U.S. patent application number 15/803790 was filed with the patent office on 2018-03-01 for compositions comprising semaphorins for the treatment of cancer and methods of selection thereof.
This patent application is currently assigned to RAPPAPORT FAMILY INSTITUTE FOR RESEARCH IN THE MEDICAL SCIENCES. The applicant listed for this patent is RAPPAPORT FAMILY INSTITUTE FOR RESEARCH IN THE MEDICAL SCIENCES. Invention is credited to Ofra Kessler, Boaz Kigel, Gera NEUFELD, Asya Varshavsky.
Application Number | 20180055907 15/803790 |
Document ID | / |
Family ID | 56887188 |
Filed Date | 2018-03-01 |
United States Patent
Application |
20180055907 |
Kind Code |
A1 |
NEUFELD; Gera ; et
al. |
March 1, 2018 |
COMPOSITIONS COMPRISING SEMAPHORINS FOR THE TREATMENT OF CANCER AND
METHODS OF SELECTION THEREOF
Abstract
A method of selecting a semaphorin for treating cancer in a
subject is disclosed. The method comprises determining an
expression of a semaphorin receptor on tumor cells of a tumor
sample of the subject wherein an amount of the semaphorin receptor
is indicative of the semaphorin suitable for treating the cancer in
the subject. Kits for treating cancer and pharmaceutical
compositions comprising semaphorins are also disclosed.
Inventors: |
NEUFELD; Gera; (Haifa,
IL) ; Kigel; Boaz; (Kiryat-tivon, IL) ;
Kessler; Ofra; (Haifa, IL) ; Varshavsky; Asya;
(Haifa, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RAPPAPORT FAMILY INSTITUTE FOR RESEARCH IN THE MEDICAL
SCIENCES |
Haifa |
|
IL |
|
|
Assignee: |
RAPPAPORT FAMILY INSTITUTE FOR
RESEARCH IN THE MEDICAL SCIENCES
Haifa
IL
|
Family ID: |
56887188 |
Appl. No.: |
15/803790 |
Filed: |
November 5, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14847025 |
Sep 8, 2015 |
9808506 |
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15803790 |
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13941574 |
Jul 15, 2013 |
9155781 |
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14847025 |
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13313063 |
Dec 7, 2011 |
8513194 |
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13941574 |
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12738634 |
Apr 18, 2010 |
8088735 |
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PCT/IL2008/001307 |
Oct 2, 2008 |
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13313063 |
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61071560 |
May 6, 2008 |
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61071053 |
Apr 10, 2008 |
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61006496 |
Jan 16, 2008 |
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60960910 |
Oct 19, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/52 20130101;
A61K 38/177 20130101; A61K 38/1709 20130101; G01N 33/57484
20130101; G01N 2333/71 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method of treating cancer in a subject in need thereof, the
method comprising administering to the subject a therapeutically
effective amount of a composition comprising an amino acid sequence
at least 70% identical to the sequence set forth in SEQ ID NO: 32,
thereby treating the cancer.
2. The method of claim 1, wherein said cancer is melanoma.
3. The method claim 1, wherein the amino acid sequence is at least
95% identical to the sequence set forth in SEQ ID NO: 32
4. The method claim 1, wherein the amino acid sequence is
semaphorin Sema3G.
5. The method of claim 1, wherein said administering comprises
systemic administration.
6. The method of claim 1, wherein said administering comprises
local administration.
7. The method of claim 1, wherein said amino acid sequence is
attached to sustained-release enhancing agent.
8. The method of claim 7, wherein said sustained-release enhancing
agent is selected from the group consisting of hyaluronic acid
(HA), alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA),
polyethylene glycol (PEG), glyme and polyisopropylacrylamide.
9. A method of treating cancer by inhibiting angiogenesis to a
tumor in a subject in need thereof, the method comprising
administering to the subject a therapeutically effective amount of
a composition comprising an amino acid sequence at least 70%
identical to the sequence set forth in SEQ ID NO: 32, thereby
treating the cancer.
10. The method claim 9, wherein the amino acid sequence is at least
95% identical to the sequence set forth in SEQ ID NO: 32
11. The method claim 9, wherein the amino acid sequence is
semaphorin Sema3G.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/847,025 filed on Sep. 5, 2015, which is a
continuation-in-part of U.S. patent application Ser. No. 13/941,574
filed on Jul. 15, 2013, which is a division of U.S. patent
application Ser. No. 13/313,063 filed on Dec. 7, 2011, which is a
division of U.S. patent application Ser. No. 12/738,634 filed on
Apr. 18, 2010, now U.S. Pat. No. 8,088,735 which is a National
Phase of PCT Patent Application No. PCT/IL2008/001307 having
International filing date of Oct. 2, 2008, which claims the benefit
of priority under 35 USC 119(e) of U.S. Provisional Patent
Application Nos. 61/071,560 filed on May 6, 2008, 61/071,053 filed
on Apr. 10, 2008, 61/006,496 filed on Jan. 16, 2008, and 60/960,910
filed on Oct. 19, 2007.
[0002] The contents of all of the above applications are
incorporated by reference as if fully set forth herein.
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention, in some embodiments thereof, relates
to methods and compositions for treating angiogenesis-related
diseases such as cancer, and methods of selection thereof.
[0004] The neuropilin-1 (np1) and the neuropilin-2 (np2) receptors
were originally characterized as functional receptors for axon
guidance factors belonging to the class-3 semaphorin (sema3)
family. It was subsequently realized that the neuropilins are
expressed by endothelial cells and by many types of cancer cells.
It was also found that the neuropilins function in addition as
receptors for several angiogenic factors belonging to the VEGF
family and as receptors for the angiogenesis/metastasis inducing
growth factor hepatocyte growth factor/scatter factor (HGF/SF), and
that they function as potent enhancers of their pro-angiogenic
activity.
[0005] Most of the sema3s, with the exception of sema3E which binds
to PlexD1, bind to one of the two neuropilin receptors or to both.
Neuropilins form spontaneous complexes with several members of the
plexin receptor family. In these complexes the sema3s bind to
neuropilins while the plexins function as the signal transducing
elements. The four type-A plexins (plexins-A1 to plexin-A4) as well
as plexin-D1 form complexes with neuropilins and participate in
neuropilin mediated signal transduction.
[0006] Semaphorins sema3B and sema3F were also characterized as
tumor suppressors whose loss contributes to the development of lung
cancer [Tomizawa, Y., 2001, Proc. Natl. Acad. Sci. U.S.A.
98:13954-13959; Xiang, R., 2002. Cancer Res. 62:2637-2643].
[0007] The identification of neuropilins in endothelial cells
suggested that class-3 semaphorins may be able to regulate
angiogenesis. Indeed, the class-3 semaphorin sema3F, a np2 agonist,
functions as a repellent of endothelial cells, induces apoptosis of
endothelial cells upon prolonged stimulation [Bielenberg, D. R., et
al., 2004, J. Clin. Invest 114:1260-1271; Guttmann-Raviv, N., et
al., 2007, J. Biol. Chem. 282:26294-26305] and inhibits
angiogenesis and tumor progression in-vivo [Bielenberg, D. R., et
al., 2004, J. Clin. Invest 114:1260-1271; Kessler, O., et al 2004.
Cancer Res. 64:1008-1015. The np1 agonist sema3A was also shown to
inhibit in-vitro and in-vivo angiogenesis [Miao, H. Q., 1999. J.
Cell Biol. 146:233-242. Bates, D., 2003, Dev. Biol. 255:77-98;
Acevedo, L. M., 2008. Blood. 111:2674-2680]. Sema3E was also
characterized as a repulsive agent that inhibits the invasion of
PlexD1 expressing blood vessels into somites during embryonic
development [Gu C. et al., 2005, Science 307:265-268].
[0008] In contrast, existing data suggests that sema3C functions as
a pro-tumorigenic and pro-angiogenic agent [Herman J G, et al, Int.
J. Oncol. 2007; 30:1231-1238; Banu N, FASEB J. 2006;
20:2150-2152].
[0009] The fact that neuropilins and plexins such as PlexD1 are
also expressed by many types of tumor cells indicates that
semaphorins may also affect the behavior of tumor cells directly.
Indeed, sema3s such as sema3F and sema3B have been observed to
inhibit the adhesion, migration or the proliferation of tumor cells
expressing appropriate semaphorin receptors [Tomizawa, Y., 2001,
Proc. Natl. Acad. Sci. U.S.A. 98:13954-13959; Xiang, R., 2002.
Cancer Res. 62:2637-2643; Bielenberg, D. R., et al., 2004, J. Clin.
Invest 114:1260-1271; Nasarre, 2006, Neoplasia. 7:180-189]. In
contrast, however, the cleavage product of Sema3E, was shown to be
an inducer of tumor invasiveness and tumor metastasis [Christensen,
C, 2005, Cancer Res. 65, 6167-6177].
SUMMARY OF THE INVENTION
[0010] According to an aspect of some embodiments of the present
invention there is provided a method of selecting a semaphorin for
treating cancer in a subject, the method comprising determining an
expression of a semaphorin receptor on tumor cells of a tumor
sample of the subject wherein an amount of the semaphorin receptor
is indicative of the semaphorin suitable for treating the cancer in
the subject.
[0011] According to an aspect of some embodiments of the present
invention there is provided a method of treating cancer in a
subject in need thereof, the method comprising:
[0012] (a) selecting a semaphorin for treating cancer in the
subject according to the method of the present invention; and
[0013] (b) contacting cancerous cells of the subject with a
therapeutically effective amount of an agent capable of
upregulating said semaphorin, thereby treating the cancer.
[0014] According to an aspect of some embodiments of the present
invention there is provided a kit for treating cancer, the kit
comprising at least one agent capable of identifying a semaphorin
receptor sub-type and at least one semaphorin.
[0015] According to an aspect of some embodiments of the present
invention there is provided a method of treating a disease
associated with angiogenesis in a subject in need thereof, the
method comprising administering to the subject a therapeutically
effective amount of a semaphorin selected from the group consisting
of Sema3D, Sema3E and Sema3G, thereby treating the disease
associated with angiogenesis.
[0016] According to an aspect of some embodiments of the present
invention there is provided a use of a semaphorin selected from the
group consisting of Sema3D, Sema3E and Sema3G for the treatment of
a disease associated with angiogenesis.
[0017] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as an active ingredient a semaphorin selected from the group
consisting of Sema3D, Sema3E and Sema 3G and a pharmaceutically
acceptable carrier.
[0018] According to some embodiments of the invention, when the
semaphorin receptor comprises NP1, the semaphorin comprises Sema3A
or Sema3D.
[0019] According to some embodiments of the invention, when the
semaphorin receptor comprises NP2, the semaphorin comprises Sema3G
or Sema3F.
[0020] According to some embodiments of the invention, when the
semaphorin receptor comprises PlexD1, the semaphorin comprises
Sema3E.
[0021] According to some embodiments of the invention, the agent is
an antibody.
[0022] According to some embodiments of the invention, the
determining is effected using an antibody.
[0023] According to some embodiments of the invention, the
semaphorin is a class 3 semaphorin.
[0024] According to some embodiments of the invention, the class 3
semaphorin is selected from the group consisting of sema3A, sema3C,
sema3D, sema3E and sema3G.
[0025] According to some embodiments of the invention, the
contacting is effected in vivo.
[0026] According to some embodiments of the invention, the
contacting is effected ex vivo.
[0027] According to some embodiments of the invention, the agent is
a polynucleotide agent comprising a nucleic acid sequence encoding
the semaphorin.
[0028] According to some embodiments of the invention, the
semaphorin receptor is selected from the group consisting of Np1,
Np2, PlexA1-4 and PlexD.
[0029] According to some embodiments of the invention, the
semaphorin is sema3E.
[0030] According to some embodiments of the invention, the sema3E
is a pro-protein convertase resistant Sema3E.
[0031] According to some embodiments of the invention, the disease
associated with angiogenesis is selected from the group consisting
of cancer, arthritis, rheumatoid arthritis, atherosclerotic
plaques, corneal graft neovascularization, hypertrophic or keloid
scars, proliferative retinopathy, diabetic retinopathy, macular
degeneration, granulation, neovascular glaucoma and uveitis.
[0032] In some embodiments of the invention, there is provided a
method of treating cancer in a subject in need thereof, the method
comprising administering to the subject a therapeutically effective
amount of a composition comprising an amino acid sequence at least
70% identical to the sequence set forth in SEQ ID NO: 32, thereby
treating the cancer.
[0033] According to one embodiment the cancer is melanoma.
[0034] In one embodiment the amino acid sequence is at least 95%
identical to the sequence set forth in SEQ ID NO: 32
[0035] In one embodiment the amino acid sequence is semaphorin
Sema3G.
