is an excellent model system in which to
investigate presenilin biology in the context of AD. Thus, as is
shown by cotransfection experiments, as well as in stable
transformants, expression of 6myc-N-sel10 or PS1-C-FLAG increases
A.beta. processing. An increase in A.beta. processing was seen in
both HEK293 cells and IMR32 cells after cotransfection of
6myc-N-sel10 or PS1-C-FLAG with APP695NL-KK. In stable
transformants of HEK293 cells expressing 6myc-Sel10 or PS1-C-FLAG,
an increase in endogenous A.beta. processing was observed, as well
as an increase in A.beta. processing after transfection with
APP695NL-KK. This suggests that inhibitors of either sel-10 and/or
PS1, may decrease A.beta. processing, and could have therappeutic
potential for Alzheimer's disease.
[0104] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0105] 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.
[0106] The entire disclosure of all publications cited herein are
hereby incorporated by reference.
1TABLE 1 Effect of 6myc-N-sel-10 and PS1-C-FLAG transient
transfection on Ab levels in HEK293 cell supernatants. Plasmids
Transfected Ab1-42 ng/ml Ab1-40 ng/ml Ab1-42/total Ab ng/ml pcDNA3
81 .+-. 20 231 .+-. 50 0.26 .+-. 0.05 6myc-N-sel-10 67 .+-. 7 246
.+-. 34 0.21 .+-. 0.03 PS1-C-FLAG 75 .+-. 18 227 .+-. 45 0.25 .+-.
0.03 PS1-C-FLAG + 6myc-N-sel-10 77 .+-. 21 220 .+-. 26 0.25 .+-.
0.03 APP695NL-KK 141 .+-. 27 896 .+-. 103 0.14 .+-. 0.02
APP695NL-KK + 6myc-N-sel-10 308 .+-. 17 2576 .+-. 190 0.11 .+-.
0.00 APP695NL-KK + PS1-C-FLAG 364 .+-. 39 3334 .+-. 337 0.09 .+-.
0.00 APP695NL-KK + PS1-C-FLAG + 6myc-N-sel-10 550 .+-. 20 5897 .+-.
388 0.09 .+-. 0.00
[0107]
2TABLE 2 Effect of 6myc-N-sel-10 and PS1-C-FLAG transient
transfection on Ab levels in IMR32 cell supernatants. Plasmids
Transfected Ab1-42 ng/ml Ab1-40 ng/ml Ab1-42/total Ab ng/ml pcDNA3
65 .+-. 3 319 .+-. 146 0.19 .+-. 0.06 6myc-N-sel-10 63 .+-. 0 246
.+-. 53 0.21 .+-. 0.04 PS1-C-FLAG 67 .+-. 6 307 .+-. 79 0.18 .+-.
0.04 PS1-C-FLAG + 6myc-N-sel-10 67 .+-. 6 302 .+-. 94 0.20 .+-.
0.08 APP695NL-KK 66 .+-. 5 348 .+-. 110 0.17 .+-. 0.05 APP695NL-KK
+ 6myc-N-sel-10 75 .+-. 18 448 .+-. 141 0.15 .+-. 0.03 APP695NL-KK
+ PS1-C-FLAG 63 .+-. 26 466 .+-. 72 0.12 .+-. 0.02 APP695NL-KK +
PS1-C-FLAG + 6myc-N-sel-10 81 .+-. 26 565 .+-. 179 0.12 .+-.
0.01
[0108]
3TABLE 3 Endogenous and exogenous Ab1-40 and Ab1-42 levels in
supernatants from stable transformants of HEK293 cells. APP695NL-
GFP Transfection KK Transfection Ab1-40 Ab1-42 Ab1-40 Ab1-42 Stable
Line ng/ml ng/ml ng/ml ng/ml 6myc-N- 297 .+-. 29 109 .+-. 17 4877
.+-. 547 750 .+-. 32 sel10/2 6myc-N- 168 .+-. 18 85 .+-. 11 8310
.+-. 308 1391 .+-. 19 sel10/6 PS1-C-FLAG/2 97 .+-. 6 68 .+-. 8 3348
.+-. 68 493 .+-. 21 PS1-C-FLAG/8 118 .+-. 11 85 .+-. 17 3516 .+-.
364 515 .+-. 36 PS1-C-FLAG/9 83 .+-. 20 67 .+-. 16 2369 .+-. 73 350
.+-. 12 PS1-C- 152 .+-. 17 68 .+-. 13 4771 .+-. 325 599 .+-. 25
FLAG/11 PS1-C- 141 .+-. 12 50 .+-. 10 4095 .+-. 210 449 .+-. 21
FLAG/12 PS1-C- 270 .+-. 139 61 .+-. 28 6983 .+-. 304 745 .+-. 41
FLAG/13 pcDNA3/1 43 .+-. 13 75 .+-. 15 1960 .+-. 234 61 .+-. 6
[0109]
4TABLE 4 Sel-10 constructs with epitope tags at the N or C terminus
increase A.beta. 1-40 and A.beta. 1-42. 10 construct A.beta. 1-40 %
increase P-value A.beta. 1-42 % increase P-value pcDNA 4240 .+-.
102 614 .+-. 10 6myc-N-sel-10 7631 .+-. 465 80% 3.7 .times.
10.sup.-6 1136 .+-. 73 46% 7.9 .times. 10.sup.-6 sel-10-C-mychis
5485 .+-. 329 29% 1.8 .times. 10.sup.-4 795 .+-. 50 29% 4.0 .times.
10.sup.-4 sel-10-C-V5his 6210 .+-. 498 46% 1.2 .times. 10.sup.-4
906 .+-. 73 48% 2.1 .times. 10.sup.-4
[0110]
Sequence CWU 1
1
27 1 3550 DNA Homo sapiens unsure (2485) unsure (3372) 1 ctcattattc
cctcgagttc ttctcagtca agctgcatgt atgtatgtgt gtcccgagaa 60
gcggtttgat actgagctgc atttgccttt actgtggagt tttgttgccg gttctgctcc
120 ctaatcttcc ttttctgacg tgcctgagca tgtccacatt agaatctgtg
acatacctac 180 ctgaaaaagg tttatattgt cagagactgc caagcagccg
gacacacggg ggcacagaat 240 cactgaaggg gaaaaataca gaaaatatgg
gtttctacgg cacattaaaa atgatttttt 300 acaaaatgaa aagaaagttg
gaccatggtt ctgaggtccg ctctttttct ttgggaaaga 360 aaccatgcaa
agtctcagaa tatacaagta ccactgggct tgtaccatgt tcagcaacac 420
caacaacttt tggggacctc agagcagcca atggccaagg gcaacaacga cgccgaatta
480 catctgtcca gccacctaca ggcctccagg aatggctaaa aatgtttcag
agctggagtg 540 gaccagagaa attgcttgct ttagatgaac tcattgatag
ttgtgaacca acacaagtaa 600 aacatatgat gcaagtgata gaaccccagt
ttcaacgaga cttcatttca ttgctcccta 660 aagagttggc actctatgtg
ctttcattcc tggaacccaa agacctgcta caagcagctc 720 agacatgtcg
ctactggaga attttggctg aagacaacct tctctggaga gagaaatgca 780
aagaagaggg gattgatgaa ccattgcaca tcaagagaag aaaagtaata aaaccaggtt
840 tcatacacag tccatggaaa agtgcataca tcagacagca cagaattgat
actaactgga 900 ggcgaggaga actcaaatct cctaaggtgc tgaaaggaca
tgatgatcat gtgatcacat 960 gcttacagtt ttgtggtaac cgaatagtta
gtggttctga tgacaacact ttaaaagttt 1020 ggtcagcagt cacaggcaaa
tgtctgagaa cattagtggg acatacaggt ggagtatggt 1080 catcacaaat
gagagacaac atcatcatta gtggatctac agatcggaca ctcaaagtgt 1140
ggaatgcaga gactggagaa tgtatacaca ccttatatgg gcatacttcc actgtgcgtt
1200 gtatgcatct tcatgaaaaa agagttgtta gcggttctcg agatgccact
cttagggttt 1260 gggatattga gacaggccag tgtttacatg ttttgatggg
tcatgttgca gcagtccgct 1320 gtgttcaata tgatggcagg agggttgtta
gtggagcata tgattttatg gtaaaggtgt 1380 gggatccaga gactgaaacc
tgtctacaca cgttgcaggg gcatactaat agagtctatt 1440 cattacagtt
tgatggtatc catgtggtga gtggatctct tgatacatca atccgtgttt 1500
gggatgtgga gacagggaat tgcattcaca cgttaacagg gcaccagtcg ttaacaagtg
1560 gaatggaact caaagacaat attcttgtct ctgggaatgc agattctaca
gttaaaatct 1620 gggatatcaa aacaggacag tgtttacaaa cattgcaagg
tcccaacaag catcagagtg 1680 ctgtgacctg tttacagttc aacaagaact
ttgtaattac cagctcagat gatggaactg 1740 taaaactatg ggacttgaaa
acgggtgaat ttattcgaaa cctagtcaca ttggagagtg 1800 gggggagtgg
gggagttgtg tggcggatca gagcctcaaa cacaaagctg gtgtgtgcag 1860
ttgggagtcg gaatgggact gaagaaacca agctgctggt gctggacttt gatgtggaca
1920 tgaagtgaag agcagaaaag atgaatttgt ccaattgtgt agacgatata
ctccctgccc 1980 ttccccctgc aaaaagaaaa aaagaaaaga aaaagaaaaa
aatcccttgt tctcagtggt 2040 gcaggatgtt ggcttggggc aacagattga
aaagacctac agactaagaa ggaaaagaag 2100 aagagatgac aaaccataac
tgacaagaga ggcgtctgct gtctcatcac ataaaaggct 2160 tcacttttga
ctgagggcag ctttgcaaaa tgagactttc taaatcaaac caggtgcaat 2220
tatttcttta ttttcttctc cagtggtcat tggggcagtg ttaatgctga aacatcatta
2280 cagattctgc tagcctgttc ttttaccact gacagctaga cacctagaaa
ggaactgcaa 2340 taatatcaaa acaagtactg gttgactttc taattagaga
gcatctgcaa caaaaagtca 2400 tttttctgga gtggaaaagc ttaaaaaaat
tactgtgaat tgtttttgta cagttatcat 2460 gaaaagcttt tttttttatt
ttttngccaa ccattgccaa tgtcaatcaa tcacagtatt 2520 agcctctgtt
