U.S. patent application number 12/709296 was filed with the patent office on 2011-03-10 for function and regulation of adamts-1.
This patent application is currently assigned to THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Qin Yu.
Application Number | 20110059065 12/709296 |
Document ID | / |
Family ID | 37884617 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110059065 |
Kind Code |
A1 |
Yu; Qin |
March 10, 2011 |
FUNCTION AND REGULATION OF ADAMTS-1
Abstract
The present invention relates to ADAMTS-1 and uses thereof. The
present invention also relates to fragments of ADAMTS-1 and methods
of inhibiting cell growth and metastasis. The present invention
also provide methods of identifying inhibitors and activators
relating to the function of ADAMTS-1.
Inventors: |
Yu; Qin; (New York,
NY) |
Assignee: |
THE TRUSTEES OF THE UNIVERSITY OF
PENNSYLVANIA
Philadelphia
PA
|
Family ID: |
37884617 |
Appl. No.: |
12/709296 |
Filed: |
February 19, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11104075 |
Apr 12, 2005 |
7696307 |
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12709296 |
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60561429 |
Apr 12, 2004 |
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60650027 |
Feb 4, 2005 |
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Current U.S.
Class: |
424/130.1 ;
424/94.67; 435/226; 435/7.1; 435/7.2; 514/19.3; 514/44R;
536/23.2 |
Current CPC
Class: |
C07K 16/40 20130101;
C12N 9/6489 20130101; G01N 33/57496 20130101; G01N 2500/00
20130101; C12Q 1/37 20130101; A61P 35/00 20180101; G01N 33/5011
20130101 |
Class at
Publication: |
424/130.1 ;
435/226; 424/94.67; 536/23.2; 514/44.R; 435/7.1; 435/7.2;
514/19.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 9/64 20060101 C12N009/64; A61K 38/46 20060101
A61K038/46; C07H 21/00 20060101 C07H021/00; A61K 31/7088 20060101
A61K031/7088; G01N 33/53 20060101 G01N033/53; A61K 38/02 20060101
A61K038/02; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with U.S. Government support (NIH
Grants No. RO1HL074117) and the U.S. Government may therefore have
certain rights in the invention.
Claims
1. An isolated polypeptide fragment of ADAMTS-1 that inhibits tumor
growth and/or metastasis, wherein the fragment consists of SEQ ID
NOs: 5, 7, 9, and/or 11.
2. A pharmaceutical composition comprising a polypeptide of claim
1.
3. A composition comprising at least two different polypeptide
fragment of ADAMTS-1 that inhibit cell proliferation or metastasis,
wherein said fragment comprises SEQ ID NOs: 5, 7, 9, and/or 11.
4. The composition of claim 3 wherein said composition is a
pharmaceutical composition.
5. An isolated polynucleotide encoding a polypeptide fragment of
ADAMTS-1 wherein said fragment inhibits tumor growth and/or
metastasis, wherein said polynucleotide comprises SEQ ID NO: 6, 8,
10, or 12.
6. A pharmaceutical composition comprising the isolated
polynucleotide of claim 6.
7. The isolated polynucleotide of claim 6 wherein said
polynucleotide is a vector or plasmid.
8. A method for identifying an inhibitor or an activator of
ADAMTS-1 auto-cleavage comprising performing a test assay
comprising: a) contacting ADAMTS-1 with a test compound under
conditions in which ADAMTS-1 undergoes cleavage in the absence of a
test compound; b) measuring cleavage level of ADAMTS-1; and c)
comparing the cleavage level to cleavage level of ADAMTS-1 in the
absence of the test compound, wherein a decrease in auto-cleavage
indicates that the test compound is a cleavage inhibitor or wherein
an increase in auto-cleavage indicates that the test compound is a
cleavage activator.
9. The method of claim 8 wherein said the test compound is
contacted with a cell comprising ADAMTS-1.
10. The method of claim 9, further comprising performing a negative
control assay which comprises contacting a cell that does not
comprise ADAMNTS-1 or a cell that comprises a cleavage resistant
mutant of ADAMTS-1.
11. The method of claim 9, further comprising performing a positive
control assay which comprises contacting a cell comprising ADAMTS-1
a positive control compound and measuring cleavage.
12. The method of claim 8, further comprising measuring the
cleavage of ADAMTS-1 in the absence of the test compound.
13. A method for identifying a heparin inhibitor comprising: a)
contacting a composition comprising heparin and ADAMTS-1 with a
test compound under conditions in which ADAMTS-1 undergoes
auto-cleavage and/or proteolytic cleavage in absence of heparin; b)
measuring cleavage level of ADAMTS-1; and c) comparing cleavage
level of ADAMTS-1 in the absence of the test compound; wherein an
increase in the cleavage of ADAMTS-1 indicates that the compound is
a heparin inhibitor.
14. A method of identifying a metalloproteinase inhibitor
comprising: a) contacting a ADAMTS-1 polypeptide or fragment
thereof comprising metalloproteinase activity with a test compound
under conditions which metalloproteinase activity is detected in
the absence of the test compound. b) measuring metalloproteinase
activity level of ADAMTS-1; and c) comparing the metalloproteinase
activity level of ADAMTS-1 in the presence or absence of the test
compound, wherein a decrease in metalloproteinase activity
indicates that the test compound is a metalloproteinase
inhibitor.
15. The method of claim 14 wherein said fragment comprises SEQ ID
NO: 5, 7, 9, and/or 11.
16. The method of claim 14 wherein the metalloproteinase activity
of ADAMTS-1 is compared to a fragment or mutant of ADAMTS-1 that
has no metalloproteinase activity.
17. The method of claim 16 wherein said fragment or mutant of
ADAMTS-1 that has no metalloproteinase activity comprises SEQ ID
NO: 31, 33, 35, and/or 36.
18. A method of treating cancer in an individual comprising
administering to the individual a therapeutically effective amount
of a polypeptide fragment of ADAMTS-1 and/or a nucleic acid that
encodes a polypeptide fragment of ADAMTS-1 that inhibits cell
proliferation and/or metastasis.
19. The method of claim 18 wherein the polypeptide fragment
comprises a TSP type-I motif.
20. The method of claim 18 wherein the fragment comprises SEQ ID
NO: 5, 7, 9 and/or 11.
21. The method of claim 18 wherein the nucleic acid molecule
encoding the polypeptide fragment comprises SEQ ID NO: 6, 8, 10,
and/or 11.
22. The method of claim 18 wherein said polypeptide fragment of
ADAMTS-1 comprises the spacer/Cys-rich and/or spacer domain of
ADAMTS-1 or a nucleic acid molecule encoding a polypeptide fragment
of ADAMTS-1 comprising the spacer/Cys-rich and/or spacer domain of
ADAMTS-1.
23. The method of claim 22 wherein said fragment comprises SEQ ID
NO: 21 and/or 23.
24. The method of claim 22 wherein said nucleic acid molecule
comprises SEQ ID NO: 22 and/or 24.
25. A method of treating cancer comprising administering an
inhibitor of the metalloproteinase activity of ADAMTS-1.
26. The method of claim 25 wherein the inhibitor is a
metalloproteinase defective polypeptide of ADAMTS-1 or a nucleic
acid molecule encoding a metalloproteinase defective polypeptide of
ADAMTS-1.
27. The method of claim 26 wherein the metalloproteinase defective
polypeptide of comprises SEQ ID NO: 29, 31, 33, and/or 35.
28. The method of claim 26 wherein the nucleic acid molecule
comprises SEQ ID NO: 30, 32, 34, and/or 36.
29. The method of claim 25 wherein said inhibitor is an antibody
that binds to ADAMTS-1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/561,429, filed Apr. 12, 2004 and U.S.
Provisional Application Ser. No. 60/650,027 filed Feb. 4, 2005 each
of which is herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] The members of ADAMTS (A Disintegrin And Metalloproteinase
with ThromboSpondin motifs) family belong to ADAM (A Disintegrin
And Metalloproteinase) family of multifunctional proteins that
display a significant sequence homology with snake venom
metalloproteinases. The amino-terminal half of ADAMTS is similar to
that of ADAM, which contains propeptide, metalloproteinase,
disintegrin, and cysteine-rich domains; while the C-terminal half
of ADAMTS is completely different and contains thrombospondin type
I-like (TSP) motifs that are originally found in thrombospondin 1
and 2 and spacer region. At least 18 members of ADAMTS have been
identified. ADAMTS-1 is the first member identified and is
expressed in many embryonic tissues and in tumors. Disruption of
ADAMTS-1 gene results in reduced growth, abnormalities in uteral,
adrenal, and dipose tissues, and female infertility.
[0004] ADAMTS-1 cleaves aggrecan and versican in vitro, however,
physiologic substrates of ADAMTS-1 remain to be identified. In
addition, ADAMTS-1 is cleaved at the spacer region by matrix
metalloproteinases (MMPs). The role of ADAMTS-1 in tumor growth and
metastasis is not well established. ADAMTS-1 was found to display
anti-angiogenic and anti-tumor activity, however, increased
expression of ADAMTS-1 was correlated to the enhanced metastatic
potential of pancreatic cancers, and studies have shown that
ADAMTS-1 is one of the genes up-regulated in the breast cancer with
elevated metastatic activity.
[0005] Thus, there is a need to clarify the biologic role of
ADAMTS-1. Furthermore, there is a need to identify compounds and/or
compositions that can be used to treat cancer or inhibit cell
growth.
SUMMARY OF THE INVENTION
[0006] In some embodiments, the present invention provides isolated
polypeptide fragments of ADAMTS-1 that inhibits tumor growth and
metastasis.
[0007] In some embodiments, the present invention provides
compositions comprising at least two different polypeptide fragment
of ADAMTS-1 that inhibit cell growth and/or metastasis.
[0008] In some embodiments, the present invention provides isolated
polynucleotides encoding a polypeptide fragment of ADAMTS-1 wherein
the fragment inhibits metastasis In some embodiments, the present
invention provides methods for identifying an inhibitor or an
activator of ADAMTS-1 cleavage.
[0009] In some embodiments, the present invention provides methods
for identifying a heparin inhibitor.
[0010] In some embodiments, the present invention provides methods
of identifying an inhibitor of the metalloproteinase activity of
ADAMTS-1.
[0011] In some embodiments, the present invention provides methods
of inhibiting metastasis comprising contacting the cell with a
polypeptide fragment of ADAMTS-1 that inhibits metastasis and/or a
nucleic acid that encodes a polypeptide fragment of ADAMTS-1 that
inhibits cell proliferation or metastasis.
[0012] In some embodiments, the present invention provides methods
of treating cancer in an individual comprising administering to the
individual a therapeutically effective amount of a polypeptide
fragment of ADAMTS-1 and/or a nucleic acid that encodes a
polypeptide fragment of ADAMTS-1 that inhibits cell proliferation
or metastasis.
[0013] In some embodiments, the present invention provides methods
of treating cancer comprising administering an inhibitor of the
metalloproteinase activity of ADAMTS-1.
[0014] In some embodiments, the present invention provides methods
of treating cancer comprising administering a therapeutically
effective amount of a composition comprising a polypeptide fragment
of ADAMTS-1 comprising the spacer/Cys-rich domain or the spacer
domain of ADAMTS-1 or a nucleic acid molecule encoding a
polypeptide fragment of ADAMTS-1 comprising the spacer/Cys-rich
domain or the spacer domain of ADAMTS-1.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1. ADAMTS-1 undergoes auto-proteolytic cleavage and the
self-cleavage of ADAMTS-1 is regulated. A. Diagram of the
expression constructs. B. Proteolytic cleavage of ADAMTS-1 requires
its own metalloproteinase activity. Cell culture supernatants
derived from the Cos-7 cells transfected with v5-epitope-tagged
ADAMTS-1 (lane 1-2) or ADAMTS-1E/Q (lane 3-4) were analyzed by
Western blot with anti-v5 antibody. The arrows indicate the
C-terminal cleavage fragments of ADAMTS-1. C. Cleavage of ADAMTS-1
is blocked by heparin and HS. TA3.sub.ADAMTS-1 cells were cultured
in the absence (lane 1) or presence of 100 .mu.g/ml of heparin
(lane 2), HS (lane 3), hyaluronan (lane 4), or CS (lane 5) for 48
hours and the cell culture supernatants were analyzed by Western
blot with anti-v5 antibody. The C-terminal fragments are indicated
by arrows. D. The clonal TA3 transfectants expressing different
cDNA products are shown. E. Auto-proteolytic cleavage capacity of
ADAMTS-1 was assessed by incubating 100 ng of purified ADAMTS-1 in
Tris buffer at 37.degree. C. for 0 min (lane 1), 30 min (lane 2), 1
(lane 3), 2 (lane 4), 4 (lane 5), 8 (lane 6), and 12 (lane 7)
hours. The reaction products were assessed by Western blot with
anti-v5 antibody.
[0016] FIG. 2. ADMATS-1 promotes pulmonary metastasis, while
ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCFa inhibits the process. A.
Representative gross pictures of the mouse lungs two-three weeks
after i.v. injection of TA3.sub.wtb (a-c) or TA3.sub.ADAMTS-1
(d-f), or TA3.sub.ADAMTS-1NTCF (g-i) cells. B. Survival rate of the
experimental mice which were injected with the TA3 transfectants
intravenously. Total of thirty mice were used for each type of
transfectants. C. Pulmonary metastatic burden is expressed by
weight of the lungs derived from the experimental mice 11 days and
18 days after the i.v. injection of the TA3 transfectants. D. The
representative H&E stained lung sections were derived the
experimental mice injected with TA3.sub.wtb (a), TA3.sub.ADAMTS-1
(b), TA3.sub.ADAMTS-1NTCF (c), and TA3.sub.ADAMTS-1CTCF (d) cells.
Bar, 100 .mu.m. E. Western blot analysis of v5-epitope tagged
ADAMTS-1 protein expressed by TA3.sub.ADAMTS-1 cells in vivo using
anti-v5 mAb. The proteins were derived from different pulmonary
metastases derived from TA3.sub.ADAMTS-1 cells. The arrow indicates
the mature proteolytically active ADAMST-1, and the arrowhead marks
pro-ADAMTS-1.
[0017] FIG. 3. ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF blocks
pulmonary metastasis by inhibiting proliferation and survival of
tumor cells and by inhibiting tumor angiogenesis. The experimental
mouse lungs were sectioned six days after iv injection of
TA3.sub.wtb (A, a-d), TA3.sub.ADAMTS-1 (A, e-h),
TA3.sub.ADAMTS-1NTCF (A, i-l), and TA3.sub.ADAMTS-1CTCF (A, m-p).
These sections were stained with H&E (A-a, e, i, m), or reacted
with Apoptag to detect apoptotic tumor cells in mouse lung
parenchyma (A-b, f, j, n), anti-Brdu antibody to detect the
proliferating tumor cells (A-c, g, k, o), or with anti-vWF antibody
to reveal blood vessels within the macro- or micro-metastases (A-d,
h, l, p). Bar: 120 .mu.m. The quantitative data that reveals the
effects of ADAMTS-1 and the fragments of ADAMTS-1 on tumor cell
apoptosis and proliferation and on tumor angiogenesis are shown in
panels B-a, b, c.
[0018] FIG. 4. ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF block
activation of EGFR and ErbB-2 in vivo, while ADAMTS-1 promotes
activation of these receptors and shedding of AR and HB-EGF
precursors. A. Tumor cell tracking assay was performed to determine
the pulmonary extravasation of TA3 transfectants. 24 hours after
i.v. injection of the green fluorescein labeled TA3 transfectants,
the mice lung were fixed and sectioned. TA3.sub.wtb (A-a),
TA3.sub.ADAMTS-1 (A-b), TA3.sub.ADAMTS-1NTCF (A-c), and
TA3.sub.ADAMTS-1CTCF (A-d) cells in the lung parenchyma were shown.
B. The pulmonary extravasation rates of the TA3 transfectants were
expressed as average number of the tumor cells per microscopic
field. C. Activation of EGFR and ErbB-2 in vivo:
immunoprecipitation with anti-EGFR(C-a) or anti-ErbB-2 (C-b)
antibody was performed using the protein lysates derived from the
mouse lungs which were implanted without (lanes 1-3) or with
TA3.sub.wtb (lanes 4-6), TA3.sub.ADAMTS-1 (lanes 7-9),
TA3.sub.ADAMTS-1NTCF (lane 10-12), and TA3.sub.ADAMTS-1CTCF (lanes
13-15) 24 hours prior. To normalize number of the tumor cells that
were included in the protein lysates, based on the tumor cell
extravasation rates (B), 100 .mu.g of the lung lysates with or
without TA3.sub.wtb cells, 71 .mu.g of the lung lysates containing
TA3.sub.ADAMTS-1 cells, and 143 .mu.g of the lung lysates
containing TA3.sub.ADAMTS-1NTCF cells, and 130 .mu.g of the lysates
containing TA3.sub.ADAMTS-1CTCF cells have been used. The
precipitated proteins were analyzed by Western blotting with
anti-phosphotyrosine antibody to detect phosphor-EGFR(C-a, upper
panel) or phosphor-ErbB-2 (C-b, upper panel), or with
anti-EGFR(C-a, bottom panel) or anti-ErbB-2 (C-b, bottom panel)
antibody to detect total amount of EGFR or ErbB-2, respectively. D.
ADAMTS-1 promotes shedding of AR (D-a), HB-EGF (D-b), but not
epigen (D-c), and the constitutive shedding of AR and HB-EGF is
blocked or inhibited by ADAMTS-1E/Q, respectively (lane 4 in D-a
and -b). Cos-7 cells were co-transfected with the expression
constructs containing cDNA inserts that encode AR, HB-EGF, or
epigen precursors without (lane 1) or with ADAMTS-1.sub.NTCF (lane
2), ADAMTS-1.sub.CTCF (lane 3), ADAMTS-1E/Q (lane 4), or ADAMTS-1
(lane 5), and the concentrated serum-free culture media derived
from these co-transfected Cos-7 cells were analyzed using anti-AR,
HB-EGF, or epigen antibody.
[0019] FIG. 5. ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF inhibit
activation of Erk1/2 kinases induced by soluble AR and HB-EGF. A.
soluble AR (5 ng/ml) or HB-EGF (4 ng/ml) were applied to the
serum-starved MCF-10A cells for 25 min in the absence (lane 3-4) or
presence of the neutralizing antibody against AR (A, lane 5-6) or
HB-EGF (B, lane 5-6) or in the presence of 400 ng of purified
full-length ADAMTS-1 (lane 7-8), ADAMTS-1.sub.NTCF (lane 9-10), or
ADAMTS-1.sub.CTCF (lane 11-12). Serum free medium alone was used in
lane 1-2. The MCF-10A cells were lysed and equal amount of the
proteins were analyzed by Western blotting with anti-phospho-Erk1/2
to detect phosphor-Erk112 (upper panels in A and B) or with
anti-Eric antibody to detect total amount of Erk1/2 (bottom panels
in A and B).
[0020] FIG. 6. ADAMTS-1.sub.NTCF, and ADAMTS-1.sub.CTCF blocks
activation of Erk1/2 kinases in HUVECs induce by AR, HB-EGF, or
VEGF.sub.165. VEGF.sub.165 (A, 15 ng/ml), bFGF (B, 15 ng/ml),
HB-EGF (C, 4 ng/ml), or AR (D, 5 ng/ml) was used alone (lane 2) or
in the presence of full-length ADAMTS-1 and ADAMTS-1 fragments
(lanes 3-6). The serum-starved HUVECs were applied with SFM alone
(lane 1), or containing different GFs alone (lane 2) or the GFs
plus 400 ng of ADAMTS-1 (lane 3), ADAMTS-1.sub.minusTSP (lane 4),
ADAMTS-1.sub.NTCF (lane 5) or ADAMTS-1.sub.CTCF (lane 6). Equal
amount of the proteins derived from these HUVECs were analyzed
Western blotting with anti-phospho-Erk1/2 to detect phosphor-Erk1/2
(upper panels in A-D) or with anti-Erk antibody to detect total
amount of Erk1/2 (bottom panels in A-D).
[0021] FIG. 7. Full-length ADMATS-1 promotes pulmonary metastasis
of TA3 cells, while ADAMTS-1E/Q, ADAMTS-1.sub.NTF, or
ADAMTS-1.sub.CTF inhibits the process. A. Schematic diagram of the
domain organization of full-length ADAMTS-1 and the different
deletional and point-mutated ADAMTS-1 that were used in FIGS. 1, 2,
3, and 8. B. The expression levels of full-length ADAMTS-1 (lane
1), ADAMTS-1E/Q (lane 2), ADAMTS-1.sub.NTF (lane 3),
ADAMTS-1.sub.minusTSP-1 (lane 4), or ADAMTS-1.sub.CTF (lane 5) by
the pooled populations of TA3 transfectants. C. Survival rates of
the experimental mice which were injected with the different TA3
transfectants intravenously. A total of 12 mice were used for each
type of transfectants. D. Pulmonary metastatic burden was expressed
by the weight of the tumor bearing mouse lungs derived from the
experimental mice 12 and 20 days after the i.v. injection of the
TA3 transfectants.
[0022] FIG. 8. The spacer/Cys-rich domain of ADAMTS-1 plays a major
role in binding of ADAMTS-1 to the ECM and the cells. Western
blotting was performed using anti-v5 antibody to determine the
distribution patterns of the v5-epitope tagged full-length ADAMTS-1
(lane 1), ADAMTS-1E/Q (lane 2), ADAMTS-1.sub.NTF+spacer/Cys-rich
(lane 3), ADAMTS-1.sub.NTF (lane 4), ADAMTS-1.sub.minusTSP-1 (lane
5), ADAMTS-1.sub.CTF (lane 6), ADAMTS-1.sub.CTF+spacer (lane 7),
and ADAMTS-1.sub.3TSP-1 (lane 8) in the cell culture supernatants
(A), the ECM materials (B), and the EDTA-lifted Cos-7 cells (C)
that were transfected with the corresponding expression
constructs.
[0023] FIG. 9. ADAMTS-1 promotes invasion of TA3 cells through
Matrigel and promotes degradation of versican. A. The
representative pictures of TA3 cells that have invaded through the
coated Matrigel: transwell coated with Matrigel only (a, the yellow
and red arrow point to the 8-.mu.m pores and the coated Matrigel,
respectively), TA3.sub.wt1 (b), TA3.sub.ADAMTS-1 (c),
TA3.sub.ADAMTS-1E/Q (d), TA3.sub.ADAMTS-1NTF (e), or
TA3.sub.ADAMTS-1CTF (f) cells. B. The invasion indexes of different
TA3 transfectants were shown. C. ADAMTS-1 promotes degradation of
versican. The ECM materials were analyzed by Western blotting with
anti-DP antibody to detect the neopeptide (DPEAAE) existed in the
COOH-terminal versican cleavage fragment. The ECM materials were
derived from TA3.sub.wtb (lane 1-2), TA3.sub.ADAMTS-1minusTSP-1
(lane 3-4), TA3.sub.ADAMTS-1E/Q (lane 5-6), TA3.sub.ADAMTS-1 (lane
7-8), TA3.sub.ADAMTS-1NTF (lane 9-10), or TA3.sub.ADAMTS-1CTF (lane
11-12) cells.
[0024] FIG. 10. Expression of ADAMTS-1. Expression of ADAMTS-1 was
assessed by RT-PCR using RNAs derived from TA3 wt, TA3.sub.wt1,
Lewis lung carcinoma cells, CMT-93 colon carcinoma cells, B16F1 and
F10 cells, 3T3 fibroblasts, C.sub.2C.sub.12 myoblasts, and mouse
placenta (lanes 2-10). Expression of .beta.-actin by these cells
was used as controls. In lane 1, reverse transcriptase was not
included in RT reaction with RNA derived from TA3.sub.wt1
cells.
[0025] FIG. 11. ADAMTS-1 promotes tumor growth while the cleavage
fragments of ADAMTS-1 inhibit tumor growth. Growth rates of the
s.c. tumors derived from different TA3 transfectants are expressed
as the means of tumors volumes+/-SD. Total of fifteen mice were
used for each type of transfectants.
[0026] FIG. 12. The cleavage fragments of ADAMTS-1 blocks
subcutaneous tumor growth by inhibiting proliferation and survival
of tumor cells, and inhibiting tumor angiogenesis in vivo. The s.c.
tumors were section 12 days after implanting TA3.sub.wtb (A, a-d),
TA3ADAMTS-1 (A, e-h), TA3ADAMTS-1.sub.NTCF (A, i-l), and
TA3ADAMTS-1.sub.CTCF (A, m-p). These sections were stained with
H&E (A-a, e, I, m), or reacted with Apoptag to detect apoptotic
tumor cells in situ (A-b, f, j, n), anti-Brdu antibody to detect
proliferating tumor cells (A-c, g, k, o), or with anti-vWF antibody
to reveal blood vessels with the tumors (a-d, h, l, p). Bar: 120
.mu.m. The quantitative data that reveals the effects of ADAMTS-1
and the fragments of ADAMTS-1 on tumor cell apoptosis and
proliferation in vivo and on tumor angiogenesis are shown in panels
B-a, b, c, respectively.
[0027] FIG. 13. The ADAMTS-1 fragments block activation of EGFR and
ErbB-2; while ADAMTS-1 promotes shedding of AR and HB-EGF. A.
Immunoprecipitation with anti EGFR (A-a) or anti-ErbB-2 (A-b)
antibody was performed by using the proteins derived from the mouse
lungs received TA3.sub.wtb (lanes 1-3), TA3ADAMTS-1 (lanes 4-6),
TA3ADAMTS-1.sub.minusTSP (lanes 7-9), TA3ADAMTS-1.sub.NTCF (lanes
10-12), and TA3ADAMTS-1.sub.ctcf (lanes 13-15) intravenously 5 days
prior. The precipitated proteins were analyzed Western blotting
with anti-phospho-tyrosine antibody to detect phosphor-EGFR (A-a,
upper panel) and phosphor-ErbB-2 (A-b, upper panel), respectively
or with anti-EGFR (A-a, bottom panel) or anti-ErbB-2 (A-b, bottom
panel) antibody to detect total amount of EGFR or ErbB-2,
respectively. B. ADAMTS-1 promotes shedding of AR (B-a), HB-EGF
(B-b), but not epigen (B-c), and the shedding is blocked by
ADAMTS-1E/Q (lane 4). Cos-7 cells were co-transfected with the
expression constructs containing cDNA inserts that encode the EGF
family ligand precursors with or without (lane 1) of
TA3ADAMTS-1.sub.NTCF (lane 2), TA3ADAMTS-1.sub.CTCF (lane 3),
ADAMTS-1E/Q (lane 4), and ADAMTS-1 (lane 5). C. The cell culture
supernatants derived from the AR- (C-a) or HB-EGF (C-b) transfected
Cos-7 cells were applied to serum-starved MCF-10A cells without
(lane 5-6) or without prior absorption of the supernatants with
blocking antibodies against AR (a, lane 7-8) or HB-EGF (b, lane
7-8) in the presence of 400 ng of ADAMTS-1 (lane 9-10),
ADAMTS-1.sub.NTCF (lane 11-12), or ADAMTS-1.sub.CTCF (lane 13-14).
Serum free medium alone (lane 1-2) or containing 5 ng of AR (a,
lane 3-4) or 4 ng of HB-EGF (b, lane 3-4) was applied to serum
starved MCF-10A cells. Equal amount of the proteins derived from
the MCF-10A cells were analyzed Western blotting with
anti-phospho-Erk1/2 to detect phosphor-Erk1/2 or with anti-Erk
antibody to detect total amount of Erk1/2. D. The cleavage
fragments of ADAMTS-1 blocks activation of Erk1/2 in HUVECs induced
by VEGF 165 (D-a), TGF-.alpha. (D-c), HB-EGF (D-d_, and AR (D-e),
but not that induced by bFGF (D-b). HUVECs were applied with SFM
alone (lane 1) or containing different GFs alone (lane 2) with 400
ng of ADAMTS-1 (lane 3), ADAMTS-1.sub.minusTSP (lane 4),
ADAMTS-1.sub.NTCF (lane 5) or ADAMTS-1.sub.CTCF (lane 6). Equal
amount of the proteins derived from HUVECs were analyzed Western
blotting with anti-phospho-Erk1/2 to detect phosphor-Erk1/2 or with
anti-Erk antibody to detect total amount of Erk1/2.
[0028] FIG. 14. Domain organization of ADAMTS-1. The various
domains of ADAMTS-1 are shown.
[0029] FIG. 15. Possible mechanisms of ADAMTS-1 function. 1)
Full-length ADAMTS-1 promotes tumor growth and metastasis by
enhancing tumor cell proliferation/survival and tumor angiogenesis
through shedding/activating HB-EGF and AR transmembrane precursors
and by promoting tumor cell invasion through degrading versican; 2)
full-length ADAMTS-1 binds to their substrates through its
spacer/Cys-rich domain directly or indirectly through binding to
HSPGs. Thus, the whole or different parts of the spacer/Cys-rich
domain can be used as a dominant negative regulator of the
full-length ADAMTS-1 (by regulating the substrate-binding of
ADAMTS-1) and to regulate its own cleavage status (to promote
proteolytic cleavage of ADAMTS-1, therefore generate anti-tumor
fragments); 3) the anti-tumor activity of the ADAMTS-1 fragments
resides in the TSP-1 domains, which exerts the anti-tumor activity
by inhibiting bioactivity of several soluble heparin binding
growth/angiogenic factors including AR and HB-EGF. Thus, the whole
or parts of ADAMTS-1NTF (ADANTS-1NTFE/Q) and/or ADAMTS-1CTF can be
used to inhibit cancers.
[0030] FIG. 16 Full-length ADAMTS-1 and the ADAMTS-1 fragments
displayed opposite effects on growth and metastasis of LLC cells.
A. The expression level of the v5-epitope tagged ADAMTS-1 (lane 1),
ADAMTS-1E/Q (lane 2), ADAMTS-1.sub.NTF (lane 3),
ADAMTS-1.sub.minusTSP-1 (lane 4), ADAMTS-1.sub.CTF (lane 5),
thrombospondin-1 (lane 6) and thrombospondin-2 (lane 7) by the
pooled LLC transfectants. B. The growth rates of the s.c. tumors
derived from the different LLC transfectants are expressed as the
means of tumors volumes+/-SD. A total of 15 mice were used for each
type of transfectants. C. Survival rates of the experimental mice
after removal of the s.c. tumors derived from the different LLC
transfectants. A total of thirty mice were used for each type of
transfectants. D. Pulmonary metastatic burden is expressed by the
average weight of the lungs derived from experimental mice three
weeks after removal of the s.c. tumors.
[0031] FIG. 17 The metalloproteinase activity of ADAMTS-1.sub.NTF
is not required for its anti-tumor activity. A. The expression
level of the v5-epitope tagged ADAMTS-1.sub.NTF (lane 1-3) and
ADAMTS-1.sub.NTFE/Q (lane 4-6) by the TA3 transfectants. B.
Survival rates of the experimental mice after i.v. injection of
1.times.10.sup.6/mouse TA3 transfectants. A total of 15 mice were
used for each type of transfectants. C. Pulmonary metastatic burden
is expressed by the average weight of the lungs derived from the
experimental mice three weeks after the iv injection.
[0032] FIG. 18 The multiple amino acid sequence alignment of the
second and third repeats of thrombospondin-1 and .sub.m and cTSP-1
domains in ADAMTS-1, and the deletional and peptide generation
strategy in the TSP-1 domains of ADAMTS-1. The deletions and
generation of three different peptides s in m and cTSP-1 domains of
ADAMTS-1 are shown.
DETAILED DESCRIPTION
[0033] In the present invention it has been discovered that
ADAMTS-1 is expressed by many tumor cells and overexpression of
ADAMTS-1 promotes growth and metastasis of TA3 mammary carcinoma
cells by promoting survival, proliferation, invasiveness of the
tumor cells and tumor angiogenesis in vivo. Additionally, disclosed
herein is that ADAMTS-1 undergoes auto-proteolytic cleavage to
generate N- and C-terminal cleavage fragments that contain at least
one TSP type I motif. Auto-proteolytic cleavage of ADAMTS-1 is
blocked by heparin and heparin sulfate (HS). Although not bound by
any theory, this indicates that the self-cleavage is regulated by
HS and heparin sulfate proteoglycans (HSPGs). Thus, as described
herein, ADAMTS-1 expressed by TA3 cells is maintained in the
full-length form in vivo to exert pro-tumor growth and metastasis
activity. In contrast to the full-length ADAMTS-1, overexpression
of the N- or C-terminal fragment of ADAMTS-1 (ADAMTS-1.sub.NTCF
and/or ADAMTS-1.sub.CTCF) inhibits subcutaneous (s.c.) growth of
TA3 cells and blocks pulmonary metastasis of the cells by
inhibiting proliferation and inducing apoptosis of the tumor cells
and by inhibiting tumor angiogenesis. Additionally, the anti-tumor
effect of the ADAMTS-1 fragments requires a TSP type-I motif. The
direct evidence was provided for the first time that ADAMTS-1
promotes tumor growth and metastasis, and can serve as a target for
cancer therapy.