[0036] In one embodiment the administration is systemic
administration.
[0037] In one embodiment the administration is local
administration.
[0038] In one embodiment the said amino acid sequence is attached
to sustained-release enhancing agent.
[0039] In one embodiment the sustained-release enhancing agent is
selected from the group consisting of hyaluronic acid (HA), alginic
acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), polyethylene
glycol (PEG), glyme and polyisopropylacrylamide.
[0040] In some embodiments of the invention, there is provided a a
method of treating cancer by inhibiting angiogenesis to a tumor in
a subject in need thereof, the method comprising administering to
the subject a therapeutically effective amount of a composition
comprising an amino acid sequence at least 70% identical to the
sequence set forth in SEQ ID NO: 32, thereby treating the
cancer.
[0041] In one embodiment the amino acid sequence is at least 95%
identical to the sequence set forth in SEQ ID NO: 32
[0042] In one embodiment the amino acid sequence is semaphorin
Sema3G.
[0043] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0045] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0046] In the drawings:
[0047] FIGS. 1A-C are photographs illustrating the expression of
sema3 receptors in breast cancer derived cell lines. FIG. 1A. Cells
were grown to 80% confluency and lysed. Equal amounts of protein
were loaded and separated on SDS/PAGE gels and blotted on
nitrocellulose. Western blot analysis of np1 and np2 was performed
as described. FIGS. 1B-C. Reverse PCR analysis of plexA1-A4 and
plexD1 expression was performed according to the instruction of the
PerfectPure kit using primer pairs specific to the different
plexins.
[0048] FIGS. 2A-L are graphs and photographs illustrating the
effect of the expression of different sema3s on the development of
tumors from MDA-MB-231 cells. MDA-MB-231 cells were implanted in
the mammary fat pads of balb\c nu/nu mice as described. FIGS. 2A,
D, G, J. Western blot analysis of aliquots of conditioned medium
derived from cells expressing the different sema3s. At the end of
the experiment tumors were excised and photographed. FIGS. 2B, E,
H, K. The average volume of the developing tumors was measured as
described. FIGS. 2C, F, I, L. The average weight of the tumors at
the end of the experiment was determined as described.
[0049] FIGS. 3A-I are graphs and photographs illustrating the
effect of the expression of different sema3s on the development of
tumors from MDA-MB-435 cells. MDA-MB-435 cells were implanted in
the mammary fat pad of balb\c nu/nu mice as described. FIGS. 3A, D,
G. Western blot analysis of aliquots of conditioned medium derived
from cells expressing the different sema3s. FIGS. 3B, E, H. The
average volume of the developing tumors was measured as described.
FIGS. 3C, F, I. The average weight of the tumors at the end of the
experiment was determined as described.
[0050] FIGS. 4A-F are graphs and photographs illustrating the
effect of the expression of sema3A and sema3F on the development of
tumors from MCF-7 and MDA-MB-468 cells. MCF-7 and MDA-MB-468 cells
were implanted in the mammary fat pad of balb\c nu/nu mice as
described. FIGS. 4A, D. Western blot analysis of aliquots of
conditioned medium derived from cells expressing either sema3A or
sema3F. FIGS. 4B, E. The average volume of the developing tumors
was measured as described. FIGS. 4C, F. The average weight of the
tumors at the end of the experiment was determined as
described.
[0051] FIGS. 5A-E are graphs and photographs illustrating that
different sema3s repel endothelial cells in-vitro and reduce the
density of tumor associated blood vessels in-vivo. FIG. 5A: Control
HEK293 cells infected with an empty lentiviral vector or HEK293
cells expressing sema3A, sema3D or sema3E were seeded on top of a
monolayer of HUVEC cells as described in experimental procedures.
The HEK292 cells were labeled with the fluorescent vital dye D1 asp
prior to seeding. Shown are composite pictures taken by phase and
fluorescent microscopy. FIG. 5B. Control HEK293 cells infected with
an empty lentiviral vector or HEK293 cells expressing sema3F,
sema3G were seeded on a monolayer of PAE cells expressing np2 and
plexA1 as described in the Materials and Methods. The HEK293 cells
were stained with D1 asp and photographed as described herein
above. FIG. 5C. The average area of blood vessels per microscopic
field was determined in sections derived from tumors that developed
from control MDA-MB-231 cells or from MDA-MB-231 cells expressing
different sema3s as described herein. Since the tumors that did
develop from sema3A expressing cells were extremely small, the
density of blood vessels in them could not be determined. FIG. 5D.
The average area of blood vessels per microscopic field was
determined in tumors derived from control MCF-7 cells or from MCF-7
cells expressing sema3A or sema3F as described herein above. FIG.
5E. The average area of blood vessels per microscopic field was
determined in tumors that developed from control MDA-MB-435 cells
or from MDA-MB-435 cells expressing different sema3s as described
herein above. No tumors developed from sema3G expressing cells.
[0052] FIGS. 6A-D are graphs and photographs illustrating that
different sema3s inhibit the formation of soft agar colonies from
MDA-MB-231 or MDA-MB-435 cells. FIG. 6A. Single cell suspensions of
control MDA-MB-231 cells or MDA-MB-231 cells expressing different
sema3s were seeded in soft agar as described herein. Colonies were
allowed to form for 21 days. The colonies were then stained with
crystal violet and microscopic fields photographed. The average
number/field of colonies with a diameter exceeding 150 .mu.m was
then determined as described under experimental procedures. FIG.
6B. Photographs of representative microscopic fields containing
crystal violet stained colonies that developed in soft agar from
control MDA-MB-231 cells or from sema3s expressing MDA-MB-231
cells. FIG. 6C. The formation of colonies in soft agar from control
MDA-MB-435 cells or from MDA-MB-435 cells expressing different
sema3s was determined as described herein above. FIG. 6D.
Photographs of representative microscopic fields containing crystal
violet stained colonies that developed in soft agar from control
MDA-MB-435 cells or from sema3s expressing MDA-MB-435 cells.
[0053] FIGS. 7A-D are graphs and photographs illustrating that
expression of np1 in MDA-MB-435 cells enhances the growth of
resulting tumors and sema3A abrogate the enhancing effect. FIG. 7A.
A western blot comparing the expression of np1 in MDA-MB-435 cells
infected with various combinations as depicted of a control
lentiviral vector, a lentiviral vector containing the np1 cDNA and
a lentiviral vector containing the sema3A cDNA is shown at the top.
The Average tumor volume as was measured during the experiment as
described and is shown in the graph below. FIG. 7B. Photographs of
tumors excised at the end of the experiment. FIG. 7C. The average
weight of the tumors at the end of the experiment was determined.
FIG. 7D. The average area of blood vessels/field in tumor sections
was determined.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0054] The present invention, in some embodiments thereof, relates
to methods and compositions for treating angiogenesis-related
diseases such as cancer, and methods of selection thereof.
[0055] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0056] The semaphorins belonging to the class-3 semaphorin
sub-family (sema-3s) function as axon guidance factors during
embryonic development. Most of the class-3 semaphorins, with the
exception of sema3E, bind to one of the two neuropilin receptors,
which in turn form complexes with several members of the plexin
receptor family. In these complexes the neuropilins bind the
semaphorins while the plexins function as the signal transducing
elements.
[0057] Semaphorins sema3B and sema3F have been characterized as
tumor suppressors, whilst sema3F, sema3A and sema3E have each been
attributed an anti angiogenic function.
[0058] Whilst reducing the present invention to practice, the
present inventors have shown that sema3A, sema3D, sema3E and sema3G
each function as potent anti-tumorigenic agents (FIGS. 2A-L; FIGS.
3A-I; FIGS. 4A-F). Specifically, injection of breast cancer cells
expressing these semaphorins into nude mice resulted in tumors of
reduced size as compared to tumors resulting from injection of
tumor cells not expressing these semaphorins.
[0059] The invention is further based on the experiments conducted
in which cDNAs encoding sema3G were expressed in MDA-MB-345. The
cells were subsequently implanted in mammary fat pads of immune
deficient mice and allowed to form tumors. It was shown that sema3G
strongly inhibited the development of tumors as shown in example 1
and FIGS. 3H-3I. Additionally, cDNAs encoding sema3G were expressed
in MDA-MB-345 grown in vitro. The cells were subsequently placed in
soft-agar. It was shown that sema3G efficiently inhibited colony
formation as shown in example 1 and in FIG. 6D. MDA-MB-345 is known
as a model for melanoma cancer as can be seen in for example, J M
Rae et al. "MDA-MB-435 cells are derived from M14 melanoma cells--a
loss for breast cancer, but a boon for melanoma research", Breast
Cancer Res Treat. 2007 July; 104(1):13-9. E-pub 2006 Sep. 27.
[0060] Whilst further reducing the present invention to practice,
the present inventors have shown that semaphorin induced inhibition
of tumor development from specific types of breast cancer cells is
correlated with the expression of appropriate semaphorin receptors
by the tumor cells. Although, the majority of the tested
semaphorins also inhibited tumor angiogenesis, the present
inventors showed that there was no correlation between inhibition
of tumor angiogenesis and inhibition of tumor development. These
results suggest that inhibition of tumor development by semaphorins
depends on the expression of appropriate semaphorin receptors by
tumor cells, and suggest that inhibition of angiogenesis is of
lesser importance. They also suggest that tumors containing tumor
cells expressing semaphorin receptors may be amenable to inhibition
by appropriate sema3s and open the way for improved methods of
personalized medicine for cancer treatment.
[0061] Thus, according to one aspect of the present invention,
there is provided a method of selecting a semaphorin for treating
cancer in a subject. The method comprises determining an expression
of a semaphorin receptor on tumor cells of a tumor sample of the
subject wherein an amount of the semaphorin receptor is indicative
of the semaphorin suitable for treating the cancer in the
subject.
[0062] As used herein, the term "semaphorin" refers to a mammalian
polypeptide (e.g. human) belonging to the semaphorin family
(including semaphorins of class 3, 4, 5, 6 and 7). Semaphorins
typically function as signals during axon guidance and comprise a
sema domain
[0063] According to one embodiment, the semaphorin belongs to the
class-3 semaphorin sub-family. Accordingly, the semaphorin may be
semaphorin 3A (Genbank accession number NM_006080, SEQ ID NO: 26);
semaphorin 3B (Genbank accession number NM_001005914, SEQ ID NO:
27); semaphorin 3C (Genbank accession number NM_006379, SEQ ID NO:
28); semaphorin 3D (Genbank accession number NM_152754, SEQ ID NO:
29); semaphorin 3E (Genbank accession number NM_012431, SEQ ID NO:
30); semaphorin 3F (Genbank accession number NM_004186, SEQ ID NO:
31); or semaphorin 3G (Genbank accession number NM_020163, SEQ ID
NO: 32).
[0064] A semaphorin of the present invention also refers to
homologs (e.g., polypeptides which are at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 87%, at least 89%, at least 91%, at
least 93%, at least 95% or more say 100% homologous to semaphorin
sequences listed herein above as determined using BlastP software
of the National Center of Biotechnology Information (NCBI) using
default parameters). The homolog may also refer to a deletion,
insertion, or substitution variant, including an amino acid
substitution, thereof and biologically active polypeptide fragments
thereof.
[0065] The term "treating" as used herein includes abrogating,
substantially inhibiting, slowing or reversing the progression of
the cancer, substantially ameliorating clinical or aesthetical
symptoms of the cancer or substantially preventing the appearance
of clinical or aesthetical symptoms of the cancer.
[0066] Typically, the subject for whom the semaphorin is selected
is a mammalian subject e.g. a human
[0067] As used herein the term "cancer" refers to the presence of
cells possessing characteristics typical of cancer-causing cells,
for example, uncontrolled proliferation, loss of specialized
functions, immortality, significant metastatic potential,
significant increase in anti-apoptotic activity, rapid growth and
proliferation rate, and certain characteristic morphology and
cellular markers. Typically, the cancer cells are in the form of a
tumor; existing locally within an animal, or circulating in the
blood stream as independent cells, for example, leukemic cells.
