U.S. patent application number 14/183341 was filed with the patent office on 2015-07-16 for function and regulation of adamts-1.
This patent application is currently assigned to THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. The applicant listed for this patent is Qin Yu. Invention is credited to Qin Yu.
Application Number | 20150197740 14/183341 |
Document ID | / |
Family ID | 37884617 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150197740 |
Kind Code |
A9 |
Yu; Qin |
July 16, 2015 |
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) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Yu; Qin |
New York |
NY |
US |
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|
Assignee: |
THE TRUSTEES OF THE UNIVERSITY OF
PENNSYLVANIA
Philadelphia
PA
|
Prior
Publication: |
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Document Identifier |
Publication Date |
|
US 20140294846 A1 |
October 2, 2014 |
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|
Family ID: |
37884617 |
Appl. No.: |
14/183341 |
Filed: |
February 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12709296 |
Feb 19, 2010 |
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14183341 |
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11104075 |
Apr 12, 2005 |
7696307 |
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12709296 |
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60650027 |
Feb 4, 2005 |
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60561429 |
Apr 12, 2004 |
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Current U.S.
Class: |
424/139.1 ;
424/94.67; 435/226; 435/23; 435/320.1; 435/7.4; 506/11; 514/44R;
536/23.2 |
Current CPC
Class: |
G01N 33/57496 20130101;
C12Q 1/37 20130101; C07K 16/40 20130101; A61P 35/00 20180101; G01N
2500/00 20130101; G01N 33/5011 20130101; C12N 9/6489 20130101 |
International
Class: |
C12N 9/64 20060101
C12N009/64; G01N 33/50 20060101 G01N033/50; G01N 33/574 20060101
G01N033/574; C07K 16/40 20060101 C07K016/40; C12Q 1/37 20060101
C12Q001/37 |
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 position 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 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 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 mating 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 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 is a continuation of U.S. patent
application Ser. No. 12/709,296, now abandoned, which is a
divisional of U.S. patent application Ser. No. 11/104,075, filed
Apr. 12, 2005 and issued as U.S. Pat. No. 7,696,307, all of which
claim 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 adipose 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. 1A and FIG. 1B depict how ADAMTS-1 undergoes
auto-proteolytic cleavage and the self-cleavage of ADAMTS-1 is
regulated. FIG. 1A depicts a diagram of the expression constructs.
FIG. 1B depicts how 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.
[0016] FIG. 2A through FIG. 2E depict that ADMATS-1 promotes
pulmonary metastasis, while ADAMTS-1.sub.NTCF and ADAMTS-1.sub.CTCF
inhibits the process. FIG. 2A depicts 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. FIG. 2B depicts the 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. FIG. 2C depicts that 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. FIG. 2D depicts 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. FIG. 2E
depicts 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. 3A through FIG. 3C depict 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. 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. 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 FIG. 3B.
[0018] FIG. 4A through FIG. 4D depict how 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. FIG. 4A. 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-NTCF (A-c), and TA3.sub.ADAMTS-1CTCF (A-d) cells in
the lung parenchyma were shown. FIG. 4B. The pulmonary
extravasation rates of the TA3 transfectants were expressed as
average number of the tumor cells per microscopic field. FIG. 4C.
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. FIG. 4D. 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. 5A through FIG. 5D depict how the ADAMTS-1 fragments
block activation of EGFR and ErbB-2; while ADAMTS-1 promotes
shedding of AR and HB-EGF. FIG. 5A. 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. FIG. 5B. 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). FIG. 5C. 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. FIG. 5D. 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.
[0020] FIG. 6A through FIG. 6D depict how ADAMTS-1.sub.NTCF and
ADAMTS-1.sub.CTCF blocks activation of Erk1/2 kinases in HUVECs
induced 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. 7A through FIG. 7D depict 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. FIG. 7A
depicts 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. FIG. 7B depicts
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. FIG. 7C depicts the 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. FIG. 7D depicts the 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. 8A through FIG. 8C depict 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 (FIG. 8A), the ECM materials (FIG.
8B), and the EDTA-lifted Cos-7 cells (FIG. 8C) that were
transfected with the corresponding expression constructs.
[0023] FIG. 9A through FIG. 9D depict how 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).
[0024] FIG. 10 depicts expression of ADAMTS-1. Expression of
ADAMTS-1 was assessed by RT-PCR using RNAs derived from TA3wt,
TA3.sub.wt1, Lewis lung carcinoma cells, CMT-93 colon carcinoma
cells, B 16F1 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. 11A and FIG. 11B depict how 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. 12A and FIG. 12B depict 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. 13A through FIG. 13D depicts 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). 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 depicts the domain organization of ADAMTS-1. The
various domains of ADAMTS-1 are shown.
[0029] FIG. 15 depicts 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. 16A through FIG. 16D depict full-length ADAMTS-1 and
the ADAMTS-1 fragments displayed opposite effects on growth and
metastasis of LLC cells. FIG. 16A. 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. FIG.
16B. 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.
FIG. 16C. 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. FIG.
16D. 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. 17A through FIG. 17C depict the metalloproteinase
activity of ADAMTS-1.sub.NTF is not required for its anti-tumor
activity. FIG. 17A. 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. FIG. 17B 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. FIG. 17C. 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 depicts 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-SEQ ID NO:46) can be located before the
COOH-terminal v5 and His epitope tags. The HRV 3C protease
specifically claves the sequence LEVLFQ.dwnarw.GP at 40C 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-.sub.1spacer/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 metallothionein. 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 inpCEP4 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 1M. 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 lag 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. 1B 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. 1A). 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 (TA3.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. 2A-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 (FIGS. 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-1CTCF
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. 2E).
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 GF
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.sub.NTF, 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-1NTF 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
nonneoplastic 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, overexpression 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-1NTF), 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. 7C-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 how 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). [0180] 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.
[0175] 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.NTCF, 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).
[0176] 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).
[0177] 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.
[0178] 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
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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
[0184] 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).
[0185] To Determine Whether the .sub.m and/or c fTSP-1 Domain
Displays Anti-Tumor Activity
[0186] It was shown that deletion of the middle TSP-1 (mTSP-1)
domain from ADAMTS-1.sub.NTF (ADAMTS-1.sub.minusTSP-1) 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.m-TSP-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
TA3wt1 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.
[0187] 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.
[0188] Deletions and Mutations in the m or cTSP-1 Domains of
ADAMTS-1 and Establish TA3 and LLC Transfectants Expressing these
ADAMTS-1 Mutants
[0189] 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 (SEQ ID NO:47) 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 (SEQ ID NO:50) motif (FIG.
18). Thus, three deletions in .sub.m/cTSP-1 domains are made:
WGPWGPWGD (ADAMTS-1.sub.mTSP-1WXXWde1-SEQ ID NO:48) or
WV/QI/VE/GE/DWG/S (ADAMTS-1.sub.cTSP-1WXXXXWde1), WGDCSRTC
(ADAMTS-1.sub.mTSP-1WGde1-SEQ ID NO:49) or WG/SE/PCSKTC
(ADAMTS-1.sub.cTSP-1WG/Sde1-SEQ ID NO:51), CSRTCGGG
(ADAMTS-1.sub.mTSP-1CSde1-SEQ ID NO:52) or CSKTCGS/KG
(ADAMTS-1.sub.cTSP-1CSde1-SEQ ID NO:53, FIG. 18).
[0190] 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-1 and fADAMTS-1.sub.cTSP-1-1,
or fADAMTS-1.sub.cTsp-1-2, or transfected with the expression
vector alone (the control).
[0191] 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.