aatctattta ctgttgcttc catatacatt cttcaatgca tatgttgctc 2580
aaaggtggca agttgtcctg ggttctgtga gtcctgagat ggatttaatt cttgatgctg
2640 gtgctagaag taggtcttca aatatgggat tgttgtccca accctgtact
gtactcccag 2700 tggccaaact tatttatgct gctaaatgaa agaaagaaaa
aagcaaatta ttttttttat 2760 tttttttctg ctgtgacgtt ttagtcccag
actgaattcc aaatttgctc tagtttggtt 2820 atggaaaaaa gactttttgc
cactgaaact tgagccatct gtgcctctaa gaggctgaga 2880 atggaagagt
ttcagataat aaagagtgaa gtttgcctgc aagtaaagaa ttgagagtgt 2940
gtgcaaagct tattttcttt tatctgggca aaaattaaaa cacattcctt ggaacagagc
3000 tattacttgc ctgttctgtg gagaaacttt tctttttgag ggctgtggtg
aatggatgaa 3060 cgtacatcgt aaaactgaca aaatatttta aaaatatata
aaacacaaaa ttaaaataaa 3120 gttgctggtc agtcttagtg ttttacagta
tttgggaaaa caactgttac agttttattg 3180 ctctgagtaa ctgacaaagc
agaaactatt cagtttttgt agtaaaggcg tcacatgcaa 3240 acaaacaaaa
tgaatgaaac agtcaaatgg tttgcctcat tctccaagag ccacaactca 3300
agctgaactg tgaaagtggt ttaacactgt atcctaggcg atcttttttc ctccttctgt
3360 ttattttttt gnttgtttta tttatagtct gatttaaaac aatcagattc
aagttggtta 3420 attttagtta tgtaacaacc tgacatgatg gaggaaaaca
acctttaaag ggattgtgtc 3480 tatggtttga ttcacttaga aattttattt
tcttataact taagtgcaat aaaatgtgtt 3540 ttttcatgtt 3550 2 3571 DNA
Homo sapiens unsure (2506) unsure (3393) 2 ctcagcaggt caggacattt
ggtaggggaa ggttgaaaga caaaagcagc aggccttggg 60 ttctcagcct
tttaaaaact attattaaat atatattttt aaaatttagt ggttagagct 120
tttagtaatg tgcctgtatt acatgtagag agtattcgtc aaccaagagg agttttaaaa
180 tgtcaaaacc gggaaaacct actctaaacc atggcttggt tcctgttgat
cttaaaagtg 240 caaaagagcc tctaccacat caaaccgtga tgaagatatt
tagcattagc atcattgccc 300 aaggcctccc tttttgtcga agacggatga
aaagaaagtt ggaccatggt tctgaggtcc 360 gctctttttc tttgggaaag
aaaccatgca aagtctcaga atatacaagt accactgggc 420 ttgtaccatg
ttcagcaaca ccaacaactt ttggggacct cagagcagcc aatggccaag 480
ggcaacaacg acgccgaatt acatctgtcc agccacctac aggcctccag gaatggctaa
540 aaatgtttca gagctggagt ggaccagaga aattgcttgc tttagatgaa
ctcattgata 600 gttgtgaacc aacacaagta aaacatatga tgcaagtgat
agaaccccag tttcaacgag 660 acttcatttc attgctccct aaagagttgg
cactctatgt gctttcattc ctggaaccca 720 aagacctgct acaagcagct
cagacatgtc gctactggag aattttggct gaagacaacc 780 ttctctggag
agagaaatgc aaagaagagg ggattgatga accattgcac atcaagagaa 840
gaaaagtaat aaaaccaggt ttcatacaca gtccatggaa aagtgcatac atcagacagc
900 acagaattga tactaactgg aggcgaggag aactcaaatc tcctaaggtg
ctgaaaggac 960 atgatgatca tgtgatcaca tgcttacagt tttgtggtaa
ccgaatagtt agtggttctg 1020 atgacaacac tttaaaagtt tggtcagcag
tcacaggcaa atgtctgaga acattagtgg 1080 gacatacagg tggagtatgg
tcatcacaaa tgagagacaa catcatcatt agtggatcta 1140 cagatcggac
actcaaagtg tggaatgcag agactggaga atgtatacac accttatatg 1200
ggcatacttc cactgtgcgt tgtatgcatc ttcatgaaaa aagagttgtt agcggttctc
1260 gagatgccac tcttagggtt tgggatattg agacaggcca gtgtttacat
gttttgatgg 1320 gtcatgttgc agcagtccgc tgtgttcaat atgatggcag
gagggttgtt agtggagcat 1380 atgattttat ggtaaaggtg tgggatccag
agactgaaac ctgtctacac acgttgcagg 1440 ggcatactaa tagagtctat
tcattacagt ttgatggtat ccatgtggtg agtggatctc 1500 ttgatacatc
aatccgtgtt tgggatgtgg agacagggaa ttgcattcac acgttaacag 1560
ggcaccagtc gttaacaagt ggaatggaac tcaaagacaa tattcttgtc tctgggaatg
1620 cagattctac agttaaaatc tgggatatca aaacaggaca gtgtttacaa
acattgcaag 1680 gtcccaacaa gcatcagagt gctgtgacct gtttacagtt
caacaagaac tttgtaatta 1740 ccagctcaga tgatggaact gtaaaactat
gggacttgaa aacgggtgaa tttattcgaa 1800 acctagtcac attggagagt
ggggggagtg ggggagttgt gtggcggatc agagcctcaa 1860 acacaaagct
ggtgtgtgca gttgggagtc ggaatgggac tgaagaaacc aagctgctgg 1920
tgctggactt tgatgtggac atgaagtgaa gagcagaaaa gatgaatttg tccaattgtg
1980 tagacgatat actccctgcc cttccccctg caaaaagaaa aaaagaaaag
aaaaagaaaa 2040 aaatcccttg ttctcagtgg tgcaggatgt tggcttgggg
caacagattg aaaagaccta 2100 cagactaaga aggaaaagaa gaagagatga
caaaccataa ctgacaagag aggcgtctgc 2160 tgtctcatca cataaaaggc
ttcacttttg actgagggca gctttgcaaa atgagacttt 2220 ctaaatcaaa
ccaggtgcaa ttatttcttt attttcttct ccagtggtca ttggggcagt 2280
gttaatgctg aaacatcatt acagattctg ctagcctgtt cttttaccac tgacagctag
2340 acacctagaa aggaactgca ataatatcaa aacaagtact ggttgacttt
ctaattagag 2400 agcatctgca acaaaaagtc atttttctgg agtggaaaag
cttaaaaaaa ttactgtgaa 2460 ttgtttttgt acagttatca tgaaaagctt
ttttttttat tttttngcca accattgcca 2520 atgtcaatca atcacagtat
tagcctctgt taatctattt actgttgctt ccatatacat 2580 tcttcaatgc
atatgttgct caaaggtggc aagttgtcct gggttctgtg agtcctgaga 2640
tggatttaat tcttgatgct ggtgctagaa gtaggtcttc aaatatggga ttgttgtccc
2700 aaccctgtac tgtactccca gtggccaaac ttatttatgc tgctaaatga
aagaaagaaa 2760 aaagcaaatt atttttttta ttttttttct gctgtgacgt
tttagtccca gactgaattc 2820 caaatttgct ctagtttggt tatggaaaaa
agactttttg ccactgaaac ttgagccatc 2880 tgtgcctcta agaggctgag
aatggaagag tttcagataa taaagagtga agtttgcctg 2940 caagtaaaga
attgagagtg tgtgcaaagc ttattttctt ttatctgggc aaaaattaaa 3000
acacattcct tggaacagag ctattacttg cctgttctgt ggagaaactt ttctttttga
3060 gggctgtggt gaatggatga acgtacatcg taaaactgac aaaatatttt
aaaaatatat 3120 aaaacacaaa attaaaataa agttgctggt cagtcttagt
gttttacagt atttgggaaa 3180 acaactgtta cagttttatt gctctgagta
actgacaaag cagaaactat tcagtttttg 3240 tagtaaaggc gtcacatgca
aacaaacaaa atgaatgaaa cagtcaaatg gtttgcctca 3300 ttctccaaga
gccacaactc aagctgaact gtgaaagtgg tttaacactg tatcctaggc 3360
gatctttttt cctccttctg tttatttttt tgnttgtttt atttatagtc tgatttaaaa
3420 caatcagatt caagttggtt aattttagtt atgtaacaac ctgacatgat
ggaggaaaac 3480 aacctttaaa gggattgtgt ctatggtttg attcacttag
aaattttatt ttcttataac 3540 ttaagtgcaa taaaatgtgt tttttcatgt t 3571
3 627 PRT Homo sapiens 3 Met Cys Val Pro Arg Ser Gly Leu Ile Leu
Ser Cys Ile Cys Leu Tyr 1 5 10 15 Cys Gly Val Leu Leu Pro Val Leu
Leu Pro Asn Leu Pro Phe Leu Thr 20 25 30 Cys Leu Ser Met Ser Thr
Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys 35 40 45 Gly Leu Tyr Cys
Gln Arg Leu Pro Ser Ser Arg Thr His Gly Gly Thr 50 55 60 Glu Ser
Leu Lys Gly Lys Asn Thr Glu Asn Met Gly Phe Tyr Gly Thr 65 70 75 80
Leu Lys Met Ile Phe Tyr Lys Met Lys Arg Lys Leu Asp His Gly Ser 85
90 95 Glu Val Arg Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser
Glu 100 105 110 Tyr Thr Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr
Pro Thr Thr 115 120 125 Phe Gly Asp Leu Arg Ala Ala Asn Gly Gln Gly
Gln Gln Arg Arg Arg 130 135 140 Ile Thr Ser Val Gln Pro Pro Thr Gly
Leu Gln Glu Trp Leu Lys Met 145 150 155 160 Phe Gln Ser Trp Ser Gly
Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu 165 170 175 Ile Asp Ser Cys
Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile 180 185 190 Glu Pro
Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu 195 200 205
Ala Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala 210
215 220 Ala Gln Thr Cys Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu
Leu 225 230 235 240 Trp Arg Glu Lys Cys Lys Glu Glu Gly Ile Asp Glu