[0068] Specific examples of cancer for which semaphorins may be
selected according to this aspect of the present invention include,
but are not limited to, adrenocortical carcinoma, hereditary;
bladder cancer; breast cancer; breast cancer, ductal; breast
cancer, invasive intraductal; breast cancer, sporadic; breast
cancer, susceptibility to; breast cancer, type 4; breast cancer,
type 4; breast cancer-1; breast cancer-3; breast-ovarian cancer;
Burkitt's lymphoma; cervical carcinoma; colorectal adenoma;
colorectal cancer; colorectal cancer, hereditary nonpolyposis, type
1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal
cancer, hereditary nonpolyposis, type 3; colorectal cancer,
hereditary nonpolyposis, type 6; colorectal cancer, hereditary
nonpolyposis, type 7; dermatofibrosarcoma protuberans; endometrial
carcinoma; esophageal cancer; gastric cancer, fibrosarcoma,
glioblastoma multiforme; glomus tumors, multiple; hepatoblastoma;
hepatocellular cancer; hepatocellular carcinoma; leukemia, acute
lymphoblastic; leukemia, acute myeloid; leukemia, acute myeloid,
with eosinophilia; leukemia, acute nonlymphocytic; leukemia,
chronic myeloid; Li-Fraumeni syndrome; liposarcoma, lung cancer;
lung cancer, small cell; lymphoma, non-Hodgkin's; lynch cancer
family syndrome II; male germ cell tumor; mast cell leukemia;
medullary thyroid; medulloblastoma; melanoma, meningioma; multiple
endocrine neoplasia; myeloid malignancy, predisposition to;
myxosarcoma, neuroblastoma; osteosarcoma; ovarian cancer; ovarian
cancer, serous; ovarian carcinoma; ovarian sex cord tumors;
pancreatic cancer; pancreatic endocrine tumors; paraganglioma,
familial nonchromaffin; pilomatricoma; pituitary tumor, invasive;
prostate adenocarcinoma; prostate cancer; renal cell carcinoma,
papillary, familial and sporadic; retinoblastoma; rhabdoid
predisposition syndrome, familial; rhabdoid tumors;
rhabdomyosarcoma; small-cell cancer of lung; soft tissue sarcoma,
squamous cell carcinoma, head and neck; T-cell acute lymphoblastic
leukemia; Turcot syndrome with glioblastoma; tylosis with
esophageal cancer; uterine cervix carcinoma, Wilms' tumor, type 2;
and Wilms' tumor, type 1, and the like. According to a particular
embodiment of this aspect of the present invention, the cancer is
breast cancer.
[0069] According to a particular embodiment of this aspect of the
present invention, the cancer is melanoma cancer.
[0070] As mentioned, the method of the present invention is
effected by determining an expression of a semaphorin receptor on
tumor cells of a tumor sample of a subject.
[0071] As used herein, the phrase "semaphorin receptor" refers to a
cell-surface polypeptide that is capable of binding to a semaphorin
and transducing a response. Exemplary semaphorin receptors include,
neuropilins, plexins and integrins.
[0072] Thus, for example, the neuropilin receptor may be a
neuropilin 1 receptor (NP1; e.g. NM_001024628; SEQ ID NO: 17) or a
neuropilin 2 receptor (NP2; e.g. NM_201279; SEQ ID NO: 18).
[0073] The plexin receptor may be a plexinA1 receptor (PlexA1; e.g.
NM_032242; SEQ ID NO: 19), a plexinA2 receptor (PlexA2; e.g.
NM_025179; SEQ ID NO: 20), a plexinA3 receptor (PlexA3; e.g.
NM_017514; SEQ ID NO: 21), a plexinA4 receptor (PlexA4; e.g.
NM_020911, SEQ ID NO: 22; NM_001105543, SEQ ID NO: 23; NM_181775,
SEQ ID NO: 24) or a plexinD receptor (PlexD; e.g. NM_015103, SEQ ID
NO: 25).
[0074] Methods of determining an expression of a semaphorin
receptor are known in the art. Specifically, determining an
expression of semaphorin receptors may be effected on the RNA or
protein level as detailed below.
[0075] Methods of Detecting Expression of a Semaphorin Receptor on
the RNA Level
[0076] Northern Blot analysis: This method involves the detection
of a particular RNA i.e. a semaphoring receptor RNA in a mixture of
RNAs. An RNA sample is denatured by treatment with an agent (e.g.,
formaldehyde) that prevents hydrogen bonding between base pairs,
ensuring that all the RNA molecules have an unfolded, linear
conformation. The individual RNA molecules are then separated
according to size by gel electrophoresis and transferred to a
nitrocellulose or a nylon-based membrane to which the denatured
RNAs adhere. The membrane is then exposed to labeled DNA probes.
Probes may be labeled using radio-isotopes or enzyme linked
nucleotides. Detection may be using autoradiography, colorimetric
reaction or chemiluminescence. This method allows both quantitation
of an amount of particular RNA molecules and determination of its
identity by a relative position on the membrane which is indicative
of a migration distance in the gel during electrophoresis.
[0077] RT-PCR analysis: This method uses PCR amplification of
relatively rare RNAs molecules. First, RNA molecules are purified
from the cells and converted into complementary DNA (cDNA) using a
reverse transcriptase enzyme (such as an MMLV-RT) and primers such
as, oligo dT, random hexamers or gene specific primers. Then by
applying gene specific primers and Taq DNA polymerase, a PCR
amplification reaction is carried out in a PCR machine. Those of
skills in the art are capable of selecting the length and sequence
of the gene specific primers and the PCR conditions (i.e.,
annealing temperatures, number of cycles and the like) which are
suitable for detecting specific RNA molecules. It will be
appreciated that a semi-quantitative RT-PCR reaction can be
employed by adjusting the number of PCR cycles and comparing the
amplification product to known controls. Exemplary primers that may
be used to detect NRP1 receptors are set forth in SEQ ID NOs: 3 and
4. Exemplary primers that may be used to detect NRP2 receptors are
set forth in SEQ ID NOs: 5 and 6. Exemplary primers that may be
used to detect PLXNA1 receptors are set forth in SEQ ID NOs: 7 and
8. Exemplary primers that may be used to detect PLXNA2 receptors
are set forth in SEQ ID NOs: 9 and 10. Exemplary primers that may
be used to detect PLXNA3 receptors are set forth in SEQ ID NOs: 11
and 12. Exemplary primers that may be used to detect PLXNA4
receptors are set forth in SEQ ID NOs: 13 and 14. Exemplary primers
that may be used to detect PLXND1 receptors are set forth in SEQ ID
NOs: 15 and 16. Exemplary primers that may be used to detect CDH2
receptors are set forth in SEQ ID NOs: 1 and 2.
[0078] RNA in situ hybridization stain: In this method DNA or RNA
probes are attached to the RNA molecules present in the cells.
Generally, the cells are first fixed to microscopic slides to
preserve the cellular structure and to prevent the RNA molecules
from being degraded and then are subjected to hybridization buffer
containing the labeled probe. The hybridization buffer includes
reagents such as formamide and salts (e.g., sodium chloride and
sodium citrate) which enable specific hybridization of the DNA or
RNA probes with their target mRNA molecules in situ while avoiding
non-specific binding of probe. Those of skills in the art are
capable of adjusting the hybridization conditions (i.e.,
temperature, concentration of salts and formamide and the like) to
specific probes and types of cells. Following hybridization, any
unbound probe is washed off and the slide is subjected to either a
photographic emulsion which reveals signals generated using
radio-labeled probes or to a colorimetric reaction which reveals
signals generated using enzyme-linked labeled probes.
[0079] In situ RT-PCR stain: This method is described in Nuovo G J,
et al. [Intracellular localization of polymerase chain reaction
(PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 1993, 17:
683-90] and Komminoth P, et al. [Evaluation of methods for
hepatitis C virus detection in archival liver biopsies. Comparison
of histology, immunohistochemistry, in situ hybridization, reverse
transcriptase polymerase chain reaction (RT-PCR) and in situ
RT-PCR. Pathol Res Pract. 1994, 190: 1017-25]. Briefly, the RT-PCR
reaction is performed on fixed cells by incorporating labeled
nucleotides to the PCR reaction. The reaction is carried on using a
specific in situ RT-PCR apparatus such as the laser-capture
microdissection PixCell I LCM system available from Arcturus
Engineering (Mountainview, Calif.).
[0080] Oligonucleotide microarray--In this method oligonucleotide
probes capable of specifically hybridizing with the polynucleotides
encoding the semaphorin receptors of the present invention are
attached to a solid surface (e.g., a glass wafer). Each
oligonucleotide probe is of approximately 20-25 nucleic acids in
length. To detect the expression pattern of the polynucleotides of
the present invention in a specific cell sample (e.g., tumor
cells), RNA is extracted from the cell sample using methods known
in the art (using e.g., a TRIZOL solution, Gibco BRL, USA).
Hybridization can take place using either labeled oligonucleotide
probes (e.g., 5'-biotinylated probes) or labeled fragments of
complementary DNA (cDNA) or RNA (cRNA). Briefly, double stranded
cDNA is prepared from the RNA using reverse transcriptase (RT)
(e.g., Superscript II RT), DNA ligase and DNA polymerase I, all
according to manufacturer's instructions (Invitrogen Life
Technologies, Frederick, Md., USA). To prepare labeled cRNA, the
double stranded cDNA is subjected to an in vitro transcription
reaction in the presence of biotinylated nucleotides using e.g.,
the BioArray High Yield RNA Transcript Labeling Kit (Enzo,
Diagnostics, Affymetix Santa Clara, Calif.). For efficient
hybridization the labeled cRNA can be fragmented by incubating the
RNA in 40 mM Tris Acetate (pH 8.1), 100 mM potassium acetate and 30
mM magnesium acetate for 35 minutes at 94.degree. C. Following
hybridization, the microarray is washed and the hybridization
signal is scanned using a confocal laser fluorescence scanner which
measures fluorescence intensity emitted by the labeled cRNA bound
to the probe arrays.
[0081] For example, in the Affymetrix microarray (Affymetrix.RTM.,
Santa Clara, Calif.) each gene on the array is represented by a
series of different oligonucleotide probes, of which, each probe
pair consists of a perfect match oligonucleotide and a mismatch
oligonucleotide. While the perfect match probe has a sequence
exactly complimentary to the particular gene, thus enabling the
measurement of the level of expression of the particular gene, the
mismatch probe differs from the perfect match probe by a single
base substitution at the center base position. The hybridization
signal is scanned using the Agilent scanner, and the Microarray
Suite software subtracts the non-specific signal resulting from the
mismatch probe from the signal resulting from the perfect match
probe.
[0082] Methods of Detecting Semaphorin Receptors on the Protein
Level
[0083] Determining expression of a semaphorin receptor on the
protein level is typically effected using an antibody capable of
specifically binding with a particular semaphorin receptor.
[0084] Exemplary antibodies capable of specifically interacting
with NP1 and NP2 are widely available e.g. from Santa-Cruz
Biotechnology (Santa Cruz, Calif., Catalogue nos. sc-12122, sc
12123, sc-12125, sc-12128 and sc-50408).
[0085] Exemplary antibodies capable of specifically interacting
with plexin receptors are also widely available e.g. from
Santa-Cruz Biotechnology (Santa Cruz, Calif., Catalogue Nos.
sc-25639, sc-10138, sc-10139, sc-10144, sc-25640, sc-10143,
sc-25641, sc-10135, sc-10134, sc-28372, sc10147, sc-25642,
sc-10145, sc-67034, sc-34504, sc-34506, sc-34507, sc-46240,
sc-67144, sc-46241, sc-46242, sc46243, sc-10152, sc-10149,
sc-46244, sc-46245, sc-67145, sc-46246 and sc-46247. Antibodies are
also available from Abcam, MA, U.S.A. (Catalogue Nos. ab32960,
ab23391, ab39350, ab39357, ab39008, ab41564 and ab39715).
[0086] Preferably, the antibody specifically binds at least one
epitope of the semaphorin receptor. As used herein, the term
"epitope" refers to any antigenic determinant on an antigen to
which the paratope of an antibody binds.
[0087] Epitopic determinants usually consist of chemically active
surface groupings of molecules such as amino acids or carbohydrate
side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
[0088] The term "antibody" as used in this invention includes
intact molecules as well as functional fragments thereof, such as
Fab, F(ab')2, and Fv that are capable of binding to macrophages.
These functional antibody fragments are defined as follows: (1)
Fab, the fragment which contains a monovalent antigen-binding
fragment of an antibody molecule, can be produced by digestion of
whole antibody with the enzyme papain to yield an intact light
chain and a portion of one heavy chain; (2) Fab', the fragment of
an antibody molecule that can be obtained by treating whole
antibody with pepsin, followed by reduction, to yield an intact
light chain and a portion of the heavy chain; two Fab' fragments
are obtained per antibody molecule; (3) (Fab')2, the fragment of
the antibody that can be obtained by treating whole antibody with
the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer
of two Fab' fragments held together by two disulfide bonds; (4) Fv,
defined as a genetically engineered fragment containing the
variable region of the light chain and the variable region of the
heavy chain expressed as two chains; and (5) Single chain antibody
("SCA"), a genetically engineered molecule containing the variable
region of the light chain and the variable region of the heavy
chain, linked by a suitable polypeptide linker as a genetically
fused single chain molecule.