[0192] 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
561968PRTMus musculus 1Met Gln Pro Lys Val Pro Leu Gly Ser Arg Lys
Gln Lys Pro Cys Ser 1 5 10 15 Asp Met Gly Asp Val Gln Arg Ala Ala
Arg Ser Arg Gly Ser Leu Ser 20 25 30 Ala His Met Leu Leu Leu Leu
Leu Ala Ser Ile Thr Met Leu Leu Cys 35 40 45 Ala Arg Gly Ala His
Gly Arg Pro Thr Glu Glu Asp Glu Glu Leu Val 50 55 60 Leu Pro Ser
Leu Glu Arg Ala Pro Gly His Asp Ser Thr Thr Thr Arg 65 70 75 80 Leu
Arg Leu Asp Ala Phe Gly Gln Gln Leu His Leu Lys Leu Gln Pro 85 90
95 Asp Ser Gly Phe Leu Ala Pro Gly Phe Thr Leu Gln Thr Val Gly Arg
100 105 110 Ser Pro Gly Ser Glu Ala Gln His Leu Asp Pro Thr Gly Asp
Leu Ala 115 120 125 His Cys Phe Tyr Ser Gly Thr Val Asn Gly Asp Pro
Gly Ser Ala Ala 130 135 140 Ala Leu Ser Leu Cys Glu Gly Val Arg Gly
Ala Phe Tyr Leu Gln Gly 145 150 155 160 Glu Glu Phe Phe Ile Gln Pro
Ala Pro Gly Val Ala Thr Glu Arg Leu 165 170 175 Ala Pro Ala Val Pro
Glu Glu Glu Ser Ser Ala Arg Pro Gln Phe His 180 185 190 Ile Leu Arg
Arg Arg Arg Arg Gly Ser Gly Gly Ala Lys Cys Gly Val 195 200 205 Met
Asp Asp Glu Thr Leu Pro Thr Ser Asp Ser Arg Pro Glu Ser Gln 210 215
220 Asn Thr Arg Asn Gln Trp Pro Val Arg Asp Pro Thr Pro Gln Asp Ala
225 230 235 240 Gly Lys Pro Ser Gly Pro Gly Ser Ile Arg Lys Lys Arg
Phe Val Ser 245 250 255 Ser Pro Arg Tyr Val Glu Thr Met Leu Val Ala
Asp Gln Ser Met Ala 260 265 270 Asp Phe His Gly Ser Gly Leu Lys His
Tyr Leu Leu Thr Leu Phe Ser 275 280 285 Val Ala Ala Arg Phe Tyr Lys
His Pro Ser Ile Arg Asn Ser Ile Ser 290 295 300 Leu Val Val Val Lys
Ile Leu Val Ile Tyr Glu Glu Gln Lys Gly Pro 305 310 315 320 Glu Val
Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp 325 330 335
Gln Lys Gln His Asn Ser Pro Ser Asp Arg Asp Pro Glu His Tyr Asp 340
345 350 Thr Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser His Thr
Cys 355 360 365 Asp Thr Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp
Pro Ser Arg 370 375 380 Ser Cys Ser Val Ile Glu Asp Asp Gly Leu Gln
Ala Ala Phe Thr Thr 385 390 395 400 Ala His Glu Leu Gly His Val Phe
Asn Met Pro His Asp Asp Ala Lys 405 410 415 His Cys Ala Ser Leu Asn
Gly Val Thr Gly Asp Ser His Leu Met Ala 420 425 430 Ser Met Leu Ser
Ser Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser 435 440 445 Ala Tyr
Met Val Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu 450 455 460
Met Asp Lys Pro Gln Asn Pro Ile Lys Leu Pro Ser Asp Leu Pro Gly 465
470 475 480 Thr Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly
Glu Glu 485 490 495 Ser Lys His Cys Pro Asp Ala Ala Ser Thr Cys Thr
Thr Leu Trp Cys 500 505 510 Thr Gly Thr Ser Gly Gly Leu Leu Val Cys
Gln Thr Lys His Phe Pro 515 520 525 Trp Ala Asp Gly Thr Ser Cys Gly
Glu Gly Lys Trp Cys Val Ser Gly 530 535 540 Lys Cys Val Asn Lys Thr
Asp Met Lys His Phe Ala Thr Pro Val His 545 550 555 560 Gly Ser Trp
Gly Pro Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys 565 570 575 Gly
Gly Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro 580 585
590 Lys Asn Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser
595 600 605 Cys Asn Ile Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe
Arg Glu 610 615 620 Glu Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala
Ser Phe Gly Asn 625 630 635 640 Glu Pro Thr Val Glu Trp Thr Pro Lys
Tyr Ala Gly Val Ser Pro Lys 645 650 655 Asp Arg Cys Lys Leu Thr Cys
Glu Ala Lys Gly Ile Gly Tyr Phe Phe 660 665 670 Val Leu Gln Pro Lys
Val Val Asp Gly Thr Pro Cys Ser Pro Asp Ser 675 680 685 Thr Ser Val
Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys Asp Arg 690 695 700 Ile
Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly 705 710
715 720 Asn Gly Ser Thr Cys Lys Lys Met Ser Gly Ile Val Thr Ser Thr
Arg 725 730 735 Pro Gly Tyr His Asp Ile Val Thr Ile Pro Ala Gly Ala
Thr Asn Ile 740 745 750 Glu Val Lys His Arg Asn Gln Arg Gly Ser Arg
Asn Asn Gly Ser Phe 755 760 765 Leu Ala Ile Arg Ala Ala Asp Gly Thr
Tyr Ile Leu Asn Gly Asn Phe 770 775 780 Thr Leu Ser Thr Leu Glu Gln
Asp Leu Thr Tyr Lys Gly Thr Val Leu 785 790 795 800 Arg Tyr Ser Gly
Ser Ser Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser 805 810 815 Pro Leu
Lys Glu Pro Leu Thr Ile Gln Val Leu Met Val Gly His Ala 820 825 830
Leu Arg Pro Lys Ile Lys Phe Thr Tyr Phe Met Lys Lys Lys Thr Glu 835
840 845 Ser Phe Asn Ala Ile Pro Thr Phe Ser Glu Trp Val Ile Glu Glu
Trp 850 855 860 Gly Glu Cys Ser Lys Thr Cys Gly Ser Gly Trp Gln Arg
Arg Val Val 865 870 875 880 Gln Cys Arg Asp Ile Asn Gly His Pro Ala
Ser Glu Cys Ala Lys Glu 885 890 895 Val Lys Pro Ala Ser Thr Arg Pro
Cys Ala Asp Leu Pro Cys Pro His 900 905 910 Trp Gln Val Gly Asp Trp
Ser Pro Cys Ser Lys Thr Cys Gly Lys Gly 915 920 925 Tyr Lys Lys Arg
Thr Leu Lys Cys Val Ser His Asp Gly Gly Val Leu 930 935 940 Ser Asn
Glu Ser Cys Asp Pro Leu Lys Lys Pro Lys His Tyr Ile Asp 945 950 955
960 Phe Cys Thr Leu Thr Gln Cys Ser 965 22910DNAMus 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 Ser 1 5 10 15 Asp Met Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg
Ser Phe Gly Pro 20 25 30 Val Pro Thr Leu Leu Leu Leu Ala Ala Ala
Leu Leu Ala Val Ser Asp 35 40 45 Ala Leu Gly Arg Pro Ser Glu Glu
Asp Glu Glu Leu Val Val Pro Glu 50 55 60 Leu Glu Arg Ala Pro Gly
His Gly Thr Thr Arg Leu Arg Leu His Ala 65 70 75 80 Phe Asp Gln Gln
Leu Asp Leu Glu Leu Arg Pro Asp Ser Ser Phe Leu 85 90 95 Ala Pro
Gly Phe Thr Leu Gln Asn Val Gly Arg Lys Ser Gly Ser Glu 100 105 110
Thr Pro Leu Pro Glu Thr Asp Leu Ala His Cys Phe Tyr Ser Gly Thr 115
120 125 Val Asn Gly Asp Pro Ser Ser Ala Ala Ala Leu Ser Leu Cys Glu
Gly 130 135 140 Val Arg Gly Ala Phe Tyr Leu Leu Gly Glu Ala Tyr Phe
Ile Gln Pro 145 150 155 160 Leu Pro Ala Ala Ser Glu Arg Leu Ala Thr
Ala Ala Pro Gly Glu Lys 165 170 175 Pro Pro Ala Pro Leu Gln Phe His
Leu Leu Arg Arg Asn Arg Gln Gly 180 185 190 Asp Val Gly Gly Thr Cys
Gly Val Val Asp Asp Glu Pro Arg Pro Thr 195 200 205 Gly Lys Ala Glu
Thr Glu Asp Glu Asp Glu Gly Thr Glu Gly Glu Asp 210 215 220 Glu Gly
Ala Gln Trp Ser Pro Gln Asp Pro Ala Leu Gln Gly Val Gly 225 230 235
240 Gln Pro Thr Gly Thr Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser
245 250 255 His Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met
Ala Glu 260 265 270 Phe His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr
Leu Phe Ser Val 275 280 285 Ala Ala Arg Leu Tyr Lys His Pro Ser Ile
Arg Asn Ser Val Ser Leu 290 295 300 Val Val Val Lys Ile Leu Val Ile
His Asp Glu Gln Lys Gly Pro Glu 305 310 315 320 Val Thr Ser Asn Ala
Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp Gln 325 330 335 Lys Gln His
Asn Pro Pro Ser Asp Arg Asp Ala Glu His Tyr Asp Thr 340 345 350 Ala
Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser Gln Thr Cys Asp 355 360
365 Thr Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser
370 375 380 Cys Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr
Thr Ala 385 390 395 400 His Glu Leu Gly His Val Phe Asn Met Pro His
Asp Asp Ala Lys Gln 405 410 415 Cys Ala Ser Leu Asn Gly Val Asn Gln
Asp Ser His Met Met Ala Ser 420 425 430 Met Leu Ser Asn Leu Asp His
Ser Gln Pro Trp Ser Pro Cys Ser Ala 435 440 445 Tyr Met Ile Thr Ser
Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met 450 455 460 Asp Lys Pro
Gln Asn Pro Ile Gln Leu Pro Gly Asp Leu Pro Gly Thr 465 470 475 480
Ser Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Asp Ser 485
490 495 Lys His Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys
Thr 500 505 510 Gly Thr Ser Gly Gly Val Leu Val Cys Gln Thr Lys His
Phe Pro Trp 515 520 525 Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp
Cys Ile Asn Gly Lys 530 535 540 Cys Val Asn Lys Thr Asp Arg Lys His