Pro Leu His Ile 245 250 255 Lys Arg Arg Lys Val Ile Lys Pro Gly Phe
Ile His Ser Pro Trp Lys 260 265 270 Ser Ala Tyr Ile Arg Gln His Arg
Ile Asp Thr Asn Trp Arg Arg Gly 275 280 285 Glu Leu Lys Ser Pro Lys
Val Leu Lys Gly His Asp Asp His Val Ile 290 295 300 Thr Cys Leu Gln
Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp 305 310 315 320 Asn
Thr Leu Lys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr 325 330
335 Leu Val Gly His Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn
340 345 350 Ile Ile Ile Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp
Asn Ala 355 360 365 Glu Thr Gly Glu Cys Ile His Thr Leu Tyr Gly His
Thr Ser Thr Val 370 375 380 Arg Cys Met His Leu His Glu Lys Arg Val
Val Ser Gly Ser Arg Asp 385 390 395 400 Ala Thr Leu Arg Val Trp Asp
Ile Glu Thr Gly Gln Cys Leu His Val 405 410 415 Leu Met Gly His Val
Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg 420 425 430 Arg Val Val
Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro 435 440 445 Glu
Thr Glu Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val 450 455
460 Tyr Ser Leu Gln Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp
465 470 475 480 Thr Ser Ile Arg Val Trp Asp Val Glu Thr Gly Asn Cys
Ile His Thr 485 490 495 Leu Thr Gly His Gln Ser Leu Thr Ser Gly Met
Glu Leu Lys Asp Asn 500 505 510 Ile Leu Val Ser Gly Asn Ala Asp Ser
Thr Val Lys Ile Trp Asp Ile 515 520 525 Lys Thr Gly Gln Cys Leu Gln
Thr Leu Gln Gly Pro Asn Lys His Gln 530 535 540 Ser Ala Val Thr Cys
Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser 545 550 555 560 Ser Asp
Asp Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe 565 570 575
Ile Arg Asn Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val 580
585 590 Trp Arg Ile Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly
Ser 595 600 605 Arg Asn Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp
Phe Asp Val 610 615 620 Asp Met Lys 625 4 592 PRT Homo sapiens 4
Met Ser Thr Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys Gly Leu Tyr 1 5
10 15 Cys Gln Arg Leu Pro Ser Ser Arg Thr His Gly Gly Thr Glu Ser
Leu 20 25 30 Lys Gly Lys Asn Thr Glu Asn Met Gly Phe Tyr Gly Thr
Leu Lys Met 35 40 45 Ile Phe Tyr Lys Met Lys Arg Lys Leu Asp His
Gly Ser Glu Val Arg 50 55 60 Ser Phe Ser Leu Gly Lys Lys Pro Cys
Lys Val Ser Glu Tyr Thr Ser 65 70 75 80 Thr Thr Gly Leu Val Pro Cys
Ser Ala Thr Pro Thr Thr Phe Gly Asp 85 90 95 Leu Arg Ala Ala Asn
Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser 100 105 110 Val Gln Pro
Pro Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser 115 120 125 Trp
Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser 130 135
140 Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln
145 150 155 160 Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu
Ala Leu Tyr 165 170 175 Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu
Gln Ala Ala Gln Thr 180 185 190 Cys Arg Tyr Trp Arg Ile Leu Ala Glu
Asp Asn Leu Leu Trp Arg Glu 195 200 205 Lys Cys Lys Glu Glu Gly Ile
Asp Glu Pro Leu His Ile Lys Arg Arg 210 215 220 Lys Val Ile Lys Pro
Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr 225 230 235 240 Ile Arg
Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu Lys 245 250 255
Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile Thr Cys Leu 260
265 270 Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr
Leu 275 280 285 Lys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr
Leu Val Gly 290 295 300 His Thr Gly Gly Val Trp Ser Ser Gln Met Arg
Asp Asn Ile Ile Ile 305 310 315 320 Ser Gly Ser Thr Asp Arg Thr Leu
Lys Val Trp Asn Ala Glu Thr Gly 325 330 335 Glu Cys Ile His Thr Leu
Tyr Gly His Thr Ser Thr Val Arg Cys Met 340 345 350 His Leu His Glu
Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu 355 360 365 Arg Val
Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly 370 375 380
His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val 385
390 395 400 Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu
Thr Glu 405 410 415 Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg
Val Tyr Ser Leu 420 425 430 Gln Phe Asp Gly Ile His Val Val Ser Gly
Ser Leu Asp Thr Ser Ile 435 440 445 Arg Val Trp Asp Val Glu Thr Gly
Asn Cys Ile His Thr Leu Thr Gly 450 455 460 His Gln Ser Leu Thr Ser
Gly Met Glu Leu Lys Asp Asn Ile Leu Val 465 470 475 480 Ser Gly Asn
Ala Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr Gly 485 490 495 Gln
Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val 500 505
510 Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp
515 520 525 Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile
Arg Asn 530 535 540 Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val
Val Trp Arg Ile 545 550 555 560 Arg Ala Ser Asn Thr Lys Leu Val Cys
Ala Val Gly Ser Arg Asn Gly 565 570 575 Thr Glu Glu Thr Lys Leu Leu
Val Leu Asp Phe Asp Val Asp Met Lys 580 585 590 5 553 PRT Homo
sapiens 5 Met Gly Phe Tyr Gly Thr Leu Lys Met Ile Phe Tyr Lys Met
Lys Arg 1 5 10 15 Lys Leu Asp His Gly Ser Glu Val Arg Ser Phe Ser
Leu Gly Lys Lys 20 25 30 Pro Cys Lys Val Ser Glu
Tyr Thr Ser Thr Thr Gly Leu Val Pro Cys 35 40 45 Ser Ala Thr Pro
Thr Thr Phe Gly Asp Leu Arg Ala Ala Asn Gly Gln 50 55 60 Gly Gln
Gln Arg Arg Arg Ile Thr Ser Val Gln Pro Pro Thr Gly Leu 65 70 75 80
Gln Glu Trp Leu Lys Met Phe Gln Ser Trp Ser Gly Pro Glu Lys Leu 85
90 95 Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys Glu Pro Thr Gln Val
Lys 100 105 110 His Met Met Gln Val Ile Glu Pro Gln Phe Gln Arg Asp
Phe Ile Ser 115 120 125 Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val Leu
Ser Phe Leu Glu Pro 130 135 140 Lys Asp Leu Leu Gln Ala Ala Gln Thr
Cys Arg Tyr Trp Arg Ile Leu 145 150 155 160 Ala Glu Asp Asn Leu Leu
Trp Arg Glu Lys Cys Lys Glu Glu Gly Ile 165 170 175 Asp Glu Pro Leu
His Ile Lys Arg Arg Lys Val Ile Lys Pro Gly Phe 180 185 190 Ile His
Ser Pro Trp Lys Ser Ala Tyr Ile Arg Gln His Arg Ile Asp 195 200 205
Thr Asn Trp Arg Arg Gly Glu Leu Lys Ser Pro Lys Val Leu Lys Gly 210
215 220 His Asp Asp His Val Ile Thr Cys Leu Gln Phe Cys Gly Asn Arg
Ile 225 230 235 240 Val Ser Gly Ser Asp Asp Asn Thr Leu Lys Val Trp
Ser Ala Val Thr 245 250 255 Gly Lys Cys Leu Arg Thr Leu Val Gly His
Thr Gly Gly Val Trp Ser 260 265 270 Ser Gln Met Arg Asp Asn Ile Ile
Ile Ser Gly Ser Thr Asp Arg Thr 275 280 285 Leu Lys Val Trp Asn Ala
Glu Thr Gly Glu Cys Ile His Thr Leu Tyr 290 295 300 Gly His Thr Ser
Thr Val Arg Cys Met His Leu His Glu Lys Arg Val 305 310 315 320 Val
Ser Gly Ser Arg Asp Ala Thr Leu Arg Val Trp Asp Ile Glu Thr 325 330
335 Gly Gln Cys Leu His Val Leu Met Gly His Val Ala Ala Val Arg Cys
340 345 350 Val Gln Tyr Asp Gly Arg Arg Val Val Ser Gly Ala Tyr Asp
Phe Met 355 360 365 Val Lys Val Trp Asp Pro Glu Thr Glu Thr Cys Leu