[0089] Methods of producing polyclonal and monoclonal antibodies as
well as fragments thereof which bind to specific semaphorin
receptors are well known in the art (See for example, Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York, 1988, incorporated herein by reference).
[0090] Methods of detecting semaphorin receptors include
immunoassays which include but are not limited to competitive and
non-competitive assay systems using techniques such as Western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, and immunoprecipitation assays and
immunohistochemical assays as detailed herein below.
[0091] Enzyme linked immunosorbent assay (ELISA): This method
involves fixation of a sample (e.g., fixed cells or a proteinaceous
solution) containing a protein substrate to a surface such as a
well of a microtiter plate. A substrate specific antibody coupled
to an enzyme is applied and allowed to bind to the substrate.
Presence of the antibody is then detected and quantitated by a
colorimetric reaction employing the enzyme coupled to the antibody.
Enzymes commonly employed in this method include horseradish
peroxidase and alkaline phosphatase. If well calibrated and within
the linear range of response, the amount of substrate present in
the sample is proportional to the amount of color produced. A
substrate standard is generally employed to improve quantitative
accuracy.
[0092] Western blot: This method involves separation of a substrate
from other protein by means of an acrylamide gel followed by
transfer of the substrate to a membrane (e.g., nylon or PVDF).
Presence of the substrate is then detected by antibodies specific
to the substrate, which are in turn detected by antibody binding
reagents. Antibody binding reagents may be, for example, protein A,
or other antibodies. Antibody binding reagents may be radiolabeled
or enzyme linked as described hereinabove. Detection may be by
autoradiography, colorimetric reaction or chemiluminescence. This
method allows both quantitation of an amount of substrate and
determination of its identity by a relative position on the
membrane which is indicative of a migration distance in the
acrylamide gel during electrophoresis.
[0093] Radio-immunoassay (RIA): In one version, this method
involves precipitation of the desired protein (i.e., the substrate)
with a specific antibody and radiolabeled antibody binding protein
(e.g., protein A labeled with I.sup.125) immobilized on a
precipitable carrier such as agarose beads. The number of counts in
the precipitated pellet is proportional to the amount of
substrate.
[0094] In an alternate version of the RIA, a labeled substrate and
an unlabeled antibody binding protein are employed. A sample
containing an unknown amount of substrate is added in varying
amounts. The decrease in precipitated counts from the labeled
substrate is proportional to the amount of substrate in the added
sample.
[0095] Fluorescence activated cell sorting (FACS): This method
involves detection of a substrate in situ in cells by substrate
specific antibodies. The substrate specific antibodies are linked
to fluorophores. Detection is by means of a cell sorting machine
which reads the wavelength of light emitted from each cell as it
passes through a light beam. This method may employ two or more
antibodies simultaneously.
[0096] Immunohistochemical analysis: This method involves detection
of a substrate in situ in fixed cells by substrate specific
antibodies. The substrate specific antibodies may be enzyme linked
or linked to fluorophores. Detection is by microscopy and
subjective or automatic evaluation. If enzyme linked antibodies are
employed, a colorimetric reaction may be required. It will be
appreciated that immunohistochemistry is often followed by
counterstaining of the cell nuclei using for example Hematoxyline
or Giemsa stain.
[0097] In situ activity assay: According to this method, a
chromogenic substrate is applied on the cells containing an active
enzyme and the enzyme catalyzes a reaction in which the substrate
is decomposed to produce a chromogenic product visible by a light
or a fluorescent microscope.
[0098] It will be appreciated that the tumor cells of the subject
are obtained from a tumor sample e.g. during a tumor biopsy.
[0099] As mentioned, the amount of the semaphorin receptor is
indicative of the semaphorin suitable for treating the cancer in
the subject.
[0100] It will be appreciated that the amount of the semaphorin
receptor should be sufficient to transduce a biological response
(i.e. tumor inhibition). The amount of receptor sufficient to
generate such a response is typically dependent on the affinity of
the semaphorin for that receptor. Thus, for example if a semaphorin
has a high affinity for a receptor (e.g. comprises a Km of about
10.sup.-7 M, 10.sup.-8 M, 10.sup.-9 M, 10.sup.-10 M or even
10.sup.-11M), the amount of receptor does not have to be as great
the amount of receptor for which the semaphorin has a low affinity
receptor (e.g. comprises a Km of about 10.sup.-6 M 10.sup.-5M,
10.sup.-4 M, 10.sup.-3 M).
[0101] According to one embodiment, the amount of receptor on the
tumor cells is at least 20% the total number of semaphorin
receptors on the tumor cells. According to another embodiment, the
amount of receptor on the tumor cells is at least 30% the total
number of semaphorin receptors on the tumor cells. According to
another embodiment, the amount of receptor on the tumor cells is at
least 40% the total number of semaphorin receptors on the tumor
cells. According to another embodiment, the amount of receptor on
the tumor cells is at least 50% the total number of semaphorin
receptors on the tumor cells. According to another embodiment, the
amount of receptor on the tumor cells is at least 60% the total
number of semaphorin receptors on the tumor cells. According to
another embodiment, the amount of receptor on the tumor cells is at
least 70% the total number of semaphorin receptors on the tumor
cells. According to another embodiment, the amount of receptor on
the tumor cells is at least 80% the total number of semaphorin
receptors on the tumor cells.
[0102] Accordingly, the present inventors have found that if a
sufficient quantity of NP1 receptors are located on the tumor
cells, the most preferable semaphorin for treatment comprises
Sema3A or Sema3D. If a sufficient quantity of NP2 receptors are
located on the tumor cells, the most preferable semaphorin for
treatment comprises Sema3G or Sema3F. If a sufficient quantity of
PlexD1 receptors are located on the tumor cells, the most
preferable semaphorin for treatment comprises Sema3E.
[0103] It will be appreciated that selection of the semaphorin is
not only based on the quantity of a receptor, but also expression
profile of a plurality of semaphorin receptors subtypes. For
example, it is known that neuropilins form spontaneous complexes
with several members of the plexin receptor family. Accordingly,
selection of the semaphorin may also be effected based on the
expression pattern of both the neuropilin receptor and the plexin
receptor.
[0104] The present inventors have also found that an additional
method for selecting a semaphorin for treating a cancer.
Semaphorins that were shown to inhibit the anchorage independent
growth of a particular tumor cell were also shown to be effective
at inhibiting tumor formation. A method of measuring anchorage
independent growth of tumor cells is described in the Materials and
Methods section of the Examples herein below involving measurement
of colonies in soft agar.
[0105] It will be appreciated that the agents used for detecting
semaphorin receptor expression may be provided as a kit, such as an
FDA-approved kit, which may contain one or more unit dosage form
containing the active agent (e.g. antibody or probe capable of
specifically interacting with a semaphorin subtype). The kit may
also comprise other agents useful for analyzing semaphorin receptor
expression (e.g. suitable buffers, control antibodies or probes).
In addition, the kit may comprise agents used for measuring tumor
colonies in soft agar.
[0106] The kit may be accompanied by instructions for
administration. The kit may also be accompanied by a notice in a
form prescribed by a governmental agency regulating the
manufacture, use, or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions for human or veterinary administration. Such notice,
for example, may include labeling approved by the U.S. Food and
Drug Administration.
[0107] Following selection, treatment of the cancer may be
initiated by contacting (either in vivo or ex vivo) the cancer
cells with an agent capable of upregulating the appropriate
semaphorin.
[0108] Accordingly, the present invention contemplates
administration of therapeutically effective amounts of semaphorins
themselves, or administration of polynucleotides encoding the
semaphorins (i.e. gene therapy) to subjects in need thereof in
order to treat cancer.
[0109] The semaphorins polypeptides may comprise the full length
sequences of those set forth in SEQ ID NOs: 26-32. Alternatively
the semaphorins may be homologs (e.g., polypeptides which are at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 87%, at least
89%, at least 91%, at least 93%, at least 95% or more say 100%
homologous to semaphorin sequences listed herein above as
determined using BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters)
comprising semaphorin activity. The homolog may also refer to a
deletion, insertion, or substitution variant, including an amino
acid substitution, thereof and biologically active polypeptide
fragments thereof.
[0110] The term "polypeptide" as used herein refers to a polymer of
natural or synthetic amino acids, encompassing native peptides
(either degradation products, synthetically synthesized
polypeptides or recombinant polypeptides) and peptidomimetics
(typically, synthetically synthesized peptides), as well as
peptoids and semipeptoids which are polypeptide analogs, which may
have, for example, modifications rendering the peptides even more
stable while in a body or more capable of penetrating into
cells.
[0111] Such modifications include, but are not limited to N
terminus modification, C terminus modification, polypeptide bond
modification, including, but not limited to, CH2-NH, CH2-S,
CH2-S.dbd.O, O.dbd.C--NH, CH2-O, CH2-CH2, S.dbd.C--NH, CH.dbd.CH or
CF.dbd.CH, backbone modifications, and residue modification.
Methods for preparing peptidomimetic compounds are well known in
the art and are specified, for example, in Quantitative Drug
Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press
(1992), which is incorporated by reference as if fully set forth
herein. Further details in this respect are provided
hereinunder.
[0112] Polypeptide bonds (--CO--NH--) within the polypeptide may be
substituted, for example, by N-methylated bonds (--N(CH3)-CO--),
ester bonds (--C(R)H--C--O--O--C(R)--N--), ketomethylen bonds
(--CO--CH2-), .alpha.-aza bonds (--NH--N(R)--CO--), wherein R is
any alkyl, e.g., methyl, carba bonds (--CH2-NH--), hydroxyethylene
bonds (--CH(OH)--CH2-), thioamide bonds (--CS--NH--), olefinic
double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--),
polypeptide derivatives (--N(R)--CH2-CO--), wherein R is the
"normal" side chain, naturally presented on the carbon atom.
[0113] These modifications can occur at any of the bonds along the
polypeptide chain and even at several (2-3) at the same time.
[0114] Natural aromatic amino acids, Trp, Tyr and Phe, may be
substituted for synthetic non-natural acid such as Phenylglycine,
TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe,
halogenated derivatives of Phe or o-methyl-Tyr.
[0115] In addition to the above, the polypeptides of the present
invention may also include one or more modified amino acids or one
or more non-amino acid monomers (e.g. fatty acids, complex
carbohydrates etc).
[0116] As used herein in the specification and in the claims
section below the term "amino acid" or "amino acids" is understood
to include the 20 naturally occurring amino acids; those amino
acids often modified post-translationally in vivo, including, for
example, hydroxyproline, phosphoserine and phosphothreonine; and
other unusual amino acids including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid"
includes both D- and L-amino acids (stereoisomers).
[0117] Tables 1 and 2 below list naturally occurring amino acids
(Table 1) and non-conventional or modified amino acids (Table 2)
which can be used with the present invention.