Phe Asp Thr Pro Phe His Gly 545 550 555 560 Ser Trp Gly Met Trp Gly
Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly 565 570 575 Gly Gly Val Gln
Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro Lys 580 585 590 Asn Gly
Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys 595 600 605
Asn Leu Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe Arg Glu Glu 610
615 620 Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe Gly Ser
Gly 625 630 635 640 Pro Ala Val Glu Trp Ile Pro Lys Tyr Ala Gly Val
Ser Pro Lys Asp 645 650 655 Arg Cys Lys Leu Ile Cys Gln Ala Lys Gly
Ile Gly Tyr Phe Phe Val 660 665 670 Leu Gln Pro Lys Val Val Asp Gly
Thr Pro Cys Ser Pro Asp Ser Thr 675 680 685 Ser Val Cys Val Gln Gly
Gln Cys Val Lys Ala Gly Cys Asp Arg Ile 690 695 700 Ile Asp Ser Lys
Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly Asn 705 710 715 720 Gly
Ser Thr Cys Lys Lys Ile Ser Gly Ser Val Thr Ser Ala Lys Pro 725 730
735 Gly Tyr His Asp Ile Ile Thr Ile Pro Thr Gly Ala Thr Asn Ile Glu
740 745 750 Val Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn Asn Gly Ser
Phe Leu 755 760 765 Ala Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu Asn
Gly Asp Tyr Thr 770 775 780 Leu Ser Thr Leu Glu Gln Asp Ile Met Tyr
Lys Gly Val Val Leu Arg 785 790 795 800 Tyr Ser Gly Ser Ser Ala Ala
Leu Glu Arg Ile Arg Ser Phe Ser Pro 805 810 815 Leu Lys Glu Pro Leu
Thr Ile Gln Val Leu Thr Val Gly Asn Ala Leu 820 825 830 Arg Pro Lys
Ile Lys Tyr Thr Tyr Phe Val Lys Lys Lys Lys Glu Ser 835 840 845 Phe
Asn Ala Ile Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp Gly 850 855
860 Glu Cys Ser Lys Ser Cys Glu Leu Gly Trp Gln Arg Arg Leu Val Glu
865 870 875
880 Cys Arg Asp Ile Asn Gly Gln Pro Ala Ser Glu Cys Ala Lys Glu Val
885 890 895 Lys Pro Ala Ser Thr Arg Pro Cys Ala Asp His Pro Cys Pro
Gln Trp 900 905 910 Gln Leu Gly Glu Trp Ser Ser Cys Ser Lys Thr Cys
Gly Lys Gly Tyr 915 920 925 Lys Lys Arg Ser Leu Lys Cys Leu Ser His
Asp Gly Gly Val Leu Ser 930 935 940 His Glu Ser Cys Asp Pro Leu Lys
Lys Pro Lys His Phe Ile Asp Phe 945 950 955 960 Cys Thr Met Ala Glu
Cys Ser 965 42907DNAHomo 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 Ala 1 5 10 15 His Met Leu Leu
Leu Leu Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20 25 30 Arg Gly
Ala His Gly Arg Pro Thr Glu Lys Lys Lys Thr Glu Ser Phe 35 40 45
Asn Ala Ile Pro Thr Phe Ser Glu Trp Val Ile Glu Glu Trp Gly Glu 50
55 60 Cys Ser Lys Thr Cys Gly Ser Gly Trp Gln Arg Arg Val Val Gln
Cys 65 70 75 80 Arg Asp Ile Asn Gly His Pro Ala Ser Glu Cys Ala Lys
Glu Val Lys 85 90 95 Pro Ala Ser Thr Arg Pro Cys Ala Asp Leu Pro
Cys Pro His Trp Gln 100 105 110 Val Gly Asp Trp Ser Pro Cys Ser Lys
Thr Cys Gly Lys Gly Tyr Lys 115 120 125 Lys Arg Thr Leu Lys Cys Val
Ser His Asp Gly Gly Val Leu Ser Asn 130 135 140 Glu Ser Cys Asp Pro
Leu Lys Lys Pro Lys His Tyr Ile Asp Phe Cys 145 150 155 160 Thr Leu
Thr Gln Cys Ser 165 6504DNAMus 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 Ser 1 5 10 15 Asp
Met Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro 20 25
30 Val Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp
35 40 45 Ala Leu Gly Arg Pro Ser Glu Lys Lys Lys Glu Ser Phe Asn
Ala Ile 50 55 60 Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp Gly
Glu Cys Ser Lys 65 70 75 80 Ser Cys Glu Leu Gly Trp Gln Arg Arg Leu
Val Glu Cys Arg Asp Ile 85 90 95 Asn Gly Gln Pro Ala Ser Glu Cys
Ala Lys Glu Val Lys Pro Ala Ser 100 105 110 Thr Arg Pro Cys Ala Asp
His Pro Cys Pro Gln Trp Gln Leu Gly Glu 115 120 125 Trp Ser Ser Cys
Ser Lys Thr Cys Gly Lys Gly Tyr Lys Lys Arg Ser 130 135 140 Leu Lys
Cys Leu Ser His Asp Gly Gly Val Leu Ser His Glu Ser Cys 145 150 155
160 Asp Pro Leu Lys Lys Pro Lys His Phe Ile Asp Phe Cys Thr Met Ala
165 170 175 Glu Cys Ser 8543DNAHomo 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 Ala 1 5 10 15 His Met Leu Leu
Leu Leu Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20 25 30 Arg Gly
Ala His Gly Arg Pro Thr Glu Glu Asp Glu Glu Leu Val Leu 35 40 45
Pro Ser Leu Glu Arg Ala Pro Gly His Asp Ser Thr Thr Thr Arg Leu 50
55 60 Arg Leu Asp Ala Phe Gly Gln Gln Leu His Leu Lys Leu Gln Pro
Asp 65 70 75 80 Ser Gly Phe Leu Ala Pro Gly Phe Thr Leu Gln Thr Val
Gly Arg Ser 85 90 95 Pro Gly Ser Glu Ala Gln His Leu Asp Pro Thr
Gly Asp Leu Ala His 100 105 110 Cys Phe Tyr Ser Gly Thr Val Asn Gly
Asp Pro Gly Ser Ala Ala Ala 115 120 125 Leu Ser Leu Cys Glu Gly Val
Arg Gly Ala Phe Tyr Leu Gln Gly Glu 130 135 140 Glu Phe Phe Ile Gln
Pro Ala Pro Gly Val Ala Thr Glu Arg Leu Ala 145 150 155 160 Pro Ala
Val Pro Glu Glu Glu Ser Ser Ala Arg Pro Gln Phe His Ile 165 170 175
Leu Arg Arg Arg Arg Arg Gly Ser Gly Gly Ala Lys Cys Gly Val Met 180
185 190 Asp Asp Glu Thr Leu Pro Thr Ser Asp Ser Arg Pro Glu Ser Gln
Asn 195 200 205 Thr Arg Asn Gln Trp Pro Val Arg Asp Pro Thr Pro Gln
Asp Ala Gly 210 215 220 Lys Pro Ser Gly Pro Gly Ser Ile Arg Lys Lys
Arg Phe Val Ser Ser 225 230 235 240 Pro Arg Tyr Val Glu Thr Met Leu
Val Ala Asp Gln Ser Met Ala Asp 245 250 255 Phe His Gly Ser Gly Leu
Lys His Tyr Leu Leu Thr Leu Phe Ser Val 260 265 270 Ala Ala Arg Phe
Tyr Lys His Pro Ser Ile Arg Asn Ser Ile Ser Leu 275 280 285 Val Val
Val Lys Ile Leu Val Ile Tyr Glu Glu Gln Lys Gly Pro Glu 290 295 300
Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp Gln 305
310 315 320 Lys Gln His Asn Ser Pro Ser Asp Arg Asp Pro Glu His Tyr
Asp Thr 325 330 335 Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser
His Thr Cys Asp 340 345 350 Thr Leu Gly Met Ala Asp Val Gly Thr Val
Cys Asp Pro Ser Arg Ser 355 360 365 Cys Ser Val Ile Glu Asp Asp Gly
Leu Gln Ala Ala Phe Thr Thr Ala 370 375 380 His Glu Leu Gly His Val
Phe Asn Met Pro His Asp Asp Ala Lys His 385 390 395 400 Cys Ala Ser
Leu Asn Gly Val Thr Gly Asp Ser His Leu Met Ala Ser 405 410 415 Met
Leu Ser Ser Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala 420 425
430 Tyr Met Val Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met
435 440 445 Asp Lys Pro Gln Asn Pro Ile Lys Leu Pro Ser Asp Leu Pro
Gly Thr 450 455 460 Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe
Gly Glu Glu Ser 465 470 475 480 Lys His Cys Pro Asp Ala Ala Ser Thr
Cys Thr Thr Leu Trp Cys Thr 485 490 495 Gly Thr Ser Gly Gly Leu Leu
Val Cys Gln Thr Lys His Phe Pro Trp 500 505 510 Ala Asp Gly Thr Ser
Cys Gly Glu Gly Lys Trp Cys Val Ser Gly Lys 515 520 525 Cys Val Asn
Lys Thr Asp Met Lys His Phe Ala Thr Pro Val His Gly 530 535 540 Ser
Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly 545 550
555 560 Gly Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro
Lys 565 570 575 Asn Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr
Arg Ser Cys 580 585 590 Asn Ile Glu Asp 595 101794DNAMus 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 Val 1 5 10 15 Pro
Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp Ala 20 25
30 Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu Leu Val Val Pro Glu Leu
35 40 45 Glu Arg Ala Pro Gly His Gly Thr Thr Arg Leu Arg Leu His
Ala Phe 50 55 60 Asp Gln Gln Leu Asp Leu Glu Leu Arg Pro Asp Ser
Ser Phe Leu Ala 65 70 75 80 Pro Gly Phe Thr Leu Gln Asn Val Gly Arg
Lys Ser Gly Ser Glu Thr 85 90 95 Pro Leu Pro Glu Thr Asp Leu Ala
His Cys Phe Tyr Ser Gly Thr Val 100 105 110 Asn Gly Asp Pro Ser Ser
Ala Ala Ala Leu Ser Leu Cys Glu Gly Val 115 120 125 Arg Gly Ala Phe
Tyr Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro Leu 130 135 140 Pro Ala
Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu Lys Pro 145 150 155
160 Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg Asn Arg Gln Gly Asp
165 170 175 Val Gly Gly Thr Cys Gly Val Val Asp Asp Glu Pro Arg Pro
Thr Gly 180 185 190 Lys Ala Glu Thr Glu Asp Glu Asp Glu Gly Thr Glu