[0034] For the first time, it has been demonstrated that unlike
full-length ADAMTS-1 which promotes shedding of the EGF family
ligands including amphiregulin (AR) and heparin-binding EGF
(HB-EGF) and activation of EGF receptor (EGFR) and ErbB-2, the
cleavage fragments of ADAMTS-1 inhibits activation of EGFR and
ErbB-2 in vivo, and interferes with Erk1/2 kinases activation
induced by soluble AR. HB-EGF, and/or VEGF in mammary epithelial
cells and endothelial cells. These different effects likely
underlie the opposite roles of ADAMTS-1 and its cleavage fragments
in tumor growth and metastasis, suggesting the ADAMTS-1 fragments
and the inhibitors of ADAMTS-1 can be most successfully used to
treat the cancers overexpressing these heparin binding growth and
angiogenic factors and with activated erbB-signaling pathways.
[0035] The term "ADAMTS-1.sub.NTCF" can also be referred to as
"ADAMTS-1.sub.NTF". The term "ADAMTS-1.sub.CTCF" can also be
referred to as "ADAMTS-1.sub.CTF". In some embodiments,
ADAMTS-1.sub.NTCF comprises SEQ ID NO: 9 and/or 11. In some
embodiments, ADAMTS-1.sub.CTCF comprises SEQ ID NO: 5 and/or 7.
[0036] The discovery that ADAMTS-1 can be cleaved into at least two
fragments has led to the following invention. In some embodiments,
the present invention provides an isolated polypeptide comprising a
fragment of ADAMTS-1 that inhibits cell growth or cell survival
and/or metastasis.
[0037] As used herein, the term "isolated polypeptide fragment"
refers to a polypeptide fragment that is free of the full length
protein. In some embodiments, the isolated polypeptide is also free
of nucleic acid molecules. In some embodiments, the isolated
polypeptide is free of cellular membranes. In some embodiments, the
isolated polypeptide has been purified away from cellular
components. In some embodiments, the polypeptide comprises a
fragment of SEQ ID NO: 1 and/or SEQ ID NO: 3. In some embodiments,
the fragment of ADAMTS-1 comprises SEQ ID NO: 5, 7, 9, and/or 11.
The fragment of ADAMTS-1 can be any length such that it is not the
full-length ADAMTS-1 protein. In some embodiments, the fragment
comprises about 100 to about 150, about 100 to about 200, about 100
to about 300, about 100 to about 400, about 100 to about 500, about
100 to about 600, about 100 to about 700, about 100 to about 800,
about 100 to about 900, or about 100 to 950 amino acid residues. In
some embodiments, the fragments of ADAMTS-1 comprise modifications
of the polypeptide sequence. The modification can be any
modification including, but not limited to, mutations, insertions,
substitutions, deletions, and the like. In some embodiments, the
fragment comprises a mutation of Glu to Gln. In some embodiments,
the mutation of Glu to Gln occurs at a position corresponding to
position 386 (in mouse ADAMTS-1) in the full length protein. One of
skill in the art can determine what position in a fragment
corresponds to position 386 in the full length protein (e.g.
position 385 in human ADAMTS-1). One of skill in the art can do
this by, for example, performing an alignment using any alignment
software or BLAST software using default settings. Examples of
software that can be used include, but are not limited to, BLAST,
GCG, and MacVector.TM.. In some embodiments, the polypeptide
fragment containing a mutation comprises SEQ ID NO: 33 and/or 35 or
a nucleic acid molecule encoding the same. In some embodiments, the
nucleic acid molecule encoding the fragment comprises SEQ ID NOs:
34 and/or 36.
[0038] In some embodiments the fragments of ADAMTS-1 are linked to
a non-ADAMTS-1 molecule. In some embodiments, the non-ADAMTS-1
molecule is a toxin, peptide, polypeptide, small molecule, drug,
and the like. In some embodiments, the non-ADAMTS-1 molecule is a
6-His-tag, GST polypeptide, HA tag, the Fc fragment of human IgG
and the like. In some embodiments, the proteinase cleavage sites
will be put before the tag sequences, so that after purification
these tags can be removed by proteolytic cleavage. For example, the
HRV 3C (human rhinovirus type 14 3C) protease cleavage site
(LEVLFQ.dwnarw.GP) can be located before the COOH-terminal v5 and
His epitope tags. The HRV 3C protease specifically claves the
sequence LEVLFQ.dwnarw.GP at 40 C and were used to efficiently
removal the COOH-terminal tags (Novagen).
[0039] In some embodiments, the fragment of ADAMTS-1 is fused to
another polypeptide that is derived from a protein that is not
ADAMTS-1. In some embodiments two fragments from ADAMTS-1 are fused
or linked together. In some embodiments, the two fragments are
identical. In some embodiments, the fragments are different from
one another. The fragments that can be linked or fused together are
ADAMTS-1.sub.CTCF (SEQ ID NO: 5 and/or SEQ ID NO:7),
ADAMTS-1.sub.NTCF (SEQ ID NO: 9 and/or 11), and ADAMTS-1.sub.spacer
or ADAMTS-1.sub.spacer/Cys-rich to achieve maximal anti-tumor
efficiency, however any two fragments from ADAMTS-1 can be fused
together.
[0040] In some embodiments, the present invention provides nucleic
acid molecules encoding a fragments of ADAMTS-1. In some
embodiments, the fragments of ADAMTS-1 that inhibits cell
proliferation or metastasis comprise a TSP type-I motif.
[0041] A fragment that inhibits cell proliferation or metastasis
can also be referred to as a fragment that inhibits cancer or a
fragment can be used to treat cancer.
[0042] As used herein, the term "inhibit cell proliferation" refers
to any measurement of cell proliferation. A fragment, compound, or
composition that causes a cell to undergo necrosis or apoptosis is
considered to inhibit cell proliferation. Cell proliferation can
also be referred to as cell growth or cell division.
[0043] Methods of measuring cell proliferation, division, and
metastasis are routine and any method can be used.
[0044] For example, one can measure cell invasion using Matrigel in
vitro. Metastasis can also be measured and/or observed in vivo by
injecting a mouse with a tumor cell and determining if the cell
spreads to a different location away from the sight of injection.
Metastasis can also be measured by measuring or observing tumor
burden or tumor growth in areas that are distinct from the primary
tumor location. Cell proliferation can be measured, for example, by
counting cells. Cell division can be measured, for example, by
monitoring what phase of the cell cycle a cell or a population of
cells is in by using flow cytometry or FACS. Determining if a cell
or cell population is dividing is routine.
[0045] In some embodiments, the present invention provides a
fragment of ADAMTS-1 that lacks a TSP motif. In some embodiments,
the present invention provides a deletion of ADAMTS-1 that lacks a
TSP motif. In some embodiments, a polypeptide of ADAMTS-1 that
lacks a TSP motif comprises SEQ ID NO:13 and/or SEQ ID NO:15. The
term "ADAMTS-1.sub.minus TSP" can also be referred to as
"ADAMTS-1.sub.minus TSP-1". In some embodiments, the present
invention provides a nucleic acid molecule that encodes for a
ADAMTS-1 polypeptide that lacks a TSP motif. In some embodiments
the nucleic acid molecule is isolated. In some embodiments the
nucleic acid molecule comprises SEQ ID NO: 14 and/or SEQ ID NO:
16.
[0046] In some embodiments, the present invention provides an
isolated nucleic acid molecule (polynucleotide) encoding a
polypeptide fragment of ADAMTS-1.
[0047] As used herein the term "isolated nucleic acid molecule
encoding a polypeptide fragment of ADAMTS-1" refers to a nucleic
acid molecule is free of a nucleic acid molecule encoding full
length ADAMTS-1.
[0048] In some embodiments, a fragment encoded by the nucleic acid
molecule can inhibit cell proliferation and/or metastasis. In some
embodiments, the nucleic acid molecule comprises a fragment of a
nucleic acid molecule encoding a polypeptide comprising SEQ ID NO:1
and/or SEQ ID NO: 2. In some embodiments the nucleic acid molecule
comprises a fragment of SEQ ID NO: 3 and/or SEQ ID NO: 4 In some
embodiments, the nucleic acid molecule encodes a polypeptide
comprising SEQ ID NOs: 5, 7, 9, and/or 11. In some embodiments, the
nucleic acid molecule comprises SEQ ID NOs: 6, 8, 10, and/or 12. In
some embodiments, the nucleic acid molecule encoding a fragment of
ADAMTS-1 is operably linked to a promoter. In some embodiments, the
promoter can facilitate the expression in a prokaryotic cell and/or
eukaryotic cell. The promoter can be any promoter that can drive
the expression of the nucleic acid molecule. Examples of promoters
include, but are not limited to, CMV, SV40, pEF, actin promoter,
and the like. In some embodiments, the nucleic acid molecule is DNA
or RNA. In some embodiments, the nucleic acid molecule is a virus,
vector, or plasmid. In some embodiments, the expression of the
nucleic acid molecule is regulated such that it can be turned on or
off based on the presence or absence of a regulatory substance.
Examples of such a system include, but is not limited to a
tetracycline-ON/OFF system.
[0049] In some embodiments, the nucleic acid molecule is a
recombinant viral vector. "A recombinant viral vector" refers to a
construct, based upon the genome of a virus, that can be used as a
vehicle for the delivery of nucleic acids encoding proteins,
polypeptides, or peptides of interest. Recombinant viral vectors
are well known in the art and are widely reported. Recombinant
viral vectors include, but are not limited to, retroviral vectors,
adenovirus vectors, adeno-associated virus vectors, and lenti-virus
vectors, which are prepared using routine methods and starting
materials.
[0050] Using standard techniques and readily available starting
materials, a nucleic acid molecule may be prepared. The nucleic
acid molecule may be incorporated into an expression vector which
is then incorporated into a host cell. Host cells for use in well
known recombinant expression systems for production of proteins are
well known and readily available. Examples of host cells include
bacteria cells such as E. coli, yeast cells such as S. cerevisiae,
insect cells such as S. frugiperda, non-human mammalian tissue
culture cells Chinese hamster ovary (CHO) cells or Cos-7 cells, and
human tissue culture cells such as 293 cells or HeLa cells.
[0051] In some embodiments, for example, one having ordinary skill
in the art can, using well known techniques, insert DNA molecules
into a commercially available expression vector for use in well
known expression systems. For example, the commercially available
plasmid pSE420 (Invitrogen, San Diego, Calif.) may be used for
production of immunomodulating proteins in E. coli. The
commercially available plasmid pYES2 (Invitrogen, San Diego,
Calif.) may, for example, be used for production in S. cerevisiae
strains of yeast. The commercially available MAXBAC.TM. complete
baculovirus expression system (Invitrogen, San Diego, Calif.) may,
for example, be used for production in insect cells. The
commercially available plasmid pcDNAI, pcDNA3, or PEF6/v5-His
(Invitrogen, San Diego, Calif.) may, for example, be used for
production in mammalian cells such as Cos-7 and CHO cells. One
having ordinary skill in the art can use these commercial
expression vectors and systems or others to produce proteins by
routine techniques and readily available starting materials. (See
e.g., Sambrook et al., eds., 2001, supra) Thus, the desired
proteins or fragments can be prepared in both prokaryotic and
eukaryotic systems, resulting in a spectrum of processed forms of
the protein or fragments.
[0052] One having ordinary skill in the art may use other
commercially available expression vectors and systems or produce
vectors using well known methods and readily available starting
materials. Expression systems containing the requisite control
sequences, such as promoters and polyadenylation signals, and
preferably enhancers, are readily available and known in the art
for a variety of hosts (See e.g., Sambrook et al., eds., 2001,
supra).
[0053] In some embodiments, the nucleic acid molecules can also be
prepared as a genetic construct. "Genetic constructs" include
regulatory elements necessary for gene expression of a nucleic acid
molecule. The elements include: a promoter, an initiation codon, a
stop codon, and a polyadenylation signal. In addition, enhancers
can be used for gene expression of the sequence that encodes the
protein or fragment. It is necessary that these elements be
operably linked to the sequence that encodes the desired
polypeptide and that the regulatory elements are operably in the
individual or cell to whom they are administered. Initiation codons
and stop codon are generally considered to be part of a nucleotide
sequence that encodes the desired protein. However, it is necessary
that these elements are functional in the individual or cell to
which the gene construct is administered. The initiation and
termination codons must be in frame with the coding sequence.
Promoters and polyadenylation signals used must be functional
within the cells. Examples of promoters useful to practice the
present invention include but are not limited to promoters from
Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter,
Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal
Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV)
such as the CMV immediate early promoter, Epstein Barr Virus (EBV),
Rous Sarcoma Virus (RSV) as well as promoters from human genes such
as human Actin, human Myosin, human Hemoglobin, human muscle
creatine and human metalothionein. Examples of polyadenylation
signals useful to practice the present invention include but are
not limited to SV40 polyadenylation signals and LTR polyadenylation
signals. In some embodiments, the SV40 polyadenylation signal which
is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to as
the SV40 polyadenylation signal, is used. In addition to the
regulatory elements required for DNA expression, other elements may
also be included in the DNA molecule. Such additional elements
include enhancers. The enhancer may be selected from the group
including but not limited to: human Actin, human Myosin, human
Hemoglobin, human muscle creatine and viral enhancers such as those
from CMV, RSV and EBV. Genetic constructs can be provided with
mammalian origin of replication in order to maintain the construct
extrachromosomally and produce multiple copies of the construct in
the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego,
Calif.) contain the Epstein Barr virus origin of replication and
nuclear antigen EBNA-1 coding region which produces high copy
episomal replication without integration. In some embodiments, the
nucleic acid molecule is free of infectious particles.
[0054] In some embodiments, the present invention provides
compositions comprising at least one polypeptide fragment of
ADAMTS-1 that inhibits cell proliferation or cell growth or
metastasis. In some embodiments, the composition comprises a
fragment comprising SEQ ID NO:1 and/or SEQ ID NO:2. In some
embodiments, the composition comprises a fragment comprising SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO: 9, and/or SEQ ID NO: 11. In some
embodiments, the composition comprises two or at least two
polypeptide fragments of ADAMTS-1. In some embodiments, the
fragment comprises the TSP-type I motif. In some embodiments, the
composition is a pharmaceutical composition.
[0055] As used herein, the term "fragment of ADAMTS-1 that inhibits
cell proliferation or metastasis" refers to a fragment of ADAMTS-1
that can inhibit cell growth, cell division, or cell proliferation.
In some embodiments, the fragment inhibits cell growth, cell
division, cell proliferation, or metastasis by 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some embodiments, the
fragment can inhibit cell growth, cell division, cell
proliferation, or metastasis by at least 10%, at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, or at least 99%. One of
skill in the art can determine the level of inhibition by, for
example, comparing the property or properties of the cell or
population of cells in the absence of the fragment.
[0056] A fragment of ADAMTS-1 that inhibits cell proliferation,
cell growth, cell division, or metastasis can be identified using
any known growth, proliferation, or division assay. For example,
one of skill in the art can contact a fragment of ADAMTS-1 with a
cell either in vitro or in vivo and determine whether the cell's
growth, proliferation, divisions, or metastasis have been
inhibited. One of skill in the art could also use a nucleic acid
molecule encoding a fragment of ADAMTS-1 and introduce the nucleic
acid molecule into a cell or organism such that the fragment is
expressed. The cell's or population of cell's property or
properties could then be measured and it could be determined
whether the fragment encoded by the nucleic acid molecule can
inhibit cell growth (proliferation), cell division, or
metastasis.
[0057] The fragments of ADAMTS-1 that can be used to inhibit cell
proliferation or growth can be produced by a cleavage event of
ADAMTS-1. In some embodiments, the cleavage produces fragments of
ADAMTS-1 comprising SEQ ID NOs: 5, 7, 9 and/or 11 or encoded by
nucleic acid molecules comprising SEQ ID NO: 6, 8, 10, and/or
12.
[0058] In some embodiments, the present invention provides methods
of inhibiting the cleavage of ADAMTS-1 in a cell comprising
contacting the cell with a cleavage inhibiting factor. A "cleavage
inhibiting factor" is a compound or composition that can inhibit
the cleavage of ADAMTS-1. In some embodiments, the cleavage of
ADAMTS-1 is auto-cleavage or cleavage that is facilitated by a
protease that is not ADAMTS-1. In some embodiments, the cleavage
inhibiting factor is heparin or heparan sulfate proteoglycans
(HSPGs). Heparin or derivatives of heparin were found to inhibit
the cleavage of ADAMTS-1 as described herein. In some embodiments,
the present invention provides methods of promoting cleavage of
ADAMTS-1 comprising contacting ADAMTS-1 with a ADAMTS-1 cleavage
activating factor. In some embodiments, the cleavage activating
factor is a compound that inhibits and/or sequesters heparin. In
some embodiments, the factor that inhibits heparin is heparinase,
platelet factor 4 (PF4-a), protamine, or polybrene. A "cleavage
activating factor" is a compound or composition that enhances,
induces, or increases the level of cleavage of ADAMTS-1. In some
embodiments, the cleavage of ADAMTS-1 can be auto-cleavage. In some
embodiments, the cleavage of ADAMTS-1 can be facilitated by a
protease that is not ADAMTS-1.
[0059] In some embodiments, the present invention provides methods
of inhibiting cell proliferation or metastasis comprising
contacting the cell with a fragment of ADAMTS-1 that inhibits cell
proliferation or metastasis. In some embodiments, the fragment of
ADAMTS-1 comprises a fragment of SEQ ID NO:1 and/or SEQ ID NO:3. In
some embodiments, the fragment comprises SEQ ID NOs: 5, 7, 9 and/or
11. One of skill in the art can determine if the fragment inhibits
cell proliferation or metastasis of a cell or population of cells
by measuring the growth or metastasis in the presence and/or
absence of the fragment of ADAMTS-1.
[0060] As used herein, the term "cell" refers to any cell. In some
embodiments, the cell is a human cell or a mouse cell. In some
embodiments, the cell is a tumor cell, inflammatory cells, or
keratinocytes. In some embodiments, the cell is a primary tumor
cell. As used herein, the term "primary tumor cell" refers to a
cell that has been excised from a tumor from an individual or
animal and has not been propagated through more than 10 cell
divisions.
[0061] The discovery that fragments of ADAMTS-1 can inhibit cell
growth and/or metastasis demonstrates that in some embodiments, the
present invention provides methods of treating cancer in an
individual comprising administering to the individual a
therapeutically effective amount of a fragment of ADAMTS-1 that is
able to inhibit cell proliferation or metastasis. The fragments can
also be said to inhibit tumor growth and the like. In some
embodiments, the fragment comprises a fragment of SEQ ID NO:1
and/or SEQ ID NO: 3. In some embodiments, the fragment comprises
SEQ ID NOs: 5, 7, 9 and/or 11. In some embodiments, the fragment of
ADAMTS-1 is co-administered with at least one other cancer
treatment. The fragment of ADAMTS-1 can be either administered
prior to, subsequently to, or at the same time as the other cancer
treatment. The fragment(s) of ADAMTS-1 can be co-administered with
any other cancer treatment, including, but not limited to, surgery,
chemotherapy, antibodies, small molecules, radiation, and the like.
In some embodiments, the fragment of ADAMTS-1 that is used to treat
the cancer in an individual is a fragment of ADAMTS-1 that is able
to inhibit cell proliferation, metastasis, or angiogenesis. In some
embodiments, the fragment inhibits cell proliferation and/or
metastasis in vitro.
[0062] Since it has been discovered that the full length ADAMTS-1
is pro-cancer while the cleavage fragments of ADAMTS-1 have
anti-cancer properties, the present invention provides methods of
treating cancer in an individual comprising administering to the
individual a composition that induces the cleavage of ADAMTS-1. In
some embodiments, the composition that induces the cleavage of
ADAMTS-1 is a heparin inhibitor. Examples of heparin inhibitors
include, but are not limited to, heparinase, platelet factor 4
(PF4-a), protamine, polybrene, the heparin-binding domain/peptide
derived from HSPGs, and the like. In some embodiments, the cleavage
of ADAMTS-1 results in the production of at least one fragment of
ADAMTS-1 that can inhibit cell proliferation or metastasis. In some
embodiments, the cleavage of ADAMTS-1 results in the production of
two or at least two fragments of ADAMTS-1 that can inhibit cell
proliferation or metastasis. In some embodiments, the fragments
that are produced by the cleavage of ADAMTS-1 comprise SEQ ID NOs:
5, 7, 9 and/or 11.
[0063] In some embodiments, the present invention provides methods
of inhibiting metastasis in an individual comprising administering
the individual a fragment or mutant of ADAMTS-1 that inhibits
metastasis and/or angiogenesis. In some embodiments, the mutant of
ADAMTS-1 is a metalloproteinase defective mutant. In some
embodiments, the fragment of ADAMTS-1 that inhibits metastasis
comprises SEQ ID NO: 5, 7, 9, and/or 11. In some embodiments, the
fragment or mutant of ADAMTS-1 that inhibits metastasis, cell
growth or proliferation and/or angiogenesis comprises SEQ ID NO: 5,
7, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, and/or 35.
[0064] In some embodiments, a method of treating cancer can refer
to a method of inhibiting cell growth, division, inducing cell
death (e.g. apoptosis and/or necrosis), promoting metastasis and
angiogenesis, or combinations thereof.
[0065] The fragments or mutants of the present invention can also
be administered in the form of a nucleic acid molecule that encodes
for the fragments or for the mutants. In some embodiments, the
nucleic acid molecule comprises SEQ ID NO: 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, and/or 36.
[0066] The present invention also provides antibodies or fragments
of antibodies that can specifically bind to and block the
pro-cancer activity of ADAMTS-1. In some embodiments, the antibody
specifically binds to ADAMTS-1 comprising SEQ ID NO: 1, 2, 3, or
4
[0067] As used herein, the term "specifically binds to" in
reference to an antibody refers to an antibody that will bind to
one peptide or protein with higher affinity than another peptide.
In some embodiments, the antibody that specifically binds to a
peptide or polypeptide will not bind to more than one peptide. In
some embodiments, the specific antibody binds with a K.sub.d that
is 10.times., 100.times., 1000.times. greater to one peptide over
another. Methods of making and identifying specific antibodies are
routine.
[0068] The present invention also provides for antibodies that
binds to full-length ADAMTS-1 to inhibit cell proliferation,
division, growth, or metastasis. In some embodiments, the
polypeptide comprises SEQ ID NO: 1, 2, 3, or 4.
[0069] The present invention also provides methods of inducing the
cleavage of ADAMTS-1 in a cell comprising contacting the cell with
a heparin inhibitor. Examples of heparin inhibitors include, but
are not limited to heparinase, platelet factor 4 (PF4-a),
protamine, polybrene, and the like.
[0070] The present invention also provides for methods for
identifying an inhibitor or an activator of ADAMTS-1 cleavage
comprising performing a test assay comprising contacting ADAMTS-1
with a test compound; and measuring the cleavage of ADAMTS-1,
wherein a decrease in cleavage indicates that the test compound is
a cleavage inhibitor or wherein an increase in cleavage indicates
that the test compound is a cleavage activator. In some
embodiments, the effect of the test compound is compared what
occurs in the absence of any test compound. In some embodiments,
the compound is contacted with ADAMTS-1 under conditions in which
ADAMTS-1 is cleaved. In some embodiments, ADAMTS-1 undergoes
auto-cleavage (e.g. where the enzyme cleaves itself). In some
embodiments, the method comprising contacting a test compound with
ADAMTS-1 under conditions where ADAMTS-1 can be cleaved. These
conditions can be any conditions and can be modified such that
ADAMTS-1 is able to be cleaved either by itself (auto-cleavage) or
by another molecule. Conditions that can be modified include, but
are not limited to, pH, ion concentration, metal concentration, and
the like.
[0071] In some embodiments the methods comprise contacting more
than one test compound, in parallel. In some embodiments, the
methods comprises contacting 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 40, 50, 100, 1000, at least 2, at least 5, at least 10, at
least 50, at least 100, or at least 1000 test compounds in
parallel. In some embodiments, the present invention is used in
High Throughput Screening of compounds and complete combinatorial
libraries can be assayed, e.g., up to thousands of compounds.
Methods of how to perform high throughput screenings are well known
in the art. The methods can also be automated such that a robot can
perform the experiments. The present invention can be adapted for
the screening of large numbers of compounds, such as combinatorial
libraries of compounds. Indeed, compositions and methods allowing
efficient and simple screening of several compounds in short
periods of time are provided. The instant methods can be partially
or completely automated, thereby allowing efficient and
simultaneous screening of large sets of compounds.
[0072] In some embodiments, the method of the present invention
comprises the step of contacting a cell expressing v5-epitope
tagged ADAMTS-1 (such as TA3.sub.ADAMTS-1) in the presence of a
test compound. The cells can be observed to determine if the test
compound inhibits or promotes the cleavage of ADAMTS-1. A control
may be provided in which the cell is not contacted with a test
compound. A further control may be provided in which test compound
is contacted with cells that either do not express ADAMTS-1 or in
which ADAMTS-1 cannot be cleaved (the cleavage-resistant ADAMTS-1
mutant). If the cells contacted with the test compound increase the
cleavage of ADAMTS-1 then pro-cleavage activity is indicated for
the test compound. If the cells contacted with the test compound
decrease the cleavage of ADAMTS-1 then anti-cleavage activity is
indicated for the test compound.
[0073] Positive and negative controls may be performed in which
known amounts of test compound and no compound, respectively, are
added to the assay. One skilled in the art would have the necessary
knowledge to perform the appropriate controls.
[0074] The test compound can be any product in isolated form or in
mixture with any other material (e.g., any other product(s)). The
compound may be defined in terms of structure and/or composition,
or it may be undefined. For instance, the compound may be an
isolated and structurally-defined product, an isolated product of
unknown structure, a mixture of several known and characterized
products or an undefined composition comprising one or several
products. Examples of such undefined compositions include for
instance tissue samples, biological fluids, cell supernatants,
vegetal preparations, etc. The test compound may be any organic or
inorganic product, including a polypeptide (or a protein or
peptide), a nucleic acid, a lipid, a polysaccharide, a chemical
product, or any mixture or derivatives thereof. The compounds may
be of natural origin or synthetic origin, including libraries of
compounds.
[0075] In some embodiments, the activity of the test compound(s) is
unknown, and the method of this invention is used to identify
compounds exhibiting the selected property (e.g., ADAMTS-1
cleavage). However, in some embodiments instances where the
activity (or type of activity) of the test compound(s) is known or
expected, the method can be used to further characterize the
activity (in terms of specificity, efficacy, etc.) and/or to
optimize the activity, by assaying derivatives of the test
compounds.
[0076] The amount (or concentration) of test compound can be
adjusted by the user, depending on the type of compound (its
toxicity, cell penetration capacity, etc.), the number of cells,
the length of incubation period, the amount of ADAMTS-1, etc. In
some embodiments, the compound can be contacted in the presence of
an agent that facilitates penetration or contact with a cell
comprising ADAMTS-1. The test compound is provided, in some
embodiments, in solution. Serial dilutions of test compounds may be
used in a series of assays. In some embodiments, test compound(s)
may be added at concentrations from 0.01 .mu.M to 1 M. In some
embodiments, a range of final concentrations of a test compound is
from 10 .mu.M to 100 .mu.M. One such test compound that is
effective to activate cleavage of ADAMTS-1 in a cell is a heparin
inhibitor.
[0077] In some embodiments, the method comprises measuring ADAMTS-1
cleavage in the presence of the test compound. If the test compound
is found to cleave ADAMTS-1 it is indicative that the test compound
is pro-cleavage ADAMTS-1 agent. Since the cleavage fragments of
ADAMTS-1 agent, a pro-cleavage fragment can also be considered an
anti-cancer agent.
[0078] ADAMTS-1 cleavage can be measured by any means that
demonstrates that the cleavage of ADAMTS-1 has been modulated
(increased or decreased) in the presence of the test compound.
Examples of how to measure ADAMTS-1 cleavage include measuring an
increase or decrease in the cleavage fragments of ADAMTS-1. The
cleavage fragments can be measured by any means including, but not
limited to, Western Blot, ELISA, Sandwich Assay, and the like.
Methods of measuring the levels protein cleavage fragments are
routine to one of ordinary skill in the art.
[0079] In some embodiments, the test compound activates the
cleavage of ADAMTS-1 by at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 100%, at least 200%. In some
embodiments, the test compound inhibits the cleavage of ADAMTS-1 by
at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, or at
least 99%. In some embodiments, the cleavage of ADAMTS-1 is
compared to cleavage of ADAMTS-1 observed in the absence of the
test compound.
[0080] In some embodiments, the methods further comprise performing
a control assay. In some embodiments, the control assay comprising
contacting a cell with a negative or positive control and
measuring, including, but not limited to, ADAMTS-1 cleavage. In
some embodiments, the control compound is compared to the test
compound. In some embodiments, the control compound is a negative
control (e.g. a compound that does not inhibit or activate ADAMTS-1
cleavage). A negative control can also be the absence of a test
compound or the vehicle (e.g. solvent) that the test compound is
contacted with the cell. In some embodiments, the control compound
is a positive control (e.g. a compound that inhibits or activates
ADAMTS-1 cleavage).
[0081] In some embodiments, the test compound is a small molecule,
a peptide (including the peptides from the heparin-binding proteins
and HSPGs), an antibody, a cellular fraction, a protease, or a
mixture thereof. As discussed above, the test compound can be
contacted with a cell comprising ADAMTS-1, but the test compound
can be contacted with ADAMTS-1. For example, ADAMTS-1 can be
expressed as a protein and either be purified or not be purified,
but is isolated from a cell. For the purposes of the screening
assays to identify test compounds that can inhibit or activate the
cleavage of ADAMTS-1, an isolated protein is a protein that is
separated from a cell. The protein can be purified from other
components in the cell, but it does not have to be. In some
embodiments, an isolated ADAMTS-1 protein results from a cell being
lysed which releases all the contents of the cell. The cleavage of
ADAMTS-1 can then be measured or monitored in a non-cellular
environment. The test compound is then contacted with ADAMTS-1 to
determine if the test compound can inhibit or activate the cleavage
of ADAMTS-1.
[0082] In some embodiments, the methods further comprise performing
a negative control assay which comprises contacting a cell that
does not comprise ADAMNTS-1 or a cell that comprises a cleavage
resistant mutant of ADAMTS-1. In some embodiments, the negative
control assay comprises contacting an isolated cleavage resistant
mutant of ADAMTS-1.
[0083] The present invention also provides methods for identifying
an anti-cancer agent comprising performing a test assay comprising
contacting a cell comprising ADAMTS-1 with a test compound; and
measuring the cleavage of ADAMTS-1, wherein an increase in cleavage
indicates that the test compound is an anti-cancer compound. In
some embodiments, the cleavage in the presence of the test compound
is compared to an assay where the cell is comprising ADAMTS-1 is
not contacted with the test compound.
[0084] As used herein, "a cell comprising ADAMTS-1" refers to a
cell expressing the protein ADAMTS-1. The cell can be either be
expressing the protein endogenously (e.g. from within its native
genome) or exogenously. An exogenously expressed protein is a
protein in a cell that would not normally be present except for
some modification. The exogenously expressed protein can be, for
example, transfected into a cell either stably or transiently.
[0085] The present invention also provides methods of inhibiting
angiogenesis in an individual comprising administering to the
individual a fragment of ADAMTS-1. In some embodiments, the
fragment of ADAMTS-1 comprises ADAMTS-1.sub.CTCF or
ADAMTS-1.sub.NTCF (SEQ ID NOs: 5, 7, 9 and/or 11). In some
embodiments a nucleic acid molecule encoding the fragments is
administered. In some embodiments, the nucleic acid molecule
comprises SEQ ID NOs: 6, 8, 10, and/or 12.
[0086] The present invention provides methods of inhibiting the
growth or metastasis of a tumor. In some embodiments, the tumor is
vascularized or non-vascularized.
[0087] The present invention also provides methods of treating
cancer comprising inhibiting the metalloproteinase activity of
ADAMTS-1. In some embodiments, the metalloproteinase activity of
ADAMTS-1 is inhibited by administering a metalloproteinase
defective full-length ADAMTS-1 or the ADAMTS-1 fragments containing
its substrate-binding domain such as ADAMTS-1.sub.spacer/Cys-rich
or ADAMTS-1.sub.spacer, which can act as the dominant negative
mutants of ADAMTS-1 and inhibit the activity of the wild-type
protein. In some embodiments, the metalloproteinase defective
ADAMTS-1 comprises SEQ ID NO: 29, 31, 33, and/or 35. In some
embodiments, the metalloproteinase activity is inhibited by an
antibody or a small molecule that binds to ADAMTS-1. In some
embodiments, the metalloproteinase activity is inhibited by an
antibody or a small molecule that binds to the metalloproteinase
active site of ADAMTS-1.