TABLE-US-00001 TABLE 1 Three-Letter Amino Acid Abbreviation
One-letter Symbol alanine Ala A Arginine Arg R Asparagine Asn N
Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic Acid
Glu E glycine Gly G Histidine His H isoleucine Iie I leucine Leu L
Lysine Lys K Methionine Met M phenylalanine Phe F Proline Pro P
Serine Ser S Threonine Thr T tryptophan Trp W tyrosine Tyr Y Valine
Val V Any amino acid as above Xaa X
TABLE-US-00002 TABLE 2 Non-conventional Non-conventional amino acid
Code amino acid Code .alpha.-aminobutyric acid Abu
L-N-methylalanine Nmala .alpha.-amino-.alpha.-methylbutyrate Mgabu
L-N-methylarginine Nmarg aminocyclopropane- Cpro
L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid
Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys
aminonorbornyl- Norb L-N-methylglutamine Nmgin carboxylate
L-N-methylglutamic Nmglu acid cyclohexylalanine Chexa
L-N-methylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N- Nmphe
methylphenylalanine D-isoleucine Dile L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t- Nmtbug butylglycine D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl- Maib aminoisobutyrate D-valine Dval
.alpha.-methyl-.gamma.- Mgabu aminobutyrate D-.alpha.-methylalanine
Dmala .alpha. ethylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.- Mcpen
methylcyclopentylalanine D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.- Manap napthylalanine
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3- Norn aminopropyl)glycine
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.- Nmaabu
methylbutyrate D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine
Anap D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2- Ngln carbamylethyl)glycine
D-.alpha.-methylornithine Dmorn N- Nasn (carbamylmethyl)glycine
D-.alpha.-methylphenylalanine Dmphe N-(2- Nglu carboxyethyl)glycine
D-.alpha.-methylproline Dmpro N- Nasp (carboxymethyl)glycine
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cyclododeclglycine Ncdod
D-.alpha.-methylalnine Dnmala N-cyclooctylglycine Ncoct
D-.alpha.-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-.alpha.-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-.alpha.-methylasparatate Dnmasp N-(2,2- Nbhm
diphenylethyl)glycine D-.alpha.-methylcysteine Dnmcys N-(3,3- Nbhe
diphenylpropyl)glycine D-N-methylleucine Dnmleu N-(3-indolylyethyl)
Nhtrp glycine D-N-methyllysine Dnmlys N-methyl-.gamma.- Nmgabu
aminobutyrate N-methylcyclohexylalanine Nmchex D-N-methylmethionine
Dnmmet D-N-methylornithine Dnmorn N- Nmcpen
methylcyclopentylalanine N-methylglycine Nala D-N- Dnmphe
methylphenylalanine N-methylaminoisobutyrate Nmaib
D-N-methylproline Dnmpro N-(1-methylpropyl)glycine Nile
D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nile
D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu
D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(1- Nva
methylethyl)glycine D-N-methyltyrosine Dnmtyr N-methyla- Nmanap
napthylalanine D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p- Nhtyr hydroxyphenyl)glycine
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t- Mtbug
butylglycine L-.alpha.-methylcysteine Mcys L-methylethylglycine
Metg L-.alpha. thylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.- Mhphe
methylhomophenylalanine L-.alpha.-methylisoleucine Mile N-(2- Nmet
methylthioethyl)glycine D-N-methylglutamine Dnmgln N-(3- Narg
guanidinopropyl)glycine D-N-methylglutamate Dnmglu N-(1- Nthr
hydroxyethyl)glycine D-N-methylhistidine Dnmhis
N-(hydroxyethyl)glycine Nser D-N-methylisoleucine Dnmile N- Nhis
(imidazolylethyl)glycine D-N-methylleucine Dnmleu N-(3- Nhtrp
indolylyethyl)glycine D-N-methyllysine Dnmlys N-methyl-.gamma.-
Nmgabu aminobutyrate N-methylcyclohexylalanine Nmchex
D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N- Nmcpen
methylcyclopentylalanine N-methylglycine Nala D-N- Dnmphe
methylphenylalanine N-methylaminoisobutyrate Nmaib
D-N-methylproline Dnmpro N-(1-methylpropyl)glycine Nile
D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu
D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(1- Nval
methylethyl)glycine D-N-methyltyrosine Dnmtyr N-methyla- Nmanap
napthylalanine D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p- Nhtyr hydroxyphenyl)glycine
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t- Mtbug
butylglycine L-.alpha.-methylcysteine Mcys L-methylethylglycine
Metg L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha. ethylhistidine Mhis L-.alpha.- Mhphe
methylhomophenylalanine L-.alpha. thylisoleucine Mile N-(2- Nmet
methylthioethyl)glycine L-.alpha.-methylleucine Mleu
L-.alpha.-methyllysine Mlys L-.alpha.-methylmethionine Mmet
L-.alpha.-methylnorleucine Mnle L-.alpha.-methylnorvaline Mnva
L-.alpha.-methylornithine Morn L-.alpha.-methylphenylalanine Mphe
L-.alpha.-methylproline Mpro L-.alpha.-methylserine mser
L-.alpha.-methylthreonine Mthr L-.alpha. ethylvaline Mtrp
L-.alpha.-methyltyrosine Mtyr L-.alpha.-methylleucine Mval L-N-
Nmhphe nbhm methylhomophenylalanine N-(N-(2,2-diphenylethyl)
N-(N-(3,3- diphenylpropyl) carbamylmethyl-glycine Nnbhm
carbamylmethyl(1)glycine Nnbhe 1-carboxy-1-(2,2-diphenyl- Nmbc
hylamino)cyclopropane
[0118] As mentioned herein above, the semaphorin of the present
invention may comprise a conservative or non-conservative
substitution.
[0119] The term "conservative substitution" as used herein, refers
to the replacement of an amino acid present in the native sequence
in the peptide with a naturally or non-naturally occurring amino or
a peptidomimetics having similar steric properties. Where the
side-chain of the native amino acid to be replaced is either polar
or hydrophobic, the conservative substitution should be with a
naturally occurring amino acid, a non-naturally occurring amino
acid or with a peptidomimetic moiety which is also polar or
hydrophobic (in addition to having the same steric properties as
the side-chain of the replaced amino acid).
[0120] As naturally occurring amino acids are typically grouped
according to their properties, conservative substitutions by
naturally occurring amino acids can be easily determined bearing in
mind the fact that in accordance with the invention replacement of
charged amino acids by sterically similar non-charged amino acids
are considered as conservative substitutions.
[0121] For producing conservative substitutions by non-naturally
occurring amino acids it is also possible to use amino acid analogs
(synthetic amino acids) well known in the art. A peptidomimetic of
the naturally occurring amino acid is well documented in the
literature known to the skilled practitioner.
[0122] When affecting conservative substitutions the substituting
amino acid should have the same or a similar functional group in
the side chain as the original amino acid.
[0123] The phrase "non-conservative substitutions" as used herein
refers to replacement of the amino acid as present in the parent
sequence by another naturally or non-naturally occurring amino
acid, having different electrochemical and/or steric properties.
Thus, the side chain of the substituting amino acid can be
significantly larger (or smaller) than the side chain of the native
amino acid being substituted and/or can have functional groups with
significantly different electronic properties than the amino acid
being substituted. Examples of non-conservative substitutions of
this type include the substitution of phenylalanine or
cycohexylmethyl glycine for alanine, isoleucine for glycine, or
--NH--CH[(--CH.sub.2).sub.5-COOH]--CO-- for aspartic acid. Those
non-conservative substitutions which fall under the scope of the
present invention are those which still constitute a peptide having
semaphorin-like properties.
[0124] As mentioned, the semaphorins of the present invention may
comprise substitutions. According to one embodiment, the semaphorin
may be engineered to resist cleavage by furin-like pro-protein
convertases. Thus, for example the pro-protein convertase
recognition sequence RFRR (SEQ ID NO: 39) may be mutated into the
sequence KFKK (SEQ ID NO: 40). This has been effected for
semaphorin-3B, where the authors showed that this mutation
conferred partial resistance to pro-protein convertases of cancer
cells without affecting the biological activity of full length
semaphorin-3B [Varshaysky A, Kessler O, Abramovitch S, Kigel B,
Zaffryar S, et al (2008) Cancer Res 68:6922-6931]. Exemplary
polypeptide and polynucleotide sequences of semaphorins that are at
least partially resistant to pro-protein convertase are set forth
in SEQ ID NOs: 33-38.
[0125] As mentioned, the N and C termini of the peptides of the
present invention may be protected by functional groups. Suitable
functional groups are described in Green and Wuts, "Protecting
Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and
7, 1991, the teachings of which are incorporated herein by
reference. Preferred protecting groups are those that facilitate
transport of the compound attached thereto into a cell, for
example, by reducing the hydrophilicity and increasing the
lipophilicity of the compounds.
[0126] These moieties can be cleaved in vivo, either by hydrolysis
or enzymatically, inside the cell. Hydroxyl protecting groups
include esters, carbonates and carbamate protecting groups Amine
protecting groups include alkoxy and aryloxy carbonyl groups, as
described above for N-terminal protecting groups. Carboxylic acid
protecting groups include aliphatic, benzylic and aryl esters, as
described above for C-terminal protecting groups. In one
embodiment, the carboxylic acid group in the side chain of one or
more glutamic acid or aspartic acid residue in a peptide of the
present invention is protected, preferably with a methyl, ethyl,
benzyl or substituted benzyl ester.
[0127] Examples of N-terminal protecting groups include acyl groups
(--CO--R1) and alkoxy carbonyl or aryloxy carbonyl groups
(--CO--O--R1), wherein R1 is an aliphatic, substituted aliphatic,
benzyl, substituted benzyl, aromatic or a substituted aromatic
group. Specific examples of acyl groups include acetyl,
(ethyl)-CO--, n-propyl-CO--, iso-propyl-CO--, n-butyl-CO--,
sec-butyl-CO--, t-butyl-CO--, hexyl, lauroyl, palmitoyl, myristoyl,
stearyl, oleoyl phenyl-CO--, substituted phenyl-CO--,
benzyl-CO--and (substituted benzyl)-CO--. Examples of alkoxy
carbonyl and aryloxy carbonyl groups include CH3-O--CO--,
(ethyl)-O--CO--, n-propyl-O--CO--, iso-propyl-O--CO--,
n-butyl-O--CO--, sec-butyl-O--CO--, t-butyl-O--CO--,
phenyl-O--CO--, substituted phenyl-O--CO--and benzyl-O--CO--,
(substituted benzyl)-O--CO--. Adamantan, naphtalen, myristoleyl,
tuluen, biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl,
cyclohexane, norbomane, Z-caproic. In order to facilitate the
N-acylation, one to four glycine residues can be present in the
N-terminus of the molecule.
[0128] The carboxyl group at the C-terminus of the compound can be
protected, for example, by an amide (i.e., the hydroxyl group at
the C-terminus is replaced with --NH.sub.2, --NHR.sub.2 and
--NR.sub.2R.sub.3) or ester (i.e. the hydroxyl group at the
C-terminus is replaced with --OR.sub.2). R.sub.2 and R.sub.3 are
independently an aliphatic, substituted aliphatic, benzyl,
substituted benzyl, aryl or a substituted aryl group. In addition,
taken together with the nitrogen atom, R.sub.2 and R.sub.3 can form
a C4 to C8 heterocyclic ring with from about 0-2 additional
heteroatoms such as nitrogen, oxygen or sulfur. Examples of
suitable heterocyclic rings include piperidinyl, pyrrolidinyl,
morpholino, thiomorpholino or piperazinyl. Examples of C-terminal
protecting groups include --NH.sub.2, --NHCH.sub.3,
--N(CH.sub.3).sub.2, --NH(ethyl), --N(ethyl).sub.2, --N(methyl)
(ethyl), --NH(benzyl), --N(C1-C4 alkyl)(benzyl), --NH(phenyl),
--N(C1-C4 alkyl) (phenyl), --OCH.sub.3, --O-(ethyl),
--O-(n-propyl), --O-(n-butyl), --O-(iso-propyl), --O-(sec-butyl),
--O-(t-butyl), --O-benzyl and --O-phenyl.
[0129] The sempahorins of the present invention may also comprise
non-amino acid moieties, such as for example, hydrophobic moieties
(various linear, branched, cyclic, polycyclic or hetrocyclic
hydrocarbons and hydrocarbon derivatives) attached to the peptides;
various protecting groups, especially where the compound is linear,
which are attached to the compound's terminals to decrease
degradation. Chemical (non-amino acid) groups present in the
compound may be included in order to improve various physiological
properties such; decreased degradation or clearance; decreased
repulsion by various cellular pumps, improve immunogenic
activities, improve various modes of administration (such as
attachment of various sequences which allow penetration through
various barriers, through the gut, etc.); increased specificity,
increased affinity, decreased toxicity and the like.
[0130] According to one embodiment, the sempahorins of the present
invention are attached to a sustained-release enhancing agent.
Exemplary sustained-release enhancing agents include, but are not
limited to hyaluronic acid (HA), alginic acid (AA),
polyhydroxyethyl methacrylate (Poly-HEMA), polyethylene glycol
(PEG), glyme and polyisopropylacrylamide.
[0131] Attaching the amino acid sequence component of the
semaphorins of the invention to other non-amino acid agents may be
by covalent linking, by non-covalent complexion, for example, by
complexion to a hydrophobic polymer, which can be degraded or
cleaved producing a compound capable of sustained release; by
entrapping the amino acid part of the semaphorin in liposomes or
micelles to produce the final semaphorin of the invention. The
association may be by the entrapment of the amino acid sequence
within the other component (liposome, micelle) or the impregnation
of the amino acid sequence within a polymer to produce the final
peptide of the invention.
[0132] The semaphorins of the present invention can be
biochemically synthesized such as by using standard solid phase
techniques. These methods include exclusive solid phase synthesis,
partial solid phase synthesis methods, fragment condensation,
classical solution synthesis. Solid phase peptide synthesis
procedures are well known in the art and further described by John
Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide
Syntheses (2nd Ed., Pierce Chemical Company, 1984).
[0133] Recombinant techniques may also be used to generate the
semaphorins of the present invention. These techniques may be
preferred due to the number of amino acids in a semaphorin
polypeptide and the large amounts required thereof. Such
recombinant techniques are described by Bitter et al., (1987)
Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in
Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514,
Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984)
EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843,
Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach &
Weissbach, 1988, Methods for Plant Molecular Biology, Academic
Press, NY, Section VIII, pp 421-463.