Gly Glu Asp Glu 195 200 205 Gly Ala Gln Trp Ser Pro Gln Asp Pro Ala
Leu Gln Gly Val Gly Gln 210 215 220 Pro Thr Gly Thr Gly Ser Ile Arg
Lys Lys Arg Phe Val Ser Ser His 225 230 235 240 Arg Tyr Val Glu Thr
Met Leu Val Ala Asp Gln Ser Met Ala Glu Phe 245 250 255 His Gly Ser
Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val Ala 260 265 270 Ala
Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu Val 275 280
285 Val Val Lys Ile Leu Val Ile His Asp Glu Gln Lys Gly Pro Glu Val
290 295 300 Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp
Gln Lys 305 310 315 320 Gln His Asn Pro Pro Ser Asp Arg Asp Ala Glu
His Tyr Asp Thr Ala 325 330 335 Ile Leu Phe Thr Arg Gln Asp Leu Cys
Gly Ser Gln Thr Cys Asp Thr 340 345 350 Leu Gly Met Ala Asp Val Gly
Thr Val
Cys Asp Pro Ser Arg Ser Cys 355 360 365 Ser Val Ile Glu Asp Asp Gly
Leu Gln Ala Ala Phe Thr Thr Ala His 370 375 380 Glu Leu Gly His Val
Phe Asn Met Pro His Asp Asp Ala Lys Gln Cys 385 390 395 400 Ala Ser
Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser Met 405 410 415
Leu Ser Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala Tyr 420
425 430 Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met
Asp 435 440 445 Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp Leu Pro
Gly Thr Ser 450 455 460 Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe
Gly Glu Asp Ser Lys 465 470 475 480 His Cys Pro Asp Ala Ala Ser Thr
Cys Ser Thr Leu Trp Cys Thr Gly 485 490 495 Thr Ser Gly Gly Val Leu
Val Cys Gln Thr Lys His Phe Pro Trp Ala 500 505 510 Asp Gly Thr Ser
Cys Gly Glu Gly Lys Trp Cys Ile Asn Gly Lys Cys 515 520 525 Val Asn
Lys Thr Asp Arg Lys His Phe Asp Thr Pro Phe His Gly Ser 530 535 540
Trp Gly Met Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly 545
550 555 560 Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro
Lys Asn 565 570 575 Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr
Arg Ser Cys Asn 580 585 590 Leu Glu Asp 595 121842DNAHomo 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 Ala 1 5 10 15 His Met
Leu Leu Leu Leu Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20 25 30
Arg Gly Ala His Gly Arg Pro Thr Glu Glu Asp Glu Glu Leu Val Leu 35
40 45 Pro Ser Leu Glu Arg Ala Pro Gly His Asp Ser Thr Thr Thr Arg
Leu 50 55 60 Arg Leu Asp Ala Phe Gly Gln Gln Leu His Leu Lys Leu
Gln Pro Asp 65 70 75 80 Ser Gly Phe Leu Ala Pro Gly Phe Thr Leu Gln
Thr Val Gly Arg Ser 85 90 95 Pro Gly Ser Glu Ala Gln His Leu Asp
Pro Thr Gly Asp Leu Ala His 100 105 110 Cys Phe Tyr Ser Gly Thr Val
Asn Gly Asp Pro Gly Ser Ala Ala Ala 115 120 125 Leu Ser Leu Cys Glu
Gly Val Arg Gly Ala Phe Tyr Leu Gln Gly Glu 130 135 140 Glu Phe Phe
Ile Gln Pro Ala Pro Gly Val Ala Thr Glu Arg Leu Ala 145 150 155 160
Pro Ala Val Pro Glu Glu Glu Ser Ser Ala Arg Pro Gln Phe His Ile 165
170 175 Leu Arg Arg Arg Arg Arg Gly Ser Gly Gly Ala Lys Cys Gly Val
Met 180 185 190 Asp Asp Glu Thr Leu Pro Thr Ser Asp Ser Arg Pro Glu
Ser Gln Asn 195 200 205 Thr Arg Asn Gln Trp Pro Val Arg Asp Pro Thr
Pro Gln Asp Ala Gly 210 215 220 Lys Pro Ser Gly Pro Gly Ser Ile Arg
Lys Lys Arg Phe Val Ser Ser 225 230 235 240 Pro Arg Tyr Val Glu Thr
Met Leu Val Ala Asp Gln Ser Met Ala Asp 245 250 255 Phe His Gly Ser
Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val 260 265 270 Ala Ala
Arg Phe Tyr Lys His Pro Ser Ile Arg Asn Ser Ile Ser Leu 275 280 285
Val Val Val Lys Ile Leu Val Ile Tyr Glu Glu Gln Lys Gly Pro Glu 290
295 300 Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp
Gln 305 310 315 320 Lys Gln His Asn Ser Pro Ser Asp Arg Asp Pro Glu
His Tyr Asp Thr 325 330 335 Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys
Gly Ser His Thr Cys Asp 340 345 350 Thr Leu Gly Met Ala Asp Val Gly
Thr Val Cys Asp Pro Ser Arg Ser 355 360 365 Cys Ser Val Ile Glu Asp
Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala 370 375 380 His Glu Leu Gly
His Val Phe Asn Met Pro His Asp Asp Ala Lys His 385 390 395 400 Cys
Ala Ser Leu Asn Gly Val Thr Gly Asp Ser His Leu Met Ala Ser 405 410
415 Met Leu Ser Ser Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala
420 425 430 Tyr Met Val Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys
Leu Met 435 440 445 Asp Lys Pro Gln Asn Pro Ile Lys Leu Pro Ser Asp
Leu Pro Gly Thr 450 455 460 Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe
Thr Phe Gly Glu Glu Ser 465 470 475 480 Lys His Cys Pro Asp Ala Ala
Ser Thr Cys Thr Thr Leu Trp Cys Thr 485 490 495 Gly Thr Ser Gly Gly
Leu Leu Val Cys Gln Thr Lys His Phe Pro Trp 500 505 510 Ala Asp Gly
Thr Ser Cys Gly Glu Gly Lys Trp Cys Val Ser Gly Lys 515 520 525 Cys
Val Asn Lys Thr Asp Met Lys His Phe Ala Thr Pro Val His Gly 530 535
540 Ser 545 141641DNAMus 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 Ser 1 5 10 15 Asp Met Gly Asn Ala
Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro 20 25 30 Val Pro Thr
Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp 35 40 45 Ala
Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu Leu Val Val Pro Glu 50 55
60 Leu Glu Arg Ala Pro Gly His Gly Thr Thr Arg Leu Arg Leu His Ala
65 70 75 80 Phe Asp Gln Gln Leu Asp Leu Glu Leu Arg Pro Asp Ser Ser
Phe Leu 85 90 95 Ala Pro Gly Phe Thr Leu Gln Asn Val Gly Arg Lys
Ser Gly Ser Glu 100 105 110 Thr Pro Leu Pro Glu Thr Asp Leu Ala His
Cys Phe Tyr Ser Gly Thr 115 120 125 Val Asn Gly Asp Pro Ser Ser Ala
Ala Ala Leu Ser Leu Cys Glu Gly 130 135 140 Val Arg Gly Ala Phe Tyr
Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro 145 150 155 160 Leu Pro Ala
Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu Lys 165 170 175 Pro
Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg Asn Arg Gln Gly 180 185
190 Asp Val Gly Gly Thr Cys Gly Val Val Asp Asp Glu Pro Arg Pro Thr
195 200 205 Gly Lys Ala Glu Thr Glu Asp Glu Asp Glu Gly Thr Glu Gly
Glu Asp 210 215 220 Glu Gly Ala Gln Trp Ser Pro Gln Asp Pro Ala Leu
Gln Gly Val Gly 225 230 235 240 Gln Pro Thr Gly Thr Gly Ser Ile Arg
Lys Lys Arg Phe Val Ser Ser 245 250 255 His Arg Tyr Val Glu Thr Met
Leu Val Ala Asp Gln Ser Met Ala Glu 260 265 270 Phe His Gly Ser Gly
Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val 275 280 285 Ala Ala Arg
Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu 290 295 300 Val
Val Val Lys Ile Leu Val Ile His Asp Glu Gln Lys Gly Pro Glu 305 310
315 320 Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp
Gln 325 330 335 Lys Gln His Asn Pro Pro Ser Asp Arg Asp Ala Glu His
Tyr Asp Thr 340 345 350 Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly
Ser Gln Thr Cys Asp 355 360 365 Thr Leu Gly Met Ala Asp Val Gly Thr
Val Cys Asp Pro Ser Arg Ser 370 375 380 Cys Ser Val Ile Glu Asp Asp
Gly Leu Gln Ala Ala Phe Thr Thr Ala 385 390 395 400 His Glu Leu Gly
His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln 405 410 415 Cys Ala
Ser Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser 420 425 430
Met Leu Ser Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala 435
440 445 Tyr Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu
Met 450 455 460 Asp Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp Leu
Pro Gly Thr 465 470 475 480 Ser Tyr Asp Ala Asn Arg Gln Cys Gln Phe
Thr Phe Gly Glu Asp Ser 485 490 495 Lys His Cys Pro Asp Ala Ala Ser
Thr Cys Ser Thr Leu Trp Cys Thr 500 505 510 Gly Thr Ser Gly Gly Val
Leu Val Cys Gln Thr Lys His Phe Pro Trp 515 520 525 Ala Asp Gly Thr
Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn Gly Lys 530 535 540 Cys Val
Asn Lys Thr Asp Arg Lys His Phe Asp Thr Pro Phe His Gly 545 550 555
560 Ser 161689DNAHomo 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 Ala 1 5 10 15 His Met Leu Leu Leu Leu Leu Ala