His Thr Leu Gln 370 375 380 Gly His Thr Asn Arg Val Tyr Ser Leu Gln
Phe Asp Gly Ile His Val 385 390 395 400 Val Ser Gly Ser Leu Asp Thr
Ser Ile Arg Val Trp Asp Val Glu Thr 405 410 415 Gly Asn Cys Ile His
Thr Leu Thr Gly His Gln Ser Leu Thr Ser Gly 420 425 430 Met Glu Leu
Lys Asp Asn Ile Leu Val Ser Gly Asn Ala Asp Ser Thr 435 440 445 Val
Lys Ile Trp Asp Ile Lys Thr Gly Gln Cys Leu Gln Thr Leu Gln 450 455
460 Gly Pro Asn Lys His Gln Ser Ala Val Thr Cys Leu Gln Phe Asn Lys
465 470 475 480 Asn Phe Val Ile Thr Ser Ser Asp Asp Gly Thr Val Lys
Leu Trp Asp 485 490 495 Leu Lys Thr Gly Glu Phe Ile Arg Asn Leu Val
Thr Leu Glu Ser Gly 500 505 510 Gly Ser Gly Gly Val Val Trp Arg Ile
Arg Ala Ser Asn Thr Lys Leu 515 520 525 Val Cys Ala Val Gly Ser Arg
Asn Gly Thr Glu Glu Thr Lys Leu Leu 530 535 540 Val Leu Asp Phe Asp
Val Asp Met Lys 545 550 6 545 PRT Homo sapiens 6 Met Ile Phe Tyr
Lys Met Lys Arg Lys Leu Asp His Gly Ser Glu Val 1 5 10 15 Arg Ser
Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr 20 25 30
Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly 35
40 45 Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile
Thr 50 55 60 Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys
Met Phe Gln 65 70 75 80 Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu
Asp Glu Leu Ile Asp 85 90 95 Ser Cys Glu Pro Thr Gln Val Lys His
Met Met Gln Val Ile Glu Pro 100 105 110 Gln Phe Gln Arg Asp Phe Ile
Ser Leu Leu Pro Lys Glu Leu Ala Leu 115 120 125 Tyr Val Leu Ser Phe
Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln 130 135 140 Thr Cys Arg
Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg 145 150 155 160
Glu Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile Lys Arg 165
170 175 Arg Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys Ser
Ala 180 185 190 Tyr Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg
Gly Glu Leu 195 200 205 Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp
His Val Ile Thr Cys 210 215 220 Leu Gln Phe Cys Gly Asn Arg Ile Val
Ser Gly Ser Asp Asp Asn Thr 225 230 235 240 Leu Lys Val Trp Ser Ala
Val Thr Gly Lys Cys Leu Arg Thr Leu Val 245 250 255 Gly His Thr Gly
Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile 260 265 270 Ile Ser
Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr 275 280 285
Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys 290
295 300 Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp Ala
Thr 305 310 315 320 Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu
His Val Leu Met 325 330 335 Gly His Val Ala Ala Val Arg Cys Val Gln
Tyr Asp Gly Arg Arg Val 340 345 350 Val Ser Gly Ala Tyr Asp Phe Met
Val Lys Val Trp Asp Pro Glu Thr 355 360 365 Glu Thr Cys Leu His Thr
Leu Gln Gly His Thr Asn Arg Val Tyr Ser 370 375 380 Leu Gln Phe Asp
Gly Ile His Val Val Ser Gly Ser Leu Asp Thr Ser 385 390 395 400 Ile
Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr Leu Thr 405 410
415 Gly His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu
420 425 430 Val Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile
Lys Thr 435 440 445 Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys
His Gln Ser Ala 450 455 460 Val Thr Cys Leu Gln Phe Asn Lys Asn Phe
Val Ile Thr Ser Ser Asp 465 470 475 480 Asp Gly Thr Val Lys Leu Trp
Asp Leu Lys Thr Gly Glu Phe Ile Arg 485 490 495 Asn Leu Val Thr Leu
Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg 500 505 510 Ile Arg Ala
Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn 515 520 525 Gly
Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met 530 535
540 Lys 545 7 540 PRT Homo sapiens 7 Met Lys Arg Lys Leu Asp His
Gly Ser Glu Val Arg Ser Phe Ser Leu 1 5 10 15 Gly Lys Lys Pro Cys
Lys Val Ser Glu Tyr Thr Ser Thr Thr Gly Leu 20 25 30 Val Pro Cys
Ser Ala Thr Pro Thr Thr Phe Gly Asp Leu Arg Ala Ala 35 40 45 Asn
Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser Val Gln Pro Pro 50 55
60 Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser Trp Ser Gly Pro
65 70 75 80 Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys Glu
Pro Thr 85 90 95 Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln
Phe Gln Arg Asp 100 105 110 Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala
Leu Tyr Val Leu Ser Phe 115 120 125 Leu Glu Pro Lys Asp Leu Leu Gln
Ala Ala Gln Thr Cys Arg Tyr Trp 130 135 140 Arg Ile Leu Ala Glu Asp
Asn Leu Leu Trp Arg Glu Lys Cys Lys Glu 145 150 155 160 Glu Gly Ile
Asp Glu Pro Leu His Ile Lys Arg Arg Lys Val Ile Lys 165 170 175 Pro
Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr Ile Arg Gln His 180 185
190 Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu Lys Ser Pro Lys Val
195 200 205 Leu Lys Gly His Asp Asp His Val Ile Thr Cys Leu Gln Phe
Cys Gly 210 215 220 Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr Leu
Lys Val Trp Ser 225 230 235 240 Ala Val Thr Gly Lys Cys Leu Arg Thr
Leu Val Gly His Thr Gly Gly 245 250 255 Val Trp Ser Ser Gln Met Arg
Asp Asn Ile Ile Ile Ser Gly Ser Thr 260 265 270 Asp Arg Thr Leu Lys
Val Trp Asn Ala Glu Thr Gly Glu Cys Ile His 275 280 285 Thr Leu Tyr
Gly His Thr Ser Thr Val Arg Cys Met His Leu His Glu 290 295 300 Lys
Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu Arg Val Trp Asp 305 310
315 320 Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly His Val Ala
Ala 325 330 335 Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val Ser
Gly Ala Tyr 340 345 350 Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr
Glu Thr Cys Leu His 355 360 365 Thr Leu Gln Gly His Thr Asn Arg Val
Tyr Ser Leu Gln Phe Asp Gly 370 375 380 Ile His Val Val Ser Gly Ser
Leu Asp Thr Ser Ile Arg Val Trp Asp 385 390 395 400 Val Glu Thr Gly
Asn Cys Ile His Thr Leu Thr Gly His Gln Ser Leu 405 410 415 Thr Ser
Gly Met Glu Leu Lys Asp Asn Ile Leu Val Ser Gly Asn Ala 420 425 430
Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr Gly Gln Cys Leu Gln 435
440 445 Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val Thr Cys Leu
Gln 450 455 460 Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp Gly
Thr Val Lys 465 470 475 480 Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile
Arg Asn Leu Val Thr Leu 485 490 495 Glu Ser Gly Gly Ser Gly Gly Val
Val Trp Arg Ile Arg Ala Ser Asn 500 505 510 Thr Lys Leu Val Cys Ala
Val Gly Ser Arg Asn Gly Thr Glu Glu Thr 515 520 525 Lys Leu Leu Val
Leu Asp Phe Asp Val Asp Met Lys 530 535 540 8 589 PRT Homo sapiens
8 Met Ser Lys Pro Gly Lys Pro Thr Leu Asn His Gly Leu Val Pro Val 1
5 10 15 Asp Leu Lys Ser Ala Lys Glu Pro Leu Pro His Gln Thr Val Met
Lys 20 25 30 Ile Phe Ser Ile Ser Ile Ile Ala Gln Gly Leu Pro Phe
Cys Arg Arg 35 40 45 Arg Met Lys Arg Lys Leu Asp His Gly Ser Glu
Val Arg Ser Phe Ser 50 55 60 Leu Gly Lys Lys Pro Cys Lys Val Ser