[0088] The present invention also provides methods of identifying
inhibitors of ADAMTS-1 metalloproteinase activity comprising
contacting a fragment of or full-length ADAMTS-1 that has
metalloproteinase activity with a test compound and determining if
the metalloproteinase activity is inhibited. (In some embodiments,
the fragment of ADAMTS-1 comprises SEQ ID NO: 5, 7, 9, and/or 11.)
In some embodiments, the activity in the presence of the test
compound is compared to the activity in the absence of the test
compound. In some embodiments, the method comprises comparing the
activity with a positive control assay and/or a negative control
assay. In some embodiments, the method comprises comparing the
activity of the fragment to a fragment that is defective in
metalloproteinase activity. A fragment can be defective in
metalloproteinase because of a mutation, substitution, deletion, or
insertion. In some embodiments, the fragment is defective in
metalloproteinase activity due to a Glu to Gln mutation. In some
embodiments, the fragment that lacks metalloproteinase activity
comprises SEQ ID NO: 33 and/or 35. In some embodiments, the
fragment that lacks metalloproteinase activity is encoded by a
nucleic acid molecule comprising SEQ ID NO: 34 and/or 36.
[0089] Methods of measuring metalloproteinase activity (e.g.
ADAMTS-1 activity) are routine. For example, the cleavage of
substrates of ADAMTS-1 can be measured and compared in the absence
and presence of a test compound. However, any method or means can
be used to measure metalloproteinase activity of ADAMTS-1.
Substrates of ADAMTS-1 are known in the art and can be measured. In
some embodiments, the substrate of the metalloproteinase is
aggrecan or versican.
[0090] In some embodiments, the present invention provides methods
of treating cancer comprising administering to an individual a
compound that is a ADAMTS-1 metalloproteinase activity inhibitor.
In some embodiments, the inhibitor is a dominant negative mutant of
ADAMTS-1. In some embodiments, the inhibitor is a polypeptide or
comprising SEQ ID NO: 33 and/or 35. In some embodiments, the
inhibitor is encoded by a nucleic acid molecule comprising SEQ ID
NO: 34 and/or 36.
[0091] Other fragments or mutants of ADAMTS-1 can also be used to
treat cancer because they also act as a dominant negative regulator
of ADAMTS-1 and, thus, be able to inhibit the function of ADAMTS-1.
Accordingly, the present invention provides methods of treating
cancer comprising administering a therapeutically effective amount
of a composition comprising a fragment of ADAMTS-1 comprising the
spacer/Cys-rich and/or Spacer domain of ADAMTS-1.
[0092] The present invention also provides polypeptide fragments of
ADAMTS-1 comprising the spacer/Cys-rich and/or spacer domain of
ADAMTS-1. In some embodiments, the fragment comprises SEQ ID NO:
17, 19, 21, and/or 23. In some embodiments, the fragments are
encoded by nucleic acid molecules comprising 18, 20, 22, and/or
24.
[0093] The present invention also provides for fragments of
ADAMTS-1 that bind to the extracellular matrix (ECM). According, in
some embodiments, the present invention provides an ECM binding
fragment of ADAMTS-1. An "ECM binding fragment of ADAMTS-1" is a
fragment of ADAMTS-1 that binds to the ECM. In some embodiments,
the ECM binding fragment of ADAMTS-1 comprises SEQ ID NO: 17, 19,
21, and/or 23. In some embodiments, a nucleic acid molecule encodes
for an ECM binding fragment of ADAMTS-1. In some embodiments, the
ECM binding fragment comprises SEQ ID NO: 18, 20, 22, and/or
24.
[0094] In some embodiments, the present invention provides nucleic
acid molecules encoding any fragment of ADAMTS-1 described herein.
In some embodiments, the nucleic acid molecule comprises SEQ ID
NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 16, 28, 30, 32,
34, 36, or combinations thereof.
[0095] In some embodiments, the present inventions provides
polypeptides comprising at least a fragment of ADAMTS-1 as
described herein. In some embodiments, the polypeptides comprise
SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 33, 35, or
combinations thereof. In some embodiments, the present invention
provides polypeptides that comprise mutations that inactivate the
metalloproteinase activity of ADAMTS-1. In some embodiments, the
mutation is a Glu to Gln mutation that corresponds to position 386
(mouse) (or 385 in human) of the wild-type ADAMTS-1. In some
embodiments, the mutant ADAMTS-1 comprises SEQ ID NO: 29 and/or 31.
In some embodiments a nucleic acid molecule encoding such mutants
is provided. In some embodiments, the nucleic acid molecule
comprises SEQ ID NO: 30 and/or 32.
[0096] The present invention also provides methods for identifying
a compound that induces the cleavage of ADAMTS-1 comprising
performing a test assay comprising identifying a compound that
inhibits heparin, wherein if a compound inhibits heparin, the
compound would be said to induce the cleavage of ADAMTS-1. Since
heparin inhibitors induce the cleavage of ADAMTS-1 heparin
inhibitors would be able to activate the cleavage of ADAMTS-1.
Thus, a compound that is identified as heparin inhibitor would be
said to be able to induce the cleavage of ADAMTS-1. In some
embodiments, the effect of heparin is a protective effect.
[0097] The present invention also provides methods of identifying a
heparin inhibitor comprising contacting heparin and ADAMTS-1 with a
test compound under conditions that ADAMTS-1 is cleaved in the
absence of heparin and determining if the test compound inhibits
heparin. As described herein, heparin inhibits the cleavage of
ADAMTS-1. Therefore, a test compound that inhibits heparin will
allow ADAMTS-1 to be cleaved by another protein or by itself. A
test compound is said to be a heparin inhibitor if ADAMTS-1 is
cleaved in the presence of heparin. In some embodiments, the
heparin and ADAMTS-1 are free of cellular proteins. In some
embodiments, the heparin and ADAMTS-1 is free of extracellular
matrix.
[0098] A fragment of ADAMTS-1, a nucleic acid molecule encoding a
fragment of ADAMTS-1, a compound that inhibits or activates the
cleavage of ADAMTS-1 can be administered by any means to the
individual whether in the form of a composition or a pharmaceutical
composition. Methods of administration are known to one of skill in
the art. For example, the agent can be prepared as a pharmaceutical
composition. In some embodiments, the pharmaceutical composition
comprises a pharmaceutically acceptable carrier or diluent. In some
embodiments, the pharmaceutical compositions are sterile and/or
pyrogen free. The pharmaceutical composition comprising the
molecule and a pharmaceutically acceptable carrier or diluent may
be formulated by one having ordinary skill in the art with
compositions selected depending upon the chosen mode of
administration. Suitable pharmaceutical carriers are described in
the most recent edition of Remington's Pharmaceutical Sciences, A.
Osol, a standard reference text in this field.
[0099] For parenteral administration, the composition can be, for
example, formulated as a solution, suspension, emulsion or
lyophilized powder in association with a pharmaceutically
acceptable parenteral vehicle. Examples of such vehicles are water,
saline, Ringer's solution, dextrose solution, and 5% human serum
albumin. Liposomes and nonaqueous vehicles such as fixed oils may
also be used. The vehicle or lyophilized powder may contain
additives that maintain isotonicity (e.g., sodium chloride,
mannitol) and chemical stability (e.g., buffers and preservatives).
The formulation is sterilized by commonly used techniques. For
example, a parenteral composition suitable for administration by
injection is prepared by dissolving 1.5% by weight of active
ingredient in 0.9% sodium chloride solution.
[0100] The pharmaceutical compositions according to the present
invention may be administered as a single doses or in multiple
doses. The pharmaceutical compositions of the present invention may
be administered either as individual therapeutic agents or in
combination with other therapeutic agents. The treatments of the
present invention may be combined with conventional therapies,
which may be administered sequentially or simultaneously.
[0101] The pharmaceutical compositions may be administered by any
means that enables the agent to reach the agent's site of action in
the body of a mammal. Because the compositions may be subject to
being digested when administered orally, parenteral administration,
i.e., intravenous, subcutaneous, intramuscular, would ordinarily be
used to optimize absorption. In addition, the pharmaceutical
compositions of the present invention may be injected at a site at
or near hyperproliferative growth. For example, administration may
be by direct injection into a solid tumor mass or in the tissue
directly adjacent thereto. The composition may also be formulated
with a pharmaceutically acceptable topical carrier and the
formulation may be administered topically as a creme, lotion or
ointment for example.
[0102] The dosage administered varies depending upon factors such
as: pharmacodynamic characteristics; its mode and route of
administration; age, health, and weight of the recipient; nature
and extent of symptoms; kind of concurrent treatment; and frequency
of treatment. Usually, a daily dosage of a composition to treat
cancer is used in an amount effect to have an anti-cancer effect.
In some embodiments, the daily dosage is used in an amount to
cleave ADAMTS-1 into a fragment that can inhibit cell proliferation
or cell growth (e.g tumor growth). In some embodiments, the dosage
can be about 1 .mu.g to 100 milligrams per kilogram of body weight.
Ordinarily 0.5 to 50, and preferably 1 to 10 milligrams per
kilogram per day given in divided doses 1 to 6 times a day or in
sustained release form is effective to obtain desired results.
EXAMPLES
[0103] The invention is now described with reference to the
following examples. These examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these examples, but rather should be construed
to encompass any and all variations which become evident as a
result of the teaching provided herein. Those of skill in the art
will readily recognize a variety of non-critical parameters that
could be changed or modified to yield essentially similar
results.
Example 1
Materials and Methods
[0104] Cell Lines and Reagents
[0105] Human umbilical vein endothelial cells (HUVECs) were
obtained from Cambrex (Walkersville, Md.). TA3 transfectants were
maintained as described previously (11, 12). Anti-v5 epitope
(Invitrogen), -vWF (Dako), -phosphorylated tyrosine (BD
Transduction Lab), -EGFR, -ErbB-2, -Erk1/2, and -phospho-Erk1/2
(Santa Cruz) antibodies, and Brdu-cell proliferation kit (Roche)
and Apoptag kit (Chemicon) were used in the experiments.
[0106] Reverse Transcriptase-polymerase Chain Reaction (RT-PCR),
Mutagenesis, and Expression Constructions
[0107] Expression of ADAMTS-1 was assessed by RT-PCR as described
(13). Full-length mouse ADAMTS-1 was obtained by RT-PCR with a
primer pair consisting of 24 nucleotides corresponding to the 3' or
5' extremity of the coding sequence of ADAMTS-1 (accession number
NM.sub.--009621). The stop codon was omitted from the reverse
primers to fuse ADAMTS-1 to the C-terminal v5 epitope tag existed
in the expression vector (pEF6/v5-HisTOPO, Invitrogen). Various
mutation and deletion of ADAMTS-1 were generated as detailed in
FIG. 1A using the QuikChange.TM. site and ExSite PCR-based
site-directed mutagenesis kits (Stratagene).
[0108] Transfection
[0109] Lipofectamine (Invitrogen) was used to transfect TA3.sub.wt1
cells with empty expression vector alone or the expression
constructs containing cDNA inserts encoding ADAMTS-1 and various
mutant or fragments of ADAMTS-1 (FIG. 1A). TA3 transfectants were
selected and the expression level of v5-tagged full-length and
fragments of ADAMTS-1 was determined by Western blotting with
anti-v5 antibody (Invitrogen).
[0110] ADAMTS-1 Production and Purification, Proteolytic Cleavage
Assay, and Western Blot Analysis
[0111] Cell culture supernatants derived from Cos-7 and TA3
transfectants expressing v5-epitope tagged wild type ADAMTS-1 or
ADAMTS-1 mutants (FIG. 1A) were collected and purified through
Ni.sup.+-Probond (Invitrogen) and anti-v5 antibody conjugated
affinity columns (Sigma). Auto-proteolytic cleavage capacity of
ADAMTS-1 was assessed by in vitro proteolytic cleavage assay using
purified ADAMTS-1. In this assay, 100 ng of ADAMTS-1 was incubated
in 50 mM Tris-acetate buffer (pH 7.3) containing 5 mM CaCl.sub.2
and 0.1M NaCl at 37.degree. C. for 30 min, 1, 2, 4, 8 and 12 hours,
and reaction was stopped by addition of 8.times.SDS sample buffer.
The reaction products were analyzed by Western blot with anti-v5
mAb.
[0112] To assess ADAMTS-1 cleavage in cellular context and to
determine how the cleavage is regulated, Cos-7 or TA3 transfectants
expressing ADAMTS-1 or ADAMTS-1E/Q was cultured for 48 hours in the
absence or presence of different reagents as detailed in the figure
legend, and the cell culture supernatants were collected and
analyzed by Western blot with anti-v5 antibody.
[0113] Tumor Cell Tracking and Pulmonary Metastasis
[0114] To track TA3 transfectants during early pulmonary
metastasis, the TA3 transfectants were labeled with green
5-chloromethyl-fluorescein diacetate (CMFDA, Molecular Probes,
Inc.) as described (13), and the CMFDA-labeled TA3 transfectants
(1.times.10.sup.6 cells/mouse) were injected into the tail vein of
A/Jax syngenic mice (the Jackson Lab). The mice were sacrificed 24
hours after the injection, and lungs were removed, fixed, and
sectioned. The localization of tumor cells in mouse lung parenchyma
were revealed under fluorescence microscope, and the extent of
tumor cell extravasation was determine by counting number of the
tumor cells in five randomly selected 10.times. microscopic
fields.
[0115] Experimental pulmonary metastasis was carried out as
detailed previously (13), and five independent clonal TA3
transfectants expressing ADAMTS-1, ADAMTS-1.sub.CTCF,
ADAMTS-1.sub.NTCF or ADAMTS-1.sub.minusTSP, or transfected with the
empty expression vector were used. For each type of the experiment,
six mice were injected with each clonal transfectant and two
independent experiments were performed. The experimental mice were
observed daily after the i.v. injection and duration of mouse
survival was recorded. The survival rate of these mice was
calculated as the following: survival rate (%)=(number of mice are
still alive/total number of the experimental mice).times.100%. The
mice that are free of symptom 60 days after the i.v. injection were
sacrificed and their lungs were examined. In the second set of
experiments, 11 and 18 days after i.v. injection, pulmonary
metastatic burden was assessed by measuring weight of the mouse
lungs.
[0116] Histology and Immunohistochemistry
[0117] To determine the tumor cell proliferation rate in vivo,
5-Bromo-2'-deoxy-uridine (Brdu) was injected into mice four hours
prior to sacrifice of the experimental mice. The mouse lungs were
fixed, sectioned, and stained with H&E as described (11). In
addition, the sections were reacted with anti-von Willebrand factor
(vWF) antibody to assess tumor angiogenesis, with anti-Brdu
antibody to detect proliferating cells or with Apoptag kit to
detect apoptotic cells in situ. Total number of the tumor cells and
number of the tumor cells that are positive for anti-Brdu antibody
or TUNEL-staining were counted in five randomly selected 400.times.
microscopic fields within the pulmonary macro- and
micro-metastases. More than 2,000 cells were counted in total for
each type of transfectants. The proliferation and apoptosis rate
was calculated as the following: proliferation or apoptosis
rate=(number of the anti-Brdu or TUNEL-positive cells per
microscopic field/total number of the tumor cells per microscopic
field).times.100%. To determine blood vessel number, the
vWF-positive blood vessels were countered in six randomly selected
200.times. microscopic fields within macro- or micro-metastases.
The number of blood vessels/microscopic field was expressed as
means+/-S.D.
[0118] EGFR and ErbB-2 Phosphorylation
[0119] RIPA buffer (50 mM Tris-HCl, PH 7.4, 50 mM NaCl, 1%
Triton-X100, 2 mM EDTA, 2 mM sodium orthovanadate, 2 mM sodium
fluoride, 2 mM phenylmethylsulfonyl fluoride, 1 mM Leupeptin, 1 mM
Pepstain A, and 10 .mu.g/ml aprotinin) was used to extract the lung
tissues derived from the mice that were injected with or without
different TA3 transfectants (1.times.10.sup.6/mouse) intravenously
24 hours prior. The proteins were used in the immunoprecipitation
reactions to pull-down EGFR and ErbB-2 using the agarose beads
conjugated with anti-EGFR or anti-ErbB-2 antibody (Santa Cruz). The
precipitated proteins were analyzed by Western blotting with
anti-phosphotyrosine antibody (BD Bioscience) to detect
phosphor-EGFR and phosphor-ErbB-2, or with anti-EGFR or anti-ErbB-2
antibody (Santa Cruz) to detect total amount of EGFR or ErbB-2,
respectively.
[0120] Shedding of the EGF Family GFs and Activation of Erk1/2
Kinases
[0121] Shedding of the transmembrane precursors of AR, HB-EGF, and
epigen by ADAMTS-1, its mutant and fragments were assessed by
co-transfection of Cos-7 cells with the expression constructs
containing cDNA inserts that encode these EGF family precursors and
various ADAMTS-1 constructs as detailed in the figure legend. The
concentrated cell culture supernatants of the co-transfected COS-7
cells were analyzed by Western blotting to detect the soluble GFs
using the corresponding antibodies (R&D Systems).
[0122] Ability of the ADAMTS-1 fragments to inhibit activation of
Erk1/2 kinase induced by soluble AR (5 ng/ml) and HB-EGF (4 ng/ml)
were assessed by applying the serum starved MCF-10A cells with
purified soluble AR or HB-EGF in the absence or presence of their
corresponding neutralization antibodies or purified full-length
ADAMTS-1 or the ADAMTS-1 fragments (400 ng). MCF-10A cells were
then lysed and equal amount of the proteins were analyzed by
Western blotting with anti-phospho-Erk1/2 to detect phosphor-Erk1/2
or with anti-Erk antibody to detect total amount of Erk1/2
protein.
[0123] HUVECs were cultured until subconfluence and switched to
serum-free medium (SFM) for overnight. VEGF.sub.165 (10 ng), bFGF
(10 ng), AR (5 ng), and HB-EGF (4 ng) were applied to the
serum-starved HUVECs in the absence or presence of 400 ng of
ADAMTS-1, ADAMTS-1.sub.minusTSP, ADAMTS-1.sub.NTCF, or
ADAMTS-1.sub.CTCF for 20 minutes. The HUVECs were lysed and equal
amount of the proteins were subjected to Western blotting with
anti-phospho-Erk1/2 or anti-Erk (Santa Cruz) to detect
phosphor-Erk1/2 or total amount of Erk, respectively.
Example 2
ADAMTS-1 Undergoes Auto-Proteolytic Cleavage and the Self-Cleavage
of ADAMTS-1 is Regulated
[0124] Previous results have shown that ADAMTS-1 is cleaved within
the spacer region and several matrix metalloproteinases (MMPs) are
responsible for the cleavage (9). Since several other members of
ADAMTS family undergo auto-proteolytic cleavage and ADAMTS-1 is an
active metalloproteinase, the possibility that the cleavage of
ADAMTS-1 can be mediated by its own metalloproteinase activity was
investigated. To achieve that, a protease-dead mutant of ADAMTS-1
was generated by switching E.sub.386 to Q (ADAMTS-1E/Q) in the
Zinc-binding pocket of the metalloproteinase domain. The expression
constructs containing v5-epitope tagged wild type ADAMTS-1 or
ADAMTS-1E/Q were used to transfect Cos-7 cells. The cell culture
supernatants of the transiently transfected Cos-7 cells were
analyzed and the results showed that only wild type ADAMTS-1 but
not ADAMTS-1E/Q is cleaved to generate the C-terminal cleavage
fragments (FIG. 1B, arrows), suggesting that the metalloproteinase
activity of ADAMTS-1 is required for the cleavage.
[0125] In order to produce full-length ADMATS-1, the regulation of
ADAMTS-1 cleavage was investigated. Different reagents were applied
to a stable TA3 transfectant expressing ADAMTS-1, and the cell
culture supernatants were analyzed 48 hours late. The result showed
that heparin and heparan sulfate (HS) completely block the
proteolytic cleavage of ADAMTS-1 (FIG. 1C), while the control
glycosaminoglycans (GAGs), chondroitin sulfate (CS) and hyaluronan
(HA), and displayed no effect on the cleavage. This result suggests
that auto-proteolytic cleavage of ADAMTS-1 is regulated by
synthesis and degradation rate of HS/heparan sulfate proteoglycans
(HSPGs) in the microenvironment where ADAMTS-1 is produced and
HS/HSPGs likely play important role in regulating ADAMTS-1
function.
[0126] Full-length ADAMTS-1 protein was produced by Cos-7 cells
transfected with the expression construct containing ADAMTS-1 cDNA
in the presence of heparin. Cell culture media of the transfected
Cos-7 cells were collected and purified through the affinity
columns. Purified ADAMTS-1 was used in a proteolytic cleavage assay
and the result showed that ADAMTS-1 was auto-proteolytically
cleaved to release v5-epitope tagged C-terminal cleavage fragments
that have molecular weight similar to that generated in the cell
culture condition (FIG. 1E).
[0127] ADAMTS-1 Promotes Metastasis, while ADAMTS-1.sub.NTCF and
ADAMTS-1.sub.CTCF Block the Process
[0128] ADAMTS-1 was found to inhibit bFGF-induced vascularization
in the cornea pocket assay and VEGF-induced angiogenesis in the
chorioallantoic membrane assay and tumor growth in vivo. However, a
study analyzing clinical pancreatic cancer samples demonstrated
that increased expression of ADAMTS-1 is correlated to enhanced
metastatic potential and worse prognosis, implying that ADAMTS-1
facilitates tumor metastasis. In addition, studies have shown that
ADAMTS-1 is one of the genes up-regulated in the breast cancer with
elevated metastatic activity. To determine the exact roles of
ADAMTS-1 and its cleavage fragments in tumor metastasis and the
underlying mechanism, set to investigate how overexpression of
full-length and the fragments of ADAMTS-1 affects metastasis of TA3
mammary carcinoma (TA3) cells. As shown in FIGS. 1 B and C, the
C-terminal cleavage fragments of ADAMTS-1 are heterogenic in their
molecular weight, suggesting that ADAMTS-1 are cleaved at more than
one sites within the spacer/Cys-rich region (FIG. 1A, arrows). The
molecular weight of the shortest C-terminal cleavage fragments is
similar to that of the expressed C-terminal fragment of ADAMTS-1
containing the last two TSP-1 type I motifs (ADAMTS-1.sub.CTCF:
amino acids 842-951, FIG. 1A, D), suggesting that in addition to
the previous identified cleavage site in the spacer region (FIG.
1A, the bigger arrow), there is at least one additional cleavage
site at the junction between spacer region and the second TSP-1
type I motif (FIG. 1A, the smaller arrow).
[0129] It was difficult to express the N-terminal fragments of
ADAMTS-1 containing different parts of the spacer and/or Cys-rich
domains (data not shown). In addition, studies have shown that
auto-proteolytic cleavage of ADAMTS-4 occurs at the multiple sites
within its spacer/Cyr-rich region, and the shortest N-terminal
cleavage fragment of ADAMTS-4 is generated by cleavage around the
junction between the Cys-rich domain and the TSP-1 type I motif.
Thus, two expression constructs containing N-terminal fragments of
ADAMTS-1 were made, which expressed well in TA3 cells. These
constructs contain the N-terminal domains of ADAMTS-1 until the end
of the first TSP-1 type I motif (ADAMTS-1.sub.NTCF, amino acids
1-596, FIG. 1A, D) or until the end of the disintegrin domain
(ADAMTS-1.sub.minusTSP, amino acids 1-545, FIG. 1A, D).
ADAMTS-1.sub.NTCF likely represents the shortest N-terminal
cleavage fragment of ADAMTS-1.
[0130] In order to assess the effects of ADAMTS-1 and its fragments
on tumor metastasis reliably, the heterogeneity of TA3 cells was
eliminated by transfecting the cells with empty expression vector
containing neomycin-resistant gene. A clonal TA3 cell (TA3.sub.wt1)
that undergoes aggressive pulmonary metastasis after intravenous
(i.v.) injection was selected (data not shown). Our RT-PCR result
showed that like its wild type counterpart, TA3.sub.wt1 cells
express ADAMTS-1 endogenously (data not shown). TA3.sub.wt1 was
used to transfect several expression constructs that contain
blasticidin-resistant gene and different ADAMTS-1 cDNA inserts
(FIG. 1 A). Five independent clonal TA3 transfectants that were
transfected with the empty expression vector alone (TA3.sub.wtb) or
expressing the following gene products (FIG. 1D) were identified
and used in pulmonary metastasis experiments: wild type ADAMTS-1
(TA3.sub.ADAMTS-1), ADAMTS-1.sub.NTCF (TA3.sub.ADAMTS-1NTCF),
ADAMTS-1.sub.CTCF (TA 3.sub.ADAMTS-1CTCF), and
ADAMTS-1.sub.minusTSP (TA3.sub.ADAMTS-1minusTSP). These TA3
transfectants displayed similar growth rate in cell culture
condition (data not shown).
[0131] Our results showed that overexpression of ADAMTS-1
significantly accelerated pulmonary metastasis and shortened the
survival time of the mice (FIG. 2 A-C). On the contrary,
ADAMTS-1.sub.NTCF or ADAMTS-1.sub.CTCF, but not
ADAMTS-1.sub.minusTSP blocks pulmonary metastasis of the TA3
transfectants (FIG. 2A-C), suggesting that the inhibitory effect of
these ADAMTS-1 fragments is likely derived from the TSP type I
motifs which exist in ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF, but
not in ADAMTS-1.sub.minusTSP; and the anti-tumor activity is likely
masked in full-length ADAMTS-1.
[0132] The metastatic tumors derived from TA3.sub.wtb,
TA3.sub.ADAMTS-1, and TA3.sub.ADAMTS-1minusTSP cells are invasive
and fused together (FIG. 2A, D, a-b, and data not shown), which
made it difficult to determine accurate number of the metastatic
lesions. Thus, metastatic burden of the experimental mice was
quantified by average weight of the experimental mouse lungs (FIG.
2C). Because there is a significant difference in survival time of
these mice and the mice usually succumb to pulmonary metastasis
when metastatic burden causes the lung weight to reach 1-1.2 grams,
the metastatic burden of the remaining survival mice at day 11 and
day 18 after i.v. injection of the TA3 transfectants was measured.
At least fifteen experimental mouse lungs were measured for each
type of the transfectants at each time point. Our results showed
that overexpression of ADAMTS-1 accelerated time that is need to
reach the maximal metastatic burden and shortened the survival time
of the mice, while overexpression of ADAMTS-1.sub.NTCF or
ADAMTS-1.sub.CTCF blocked pulmonary metastasis and render most of
the experimental mice free of metastatic disease (FIG. 2B-C).
[0133] Histologic analysis of the lung sections showed that
TA3.sub.wtb, TA3.sub.ADAMTS-1, and TA3.sub.ADAMTS-1minusTSP cells
are invasive and fill up the pulmonary space (FIG. 2D-a-b, and data
not shown). On the contrary, only micrometastasis were detected in
the lungs received TA3.sub.ADAMTS-1NTCF or TA3.sub.ADAMTS-1 cells
(FIG. 2D-c, d, arrows). To assess whether ADAMTS-1 expressed by the
transfected TA3 cells is cleaved in vivo, different pulmonary
tumors derived from TA3.sub.ADAMTS-1 cells were lysed and the
proteins were analyzed by Western blotting with anti-v5 antibody,
which recognizes the v5-tagged ADAMTS-1. The result showed that
ADAMTS-1 protein is maintained in full-length form in vivo and no
cleavage fragments of ADAMTS-1 were detected (FIG. 2 E). This
result suggests that proteolytic cleavage of ADAMTS-1 regulates
ADAMTS-1 function and the cleavage status of ADAMTS-1 in vivo
determine its effect (stimulatory or inhibitory) on tumor
metastasis.
[0134] ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF Blocks Pulmonary
Metastasis by Inhibiting Proliferation and Inducing Apoptosis of
Tumor Cells, and by Repressing Tumor Angiogenesis
[0135] To determine the mechanism underlying the pro-tumor effect
of full-length of ADAMTS-1 and the anti-tumor effect of
ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF, proliferation and
apoptosis rates of the tumor cells and the extent of tumor
angiogenesis during pulmonary metastasis of TA3 transfectants were
analyzed. A 5-Bromo-2'-deoxy-uridine (Brdu) incorporation assay and
in situ detection of apoptotic cells on the sections derived from
the experimental mouse lungs (six days after i.v. injection of TA3
transfectants) was performed. Results demonstrated that expression
of ADAMTS-1.sub.NTCF or ADAMTS-1.sub.CTCF, but not that of
ADAMTS-1.sub.minusTSP, inhibits proliferation and promotes
apoptosis of the tumor cells, and inhibits tumor angiogenesis;
while overexpression of full-length exogenous ADAMTS-1 on the top
of endogenous ADAMTS-1 has weak effect on tumor cell proliferation
and apoptosis and promotes tumor angiogenesis in vivo (FIG. 3).
These results imply that ADAMTS-1 plays a role in
releasing/activating growth/survival/angiogenesis factors in the
microenvironments, while ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF
blocks/interferes activities of the factors that promote tumor cell
proliferation and survival and tumor angiogenesis.
[0136] ADAMTS-1 Promotes Extravasation of the Tumor Cells and
Activation of EGFR and ErbB-2 In Vivo, and Promotes Shedding of AR
and HB-EGF
[0137] Activation of EGFR and ErbB-2 is known to promote
proliferation and survival of breast carcinoma cells and is
essential for progression of breast cancers. To determine whether
ADAMTS-1 promotes activation of EGFR and/or ErbB-2 in vivo,
activity of EGFR and ErbB-2 in the lungs where TA3.sub.wtb,
TA3.sub.ADAMTS-1, TA3.sub.ADAMTS-1NTCF, or TA3.sub.ADAMTS-1CTCF
cells were injected intravenously 24 hours prior was assessed. In
order to normalizing amount of the tumor cells that were included
in the protein lysates and used in the immunoprecipitation, a tumor
cells tracking assay to determine the pulmonary extravasation rate
of TA3 transfectants that were injected intravenously into their
syngenic mice 24 hours prior was performed. The result showed that
overexpression of ADAMTS-1 promotes tumor cell extravasation into
lung parenchyma, while expression of TA3.sub.ADAMTS-1NTCF, or
TA3.sub.ADAMTS-1CTCF inhibits the process (FIG. 4A-B).
[0138] Normal mouse lungs and the mouse lungs that received TA3
transfectants intravenously 24 hour prior were lysed, and the
protein lysates that statistically contain the same amount of the
tumor cells were used in immunoprecipitation to pull-down EGFR or
ErbB-2 and anti-phosphotyrosine antibody was used to detect
phosphor-EGFR or phosphor-ErbB-2. The result showed that
overexpression of ADAMTS-1 promotes activation of EGFR and ErbB-2
(FIG. 4C). On the contrary, expression of ADAMTS-1.sub.NTCF or
ADAMTS-1.sub.CTCF blocks activation of EGFR and ErbB-2 (FIG.
4C).
[0139] Whether increased activation of EGFR and ErbB-2 induced by
ADAMTS-1 is achieved via shedding/activating EGF family OF
precursors, the ligands of ErbB receptor tyrosine kinases which
include EGFR, ErbB-2, -3, and -4. EGF family GFs include EGF,
transforming growth factors-.alpha. (TGF-.alpha.), HB-EGF, AR,
betacellulin, epiregulin, neuregulin, and epigen, and are shed from
cell surface was investigated. Increasing amount of data suggests
that shedding of the EGF family GF precursors are essential in
regulating availability and bioactivity of these factors and in
activation of the ErbB signaling pathways. The members of ADAM
family, especially ADAM17 have been shown to play major but not
sole role in shedding of these factors.
[0140] To determine whether ADAMTS-1 play an important role in
constitutive shedding EGF family GFs especially the ones that bind
to heparin, Cos-7 cells with several EGF family GF precursors that
are expressed by TA3 cells (data not shown) including HB-EGF, AR,
and epigen with were co-transfected with empty expression vector or
the expression constructs containing full-length ADAMTS-1,
ADAMTS-1E/Q or various ADAMTS-1 fragments. Serum-free cell culture
medium (SFM) of the co-transfected Cos-7 cells were collected,
concentrated and analyzed. Cos-7 cells express endogenous ADAMTS-1
(data not shown). Overexpression of exogenous ADAMTS-1 promotes
shedding of AR and HB-EGF but not shedding of epigen (FIG. 4D).
More importantly, overexpress ADAMTS-1E/Q which acts as a dominant
negative regulator of endogenous ADAMTS-1 completely blocks the
shedding of AR and inhibits the shedding of HB-EGF, while ADAMTS-1
fragments displayed no significant effect on the shedding (FIG.
4D). These data suggest that ADAMTS-1 promotes activation of EGFR
and ErbB-2 by promoting shedding and activation of the EGF family
GFs.
[0141] ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF Inhibit Activation
of Erk1/2 Kinases Induced by the EGF Family GFs
[0142] Since ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF display no
significant inhibitory effect on shedding of AR and HB-EGF, it was
investigated as to whether ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF
inhibits activation of EGFR and ErbB-2 by interfering activity of
the soluble EGF family GFs. To assess that, purified soluble AR or
HB-EGF was applied to MCF-10A mammary epithelial cells in the
presence and absence of the naturalizing antibodies to HB-EGF or
AR, ADAMTS-1.sub.NTCF, ADAMTS-1.sub.CTCF, or full-length ADAMTS-1.