Ser Ile Thr Met Leu Leu Cys Ala 20 25 30 Arg Gly Ala His Gly Arg
Pro Thr Glu Lys Lys Met Ser Gly Ile Val 35 40 45 Thr Ser Thr Arg
Pro Gly Tyr His Asp Ile Val Thr Ile Pro Ala Gly 50 55 60 Ala Thr
Asn Ile Glu Val Lys His Arg Asn Gln Arg Gly Ser Arg Asn 65 70 75 80
Asn Gly Ser Phe Leu Ala Ile Arg Ala Ala Asp Gly Thr Tyr Ile Leu 85
90 95 Asn Gly Asn Phe Thr Leu Ser Thr Leu Glu Gln Asp Leu Thr Tyr
Lys 100 105 110 Gly Thr Val Leu Arg Tyr Ser Gly Ser Ser Ala Ala Leu
Glu Arg Ile 115 120 125 Arg Ser Phe Ser Pro Leu Lys Glu Pro Leu Thr
Ile Gln
Val Leu Met 130 135 140 Val Gly His Ala Leu Arg Pro Lys Ile Lys Phe
Thr Tyr Phe Met 145 150 155 18483DNAMus 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 Ser 1 5 10 15 Asp Met Gly Asn
Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro 20 25 30 Val Pro
Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp 35 40 45
Ala Leu Gly Arg Pro Ser Glu Lys Lys Ile Ser Gly Ser Val Thr Ser 50
55 60 Ala Lys Pro Gly Tyr His Asp Ile Ile Thr Ile Pro Thr Gly Ala
Thr 65 70 75 80 Asn Ile Glu Val Lys Gln Arg Asn Gln Arg Gly Ser Arg
Asn Asn Gly 85 90 95 Ser Phe Leu Ala Ile Lys Ala Ala Asp Gly Thr
Tyr Ile Leu Asn Gly 100 105 110 Asp Tyr Thr Leu Ser Thr Leu Glu Gln
Asp Ile Met Tyr Lys Gly Val 115 120 125 Val Leu Arg Tyr Ser Gly Ser
Ser Ala Ala Leu Glu Arg Ile Arg Ser 130 135 140 Phe Ser Pro Leu Lys
Glu Pro Leu Thr Ile Gln Val Leu Thr Val Gly 145 150 155 160 Asn Ala
Leu Arg Pro Lys Ile Lys Tyr Thr Tyr Phe Val 165 170 20525DNAHomo
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 Ala 1 5 10 15 His Met Leu Leu
Leu Leu Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20 25 30 Arg Gly
Ala His Gly Arg Pro Thr Glu Cys Pro Asp Asn Asn Gly Lys 35 40 45
Thr Phe Arg Glu Glu Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala 50
55 60 Ser Phe Gly Asn Glu Pro Thr Val Glu Trp Thr Pro Lys Tyr Ala
Gly 65 70 75 80 Val Ser Pro Lys Asp Arg Cys Lys Leu Thr Cys Glu Ala
Lys Gly Ile 85 90 95 Gly Tyr Phe Phe Val Leu Gln Pro Lys Val Val
Asp Gly Thr Pro Cys 100 105 110 Ser Pro Asp Ser Thr Ser Val Cys Val
Gln Gly Gln Cys Val Lys Ala 115 120 125 Gly Cys Asp Arg Ile Ile Asp
Ser Lys Lys Lys Phe Asp Lys Cys Gly 130 135 140 Val Cys Gly Gly Asn
Gly Ser Thr Cys Lys Lys Met Ser Gly Ile Val 145 150 155 160 Thr Ser
Thr Arg Pro Gly Tyr His Asp Ile Val Thr Ile Pro Ala Gly 165 170 175
Ala Thr Asn Ile Glu Val Lys His Arg Asn Gln Arg Gly Ser Arg Asn 180
185 190 Asn Gly Ser Phe Leu Ala Ile Arg Ala Ala Asp Gly Thr Tyr Ile
Leu 195 200 205 Asn Gly Asn Phe Thr Leu Ser Thr Leu Glu Gln Asp Leu
Thr Tyr Lys 210 215 220 Gly Thr Val Leu Arg Tyr Ser Gly Ser Ser Ala
Ala Leu Glu Arg Ile 225 230 235 240 Arg Ser Phe Ser Pro Leu Lys Glu
Pro Leu Thr Ile Gln Val Leu Met 245 250 255 Val Gly His Ala Leu Arg
Pro Lys Ile Lys Phe Thr Tyr Phe Met 260 265 270 22819DNAMus
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 Ser 1 5 10 15 Asp Met Gly Asn Ala Glu Arg Ala
Pro Gly Ser Arg Ser Phe Gly Pro 20 25 30 Val Pro Thr Leu Leu Leu
Leu Ala Ala Ala Leu Leu Ala Val Ser Asp 35 40 45 Ala Leu Gly Arg
Pro Ser Glu Cys Pro Asp Asn Asn Gly Lys Thr Phe 50 55 60 Arg Glu
Glu Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe 65 70 75 80
Gly Ser Gly Pro Ala Val Glu Trp Ile Pro Lys Tyr Ala Gly Val Ser 85
90 95 Pro Lys Asp Arg Cys Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly
Tyr 100 105 110 Phe Phe Val Leu Gln Pro Lys Val Val Asp Gly Thr Pro
Cys Ser Pro 115 120 125 Asp Ser Thr Ser Val Cys Val Gln Gly Gln Cys
Val Lys Ala Gly Cys 130 135 140 Asp Arg Ile Ile Asp Ser Lys Lys Lys
Phe Asp Lys Cys Gly Val Cys 145 150 155 160 Gly Gly Asn Gly Ser Thr
Cys Lys Lys Ile Ser Gly Ser Val Thr Ser 165 170 175 Ala Lys Pro Gly
Tyr His Asp Ile Ile Thr Ile Pro Thr Gly Ala Thr 180 185 190 Asn Ile
Glu Val Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn Asn Gly 195 200 205
Ser Phe Leu Ala Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu Asn Gly 210
215 220 Asp Tyr Thr Leu Ser Thr Leu Glu Gln Asp Ile Met Tyr Lys Gly
Val 225 230 235 240 Val Leu Arg Tyr Ser Gly Ser Ser Ala Ala Leu Glu
Arg Ile Arg Ser 245 250 255 Phe Ser Pro Leu Lys Glu Pro Leu Thr Ile
Gln Val Leu Thr Val Gly 260 265 270 Asn Ala Leu Arg Pro Lys Ile Lys
Tyr Thr Tyr Phe Val 275 280 285 24861PRTHomo sapiens 24Gly Cys Ala
Ala Thr Gly Cys Ala Gly Cys Gly Ala Gly Cys Thr Gly 1 5 10 15 Thr
Gly Cys Cys Cys Gly Ala Gly Gly Gly Gly Thr Thr Cys Gly Gly 20 25
30 Ala Ala Gly Gly Cys Gly Cys Ala Ala Gly Cys Thr Gly Gly Gly Cys
35 40 45 Ala Gly Cys Gly Ala Cys Ala Thr Gly Gly Gly Gly Ala Ala
Cys Gly 50 55 60 Cys Gly Gly Ala Gly Cys Gly Gly Gly Cys Thr Cys
Cys Gly Gly Gly 65 70 75 80 Gly Thr Cys Thr Cys Gly Gly Ala Gly Cys
Thr Thr Thr Gly Gly Gly 85 90 95 Cys Cys Cys Gly Thr Ala Cys Cys
Cys Ala Cys Gly Cys Thr Gly Cys 100 105 110 Thr Gly Cys Thr Gly Cys
Thr Cys Gly Cys Cys Gly Cys Gly Gly Cys 115 120 125 Gly Cys Thr Ala
Cys Thr Gly Gly Cys Cys Gly Thr Gly Thr Cys Gly 130 135 140 Gly Ala
Cys Gly Cys Ala Cys Thr Cys Gly Gly Gly Cys Gly Cys Cys 145 150 155
160 Cys Cys Thr Cys Cys Gly Ala Gly Thr Gly Thr Cys Cys Ala Gly Ala
165 170 175 Cys Ala Ala Thr Ala Ala Thr Gly Gly Ala Ala Ala Ala Ala
Cys Cys 180 185 190 Thr Thr Thr Ala Gly Ala Gly Ala Gly Gly Ala Ala
Cys Ala Ala Thr 195 200 205 Gly Thr Gly Ala Ala Gly Cys Ala Cys Ala
Cys Ala Ala Cys Gly Ala 210 215 220 Gly Thr Thr Thr Thr Cys Ala Ala
Ala Ala Gly Cys Thr Thr Cys Cys 225 230 235 240 Thr Thr Thr Gly Gly
Gly Ala Gly Thr Gly Gly Gly Cys Cys Thr Gly 245 250 255 Cys Gly Gly
Thr Gly Gly Ala Ala Thr Gly Gly Ala Thr Thr Cys Cys 260 265 270 Cys
Ala Ala Gly Thr Ala Cys Gly Cys Thr Gly Gly Cys Gly Thr Cys 275 280
285 Thr Cys Ala Cys Cys Ala Ala Ala Gly Gly Ala Cys Ala Gly Gly Thr
290 295 300 Gly Cys Ala Ala Gly Cys Thr Cys Ala Thr Cys Thr Gly Cys
Cys Ala 305 310 315 320 Ala Gly Cys Cys Ala Ala Ala Gly Gly Cys Ala
Thr Thr Gly Gly Cys 325 330 335 Thr Ala Cys Thr Thr Cys Thr Thr Cys
Gly Thr Thr Thr Thr Gly Cys 340 345 350 Ala Gly Cys Cys Cys Ala Ala
Gly Gly Thr Thr Gly Thr Ala Gly Ala 355 360 365 Thr Gly Gly Thr Ala
Cys Thr Cys Cys Ala Thr Gly Thr Ala Gly Cys 370 375 380 Cys Cys Ala
Gly Ala Thr Thr Cys Cys Ala Cys Cys Thr Cys Thr Gly 385 390 395 400
Thr Cys Thr Gly Thr Gly Thr Gly Cys Ala Ala Gly Gly Ala Cys Ala 405
410 415 Gly Thr Gly Thr Gly Thr Ala Ala Ala Ala Gly Cys Thr Gly Gly
Thr 420 425 430 Thr Gly Thr Gly Ala Thr Cys Gly Cys Ala Thr Cys Ala
Thr Ala Gly 435 440 445 Ala Cys Thr Cys Cys Ala Ala Ala Ala Ala Gly
Ala Ala Gly Thr Thr 450 455 460 Thr Gly Ala Thr Ala Ala Ala Thr Gly
Thr Gly Gly Thr Gly Thr Thr 465 470 475 480 Thr Gly Cys Gly Gly Gly
Gly Gly Ala Ala Ala Thr Gly Gly Ala Thr 485 490 495 Cys Thr Ala Cys
Thr Thr Gly Thr Ala Ala Ala Ala Ala Ala Ala Thr 500 505 510 Ala Thr
Cys Ala Gly Gly Ala Thr Cys Ala Gly Thr Thr Ala Cys Thr 515 520 525
Ala Gly Thr Gly Cys Ala Ala Ala Ala Cys Cys Thr Gly Gly Ala Thr 530
535 540 Ala Thr Cys Ala Thr Gly Ala Thr Ala Thr Cys Ala Thr Cys Ala
Cys 545 550 555 560 Ala Ala Thr Thr Cys Cys Ala Ala Cys Thr Gly Gly
Ala Gly Cys Cys 565 570 575 Ala Cys Cys Ala Ala Cys Ala Thr Cys Gly
Ala Ala Gly Thr Gly Ala 580 585 590 Ala Ala Cys Ala Gly Cys Gly Gly
Ala Ala Cys Cys Ala Gly Ala Gly 595 600 605 Gly Gly Gly Ala Thr Cys
Cys Ala Gly Gly Ala Ala Cys Ala Ala Thr 610 615 620 Gly Gly Cys Ala
Gly Cys Thr Thr Thr Cys Thr Thr Gly Cys Cys Ala 625 630 635 640 Thr
Cys Ala Ala Ala Gly Cys Thr Gly Cys Thr Gly Ala Thr Gly Gly 645 650
655 Cys Ala Cys Ala Thr Ala Thr Ala Thr Thr Cys Thr Thr Ala Ala Thr
660 665 670 Gly Gly Thr Gly Ala Cys Thr Ala Cys Ala Cys Thr Thr Thr
Gly Thr 675 680 685 Cys Cys Ala Cys Cys Thr Thr Ala Gly Ala Gly Cys