Glu Tyr Thr Ser Thr Thr Gly 65 70 75 80 Leu Val Pro Cys Ser Ala Thr
Pro Thr Thr Phe Gly Asp Leu Arg Ala 85 90 95 Ala Asn Gly Gln Gly
Gln Gln Arg Arg Arg Ile Thr Ser Val Gln Pro 100 105 110 Pro Thr Gly
Leu Gln Glu Trp Leu Lys Met Phe Gln Ser Trp Ser Gly 115 120 125 Pro
Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys Glu Pro 130 135
140 Thr Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln Phe Gln Arg
145 150 155 160 Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr
Val Leu Ser 165 170 175 Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala
Gln Thr Cys Arg Tyr 180 185 190 Trp Arg Ile Leu Ala Glu Asp Asn Leu
Leu Trp Arg Glu Lys Cys Lys 195 200 205 Glu Glu Gly Ile Asp Glu Pro
Leu His Ile Lys Arg Arg Lys Val Ile 210 215 220 Lys Pro Gly Phe Ile
His Ser Pro Trp Lys Ser Ala Tyr Ile Arg Gln 225 230 235 240 His Arg
Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu Lys Ser Pro Lys 245 250 255
Val Leu Lys Gly His Asp Asp His Val Ile Thr Cys Leu Gln Phe Cys 260
265 270 Gly Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr Leu Lys Val
Trp 275 280 285 Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val Gly
His Thr Gly 290 295 300 Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile
Ile Ile Ser Gly Ser 305 310 315 320 Thr Asp Arg Thr Leu Lys Val Trp
Asn Ala Glu Thr Gly Glu Cys Ile 325 330 335 His Thr Leu Tyr Gly His
Thr Ser Thr Val Arg Cys Met His Leu His 340 345 350 Glu Lys Arg Val
Val Ser Gly Ser Arg Asp Ala Thr Leu Arg Val Trp 355 360 365 Asp Ile
Glu Thr Gly Gln Cys Leu His Val Leu Met Gly His Val Ala 370 375 380
Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val Ser Gly Ala 385
390 395 400 Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu Thr
Cys Leu 405 410 415 His Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser
Leu Gln Phe Asp 420 425 430 Gly Ile His Val Val Ser Gly Ser Leu Asp
Thr Ser Ile Arg Val Trp 435 440 445 Asp Val Glu Thr Gly Asn Cys Ile
His Thr Leu Thr Gly His Gln Ser 450 455 460 Leu Thr Ser Gly Met Glu
Leu Lys Asp Asn Ile Leu Val Ser Gly Asn 465 470 475 480 Ala Asp Ser
Thr Val Lys Ile Trp Asp Ile Lys Thr Gly Gln Cys Leu 485 490 495 Gln
Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val Thr Cys Leu 500 505
510 Gln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp Gly Thr Val
515 520 525 Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg Asn Leu
Val Thr 530 535 540 Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg
Ile Arg Ala Ser 545 550 555 560 Asn Thr Lys Leu Val Cys Ala Val Gly
Ser Arg Asn Gly Thr Glu Glu 565 570 575 Thr Lys Leu Leu Val Leu Asp
Phe Asp Val Asp Met Lys 580 585 9 559 PRT Homo sapiens 9 Met Lys
Ile Phe Ser Ile Ser Ile Ile Ala Gln Gly Leu Pro Phe Cys 1 5 10 15
Arg Arg Arg Met Lys Arg Lys Leu Asp His Gly Ser Glu Val Arg Ser 20
25 30 Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr Ser
Thr 35 40 45 Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr Phe
Gly Asp Leu 50 55 60 Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg
Arg Ile Thr Ser Val 65 70 75 80 Gln Pro Pro Thr Gly Leu Gln Glu Trp
Leu Lys Met Phe Gln Ser Trp 85 90 95 Ser Gly Pro Glu Lys Leu Leu
Ala Leu Asp Glu Leu Ile Asp Ser Cys 100 105 110 Glu Pro Thr Gln Val
Lys His Met Met Gln Val Ile Glu Pro Gln Phe 115 120 125 Gln Arg Asp
Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val 130 135 140 Leu
Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln Thr Cys 145 150
155 160 Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg Glu
Lys 165 170 175 Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile Lys
Arg Arg Lys 180 185 190 Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp
Lys Ser Ala Tyr Ile 195 200 205 Arg Gln His Arg Ile Asp Thr Asn Trp
Arg Arg Gly Glu Leu Lys Ser 210 215 220 Pro Lys Val Leu Lys Gly His
Asp Asp His Val Ile Thr Cys Leu Gln 225 230 235 240 Phe Cys Gly Asn
Arg Ile Val Ser Gly Ser Asp Asp Asn Thr Leu Lys
245 250 255 Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val
Gly His 260 265 270 Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn
Ile Ile Ile Ser 275 280 285 Gly Ser Thr Asp Arg Thr Leu Lys Val Trp
Asn Ala Glu Thr Gly Glu 290 295 300 Cys Ile His Thr Leu Tyr Gly His
Thr Ser Thr Val Arg Cys Met His 305 310 315 320 Leu His Glu Lys Arg
Val Val Ser Gly Ser Arg Asp Ala Thr Leu Arg 325 330 335 Val Trp Asp
Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly His 340 345 350 Val
Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val Ser 355 360
365 Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu Thr
370 375 380 Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser
Leu Gln 385 390 395 400 Phe Asp Gly Ile His Val Val Ser Gly Ser Leu
Asp Thr Ser Ile Arg 405 410 415 Val Trp Asp Val Glu Thr Gly Asn Cys
Ile His Thr Leu Thr Gly His 420 425 430 Gln Ser Leu Thr Ser Gly Met
Glu Leu Lys Asp Asn Ile Leu Val Ser 435 440 445 Gly Asn Ala Asp Ser
Thr Val Lys Ile Trp Asp Ile Lys Thr Gly Gln 450 455 460 Cys Leu Gln
Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val Thr 465 470 475 480
Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp Gly 485
490 495 Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg Asn
Leu 500 505 510 Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp
Arg Ile Arg 515 520 525 Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly
Ser Arg Asn Gly Thr 530 535 540 Glu Glu Thr Lys Leu Leu Val Leu Asp
Phe Asp Val Asp Met Lys 545 550 555 10 540 PRT Homo sapiens 10 Met
Lys Arg Lys Leu Asp His Gly Ser Glu Val Arg Ser Phe Ser Leu 1 5 10
15 Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr Ser Thr Thr Gly Leu
20 25 30 Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly Asp Leu Arg
Ala Ala 35 40 45 Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser
Val Gln Pro Pro 50 55 60 Thr Gly Leu Gln Glu Trp Leu Lys Met Phe
Gln Ser Trp Ser Gly Pro 65 70 75 80 Glu Lys Leu Leu Ala Leu Asp Glu
Leu Ile Asp Ser Cys Glu Pro Thr 85 90 95 Gln Val Lys His Met Met
Gln Val Ile Glu Pro Gln Phe Gln Arg Asp 100 105 110 Phe Ile Ser Leu
Leu Pro Lys Glu Leu Ala Leu Tyr Val Leu Ser Phe 115 120 125 Leu Glu
Pro Lys Asp Leu Leu Gln Ala Ala Gln Thr Cys Arg Tyr Trp 130 135 140
Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg Glu Lys Cys Lys Glu 145
150 155 160 Glu Gly Ile Asp Glu Pro Leu His Ile Lys Arg Arg Lys Val
Ile Lys 165 170 175 Pro Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr
Ile Arg Gln His 180 185 190 Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu
Leu Lys Ser Pro Lys Val 195 200 205 Leu Lys Gly His Asp Asp His Val
Ile Thr Cys Leu Gln Phe Cys Gly 210 215 220 Asn Arg Ile Val Ser Gly
Ser Asp Asp Asn Thr Leu Lys Val Trp Ser 225 230 235 240 Ala Val Thr
Gly Lys Cys Leu Arg Thr Leu Val Gly His Thr Gly Gly 245 250 255 Val
Trp Ser Ser Gln Met Arg Asp Asn Ile Ile Ile Ser Gly Ser Thr 260 265