[0134] To produce an expression vector for the expression of the
semaphorins of the present invention, a polynucleotide encoding the
semaphorins of the present invention are ligated into a nucleic
acid expression vector, which comprises the polynucleotide sequence
under the transcriptional control of a cis-regulatory sequence
(e.g., promoter sequence) suitable for directing constitutive,
tissue specific or inducible transcription of the semaphorins of
the present invention in the host cells.
[0135] The phrase "an isolated polynucleotide" refers to a single
or double stranded nucleic acid sequence which is isolated and
provided in the form of an RNA sequence, a complementary
polynucleotide sequence (cDNA), a genomic polynucleotide sequence
and/or a composite polynucleotide sequences (e.g., a combination of
the above).
[0136] As used herein the phrase "complementary polynucleotide
sequence" refers to a sequence, which results from reverse
transcription of messenger RNA using a reverse transcriptase or any
other RNA dependent DNA polymerase. Such a sequence can be
subsequently amplified in vivo or in vitro using a DNA dependent
DNA polymerase.
[0137] As used herein the phrase "genomic polynucleotide sequence"
refers to a sequence derived (isolated) from a chromosome and thus
it represents a contiguous portion of a chromosome.
[0138] As used herein the phrase "composite polynucleotide
sequence" refers to a sequence, which is at least partially
complementary and at least partially genomic. A composite sequence
can include some exonal sequences required to encode the semaphorin
of the present invention, as well as some intronic sequences
interposing therebetween. The intronic sequences can be of any
source, including of other genes, and typically will include
conserved splicing signal sequences. Such intronic sequences may
further include cis acting expression regulatory elements.
[0139] As mentioned hereinabove, polynucleotide sequences of the
present invention are inserted into expression vectors (i.e., a
nucleic acid construct) to enable expression of the recombinant
semaphorin. The expression vector of the present invention may
include additional sequences which render this vector suitable for
replication and integration in prokaryotes, eukaryotes, or
preferably both (e.g., shuttle vectors). Typical cloning vectors
contain transcription and translation initiation sequences (e.g.,
promoters, enhances) and transcription and translation terminators
(e.g., polyadenylation signals).
[0140] A variety of prokaryotic or eukaryotic cells can be used as
host-expression systems to express the semaphorins of the present
invention. These include, but are not limited to, microorganisms,
such as bacteria transformed with a recombinant bacteriophage DNA,
plasmid DNA or cosmid DNA expression vector containing the
semaphorin coding sequence; yeast transformed with recombinant
yeast expression vectors containing the semaphorin coding sequence;
plant cell systems infected with recombinant virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or transformed with recombinant plasmid expression
vectors, such as Ti plasmid, containing the semaphorin coding
sequence.
[0141] Other than containing the necessary elements for the
transcription and translation of the inserted coding sequence
(encoding the semaphorin), the expression construct of the present
invention can also include sequences engineered to optimize
stability, production, purification, yield or activity of the
expressed semaphorin.
[0142] Various methods can be used to introduce the expression
vector of the present invention into the host cell system. Such
methods are generally described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New
York (1989, 1992), in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989),
Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.
(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich.
(1995), Vectors: A Survey of Molecular Cloning Vectors and Their
Uses, Butterworths, Boston, Mass. (1988) and Gilboa et al.
[Biotechniques 4 (6): 504-512, 1986] and include, for example,
stable or transient transfection, lipofection, electroporation and
infection with recombinant viral vectors. In addition, see U.S.
Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection
methods.
[0143] Transformed cells are cultured under effective conditions,
which allow for the expression of high amounts of recombinant
peptide. Effective culture conditions include, but are not limited
to, effective media, bioreactor, temperature, pH and oxygen
conditions that permit protein production. An effective medium
refers to any medium in which a cell is cultured to produce the
recombinant semaphorin of the present invention. Such a medium
typically includes an aqueous solution having assimilable carbon,
nitrogen and phosphate sources, and appropriate salts, minerals,
metals and other nutrients, such as vitamins. Cells of the present
invention can be cultured in conventional fermentation bioreactors,
shake flasks, test tubes, microtiter dishes and petri plates.
Culturing can be carried out at a temperature, pH and oxygen
content appropriate for a recombinant cell. Such culturing
conditions are within the expertise of one of ordinary skill in the
art.
[0144] Depending on the vector and host system used for production,
resultant semaphorins of the present invention may either remain
within the recombinant cell, secreted into the fermentation medium,
secreted into a space between two cellular membranes, such as the
periplasmic space in E. coli; or retained on the outer surface of a
cell or viral membrane.
[0145] Following a predetermined time in culture, recovery of the
recombinant semaphorin is effected.
[0146] The phrase "recovering the recombinant semaphorin" used
herein refers to collecting the whole fermentation medium
containing the semaphorin and need not imply additional steps of
separation or purification.
[0147] Thus, the semaphorins of the present invention can be
purified using a variety of standard protein purification
techniques, such as, but not limited to, affinity chromatography,
ion exchange chromatography, filtration, electrophoresis,
hydrophobic interaction chromatography, gel filtration
chromatography, reverse phase chromatography, concanavalin A
chromatography, chromatofocusing and differential
solubilization.
[0148] To facilitate recovery, the expressed coding sequence can be
engineered to encode a semaphorin fused to a cleavable moiety. Such
a fusion protein can be designed so that the semaphorin can be
readily isolated by affinity chromatography; e.g., by
immobilization on a column specific for the cleavable moiety. Where
a cleavage site is engineered between the semaphorin and the
cleavable moiety, the semaphorin can be released from the
chromatographic column by treatment with an appropriate enzyme or
agent that specifically cleaves the fusion protein at this site
[e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and
Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].
[0149] The semaphorin of the present invention is preferably
retrieved in "substantially pure" form.
[0150] As used herein, the phrase "substantially pure" refers to a
purity that allows for the effective use of the semaphorin in the
applications described herein.
[0151] In addition to being synthesizable in host cells, the
semaphorin of the present invention can also be synthesized using
in vitro expression systems. These methods are well known in the
art and the components of the system are commercially
available.
[0152] As mentioned, the semaphorin may be administered to the
subject in need thereof as polynucleotides where they are expressed
in vivo i.e. gene therapy.
[0153] The phrase "gene therapy" as used herein refers to the
transfer of genetic material (e.g. DNA or RNA) of interest into a
host to treat or prevent a genetic or acquired disease or condition
or phenotype. The genetic material of interest encodes a product
(e.g. a protein, polypeptide, peptide, functional RNA, antisense)
whose production in vivo is desired. For example, the genetic
material of interest can encode a hormone, receptor, enzyme,
polypeptide or peptide of therapeutic value. For review see, in
general, the text "Gene Therapy" (Advanced in Pharmacology 40,
Academic Press, 1997).
[0154] Two basic approaches to gene therapy have evolved: (1) ex
vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells
are removed from a patient, and while being cultured are treated in
vitro. Generally, a functional replacement gene is introduced into
the cell via an appropriate gene delivery vehicle/method
(transfection, transduction, homologous recombination, etc.) and an
expression system as needed and then the modified cells are
expanded in culture and returned to the host/patient. These
genetically reimplanted cells have been shown to express the
transfected genetic material in situ. The cells may be autologous
or non-autologous to the subject. Since non-autologous cells are
likely to induce an immune reaction when administered to the body
several approaches have been developed to reduce the likelihood of
rejection of non-autologous cells. These include either suppressing
the recipient immune system or encapsulating the non-autologous
cells in immunoisolating, semipermeable membranes before
transplantation.
[0155] In in vivo gene therapy, target cells are not removed from
the subject rather the genetic material to be transferred is
introduced into the cells of the recipient organism in situ, that
is within the recipient. These genetically altered cells have been
shown to express the transfected genetic material in situ.
[0156] To confer specificity, preferably the nucleic acid
constructs used to express the semaphorins of the present invention
comprise cell-specific promoter sequence elements.
[0157] Recombinant viral vectors are useful for in vivo expression
of the semaphorins of the present invention since they offer
advantages such as lateral infection and targeting specificity.
Lateral infection is inherent in the life cycle of, for example,
retrovirus and is the process by which a single infected cell
produces many progeny virions that bud off and infect neighboring
cells. The result is that a large area becomes rapidly infected,
most of which was not initially infected by the original viral
particles. This is in contrast to vertical-type of infection in
which the infectious agent spreads only through daughter progeny.
Viral vectors can also be produced that are unable to spread
laterally. This characteristic can be useful if the desired purpose
is to introduce a specified gene into only a localized number of
targeted cells.
[0158] The present inventors have shown that as well as having a
direct effect on tumor cells, semaphorins also affect angiogenesis
by interacting with receptors on endothelial cells.
[0159] Thus, as well as treating cancer, the semaphorins of the
present invention may also treat other angiogenesis related
disorders.
[0160] Angiogenesis-related diseases include, but are not limited
to, inflammatory, autoimmune, and infectious diseases;
angiogenesis-dependent cancer, including, for example, solid
tumors, blood born tumors such as leukemias, and tumor metastases;
benign tumors, for example hemangiomas, acoustic neuromas,
neurofibromas, trachomas, and pyogenic granulomas; rheumatoid
arthritis; psoriasis; eczema; ocular angiogenic diseases, for
example, diabetic retinopathy, retinopathy of prematurity, macular
degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia, rubeosis; Osler-Webber Syndrome;
myocardial angiogenesis; plaque neovascularization; telangiectasia;
hemophiliac joints; angiofibroma; and wound granulation. In
addition, compositions of this invention can be used to treat
diseases such as, but not limited to, intestinal adhesions,
atherosclerosis, scleroderma, warts, and hypertrophic scars (i.e.,
keloids). Compositions of this invention may also be useful in the
treatment of diseases that have angiogenesis as a pathologic
consequence such as cat scratch disease (Rochele minalia quintosa),
ulcers (Helobacter pylori), tuberculosis, and leprosy.
[0161] The semaphorins or polynucleotides encoding same may be
administered to a subject per se or they may be part of a
pharmaceutical composition.
[0162] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0163] Herein the term "active ingredient" refers to the semaphorin
accountable for the biological effect.
[0164] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0165] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0166] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0167] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intracardiac, e.g., into the right or left
ventricular cavity, into the common coronary artery, intravenous,
inrtaperitoneal, intranasal, or intraocular injections.
[0168] Conventional approaches for drug delivery to the central
nervous system (CNS) include: neurosurgical strategies (e.g.,
intracerebral injection or intracerebroventricular infusion);
molecular manipulation of the agent (e.g., production of a chimeric
fusion protein that comprises a transport peptide that has an
affinity for an endothelial cell surface molecule in combination
with an agent that is itself incapable of crossing the BBB) in an
attempt to exploit one of the endogenous transport pathways of the
BBB; pharmacological strategies designed to increase the lipid
solubility of an agent (e.g., conjugation of water-soluble agents
to lipid or cholesterol carriers); and the transitory disruption of
the integrity of the BBB by hyperosmotic disruption (resulting from
the infusion of a mannitol solution into the carotid artery or the
use of a biologically active agent such as an angiotensin peptide).
However, each of these strategies has limitations, such as the
inherent risks associated with an invasive surgical procedure, a
size limitation imposed by a limitation inherent in the endogenous
transport systems, potentially undesirable biological side effects
associated with the systemic administration of a chimeric molecule
comprised of a carrier motif that could be active outside of the
CNS, and the possible risk of brain damage within regions of the
brain where the BBB is disrupted, which renders it a suboptimal
delivery method.
[0169] Alternately, one may administer the pharmaceutical
composition in a local rather than systemic manner, for example,
via injection of the pharmaceutical composition directly into a
tissue region of a patient.
[0170] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0171] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0172] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0173] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0174] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0175] Pharmaceutical compositions which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0176] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner
[0177] For administration by nasal inhalation, the active
ingredients for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0178] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0179] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0180] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0181] The pharmaceutical composition of the present invention may
also be formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0182] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a therapeutically effective
amount means an amount of active ingredients (semaphorins)
effective to prevent, alleviate or ameliorate symptoms of a
disorder (e.g., cancer) or prolong the survival of the subject
being treated.
[0183] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0184] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
concentration or titer. Such information can be used to more
accurately determine useful doses in humans
[0185] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1).
[0186] Dosage amount and interval may be adjusted individually to
ensure levels of the active ingredient are sufficient to induce or
suppress the biological effect (minimal effective concentration,
MEC). The MEC will vary for each preparation, but can be estimated
from in vitro data. Dosages necessary to achieve the MEC will
depend on individual characteristics and route of administration.
Detection assays can be used to determine plasma
concentrations.
[0187] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0188] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0189] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert. Compositions comprising a preparation of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition, as is further detailed
above.
[0190] As used herein the term "about" refers to .+-.10%
[0191] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0192] The term "consisting of" means "including and limited
to".
[0193] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0194] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0195] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0196] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0197] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion. Reference is now made to
the following examples, which together with the above descriptions,
illustrate the invention in a non limiting fashion.