Ala Ala Gly Ala 690 695 700 Cys Ala Thr Thr Ala Thr Gly Thr Ala Cys
Ala Ala Ala Gly Gly Thr 705 710 715 720 Gly Thr Thr Gly Thr Cys Thr
Thr Gly Ala Gly Gly Thr Ala Cys Ala 725 730 735 Gly Cys Gly Gly Cys
Thr Cys Cys Thr Cys Thr Gly Cys Gly Gly Cys 740 745 750 Ala Thr Thr
Gly Gly Ala Ala Ala Gly Ala Ala Thr Thr Cys Gly Cys 755 760 765 Ala
Gly Cys Thr Thr Thr Ala Gly Cys Cys Cys Thr Cys Thr Cys Ala 770 775
780 Ala Ala Gly Ala Gly Cys Cys Cys Thr Thr Gly Ala Cys Cys Ala Thr
785 790 795 800 Cys Cys Ala Gly Gly Thr Thr Cys Thr Thr Ala Cys Thr
Gly Thr Gly 805 810 815 Gly Gly Cys Ala Ala Thr Gly Cys Cys Cys Thr
Thr Cys Gly Ala Cys 820 825 830 Cys Thr Ala Ala Ala Ala Thr Thr Ala
Ala Ala Thr Ala Cys Ala Cys 835 840 845 Cys Thr Ala Cys Thr Thr Cys
Gly Thr Ala Thr Ala Ala 850 855 860 25447PRTMus musculus 25Met Gly
Asp Val Gln Arg Ala Ala Arg Ser Arg Gly Ser Leu Ser Ala 1 5 10 15
His Met Leu Leu Leu Leu Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20
25 30 Arg Gly Ala His Gly Arg Pro Thr Glu Trp Gly Pro Trp Gly Pro
Trp 35 40 45 Gly Asp Cys Ser Arg Thr Cys Gly Gly Gly Val Gln Tyr
Thr Met Arg 50 55 60 Glu Cys Asp Asn Pro Val Pro Lys Asn Gly Gly
Lys Tyr Cys Glu Gly 65 70 75 80 Lys Arg Val Arg Tyr Arg Ser Cys Asn
Ile Glu Asp Cys Pro Asp Asn 85 90 95 Asn Gly Lys Thr Phe Arg Glu
Glu Gln Cys Glu Ala His Asn Glu Phe 100 105 110 Ser Lys Ala Ser Phe
Gly Asn Glu Pro Thr Val Glu Trp Thr Pro Lys 115 120 125 Tyr Ala Gly
Val Ser Pro Lys Asp Arg Cys Lys Leu Thr Cys Glu Ala 130 135 140 Lys
Gly Ile Gly Tyr Phe Phe Val Leu Gln Pro Lys Val Val Asp Gly 145 150
155 160 Thr Pro Cys Ser Pro Asp Ser Thr Ser Val Cys Val Gln Gly Gln
Cys 165 170 175 Val Lys Ala Gly Cys Asp Arg Ile Ile Asp Ser Lys Lys
Lys Phe Asp 180 185 190 Lys Cys Gly Val Cys Gly Gly Asn Gly Ser Thr
Cys Lys Lys Met Ser 195 200 205 Gly Ile Val Thr Ser Thr Arg Pro Gly
Tyr His Asp Ile Val Thr Ile 210 215 220 Pro Ala Gly Ala Thr Asn Ile
Glu Val Lys His Arg Asn Gln Arg Gly 225 230 235 240 Ser Arg Asn Asn
Gly Ser Phe Leu Ala Ile Arg Ala Ala Asp Gly Thr 245 250 255 Tyr Ile
Leu Asn Gly Asn Phe Thr Leu Ser Thr Leu Glu Gln Asp Leu 260 265 270
Thr Tyr Lys Gly Thr Val Leu Arg Tyr Ser Gly Ser Ser Ala Ala Leu 275
280 285 Glu Arg Ile Arg Ser Phe Ser Pro Leu Lys Glu Pro Leu Thr Ile
Gln 290 295 300 Val Leu Met Val Gly His Ala Leu Arg Pro Lys Ile Lys
Phe Thr Tyr 305 310 315 320 Phe Met Lys Lys Lys Thr Glu Ser Phe Asn
Ala Ile Pro Thr Phe Ser 325 330 335 Glu Trp Val Ile Glu Glu Trp Gly
Glu Cys Ser Lys Thr Cys Gly Ser 340 345 350 Gly Trp Gln Arg Arg Val
Val Gln Cys Arg Asp Ile Asn Gly His Pro 355 360 365 Ala Ser Glu Cys
Ala Lys Glu
Val Lys Pro Ala Ser Thr Arg Pro Cys 370 375 380 Ala Asp Leu Pro Cys
Pro His Trp Gln Val Gly Asp Trp Ser Pro Cys 385 390 395 400 Ser Lys
Thr Cys Gly Lys Gly Tyr Lys Lys Arg Thr Leu Lys Cys Val 405 410 415
Ser His Asp Gly Gly Val Leu Ser Asn Glu Ser Cys Asp Pro Leu Lys 420
425 430 Lys Pro Lys His Tyr Ile Asp Phe Cys Thr Leu Thr Gln Cys Ser
435 440 445 261347DNAMus 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 Ser 1 5 10 15 Asp Met Gly
Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro 20 25 30 Val
Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp 35 40
45 Ala Leu Gly Arg Pro Ser Glu Cys Pro Asp Asn Asn Gly Lys Thr Phe
50 55 60 Arg Glu Glu Gln Cys Glu Ala His Asn Glu Phe Ser Lys Ala
Ser Phe 65 70 75 80 Gly Ser Gly Pro Ala Val Glu Trp Ile Pro Lys Tyr
Ala Gly Val Ser 85 90 95 Pro Lys Asp Arg Cys Lys Leu Ile Cys Gln
Ala Lys Gly Ile Gly Tyr 100 105 110 Phe Phe Val Leu Gln Pro Lys Val
Val Asp Gly Thr Pro Cys Ser Pro 115 120 125 Asp Ser Thr Ser Val Cys
Val Gln Gly Gln Cys Val Lys Ala Gly Cys 130 135 140 Asp Arg Ile Ile
Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys 145 150 155 160 Gly
Gly Asn Gly Ser Thr Cys Lys Lys Ile Ser Gly Ser Val Thr Ser 165 170
175 Ala Lys Pro Gly Tyr His Asp Ile Ile Thr Ile Pro Thr Gly Ala Thr
180 185 190 Asn Ile Glu Val Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn
Asn Gly 195 200 205 Ser Phe Leu Ala Ile Lys Ala Ala Asp Gly Thr Tyr
Ile Leu Asn Gly 210 215 220 Asp Tyr Thr Leu Ser Thr Leu Glu Gln Asp
Ile Met Tyr Lys Gly Val 225 230 235 240 Val Leu Arg Tyr Ser Gly Ser
Ser Ala Ala Leu Glu Arg Ile Arg Ser 245 250 255 Phe Ser Pro Leu Lys
Glu Pro Leu Thr Ile Gln Val Leu Thr Val Gly 260 265 270 Asn Ala Leu
Arg Pro Lys Ile Lys Tyr Thr Tyr Phe Val Lys Lys Lys 275 280 285 Lys
Glu Ser Phe Asn Ala Ile Pro Thr Phe Ser Ala Trp Val Ile Glu 290 295
300 Glu Trp Gly Glu Cys Ser Lys Ser Cys Glu Leu Gly Trp Gln Arg Arg
305 310 315 320 Leu Val Glu Cys Arg Asp Ile Asn Gly Gln Pro Ala Ser
Glu Cys Ala 325 330 335 Lys Glu Val Lys Pro Ala Ser Thr Arg Pro Cys
Ala Asp His Pro Cys 340 345 350 Pro Gln Trp Gln Leu Gly Glu Trp Ser
Ser Cys Ser Lys Thr Cys Gly 355 360 365 Lys Gly Tyr Lys Lys Arg Ser
Leu Lys Cys Leu Ser His Asp Gly Gly 370 375 380 Val Leu Ser His Glu
Ser Cys Asp Pro Leu Lys Lys Pro Lys His Phe 385 390 395 400 Ile Asp
Phe Cys Thr Met Ala Glu Cys Ser 405 410 281389DNAHomo 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 Ala 1 5 10 15 His Met
Leu Leu Leu Leu Leu Ala Ser Ile Thr Met Leu Leu Cys Ala 20 25 30
Arg Gly Ala His Gly Arg Pro Thr Glu Glu Asp Glu Glu Leu Val Leu 35
40 45 Pro Ser Leu Glu Arg Ala Pro Gly His Asp Ser Thr Thr Thr Arg
Leu 50 55 60 Arg Leu Asp Ala Phe Gly Gln Gln Leu His Leu Lys Leu
Gln Pro Asp 65 70 75 80 Ser Gly Phe Leu Ala Pro Gly Phe Thr Leu Gln
Thr Val Gly Arg Ser 85 90 95 Pro Gly Ser Glu Ala Gln His Leu Asp
Pro Thr Gly Asp Leu Ala His 100 105 110 Cys Phe Tyr Ser Gly Thr Val
Asn Gly Asp Pro Gly Ser Ala Ala Ala 115 120 125 Leu Ser Leu Cys Glu
Gly Val Arg Gly Ala Phe Tyr Leu Gln Gly Glu 130 135 140 Glu Phe Phe
Ile Gln Pro Ala Pro Gly Val Ala Thr Glu Arg Leu Ala 145 150 155 160
Pro Ala Val Pro Glu Glu Glu Ser Ser Ala Arg Pro Gln Phe His Ile 165
170 175 Leu Arg Arg Arg Arg Arg Gly Ser Gly Gly Ala Lys Cys Gly Val
Met 180 185 190 Asp Asp Glu Thr Leu Pro Thr Ser Asp Ser Arg Pro Glu
Ser Gln Asn 195 200 205 Thr Arg Asn Gln Trp Pro Val Arg Asp Pro Thr
Pro Gln Asp Ala Gly 210 215 220 Lys Pro Ser Gly Pro Gly Ser Ile Arg
Lys Lys Arg Phe Val Ser Ser 225 230 235 240 Pro Arg Tyr Val Glu Thr
Met Leu Val Ala Asp Gln Ser Met Ala Asp 245 250 255 Phe His Gly Ser
Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val 260 265 270 Ala Ala
Arg Phe Tyr Lys His Pro Ser Ile Arg Asn Ser Ile Ser Leu 275 280 285
Val Val Val Lys Ile Leu Val Ile Tyr Glu Glu Gln Lys Gly Pro Glu 290
295 300 Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp
Gln 305 310 315 320 Lys Gln His Asn Ser Pro Ser Asp Arg Asp Pro Glu
His Tyr Asp Thr 325 330 335 Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys
Gly Ser His Thr Cys Asp 340 345 350 Thr Leu Gly Met Ala Asp Val Gly
Thr Val Cys Asp Pro Ser Arg Ser 355 360 365 Cys Ser Val Ile Glu Asp
Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala 370 375 380 His Gln Leu Gly
His Val Phe Asn Met Pro His Asp Asp Ala Lys His 385 390 395 400 Cys
Ala Ser Leu Asn Gly Val Thr Gly Asp Ser His Leu Met Ala Ser 405 410
415 Met Leu Ser Ser Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala
420 425 430 Tyr Met Val Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys
Leu Met 435 440 445 Asp Lys Pro Gln Asn Pro Ile Lys Leu Pro Ser Asp
Leu Pro Gly Thr 450 455 460 Leu Tyr Asp Ala Asn Arg Gln Cys Gln Phe
Thr Phe Gly Glu Glu Ser 465 470 475 480 Lys His Cys Pro Asp Ala Ala
Ser Thr Cys Thr Thr Leu Trp Cys Thr 485 490 495 Gly Thr Ser Gly Gly
Leu Leu Val Cys Gln Thr Lys His Phe Pro Trp 500 505 510 Ala Asp Gly
Thr Ser Cys Gly Glu Gly Lys Trp Cys Val Ser Gly Lys 515 520 525 Cys
Val Asn Lys Thr Asp Met Lys His Phe Ala Thr Pro Val His Gly 530 535