270 Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr Gly Glu Cys Ile His
275 280 285 Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys Met His Leu
His Glu 290 295 300 Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu
Arg Val Trp Asp 305 310 315 320 Ile Glu Thr Gly Gln Cys Leu His Val
Leu Met Gly His Val Ala Ala 325 330 335 Val Arg Cys Val Gln Tyr Asp
Gly Arg Arg Val Val Ser Gly Ala Tyr 340 345 350 Asp Phe Met Val Lys
Val Trp Asp Pro Glu Thr Glu Thr Cys Leu His 355 360 365 Thr Leu Gln
Gly His Thr Asn Arg Val Tyr Ser Leu Gln Phe Asp Gly 370 375 380 Ile
His Val Val Ser Gly Ser Leu Asp Thr Ser Ile Arg Val Trp Asp 385 390
395 400 Val Glu Thr Gly Asn Cys Ile His Thr Leu Thr Gly His Gln Ser
Leu 405 410 415 Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu Val Ser
Gly Asn Ala 420 425 430 Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr
Gly Gln Cys Leu Gln 435 440 445 Thr Leu Gln Gly Pro Asn Lys His Gln
Ser Ala Val Thr Cys Leu Gln 450 455 460 Phe Asn Lys Asn Phe Val Ile
Thr Ser Ser Asp Asp Gly Thr Val Lys 465 470 475 480 Leu Trp Asp Leu
Lys Thr Gly Glu Phe Ile Arg Asn Leu Val Thr Leu 485 490 495 Glu Ser
Gly Gly Ser Gly Gly Val Val Trp Arg Ile Arg Ala Ser Asn 500 505 510
Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn Gly Thr Glu Glu Thr 515
520 525 Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met Lys 530 535 540
11 34 DNA Artificial Sequence Description of Artificial Sequence
Oligonucleotide primer 11 cgggatccac catggatgat ggatcgatga cacc 34
12 33 DNA Artificial Sequence Description of Artificial Sequence
Oligonucleotide primer 12 ggaattcctt aagggtatac agcatcaaag tcg 33
13 25 DNA Artificial Sequence Description of Artificial Sequence
Oligonucleotide primer 13 tcacttcatg tccacatcaa agtcc 25 14 26 DNA
Artificial Sequence Description of Artificial Sequence
Oligonucleotide primer 14 ggtaattaca agttcttgtt gaactg 26 15 22 DNA
Artificial Sequence Description of Artificial Sequence
Oligonucleotide primer 15 ccctgcaacg tgtgtagaca gg 22 16 24 DNA
Artificial Sequence Description of Artificial Sequence
Oligonucleotide primer 16 ccagtctctg cattccacac tttg 24 17 23 DNA
Artificial Sequence Description of Artificial Sequence
Oligonucleotide primer 17 ctcagacagg tcaggacatt tgg 23 18 33 DNA
Artificial Sequence Description of Artificial Sequence
Oligonucleotide primer 18 ggaattccat gaaaagattg gaccatggtt ctg 33
19 34 DNA Artificial Sequence Description of Artificial Sequence
Oligonucleotide primer 19 ggaattcctc acttcatgtc acatcaaagt ccag 34
20 1881 DNA Artificial Sequence Description of Artificial Sequence
6 myc tagged homo sapiens 20 atggagcaaa agctcatttc tgaagaggac
ttgaatgaaa tggagcaaaa gctcatttct 60 gaagaggact tgaatgaaat
ggagcaaaag ctcatttctg aagaggactt gaatgaaatg 120 gagcaaaagc
tcatttctga agaggacttg aatgaaatgg agcaaaagct catttctgaa 180
gaggacttga atgaaatgga gagcttgggc gacctcacca tggagcaaaa gctcatttct
240 gaagaggact tgaattccat gaaaagaaag ttggaccatg gttctgaggt
ccgctctttt 300 tctttgggaa agaaaccatg caaagtctca gaatatacaa
gtaccactgg gcttgtacca 360 tgttcagcaa caccaacaac ttttggggac
ctcagagcag ccaatggcca agggcaacaa 420 cgacgccgaa ttacatctgt
ccagccacct acaggcctcc aggaatggct aaaaatgttt 480 cagagctgga
gtggaccaga gaaattgctt gctttagatg aactcattga tagttgtgaa 540
ccaacacaag taaaacatat gatgcaagtg atagaacccc agtttcaacg agacttcatt
600 tcattgctcc ctaaagagtt ggcactctat gtgctttcat tcctggaacc
caaagacctg 660 ctacaagcag ctcagacatg tcgctactgg agaattttgg
ctgaagacaa ccttctctgg 720 agagagaaat gcaaagaaga ggggattgat
gaaccattgc acatcaagag aagaaaagta 780 ataaaaccag gtttcataca
cagtccatgg aaaagtgcat acatcagaca gcacagaatt 840 gatactaact
ggaggcgagg agaactcaaa tctcctaagg tgctgaaagg acatgatgat 900
catgtgatca catgcttaca gttttgtggt aaccgaatag ttagtggttc tgatgacaac
960 actttaaaag tttggtcagc agtcacaggc aaatgtctga gaacattagt
gggacataca 1020 ggtggagtat ggtcatcaca aatgagggac aacatcatca
ttagtggatc tacagatcgg 1080 acactcaaag tgtggaatgc agagactgga
gaatgtatac acaccttata tgggcatact 1140 tccactgtgc gttgtatgca
tcttcatgaa aaaagagttg ttagcggttc tcgagatgcc 1200 actcttaggg
tttgggatat tgagacaggc cagtgtttac atgttttgat gggtcatgtt 1260
gcagcagtcc gctgtgttca atatgatggc aggagggttg ttagtggagc atatgatttt
1320 atggtaaagg tgtgggatcc agagactgaa acctgtctac acacgttgca
ggggcatact 1380 aatagagtct attcattaca gtttgatggt atccatgtgg
tgagtggatc tcttgataca 1440 tccatccgtg tttgggatgt ggagacaggg
aattgcattc acacgttaac agggcaccag 1500 tcgttaacaa gtggaatgga
actcaaagac aatattcttg tctctgggaa tgcagattct 1560 acagttaaaa
tctgggatat caaaacagga cagtgtttac aaacattgca aggtcccaac 1620
aagcatcaga gtgctgtgac ctgtttacag ttcaacaaga actttgtaat taccagctca
1680 gatgatggaa ctgtaaaact atgggacttg aaaacgggtg aatttattcg
aaacctagtc 1740 acattggaga gtggggggag tgggggagtt gtgtggcgga
tcagagcctc aaacacaaag 1800 ctggtgtgtg cagttgggag tcggaatggg
actgaagaaa ccaagctgct ggtgctggac 1860 tttgatgtgg acatgaagtg a 1881
21 626 PRT Artificial Sequence Description of Artificial Sequence 6
myc tagged homo sapien 21 Met Glu Gln Lys Leu Ile Ser Glu Glu Asp
Leu Asn Glu Met Glu Gln 1 5 10 15 Lys Leu Ile Ser Glu Glu Asp Leu
Asn Glu Met Glu Gln Lys Leu Ile 20 25 30 Ser Glu Glu Asp Leu Asn
Glu Met Glu Gln Lys Leu Ile Ser Glu Glu 35 40 45 Asp Leu Asn Glu
Met Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 50 55 60 Glu Met
Glu Ser Leu Gly Asp Leu Thr Met Glu Gln Lys Leu Ile Ser 65 70 75 80
Glu Glu Asp Leu Asn Ser Met Lys Arg Lys Leu Asp His Gly Ser Glu 85
90 95 Val Arg Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu
Tyr 100 105 110 Thr Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro
Thr Thr Phe 115 120 125 Gly Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln
Gln Arg Arg Arg Ile 130 135 140 Thr Ser Val Gln Pro Pro Thr Gly Leu
Gln Glu Trp Leu Lys Met Phe 145 150 155 160 Gln Ser Trp Ser Gly Pro
Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile 165 170 175 Asp Ser Cys Glu
Pro Thr Gln Val Lys His Met Met Gln Val Ile Glu 180 185 190 Pro Gln
Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala 195 200 205
Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala 210
215 220 Gln Thr Cys Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu
Trp 225 230 235 240 Arg Glu Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro
Leu His Ile Lys 245 250 255 Arg Arg Lys Val Ile Lys Pro Gly Phe Ile
His Ser Pro Trp Lys Ser 260 265 270 Ala Tyr Ile Arg Gln His Arg Ile
Asp Thr Asn Trp Arg Arg Gly Glu 275 280 285 Leu Lys Ser Pro Lys Val
Leu Lys Gly His Asp Asp His Val Ile Thr 290 295 300 Cys Leu Gln Phe
Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp Asn 305 310 315 320 Thr
Leu Lys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu 325 330
335 Val Gly His Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile
340 345 350 Ile Ile Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn
Ala Glu 355 360 365 Thr Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr
Ser Thr Val Arg 370 375 380 Cys Met His Leu His Glu Lys Arg Val Val
Ser Gly Ser Arg Asp Ala 385 390 395 400 Thr Leu Arg Val Trp Asp Ile
Glu Thr Gly Gln Cys Leu His Val Leu 405 410 415 Met Gly His Val Ala
Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg 420 425 430 Val Val Ser
Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu 435 440 445 Thr
Glu Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val Tyr 450 455
460 Ser Leu Gln Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp Thr
465 470 475 480 Ser Ile Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile
His Thr Leu 485 490 495 Thr Gly His Gln Ser Leu Thr Ser Gly Met Glu
Leu Lys Asp Asn Ile 500 505 510 Leu Val Ser Gly Asn Ala Asp Ser Thr
Val Lys Ile Trp Asp Ile Lys 515 520 525 Thr Gly Gln Cys Leu Gln Thr
Leu Gln Gly Pro Asn Lys His Gln Ser 530 535 540 Ala Val Thr Cys Leu
Gln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser 545 550 555 560 Asp Asp
Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile 565 570 575
Arg Asn Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp 580
585 590 Arg Ile Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser
Arg 595 600 605 Asn Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe
Asp Val Asp 610 615 620 Met Lys 625 22 31 DNA Artificial Sequence
Description of Artificial Sequence Oligonucleotide primer 22
gggtacccct cattattccc tcgagttctt c 31 23 29 DNA Artificial Sequence
Description of Artificial Sequence Oligonucleotide primer 23
ggaattcctt catgtccaca tcaaagtcc 29 24 2010 DNA Artificial Sequence
Description of Artificial Sequence V5HIS tagged homo sapien 24
atgtgtgtcc cgagaagcgg tttgatactg agctgcattt gcctttactg tggagttttg
60 ttgccggttc tgctccctaa tcttcctttt ctgacgtgcc tgagcatgtc
cacattagaa 120 tctgtgacat acctacctga aaaaggttta tattgtcaga
gactgccaag cagccggaca 180 cacgggggca cagaatcact gaaggggaaa
aatacagaaa atatgggttt ctacggcaca 240 ttaaaaatga ttttttacaa
aatgaaaaga aagttggacc atggttctga ggtccgctct 300 ttttctttgg
gaaagaaacc atgcaaagtc tcagaatata caagtaccac tgggcttgta 360
ccatgttcag caacaccaac aacttttggg gacctcagag cagccaatgg ccaagggcaa
420 caacgacgcc gaattacatc tgtccagcca cctacaggcc tccaggaatg
gctaaaaatg 480 tttcagagct ggagtggacc agagaaattg cttgctttag
atgaactcat tgatagttgt 540 gaaccaacac aagtaaaaca tatgatgcaa
gtgatagaac cccagtttca acgagacttc 600 atttcattgc tccctaaaga
gttggcactc tatgtgcttt cattcctgga acccaaagac 660 ctgctacaag
cagctcagac atgtcgctac tggagaattt tggctgaaga caaccttctc 720
tggagagaga aatgcaaaga agaggggatt gatgaaccat tgcacatcaa gagaagaaaa
780 gtaataaaac caggtttcat acacagtcca tggaaaagtg catacatcag
acagcacaga 840 attgatacta actggaggcg aggagaactc aaatctccta
aggtgctgaa aggacatgat 900 gatcatgtga tcacatgctt acagttttgt
ggtaaccgaa tagttagtgg ttctgatgac 960 aacactttaa aagtttggtc
agcagtcaca ggcaaatgtc tgagaacatt agtgggacat 1020 acaggtggag
tatggtcatc acaaatgaga gacaacatca tcattagtgg atctacagat 1080
cggacactca aagtgtggaa tgcagagact ggagaatgta tacacacctt atatgggcat
1140 acttccactg tgcgttgtat gcatcttcat gaaaaaagag ttgttagcgg
ttctcgagat 1200 gccactctta gggtttggga tattgagaca ggccagtgtt
tacatgtttt gatgggtcat 1260 gttgcagcag tccgctgtgt tcaatatgat
ggcaggaggg ttgttagtgg agcatatgat 1320 tttatggtaa aggtgtggga
tccagagact gaaacctgtc tacacacgtt gcaggggcat 1380 actaatagag
tctattcatt acagtttgat ggtatccatg tggtgagtgg atctcttgat 1440
acatcaatcc gtgtttggga tgtggagaca gggaattgca ttcacacgtt aacagggcac
1500 cagtcgttaa caagtggaat ggaactcaaa gacaatattc ttgtctctgg
gaatgcagat 1560 tctacagtta aaatctggga tatcaaaaca ggacagtgtt
tacaaacatt gcaaggtccc 1620 aacaagcatc agagtgctgt gacctgttta
cagttcaaca agaactttgt aattaccagc 1680 tcagatgatg gaactgtaaa
actatgggac ttgaaaacgg gtgaatttat tcgaaaccta 1740 gtcacattgg
agagtggggg gagtggggga gttgtgtggc ggatcagagc ctcaaacaca 1800
aagctggtgt gtgcagttgg gagtcggaat gggactgaag aaaccaagct gctggtgctg
1860 gactttgatg tggacatgaa ggaattctgc agatatccag cacagtggcg
gccgctcgag 1920 tctagagggc ccttcgaagg taagcctatc cctaaccctc
tcctcggtct cgattctacg 1980 cgtaccggtc atcatcacca
tcaccattga 2010 25 669 PRT Artificial Sequence Description of
Artificial Sequence V5HIS tagged homo sapien 25 Met Cys Val Pro Arg
Ser Gly Leu Ile Leu Ser Cys Ile Cys Leu Tyr 1 5 10 15 Cys Gly Val
Leu Leu Pro Val Leu Leu Pro Asn Leu Pro Phe Leu Thr 20 25 30 Cys
Leu Ser Met Ser Thr Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys 35 40
45 Gly Leu Tyr Cys Gln Arg Leu Pro Ser Ser Arg Thr His Gly Gly Thr
50 55 60 Glu Ser Leu Lys Gly Lys Asn Thr Glu Asn Met Gly Phe Tyr
Gly Thr 65 70 75 80 Leu Lys Met Ile Phe Tyr Lys Met Lys Arg Lys Leu
Asp His Gly Ser 85 90 95 Glu Val Arg Ser Phe Ser Leu Gly Lys Lys
Pro Cys Lys Val Ser Glu 100 105 110 Tyr Thr Ser Thr Thr Gly Leu Val
Pro Cys Ser Ala Thr Pro Thr Thr 115 120 125 Phe Gly Asp Leu Arg Ala
Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg 130 135 140 Ile Thr Ser Val
Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met 145 150 155 160 Phe
Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu 165 170
175 Ile Asp Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile
180 185 190 Glu Pro Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys
Glu Leu 195 200 205 Ala Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp
Leu Leu Gln Ala 210 215 220 Ala Gln Thr Cys Arg Tyr Trp Arg Ile Leu
Ala Glu Asp Asn Leu Leu 225 230 235 240 Trp Arg Glu Lys Cys Lys Glu
Glu Gly Ile Asp Glu Pro Leu His Ile 245 250 255 Lys Arg Arg Lys Val
Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys 260 265 270 Ser Ala Tyr
Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly 275 280 285 Glu
Leu Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile 290 295
300 Thr Cys Leu Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp
305 310 315 320 Asn Thr Leu Lys Val Trp Ser Ala Val Thr Gly Lys Cys
Leu Arg Thr 325 330 335 Leu Val Gly His Thr Gly Gly Val Trp Ser Ser
Gln Met Arg Asp Asn 340 345 350 Ile Ile Ile Ser Gly Ser Thr Asp Arg
Thr Leu Lys Val Trp Asn Ala 355 360 365 Glu Thr Gly Glu Cys Ile His
Thr Leu Tyr Gly His Thr Ser Thr Val 370 375 380 Arg Cys Met His Leu
His Glu Lys Arg Val Val Ser Gly Ser Arg Asp 385 390 395 400 Ala Thr
Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val 405 410 415
Leu Met Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg 420
425 430 Arg Val Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp
Pro 435 440 445 Glu Thr Glu Thr Cys Leu His Thr Leu Gln Gly His Thr
Asn Arg Val 450 455 460 Tyr Ser Leu Gln Phe Asp Gly Ile His Val Val
Ser Gly Ser Leu Asp 465 470 475 480 Thr Ser Ile Arg Val Trp Asp Val
Glu Thr Gly Asn Cys Ile His Thr 485 490 495 Leu Thr Gly His Gln Ser
Leu Thr Ser Gly Met Glu Leu Lys Asp Asn 500 505 510 Ile Leu Val Ser
Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile 515 520 525 Lys Thr
Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln 530 535 540
Ser Ala Val Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser 545
550 555 560 Ser Asp Asp Gly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly
Glu Phe 565 570 575 Ile Arg Asn Leu Val Thr Leu Glu Ser Gly Gly Ser
Gly Gly Val Val 580 585 590 Trp Arg Ile Arg Ala Ser Asn Thr Lys Leu
Val Cys Ala Val Gly Ser 595 600 605 Arg Asn Gly Thr Glu Glu Thr Lys
Leu Leu Val Leu Asp Phe Asp Val 610 615 620 Asp Met Lys Glu Phe Cys
Arg Tyr Pro Ala Gln Trp Arg Pro Leu Glu 625 630 635 640 Ser Arg Gly
Pro Phe Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly 645 650 655 Leu
Asp Ser Thr Arg Thr Gly His His His His His His 660 665 26 2001 DNA
Artificial Sequence Description of Artificial Sequence MYCHIS
tagged homo sapiens 26 atgtgtgtcc cgagaagcgg tttgatactg agctgcattt
gcctttactg tggagttttg 60 ttgccggttc tgctccctaa tcttcctttt
ctgacgtgcc tgagcatgtc cacattagaa 120 tctgtgacat acctacctga
aaaaggttta tattgtcaga gactgccaag cagccggaca 180 cacgggggca
cagaatcact gaaggggaaa aatacagaaa atatgggttt ctacggcaca 240
ttaaaaatga ttttttacaa aatgaaaaga aagttggacc atggttctga ggtccgctct
300 ttttctttgg gaaagaaacc atgcaaagtc tcagaatata caagtaccac
tgggcttgta 360 ccatgttcag caacaccaac aacttttggg gacctcagag
cagccaatgg ccaagggcaa 420 caacgacgcc gaattacatc tgtccagcca
cctacaggcc tccaggaatg gctaaaaatg 480 tttcagagct ggagtggacc
agagaaattg cttgctttag atgaactcat tgatagttgt 540 gaaccaacac
aagtaaaaca tatgatgcaa gtgatagaac cccagtttca acgagacttc 600
atttcattgc tccctaaaga gttggcactc tatgtgcttt cattcctgga acccaaagac
660 ctgctacaag cagctcagac atgtcgctac tggagaattt tggctgaaga
caaccttctc 720 tggagagaga aatgcaaaga agaggggatt gatgaaccat
tgcacatcaa gagaagaaaa 780 gtaataaaac caggtttcat acacagtcca
tggaaaagtg catacatcag acagcacaga 840 attgatacta actggaggcg
aggagaactc aaatctccta aggtgctgaa aggacatgat 900 gatcatgtga
tcacatgctt acagttttgt ggtaaccgaa tagttagtgg ttctgatgac 960
aacactttaa aagtttggtc agcagtcaca ggcaaatgtc tgagaacatt agtgggacat
1020 acaggtggag tatggtcatc acaaatgaga gacaacatca tcattagtgg
atctacagat 1080 cggacactca aagtgtggaa tgcagagact ggagaatgta
tacacacctt atatgggcat 1140 acttccactg tgcgttgtat gcatcttcat
gaaaaaagag ttgttagcgg ttctcgagat 1200 gccactctta gggtttggga
tattgagaca ggccagtgtt tacatgtttt gatgggtcat 1260 gttgcagcag
tccgctgtgt tcaatatgat ggcaggaggg ttgttagtgg agcatatgat 1320
tttatggtaa aggtgtggga tccagagact gaaacctgtc tacacacgtt gcaggggcat
1380 actaatagag tctattcatt acagtttgat ggtatccatg tggtgagtgg
atctcttgat 1440 acatcaatcc gtgtttggga tgtggagaca gggaattgca
ttcacacgtt aacagggcac 1500 cagtcgttaa caagtggaat ggaactcaaa
gacaatattc ttgtctctgg gaatgcagat 1560 tctacagtta aaatctggga
tatcaaaaca ggacagtgtt tacaaacatt gcaaggtccc 1620 aacaagcatc
agagtgctgt gacctgttta cagttcaaca agaactttgt aattaccagc 1680
tcagatgatg gaactgtaaa actatgggac ttgaaaacgg gtgaatttat tcgaaaccta
1740 gtcacattgg agagtggggg gagtggggga gttgtgtggc ggatcagagc
ctcaaacaca 1800 aagctggtgt gtgcagttgg gagtcggaat gggactgaag
aaaccaagct gctggtgctg 1860 gactttgatg tggacatgaa ggaattctgc
agatatccag cacagtggcg gccgctcgag 1920 tctagagggc ccttcgaaca
aaaactcatc tcagaagagg atctgaatat gcataccggt 1980 catcatcacc
atcaccattg a 2001 27 666 PRT Artificial Sequence Description of
Artificial Sequence MYCHIS tagged homo sapiens 27 Met Cys Val Pro
Arg Ser Gly Leu Ile Leu Ser Cys Ile Cys Leu Tyr 1 5 10 15 Cys Gly
Val Leu Leu Pro Val Leu Leu Pro Asn Leu Pro Phe Leu Thr 20 25 30
Cys Leu Ser Met Ser Thr Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys 35
40 45 Gly Leu Tyr Cys Gln Arg Leu Pro Ser Ser Arg Thr His Gly Gly
Thr 50 55 60 Glu Ser Leu Lys Gly Lys Asn Thr Glu Asn Met Gly Phe
Tyr Gly Thr 65 70 75 80 Leu Lys Met Ile Phe Tyr Lys Met Lys Arg Lys
Leu Asp His Gly Ser 85 90 95 Glu Val Arg Ser Phe Ser Leu Gly Lys
Lys Pro Cys Lys Val Ser Glu 100 105 110 Tyr Thr Ser Thr Thr Gly Leu
Val Pro Cys Ser Ala Thr Pro Thr Thr 115 120 125 Phe Gly Asp Leu Arg
Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg 130 135 140 Ile Thr Ser
Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met 145 150 155 160
Phe Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu 165
170 175 Ile Asp Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val
Ile 180 185 190 Glu Pro Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro
Lys Glu Leu 195 200 205 Ala Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys
Asp Leu Leu Gln Ala 210 215 220 Ala Gln Thr Cys Arg Tyr Trp Arg Ile
Leu Ala Glu Asp Asn Leu Leu 225 230 235 240 Trp Arg Glu Lys Cys Lys
Glu Glu Gly Ile Asp Glu Pro Leu His Ile 245 250 255 Lys Arg Arg Lys
Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys 260 265 270 Ser Ala
Tyr Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly 275 280 285
Glu Leu Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile 290
295 300 Thr Cys Leu Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp
Asp 305 310 315 320 Asn Thr Leu Lys Val Trp Ser Ala Val Thr Gly Lys
Cys Leu Arg Thr 325 330 335 Leu Val Gly His Thr Gly Gly Val Trp Ser
Ser Gln Met Arg Asp Asn 340 345 350 Ile Ile Ile Ser Gly Ser Thr Asp
Arg Thr Leu Lys Val Trp Asn Ala 355 360 365 Glu Thr Gly Glu Cys Ile
His Thr Leu Tyr Gly His Thr Ser Thr Val 370 375 380 Arg Cys Met His
Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp 385 390 395 400 Ala
Thr Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val 405 410
415 Leu Met Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg
420 425 430 Arg Val Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp
Asp Pro 435 440 445 Glu Thr Glu Thr Cys Leu His Thr Leu Gln Gly His
Thr Asn Arg Val 450 455 460 Tyr Ser Leu Gln Phe Asp Gly Ile His Val
Val Ser Gly Ser Leu Asp 465 470 475 480 Thr Ser Ile Arg Val Trp Asp
Val Glu Thr Gly Asn Cys Ile His Thr 485 490 495 Leu Thr Gly His Gln
Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn 500 505 510 Ile Leu Val
Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile 515 520 525 Lys
Thr Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln 530 535
540 Ser Ala Val Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser
545 550 555 560 Ser Asp Asp Gly Thr Val Lys Leu Trp Asp Leu Lys Thr
Gly Glu Phe 565 570 575 Ile Arg Asn Leu Val Thr Leu Glu Ser Gly Gly
Ser Gly Gly Val Val 580 585 590 Trp Arg Ile Arg Ala Ser Asn Thr Lys
Leu Val Cys Ala Val Gly Ser 595 600 605 Arg Asn Gly Thr Glu Glu Thr
Lys Leu Leu Val Leu Asp Phe Asp Val 610 615 620 Asp Met Lys Glu Phe
Cys Arg Tyr Pro Ala Gln Trp Arg Pro Leu Glu 625 630 635 640 Ser Arg
Gly Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 645 650 655
Met His Thr Gly His His His His His His 660 665
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