This result showed that the neutralizing antibodies,
ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF, but not full-length
ADAMTS-1 inhibit Erk1/2 kinase activation induced by soluble AR and
HB-EGF (FIG. 5). This result suggests that ADAMTS-1.sub.NTCF or
ADAMTS-1.sub.CTCF inhibits activation of EGFR and ErbB-2 by
inhibiting their ligand activity likely via interfering the binding
between ligands and their receptors and that the different effects
of ADAMTS-1 and its cleavage fragments on availability and activity
of soluble AR and HB-EGF underlie their opposite roles in tumor
metastasis.
[0143] To further determine the molecular mechanism underlying the
anti-angiogenic activity of ADAMTS-1.sub.NTCF and
ADAMTS-1.sub.CTCF, it was investigated how these fragments affect
activities of several important growth/angiogenic factors that are
known to regulate angiogenesis. Bioactivity of VEGF.sub.165, basic
FGF (bFGF), HB-EGF, and AR were revealed by their ability to induce
activation of Erk1/2 kinases in HUVECs in the presence or absence
of purified ADAMTS-1 or the ADAMTS-1 fragments. Our results showed
that ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF but not full-length
ADAMTS-1 or ADAMTS-1.sub.minusTSP block activation of Erk1/2
kinases induced by VEGF.sub.165, HB-EGF, and AR but not that
induced by bFGF (FIG. 6). These results suggest that
ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF block tumor angiogenesis by
sequestering the activities of several important heparin binding
factors that are essential for endothelial cells proliferation and
survival.
[0144] ADAMTS-1 was found to inhibit tumor growth by blocking tumor
angiogenesis; however, this study did not investigate whether the
anti-tumor activity is derived from the full-length ADAMTS-1, its
cleavage fragments, or both. On the contrary, increased expression
of ADAMTS-1 was correlated to the increased metastatic potential in
the clinic tumor samples. The current study was designed to better
understand the role of full-length and the cleavage fragments of
ADAMTS-1 in tumor metastasis and to elucidate the underlying
mechanisms. It was demonstrated that overexpression of ADAMTS-1
promotes tumor metastasis by promoting tumor cell extravasation and
tumor angiogenesis. It is well established that tumor cell
extravasation is a critical step during tumor metastasis and
studies have shown that ADAMTS-1 is capable of degrading aggrecan
and versican. The ability of ADAMTS-1 to degrade aggrecan/versican
and other not yet identified ECM components is likely responsible
for the enhanced extravasation ability of TA3.sub.ADAMTS-1 cells.
Furthermore, as described herein, ADAMTS-1 promotes shedding of AR
and HB-EGF, which in turn promotes activation of EGFR and ErbB-2
and proliferation and survival of the tumor cells in vivo.
[0145] In the current study, it was demonstrated that ADAMTS-1
undergoes auto-proteolytic cleavage and overexpression of the
cleavage fragments of ADAMTS-1 (ADAMTS-1.sub.NTCF and
ADAMTS-1.sub.CTCF) block metastasis of TA3 cells by inhibiting
extravasation, proliferation and survival of the tumor cells, and
by repressing tumor angiogenesis via interfering activities of
several important heparin binding growth/angiogenic factors.
Furthermore, it was demonstrated that auto-proteolytic cleavage of
ADAMTS-1 is blocked by HS, which suggests that the level of HS/HSPG
in the microenvironment likely regulates which form of ADAMTS-1
(full-length or the cleavage fragments) presents predominantly in
the microenvironment to exert pro- or anti-tumor activity,
respectively. Thus, the roles of ADAMTS-1 and its cleavage
fragments in tumor metastasis, provided the regulatory mechanism of
ADAMTS-1 function (by auto-proteolytic cleavage and HS/HSPGs), and
revealed the mechanisms underlying the function of ADAMTS-1 and the
ADAMTS-1 cleavage fragments (by regulating availability and
activity of the EGF family GFs and ErbB signaling pathway).
[0146] Shedding EGF Family GFs by ADAMTS-1
[0147] Although functional differences between mature soluble EGF
family GFs and their transmembrane precursors are not
well-established, the phenotype similarity between TGF-.alpha.- and
ADAM17-null mice and between HB-EGF-null and HB-EGF cleavage
resistant mice clearly suggested that shedding of these precursors
is essential for availability and activity of these factors.
Several members of ADAM family including ADAM 9, 10, 12, 17 have
been implicated in shedding of HB-EGF and AR. The studies using the
cells derived from ADAM-9, -10, -12, -15, and/or -17 null-mice have
suggested that ADAM17 are the major but not the sole sheddase of AR
and HB-EGF, and other member(s) of ADAM and/or ADAMTS family
is(are) likely play important roles as well, especially in the
non-PMA-induced/metalloproteinase inhibitor sensitive/constitutive
shedding of these factors.
[0148] Several members of EGF family GFs including HB-EGF and AR
bind to HS/HSPGs. ADAMTS-1 binds to HS as well through the spacer
region and the TSP type I motifs, which brings the proteinase
domain of ADAMTS-1 close to the HS/HSPG bound factors and makes
ADAMTS-1 as an ideal sheddase to cleave these HS/HSPG binding GF
precursors. The present disclosure has provided evidences that
ADAMTS-1 promotes shedding of AR and HB-EGF and ADAMTS-1 may be a
major sheddase that is responsible for constitutive shedding of AR
and HB-EGF. Soluble AR and HB-EGF shed by ADADMTS-1 can in turn
promote tumor cell survival and proliferation and tumor
angiogenesis in vivo.
[0149] As discussed herein, it is shown that ADAMTS-1 but not the
ADAMTS-1 fragments promotes shedding of AR and HB-EGF, suggesting
that the intact spacer/Cys-rich domain is required for the shedding
and the spacer/Cys-rich domain contains substrate
recognition/binding site(s) which is(are) destroyed by the
auto-proteolytic cleavage in this region. Since all the members of
ADAMTS family have similar domain organization, in addition to
ADAMTS-1, other members of the ADAMTS family may also involve in
regulating availability and activity of HS/HSPG-binding
factors.
[0150] The Anti-Tumor Activity of the ADAMTS-1 Fragments is Masked
in the Full-Length Molecule.
[0151] As described herein, it is demonstrated that in contrast to
the effect of full-length ADAMTS-1, the ADAMTS-1 cleavage fragments
(ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF) block pulmonary
metastasis of TA3 cells. How can auto-proteolytic cleavage convert
a pro-tumor factor into anti-tumor ones? The results suggest that
auto-proteolytic cleavage destroys the substrate binding domain in
the spacer/Cys-rich region that is likely required for binding to
AR and HB-EGF precursors. In addition, it is described herein that
the N-terminal deletion fragment of ADAMTS-1 in which all the TSP
type I motifs were deleted (ADAMTS-1.sub.minusTSP) displayed no
anti-tumor activity, suggesting that the anti-tumor activity of
ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF is derived from the TSP
type I motif. Even though full-length ADAMTS-1 contains all three
TSP type I motifs, they are likely masked and unable to exert
anti-tumor activity. Auto-proteolytic cleavage of ADAMTS-1 at the
spacer/Cys-rich region not only renders the N-terminal cleavage
fragment (ADAMTS-1.sub.NTCF) that contains the metalloproteinase
domain incapable of binding to and shedding AR and HB-EGF
precursors (FIG. 4D), but also exposes the cryptic anti-tumor
domains in both N- and C-terminal cleavage fragments. In addition
to ADAMTS-1, ADAMTS-4, and -12 undergo proteolytic cleavage at
their spacer/Cys-rich region as well. The auto-proteolytic cleavage
may be a general mechanism that regulates function of many ADAMTS
family members, and our results provided the first example of this
type of regulatory mechanism.
[0152] As described herein, it has been shown that
ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF inhibits activation of
Erk1/2 kinase induced by AR, HB-EGF, or VEGF (FIG. 5-6). A recent
study has shown that ADAMTS-1 inhibits VEGF activity by blocking
the binding between VEGF and their receptor. Although additional
study is required to determine the exact biochemical mechanism
underlying the inhibitory effect of ADAMTS-1.sub.NTCF and
ADAMTS-1.sub.CTCF, they likely exert their inhibitory effect by
sequestering these soluble GFs from their receptors.
[0153] The Function of ADAMTS-1 is Regulated by HS/HSPGs
[0154] As described herein, heparin/HS blocks auto-proteolytic
cleavage of ADAMTS-1, and full-length ADAMTS-1 and the ADAMTS-1
cleavage fragments (ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF)
displayed opposite effects on tumor metastasis. Thus, ability of
ADAMTS-1 to inhibit or promote tumor metastasis is dependent on the
ability of tumor cells and their surrounding microenvironment to
cleave ADAMTS-1. In other words, in a microenvironment that is
highly enriched HS and HSPGs, binding of ADAMTS-1 to HS/HSPGs
protects the proteolytic cleavage sites in the spacer/Cys-rich
region which keeps ADAMTS-1 in the full-length form and in turn
binds and cleaves its substrates including transmembrane AR and
HB-EGF. In that situation, full-length ADAMTS-1 exerts pro-tumor
activity by releasing and activating pro-proliferation, -survival,
and -angiogenic factors. In addition, the anti-tumor activity
derived from TSP type I motifs is likely masked in full-length
ADAMTS-1. On the contrary, in a microenvironment that is lack of or
low in HS/HSPGs, ADAMTS-1 is likely cleaved to generate the
cleavage fragments that are without the substrate (AR and HB-EGF)
binding site(s) and contain unmask the anti-tumor TSP type I
motifs.
Example 3
The Pro-Tumor Effect of Full-Length ADAMTS-1 and the Anti-Tumor
Effect of the ADAMTS-1 Fragments were Confirmed in Lewis Lung
Carcinoma (LLC) Cells
[0155] To confirm the effects of full-length ADAMTS-1 and the
ADAMTS-1 fragments on tumor growth and metastasis, and compare
their effects with that of thrombospondin-1 and -2, LLC
transfectants were established that were transfected with empty
expression vectors (LLC.sub.wtb) or expressing full-length
ADAMTS-1, ADAMTS-1E/Q, ADAMTS-1, ADAMTS-1.sub.CTF,
thrombospondin-1, or -2 (FIG. 16). ADAMTS-1, thrombospondin-1 and
-2 are the members of thrombospondin type I repeat superfamily
(TRS). Thrombospondin-1 is a 450 kDa homotrimeric ECM protein and
is considered as a potent anti-tumor molecule. Studies have shown
that systemic injection or overexpression of thrombospondin-1
inhibits the in vivo growth of several tumor cells including LLC
cells (70, 78, 79). A subline of LLC cell (LLC.sub.wt) that
undergoes spontaneous pulmonary metastasis after removal of the
primary subcutaneous (s.c.) tumors were used to establish these
transfectants. LLC.sub.wt cells express a low level of endogenous
ADAMTS-1 as assessed by RT-PCR and Western blot analysis (data not
shown). Five independent clonal LLC transfectants expressing a high
to intermediate level of the same gene products were randomly
selected, pooled (FIG. 16), and used in the s.c. tumor growth and
spontaneous pulmonary metastasis experiments following the
established protocols (80-82, 108, 110).
[0156] The results showed that expression of full-length ADAMTS-1
promotes while expression ADAMTS-1.sub.NTF or ADAMTS-1.sub.CTF and
to a less extent that of ADAMTS-1E/Q inhibits s.c. growth and
spontaneous pulmonary metastasis of the LLC transfectants (FIG.
16). More importantly, even though the LLC transfectants express a
higher level of thrombospondin-1 or -2 comparing to that of
ADAMTS-1NTF and ADAMTS-1CTF, the inhibitory effect derived from the
ADAMTS-1 fragments is stronger than that derived from
thrombospondin-1 or -2 (FIG. 16), suggesting that the ADAMTS-1
fragments and their derivatives have unique features and a great
potential to be used as the potent anti-cancer agents.
[0157] The Metalloproteinase Activity in ADAMTS-1.sub.NTF is not
Required for its Anti-Tumor Activity:
[0158] To investigate whether metalloproteinase activity in
ADAMTS-1.sub.NTF is required for the anti-tumor activity of
ADAMTS-1.sub.NTF, a protease-dead ADAMTS-1NTFE/Q mutant was
established, in which E386 is switched to Q in the Zinc-binding
pocket of the metalloproteinase domain. The expression constructs
were used to transfect TA3 mouse mammary carcinoma cells. Three
independent positive colonies that express ADAMTS-1NTFE/Q or
ADAMTS-1NIT or transfected with the empty expression vectors (FIG.
17) were used in the pulmonary tumor metastasis experiments. Our
results showed that ADAMTS-1.sub.NTFE/Q behaved like
ADAMTS-1.sub.NTF and significantly promoted the survival of the
experimental mice and inhibited the pulmonary tumor metastasis
(FIG. 17). This result suggests that the metalloproteinase domain
of ADAMTS-1 does not contribute to the anti-tumor effect of
ADAMTS-1.sub.NTF.
Example 4
[0159] The anti-tumor and anti-angiogenic activity of
thrombospondin-1 has been well established and the anti-tumor
activity has been mapped to the several domains including the TSP
type I repeats. All the members of the ADAMTS family contain at
least one TSP-1 motif and belong to the thrombospondin type I
repeat (TSR) superfamily (73). Since identification of ADAMTS-1
(22), several studies have been performed to investigate the role
of ADAMTS-1 in tumor growth and metastasis, and the results
obtained appeared to contradict each other. In pancreatic cancer
samples, a higher ADAMTS-1 mRNA level was correlate to the severe
lymph node metastasis or retroperitoneal invasion and worse
prognosis, suggesting that ADAMST-1 likely promotes pancreatic
cancer invasion and metastasis. However, ADAMTS-1 mRNA is
down-regulated in the breast carcinoma samples comparing to the
normeoplastic mammary tissues but with no strong links between the
ADAMTS-1 mRNA level and the clinicopathological features of these
breast cancer cases studied. These studies have only measured
ADAMTS-1 mRNA level but not the protein level and the proteolytic
activity of ADAMTS-1, both of which are more relevant to the
ADAMTS-1 function.
[0160] In addition, ADAMTS-1 was found to inhibit tumor growth by
blocking tumor angiogenesis, which is likely achieved by
sequestering VEGF.sub.165 from its receptor, and the
metalloproteinase activity of ADAMTS-1 is required for the observed
anti-angiogenesis and anti-tumor growth activity. In contrast to
this finding, overexpressioin of ADAMTS-1 was found to promote
subcutaneous growth of the transfected CHO cells but inhibit
experimental metastasis of the same transfectants. However, these
studies have neither considered the fact that ADAMTS-1 is
proteolytically cleaved, nor investigated the cleavage status of
ADAMTS-1 in vivo (in subcutaneous and pulmonary microenvironments),
and did not consider the possibility that the requirement of the
metalloproteinase activity of ADAMTS-1 for its anti-tumor effect
may merely reflect to the fact that the anti-tumor effect is
actually derived from the auto-proteolytic cleavage fragments but
not the full-length ADAMTS-1, and that the metalloproteinase
activity of ADAMTS-1 is required for generating these ADAMTS-1
fragments.
[0161] To test this possibility, the how and why full-length and
the ADAMTS-1 fragments affect tumor growth and metastasis was
investigated. As described herein, it is demonstrated that
overexpression of full-length ADAMTS-1, which is maintained in the
full-length form during metastasis of TA3 mammary carcinoma cells,
promotes the tumor metastasis, and that ADAMTS-1 promotes shedding
of AR and HB-EGF precursors and activation of EGFR and ErbB-2 in
vivo. In addition, for the first time that ADAMTS-1 undergoes
auto-proteolytic cleavage to generate the NH.sub.2- and
COOH-terminal fragments that contain at least one TSP-1 motif is
shown. In contrast to that of full-length ADAMTS-1, overexpression
of the fragments of ADAMTS-1 (ADAMTS-1.sub.NTCF and
ADAMTS-1.sub.CTCF) that mimic the proteolytic cleavage fragments of
ADAMTS-1 blocks pulmonary metastasis of TA3 cells by inhibiting
tumor cell extravasation, proliferation and survival, and by
repressing tumor angiogenesis. It is demonstrated that the
anti-metastatic activity of the ADAMTS-1 fragments requires the
TSP-1 motif, which is likely masked in the full-length molecule,
and that ADAMTS-1.sub.NTF and ADAMTS-1.sub.CTF inhibit activation
of EGFR and ErbB-2 in vivo and inhibits the Erk1/2 kinase
activation induced by soluble AR and HB-EGF.
[0162] Furthermore, it is demonstrated that the proteolytic
cleavage of ADAMTS-1 is blocked by heparin, and HS, suggesting that
the binding of ADAMTS-1 to heparan sulfate proteoglycans (HSPGs)
masks the auto-proteolytic cleavage site(s) in the spacer/Cys-rich
domain and keep ADAMTS-1 in the full-length form to cleave their
substrates. On the other hand, the auto-proteolytic cleavage of
ADAMTS-1 in the spacer/Cys-rich domain likely destroys the
substrate binding sites and unmasks the anti-tumor TSP-1 domain,
which renders the anti-tumor activity to the ADAMTS-1 fragments.
Thus, the level of HSPGs in the microenvironment likely regulates
the form of ADAMTS-1 (full-length or the cleavage fragments) that
exists predominantly in the microenvironment to exert pro- or
anti-tumor activity, respectively. It is demonstrated that ADAMTS-1
expressed by TA3 cells is maintained in the full-length form in
vivo to exert pro-metastasis activity. Thus, the results have
reconciled the apparent contradiction in the previous results and
demonstrated that the cleavage status of ADAMTS-1 determines its
effect (stimulatory or inhibitory) on tumor growth and
metastasis.
[0163] The results described herein suggested that ADAMTS-1 plays
the multiple roles in tumor growth and metastasis and is a prime
target for cancer therapy, and that the ADAMTS-1 fragments have
great potential as the potent anti-cancer agents that inhibit not
only tumor cell proliferation/survival/invasion, but also tumor
angiogenesis.
[0164] The Pro-Metastatic Activity of Full-Length ADAMTS-1 Requires
its Metalloproteinase Activity
[0165] To determine whether the metalloproteinase activity of
ADAMTS-1 is required for the pro-metastatic activity of ADAMTS-1,
TA3 transfectants expressing the protease-dead mutant of ADAMTS-1
(ADAMTS-1E/Q), which harbors an E.sub.386 to Q point mutation in
the Zinc-binding pocket of the metalloproteinase domain were
generated. The study has shown that this mutant lacks the catalytic
activity.
[0166] In order to assess the effects of ADAMTS-1E/Q on tumor
metastasis reliably, the established clonal TA3 cell line,
TA3.sub.wt1 was used. Like its parental cells, TA3.sub.wt1 cells
express ADAMTS-1 endogenously and undergo pulmonary metastasis
after intravenous (i.v.) injection. Five independent clonal TA3
transfectants expressing a high to intermediate level of
ADAMTS-1E/Q were randomly selected and used as the pooled
population (TA3.sub.ADAMTS-1E/Q, FIG. 7B) in the pulmonary
metastasis experiments. Five independent clonal TA3 transfectants
transfected with the empty expression vectors or expressing the
following same gene products were used as the pooled population as
well: full-length ADAMTS-1 (TA3.sub.ADAMTS-1), ADAMTS-1.sub.NTF
(TA3.sub.ADAMTS-1NFT), ADAMTS-1.sub.CTF (TA3.sub.ADAMTS-1CTF), and
ADAMTS-1.sub.minusTSP-1 (TA3.sub.ADAMTS-1minusTSP-1). These pooled
TA3 transfectants express a similar level of the transfected gene
products (FIG. 7B) and displayed a similar growth rate in the cell
culture condition with 10% FBS (data not shown).
[0167] It was confirmed that the expression of full-length ADAMTS-1
promotes the pulmonary metastasis of TA3 cells and shortens the
survival time of the mice, while ADAMTS-1.sub.NTF or
ADAMTS-1.sub.CTF, but not ADAMTS-1.sub.minusTSP-1 blocks the
pulmonary metastasis of the transfectants (FIG. 7 C-D). In
addition, the expression of ADAMTS-1E/Q inhibits the pulmonary
metastasis of the transfectants and extends the survival time of
the mice (FIG. 7C-D), suggesting that the metalloproteinase
activity is required for the pro-metastatic activity of full-length
ADAMTS-1. The metastatic burden was quantified by the average
weight of the experimental mouse lungs (FIG. 7D). Because there is
a significant difference in the survival time of the experimental
mice which succumb to pulmonary metastasis when metastatic burden
causes the lung weight to reach 1-1.2 gram, the metastatic burden
was measured in the remaining survival mice at day 12 and 20 after
i.v. injection of these TA3 transfectants. At least 12 mouse lungs
were weighted for each type of the transfectants at each time
point. We confirmed that overexpression of full-length ADAMTS-1
accelerated the time that is required to reach the maximal
metastatic burden and shortened the survival time of the mice,
while overexpression of ADAMTS-1E/Q, ADAMTS-1.sub.NTF, or
ADAMTS-1.sub.CTF but not ADAMTS-1.sub.minusTSP-1 reduced the
metastatic burden (FIG. 7D). Furthermore, it was demonstrated that
the inhibitory effect derived from ADAMTS-1.sub.NTF or
ADAMTS-1.sub.CTF is stronger than that derived from ADAMTS-1E/Q,
implying that the underlying mechanisms for their anti-metastatic
effects may be different. This hypothesis was supported by the
results obtained previously, which indicated that ADAMTS-1E/Q but
not ADAMTS-1.sub.NTF and ADAMTS-1.sub.CTF serves as a dominant
negative regulator of full-length endogenous ADAMTS-1 by inhibiting
the shedding of HB-EGF and AR transmembrane precursors (FIG. 4).
Together, these data suggest that like full-length ADAMTS-1, the
anti-tumor TSP-1 domains in ADAMTS-1E/Q are masked, and that the
anti-tumor activity is likely derived from the intact
spacer/Cys-rich domain, which competes with ADAMTS-1 for the
binding to its substrates.
[0168] The Spacer/Cys-Rich Domain is Essential for Binding of
ADAMTS-1 to the Cell Surface and the ECM
[0169] The ADAMTS-1 substrates identified so far are versican,
aggrecan, and HB-EGF and AR precursors, which are located on the
ECM and the cell surface, respectively. To determine the domain(s)
of ADAMTS-1 that mediate(s) the substrate binding, we first assess
the ECM and the cell binding capacity of the different deletional
mutants of ADAMTS-1 (see FIG. 7A). All the constructs contain the
COOH-terminal v5-epitope tags for easy identification and
purification, and the constructs were transfected into COS-7 cells.
72 hours after the transfection, the proteins derived from the cell
culture supernatants, the ECM materials deposited by the
transfected cells, and the lysates of the transfected cells were
analyzed by Western blotting with anti-v5 epitope antibody as
described (106, 109). The results showed that the spacer/Cys-rich
domain is essential for the binding of ADAMTS-1 to the ECM and the
cells (FIG. 8), suggesting that the spacer/Cys-rich domain likely
mediates the substrate binding of ADAMTS-1.
[0170] ADAMTS-1 Promotes Invasion of TA3 Cells Through Matrigel
[0171] It is well established that the pericellular proteolysis
mediated by MMPs is essential for tumor invasion. As a member of
the Zinc.sup.2+-dependent metalloproteinase family, ADAMTS-1 plays
an important role in degrading versican, an important component of
the ECM and the blood vessel walls. We have shown that ADAMTS-1
promotes extravasation of TA3 cells into lung parenchyma (FIG. 4).
To determine bow full-length ADAMTS-1 and the fragments of ADAMTS-1
affect tumor cell invasion through Matrigel which mimics the
basement membrane as the barriers of tumor cell invasion, an
invasion assay by using Transwell cell culture chambers with
8-.mu.m pores (Costar) coated with a layer of Matrigel
(Collaborative Biomedical) was performed. The DMEM containing 2%
FBS was be added into the lower chambers of the Transwells.
2.times.10.sup.5 of the different TA3 transfectants were seeded on
top of the Transwell in triplicate and incubated for 24 hours. The
bottom filters were then be fixed and stained. The cells on the top
chambers were removed by wiping with cotton swabs, and the stained
cells (blue color) that have migrated through the Matrigel were
counted under a microscope. Six randomly selected 100.times.
microscopic fields will be countered. The invasion index of the
different TA3 transfectants was calculated as following
formula:
Invasion Index = 100 % .times. Average numbers of cells in lower
camber / microscopic field Numbers of cells seeded on upper camber
/ microscopic field ##EQU00001##
[0172] The results showed that TA3.sub.ADAMTS-1 cells displayed
approximately two time higher invasion index than TA3.sub.wt1 and
TA3.sub.ADAMTS-1minusTSP-1 cells, and four-eight time higher
invasion index compared to TA3.sub.ADAMTS-1NTF/TA3.sub.ADAMTS-1CTF
and TA3.sub.ADAMTS-1E/Q cells, respectively (FIG. 9A-B). These
results further confirmed that ADAMTS-1 promotes tumor cell
invasion, while TA3.sub.ADAMTS-1E/Q and to a less extent
TA3.sub.ADAMTS-1NTF, or TA3.sub.ADAMTS-1CTF inhibits the process.
To determine whether ADAMTS-1 promotes TA3 cell invasion by
degrading versican or inhibiting the pro-migratory effect of
soluble HB-EGF and AR, the confluence TA3 transfectants were lifted
by the EDTA solution and the ECM materials remained on the cell
culture dishes were extracted and analyzed by Western blotting with
anti-DP antibody, which detects the cleavage fragments of versican.
The result showed that increased expression of exogenous ADAMTS-1
but not ADAMTS-1.sub.minusTSP-1 on top of the endogenous ADAMTS-1
promotes degradation of versican, while expression of ADAMTS-1E/Q
but not ADAMTS-1.sub.NTF, ADAMTS-1.sub.CTF inhibits the degradation
(FIG. 9C). These data suggest that ADAMTS-1E/Q inhibits TA3 cell
invasion by blocking the ADAMTS-1 mediated versican degradation,
while the weaker inhibitory effect of the ADAMTS-1 fragments is
likely derived from their indirect effect on activity of
HB-EGF/AR.
Example 5
[0173] Following the experimental procedures described in Example
1, addition data was generated showing additional differences
between full length ADAMTS-1 and its cleavage products,
ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF. Overexpression of ADAMTS-1
promotes growth of TA3 mammary carcinoma (TA3) cells while
overexpression of the N- or C-terminal fragment of ADAMTS-1 blocks
growth of TA3 cells by inhibiting proliferation and inducing
apoptosis of the tumor cells and by inhibiting tumor angiogenesis.
ADAMTS-1 expressed by TA3 cells maintained in the full-length form
in vivo exerted pro-tumor growth and metastasis activity. In
contrast to the of full-length ADAMTS-1, overexpression of the N-
or C-terminal fragment of ADAMTS-1 (ADAMTS-1.sub.NTCF and
ADAMTS-1.sub.CTCF) inhibits subcutaneous (s.c.) growth of TA3
cells. In addition, unlike full-length ADAMTS-1 which promotes
shedding of the EGF family ligands including amphiregulin (AR) and
heparin-binding EGH (HB-EGF) and activation of EGF receptor (EGFR)
and ErbB-2, the ADAMTS-1 fragments inhibits activation of EGFR and
ErbB-2 in vivo.
[0174] RT-PCR results showed that like wild type TA3 cells,
TA3.sub.wt1 cells express ADAMTS-1 endogenously as do several other
tumor cell lines (FIG. 10). Growth rates of the s.c. solid tumors
derived from different TA3 transfectants were measured and the
result showed that overexpression of ADAMTS-1 promotes tumor
growth, while overexpression of ADAMTS-1.sub.NTCF and
ADAMTS-1.sub.CTCF, but not that of ADAMTS-1.sub.minusTSP
significantly inhibits tumor growth (FIG. 11). These results
suggest that the inhibitory effect of the ADAMTS-1 fragments is
likely derived from the TSP type I motifs, which exist in
ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF, but not in
ADAMTS-1.sub.minusTSP. The data show that ADAMTS-1.sub.NTCF or
ADAMTS-1.sub.CTCF, blocks pulmonary metastasis of
TA3ADAMTS-1.sub.NTCF or TA3ADAMTS-1.sub.CTCF cells (FIG. 11, B).
The metastatic burden of the experimental mice was quantified by
average weight of the experimental mouse lungs received different
TA3 transfectants (FIG. 11, B). Results showed that overexpression
of ADAMTS-1.sub.NTCF or ADAMTS-1.sub.CTCF dramatically reduced
metastatic burden of the mice received the corresponding TA3
transfectants, and render most of the experimental mice free of
metastatic disease and significantly extended survival time of
these mice (FIG. 11, B).
[0175] The ADAMTS-1 fragments blocks tumor growth by inhibiting
proliferating and inducing apoptosis of tumor cells, and inhibiting
tumor angiogenesis. To determine the cellular basis of the
pro-tumor effect of full-length of ADAMTS-1 and the anti-tumor
effect of ADAMTS-1 fragments, proliferation and apoptosis rates of
the tumor cells and tumor angiogenesis during s.c. growth were
analyzed. Brdu (5-Bomo-2'-deoxy-uridine) incorporation assay and in
situ detection of apoptotic cells were performed on the sections
derived from s.c. solid tumors (twelve days after implanting the
TA3 cells). Results demonstrated that expression of
ADAMTS-1.sub.NTCF and to a less extent that of ADAMTS-1.sub.CFCF,
but not expression of ADAMTS-1.sub.minusTSP, inhibits proliferation
and promotes apoptosis of the tumor cells, and inhibits
angiogenesis in the subcutaneous space; while expression of
exogenous ADAMTS-1 mildly enhances proliferation rate and reduces
apoptosis rate of the tumor cells, and promotes tumor angiogenesis
in vivo (FIG. 12) These results suggest that ADAMTS-1 may play an
important role in releasing/activating growth/survival factors in
the microenvironments, while the cleavage fragments of ADAMTS-1 may
block the activities of the factors that promote tumor cell
proliferation and survival and tumor angiogenesis.
[0176] Activation of EGFR and ErbB-2 is known to promote
proliferation and survival of breast carcinoma cells and play
essential roles in progression of breast cancers. To determine
whether activation of EGFR and/or ErbB-2 underlies the pro-tumor
activity of ADAMTS-1, we assessed activity of EGFR and ErbB-2 in
the lungs where TA3.sub.wtb, TA3.sub.ADAMTS-1,
TA3ADAMTS-1.sub.NTCF, TA3ADAMTS-1.sub.CTCF, or
ADAMTS-1.sub.minusTSP cells were injected five days prior. The
result showed that expression of ADAMTS-1 by TA3 cells promotes
activation of EGFR and ErbB-2 in vivo (FIG. 13, A). On the
contrary, expression of ADAMTS-1.sub.NTCF or ADAMTS-1.sub.CTCF, but
not ADAMTS-1.sub.minusTSP which lacks TSP type I motifs, blocks
activation of EGFR and ErbB-2 in vivo (FIG. 13, A).
[0177] Experiments were done to assess whether induces activation
of EGFR and ErbB-2 by ADAMTS-1 is achieved via promoting shedding
EGF family ligands. EGF family ligands are produced as
transmembrane precursors, which are shed and released from cell
surface as soluble mature form. Several EGF family ligands are
known to be shed-activated by the ADAM family proteinases including
ADAM-17. However, studies using the cells derived from ADAM-17
null-mouse suggested that ADAM-17 is not the sole proteinase that
is responsible for shedding of TGF-.alpha. and other member(s) of
ADAM family is (are) likely to play a role as well. To determine
whether ADAMTS-1 play a role in shedding EGF family ligands
especially the ones that bind to heparin, several EGF family
ligands were co-transfected including HB-EGF, TGF-.alpha., AR, and
epigen which are expressed by TA3 cells (data not shown) with or
without the full-length ADAMTS-1, ADAMTS-1E/Q and the ADAMTS-1
fragments. The serum-free cell culture medium of the transfected
cells were collected and concentrated and analyzed. The results
showed that ADAMTS-1 promotes shedding of AR and HB-EGF but not
shedding of TGF-.alpha. and epigen; while ADAMTS-1E/Q blocks the
shedding. The ADAMTS-1 fragments displayed no effect on the
shedding (FIG. 13, B and data not shown).
[0178] To determine whether the ADAMTS-1 fragments affect the
signal transduction pathways activated by HB-EGF and AR, the
serum-free cell culture media (SFM) derived from the co-transfected
cells were applied to MCF-10A mammary epithelial cells to determine
their ability to induce Erk1/2 kinase activation. The results
showed that soluble HB-EGF and AR in the SFM induces activation of
Erk1/2 kinases, which is specifically blocked by the corresponding
blocking antibodies or the ADAMTS-1 fragments, but not by the
full-length ADAMTS-1 (FIG. 13, C). This result suggests that the
ADAMTS-1 fragments inhibit activation of EGFR and ErbB-2 by
interfering with their ligand activity; and the effects of ADAMTS-1
and its cleavage fragments on the availability and activity of EGF
family ligands likely underlies their roles in tumor growth and
metastasis.