[0198] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
EXAMPLE 1
Class-3 Semaphorins Inhibit the Development of Cancer Derived
Tumors by Targeting Receptor Expressing Tumor Cells Materials and
Methods
[0199] Materials: Antibodies directed against .beta.-actin, and
against the myc and FLAG epitope tags as well as chemicals were
from Sigma (St. Louis, Mich.). Media and sera for cell culture were
from Biological-Industries Inc. (Kibbutz Beth-Haemek, Israel).
Fugene-6 was obtained from Roche Ltd (Switzerland). Antibodies
directed against np1 and np2 were purchased from Santa-Cruz inc.
(San-Diego, Calif.). The cDNAs encoding different semaphorins were
cloned to the NSPI-CMV-MCS-myc-His lentiviral expression vector
containing SV40 promoter driving Puromycin selection marker.
Antibodies directed against CD-31 were from BD biosciences
Pharmingen. The PerfectPure RNA reverse PCR kit was from 5-Prime
(Gaithersburg, Md.).
[0200] Expression plasmids: The cDNAs of sema3A, sema3F and sema3E
were sub-cloned into the NSPI-CMV-myc-his lentiviral expression
vector. The sema3G cDNA was cloned from HUVEC mRNA using RT-PCR.
The sema3D cDNA was cloned using RT-PCR from HUVEC cells treated
with 30 ng/ml of VEGF for 6 hours. The cDNAs encoding sema3D and
sema3G were also subcloned into the NSPI expression vector. cDNA's
containing the myc epitope tag were added in frame upstream to the
stop codon of sema3D, sema3E, sema3F and sema3G. A FLAG epitope tag
was added upstream to the stop codon of sema3A as described
[Guttman-Raviv et al., 2007, J. Biol. Chem. 282:26294-26305].
[0201] Generation of recombinant lentiviruses and letiviral
mediated infection of cells: HEK293-T cells were seeded in 100 mm
tissue culture dishes (2.5.times.10.sup.6 cells per dish). A day
after seeding, the cells were co-transfected with the appropriate
lentiviral expression plasmid (8 .mu.g), with the packaging vector
pCMVdR8.91 (5 .mu.g), and with a plasmid encoding the vesicular
stomatitis virus coat envelope pMD2-VSVG (2 .mu.g) using Fugene-6
according to the instructions of the vendor. Conditioned medium
containing infective lentiviral particles was collected 48 hours
and 72 hours post transfection. Polybrene (8 .mu.g/ml) was added to
the conditioned medium and incubated 8 hours with the target
cells.
[0202] Cell lines: Mycoplasma free MDA-MB-231, MDA-MB-435,
MDA-MB-468 and MCF7 cancer derived cells were obtained from the
ATCC. The cells were cultured in DMEM containing 4.5 mg/ml glucose
supplemented with 10% FCS and antibiotics. HUVEC, PAE, HEK293 and
HEK293-T cells were cultured as previously described [Kessler O. et
al, 2004, Cancer Res. 64:1008-1015]. HUVEC were used between
passages 3-7.
[0203] In-vivo tumor formation assays: Cells expressing semaphorins
or control cells infected with empty lentiviral vectors were
implanted (5.times.10.sup.6/mouse) into the mammary fat pads of 4-6
week old balb\c nu/nu female mice (Harlan laboratories). In most
experiments we groups of 9 animals/experiment were used. The tumors
were measured twice a week using a caliper. The tumor volume (V)
was determined using the formula, V=0.52.times.A.sup.2.times.B in
which A is the short diameter and B is the long one. When
MDA-MB-231 tumors reached an average volume of 200-300 mm.sup.3,
they were excised and weighted. Each experiment was repeated at
least twice to confirm the results. Estrogen pellets were used in
experiments in which the development of tumors from MCF-7 cells was
determined as previously described [Akiri G et al., 2003, Cancer
Res. 63:1657-1666].
[0204] Immunohistochemistry: Tumors were embedded in OCT and frozen
in 2-methylbutane cooled by liquid nitrogen. They were then
sectioned into 30 .mu.m thick sections using a cryostat. Sections
were blocked with cold acetone, and reacted with an antibody
directed against the endothelial marker CD-31, counterstained with
hematoxilin and photographed. Eight different microscopic fields
derived from different sections of three different tumors were
photographed. These photographs were taken from areas in which the
density of blood vessels was highest (hot spot method) [Vermeulen,
P. B., 1996, Eur. J. Cancer 32A:2474-2484]. The area of the blood
vessels in fields of equal area was quantified using the Image Pro
Plus software.
[0205] Western Blots: Cell lysates were prepared and the
concentration of protein determined as previously described
[Guttman-Raviv, 2007, J. Biol. Chem. 282:26294-26305]. In order to
determine the concentration of secreted sema3s in conditioned
mediums of the various cell lines, cells were seeded in 12 well
dishes at a concentration of 2.times.10.sup.5 cells/well. The cells
were incubated for 48 hours in 0.4 ml of serum-free medium.
Aliquots of equal volume were analyzed by western blot analysis for
the presence of sema3s using antibodies directed against the
appropriate myc or FLAG epitope tags as previously described
described [Guttman-Raviv, 2007, J. Biol. Chem. 282:26294-26305].
None of the expressed semaphorins affected the proliferation rate
or the survival of the various cell lines (data not shown).
[0206] Proliferation assay: Tumor cells (10.sup.4 cells/well) were
seeded in triplicate in 24 well dishes. Adherent cells were
trypsinized and counted every 24 hours for 4 days, using a coulter
counter. The data was plotted on a semi-log graph in which the
slope of the graph represents the growth rate of the cell line.
[0207] Adhesion assay: In cell adhesion experiments uncoated 12
well cell culture dishes were used as well as non-adhesive 12 well
dishes coated with fibronectin (5 .mu.l/ml). Tumor cells (10.sup.5
cells/well) were seeded in triplicates in growth media. The cells
were washed twice with PBS, trypsinized to release adherent cells,
and counted with a coulter counter. The cells were counted 5, 10,
20 and 45 minutes after they were seeded. The percentage of
adherent cells relative to the number of seeded cells was then
calculated and plotted. The time required for the adherence of 50%
of the seeded cells was used as a measure to compare the adhesive
properties of control cells and of the semaphorin expressing
cells.
[0208] Endothelial cells repulsion assay: Cell repulsion assays
were performed essentially as described [Guttman-Raviv, 2007, J.
Biol. Chem. 282:26294-26305].
[0209] Soft-agar colony formation assay: A first layer of agar
containing 2 ml of 0.5% low melting agar (Bio-Rad) dissolved in
growth media was poured into the wells of a 6 well cell culture
dish and allowed to polymerize at 4.degree. C. for 20 minutes. A
second layer (1 ml) containing 0.3% of low melting agar dissolved
on growth media containing cells (3.times.10.sup.3/ml) was placed
on top of the first layer and allowed to set at 4.degree. C. for 20
minutes. Growth medium (2 ml) was added on top of the second layer
and the cells were incubated in a humidified incubator at
37.degree. C. for 21 days with a twice a week change of medium. At
the end of the experiment, colonies were stained for 1 hour with
0.005% crystal violet, and incubated with PBS overnight to remove
excess crystal violet. The colonies were photographed and colonies
with a diameter of 150 .mu.m or more were counted using the
Image-pro morphometric software.
[0210] Statistical analysis: Statistical analysis was performed
using the upaired data with unequal variance student's T-test.
Error bars represent the standard error of the mean. Statistical
significance is presented in the following manner: *p<0.05,
**p<0.01 and ***p<0.001.
[0211] Results
[0212] Expression patterns of class-3 semaphorin receptors in
breast cancer derived cell lines: Semaphorins may affect the
development of tumors by several mechanisms which include direct
effects on the tumor cells, effects on angiogenesis and effects on
stromal cells. In order to find out if the class-3 semaphorins
sema3A, sema3D, sema3E, sema3F and sema3G can influence the
formation of tumors from breast cancer cells by directly
influencing tumor cell behavior, the present inventors first
determined the expression patterns of known sema3s receptors in the
cells. The different types of breast cancer derived cells differed
in their expression of sema3 receptors. MDA-MB-231 cells express
predominantly np1, a receptor for sema3A and sema3D, but very
little np2 if at all. MDA-MB-435 cells on the other hand express
predominantly np2, a receptor for sema3F and sema3G and very little
if any np1. MCF-7 cells express np1 (although at lower levels as
compared to the MDA-MB-231 cells) and do not express np2 (FIG.
1A).
[0213] Because of their short intracellular domains the neuropilins
cannot transduce sema3 signals on their own and form complexes with
plexins in which the plexins serve as the signal transducing
elements. All four cell lines expressed plexA1 and all but the
[0214] MDA-MB-468 cells also expressed plexA2. However, none of the
breast cancer derived cell lines expressed plexA4 and only the
MCF-7 cells expressed low levels of plexA3 (FIGS. 1B-C). The mRNA
encoding sema3E receptor PlexD1 was expressed in MDA-MB-231 and
MCF-7 while MDA-MB-435 cells expressed lower concentrations and
MDA-MB-468 cells did not express at all (FIGS. 1B-C).
[0215] The effects of different sema3s on the development of tumors
from MDA-MB-231, MDA-MB-435, MDA-MB-468 and MCF-7 breast cancer
cells: In order to determine the effects of sema3A, sema3D, sema3E,
sema3F and sema3G in the breast cancer cell lines, the full length
cDNAs encoding the five semaphorins (or a control of empty
expression vector) were expressed in the cells using lentiviral
vectors carrying a selection marker that conveys resistance to
puromycin. Pools of infected cells were selected and examined for
semaphorin expression by western blot analysis using antibodies
directed against epitope tags incorporated into the recombinant
semaphorins. Sema3s contain conserved cleavage sites for furin like
pro-protein convertases and in the case of sema3E the cleaved
product possess pro-metastatic properties. However, there was only
minimal cleavage of any of the recombinant semaphorins in
MDA-MB-231 cells or in the MDA-MB-435 cells (data not shown). The
MDA-MB-231 cells were subsequently implanted in the mammary fat
pads of immune deficient mice, and allowed to develop into tumors.
In the case of the MDA-MB-231 cells, all the semaphorins that were
tested were efficiently expressed (FIGS. 2A, D, G and J).
Expression of the np1 agonist sema3A inhibited almost completely
the development of tumors from these cells (FIGS. 2B-C). Sema3D is
an agonist for np1 and for np2. Sema3D inhibited tumor formation
completely in one experiment (data not shown) and in another
experiment inhibited strongly though not completely the development
of tumors (FIGS. 2E-F). In contrast, the np2 agonist sema3G was not
able to inhibit the development of tumors from MDA-MB-231 cells
(FIGS. 2K-L). Expression of the np2 agonist sema3F on the other
hand, inhibited significantly the development of tumors from these
cells despite the lack of np2 receptors. The tumors that developed
from the sema3F expressing MDA-MB-231 cells (FIG. 2C) appeared much
less bloody than the control tumors suggesting that sema3F
inhibited tumor angiogenesis (FIG. 2A). Expression of the PlexD1
agonist sema3E also inhibited significantly the development of
tumors from MDA-MB-231 cells but the resulting tumors did not look
starved of blood vessels (FIG. 2G-I).
[0216] A different picture emerges when the effects of these
semaphorins on the development of tumors from np2 expressing
MDA-MB-435 cells are examined. When control cells are injected into
the mammary fat pads of nu/nu balb/c mice they develop into small
tumors that stop growing when they reach an average volume of
50-100 mm.sup.3 (FIGS. 3B, E and H). In contrast, there is no such
limitation on the development of tumors from any of the other
breast cancer derived cell lines used in this study. Expression of
the np2 agonist sema3F inhibited significantly the development of
tumors from these cells (FIGS. 3B and C) and the np2 agonist sema3G
was an even stronger inhibitor (FIGS. 3H-I). In contrast,
expression of the np1 specific sema3A did not inhibit the
development of tumors from these cells (FIGS. 3B-C), while sema3D,
a semaphorin that binds to both neuropilins, also inhibited their
development significantly but less potently than sema3G (FIGS.
3E-F). MDA-MB-435 cells also express the sema3E receptor PlexD1,
although at a lower expression levels than those found in
MDA-MB-231 cells (FIGS. 1A-B). Expression of sema3E did not inhibit
the formation of tumors from the MDA-MB-435 cells. This was not due
to cleavage by furin like pro-protein convertases since less than
5% of the sema3E found in the conditioned medium of these cells was
cleaved (data not shown).