540 Ser Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly
545 550 555 560 Gly Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro
Val Pro Lys 565 570 575 Asn Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val
Arg Tyr Arg Ser Cys 580 585 590 Asn Ile Glu Asp Cys Pro Asp Asn Asn
Gly Lys Thr Phe Arg Glu Glu 595 600 605 Gln Cys Glu Ala His Asn Glu
Phe Ser Lys Ala Ser Phe Gly Asn Glu 610 615 620 Pro Thr Val Glu Trp
Thr Pro Lys Tyr Ala Gly Val Ser Pro Lys Asp 625 630 635 640 Arg Cys
Lys Leu Thr Cys Glu Ala Lys Gly Ile Gly Tyr Phe Phe Val 645 650 655
Leu Gln Pro Lys Val Val Asp Gly Thr Pro Cys Ser Pro Asp Ser Thr 660
665 670 Ser Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys Asp Arg
Ile 675 680 685 Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys
Gly Gly Asn 690 695 700 Gly Ser Thr Cys Lys Lys Met Ser Gly Ile Val
Thr Ser Thr Arg Pro 705 710 715 720 Gly Tyr His Asp Ile Val Thr Ile
Pro Ala Gly Ala Thr Asn Ile Glu 725 730 735 Val Lys His Arg Asn Gln
Arg Gly Ser Arg Asn Asn Gly Ser Phe Leu 740 745 750 Ala Ile Arg Ala
Ala Asp Gly Thr Tyr Ile Leu Asn Gly Asn Phe Thr 755 760 765 Leu Ser
Thr Leu Glu Gln Asp Leu Thr Tyr Lys Gly Thr Val Leu Arg 770 775 780
Tyr Ser Gly Ser Ser Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser Pro 785
790 795 800 Leu Lys Glu Pro Leu Thr Ile Gln Val Leu Met Val Gly His
Ala Leu 805 810 815 Arg Pro Lys Ile Lys Phe Thr Tyr Phe Met Lys Lys
Lys Thr Glu Ser 820 825 830 Phe Asn Ala Ile Pro Thr Phe Ser Glu Trp
Val Ile Glu Glu Trp Gly 835 840 845 Glu Cys Ser Lys Thr Cys Gly Ser
Gly Trp Gln Arg Arg Val Val Gln 850 855 860 Cys Arg Asp Ile Asn Gly
His Pro Ala Ser Glu Cys Ala Lys Glu Val 865 870 875 880 Lys Pro Ala
Ser Thr Arg Pro Cys Ala Asp Leu Pro Cys Pro His Trp 885 890 895 Gln
Val Gly Asp Trp Ser Pro Cys Ser Lys Thr Cys Gly Lys Gly Tyr 900 905
910 Lys Lys Arg Thr Leu Lys Cys Val Ser His Asp Gly Gly Val Leu Ser
915 920 925 Asn Glu Ser Cys Asp Pro Leu Lys Lys Pro Lys His Tyr Ile
Asp Phe 930 935 940 Cys Thr Leu Thr Gln Cys Ser 945 950
302859DNAMus 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 Val 1 5 10 15 Pro Thr Leu Leu Leu
Leu Ala Ala Ala Leu Leu Ala Val Ser Asp Ala 20 25 30 Leu Gly Arg
Pro Ser Glu Glu Asp Glu Glu Leu Val Val Pro Glu Leu 35 40 45 Glu
Arg Ala Pro Gly His Gly Thr Thr Arg Leu Arg Leu His Ala Phe 50 55
60 Asp Gln Gln Leu Asp Leu Glu Leu Arg Pro Asp Ser Ser Phe Leu Ala
65 70 75 80 Pro Gly Phe Thr Leu Gln Asn Val Gly Arg Lys Ser Gly Ser
Glu Thr 85 90 95 Pro Leu Pro Glu Thr Asp Leu Ala His Cys Phe Tyr
Ser Gly Thr Val 100 105 110 Asn Gly Asp Pro Ser Ser Ala Ala Ala Leu
Ser Leu Cys Glu Gly Val 115 120 125 Arg Gly Ala Phe Tyr Leu Leu Gly
Glu Ala Tyr Phe Ile Gln Pro Leu 130 135 140 Pro Ala Ala Ser Glu Arg
Leu Ala Thr Ala Ala Pro Gly Glu Lys Pro 145 150 155 160 Pro Ala Pro
Leu Gln Phe His Leu Leu Arg Arg Asn Arg Gln Gly Asp 165 170 175 Val
Gly Gly Thr Cys Gly Val Val Asp Asp Glu Pro Arg Pro Thr Gly 180 185
190 Lys Ala Glu Thr Glu Asp Glu Asp Glu Gly Thr Glu Gly Glu Asp Glu
195 200 205 Gly Ala Gln Trp Ser Pro Gln Asp Pro Ala Leu Gln Gly Val
Gly Gln 210 215 220 Pro Thr Gly Thr Gly Ser Ile Arg Lys Lys Arg Phe
Val Ser Ser His 225 230 235 240 Arg Tyr Val Glu Thr Met Leu Val Ala
Asp Gln Ser Met Ala Glu Phe 245 250 255 His Gly Ser Gly Leu Lys His
Tyr Leu Leu Thr Leu Phe Ser Val Ala 260 265 270 Ala Arg Leu Tyr Lys
His Pro Ser Ile Arg Asn Ser Val Ser Leu Val 275 280 285 Val Val Lys
Ile Leu Val Ile His Asp Glu Gln Lys Gly Pro Glu Val 290 295 300 Thr
Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp Gln Lys 305 310
315 320 Gln His Asn Pro Pro Ser Asp Arg Asp Ala Glu His Tyr Asp Thr
Ala 325 330 335 Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Ser Gln Thr
Cys Asp Thr 340 345 350 Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp
Pro Ser Arg Ser Cys 355 360 365 Ser Val Ile Glu Asp Asp Gly Leu Gln
Ala Ala Phe Thr Thr Ala His 370 375 380 Gln Leu Gly His Val Phe Asn
Met Pro His Asp Asp Ala Lys Gln Cys 385 390 395 400 Ala Ser Leu Asn
Gly Val Asn Gln Asp Ser His Met Met Ala Ser Met 405 410 415 Leu Ser
Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Ala Tyr 420 425 430
Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met Asp 435
440 445 Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp Leu Pro Gly Thr
Ser 450 455 460 Tyr Asp Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu
Asp Ser Lys 465 470 475 480 His Cys Pro Asp Ala Ala Ser Thr Cys Ser
Thr Leu Trp Cys Thr Gly 485 490 495 Thr Ser Gly Gly Val Leu Val Cys
Gln Thr Lys His Phe Pro Trp Ala 500 505 510 Asp Gly Thr Ser Cys Gly
Glu Gly Lys Trp Cys Ile Asn Gly Lys Cys 515 520 525 Val Asn Lys Thr
Asp Arg Lys His Phe Asp Thr Pro Phe His Gly Ser 530 535 540 Trp Gly
Met Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly 545 550 555
560 Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro Lys Asn
565 570 575 Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser
Cys Asn 580 585 590 Leu Glu Asp Cys Pro Asp Asn Asn Gly Lys Thr Phe
Arg Glu Glu Gln 595 600 605 Cys Glu Ala His Asn Glu Phe Ser Lys Ala
Ser Phe Gly Ser Gly Pro 610 615 620 Ala Val Glu Trp Ile Pro Lys Tyr
Ala Gly Val Ser Pro Lys Asp Arg 625 630 635 640 Cys Lys Leu Ile Cys
Gln Ala Lys Gly Ile Gly Tyr Phe Phe Val Leu 645 650 655 Gln Pro Lys
Val Val Asp Gly Thr Pro Cys Ser Pro Asp Ser Thr Ser 660 665 670 Val
Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys Asp Arg Ile Ile 675 680
685 Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly Asn Gly
690 695 700 Ser Thr Cys Lys Lys Ile Ser Gly Ser Val Thr Ser Ala Lys
Pro Gly 705 710 715 720 Tyr His Asp Ile Ile Thr Ile Pro Thr Gly Ala
Thr Asn Ile Glu Val 725 730 735 Lys Gln Arg Asn Gln Arg Gly Ser Arg
Asn Asn Gly Ser Phe Leu Ala 740 745 750 Ile Lys Ala Ala Asp Gly Thr
Tyr Ile Leu Asn Gly Asp Tyr Thr Leu 755 760 765 Ser Thr Leu Glu Gln
Asp Ile Met Tyr Lys Gly Val Val Leu Arg Tyr 770 775 780 Ser Gly Ser
Ser Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser Pro Leu 785 790 795 800
Lys Glu Pro Leu Thr Ile Gln Val Leu Thr Val Gly Asn Ala Leu Arg 805
810 815 Pro Lys Ile Lys Tyr Thr Tyr Phe Val Lys Lys Lys Lys Glu Ser
Phe 820 825 830 Asn Ala Ile Pro Thr Phe Ser Ala Trp Val Ile Glu Glu
Trp Gly Glu 835 840 845 Cys Ser Lys Ser Cys Glu Leu Gly Trp Gln Arg
Arg Leu Val Glu Cys 850 855 860 Arg Asp Ile Asn Gly Gln Pro Ala Ser
Glu Cys Ala Lys Glu Val Lys 865 870 875 880 Pro Ala Ser Thr Arg Pro
Cys Ala Asp His Pro Cys Pro Gln Trp Gln 885 890 895 Leu Gly Glu Trp
Ser Ser Cys Ser Lys Thr Cys Gly Lys Gly Tyr Lys 900 905 910 Lys Arg
Ser Leu Lys Cys Leu Ser His Asp Gly Gly Val Leu Ser His 915 920 925
Glu Ser Cys Asp Pro Leu Lys Lys Pro Lys His Phe Ile Asp Phe Cys 930
935 940 Thr Met Ala Glu Cys Ser 945 950 322907DNAHomo 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 Ala 1 5 10 15 His Met Leu Leu Leu Leu Leu Ala Ser Ile Thr Met
Leu Leu Cys Ala 20 25 30 Arg Gly Ala His Gly Arg Pro Thr Glu Glu
Asp Glu Glu Leu Val Leu 35 40 45 Pro Ser Leu Glu Arg Ala Pro Gly
His Asp Ser Thr Thr Thr Arg Leu 50 55 60 Arg Leu Asp Ala Phe Gly
Gln Gln Leu His Leu Lys Leu Gln Pro Asp 65 70 75 80 Ser Gly Phe Leu
Ala Pro Gly Phe Thr Leu Gln Thr Val Gly Arg Ser 85 90 95 Pro Gly
Ser Glu Ala Gln His Leu Asp Pro Thr Gly Asp Leu Ala His 100 105 110
Cys Phe Tyr Ser Gly Thr Val Asn Gly Asp Pro Gly Ser Ala Ala Ala 115
120 125 Leu Ser Leu Cys Glu Gly Val Arg Gly Ala Phe Tyr Leu Gln Gly
Glu 130 135 140 Glu Phe Phe Ile Gln Pro Ala Pro Gly Val Ala Thr Glu
Arg Leu Ala 145 150 155 160 Pro Ala Val Pro Glu Glu Glu Ser Ser Ala
Arg Pro Gln Phe His Ile 165 170 175 Leu Arg Arg Arg Arg Arg Gly Ser
Gly Gly Ala Lys Cys Gly Val Met 180 185 190 Asp Asp Glu Thr Leu Pro