[0179] The affect the fragments have on activities of several
important growth/angiogenic factors that are known to regulate
angiogenesis was investigated. Activity of VEGF.sub.165, bFGF,
HB-EGF, TGF-.alpha., and AR were revealed by their ability to
induce activation of Erk1/2 kinases in HUVECs in the presence or
absence of different purified ADAMTS-1 proteins. Results showed
that ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF but not full-length
ADAMTS-1 or ADAMTS-1.sub.minusTSP block activation of Erk1/2 kinase
induced by VEGF.sub.165, TGF-.alpha., HB-EGF, and AR
[0180] ADAMTS-1 is widely expressed by tumor cells and undergoes
auto-proteolytic cleavage. In addition, overexpression of ADAMTS-1
promotes tumor growth and metastasis by enhancing tumor cell
proliferation and survival and by promoting tumor angiogenesis
through shedding transmembrane EGF family ligands, AR and HB-EGF,
which in turn promotes activation of EGFR and ErbB-2 in vivo.
[0181] The results not only provided a potential important target
(full-length ADAMTS-1), potent novel anti-cancer reagents (the
ADAMTS-1 fragments), and the regulatory reagents for ADAMTS-1
activity (HS/HSPGs) for the treatment of cancers especially breast
cancers in the figure, but also revealed the mechanism underlying
the function of ADAMTS-1 and the ADAMTS-1 fragments.
[0182] The presence of TSP type I motif is a common feature of all
members of ADAMTS family, among them ADAMTS-1, -4, AND -12 undergo
proteolytic cleavage at their spacer/Cys-rich region, which have
potential to generate ADAMTS fragments containing unmasked TSP type
I motifs that may possess anti-tumor activity. In addition, as
indicated in this study, the auto-proteolytic cleavage may be a
general mechanism that regulates the function of ADAMTS family
members. Additional work is required to verify these hypotheses and
the results obtained in this study provide general rules that may
apply the other ADAMTS family members as well.
[0183] For subcutaneous tumor growth experiments, five independent
clonal TA3 transfectants expressing ADAMTS-1, ADAMTS-1.sub.CTCF,
ADAMTS-1.sub.NTCF or ADAMTS-1.sub.minusTSP, or transfected with the
empty expression vector were used in the in vivo experiments. For
each type of the experiment, six mice were injected with each
clonal transfectants and two independent experiments were
performed.
[0184] In tumor growth experiments, TA3 transfectants were injected
subcutaneously into syngenic A/Jax-mic as described. After solid
tumors became visible (7-10 days after the injection), the tumors
were measured by a digital caliper every other day for the next two
weeks. The largest and shortest diameters of the solid tumors were
measured. The tumor volume was calculated by using the following
formula: tumor volume=1/2.times.(shortest
diameter).sup.2.times.longest diameter (mm.sup.3).
Example 6
Determining the Exact Amino Acid Segments in the TSP-1 Domains
Containing Anti-Cancer Activity
[0185] As discussed herein, the ADAMTS-1 fragments that contain
either the middle TSP-1 motif (ADAMTS-1.sub.NTF) or the two
COOH-terminal TSP-1 modules (ADAMTS-1.sub.CTF) inhibit growth
and/or metastasis of TA3 and LLC, and their inhibitory effect is
much stronger than that caused by thrombospondin-1 and -2,
suggesting the unique molecular basis underlying the potent
inhibitory effect of the ADAMTS-1 fragments is not present in
thrombospondin-1 and -2. Also, the TSP-1 domain is required for the
anti-tumor activity of ADAMTS-1.sub.NTF. The middle TSP-1 domain
(mTSP-1, amino acids 546-596) in ADAMTS-1 is similar but not
identical to the second and third TSP-1 repeats (WXXWXXW) in
thrombospondin-1, which have been shown to contain anti-tumor and
anti-angiogenic activity (102, 111-113). Even though the
COOH-terminal TSP-1 modules (cTSP-1, amino acid 842-895 and
896-951, FIG. 9) of ADAMTS-1 do not have high homology to the TSP-1
repeats in thrombospondin-1, the ADAMST-1 fragments that contain
either the mTSP-1 or cTSP-1 domain exhibited the similar anti-tumor
activity, implying that the common unidentified unique amino acid
segments or three dimension feature (other than the WXXWXXW) in the
m/cTSP-1 domains of ADAMTS-1 may be essential for the potent
anti-tumor activity. Accordingly, the molecular basis for the
potent anti-tumor activity that is unique to the ADAMTS-1 fragments
can be identified using this domain. To achieve that, deletions in
the TSP-1 domains of ADAMTS-1 can be made and tumor growth and
metastasis assays can be performed using TA3 and LLC transfectants
expressing these ADAMTS-1 mutants (as described herein).
[0186] To Determine Whether the .sub.m and/or cfTSP-1 Domain
Displays Anti-Tumor Activity
[0187] It was shown that deletion of the middle TSP-1 (mTSP-1)
domain from ADAMTS-1.sub.NTF (ADAMTS-1.sub.minusTSP) abolishes the
potent anti-tumor activity of the fragment, suggesting the
anti-tumor activity resided in the mTSP-1 domain and that the
ADAMTS-1.sub.CTF is composed of the two COOH-terminal TSP-1
(cTSP-1) domains. Thus, whether the mTSP-1 domain and each of the
cTSP-1 domains display as potent anti-tumor activity as the
ADAMTS-1.sub.NTF and ADAMTS-1.sub.NTF fragments need to be
determined. To achieve that, three expression constructs can be
generated that contain the signal peptide plus the mTSP-1 domain
(fADAMTS-1.sub.mTSP-1), the first cTSP-1 (fADAMTS-1.sub.cTSP-1-1),
or the second cTSP-1 (fADAMTS-1.sub.cTSP-1-2) domain in
pEF/6/v5-His expression vectors. They can be used to transfect TA3
wt1 and LLCwt1 cells. Five independent TA3 or LLC transfectants
expressing a high to intermediate level of fADAMTS-1 mTSP-1,
fADAMTS-1cTSP-1-1 or fADAMTS-1cTSP-1-2 will be randomly selected
and used as the pooled populations together with the established
TA3 and LLC transfectants expressing a similar level of ADAMTS-1CTF
or ADAMTS-1NTF, or transfected with the expression vector alone in
the s.c. tumor growth and metastasis experiments.
[0188] mTSP-1 domain inhibits growth and metastasis of TA3 and LLC
cells in a similar extent as that of ADAMTS-1.sub.NTF; while
expression of each of the cTSP-1 domains display a weaker
anti-tumor effect compared to that caused by ADAMTS-1.sub.CTF,
which contains two TSP-1 modules. These small recombinant proteins
(53-56 amino acid long) are used as anti-cancers agents.
[0189] Deletions and Mutations in the m or cTSP-1 Domains of
ADAMTS-1 and Establish TA3 and LLC Transfectants Expressing These
ADAMTS-1 Mutants
[0190] Within in the TSP type I repeats of thrombospondin-1, in
addition to WXXWXXW motif, the CSVTCG motif, which binds to CD36,
has been shown to contain anti-tumor and anti-angiogenic activity.
The .sub.m/cTSP-1 domains of ADAMTS-1 contain the motifs that are
similar to WXXWXXW and/or CSVTCG motifs. In addition, the consensus
motif search (GCG genomics) has demonstrated that the most
consensus motif among the m and cTSP-1 domains of ADAMTS-1 is the
WGE/DCSKTC motif (FIG. 18). Thus, three deletions in .sub.m/cTSP-1
domains are made: WGPWGPWGD (ADAMTS-1.sub.mTSP-1WXXWdel) or
WV/QI/VE/GE/DWG/S (ADAMTS-1.sub.cTSP-1WXXXXWdel), WGDCSRTC
(ADAMTS-1.sub.mTSP-1WGdel) or WG/SE/PCSKTC
(ADAMTS-1.sub.cTSP-1WXXXXWdel), CSRTCGGG (ADAMTS-1.sub.mTSP-1CSdel)
or CSKTCGS/KG (ADAMTS-1.sub.cTSP-1CSdel, FIG. 18).
[0191] The deletional mutagenesis are performed as described using
fADAMTS-1.sub.mTSP-1 and fADAMTS-1.sub.cTSP-1-1, or
fADAMTS-1.sub.cTSP-1-2 (in pEF/6/v5-His expression vectors) as the
templates. These expression constructs are used to transfect Cos-7
cells transiently to assess the expression capacity of these
v5-epitope tagged fragments. All the deletional mutants are
established in cell lines and their proper expression in Cos-7
cells is demonstrated. These deletional constructs are used to
transfect TA3.sub.wt1 and LLC.sub.wt1 cells. Five independent TA3
or LLC transfectants expressing a high to intermediate level of
each of the mutants are used as the pooled populations in the s.c.
tumor growth and metastasis experiments together with the
established TA3 and LLC transfectants expressing ADAMTS-1.sub.NTF,
ADAMTS-1.sub.CTF, fADAMTS-1.sub.mTSP-4 and fADAMTS-1.sub.cTSP-1-1,
or fADAMTS-1.sub.cTSP-1-2, or transfected with the expression
vector alone (the control).
[0192] The disclosures of each and every patent, patent
application, publication, and accession number cited herein are
hereby incorporated herein by reference in their entirety. The
appended sequence listing is hereby incorporated herein by
reference in its entirety.
[0193] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
361968PRTMus musculus 1Met Gln Pro Lys Val Pro Leu Gly Ser Arg Lys
Gln Lys Pro Cys Ser1 5 10 15Asp Met Gly Asp Val Gln Arg Ala Ala Arg
Ser Arg Gly Ser Leu Ser 20 25 30Ala His Met Leu Leu Leu Leu Leu Ala
Ser Ile Thr Met Leu Leu Cys 35 40 45Ala Arg Gly Ala His Gly Arg Pro
Thr Glu Glu Asp Glu Glu Leu Val 50 55 60Leu Pro Ser Leu Glu Arg Ala
Pro Gly His Asp Ser Thr Thr Thr Arg65 70 75 80Leu Arg Leu Asp Ala
Phe Gly Gln Gln Leu His Leu Lys Leu Gln Pro 85 90 95Asp Ser Gly Phe
Leu Ala Pro Gly Phe Thr Leu Gln Thr Val Gly Arg 100 105 110Ser Pro
Gly Ser Glu Ala Gln His Leu Asp Pro Thr Gly Asp Leu Ala 115 120
125His Cys Phe Tyr Ser Gly Thr Val Asn Gly Asp Pro Gly Ser Ala Ala
130 135 140Ala Leu Ser Leu Cys Glu Gly Val Arg Gly Ala Phe Tyr Leu
Gln Gly145 150 155 160Glu Glu Phe Phe Ile Gln Pro Ala Pro Gly Val
Ala Thr Glu Arg Leu 165 170 175Ala Pro Ala Val Pro Glu Glu Glu Ser
Ser Ala Arg Pro Gln Phe His 180 185 190Ile Leu Arg Arg Arg Arg Arg
Gly Ser Gly Gly Ala Lys Cys Gly Val 195 200 205Met Asp Asp Glu Thr
Leu Pro Thr Ser Asp Ser Arg Pro Glu Ser Gln 210 215 220Asn Thr Arg
Asn Gln Trp Pro Val Arg Asp Pro Thr Pro Gln Asp Ala225 230 235
240Gly Lys Pro Ser Gly Pro Gly Ser Ile Arg Lys Lys Arg Phe Val Ser
245 250 255Ser Pro Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser
Met Ala 260 265 270Asp Phe His Gly Ser Gly Leu Lys His Tyr Leu Leu
Thr Leu Phe Ser 275 280 285Val Ala Ala Arg Phe Tyr Lys His Pro Ser
Ile Arg Asn Ser Ile Ser 290 295 300Leu Val Val Val Lys Ile Leu Val
Ile Tyr Glu Glu Gln Lys Gly Pro305 310 315 320Glu Val Thr Ser Asn
Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp 325 330 335Gln Lys Gln
His Asn Ser Pro Ser Asp Arg Asp Pro Glu His Tyr Asp 340 345 350Thr
Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser His Thr Cys 355 360
365Asp Thr Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg
370 375 380Ser Cys Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe
Thr Thr385 390 395 400Ala His Glu Leu Gly His Val Phe Asn Met Pro
His Asp Asp Ala Lys 405 410 415His Cys Ala Ser Leu Asn Gly Val Thr
Gly Asp Ser His Leu Met Ala 420 425 430Ser Met Leu Ser Ser Leu Asp
His Ser Gln Pro Trp Ser Pro Cys Ser 435 440 445Ala Tyr Met Val Thr
Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu 450 455 460Met Asp Lys
Pro Gln Asn Pro Ile Lys Leu Pro Ser Asp Leu Pro Gly465 470 475
480Thr Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Glu
485 490 495Ser Lys His Cys Pro Asp Ala Ala Ser Thr Cys Thr Thr Leu
Trp Cys 500 505 510Thr Gly Thr Ser Gly Gly Leu Leu Val Cys Gln Thr
Lys His Phe Pro 515 520 525Trp Ala Asp Gly Thr Ser Cys Gly Glu Gly
Lys Trp Cys Val Ser Gly 530 535 540Lys Cys Val Asn Lys Thr Asp Met
Lys His Phe Ala Thr Pro Val His545 550 555 560Gly Ser Trp Gly Pro
Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys 565 570 575Gly Gly Gly
Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro 580 585 590Lys
Asn Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser 595 600
605Cys Asn Ile Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe Arg Glu
610 615 620Glu Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe
Gly Asn625 630 635 640Glu Pro Thr Val Glu Trp Thr Pro Lys Tyr Ala
Gly Val Ser Pro Lys 645 650 655Asp Arg Cys Lys Leu Thr Cys Glu Ala
Lys Gly Ile Gly Tyr Phe Phe 660 665 670Val Leu Gln Pro Lys Val Val
Asp Gly Thr Pro Cys Ser Pro Asp Ser 675 680 685Thr Ser Val Cys Val
Gln Gly Gln Cys Val Lys Ala Gly Cys Asp Arg 690 695 700Ile Ile Asp
Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly705 710 715
720Asn Gly Ser Thr Cys Lys Lys Met Ser Gly Ile Val Thr Ser Thr Arg
725 730 735Pro Gly Tyr His Asp Ile Val Thr Ile Pro Ala Gly Ala Thr
Asn Ile 740 745 750Glu Val Lys His Arg Asn Gln Arg Gly Ser Arg Asn
Asn Gly Ser Phe 755 760 765Leu Ala Ile Arg Ala Ala Asp Gly Thr Tyr
Ile Leu Asn Gly Asn Phe 770 775 780Thr Leu Ser Thr Leu Glu Gln Asp
Leu Thr Tyr Lys Gly Thr Val Leu785 790 795 800Arg Tyr Ser Gly Ser
Ser Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser 805 810 815Pro Leu Lys
Glu Pro Leu Thr Ile Gln Val Leu Met Val Gly His Ala 820 825 830Leu
Arg Pro Lys Ile Lys Phe Thr Tyr Phe Met Lys Lys Lys Thr Glu 835 840
845Ser Phe Asn Ala Ile Pro Thr Phe Ser Glu Trp Val Ile Glu Glu Trp
850 855 860Gly Glu Cys Ser Lys Thr Cys Gly Ser Gly Trp Gln Arg Arg
Val Val865 870 875 880Gln Cys Arg Asp Ile Asn Gly His Pro Ala Ser
Glu Cys Ala Lys Glu 885 890 895Val Lys Pro Ala Ser Thr Arg Pro Cys
Ala Asp Leu Pro Cys Pro His 900 905 910Trp Gln Val Gly Asp Trp Ser
Pro Cys Ser Lys Thr Cys Gly Lys Gly 915 920 925Tyr Lys Lys Arg Thr
Leu Lys Cys Val Ser His Asp Gly Gly Val Leu 930 935 940Ser Asn Glu
Ser Cys Asp Pro Leu Lys Lys Pro Lys His Tyr Ile Asp945 950 955
960Phe Cys Thr Leu Thr Gln Cys Ser 96522910DNAMus musculus
2gccatgcagc caaaagtccc tttggggtca cgcaagcaga agccctgctc cgacatgggg
60gacgtccagc gggcagcgag atctcggggc tctctgtccg cacacatgct gttgctgctc
120ctcgcttcca taacaatgct gctatgtgcg cggggcgcac acgggcgccc
cacggaggaa 180gatgaggagc tggtcctgcc ctcgctggag cgcgccccgg
gccacgattc caccaccaca 240cgccttcgtc tggacgcctt tggccagcag
ctacatctga agttgcagcc ggacagcggt 300ttcttggcgc ctggcttcac
cctgcagact gtggggcgca gtcccgggtc cgaggcacaa 360catctggacc
ccaccgggga cctggctcac tgcttctact ctggcacggt gaacggtgat
420cccggctctg ccgcagccct cagcctctgt gaaggtgtgc gtggtgcctt
ctacctacaa 480ggagaggagt tcttcattca gccagcgcct ggagtggcca
ccgagcgcct ggcccctgcc 540gtgcccgagg aggagtcatc cgcacggccg
cagttccaca tcctgaggcg aaggcggcgg 600ggcagtggcg gcgccaagtg
cggcgtcatg gacgacgaga ccctgccaac cagcgactcg 660cgacccgaga
gccagaacac ccggaaccag tggcctgtgc gggaccccac gcctcaggac
720gcgggaaagc catcaggacc aggaagcata aggaagaagc gatttgtgtc
cagcccccgt 780tatgtggaaa ccatgctcgt ggctgaccag tccatggccg
acttccacgg cagcggtcta 840aagcattacc ttctaaccct gttctcggtg
gcagccaggt tttacaagca tcccagcatt 900aggaattcaa ttagcctggt
ggtggtgaag atcttggtca tatatgagga gcagaaggga 960ccagaagtta
cctccaatgc agctctcacc cttcggaatt tctgcaactg gcagaaacaa
1020cacaacagcc ccagtgaccg ggatccagag cactatgaca ctgcaattct
gttcaccaga 1080caggatttat gtggctccca cacgtgtgac actctcggga
tggcagatgt tggaactgta 1140tgtgacccca gcaggagctg ctcagtcata
gaagatgatg gtttgcaagc cgccttcacc 1200acagcccacg aattgggcca
tgtgtttaac atgccgcacg atgatgctaa gcactgtgcc 1260agcttgaatg
gtgtgactgg cgattctcat ctgatggcct cgatgctctc cagcttagac
1320catagccagc cctggtcacc ttgcagtgcc tacatggtca cgtccttcct
agataatgga 1380cacggggaat gtttgatgga caagccccag aatccaatca
agctcccttc tgatcttccc 1440ggtaccttgt acgatgccaa ccgccagtgt
cagtttacat tcggagagga atccaagcac 1500tgccctgatg cagccagcac
atgtactacc ctgtggtgca ctggcacctc cggtggctta 1560ctggtgtgcc
aaacaaaaca cttcccttgg gcagatggca ccagctgtgg agaagggaag
1620tggtgtgtca gtggcaagtg cgtgaacaag acagacatga agcattttgc
tactcctgtt 1680catggaagct ggggaccatg gggaccgtgg ggagactgct
caagaacctg tggtggtgga 1740gttcaataca caatgagaga atgtgacaac
ccagtcccaa agaacggagg gaagtactgt 1800gaaggcaaac gagtccgcta
caggtcctgt aacatcgagg actgtccaga caataacgga 1860aaaacgttca
gagaggagca gtgcgaggcg cacaatgagt tttccaaagc ttcctttggg
1920aatgagccca ctgtagagtg gacacccaag tacgccggcg tctcgccaaa
ggacaggtgc 1980aagctcacct gtgaagccaa aggcattggc tactttttcg
tcttacagcc caaggttgta 2040gatggcactc cctgtagtcc agactctacc
tctgtctgtg tgcaagggca gtgtgtgaaa 2100gctggctgtg atcgcatcat
agactccaaa aagaagtttg ataagtgtgg cgtttgtgga 2160ggaaacggtt
ccacatgcaa gaagatgtca ggaatagtca ctagtacaag acctgggtat
2220catgacattg tcacaattcc tgctggagcc accaacattg aagtgaaaca
tcggaatcaa 2280agggggtcca gaaacaatgg cagctttctg gctattagag
ccgctgatgg tacctatatt 2340ctgaatggaa acttcactct gtccacacta
gagcaagacc tcacctacaa aggtactgtc 2400ttaaggtaca gtggttcctc
ggctgcgctg gagagaatcc gcagctttag tccactcaaa 2460gaacccttaa
ccatccaggt tcttatggta ggccatgctc tccgacccaa aattaaattc
2520acctacttta tgaagaagaa gacagagtca ttcaacgcca ttcccacatt
ttctgagtgg 2580gtgattgaag agtgggggga gtgctccaag acatgcggct
caggttggca gagaagagta 2640gtgcagtgca gagacattaa tggacaccct
gcttccgaat gtgcaaagga agtgaagcca 2700gccagtacca gaccttgtgc
agaccttcct tgcccacact ggcaggtggg ggattggtca 2760ccatgttcca
aaacttgcgg gaagggttac aagaagagaa ccttgaaatg tgtgtcccac
2820gatgggggcg tgttatcaaa tgagagctgt gatcctttga agaagccaaa
gcattacatt 2880gacttttgca cactgacaca gtgcagttaa 29103967PRTHomo
sapiens 3Met Gln Arg Ala Val Pro Glu Gly Phe Gly Arg Arg Lys Leu
Gly Ser1 5 10 15Asp Met Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser
Phe Gly Pro 20 25 30Val Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu
Ala Val Ser Asp 35 40 45Ala Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu
Leu Val Val Pro Glu 50 55 60Leu Glu Arg Ala Pro Gly His Gly Thr Thr
Arg Leu Arg Leu His Ala65 70 75 80Phe Asp Gln Gln Leu Asp Leu Glu
Leu Arg Pro Asp Ser Ser Phe Leu 85 90 95Ala Pro Gly Phe Thr Leu Gln
Asn Val Gly Arg Lys Ser Gly Ser Glu 100 105 110Thr Pro Leu Pro Glu
Thr Asp Leu Ala His Cys Phe Tyr Ser Gly Thr 115 120 125Val Asn Gly
Asp Pro Ser Ser Ala Ala Ala Leu Ser Leu Cys Glu Gly 130 135 140Val
Arg Gly Ala Phe Tyr Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro145 150
155 160Leu Pro Ala Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu
Lys 165 170 175Pro Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg Asn
Arg Gln Gly 180 185 190Asp Val Gly Gly Thr Cys Gly Val Val Asp Asp
Glu Pro Arg Pro Thr 195 200 205Gly Lys Ala Glu Thr Glu Asp Glu Asp
Glu Gly Thr Glu Gly Glu Asp 210 215 220Glu Gly Ala Gln Trp Ser Pro
Gln Asp Pro Ala Leu Gln Gly Val Gly225 230 235 240Gln Pro Thr Gly
Thr Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser 245 250 255His Arg
Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Glu 260 265
270Phe His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val
275 280 285Ala Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val
Ser Leu 290 295 300Val Val Val Lys Ile Leu Val Ile His Asp Glu Gln
Lys Gly Pro Glu305 310 315 320Val Thr Ser Asn Ala Ala Leu Thr Leu
Arg Asn Phe Cys Asn Trp Gln 325 330 335Lys Gln His Asn Pro Pro Ser
Asp Arg Asp Ala Glu His Tyr Asp Thr 340 345 350Ala Ile Leu Phe Thr
Arg Gln Asp Leu Cys Gly Ser Gln Thr Cys Asp 355 360 365Thr Leu Gly
Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser 370 375 380Cys
Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala385 390
395 400His Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys
Gln 405 410 415Cys Ala Ser Leu Asn Gly Val Asn Gln Asp Ser His Met
Met Ala Ser 420 425 430Met Leu Ser Asn Leu Asp His Ser Gln Pro Trp
Ser Pro Cys Ser Ala 435 440 445Tyr Met Ile Thr Ser Phe Leu Asp Asn
Gly His Gly Glu Cys Leu Met 450 455 460Asp Lys Pro Gln Asn Pro Ile
Gln Leu Pro Gly Asp Leu Pro Gly Thr465 470 475 480Ser Tyr Asp Ala
Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Asp Ser 485 490 495Lys His
Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys Thr 500 505
510Gly Thr Ser Gly Gly Val Leu Val Cys Gln Thr Lys His Phe Pro Trp
515 520 525Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn
Gly Lys 530 535 540Cys Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr
Pro Phe His Gly545 550 555 560Ser Trp Gly Met Trp Gly Pro Trp Gly
Asp Cys Ser Arg Thr Cys Gly 565 570 575Gly Gly Val Gln Tyr Thr Met
Arg Glu Cys Asp Asn Pro Val Pro Lys 580 585 590Asn Gly Gly Lys Tyr
Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys 595 600 605Asn Leu Glu
Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe Arg Glu Glu 610 615 620Gln
Cys Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe Gly Ser Gly625 630
635 640Pro Ala Val Glu Trp Ile Pro Lys Tyr Ala Gly Val Ser Pro Lys
Asp 645 650 655Arg Cys Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly Tyr
Phe Phe Val 660 665 670Leu Gln Pro Lys Val Val Asp Gly Thr Pro Cys
Ser Pro Asp Ser Thr 675 680 685Ser Val Cys Val Gln Gly Gln Cys Val
Lys Ala Gly Cys Asp Arg Ile 690 695 700Ile Asp Ser Lys Lys Lys Phe
Asp Lys Cys Gly Val Cys Gly Gly Asn705 710 715 720Gly Ser Thr Cys
Lys Lys Ile Ser Gly Ser Val Thr Ser Ala Lys Pro 725 730 735Gly Tyr
His Asp Ile Ile Thr Ile Pro Thr Gly Ala Thr Asn Ile Glu 740 745
750Val Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn Asn Gly Ser Phe Leu
755 760 765Ala Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu Asn Gly Asp
Tyr Thr 770 775 780Leu Ser Thr Leu Glu Gln Asp Ile Met Tyr Lys Gly
Val Val Leu Arg785 790 795 800Tyr Ser Gly Ser Ser Ala Ala Leu Glu
Arg Ile Arg Ser Phe Ser Pro 805 810 815Leu Lys Glu Pro Leu Thr Ile
Gln Val Leu Thr Val Gly Asn Ala Leu 820 825 830Arg Pro Lys Ile Lys
Tyr Thr Tyr Phe Val Lys Lys Lys Lys Glu Ser 835 840 845Phe Asn Ala
Ile Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp Gly 850 855 860Glu
Cys Ser Lys Ser Cys Glu Leu Gly Trp Gln Arg Arg Leu Val Glu865 870
875 880Cys Arg Asp Ile Asn Gly Gln Pro Ala Ser Glu Cys Ala Lys Glu
Val 885 890 895Lys Pro Ala Ser Thr Arg Pro Cys Ala Asp His Pro Cys
Pro Gln Trp 900 905 910Gln Leu Gly Glu Trp Ser Ser Cys Ser Lys Thr
Cys Gly Lys Gly Tyr 915 920 925Lys Lys Arg Ser Leu Lys Cys Leu Ser
His Asp Gly Gly Val Leu Ser 930 935 940His Glu Ser Cys Asp Pro Leu
Lys Lys Pro Lys His Phe Ile Asp Phe945 950 955 960Cys Thr Met Ala
Glu Cys Ser 96542907DNAHomo sapiens 4gcaatgcagc gagctgtgcc
cgaggggttc ggaaggcgca agctgggcag cgacatgggg 60aacgcggagc gggctccggg
gtctcggagc tttgggcccg tacccacgct gctgctgctc 120gccgcggcgc
tactggccgt gtcggacgca ctcgggcgcc cctccgagga ggacgaggag
180ctagtggtgc cggagctgga gcgcgccccg ggacacggga ccacgcgcct
ccgcctgcac 240gcctttgacc agcagctgga tctggagctg cggcccgaca
gcagcttttt ggcgcccggc 300ttcacgctcc agaacgtggg gcgcaaatcc
gggtccgaga cgccgcttcc ggaaaccgac 360ctggcgcact gcttctactc
cggcaccgtg aatggcgatc ccagctcggc tgccgccctc 420agcctctgcg
agggcgtgcg cggcgccttc tacctgctgg gggaggcgta tttcatccag
480ccgctgcccg ccgccagcga gcgcctcgcc accgccgccc caggggagaa
gccgccggca 540ccactacagt tccacctcct gcggcggaat cggcagggcg
acgtcggcgg cacgtgcggg 600gtcgtggacg acgagccccg gccgactggg
aaagcggaga ccgaagacga ggacgaaggg 660actgagggcg aggacgaagg
ggctcagtgg tcgccgcagg acccggcact gcaaggcgta 720ggacagccca
caggaactgg aagcataaga aagaagcgat ttgtgtccag tcaccgctat
780gtggaaacca tgcttgtggc agaccagtcg atggcagaat tccacggcag
tggtctaaag 840cattaccttc tcacgttgtt ttcggtggca gccagattgt
acaaacaccc cagcattcgt 900aattcagtta gcctggtggt ggtgaagatc
ttggtcatcc acgatgaaca gaaggggccg 960gaagtgacct ccaatgctgc
cctcactctg cggaactttt gcaactggca gaagcagcac 1020aacccaccca
gtgaccggga tgcagagcac tatgacacag caattctttt caccagacag
1080gacttgtgtg ggtcccagac atgtgatact cttgggatgg ctgatgttgg
aactgtgtgt 1140gatccgagca gaagctgctc cgtcatagaa gatgatggtt
tacaagctgc cttcaccaca 1200gcccatgaat taggccacgt gtttaacatg
ccacatgatg atgcaaagca gtgtgccagc 1260cttaatggtg tgaaccagga
ttcccacatg atggcgtcaa tgctttccaa cctggaccac 1320agccagcctt
ggtctccttg cagtgcctac atgattacat catttctgga taatggtcat
1380ggggaatgtt tgatggacaa gcctcagaat cccatacagc tcccaggcga
tctccctggc 1440acctcgtacg atgccaaccg gcagtgccag tttacatttg
gggaggactc caaacactgc 1500cccgatgcag ccagcacatg tagcaccttg
tggtgtaccg gcacctctgg tggggtgctg 1560gtgtgtcaaa ccaaacactt
cccgtgggcg gatggcacca gctgtggaga agggaaatgg 1620tgtatcaacg
gcaagtgtgt gaacaaaacc gacagaaagc attttgatac gccttttcat
1680ggaagctggg gaatgtgggg gccttgggga gactgttcga gaacgtgcgg
tggaggagtc 1740cagtacacga tgagggaatg tgacaaccca gtcccaaaga
atggagggaa gtactgtgaa 1800ggcaaacgag tgcgctacag atcctgtaac
cttgaggact gtccagacaa taatggaaaa 1860acctttagag aggaacaatg
tgaagcacac aacgagtttt caaaagcttc ctttgggagt 1920gggcctgcgg
tggaatggat tcccaagtac gctggcgtct caccaaagga caggtgcaag
1980ctcatctgcc aagccaaagg cattggctac ttcttcgttt tgcagcccaa
ggttgtagat 2040ggtactccat gtagcccaga ttccacctct gtctgtgtgc
aaggacagtg tgtaaaagct 2100ggttgtgatc gcatcataga ctccaaaaag
aagtttgata aatgtggtgt ttgcggggga 2160aatggatcta cttgtaaaaa
aatatcagga tcagttacta gtgcaaaacc tggatatcat 2220gatatcatca
caattccaac tggagccacc aacatcgaag tgaaacagcg gaaccagagg
2280ggatccagga acaatggcag ctttcttgcc atcaaagctg ctgatggcac
atatattctt 2340aatggtgact acactttgtc caccttagag caagacatta
tgtacaaagg tgttgtcttg 2400aggtacagcg gctcctctgc ggcattggaa
agaattcgca gctttagccc tctcaaagag 2460cccttgacca tccaggttct
tactgtgggc aatgcccttc gacctaaaat taaatacacc 2520tacttcgtaa
agaagaagaa ggaatctttc aatgctatcc ccactttttc agcatgggtc
2580attgaagagt ggggcgaatg ttctaagtca tgtgaattgg gttggcagag
aagactggta 2640gaatgccgag acattaatgg acagcctgct tccgagtgtg
caaaggaagt gaagccagcc 2700agcaccagac cttgtgcaga ccatccctgc
ccccagtggc agctggggga gtggtcatca 2760tgttctaaga cctgtgggaa
gggttacaaa aaaagaagct tgaagtgtct gtcccatgat 2820ggaggggtgt
tatctcatga gagctgtgat cctttaaaga aacctaaaca tttcatagac
2880ttttgcacaa tggcagaatg cagttaa 29075166PRTMus musculus 5Met Gly
Asp Val Gln Arg Ala Ala Arg Ser Arg Gly Ser Leu Ser Ala1 5 10 15His
Met Leu Leu Leu Leu Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20 25
30Arg Gly Ala His Gly Arg Pro Thr Glu Lys Lys Lys Thr Glu Ser Phe
35 40 45Asn Ala Ile Pro Thr Phe Ser Glu Trp Val Ile Glu Glu Trp Gly
Glu 50 55 60Cys Ser Lys Thr Cys Gly Ser Gly Trp Gln Arg Arg Val Val
Gln Cys65 70 75 80Arg Asp Ile Asn Gly His Pro Ala Ser Glu Cys Ala
Lys Glu Val Lys 85 90 95Pro Ala Ser Thr Arg Pro Cys Ala Asp Leu Pro
Cys Pro His Trp Gln 100 105 110Val Gly Asp Trp Ser Pro Cys Ser Lys
Thr Cys Gly Lys Gly Tyr Lys 115 120 125Lys Arg Thr Leu Lys Cys Val
Ser His Asp Gly Gly Val Leu Ser Asn 130 135 140Glu Ser Cys Asp Pro
Leu Lys Lys Pro Lys His Tyr Ile Asp Phe Cys145 150 155 160Thr Leu
Thr Gln Cys Ser 1656504DNAMus musculus 6gacatggggg acgtccagcg
ggcagcgaga tctcggggct ctctgtccgc acacatgctg 60ttgctgctcc tcgcttccat
aacaatgctg ctatgtgcgc ggggcgcaca cgggcgcccc 120acggagaaga
agaagacaga gtcattcaac gccattccca cattttctga gtgggtgatt
180gaagagtggg gggagtgctc caagacatgc ggctcaggtt ggcagagaag
agtagtgcag 240tgcagagaca ttaatggaca ccctgcttcc gaatgtgcaa
aggaagtgaa gccagccagt 300accagacctt gtgcagacct tccttgccca
cactggcagg tgggggattg gtcaccatgt 360tccaaaactt gcgggaaggg
ttacaagaag agaaccttga aatgtgtgtc ccacgatggg 420ggcgtgttat
caaatgagag ctgtgatcct ttgaagaagc caaagcatta cattgacttt
480tgcacactga cacagtgcag ttaa 5047179PRTHomo sapiens 7Met Gln Arg
Ala Val Pro Glu Gly Phe Gly Arg Arg Lys Leu Gly Ser1 5 10 15Asp Met
Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro 20 25 30Val
Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp 35 40
45Ala Leu Gly Arg Pro Ser Glu Lys Lys Lys Glu Ser Phe Asn Ala Ile
50 55 60Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp Gly Glu Cys Ser
Lys65 70 75 80Ser Cys Glu Leu Gly Trp Gln Arg Arg Leu Val Glu Cys
Arg Asp Ile 85 90 95Asn Gly Gln Pro Ala Ser Glu Cys Ala Lys Glu Val
Lys Pro Ala Ser 100 105 110Thr Arg Pro Cys Ala Asp His Pro Cys Pro
Gln Trp Gln Leu Gly Glu 115 120 125Trp Ser Ser Cys Ser Lys Thr Cys
Gly Lys Gly Tyr Lys Lys Arg Ser 130 135 140Leu Lys Cys Leu Ser His
Asp Gly Gly Val Leu Ser His Glu Ser Cys145 150 155 160Asp Pro Leu
Lys Lys Pro Lys His Phe Ile Asp Phe Cys Thr Met Ala 165 170 175Glu
Cys Ser8543DNAHomo sapiens 8gcaatgcagc gagctgtgcc cgaggggttc
ggaaggcgca agctgggcag cgacatgggg 60aacgcggagc gggctccggg gtctcggagc
tttgggcccg tacccacgct gctgctgctc 120gccgcggcgc tactggccgt
gtcggacgca ctcgggcgcc cctccgagaa gaagaaggaa 180tctttcaatg
ctatccccac tttttcagca tgggtcattg aagagtgggg cgaatgttct
240aagtcatgtg aattgggttg gcagagaaga ctggtagaat gccgagacat
taatggacag 300cctgcttccg agtgtgcaaa ggaagtgaag ccagccagca
ccagaccttg tgcagaccat 360ccctgccccc agtggcagct gggggagtgg
tcatcatgtt ctaagacctg tgggaagggt 420tacaaaaaaa gaagcttgaa
gtgtctgtcc catgatggag gggtgttatc tcatgagagc 480tgtgatcctt
taaagaaacc taaacatttc atagactttt gcacaatggc agaatgcagt 540taa
5439596PRTMus musculus 9Met Gly Asp Val Gln Arg Ala Ala Arg Ser Arg
Gly Ser Leu Ser Ala1 5 10 15His Met Leu Leu Leu Leu Leu Ala Ser Ile
Thr Met Leu Leu Cys Ala 20 25 30Arg Gly Ala His Gly Arg Pro Thr Glu
Glu Asp Glu Glu Leu Val Leu 35 40 45Pro Ser Leu Glu Arg Ala Pro Gly
His Asp Ser Thr Thr Thr Arg Leu 50 55 60Arg Leu Asp Ala Phe Gly Gln
Gln Leu His Leu Lys Leu Gln Pro Asp65 70 75 80Ser Gly Phe Leu Ala
Pro Gly Phe Thr Leu Gln Thr Val Gly Arg Ser 85 90 95Pro Gly Ser Glu
Ala Gln His Leu Asp Pro Thr Gly Asp Leu Ala His 100 105 110Cys Phe
Tyr Ser Gly Thr Val Asn Gly Asp Pro Gly Ser Ala Ala Ala 115 120
125Leu Ser Leu Cys Glu Gly Val Arg Gly Ala Phe Tyr Leu Gln Gly Glu
130 135 140Glu Phe Phe Ile Gln Pro Ala Pro Gly Val Ala Thr Glu Arg
Leu Ala145 150 155 160Pro Ala Val Pro Glu Glu Glu Ser Ser Ala Arg
Pro Gln Phe His Ile 165 170 175Leu Arg Arg Arg Arg Arg Gly Ser Gly
Gly Ala Lys Cys Gly Val Met 180 185 190Asp Asp Glu Thr Leu Pro Thr
Ser Asp Ser Arg Pro Glu Ser Gln Asn 195 200 205Thr Arg Asn Gln Trp
Pro Val Arg Asp Pro Thr Pro Gln Asp Ala Gly 210 215 220Lys Pro Ser
Gly Pro Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser225 230 235
240Pro Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Asp
245 250 255Phe His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe
Ser Val 260 265 270Ala Ala Arg Phe Tyr Lys His Pro Ser Ile Arg Asn
Ser Ile Ser Leu 275 280 285Val Val Val Lys Ile Leu Val Ile Tyr Glu
Glu Gln Lys Gly Pro Glu 290 295 300Val Thr Ser Asn Ala Ala Leu Thr
Leu Arg Asn Phe Cys Asn Trp Gln305 310 315 320Lys Gln His Asn Ser
Pro Ser Asp Arg Asp Pro Glu His Tyr Asp Thr 325 330 335Ala Ile Leu
Phe Thr Arg Gln Asp Leu Cys Gly Ser His Thr Cys Asp 340 345 350Thr
Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser 355 360
365Cys Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala
370 375 380His Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala
Lys His385 390 395 400Cys Ala Ser Leu Asn Gly Val Thr Gly Asp Ser
His Leu Met Ala Ser 405 410 415Met Leu Ser Ser Leu Asp His Ser Gln
Pro Trp Ser Pro Cys Ser Ala 420 425 430Tyr Met Val Thr Ser Phe Leu
Asp Asn Gly His Gly Glu Cys Leu Met 435 440 445Asp Lys Pro Gln Asn
Pro Ile Lys Leu Pro Ser Asp Leu Pro Gly Thr 450 455 460Leu Tyr Asp
Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Glu Ser465 470 475
480Lys His Cys Pro Asp Ala Ala Ser Thr Cys Thr Thr Leu Trp Cys Thr
485 490 495Gly Thr Ser Gly Gly Leu Leu Val Cys Gln Thr Lys His Phe
Pro Trp 500 505 510Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys
Val Ser Gly Lys 515 520 525Cys Val Asn Lys Thr Asp Met Lys His Phe
Ala Thr Pro Val His Gly 530 535 540Ser Trp Gly Pro Trp Gly Pro Trp
Gly Asp Cys Ser Arg Thr Cys Gly545 550 555 560Gly Gly Val Gln Tyr
Thr Met Arg Glu Cys Asp Asn Pro Val Pro Lys 565 570 575Asn Gly Gly
Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys 580 585 590Asn
Ile Glu Asp 595101794DNAMus musculus 10gacatggggg acgtccagcg
ggcagcgaga tctcggggct ctctgtccgc acacatgctg 60ttgctgctcc tcgcttccat
aacaatgctg ctatgtgcgc ggggcgcaca cgggcgcccc 120acggaggaag
atgaggagct ggtcctgccc tcgctggagc gcgccccggg ccacgattcc
180accaccacac gccttcgtct ggacgccttt ggccagcagc tacatctgaa
gttgcagccg 240gacagcggtt tcttggcgcc tggcttcacc ctgcagactg
tggggcgcag tcccgggtcc 300gaggcacaac atctggaccc caccggggac
ctggctcact gcttctactc tggcacggtg 360aacggtgatc ccggctctgc
cgcagccctc agcctctgtg aaggtgtgcg tggtgccttc 420tacctacaag
gagaggagtt cttcattcag ccagcgcctg gagtggccac cgagcgcctg
480gcccctgccg tgcccgagga ggagtcatcc gcacggccgc agttccacat
cctgaggcga 540aggcggcggg gcagtggcgg cgccaagtgc ggcgtcatgg
acgacgagac cctgccaacc 600agcgactcgc gacccgagag ccagaacacc
cggaaccagt ggcctgtgcg ggaccccacg 660cctcaggacg cgggaaagcc
atcaggacca ggaagcataa ggaagaagcg atttgtgtcc 720agcccccgtt
atgtggaaac catgctcgtg gctgaccagt ccatggccga cttccacggc
780agcggtctaa agcattacct tctaaccctg ttctcggtgg cagccaggtt
ttacaagcat 840cccagcatta ggaattcaat tagcctggtg gtggtgaaga
tcttggtcat atatgaggag 900cagaagggac cagaagttac ctccaatgca
gctctcaccc ttcggaattt ctgcaactgg 960cagaaacaac acaacagccc
cagtgaccgg gatccagagc actatgacac tgcaattctg 1020ttcaccagac
aggatttatg tggctcccac acgtgtgaca ctctcgggat ggcagatgtt
1080ggaactgtat gtgaccccag caggagctgc tcagtcatag aagatgatgg
tttgcaagcc 1140gccttcacca cagcccacga attgggccat gtgtttaaca
tgccgcacga tgatgctaag 1200cactgtgcca gcttgaatgg tgtgactggc
gattctcatc tgatggcctc gatgctctcc 1260agcttagacc atagccagcc
ctggtcacct tgcagtgcct acatggtcac gtccttccta 1320gataatggac
acggggaatg tttgatggac aagccccaga atccaatcaa gctcccttct
1380gatcttcccg gtaccttgta cgatgccaac cgccagtgtc agtttacatt
cggagaggaa 1440tccaagcact gccctgatgc agccagcaca tgtactaccc
tgtggtgcac tggcacctcc 1500ggtggcttac tggtgtgcca aacaaaacac
ttcccttggg cagatggcac cagctgtgga 1560gaagggaagt ggtgtgtcag
tggcaagtgc gtgaacaaga cagacatgaa gcattttgct 1620actcctgttc
atggaagctg gggaccatgg ggaccgtggg gagactgctc aagaacctgt
1680ggtggtggag ttcaatacac aatgagagaa tgtgacaacc cagtcccaaa
gaacggaggg 1740aagtactgtg aaggcaaacg agtccgctac aggtcctgta
acatcgagga ctaa 179411595PRTHomo sapiens 11Met Gly Asn Ala Glu Arg
Ala Pro Gly Ser Arg Ser Phe Gly Pro Val1 5 10 15Pro Thr Leu Leu Leu
Leu Ala Ala Ala Leu Leu Ala Val Ser Asp Ala 20 25 30Leu Gly Arg Pro
Ser Glu Glu Asp Glu Glu Leu Val Val Pro Glu Leu 35 40 45Glu Arg Ala
Pro Gly His Gly Thr Thr Arg Leu Arg Leu His Ala Phe 50 55 60Asp Gln
Gln Leu Asp Leu Glu Leu Arg Pro Asp Ser Ser Phe Leu Ala65 70 75
80Pro Gly Phe Thr Leu Gln Asn Val Gly Arg Lys Ser Gly Ser Glu Thr
85 90 95Pro Leu Pro Glu Thr Asp Leu Ala His Cys Phe Tyr Ser Gly Thr
Val 100 105 110Asn Gly Asp Pro Ser Ser Ala Ala Ala Leu Ser Leu Cys
Glu Gly Val 115 120 125Arg Gly Ala Phe Tyr Leu Leu Gly Glu Ala Tyr
Phe Ile Gln Pro Leu 130 135 140Pro Ala Ala Ser Glu Arg Leu Ala Thr
Ala Ala Pro Gly Glu Lys Pro145 150 155 160Pro Ala Pro Leu Gln Phe
His Leu Leu Arg Arg Asn Arg Gln Gly Asp 165 170 175Val Gly Gly Thr
Cys Gly Val Val Asp Asp Glu Pro Arg Pro Thr Gly 180 185 190Lys Ala
Glu Thr Glu Asp Glu Asp Glu Gly Thr Glu Gly Glu Asp Glu 195 200
205Gly Ala Gln Trp Ser Pro Gln Asp Pro Ala Leu Gln Gly Val Gly Gln
210 215 220Pro Thr Gly Thr Gly Ser Ile Arg Lys Lys Arg Phe Val Ser
Ser His225 230 235 240Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln
Ser Met Ala Glu Phe 245 250 255His Gly Ser Gly Leu Lys His Tyr Leu
Leu Thr Leu Phe Ser Val Ala 260 265 270Ala Arg Leu Tyr Lys His Pro
Ser Ile Arg Asn Ser Val Ser Leu Val 275 280 285Val Val Lys Ile Leu
Val Ile His Asp Glu Gln Lys Gly Pro Glu Val 290 295 300Thr Ser Asn
Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp Gln Lys305 310 315
320Gln His Asn Pro Pro Ser Asp Arg Asp Ala Glu His Tyr Asp Thr Ala
325 330 335Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser Gln Thr Cys
Asp Thr 340 345 350Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro
Ser Arg Ser Cys 355 360 365Ser Val Ile Glu Asp Asp Gly Leu Gln Ala
Ala Phe Thr Thr Ala His 370 375 380Glu Leu Gly His Val Phe Asn Met
Pro His Asp Asp Ala Lys Gln Cys385 390 395 400Ala Ser Leu Asn Gly
Val Asn Gln Asp Ser His Met Met Ala Ser Met 405 410 415Leu Ser Asn
Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala Tyr 420 425 430Met
Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met Asp 435 440
445Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp Leu Pro Gly Thr Ser
450 455 460Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Asp
Ser Lys465 470 475 480His Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr
Leu Trp Cys Thr Gly 485 490 495Thr Ser Gly Gly Val Leu Val Cys Gln
Thr Lys His Phe Pro Trp Ala 500 505 510Asp Gly Thr Ser Cys Gly Glu
Gly Lys Trp Cys Ile Asn Gly Lys Cys 515 520 525Val Asn Lys Thr Asp
Arg Lys His Phe Asp Thr Pro Phe His Gly Ser 530 535 540Trp Gly Met
Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly545 550 555
560Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro Lys Asn
565 570 575Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser
Cys Asn 580 585 590Leu Glu Asp
595121842DNAHomo sapiens 12gcaatgcagc gagctgtgcc cgaggggttc
ggaaggcgca agctgggcag cgacatgggg 60aacgcggagc gggctccggg gtctcggagc
tttgggcccg tacccacgct gctgctgctc 120gccgcggcgc tactggccgt
gtcggacgca ctcgggcgcc cctccgagga ggacgaggag 180ctagtggtgc
cggagctgga gcgcgccccg ggacacggga ccacgcgcct ccgcctgcac
240gcctttgacc agcagctgga tctggagctg cggcccgaca gcagcttttt
ggcgcccggc 300ttcacgctcc agaacgtggg gcgcaaatcc gggtccgaga
cgccgcttcc ggaaaccgac 360ctggcgcact gcttctactc cggcaccgtg
aatggcgatc ccagctcggc tgccgccctc 420agcctctgcg agggcgtgcg
cggcgccttc tacctgctgg gggaggcgta tttcatccag 480ccgctgcccg
ccgccagcga gcgcctcgcc accgccgccc caggggagaa gccgccggca
540ccactacagt tccacctcct gcggcggaat cggcagggcg acgtcggcgg
cacgtgcggg 600gtcgtggacg acgagccccg gccgactggg aaagcggaga
ccgaagacga ggacgaaggg 660actgagggcg aggacgaagg ggctcagtgg
tcgccgcagg acccggcact gcaaggcgta 720ggacagccca caggaactgg
aagcataaga aagaagcgat ttgtgtccag tcaccgctat 780gtggaaacca
tgcttgtggc agaccagtcg atggcagaat tccacggcag tggtctaaag
840cattaccttc tcacgttgtt ttcggtggca gccagattgt acaaacaccc
cagcattcgt 900aattcagtta gcctggtggt ggtgaagatc ttggtcatcc
acgatgaaca gaaggggccg 960gaagtgacct ccaatgctgc cctcactctg
cggaactttt gcaactggca gaagcagcac 1020aacccaccca gtgaccggga
tgcagagcac tatgacacag caattctttt caccagacag 1080gacttgtgtg
ggtcccagac atgtgatact cttgggatgg ctgatgttgg aactgtgtgt
1140gatccgagca gaagctgctc cgtcatagaa gatgatggtt tacaagctgc
cttcaccaca 1200gcccatgaat taggccacgt gtttaacatg ccacatgatg
atgcaaagca gtgtgccagc 1260cttaatggtg tgaaccagga ttcccacatg
atggcgtcaa tgctttccaa cctggaccac 1320agccagcctt ggtctccttg
cagtgcctac atgattacat catttctgga taatggtcat 1380ggggaatgtt
tgatggacaa gcctcagaat cccatacagc tcccaggcga tctccctggc
1440acctcgtacg atgccaaccg gcagtgccag tttacatttg gggaggactc
caaacactgc 1500cccgatgcag ccagcacatg tagcaccttg tggtgtaccg
gcacctctgg tggggtgctg 1560gtgtgtcaaa ccaaacactt cccgtgggcg
gatggcacca gctgtggaga agggaaatgg 1620tgtatcaacg gcaagtgtgt
gaacaaaacc gacagaaagc attttgatac gccttttcat 1680ggaagctggg
gaatgtgggg gccttgggga gactgttcga gaacgtgcgg tggaggagtc
1740cagtacacga tgagggaatg tgacaaccca gtcccaaaga atggagggaa
gtactgtgaa 1800ggcaaacgag tgcgctacag atcctgtaac cttgaggact aa
184213545PRTMus musculus 13Met Gly Asp Val Gln Arg Ala Ala Arg Ser
Arg Gly Ser Leu Ser Ala1 5 10 15His Met Leu Leu Leu Leu Leu Ala Ser
Ile Thr Met Leu Leu Cys Ala 20 25 30Arg Gly Ala His Gly Arg Pro Thr
Glu Glu Asp Glu Glu Leu Val Leu 35 40 45Pro Ser Leu Glu Arg Ala Pro
Gly His Asp Ser Thr Thr Thr Arg Leu 50 55 60Arg Leu Asp Ala Phe Gly
Gln Gln Leu His Leu Lys Leu Gln Pro Asp65 70 75 80Ser Gly Phe Leu
Ala Pro Gly Phe Thr Leu Gln Thr Val Gly Arg Ser 85 90 95Pro Gly Ser
Glu Ala Gln His Leu Asp Pro Thr Gly Asp Leu Ala His 100 105 110Cys
Phe Tyr Ser Gly Thr Val Asn Gly Asp Pro Gly Ser Ala Ala Ala 115 120
125Leu Ser Leu Cys Glu Gly Val Arg Gly Ala Phe Tyr Leu Gln Gly Glu
130 135 140Glu Phe Phe Ile Gln Pro Ala Pro Gly Val Ala Thr Glu Arg
Leu Ala145 150 155 160Pro Ala Val Pro Glu Glu Glu Ser Ser Ala Arg
Pro Gln Phe His Ile 165 170 175Leu Arg Arg Arg Arg Arg Gly Ser Gly
Gly Ala Lys Cys Gly Val Met 180 185 190Asp Asp Glu Thr Leu Pro Thr
Ser Asp Ser Arg Pro Glu Ser Gln Asn 195 200 205Thr Arg Asn Gln Trp
Pro Val Arg Asp Pro Thr Pro Gln Asp Ala Gly 210 215 220Lys Pro Ser
Gly Pro Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser225 230 235
240Pro Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Asp
245 250 255Phe His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe
Ser Val 260 265 270Ala Ala Arg Phe Tyr Lys His Pro Ser Ile Arg Asn
Ser Ile Ser Leu 275 280 285Val Val Val Lys Ile Leu Val Ile Tyr Glu
Glu Gln Lys Gly Pro Glu 290 295 300Val Thr Ser Asn Ala Ala Leu Thr
Leu Arg Asn Phe Cys Asn Trp Gln305 310 315 320Lys Gln His Asn Ser
Pro Ser Asp Arg Asp Pro Glu His Tyr Asp Thr 325 330 335Ala Ile Leu
Phe Thr Arg Gln Asp Leu Cys Gly Ser His Thr Cys Asp 340 345 350Thr
Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser 355 360
365Cys Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala
370 375 380His Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala
Lys His385 390 395 400Cys Ala Ser Leu Asn Gly Val Thr Gly Asp Ser
His Leu Met Ala Ser 405 410 415Met Leu Ser Ser Leu Asp His Ser Gln
Pro Trp Ser Pro Cys Ser Ala 420 425 430Tyr Met Val Thr Ser Phe Leu
Asp Asn Gly His Gly Glu Cys Leu Met 435 440 445Asp Lys Pro Gln Asn
Pro Ile Lys Leu Pro Ser Asp Leu Pro Gly Thr 450 455 460Leu Tyr Asp
Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Glu Ser465 470 475
480Lys His Cys Pro Asp Ala Ala Ser Thr Cys Thr Thr Leu Trp Cys Thr
485 490 495Gly Thr Ser Gly Gly Leu Leu Val Cys Gln Thr Lys His Phe
Pro Trp 500 505 510Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys
Val Ser Gly Lys 515 520 525Cys Val Asn Lys Thr Asp Met Lys His Phe
Ala Thr Pro Val His Gly 530 535 540Ser545141641DNAMus musculus
14gacatggggg acgtccagcg ggcagcgaga tctcggggct ctctgtccgc acacatgctg
60ttgctgctcc tcgcttccat aacaatgctg ctatgtgcgc ggggcgcaca cgggcgcccc
120acggaggaag atgaggagct ggtcctgccc tcgctggagc gcgccccggg
ccacgattcc 180accaccacac gccttcgtct ggacgccttt ggccagcagc
tacatctgaa gttgcagccg 240gacagcggtt tcttggcgcc tggcttcacc
ctgcagactg tggggcgcag tcccgggtcc 300gaggcacaac atctggaccc
caccggggac ctggctcact gcttctactc tggcacggtg 360aacggtgatc
ccggctctgc cgcagccctc agcctctgtg aaggtgtgcg tggtgccttc
420tacctacaag gagaggagtt cttcattcag ccagcgcctg gagtggccac
cgagcgcctg 480gcccctgccg tgcccgagga ggagtcatcc gcacggccgc
agttccacat cctgaggcga 540aggcggcggg gcagtggcgg cgccaagtgc
ggcgtcatgg acgacgagac cctgccaacc 600agcgactcgc gacccgagag
ccagaacacc cggaaccagt ggcctgtgcg ggaccccacg 660cctcaggacg
cgggaaagcc atcaggacca ggaagcataa ggaagaagcg atttgtgtcc
720agcccccgtt atgtggaaac catgctcgtg gctgaccagt ccatggccga
cttccacggc 780agcggtctaa agcattacct tctaaccctg ttctcggtgg
cagccaggtt ttacaagcat 840cccagcatta ggaattcaat tagcctggtg
gtggtgaaga tcttggtcat atatgaggag 900cagaagggac cagaagttac
ctccaatgca gctctcaccc ttcggaattt ctgcaactgg 960cagaaacaac
acaacagccc cagtgaccgg gatccagagc actatgacac tgcaattctg
1020ttcaccagac aggatttatg tggctcccac acgtgtgaca ctctcgggat
ggcagatgtt 1080ggaactgtat gtgaccccag caggagctgc tcagtcatag
aagatgatgg tttgcaagcc 1140gccttcacca cagcccacga attgggccat
gtgtttaaca tgccgcacga tgatgctaag 1200cactgtgcca gcttgaatgg
tgtgactggc gattctcatc tgatggcctc gatgctctcc 1260agcttagacc
atagccagcc ctggtcacct tgcagtgcct acatggtcac gtccttccta
1320gataatggac acggggaatg tttgatggac aagccccaga atccaatcaa
gctcccttct 1380gatcttcccg gtaccttgta cgatgccaac cgccagtgtc
agtttacatt cggagaggaa 1440tccaagcact gccctgatgc agccagcaca
tgtactaccc tgtggtgcac tggcacctcc 1500ggtggcttac tggtgtgcca
aacaaaacac ttcccttggg cagatggcac cagctgtgga 1560gaagggaagt
ggtgtgtcag tggcaagtgc gtgaacaaga cagacatgaa gcattttgct
1620actcctgttc atggaagcta a 164115561PRTHomo sapiens 15Met Gln Arg
Ala Val Pro Glu Gly Phe Gly Arg Arg Lys Leu Gly Ser1 5 10 15Asp Met
Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro 20 25 30Val
Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp 35 40
45Ala Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu Leu Val Val Pro Glu
50 55 60Leu Glu Arg Ala Pro Gly His Gly Thr Thr Arg Leu Arg Leu His
Ala65 70 75 80Phe Asp Gln Gln Leu Asp Leu Glu Leu Arg Pro Asp Ser
Ser Phe Leu 85 90 95Ala Pro Gly Phe Thr Leu Gln Asn Val Gly Arg Lys
Ser Gly Ser Glu 100 105 110Thr Pro Leu Pro Glu Thr Asp Leu Ala His
Cys Phe Tyr Ser Gly Thr 115 120 125Val Asn Gly Asp Pro Ser Ser Ala
Ala Ala Leu Ser Leu Cys Glu Gly 130 135 140Val Arg Gly Ala Phe Tyr
Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro145 150 155 160Leu Pro Ala
Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu Lys 165 170 175Pro
Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg Asn Arg Gln Gly 180 185
190Asp Val Gly Gly Thr Cys Gly Val Val Asp Asp Glu Pro Arg Pro Thr
195 200 205Gly Lys Ala Glu Thr Glu Asp Glu Asp Glu Gly Thr Glu Gly
Glu Asp 210 215 220Glu Gly Ala Gln Trp Ser Pro Gln Asp Pro Ala Leu
Gln Gly Val Gly225 230 235 240Gln Pro Thr Gly Thr Gly Ser Ile Arg
Lys Lys Arg Phe Val Ser Ser 245 250 255His Arg Tyr Val Glu Thr Met
Leu Val Ala Asp Gln Ser Met Ala Glu 260 265 270Phe His Gly Ser Gly
Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val 275 280 285Ala Ala Arg
Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu 290 295 300Val
Val Val Lys Ile Leu Val Ile His Asp Glu Gln Lys Gly Pro Glu305 310
315 320Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp
Gln 325 330 335Lys Gln His Asn Pro Pro Ser Asp Arg Asp Ala Glu His
Tyr Asp Thr 340 345 350Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly
Ser Gln Thr Cys Asp 355 360 365Thr Leu Gly Met Ala Asp Val Gly Thr
Val Cys Asp Pro Ser Arg Ser 370 375 380Cys Ser Val Ile Glu Asp Asp
Gly Leu Gln Ala Ala Phe Thr Thr Ala385 390 395 400His Glu Leu Gly
His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln 405 410 415Cys Ala
Ser Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser 420 425
430Met Leu Ser Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala
435 440 445Tyr Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys
Leu Met 450 455 460Asp Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp
Leu Pro Gly Thr465 470 475 480Ser Tyr Asp Ala Asn Arg Gln Cys Gln
Phe Thr Phe Gly Glu Asp Ser 485 490 495Lys His Cys Pro Asp Ala Ala
Ser Thr Cys Ser Thr Leu Trp Cys Thr 500 505 510Gly Thr Ser Gly Gly
Val Leu Val Cys Gln Thr Lys His Phe Pro Trp 515 520 525Ala Asp Gly
Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn Gly Lys 530 535 540Cys
Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr Pro Phe His Gly545 550
555 560Ser161689DNAHomo sapiens 16gcaatgcagc gagctgtgcc cgaggggttc
ggaaggcgca agctgggcag cgacatgggg 60aacgcggagc gggctccggg gtctcggagc
tttgggcccg tacccacgct gctgctgctc 120gccgcggcgc tactggccgt
gtcggacgca ctcgggcgcc cctccgagga ggacgaggag 180ctagtggtgc
cggagctgga gcgcgccccg ggacacggga ccacgcgcct ccgcctgcac
240gcctttgacc agcagctgga tctggagctg cggcccgaca gcagcttttt
ggcgcccggc 300ttcacgctcc agaacgtggg gcgcaaatcc gggtccgaga
cgccgcttcc ggaaaccgac 360ctggcgcact gcttctactc cggcaccgtg