Thr Ser Asp Ser Arg Pro Glu Ser Gln Asn 195 200 205 Thr Arg Asn Gln
Trp Pro Val Arg Asp Pro Thr Pro Gln Asp Ala Gly 210 215 220 Lys Pro
Ser Gly Pro Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser 225 230 235
240 Pro Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Asp
245 250 255 Phe His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe
Ser Val 260 265 270 Ala Ala Arg Phe Tyr Lys His Pro Ser Ile Arg Asn
Ser Ile Ser Leu 275 280 285 Val Val Val Lys Ile Leu Val Ile Tyr Glu
Glu Gln Lys Gly Pro Glu 290 295 300 Val Thr Ser Asn Ala Ala Leu Thr
Leu Arg Asn Phe Cys Asn Trp Gln 305 310 315 320 Lys Gln His Asn Ser
Pro Ser Asp Arg Asp Pro Glu His Tyr Asp Thr 325 330 335 Ala Ile Leu
Phe Thr Arg Gln Asp Leu Cys Gly Ser His Thr Cys Asp 340 345 350 Thr
Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser 355 360
365 Cys Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala
370 375 380 His Gln Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala
Lys His 385 390 395 400 Cys Ala Ser Leu Asn Gly Val Thr Gly Asp Ser
His Leu Met Ala Ser 405 410 415 Met Leu Ser Ser Leu Asp His Ser Gln
Pro Trp Ser Pro Cys Ser Ala 420 425 430 Tyr Met Val Thr Ser Phe Leu
Asp Asn Gly His Gly Glu Cys Leu Met 435 440 445 Asp Lys Pro Gln Asn
Pro Ile Lys Leu Pro Ser Asp Leu Pro Gly Thr 450 455 460 Leu Tyr Asp
Ala Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Glu Ser 465 470 475 480
Lys His Cys Pro Asp Ala Ala Ser Thr Cys Thr Thr Leu Trp Cys Thr 485
490 495 Gly Thr Ser Gly Gly Leu Leu Val Cys Gln Thr Lys His Phe Pro
Trp 500 505 510 Ala Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Val
Ser Gly Lys 515 520 525 Cys Val Asn Lys Thr Asp Met Lys His Phe Ala
Thr Pro Val His Gly 530 535 540 Ser Trp Gly Pro Trp Gly Pro Trp Gly
Asp Cys Ser Arg Thr Cys Gly 545 550 555 560 Gly Gly Val Gln Tyr Thr
Met Arg Glu Cys Asp Asn Pro Val Pro Lys 565 570 575 Asn Gly Gly Lys
Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys 580 585 590 Asn Ile
Glu Asp 595 341794DNAMus 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 Val 1 5 10 15 Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala
Val Ser Asp Ala 20 25 30 Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu
Leu Val Val Pro Glu Leu 35 40 45 Glu Arg Ala Pro Gly His Gly Thr
Thr Arg Leu Arg Leu His Ala Phe 50 55 60 Asp Gln Gln Leu Asp Leu
Glu Leu Arg Pro Asp Ser Ser Phe Leu Ala 65 70 75 80 Pro Gly Phe Thr
Leu Gln Asn Val Gly Arg Lys Ser Gly Ser Glu Thr 85 90 95 Pro Leu
Pro Glu Thr Asp Leu Ala His Cys Phe Tyr Ser Gly Thr Val 100 105 110
Asn Gly Asp Pro Ser Ser Ala Ala Ala Leu Ser Leu Cys Glu Gly Val 115
120 125 Arg Gly Ala Phe Tyr Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro
Leu 130 135 140 Pro Ala Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly
Glu Lys Pro 145 150 155 160 Pro Ala Pro Leu Gln Phe His Leu Leu Arg
Arg Asn Arg Gln Gly Asp 165 170 175 Val Gly Gly Thr Cys Gly Val Val
Asp Asp Glu Pro Arg Pro Thr Gly 180 185 190 Lys Ala Glu Thr Glu Asp
Glu Asp Glu Gly Thr Glu Gly Glu Asp Glu 195 200 205 Gly Ala Gln Trp
Ser Pro Gln Asp Pro Ala Leu Gln Gly Val Gly Gln 210 215 220 Pro Thr
Gly Thr Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser His 225 230 235
240 Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Glu Phe
245 250 255 His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser
Val Ala 260 265 270 Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser
Val Ser Leu Val 275 280 285 Val Val Lys Ile Leu Val Ile His Asp Glu
Gln Lys Gly Pro Glu Val 290 295 300 Thr Ser Asn Ala Ala Leu Thr Leu
Arg Asn Phe Cys Asn Trp Gln Lys 305 310 315 320 Gln His Asn Pro Pro
Ser Asp Arg Asp Ala Glu His Tyr Asp Thr Ala 325 330 335 Ile Leu Phe
Thr Arg Gln Asp Leu Cys Gly Ser Gln Thr Cys Asp Thr 340 345 350 Leu
Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser Cys 355 360
365 Ser Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala His
370 375 380 Gln Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys
Gln Cys 385 390 395 400 Ala Ser Leu Asn Gly Val Asn Gln Asp Ser His
Met Met Ala Ser Met 405 410 415 Leu Ser Asn Leu Asp His Ser Gln Pro
Trp Ser Pro Cys Ser Ala Tyr 420 425 430 Met Ile Thr Ser Phe Leu Asp
Asn Gly His Gly Glu Cys Leu Met Asp 435 440 445 Lys Pro Gln Asn Pro
Ile Gln Leu Pro Gly Asp Leu Pro Gly Thr Ser 450 455 460 Tyr Asp Ala
Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Asp Ser Lys 465 470 475 480
His Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys Thr Gly 485
490 495 Thr Ser Gly Gly Val Leu Val Cys Gln Thr Lys His Phe Pro Trp
Ala 500 505 510 Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn
Gly Lys Cys 515 520 525 Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr
Pro Phe His Gly Ser 530 535 540 Trp Gly Met Trp Gly Pro Trp Gly Asp
Cys Ser Arg Thr Cys Gly Gly 545 550 555 560 Gly Val Gln Tyr Thr Met
Arg Glu Cys Asp Asn Pro Val Pro Lys Asn 565 570 575 Gly Gly Lys Tyr
Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys Asn 580 585 590 Leu Glu
Asp 595 361842DNAHomo 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
18423749PRTArtificial SequenceTSP-1 repeat 2 37Gly Gly Trp Ser His
Trp Ser Pro Trp Ser Ser Cys Ser Val Thr Cys 1 5 10 15 Gly Asp Gly
Val Ile Thr Arg Ile Arg Leu Cys Asn Ser Pro Ser Pro 20 25 30 Gln
Met Asn Gly Lys Pro Cys Glu Gly Glu Ala Arg Glu Thr Lys Ala 35 40
45 Cys 3853PRTArtificial SequenceTSP-1 repeat-3 38Gly Gly Trp Gly
Pro Trp Ser Pro Trp Asp Ile Cys Ser Val Thr Cys 1 5 10 15 Gly Gly
Gly Val Gln Arg Arg Ser Arg Leu Cys Asn Asn Pro Thr Pro 20 25 30
Gln Phe Gly Gly Lys Asp Cys Val Gly Asp Val Thr Glu Asn Gln Val 35
40 45 Cys Asn Lys Gln Asp 50 3953PRTArtificial SequenceADAMTS-1
fragment mTSP-1 39Gly Ser Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys
Ser Arg Thr Cys 1 5 10 15 Gly Gly Gly Val Gln Tyr Thr Met Arg Glu
Cys Asp Asn Pro Val Pro 20 25 30 Lys Asn Gly Gly Lys Tyr Cys Glu
Gly Lys Arg Val Arg Tyr Arg Ser 35 40 45 Cys Asn Ile Glu Asp 50
4054PRTArtificial SequenceADAMTS-1 cTSP-1-1 40Trp Val Ile Glu Glu
Trp Gly Glu Cys Ser Lys Thr Cys Gly Ser Gly 1 5 10 15 Trp Gln Arg
Arg Val Val Gln Cys Arg Asp Ile Asn Gly His Pro Ala 20 25 30 Ser
Glu Cys Ala Lys Glu Val Lys Pro Ala Ser Thr Arg Pro Cys Ala 35 40
45 Asp Leu Pro Cys Pro His 50 4156PRTArtificial SequenceADAMTS-1
cTSP1-2 41Trp Gln Val Gly Asp Trp Ser Pro Cys Ser Lys Thr Cys Gly
Lys Gly 1 5 10 15 Tyr Lys Lys Arg Thr Leu Lys Cys Val Ser His Asp
Gly Gly Val Leu 20 25 30 Ser Asn Glu Ser Cys Asp Pro Leu Lys Lys
Pro Lys His Tyr Ile Asp 35 40 45 Phe Cys Thr Leu Thr Gln Cys Ser 50
55 428PRTArtificial Sequencedeletion mutant motif 1 42Cys Ser Xaa
Thr Cys Gly Xaa Xaa 1 5 439PRTArtificial Sequencedeletion mutant
motif 2 43Trp Gly Xaa Cys Ser Lys Thr Cys Gly 1 5 444PRTArtificial
SequenceMotif 1 44Val Ile Glu Glu 1 454PRTArtificial Sequencemotif
2 45Gln Val Gly Asp 1 468PRTArtificial Sequenceprotease cleavage
site 46Leu Glu Val Leu Phe Gln Gly Pro 1 5 476PRTArtificial
Sequencemotif 3 47Cys Ser Val Thr Cys Gly 1 5 489PRTArtificial
Sequencemotif 3 48Trp Gly Pro Trp Gly Pro Trp Gly Asp 1 5
498PRTArtificial Sequencemotif 5 49Trp Gly Asp Cys Ser Arg Thr Cys
1 5 506PRTArtificial Sequencemotif 6 50Asp Cys Ser Lys Thr Cys 1 5
516PRTArtificial Sequencemotif 7 51Pro Cys Ser Lys Thr Cys 1 5
528PRTArtificial Sequencemotif 8 52Cys Ser Arg Thr Cys Gly Gly Gly
1 5 537PRTArtificial Sequencemotif 9 53Cys Ser Lys Thr Cys Gly Ser
1 5 549PRTArtificial SequenceTSP1-like domain 1 54Trp Gly Pro Trp
Gly Pro Trp Gly Asp 1 5 557PRTArtificial SequenceTSP1-like domain 2
55Trp Val Ile Glu Glu Trp Gly 1 5 567PRTArtificial
SequenceTSP1-like domain 3 56Trp Gln Val Gly Asp Trp Ser 1 5
* * * * *