aatggcgatc ccagctcggc tgccgccctc 420agcctctgcg agggcgtgcg
cggcgccttc tacctgctgg gggaggcgta tttcatccag 480ccgctgcccg
ccgccagcga gcgcctcgcc accgccgccc caggggagaa gccgccggca
540ccactacagt tccacctcct gcggcggaat cggcagggcg acgtcggcgg
cacgtgcggg 600gtcgtggacg acgagccccg gccgactggg aaagcggaga
ccgaagacga ggacgaaggg 660actgagggcg aggacgaagg ggctcagtgg
tcgccgcagg acccggcact gcaaggcgta 720ggacagccca caggaactgg
aagcataaga aagaagcgat ttgtgtccag tcaccgctat 780gtggaaacca
tgcttgtggc agaccagtcg atggcagaat tccacggcag tggtctaaag
840cattaccttc tcacgttgtt ttcggtggca gccagattgt acaaacaccc
cagcattcgt 900aattcagtta gcctggtggt ggtgaagatc ttggtcatcc
acgatgaaca gaaggggccg 960gaagtgacct ccaatgctgc cctcactctg
cggaactttt gcaactggca gaagcagcac 1020aacccaccca gtgaccggga
tgcagagcac tatgacacag caattctttt caccagacag 1080gacttgtgtg
ggtcccagac atgtgatact cttgggatgg ctgatgttgg aactgtgtgt
1140gatccgagca gaagctgctc cgtcatagaa gatgatggtt tacaagctgc
cttcaccaca 1200gcccatgaat taggccacgt gtttaacatg ccacatgatg
atgcaaagca gtgtgccagc 1260cttaatggtg tgaaccagga ttcccacatg
atggcgtcaa tgctttccaa cctggaccac 1320agccagcctt ggtctccttg
cagtgcctac atgattacat catttctgga taatggtcat 1380ggggaatgtt
tgatggacaa gcctcagaat cccatacagc tcccaggcga tctccctggc
1440acctcgtacg atgccaaccg gcagtgccag tttacatttg gggaggactc
caaacactgc 1500cccgatgcag ccagcacatg tagcaccttg tggtgtaccg
gcacctctgg tggggtgctg 1560gtgtgtcaaa ccaaacactt cccgtgggcg
gatggcacca gctgtggaga agggaaatgg 1620tgtatcaacg gcaagtgtgt
gaacaaaacc gacagaaagc attttgatac gccttttcat 1680ggaagctaa
168917159PRTMus musculus 17Met Gly Asp Val Gln Arg Ala Ala Arg Ser
Arg Gly Ser Leu Ser Ala1 5 10 15His Met Leu Leu Leu Leu Leu Ala Ser
Ile Thr Met Leu Leu Cys Ala 20 25 30Arg Gly Ala His Gly Arg Pro Thr
Glu Lys Lys Met Ser Gly Ile Val 35 40 45Thr Ser Thr Arg Pro Gly Tyr
His Asp Ile Val Thr Ile Pro Ala Gly 50 55 60Ala Thr Asn Ile Glu Val
Lys His Arg Asn Gln Arg Gly Ser Arg Asn65 70 75 80Asn Gly Ser Phe
Leu Ala Ile Arg Ala Ala Asp Gly Thr Tyr Ile Leu 85 90 95Asn Gly Asn
Phe Thr Leu Ser Thr Leu Glu Gln Asp Leu Thr Tyr Lys 100 105 110Gly
Thr Val Leu Arg Tyr Ser Gly Ser Ser Ala Ala Leu Glu Arg Ile 115 120
125Arg Ser Phe Ser Pro Leu Lys Glu Pro Leu Thr Ile Gln Val Leu Met
130 135 140Val Gly His Ala Leu Arg Pro Lys Ile Lys Phe Thr Tyr Phe
Met145 150 15518483DNAMus musculus 18gacatggggg acgtccagcg
ggcagcgaga tctcggggct ctctgtccgc acacatgctg 60ttgctgctcc tcgcttccat
aacaatgctg ctatgtgcgc ggggcgcaca cgggcgcccc 120acggagaaga
agatgtcagg aatagtcact agtacaagac ctgggtatca tgacattgtc
180acaattcctg ctggagccac caacattgaa gtgaaacatc ggaatcaaag
ggggtccaga 240aacaatggca gctttctggc tattagagcc gctgatggta
cctatattct gaatggaaac 300ttcactctgt ccacactaga gcaagacctc
acctacaaag gtactgtctt aaggtacagt 360ggttcctcgg ctgcgctgga
gagaatccgc agctttagtc cactcaaaga acccttaacc 420atccaggttc
ttatggtagg ccatgctctc cgacccaaaa ttaaattcac ctactttatg 480taa
48319173PRTHomo sapiens 19Met Gln Arg Ala Val Pro Glu Gly Phe Gly
Arg Arg Lys Leu Gly Ser1 5 10 15Asp Met Gly Asn Ala Glu Arg Ala Pro
Gly Ser Arg Ser Phe Gly Pro 20 25 30Val Pro Thr Leu Leu Leu Leu Ala
Ala Ala Leu Leu Ala Val Ser Asp 35 40 45Ala Leu Gly Arg Pro Ser Glu
Lys Lys Ile Ser Gly Ser Val Thr Ser 50 55 60Ala Lys Pro Gly Tyr His
Asp Ile Ile Thr Ile Pro Thr Gly Ala Thr65 70 75 80Asn Ile Glu Val
Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn Asn Gly 85 90 95Ser Phe Leu
Ala Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu Asn Gly 100 105 110Asp
Tyr Thr Leu Ser Thr Leu Glu Gln Asp Ile Met Tyr Lys Gly Val 115 120
125Val Leu Arg Tyr Ser Gly Ser Ser Ala Ala Leu Glu Arg Ile Arg Ser
130 135 140Phe Ser Pro Leu Lys Glu Pro Leu Thr Ile Gln Val Leu Thr
Val Gly145 150 155 160Asn Ala Leu Arg Pro Lys Ile Lys Tyr Thr Tyr
Phe Val 165 17020525DNAHomo sapiens 20gcaatgcagc gagctgtgcc
cgaggggttc ggaaggcgca agctgggcag cgacatgggg 60aacgcggagc gggctccggg
gtctcggagc tttgggcccg tacccacgct gctgctgctc 120gccgcggcgc
tactggccgt gtcggacgca ctcgggcgcc cctccgagaa aaaaatatca
180ggatcagtta ctagtgcaaa acctggatat catgatatca tcacaattcc
aactggagcc 240accaacatcg aagtgaaaca gcggaaccag aggggatcca
ggaacaatgg cagctttctt 300gccatcaaag ctgctgatgg cacatatatt
cttaatggtg actacacttt gtccacctta
360gagcaagaca ttatgtacaa aggtgttgtc ttgaggtaca gcggctcctc
tgcggcattg 420gaaagaattc gcagctttag ccctctcaaa gagcccttga
ccatccaggt tcttactgtg 480ggcaatgccc ttcgacctaa aattaaatac
acctacttcg tataa 52521271PRTMus musculus 21Met Gly Asp Val Gln Arg
Ala Ala Arg Ser Arg Gly Ser Leu Ser Ala1 5 10 15His Met Leu Leu Leu
Leu Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20 25 30Arg Gly Ala His
Gly Arg Pro Thr Glu Cys Pro Asp Asn Asn Gly Lys 35 40 45Thr Phe Arg
Glu Glu Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala 50 55 60Ser Phe
Gly Asn Glu Pro Thr Val Glu Trp Thr Pro Lys Tyr Ala Gly65 70 75
80Val Ser Pro Lys Asp Arg Cys Lys Leu Thr Cys Glu Ala Lys Gly Ile
85 90 95Gly Tyr Phe Phe Val Leu Gln Pro Lys Val Val Asp Gly Thr Pro
Cys 100 105 110Ser Pro Asp Ser Thr Ser Val Cys Val Gln Gly Gln Cys
Val Lys Ala 115 120 125Gly Cys Asp Arg Ile Ile Asp Ser Lys Lys Lys
Phe Asp Lys Cys Gly 130 135 140Val Cys Gly Gly Asn Gly Ser Thr Cys
Lys Lys Met Ser Gly Ile Val145 150 155 160Thr Ser Thr Arg Pro Gly
Tyr His Asp Ile Val Thr Ile Pro Ala Gly 165 170 175Ala Thr Asn Ile
Glu Val Lys His Arg Asn Gln Arg Gly Ser Arg Asn 180 185 190Asn Gly
Ser Phe Leu Ala Ile Arg Ala Ala Asp Gly Thr Tyr Ile Leu 195 200
205Asn Gly Asn Phe Thr Leu Ser Thr Leu Glu Gln Asp Leu Thr Tyr Lys
210 215 220Gly Thr Val Leu Arg Tyr Ser Gly Ser Ser Ala Ala Leu Glu
Arg Ile225 230 235 240Arg Ser Phe Ser Pro Leu Lys Glu Pro Leu Thr
Ile Gln Val Leu Met 245 250 255Val Gly His Ala Leu Arg Pro Lys Ile
Lys Phe Thr Tyr Phe Met 260 265 27022819DNAMus musculus
22gacatggggg acgtccagcg ggcagcgaga tctcggggct ctctgtccgc acacatgctg
60ttgctgctcc tcgcttccat aacaatgctg ctatgtgcgc ggggcgcaca cgggcgcccc
120acggagtgtc cagacaataa cggaaaaacg ttcagagagg agcagtgcga
ggcgcacaat 180gagttttcca aagcttcctt tgggaatgag cccactgtag
agtggacacc caagtacgcc 240ggcgtctcgc caaaggacag gtgcaagctc
acctgtgaag ccaaaggcat tggctacttt 300ttcgtcttac agcccaaggt
tgtagatggc actccctgta gtccagactc tacctctgtc 360tgtgtgcaag
ggcagtgtgt gaaagctggc tgtgatcgca tcatagactc caaaaagaag
420tttgataagt gtggcgtttg tggaggaaac ggttccacat gcaagaagat
gtcaggaata 480gtcactagta caagacctgg gtatcatgac attgtcacaa
ttcctgctgg agccaccaac 540attgaagtga aacatcggaa tcaaaggggg
tccagaaaca atggcagctt tctggctatt 600agagccgctg atggtaccta
tattctgaat ggaaacttca ctctgtccac actagagcaa 660gacctcacct
acaaaggtac tgtcttaagg tacagtggtt cctcggctgc gctggagaga
720atccgcagct ttagtccact caaagaaccc ttaaccatcc aggttcttat
ggtaggccat 780gctctccgac ccaaaattaa attcacctac tttatgtaa
81923285PRTHomo sapiens 23Met Gln Arg Ala Val Pro Glu Gly Phe Gly
Arg Arg Lys Leu Gly Ser1 5 10 15Asp Met Gly Asn Ala Glu Arg Ala Pro
Gly Ser Arg Ser Phe Gly Pro 20 25 30Val Pro Thr Leu Leu Leu Leu Ala
Ala Ala Leu Leu Ala Val Ser Asp 35 40 45Ala Leu Gly Arg Pro Ser Glu
Cys Pro Asp Asn Asn Gly Lys Thr Phe 50 55 60Arg Glu Glu Gln Cys Glu
Ala His Asn Glu Phe Ser Lys Ala Ser Phe65 70 75 80Gly Ser Gly Pro
Ala Val Glu Trp Ile Pro Lys Tyr Ala Gly Val Ser 85 90 95Pro Lys Asp
Arg Cys Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly Tyr 100 105 110Phe
Phe Val Leu Gln Pro Lys Val Val Asp Gly Thr Pro Cys Ser Pro 115 120
125Asp Ser Thr Ser Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys
130 135 140Asp Arg Ile Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly
Val Cys145 150 155 160Gly Gly Asn Gly Ser Thr Cys Lys Lys Ile Ser
Gly Ser Val Thr Ser 165 170 175Ala Lys Pro Gly Tyr His Asp Ile Ile
Thr Ile Pro Thr Gly Ala Thr 180 185 190Asn Ile Glu Val Lys Gln Arg
Asn Gln Arg Gly Ser Arg Asn Asn Gly 195 200 205Ser Phe Leu Ala Ile
Lys Ala Ala Asp Gly Thr Tyr Ile Leu Asn Gly 210 215 220Asp Tyr Thr
Leu Ser Thr Leu Glu Gln Asp Ile Met Tyr Lys Gly Val225 230 235
240Val Leu Arg Tyr Ser Gly Ser Ser Ala Ala Leu Glu Arg Ile Arg Ser
245 250 255Phe Ser Pro Leu Lys Glu Pro Leu Thr Ile Gln Val Leu Thr
Val Gly 260 265 270Asn Ala Leu Arg Pro Lys Ile Lys Tyr Thr Tyr Phe
Val 275 280 28524861PRTHomo sapiens 24Gly Cys Ala Ala Thr Gly Cys
Ala Gly Cys Gly Ala Gly Cys Thr Gly1 5 10 15Thr Gly Cys Cys Cys Gly
Ala Gly Gly Gly Gly Thr Thr Cys Gly Gly 20 25 30Ala Ala Gly Gly Cys
Gly Cys Ala Ala Gly Cys Thr Gly Gly Gly Cys 35 40 45Ala Gly Cys Gly
Ala Cys Ala Thr Gly Gly Gly Gly Ala Ala Cys Gly 50 55 60Cys Gly Gly
Ala Gly Cys Gly Gly Gly Cys Thr Cys Cys Gly Gly Gly65 70 75 80Gly
Thr Cys Thr Cys Gly Gly Ala Gly Cys Thr Thr Thr Gly Gly Gly 85 90
95Cys Cys Cys Gly Thr Ala Cys Cys Cys Ala Cys Gly Cys Thr Gly Cys
100 105 110Thr Gly Cys Thr Gly Cys Thr Cys Gly Cys Cys Gly Cys Gly
Gly Cys 115 120 125Gly Cys Thr Ala Cys Thr Gly Gly Cys Cys Gly Thr
Gly Thr Cys Gly 130 135 140Gly Ala Cys Gly Cys Ala Cys Thr Cys Gly
Gly Gly Cys Gly Cys Cys145 150 155 160Cys Cys Thr Cys Cys Gly Ala
Gly Thr Gly Thr Cys Cys Ala Gly Ala 165 170 175Cys Ala Ala Thr Ala
Ala Thr Gly Gly Ala Ala Ala Ala Ala Cys Cys 180 185 190Thr Thr Thr
Ala Gly Ala Gly Ala Gly Gly Ala Ala Cys Ala Ala Thr 195 200 205Gly
Thr Gly Ala Ala Gly Cys Ala Cys Ala Cys Ala Ala Cys Gly Ala 210 215
220Gly Thr Thr Thr Thr Cys Ala Ala Ala Ala Gly Cys Thr Thr Cys
Cys225 230 235 240Thr Thr Thr Gly Gly Gly Ala Gly Thr Gly Gly Gly
Cys Cys Thr Gly 245 250 255Cys Gly Gly Thr Gly Gly Ala Ala Thr Gly
Gly Ala Thr Thr Cys Cys 260 265 270Cys Ala Ala Gly Thr Ala Cys Gly
Cys Thr Gly Gly Cys Gly Thr Cys 275 280 285Thr Cys Ala Cys Cys Ala
Ala Ala Gly Gly Ala Cys Ala Gly Gly Thr 290 295 300Gly Cys Ala Ala
Gly Cys Thr Cys Ala Thr Cys Thr Gly Cys Cys Ala305 310 315 320Ala
Gly Cys Cys Ala Ala Ala Gly Gly Cys Ala Thr Thr Gly Gly Cys 325 330
335Thr Ala Cys Thr Thr Cys Thr Thr Cys Gly Thr Thr Thr Thr Gly Cys
340 345 350Ala Gly Cys Cys Cys Ala Ala Gly Gly Thr Thr Gly Thr Ala
Gly Ala 355 360 365Thr Gly Gly Thr Ala Cys Thr Cys Cys Ala Thr Gly
Thr Ala Gly Cys 370 375 380Cys Cys Ala Gly Ala Thr Thr Cys Cys Ala
Cys Cys Thr Cys Thr Gly385 390 395 400Thr Cys Thr Gly Thr Gly Thr
Gly Cys Ala Ala Gly Gly Ala Cys Ala 405 410 415Gly Thr Gly Thr Gly
Thr Ala Ala Ala Ala Gly Cys Thr Gly Gly Thr 420 425 430Thr Gly Thr
Gly Ala Thr Cys Gly Cys Ala Thr Cys Ala Thr Ala Gly 435 440 445Ala
Cys Thr Cys Cys Ala Ala Ala Ala Ala Gly Ala Ala Gly Thr Thr 450 455
460Thr Gly Ala Thr Ala Ala Ala Thr Gly Thr Gly Gly Thr Gly Thr
Thr465 470 475 480Thr Gly Cys Gly Gly Gly Gly Gly Ala Ala Ala Thr
Gly Gly Ala Thr 485 490 495Cys Thr Ala Cys Thr Thr Gly Thr Ala Ala
Ala Ala Ala Ala Ala Thr 500 505 510Ala Thr Cys Ala Gly Gly Ala Thr
Cys Ala Gly Thr Thr Ala Cys Thr 515 520 525Ala Gly Thr Gly Cys Ala
Ala Ala Ala Cys Cys Thr Gly Gly Ala Thr 530 535 540Ala Thr Cys Ala
Thr Gly Ala Thr Ala Thr Cys Ala Thr Cys Ala Cys545 550 555 560Ala
Ala Thr Thr Cys Cys Ala Ala Cys Thr Gly Gly Ala Gly Cys Cys 565 570
575Ala Cys Cys Ala Ala Cys Ala Thr Cys Gly Ala Ala Gly Thr Gly Ala
580 585 590Ala Ala Cys Ala Gly Cys Gly Gly Ala Ala Cys Cys Ala Gly
Ala Gly 595 600 605Gly Gly Gly Ala Thr Cys Cys Ala Gly Gly Ala Ala
Cys Ala Ala Thr 610 615 620Gly Gly Cys Ala Gly Cys Thr Thr Thr Cys
Thr Thr Gly Cys Cys Ala625 630 635 640Thr Cys Ala Ala Ala Gly Cys
Thr Gly Cys Thr Gly Ala Thr Gly Gly 645 650 655Cys Ala Cys Ala Thr
Ala Thr Ala Thr Thr Cys Thr Thr Ala Ala Thr 660 665 670Gly Gly Thr
Gly Ala Cys Thr Ala Cys Ala Cys Thr Thr Thr Gly Thr 675 680 685Cys
Cys Ala Cys Cys Thr Thr Ala Gly Ala Gly Cys Ala Ala Gly Ala 690 695
700Cys Ala Thr Thr Ala Thr Gly Thr Ala Cys Ala Ala Ala Gly Gly
Thr705 710 715 720Gly Thr Thr Gly Thr Cys Thr Thr Gly Ala Gly Gly
Thr Ala Cys Ala 725 730 735Gly Cys Gly Gly Cys Thr Cys Cys Thr Cys
Thr Gly Cys Gly Gly Cys 740 745 750Ala Thr Thr Gly Gly Ala Ala Ala
Gly Ala Ala Thr Thr Cys Gly Cys 755 760 765Ala Gly Cys Thr Thr Thr
Ala Gly Cys Cys Cys Thr Cys Thr Cys Ala 770 775 780Ala Ala Gly Ala
Gly Cys Cys Cys Thr Thr Gly Ala Cys Cys Ala Thr785 790 795 800Cys
Cys Ala Gly Gly Thr Thr Cys Thr Thr Ala Cys Thr Gly Thr Gly 805 810
815Gly Gly Cys Ala Ala Thr Gly Cys Cys Cys Thr Thr Cys Gly Ala Cys
820 825 830Cys Thr Ala Ala Ala Ala Thr Thr Ala Ala Ala Thr Ala Cys
Ala Cys 835 840 845Cys Thr Ala Cys Thr Thr Cys Gly Thr Ala Thr Ala
Ala 850 855 86025447PRTMus musculus 25Met Gly Asp Val Gln Arg Ala
Ala Arg Ser Arg Gly Ser Leu Ser Ala1 5 10 15His Met Leu Leu Leu Leu
Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20 25 30Arg Gly Ala His Gly
Arg Pro Thr Glu Trp Gly Pro Trp Gly Pro Trp 35 40 45Gly Asp Cys Ser
Arg Thr Cys Gly Gly Gly Val Gln Tyr Thr Met Arg 50 55 60Glu Cys Asp
Asn Pro Val Pro Lys Asn Gly Gly Lys Tyr Cys Glu Gly65 70 75 80Lys
Arg Val Arg Tyr Arg Ser Cys Asn Ile Glu Asp Cys Pro Asp Asn 85 90
95Asn Gly Lys Thr Phe Arg Glu Glu Gln Cys Glu Ala His Asn Glu Phe
100 105 110Ser Lys Ala Ser Phe Gly Asn Glu Pro Thr Val Glu Trp Thr
Pro Lys 115 120 125Tyr Ala Gly Val Ser Pro Lys Asp Arg Cys Lys Leu
Thr Cys Glu Ala 130 135 140Lys Gly Ile Gly Tyr Phe Phe Val Leu Gln
Pro Lys Val Val Asp Gly145 150 155 160Thr Pro Cys Ser Pro Asp Ser
Thr Ser Val Cys Val Gln Gly Gln Cys 165 170 175Val Lys Ala Gly Cys
Asp Arg Ile Ile Asp Ser Lys Lys Lys Phe Asp 180 185 190Lys Cys Gly
Val Cys Gly Gly Asn Gly Ser Thr Cys Lys Lys Met Ser 195 200 205Gly
Ile Val Thr Ser Thr Arg Pro Gly Tyr His Asp Ile Val Thr Ile 210 215
220Pro Ala Gly Ala Thr Asn Ile Glu Val Lys His Arg Asn Gln Arg
Gly225 230 235 240Ser Arg Asn Asn Gly Ser Phe Leu Ala Ile Arg Ala
Ala Asp Gly Thr 245 250 255Tyr Ile Leu Asn Gly Asn Phe Thr Leu Ser
Thr Leu Glu Gln Asp Leu 260 265 270Thr Tyr Lys Gly Thr Val Leu Arg
Tyr Ser Gly Ser Ser Ala Ala Leu 275 280 285Glu Arg Ile Arg Ser Phe
Ser Pro Leu Lys Glu Pro Leu Thr Ile Gln 290 295 300Val Leu Met Val
Gly His Ala Leu Arg Pro Lys Ile Lys Phe Thr Tyr305 310 315 320Phe
Met Lys Lys Lys Thr Glu Ser Phe Asn Ala Ile Pro Thr Phe Ser 325 330
335Glu Trp Val Ile Glu Glu Trp Gly Glu Cys Ser Lys Thr Cys Gly Ser
340 345 350Gly Trp Gln Arg Arg Val Val Gln Cys Arg Asp Ile Asn Gly
His Pro 355 360 365Ala Ser Glu Cys Ala Lys Glu Val Lys Pro Ala Ser
Thr Arg Pro Cys 370 375 380Ala Asp Leu Pro Cys Pro His Trp Gln Val
Gly Asp Trp Ser Pro Cys385 390 395 400Ser Lys Thr Cys Gly Lys Gly
Tyr Lys Lys Arg Thr Leu Lys Cys Val 405 410 415Ser His Asp Gly Gly
Val Leu Ser Asn Glu Ser Cys Asp Pro Leu Lys 420 425 430Lys Pro Lys
His Tyr Ile Asp Phe Cys Thr Leu Thr Gln Cys Ser 435 440
445261347DNAMus musculus 26gacatggggg acgtccagcg ggcagcgaga
tctcggggct ctctgtccgc acacatgctg 60ttgctgctcc tcgcttccat aacaatgctg
ctatgtgcgc ggggcgcaca cgggcgcccc 120acggagtggg gaccatgggg
accgtgggga gactgctcaa gaacctgtgg tggtggagtt 180caatacacaa
tgagagaatg tgacaaccca gtcccaaaga acggagggaa gtactgtgaa
240ggcaaacgag tccgctacag gtcctgtaac atcgaggact gtccagacaa
taacggaaaa 300acgttcagag aggagcagtg cgaggcgcac aatgagtttt
ccaaagcttc ctttgggaat 360gagcccactg tagagtggac acccaagtac
gccggcgtct cgccaaagga caggtgcaag 420ctcacctgtg aagccaaagg
cattggctac tttttcgtct tacagcccaa ggttgtagat 480ggcactccct
gtagtccaga ctctacctct gtctgtgtgc aagggcagtg tgtgaaagct
540ggctgtgatc gcatcataga ctccaaaaag aagtttgata agtgtggcgt
ttgtggagga 600aacggttcca catgcaagaa gatgtcagga atagtcacta
gtacaagacc tgggtatcat 660gacattgtca caattcctgc tggagccacc
aacattgaag tgaaacatcg gaatcaaagg 720gggtccagaa acaatggcag
ctttctggct attagagccg ctgatggtac ctatattctg 780aatggaaact
tcactctgtc cacactagag caagacctca cctacaaagg tactgtctta
840aggtacagtg gttcctcggc tgcgctggag agaatccgca gctttagtcc
actcaaagaa 900cccttaacca tccaggttct tatggtaggc catgctctcc
gacccaaaat taaattcacc 960tactttatga agaagaagac agagtcattc
aacgccattc ccacattttc tgagtgggtg 1020attgaagagt ggggggagtg
ctccaagaca tgcggctcag gttggcagag aagagtagtg 1080cagtgcagag
acattaatgg acaccctgct tccgaatgtg caaaggaagt gaagccagcc
1140agtaccagac cttgtgcaga ccttccttgc ccacactggc aggtggggga
ttggtcacca 1200tgttccaaaa cttgcgggaa gggttacaag aagagaacct
tgaaatgtgt gtcccacgat 1260gggggcgtgt tatcaaatga gagctgtgat
cctttgaaga agccaaagca ttacattgac 1320ttttgcacac tgacacagtg cagttaa
134727410PRTHomo sapiens 27Met Gln Arg Ala Val Pro Glu Gly Phe Gly
Arg Arg Lys Leu Gly Ser1 5 10 15Asp Met Gly Asn Ala Glu Arg Ala Pro
Gly Ser Arg Ser Phe Gly Pro 20 25 30Val Pro Thr Leu Leu Leu Leu Ala
Ala Ala Leu Leu Ala Val Ser Asp 35 40 45Ala Leu Gly Arg Pro Ser Glu
Cys Pro Asp Asn Asn Gly Lys Thr Phe 50 55 60Arg Glu Glu Gln Cys Glu
Ala His Asn Glu Phe Ser Lys Ala Ser Phe65 70 75 80Gly Ser Gly Pro
Ala Val Glu Trp Ile Pro Lys Tyr Ala Gly Val Ser 85 90 95Pro Lys Asp
Arg Cys Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly Tyr 100 105 110Phe
Phe Val Leu Gln Pro Lys Val Val Asp Gly Thr Pro Cys Ser Pro 115 120
125Asp Ser Thr Ser Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys
130 135 140Asp Arg Ile Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly
Val Cys145 150 155 160Gly Gly Asn Gly Ser Thr Cys Lys Lys Ile Ser
Gly Ser Val Thr Ser 165 170 175Ala Lys Pro Gly Tyr His Asp Ile Ile
Thr Ile Pro Thr Gly Ala Thr 180 185
190Asn Ile Glu Val Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn Asn Gly
195 200 205Ser Phe Leu Ala Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu
Asn Gly 210 215 220Asp Tyr Thr Leu Ser Thr Leu Glu Gln Asp Ile Met
Tyr Lys Gly Val225 230 235 240Val Leu Arg Tyr Ser Gly Ser Ser Ala
Ala Leu Glu Arg Ile Arg Ser 245 250 255Phe Ser Pro Leu Lys Glu Pro
Leu Thr Ile Gln Val Leu Thr Val Gly 260 265 270Asn Ala Leu Arg Pro
Lys Ile Lys Tyr Thr Tyr Phe Val Lys Lys Lys 275 280 285Lys Glu Ser
Phe Asn Ala Ile Pro Thr Phe Ser Ala Trp Val Ile Glu 290 295 300Glu
Trp Gly Glu Cys Ser Lys Ser Cys Glu Leu Gly Trp Gln Arg Arg305 310
315 320Leu Val Glu Cys Arg Asp Ile Asn Gly Gln Pro Ala Ser Glu Cys
Ala 325 330 335Lys Glu Val Lys Pro Ala Ser Thr Arg Pro Cys Ala Asp
His Pro Cys 340 345 350Pro Gln Trp Gln Leu Gly Glu Trp Ser Ser Cys
Ser Lys Thr Cys Gly 355 360 365Lys Gly Tyr Lys Lys Arg Ser Leu Lys
Cys Leu Ser His Asp Gly Gly 370 375 380Val Leu Ser His Glu Ser Cys
Asp Pro Leu Lys Lys Pro Lys His Phe385 390 395 400Ile Asp Phe Cys
Thr Met Ala Glu Cys Ser 405 410281389DNAHomo sapiens 28gcaatgcagc
gagctgtgcc cgaggggttc ggaaggcgca agctgggcag cgacatgggg 60aacgcggagc
gggctccggg gtctcggagc tttgggcccg tacccacgct gctgctgctc
120gccgcggcgc tactggccgt gtcggacgca ctcgggcgcc cctccgagtg
gggaatgtgg 180gggccttggg gagactgttc gagaacgtgc ggtggaggag
tccagtacac gatgagggaa 240tgtgacaacc cagtcccaaa gaatggaggg
aagtactgtg aaggcaaacg agtgcgctac 300agatcctgta accttgagga
ctgtccagac aataatggaa aaacctttag agaggaacaa 360tgtgaagcac
acaacgagtt ttcaaaagct tcctttggga gtgggcctgc ggtggaatgg
420attcccaagt acgctggcgt ctcaccaaag gacaggtgca agctcatctg
ccaagccaaa 480ggcattggct acttcttcgt tttgcagccc aaggttgtag
atggtactcc atgtagccca 540gattccacct ctgtctgtgt gcaaggacag
tgtgtaaaag ctggttgtga tcgcatcata 600gactccaaaa agaagtttga
taaatgtggt gtttgcgggg gaaatggatc tacttgtaaa 660aaaatatcag
gatcagttac tagtgcaaaa cctggatatc atgatatcat cacaattcca
720actggagcca ccaacatcga agtgaaacag cggaaccaga ggggatccag
gaacaatggc 780agctttcttg ccatcaaagc tgctgatggc acatatattc
ttaatggtga ctacactttg 840tccaccttag agcaagacat tatgtacaaa
ggtgttgtct tgaggtacag cggctcctct 900gcggcattgg aaagaattcg
cagctttagc cctctcaaag agcccttgac catccaggtt 960cttactgtgg
gcaatgccct tcgacctaaa attaaataca cctacttcgt aaagaagaag
1020aaggaatctt tcaatgctat ccccactttt tcagcatggg tcattgaaga
gtggggcgaa 1080tgttctaagt catgtgaatt gggttggcag agaagactgg
tagaatgccg agacattaat 1140ggacagcctg cttccgagtg tgcaaaggaa
gtgaagccag ccagcaccag accttgtgca 1200gaccatccct gcccccagtg
gcagctgggg gagtggtcat catgttctaa gacctgtggg 1260aagggttaca
aaaaaagaag cttgaagtgt ctgtcccatg atggaggggt gttatctcat
1320gagagctgtg atcctttaaa gaaacctaaa catttcatag acttttgcac
aatggcagaa 1380tgcagttaa 138929951PRTMus musculus 29Met Gly Asp Val
Gln Arg Ala Ala Arg Ser Arg Gly Ser Leu Ser Ala1 5 10 15His Met Leu
Leu Leu Leu Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20 25 30Arg Gly
Ala His Gly Arg Pro Thr Glu Glu Asp Glu Glu Leu Val Leu 35 40 45Pro
Ser Leu Glu Arg Ala Pro Gly His Asp Ser Thr Thr Thr Arg Leu 50 55
60Arg Leu Asp Ala Phe Gly Gln Gln Leu His Leu Lys Leu Gln Pro Asp65
70 75 80Ser Gly Phe Leu Ala Pro Gly Phe Thr Leu Gln Thr Val Gly Arg
Ser 85 90 95Pro Gly Ser Glu Ala Gln His Leu Asp Pro Thr Gly Asp Leu
Ala His 100 105 110Cys Phe Tyr Ser Gly Thr Val Asn Gly Asp Pro Gly
Ser Ala Ala Ala 115 120 125Leu Ser Leu Cys Glu Gly Val Arg Gly Ala
Phe Tyr Leu Gln Gly Glu 130 135 140Glu Phe Phe Ile Gln Pro Ala Pro
Gly Val Ala Thr Glu Arg Leu Ala145 150 155 160Pro Ala Val Pro Glu
Glu Glu Ser Ser Ala Arg Pro Gln Phe His Ile 165 170 175Leu Arg Arg
Arg Arg Arg Gly Ser Gly Gly Ala Lys Cys Gly Val Met 180 185 190Asp
Asp Glu Thr Leu Pro Thr Ser Asp Ser Arg Pro Glu Ser Gln Asn 195 200
205Thr Arg Asn Gln Trp Pro Val Arg Asp Pro Thr Pro Gln Asp Ala Gly
210 215 220Lys Pro Ser Gly Pro Gly Ser Ile Arg Lys Lys Arg Phe Val
Ser Ser225 230 235 240Pro Arg Tyr Val Glu Thr Met Leu Val Ala Asp
Gln Ser Met Ala Asp 245 250 255Phe His Gly Ser Gly Leu Lys His Tyr
Leu Leu Thr Leu Phe Ser Val 260 265 270Ala Ala Arg Phe Tyr Lys His
Pro Ser Ile Arg Asn Ser Ile Ser Leu 275 280 285Val Val Val Lys Ile
Leu Val Ile Tyr Glu Glu Gln Lys Gly Pro Glu 290 295 300Val Thr Ser
Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp Gln305 310 315
320Lys Gln His Asn Ser Pro Ser Asp Arg Asp Pro Glu His Tyr Asp Thr
325 330 335Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser His Thr
Cys Asp 340 345 350Thr Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp
Pro Ser Arg Ser 355 360 365Cys Ser Val Ile Glu Asp Asp Gly Leu Gln
Ala Ala Phe Thr Thr Ala 370 375 380His Gln Leu Gly His Val Phe Asn
Met Pro His Asp Asp Ala Lys His385 390 395 400Cys Ala Ser Leu Asn
Gly Val Thr Gly Asp Ser His Leu Met Ala Ser 405 410 415Met Leu Ser
Ser Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala 420 425 430Tyr
Met Val Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met 435 440
445Asp Lys Pro Gln Asn Pro Ile Lys Leu Pro Ser Asp Leu Pro Gly Thr
450 455 460Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu
Glu Ser465 470 475 480Lys His Cys Pro Asp Ala Ala Ser Thr Cys Thr
Thr Leu Trp Cys Thr 485 490 495Gly Thr Ser Gly Gly Leu Leu Val Cys
Gln Thr Lys His Phe Pro Trp 500 505 510Ala Asp Gly Thr Ser Cys Gly
Glu Gly Lys Trp Cys Val Ser Gly Lys 515 520 525Cys Val Asn Lys Thr
Asp Met Lys His Phe Ala Thr Pro Val His Gly 530 535 540Ser Trp Gly
Pro Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly545 550 555
560Gly Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro Lys
565 570 575Asn Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg
Ser Cys 580 585 590Asn Ile Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr
Phe Arg Glu Glu 595 600 605Gln Cys Glu Ala His Asn Glu Phe Ser Lys
Ala Ser Phe Gly Asn Glu 610 615 620Pro Thr Val Glu Trp Thr Pro Lys
Tyr Ala Gly Val Ser Pro Lys Asp625 630 635 640Arg Cys Lys Leu Thr
Cys Glu Ala Lys Gly Ile Gly Tyr Phe Phe Val 645 650 655Leu Gln Pro
Lys Val Val Asp Gly Thr Pro Cys Ser Pro Asp Ser Thr 660 665 670Ser
Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys Asp Arg Ile 675 680
685Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly Asn
690 695 700Gly Ser Thr Cys Lys Lys Met Ser Gly Ile Val Thr Ser Thr
Arg Pro705 710 715 720Gly Tyr His Asp Ile Val Thr Ile Pro Ala Gly
Ala Thr Asn Ile Glu 725 730 735Val Lys His Arg Asn Gln Arg Gly Ser
Arg Asn Asn Gly Ser Phe Leu 740 745 750Ala Ile Arg Ala Ala Asp Gly
Thr Tyr Ile Leu Asn Gly Asn Phe Thr 755 760 765Leu Ser Thr Leu Glu
Gln Asp Leu Thr Tyr Lys Gly Thr Val Leu Arg 770 775 780Tyr Ser Gly
Ser Ser Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser Pro785 790 795
800Leu Lys Glu Pro Leu Thr Ile Gln Val Leu Met Val Gly His Ala Leu
805 810 815Arg Pro Lys Ile Lys Phe Thr Tyr Phe Met Lys Lys Lys Thr
Glu Ser 820 825 830Phe Asn Ala Ile Pro Thr Phe Ser Glu Trp Val Ile
Glu Glu Trp Gly 835 840 845Glu Cys Ser Lys Thr Cys Gly Ser Gly Trp
Gln Arg Arg Val Val Gln 850 855 860Cys Arg Asp Ile Asn Gly His Pro
Ala Ser Glu Cys Ala Lys Glu Val865 870 875 880Lys Pro Ala Ser Thr
Arg Pro Cys Ala Asp Leu Pro Cys Pro His Trp 885 890 895Gln Val Gly
Asp Trp Ser Pro Cys Ser Lys Thr Cys Gly Lys Gly Tyr 900 905 910Lys
Lys Arg Thr Leu Lys Cys Val Ser His Asp Gly Gly Val Leu Ser 915 920
925Asn Glu Ser Cys Asp Pro Leu Lys Lys Pro Lys His Tyr Ile Asp Phe
930 935 940Cys Thr Leu Thr Gln Cys Ser945 950302859DNAMus musculus
30gacatggggg acgtccagcg ggcagcgaga tctcggggct ctctgtccgc acacatgctg
60ttgctgctcc tcgcttccat aacaatgctg ctatgtgcgc ggggcgcaca cgggcgcccc
120acggaggaag atgaggagct ggtcctgccc tcgctggagc gcgccccggg
ccacgattcc 180accaccacac gccttcgtct ggacgccttt ggccagcagc
tacatctgaa gttgcagccg 240gacagcggtt tcttggcgcc tggcttcacc
ctgcagactg tggggcgcag tcccgggtcc 300gaggcacaac atctggaccc
caccggggac ctggctcact gcttctactc tggcacggtg 360aacggtgatc
ccggctctgc cgcagccctc agcctctgtg aaggtgtgcg tggtgccttc
420tacctacaag gagaggagtt cttcattcag ccagcgcctg gagtggccac
cgagcgcctg 480gcccctgccg tgcccgagga ggagtcatcc gcacggccgc
agttccacat cctgaggcga 540aggcggcggg gcagtggcgg cgccaagtgc
ggcgtcatgg acgacgagac cctgccaacc 600agcgactcgc gacccgagag
ccagaacacc cggaaccagt ggcctgtgcg ggaccccacg 660cctcaggacg
cgggaaagcc atcaggacca ggaagcataa ggaagaagcg atttgtgtcc
720agcccccgtt atgtggaaac catgctcgtg gctgaccagt ccatggccga
cttccacggc 780agcggtctaa agcattacct tctaaccctg ttctcggtgg
cagccaggtt ttacaagcat 840cccagcatta ggaattcaat tagcctggtg
gtggtgaaga tcttggtcat atatgaggag 900cagaagggac cagaagttac
ctccaatgca gctctcaccc ttcggaattt ctgcaactgg 960cagaaacaac
acaacagccc cagtgaccgg gatccagagc actatgacac tgcaattctg
1020ttcaccagac aggatttatg tggctcccac acgtgtgaca ctctcgggat
ggcagatgtt 1080ggaactgtat gtgaccccag caggagctgc tcagtcatag
aagatgatgg tttgcaagcc 1140gccttcacca cagcccacca attgggccat
gtgtttaaca tgccgcacga tgatgctaag 1200cactgtgcca gcttgaatgg
tgtgactggc gattctcatc tgatggcctc gatgctctcc 1260agcttagacc
atagccagcc ctggtcacct tgcagtgcct acatggtcac gtccttccta
1320gataatggac acggggaatg tttgatggac aagccccaga atccaatcaa
gctcccttct 1380gatcttcccg gtaccttgta cgatgccaac cgccagtgtc
agtttacatt cggagaggaa 1440tccaagcact gccctgatgc agccagcaca
tgtactaccc tgtggtgcac tggcacctcc 1500ggtggcttac tggtgtgcca
aacaaaacac ttcccttggg cagatggcac cagctgtgga 1560gaagggaagt
ggtgtgtcag tggcaagtgc gtgaacaaga cagacatgaa gcattttgct
1620actcctgttc atggaagctg gggaccatgg ggaccgtggg gagactgctc
aagaacctgt 1680ggtggtggag ttcaatacac aatgagagaa tgtgacaacc
cagtcccaaa gaacggaggg 1740aagtactgtg aaggcaaacg agtccgctac
aggtcctgta acatcgagga ctgtccagac 1800aataacggaa aaacgttcag
agaggagcag tgcgaggcgc acaatgagtt ttccaaagct 1860tcctttggga
atgagcccac tgtagagtgg acacccaagt acgccggcgt ctcgccaaag
1920gacaggtgca agctcacctg tgaagccaaa ggcattggct actttttcgt
cttacagccc 1980aaggttgtag atggcactcc ctgtagtcca gactctacct
ctgtctgtgt gcaagggcag 2040tgtgtgaaag ctggctgtga tcgcatcata
gactccaaaa agaagtttga taagtgtggc 2100gtttgtggag gaaacggttc
cacatgcaag aagatgtcag gaatagtcac tagtacaaga 2160cctgggtatc
atgacattgt cacaattcct gctggagcca ccaacattga agtgaaacat
2220cggaatcaaa gggggtccag aaacaatggc agctttctgg ctattagagc
cgctgatggt 2280acctatattc tgaatggaaa cttcactctg tccacactag
agcaagacct cacctacaaa 2340ggtactgtct taaggtacag tggttcctcg
gctgcgctgg agagaatccg cagctttagt 2400ccactcaaag aacccttaac
catccaggtt cttatggtag gccatgctct ccgacccaaa 2460attaaattca
cctactttat gaagaagaag acagagtcat tcaacgccat tcccacattt
2520tctgagtggg tgattgaaga gtggggggag tgctccaaga catgcggctc
aggttggcag 2580agaagagtag tgcagtgcag agacattaat ggacaccctg
cttccgaatg tgcaaaggaa 2640gtgaagccag ccagtaccag accttgtgca
gaccttcctt gcccacactg gcaggtgggg 2700gattggtcac catgttccaa
aacttgcggg aagggttaca agaagagaac cttgaaatgt 2760gtgtcccacg
atgggggcgt gttatcaaat gagagctgtg atcctttgaa gaagccaaag
2820cattacattg acttttgcac actgacacag tgcagttaa 285931950PRTHomo
sapiens 31Met Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly
Pro Val1 5 10 15Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val
Ser Asp Ala 20 25 30Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu Leu Val
Val Pro Glu Leu 35 40 45Glu Arg Ala Pro Gly His Gly Thr Thr Arg Leu
Arg Leu His Ala Phe 50 55 60Asp Gln Gln Leu Asp Leu Glu Leu Arg Pro
Asp Ser Ser Phe Leu Ala65 70 75 80Pro Gly Phe Thr Leu Gln Asn Val
Gly Arg Lys Ser Gly Ser Glu Thr 85 90 95Pro Leu Pro Glu Thr Asp Leu
Ala His Cys Phe Tyr Ser Gly Thr Val 100 105 110Asn Gly Asp Pro Ser
Ser Ala Ala Ala Leu Ser Leu Cys Glu Gly Val 115 120 125Arg Gly Ala
Phe Tyr Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro Leu 130 135 140Pro
Ala Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu Lys Pro145 150
155 160Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg Asn Arg Gln Gly
Asp 165 170 175Val Gly Gly Thr Cys Gly Val Val Asp Asp Glu Pro Arg
Pro Thr Gly 180 185 190Lys Ala Glu Thr Glu Asp Glu Asp Glu Gly Thr
Glu Gly Glu Asp Glu 195 200 205Gly Ala Gln Trp Ser Pro Gln Asp Pro
Ala Leu Gln Gly Val Gly Gln 210 215 220Pro Thr Gly Thr Gly Ser Ile
Arg Lys Lys Arg Phe Val Ser Ser His225 230 235 240Arg Tyr Val Glu
Thr Met Leu Val Ala Asp Gln Ser Met Ala Glu Phe 245 250 255His Gly
Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val Ala 260 265
270Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu Val
275 280 285Val Val Lys Ile Leu Val Ile His Asp Glu Gln Lys Gly Pro
Glu Val 290 295 300Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys
Asn Trp Gln Lys305 310 315 320Gln His Asn Pro Pro Ser Asp Arg Asp
Ala Glu His Tyr Asp Thr Ala 325 330 335Ile Leu Phe Thr Arg Gln Asp
Leu Cys Gly Ser Gln Thr Cys Asp Thr 340 345 350Leu Gly Met Ala Asp
Val Gly Thr Val Cys Asp Pro Ser Arg Ser Cys 355 360 365Ser Val Ile
Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala His 370 375 380Gln
Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln Cys385 390
395 400Ala Ser Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser
Met 405 410 415Leu Ser Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys
Ser Ala Tyr 420 425 430Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly
Glu Cys Leu Met Asp 435 440 445Lys Pro Gln Asn Pro Ile Gln Leu Pro
Gly Asp Leu Pro Gly Thr Ser 450 455 460Tyr Asp Ala Asn Arg Gln Cys
Gln Phe Thr Phe Gly Glu Asp Ser Lys465 470 475 480His Cys Pro Asp
Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys Thr Gly 485 490 495Thr Ser
Gly Gly Val Leu Val Cys Gln Thr Lys His Phe Pro Trp Ala 500 505
510Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn Gly Lys Cys
515 520 525Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr Pro Phe His
Gly Ser 530 535 540Trp Gly Met Trp Gly Pro Trp Gly Asp Cys Ser Arg
Thr Cys Gly Gly545 550 555 560Gly Val Gln Tyr Thr Met Arg Glu Cys
Asp Asn Pro Val Pro Lys Asn 565 570 575Gly Gly Lys Tyr Cys Glu Gly
Lys Arg Val Arg Tyr Arg Ser Cys Asn
580 585 590Leu Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe Arg Glu
Glu Gln 595 600 605Cys Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe
Gly Ser Gly Pro 610 615 620Ala Val Glu Trp Ile Pro Lys Tyr Ala Gly
Val Ser Pro Lys Asp Arg625 630 635 640Cys Lys Leu Ile Cys Gln Ala
Lys Gly Ile Gly Tyr Phe Phe Val Leu 645 650 655Gln Pro Lys Val Val
Asp Gly Thr Pro Cys Ser Pro Asp Ser Thr Ser 660 665 670Val Cys Val
Gln Gly Gln Cys Val Lys Ala Gly Cys Asp Arg Ile Ile 675 680 685Asp
Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly Asn Gly 690 695
700Ser Thr Cys Lys Lys Ile Ser Gly Ser Val Thr Ser Ala Lys Pro
Gly705 710 715 720Tyr His Asp Ile Ile Thr Ile Pro Thr Gly Ala Thr
Asn Ile Glu Val 725 730 735Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn
Asn Gly Ser Phe Leu Ala 740 745 750Ile Lys Ala Ala Asp Gly Thr Tyr
Ile Leu Asn Gly Asp Tyr Thr Leu 755 760 765Ser Thr Leu Glu Gln Asp
Ile Met Tyr Lys Gly Val Val Leu Arg Tyr 770 775 780Ser Gly Ser Ser
Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser Pro Leu785 790 795 800Lys
Glu Pro Leu Thr Ile Gln Val Leu Thr Val Gly Asn Ala Leu Arg 805 810
815Pro Lys Ile Lys Tyr Thr Tyr Phe Val Lys Lys Lys Lys Glu Ser Phe
820 825 830Asn Ala Ile Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp
Gly Glu 835 840 845Cys Ser Lys Ser Cys Glu Leu Gly Trp Gln Arg Arg
Leu Val Glu Cys 850 855 860Arg Asp Ile Asn Gly Gln Pro Ala Ser Glu
Cys Ala Lys Glu Val Lys865 870 875 880Pro Ala Ser Thr Arg Pro Cys
Ala Asp His Pro Cys Pro Gln Trp Gln 885 890 895Leu Gly Glu Trp Ser
Ser Cys Ser Lys Thr Cys Gly Lys Gly Tyr Lys 900 905 910Lys Arg Ser
Leu Lys Cys Leu Ser His Asp Gly Gly Val Leu Ser His 915 920 925Glu
Ser Cys Asp Pro Leu Lys Lys Pro Lys His Phe Ile Asp Phe Cys 930 935
940Thr Met Ala Glu Cys Ser945 950322907DNAHomo sapiens 32gcaatgcagc
gagctgtgcc cgaggggttc ggaaggcgca agctgggcag cgacatgggg 60aacgcggagc
gggctccggg gtctcggagc tttgggcccg tacccacgct gctgctgctc
120gccgcggcgc tactggccgt gtcggacgca ctcgggcgcc cctccgagga
ggacgaggag 180ctagtggtgc cggagctgga gcgcgccccg ggacacggga
ccacgcgcct ccgcctgcac 240gcctttgacc agcagctgga tctggagctg
cggcccgaca gcagcttttt ggcgcccggc 300ttcacgctcc agaacgtggg
gcgcaaatcc gggtccgaga cgccgcttcc ggaaaccgac 360ctggcgcact
gcttctactc cggcaccgtg aatggcgatc ccagctcggc tgccgccctc
420agcctctgcg agggcgtgcg cggcgccttc tacctgctgg gggaggcgta
tttcatccag 480ccgctgcccg ccgccagcga gcgcctcgcc accgccgccc
caggggagaa gccgccggca 540ccactacagt tccacctcct gcggcggaat
cggcagggcg acgtcggcgg cacgtgcggg 600gtcgtggacg acgagccccg
gccgactggg aaagcggaga ccgaagacga ggacgaaggg 660actgagggcg
aggacgaagg ggctcagtgg tcgccgcagg acccggcact gcaaggcgta
720ggacagccca caggaactgg aagcataaga aagaagcgat ttgtgtccag
tcaccgctat 780gtggaaacca tgcttgtggc agaccagtcg atggcagaat
tccacggcag tggtctaaag 840cattaccttc tcacgttgtt ttcggtggca
gccagattgt acaaacaccc cagcattcgt 900aattcagtta gcctggtggt
ggtgaagatc ttggtcatcc acgatgaaca gaaggggccg 960gaagtgacct
ccaatgctgc cctcactctg cggaactttt gcaactggca gaagcagcac
1020aacccaccca gtgaccggga tgcagagcac tatgacacag caattctttt
caccagacag 1080gacttgtgtg ggtcccagac atgtgatact cttgggatgg
ctgatgttgg aactgtgtgt 1140gatccgagca gaagctgctc cgtcatagaa
gatgatggtt tacaagctgc cttcaccaca 1200gcccatcaat taggccacgt
gtttaacatg ccacatgatg atgcaaagca gtgtgccagc 1260cttaatggtg
tgaaccagga ttcccacatg atggcgtcaa tgctttccaa cctggaccac
1320agccagcctt ggtctccttg cagtgcctac atgattacat catttctgga
taatggtcat 1380ggggaatgtt tgatggacaa gcctcagaat cccatacagc
tcccaggcga tctccctggc 1440acctcgtacg atgccaaccg gcagtgccag
tttacatttg gggaggactc caaacactgc 1500cccgatgcag ccagcacatg
tagcaccttg tggtgtaccg gcacctctgg tggggtgctg 1560gtgtgtcaaa
ccaaacactt cccgtgggcg gatggcacca gctgtggaga agggaaatgg
1620tgtatcaacg gcaagtgtgt gaacaaaacc gacagaaagc attttgatac
gccttttcat 1680ggaagctggg gaatgtgggg gccttgggga gactgttcga
gaacgtgcgg tggaggagtc 1740cagtacacga tgagggaatg tgacaaccca
gtcccaaaga atggagggaa gtactgtgaa 1800ggcaaacgag tgcgctacag
atcctgtaac cttgaggact gtccagacaa taatggaaaa 1860acctttagag
aggaacaatg tgaagcacac aacgagtttt caaaagcttc ctttgggagt
1920gggcctgcgg tggaatggat tcccaagtac gctggcgtct caccaaagga
caggtgcaag 1980ctcatctgcc aagccaaagg cattggctac ttcttcgttt
tgcagcccaa ggttgtagat 2040ggtactccat gtagcccaga ttccacctct
gtctgtgtgc aaggacagtg tgtaaaagct 2100ggttgtgatc gcatcataga
ctccaaaaag aagtttgata aatgtggtgt ttgcggggga 2160aatggatcta
cttgtaaaaa aatatcagga tcagttacta gtgcaaaacc tggatatcat
2220gatatcatca caattccaac tggagccacc aacatcgaag tgaaacagcg
gaaccagagg 2280ggatccagga acaatggcag ctttcttgcc atcaaagctg
ctgatggcac atatattctt 2340aatggtgact acactttgtc caccttagag
caagacatta tgtacaaagg tgttgtcttg 2400aggtacagcg gctcctctgc
ggcattggaa agaattcgca gctttagccc tctcaaagag 2460cccttgacca
tccaggttct tactgtgggc aatgcccttc gacctaaaat taaatacacc
2520tacttcgtaa agaagaagaa ggaatctttc aatgctatcc ccactttttc
agcatgggtc 2580attgaagagt ggggcgaatg ttctaagtca tgtgaattgg
gttggcagag aagactggta 2640gaatgccgag acattaatgg acagcctgct
tccgagtgtg caaaggaagt gaagccagcc 2700agcaccagac cttgtgcaga
ccatccctgc ccccagtggc agctggggga gtggtcatca 2760tgttctaaga
cctgtgggaa gggttacaaa aaaagaagct tgaagtgtct gtcccatgat
2820ggaggggtgt tatctcatga gagctgtgat cctttaaaga aacctaaaca
tttcatagac 2880ttttgcacaa tggcagaatg cagttaa 290733596PRTMus
musculus 33Met Gly Asp Val Gln Arg Ala Ala Arg Ser Arg Gly Ser Leu
Ser Ala1 5 10 15His Met Leu Leu Leu Leu Leu Ala Ser Ile Thr Met Leu
Leu Cys Ala 20 25 30Arg Gly Ala His Gly Arg Pro Thr Glu Glu Asp Glu
Glu Leu Val Leu 35 40 45Pro Ser Leu Glu Arg Ala Pro Gly His Asp Ser
Thr Thr Thr Arg Leu 50 55 60Arg Leu Asp Ala Phe Gly Gln Gln Leu His
Leu Lys Leu Gln Pro Asp65 70 75 80Ser Gly Phe Leu Ala Pro Gly Phe
Thr Leu Gln Thr Val Gly Arg Ser 85 90 95Pro Gly Ser Glu Ala Gln His
Leu Asp Pro Thr Gly Asp Leu Ala His 100 105 110Cys Phe Tyr Ser Gly
Thr Val Asn Gly Asp Pro Gly Ser Ala Ala Ala 115 120 125Leu Ser Leu
Cys Glu Gly Val Arg Gly Ala Phe Tyr Leu Gln Gly Glu 130 135 140Glu
Phe Phe Ile Gln Pro Ala Pro Gly Val Ala Thr Glu Arg Leu Ala145 150
155 160Pro Ala Val Pro Glu Glu Glu Ser Ser Ala Arg Pro Gln Phe His
Ile 165 170 175Leu Arg Arg Arg Arg Arg Gly Ser Gly Gly Ala Lys Cys
Gly Val Met 180 185 190Asp Asp Glu Thr Leu Pro Thr Ser Asp Ser Arg
Pro Glu Ser Gln Asn 195 200 205Thr Arg Asn Gln Trp Pro Val Arg Asp
Pro Thr Pro Gln Asp Ala Gly 210 215 220Lys Pro Ser Gly Pro Gly Ser
Ile Arg Lys Lys Arg Phe Val Ser Ser225 230 235 240Pro Arg Tyr Val
Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Asp 245 250 255Phe His
Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val 260 265
270Ala Ala Arg Phe Tyr Lys His Pro Ser Ile Arg Asn Ser Ile Ser Leu
275 280 285Val Val Val Lys Ile Leu Val Ile Tyr Glu Glu Gln Lys Gly
Pro Glu 290 295 300Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe
Cys Asn Trp Gln305 310 315 320Lys Gln His Asn Ser Pro Ser Asp Arg
Asp Pro Glu His Tyr Asp Thr 325 330 335Ala Ile Leu Phe Thr Arg Gln
Asp Leu Cys Gly Ser His Thr Cys Asp 340 345 350Thr Leu Gly Met Ala
Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser 355 360 365Cys Ser Val
Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala 370 375 380His
Gln Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys His385 390
395 400Cys Ala Ser Leu Asn Gly Val Thr Gly Asp Ser His Leu Met Ala
Ser 405 410 415Met Leu Ser Ser Leu Asp His Ser Gln Pro Trp Ser Pro
Cys Ser Ala 420 425 430Tyr Met Val Thr Ser Phe Leu Asp Asn Gly His
Gly Glu Cys Leu Met 435 440 445Asp Lys Pro Gln Asn Pro Ile Lys Leu
Pro Ser Asp Leu Pro Gly Thr 450 455 460Leu Tyr Asp Ala Asn Arg Gln
Cys Gln Phe Thr Phe Gly Glu Glu Ser465 470 475 480Lys His Cys Pro
Asp Ala Ala Ser Thr Cys Thr Thr Leu Trp Cys Thr 485 490 495Gly Thr
Ser Gly Gly Leu Leu Val Cys Gln Thr Lys His Phe Pro Trp 500 505
510Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Val Ser Gly Lys
515 520 525Cys Val Asn Lys Thr Asp Met Lys His Phe Ala Thr Pro Val
His Gly 530 535 540Ser Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys Ser
Arg Thr Cys Gly545 550 555 560Gly Gly Val Gln Tyr Thr Met Arg Glu
Cys Asp Asn Pro Val Pro Lys 565 570 575Asn Gly Gly Lys Tyr Cys Glu
Gly Lys Arg Val Arg Tyr Arg Ser Cys 580 585 590Asn Ile Glu Asp
595341794DNAMus musculus 34gacatggggg acgtccagcg ggcagcgaga
tctcggggct ctctgtccgc acacatgctg 60ttgctgctcc tcgcttccat aacaatgctg
ctatgtgcgc ggggcgcaca cgggcgcccc 120acggaggaag atgaggagct
ggtcctgccc tcgctggagc gcgccccggg ccacgattcc 180accaccacac
gccttcgtct ggacgccttt ggccagcagc tacatctgaa gttgcagccg
240gacagcggtt tcttggcgcc tggcttcacc ctgcagactg tggggcgcag
tcccgggtcc 300gaggcacaac atctggaccc caccggggac ctggctcact
gcttctactc tggcacggtg 360aacggtgatc ccggctctgc cgcagccctc
agcctctgtg aaggtgtgcg tggtgccttc 420tacctacaag gagaggagtt
cttcattcag ccagcgcctg gagtggccac cgagcgcctg 480gcccctgccg
tgcccgagga ggagtcatcc gcacggccgc agttccacat cctgaggcga
540aggcggcggg gcagtggcgg cgccaagtgc ggcgtcatgg acgacgagac
cctgccaacc 600agcgactcgc gacccgagag ccagaacacc cggaaccagt
ggcctgtgcg ggaccccacg 660cctcaggacg cgggaaagcc atcaggacca
ggaagcataa ggaagaagcg atttgtgtcc 720agcccccgtt atgtggaaac
catgctcgtg gctgaccagt ccatggccga cttccacggc 780agcggtctaa
agcattacct tctaaccctg ttctcggtgg cagccaggtt ttacaagcat
840cccagcatta ggaattcaat tagcctggtg gtggtgaaga tcttggtcat
atatgaggag 900cagaagggac cagaagttac ctccaatgca gctctcaccc
ttcggaattt ctgcaactgg 960cagaaacaac acaacagccc cagtgaccgg
gatccagagc actatgacac tgcaattctg 1020ttcaccagac aggatttatg
tggctcccac acgtgtgaca ctctcgggat ggcagatgtt 1080ggaactgtat
gtgaccccag caggagctgc tcagtcatag aagatgatgg tttgcaagcc
1140gccttcacca cagcccacca attgggccat gtgtttaaca tgccgcacga
tgatgctaag 1200cactgtgcca gcttgaatgg tgtgactggc gattctcatc
tgatggcctc gatgctctcc 1260agcttagacc atagccagcc ctggtcacct
tgcagtgcct acatggtcac gtccttccta 1320gataatggac acggggaatg
tttgatggac aagccccaga atccaatcaa gctcccttct 1380gatcttcccg
gtaccttgta cgatgccaac cgccagtgtc agtttacatt cggagaggaa
1440tccaagcact gccctgatgc agccagcaca tgtactaccc tgtggtgcac
tggcacctcc 1500ggtggcttac tggtgtgcca aacaaaacac ttcccttggg
cagatggcac cagctgtgga 1560gaagggaagt ggtgtgtcag tggcaagtgc
gtgaacaaga cagacatgaa gcattttgct 1620actcctgttc atggaagctg
gggaccatgg ggaccgtggg gagactgctc aagaacctgt 1680ggtggtggag
ttcaatacac aatgagagaa tgtgacaacc cagtcccaaa gaacggaggg
1740aagtactgtg aaggcaaacg agtccgctac aggtcctgta acatcgagga ctaa
179435595PRTHomo sapiens 35Met Gly Asn Ala Glu Arg Ala Pro Gly Ser
Arg Ser Phe Gly Pro Val1 5 10 15Pro Thr Leu Leu Leu Leu Ala Ala Ala
Leu Leu Ala Val Ser Asp Ala 20 25 30Leu Gly Arg Pro Ser Glu Glu Asp
Glu Glu Leu Val Val Pro Glu Leu 35 40 45Glu Arg Ala Pro Gly His Gly
Thr Thr Arg Leu Arg Leu His Ala Phe 50 55 60Asp Gln Gln Leu Asp Leu
Glu Leu Arg Pro Asp Ser Ser Phe Leu Ala65 70 75 80Pro Gly Phe Thr
Leu Gln Asn Val Gly Arg Lys Ser Gly Ser Glu Thr 85 90 95Pro Leu Pro
Glu Thr Asp Leu Ala His Cys Phe Tyr Ser Gly Thr Val 100 105 110Asn
Gly Asp Pro Ser Ser Ala Ala Ala Leu Ser Leu Cys Glu Gly Val 115 120
125Arg Gly Ala Phe Tyr Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro Leu
130 135 140Pro Ala Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu
Lys Pro145 150 155 160Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg
Asn Arg Gln Gly Asp 165 170 175Val Gly Gly Thr Cys Gly Val Val Asp
Asp Glu Pro Arg Pro Thr Gly 180 185 190Lys Ala Glu Thr Glu Asp Glu
Asp Glu Gly Thr Glu Gly Glu Asp Glu 195 200 205Gly Ala Gln Trp Ser
Pro Gln Asp Pro Ala Leu Gln Gly Val Gly Gln 210 215 220Pro Thr Gly
Thr Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser His225 230 235
240Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Glu Phe
245 250 255His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser
Val Ala 260 265 270Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser
Val Ser Leu Val 275 280 285Val Val Lys Ile Leu Val Ile His Asp Glu
Gln Lys Gly Pro Glu Val 290 295 300Thr Ser Asn Ala Ala Leu Thr Leu
Arg Asn Phe Cys Asn Trp Gln Lys305 310 315 320Gln His Asn Pro Pro
Ser Asp Arg Asp Ala Glu His Tyr Asp Thr Ala 325 330 335Ile Leu Phe
Thr Arg Gln Asp Leu Cys Gly Ser Gln Thr Cys Asp Thr 340 345 350Leu
Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser Cys 355 360
365Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala His
370 375 380Gln Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys
Gln Cys385 390 395 400Ala Ser Leu Asn Gly Val Asn Gln Asp Ser His
Met Met Ala Ser Met 405 410 415Leu Ser Asn Leu Asp His Ser Gln Pro
Trp Ser Pro Cys Ser Ala Tyr 420 425 430Met Ile Thr Ser Phe Leu Asp
Asn Gly His Gly Glu Cys Leu Met Asp 435 440 445Lys Pro Gln Asn Pro
Ile Gln Leu Pro Gly Asp Leu Pro Gly Thr Ser 450 455 460Tyr Asp Ala
Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Asp Ser Lys465 470 475
480His Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys Thr Gly
485 490 495Thr Ser Gly Gly Val Leu Val Cys Gln Thr Lys His Phe Pro
Trp Ala 500 505 510Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile
Asn Gly Lys Cys 515 520 525Val Asn Lys Thr Asp Arg Lys His Phe Asp
Thr Pro Phe His Gly Ser 530 535 540Trp Gly Met Trp Gly Pro Trp Gly
Asp Cys Ser Arg Thr Cys Gly Gly545 550 555 560Gly Val Gln Tyr Thr
Met Arg Glu Cys Asp Asn Pro Val Pro Lys Asn 565 570 575Gly Gly Lys
Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys Asn 580 585 590Leu
Glu Asp 595361842DNAHomo sapiens 36gcaatgcagc gagctgtgcc cgaggggttc
ggaaggcgca agctgggcag cgacatgggg 60aacgcggagc gggctccggg gtctcggagc
tttgggcccg tacccacgct gctgctgctc 120gccgcggcgc tactggccgt
gtcggacgca ctcgggcgcc cctccgagga ggacgaggag 180ctagtggtgc
cggagctgga gcgcgccccg ggacacggga ccacgcgcct ccgcctgcac
240gcctttgacc agcagctgga tctggagctg cggcccgaca gcagcttttt
ggcgcccggc 300ttcacgctcc agaacgtggg gcgcaaatcc gggtccgaga
cgccgcttcc ggaaaccgac 360ctggcgcact gcttctactc cggcaccgtg
aatggcgatc ccagctcggc tgccgccctc 420agcctctgcg agggcgtgcg
cggcgccttc tacctgctgg gggaggcgta tttcatccag 480ccgctgcccg
ccgccagcga gcgcctcgcc accgccgccc caggggagaa gccgccggca
540ccactacagt tccacctcct gcggcggaat cggcagggcg acgtcggcgg
cacgtgcggg 600gtcgtggacg acgagccccg gccgactggg aaagcggaga
ccgaagacga ggacgaaggg 660actgagggcg aggacgaagg ggctcagtgg
tcgccgcagg acccggcact gcaaggcgta 720ggacagccca caggaactgg
aagcataaga aagaagcgat
ttgtgtccag tcaccgctat 780gtggaaacca tgcttgtggc agaccagtcg
atggcagaat tccacggcag tggtctaaag 840cattaccttc tcacgttgtt
ttcggtggca gccagattgt acaaacaccc cagcattcgt 900aattcagtta
gcctggtggt ggtgaagatc ttggtcatcc acgatgaaca gaaggggccg
960gaagtgacct ccaatgctgc cctcactctg cggaactttt gcaactggca
gaagcagcac 1020aacccaccca gtgaccggga tgcagagcac tatgacacag
caattctttt caccagacag 1080gacttgtgtg ggtcccagac atgtgatact
cttgggatgg ctgatgttgg aactgtgtgt 1140gatccgagca gaagctgctc
cgtcatagaa gatgatggtt tacaagctgc cttcaccaca 1200gcccatcaat
taggccacgt gtttaacatg ccacatgatg atgcaaagca gtgtgccagc
1260cttaatggtg tgaaccagga ttcccacatg atggcgtcaa tgctttccaa
cctggaccac 1320agccagcctt ggtctccttg cagtgcctac atgattacat
catttctgga taatggtcat 1380ggggaatgtt tgatggacaa gcctcagaat
cccatacagc tcccaggcga tctccctggc 1440acctcgtacg atgccaaccg
gcagtgccag tttacatttg gggaggactc caaacactgc 1500cccgatgcag
ccagcacatg tagcaccttg tggtgtaccg gcacctctgg tggggtgctg
1560gtgtgtcaaa ccaaacactt cccgtgggcg gatggcacca gctgtggaga
agggaaatgg 1620tgtatcaacg gcaagtgtgt gaacaaaacc gacagaaagc
attttgatac gccttttcat 1680ggaagctggg gaatgtgggg gccttgggga
gactgttcga gaacgtgcgg tggaggagtc 1740cagtacacga tgagggaatg
tgacaaccca gtcccaaaga atggagggaa gtactgtgaa 1800ggcaaacgag
tgcgctacag atcctgtaac cttgaggact aa 1842
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