[0217] The present inventors also determined how the expression of
sema3A and sema3F, the best studied np1 and np2 agonists
respectively, affects the development of tumors from
non-metastatic, estrogen dependent MCF-7 cells which express
predominantly np1. Expression of sema3A inhibited significantly
though not completely the development of tumors from these cells
while sema3F did not (FIGS. 4A-C). These effects are similar to
those observed with regard to the effect of these semaphorins on
the development of tumors from MDA-MB-231 cells (FIGS. 2A and 2C).
Taken together, these results suggest that sema3s ability to
inhibit tumor formation from a given breast cancer derived cell
type depends on the identity of the semaphorin receptors expressed
by the cells of the developing tumor, and further suggest that
sema3s should not be able to inhibit the formation of tumors from
breast cancer cells that do not express sema3 receptors.
[0218] To put this prediction to the test, sema3A and sema3F were
expressed in MDA-MB-468 breast cancer cells, which do not express
np1, np2 or PlexD1 (FIG. 1A, C). These cells form slowly growing
tumors upon injection into the mammary fat pads of nu/nu balb/c
mice. In agreement with the present prediction, neither the
expression of sema3A nor expression of sema3F significantly
inhibited the formation of tumors from these cells (FIGS.
4E-F).
[0219] In another experiment, MDA-MB-435 cells are injected into
the right posterior flank of anaesthetized immune deficient mice.
One group of mice is treated with purified Sema3G which is injected
into the peritoneal cavity twice a week (the effective
concentration will be determined experimentally but given previous
experience with other semaphorins we expect it to be around 10
mg/kg) and the other group with vehicle. Tumour size is measured
externally every 2 days using a caliper, and tumour volume is
further estimated using methods known in the art. Mice are
sacrificed after 4-6 weeks from transplant, and tumours are
weighted after dissection.
[0220] The effects of different class-3 semaphorins on tumor
angiogenesis: Sema3F was characterized in several studies as an
inhibitor of tumor angiogenesis and as a repulsive factor for
endothelial cells and sema3A was also found to function as an
inhibitor of VEGF induced angiogenesis and as a repulsive factor
for endothelial cells although not as an inhibitor of tumor
angiogenesis. To compare the repulsive properties of the different
class-3 semaphorins HEK293 cells expressing similar levels of
semaphorins were seeded on top of monolayers of human umbilical
vein derived endothelial (HUVEC) cells at clonal densities. The
HEK293 cells secreted similar concentrations of semaphorin into
their growth media as determined by western blot analysis using
antibodies directed against myc epitope tags that were fused in
frame before the stop codon of the cDNAs of the different
semaphorins (data not shown). Control cells infected with the empty
lentiviral vector did not repel the endothelial cells but sema3A,
sema3D and sema3E expressing cells repelled the endothelial cells
efficiently (FIG. 5A). However, the np2 agonists sema3F and in
particular sema3G repelled HUVEC less potently than the np1
agonists or the PlexD1 agonist sema3E (data not shown). Therefore
cells expressing either sema3F or sema3G were seeded on top of
porcine aortic endothelial (PAE) cells engineered to co-express np2
and plexA1. These cells were repelled very strongly by sema3F as
expected but were still repelled rather inefficiently by sema3G
(FIG. 5B).
[0221] In order to find out if the various sema3s that were
examined in the present example inhibit tumor angiogenesis, the
concentration of blood vessels in tumors that developed from
control and semaphorin expressing breast cancer derived cells was
determined. Since sema3A inhibited tumor formation in MDA-MB-231
cell almost completely it was not possible to determine the
concentration of blood vessels in this case. However, expression of
the np1 agonist sema3D in this cell type resulted in the formation
of tumors containing significantly lower concentrations of blood
vessels than in tumors that developed from control cells (FIG. 5C).
The reduction in the concentration of tumor associated blood
vessels was not correlated with the types of semaphorin receptors
expressed by the cancer cells since expression of the np2 agonists
sema3F and sema3G also reduced significantly the concentration of
tumor associated blood vessels in tumors developing from MDA-MB-231
cells (FIG. 5C). Quantitatively, a similar reduction in the
concentration of tumor associated blood vessels was observed
regardless of whether sema3D or sema3G were used, even though
sema3D expression inhibited tumor formation efficiently while
sema3G did not inhibit tumor formation (FIGS. 2A-L). In-contrast,
expression of sema3E, a semaphorin which inhibited the development
of tumors from MDA-MB-231 cells (FIGS. 2G-I) and which inhibits the
invasion of blood vessels into somites during early development,
did not result in a decrease in the concentration of tumor
associated blood vessels in MDA-MB-231 derived tumors (FIG.
5C).
[0222] The effects of sema3A and sema3F expression on the
concentration of tumor associated blood vessels in MCF-7 cells were
also examined. These tumors develop in the mammary fat pads of the
mice only in the presence of slow estrogen release pellets.
Expression of sema3A in these cells consistently and significantly
reduced the concentration of tumor associated blood vessels.
However, expression of sema3F did not (FIG. 5D).
[0223] In the case of the tumors that developed from the MDA-MB-435
cells, the expression of sema3A and sema3D was found to strongly
reduce the concentration of blood vessels in resulting tumors (FIG.
5E) even though tumor development from these cells was not
inhibited by these semaphorins (FIGS. 3A-I). It was not possible to
determine the blood vessel concentration in tumors that developed
from cells expressing sema3F or sema3G since the resulting tumors
were too small or non-existent as in the case of sema3G. Expression
of sema3E did produce a decrease in the concentration of blood
vessels in tumors developing from these cells, but the decrease,
although statistically significant, was small.
[0224] Taken together, these experiments indicate that although
most of the semaphorins are able to inhibit angiogenesis, as
manifested by the reduction in the concentration of blood vessels
in tumors, and even though the inhibition may contribute to the
inhibition of tumor progression, there was generally no correlation
between this ability and the inhibition of tumor development which
was mostly correlated with the expression of the appropriate
semaphorin receptors by the tumor cells.
[0225] The effects of the expression of different class-3
semaphorins on the behavior of the tumor cells in-vitro: The
experiments described hereinabove suggest that semaphorin
expression may strongly modulate the behavior of tumor cells.
Indeed, other researchers have described effects of various class-3
semaphorins on the adhesion, spreading and proliferation of various
types of tumor cells [Tomizawa, Y., et al., 2001, Proc. Natl. Acad.
Sci. U.S.A. 98:13954-13959; Bielenberg, D. R., et al., 2004, J.
Clin. Invest 114:1260-1271; Nasarre, Petal., 2005, Neoplasia.
7:180-189]. However, the proliferation of MDA-MB-231 cells
expressing either sema3A, sema3F, sema3D or sema3E was not
inhibited as compared to control cells infected with empty vector
containing lentiviruses. Similarly, the proliferation of MCF-7
cells expressing either sema3A or sema3F was not altered as
compared to controls and MDA-MB-435 cells expressing sema3A or
sema3F were also not affected as compared to control cells (data
not shown). These results indicate that the effect that these
semaphorins have on the growth of tumors in-vivo are probably not
mediated by a direct effect on the proliferation machinery of the
tumor cells. The effect of the expression of different semaphorins
on the adhesion of the various tumor cells to plastic or to
fibronectin was also examined. Neither sema3A nor sema3F expression
affected the rate or extent of adhesion of MDA-MB-231, MDA-MB-435
or MCF-7 cells regardless of whether the substrate was plastic or
fibronectin (data not shown).
[0226] The ability to form colonies in soft-agar is a hallmark that
differentiates many types of cancer cells from their normal
counterparts. Therefore, the present inventors determined whether
the expression of different class-3 semaphorins in MDA-MB-231 or
MDA-MB-435 cells affects their ability to form colonies in
soft-agar. None of the semaphorins inhibited completely the
formation of colonies by MDA-MB-231 cells. However, both sema3A and
sema3D, semaphorins that strongly inhibit tumor formation from
these cells (FIGS. 2A-L), also significantly inhibited the
formation of large colonies in soft agar (FIGS. 6A-B).
Surprisingly, sema3F also inhibited significantly the formation of
large colonies in soft agar despite the absence of np2 receptors on
these cells. However, sema3F does bind to np1, albeit with a 10
fold lower affinity, and it is possible that this inhibitory effect
is mediated by np1. Another np2 agonist, sema3G, which in contrast
to sema3F does not inhibit the development of tumors from
MDA-MB-231 cells at all (FIGS. 2J-L) and does not bind to np1, had
no effect on the development of colonies in soft agar (FIGS. 6A-B).
These results suggested that the semaphorin needs to bind to the
semaphorin receptor expressed by the tumor cells in order to be
able to inhibit soft-agar colony formation. MDA-MB-231 cells also
express the sema3E receptor PlexD1 and expression of sema3E
inhibits the formation of tumor formation from these cells (FIGS.
2G-I). However, sema3E failed to inhibit the formation of large
colonies of MDA-MB-231 cells in soft-agar (FIGS. 6A-B).
[0227] Based on these results the present inventors predicted that
sema3D, sema3F and sema3G, which inhibit tumor formation from
MDA-MB-435 cells (FIGS. 3A-I), should also inhibit efficiently the
formation of soft-agar colonies from these np2 expressing cells.
Indeed, sema3d and sema3G inhibited colony formation efficiently as
predicted. However, unexpectedly it was found that sema3F inhibited
the formation of colonies in soft agar from these cells even though
it did not inhibit the formation of tumors from these cells (FIGS.
6C-D). Another unexpected observation was that sema3E, which did
not inhibit the formation of tumors from these cells did inhibit
colony formation (FIGS. 6C-D). Lastly, it was expected that sema3A
would not affect colony formation since its receptor is not
expressed by MDA-MB-435 cells (FIGS. 6A-D). Indeed, colony
formation from MDA-MB-435 cells was not inhibited by sema3A.
Instead it was even enhanced (FIGS. 6C-D).
[0228] Taken together these results suggest that the different
semaphorins are able to modulate the behavior of MDA-MB-231 and
MDA-MB-435 cells directly, although there were exceptions to this
rule.
[0229] Expression of np1 in MDA-MB-435 cells enhances tumor
development and the enhancement is negated by co-expression of
sema3A: The experiments described above suggest that the expression
of specific class-3 semaphorin receptors by breast cancer derived
tumor cells is probably the most important factor that determines
whether a given class-3 semaphorin will be an effective inhibitor
of tumor development. To test this hypothesis the present inventors
asked whether expression of np1 in MDA-MB-435 cells would render
tumors that will develop from these cells sensitive to sema3A.
Tumors that developed from MDA-MB-435 cells that express np1 did
not stop developing when they reached a mean volume of 50-100
mm.sup.3 like wild-type MDA-MB-435 cells (FIGS. 3A-I). The np1
expressing MDA-MB-435 cells formed rapidly forming tumors when
implanted in the mammary fat pads of mice (FIGS. 7A-C). Even though
these np1 expressing cells formed tumors that appeared bloody, the
concentration of blood vessels within these tumors, as determined
by staining with an antibody directed against CD-31, was not
significantly different from that of control tumors (FIG. 7D). When
the np1 agonist sema3A was co-expressed in these cells with np1,
the cells that expressed both genes reverted to the behavior
exhibited by the parental cells and formed tumors that stopped
growing when the tumors reached a volume of 50-100 mm.sup.3 thereby
eliminating the growth advantage conferred by the presence of np1
(FIGS. 7A-C), but not that conferred by the presence of np2 which
can be inhibited by np2 agonists such as sema3F or sema3G (FIGS.
3A-I). Interestingly, the density of blood vessels in tumors that
developed from MDA-MB-435 cells expressing sema3A or sema3A+np1 was
similar and significantly lower than in tumors that developed from
MDA-MB-435 cells that do not express sema3A (FIG. 7D), suggesting
once again that inhibition of angiogenesis represents part of the
mechanism by which semaphorins modulate tumor progression, but that
it may not always be sufficient to inhibit tumor expansion.
[0230] Discussion
[0231] The present results indicate for the first time that sema3A,
sema3D, sema3E and sema3G inhibit the formation of tumors from
several cell lines derived from human breast carcinomas. It should
be noted that sema3E was previously described as a pro-metastatic
agent, but the pro-metastatic activity was associated with a
cleavage product generated by furin like pro-protein convertases
and not by the full length protein. In the present experiments
there was almost no cleavage of sema3E in the MDA-MB-231 cells or
MDA-MB-435 cells.
[0232] Many types of tumorigenic cells express one or more than one
of the class-3 semaphorin receptors np1, np2 or PlexD1. The breast
cancer derived cell types that were employed in the present
examples in order to study the anti-tumorigenic effects of the
different semaphorins, express different combinations of class-3
semaphorin receptors on their cell surfaces. Both neuropilins as
well as several types of plexins are also expressed in endothelial
cells in which they play an important role in the transduction of
VEGF induced angiogenic signals, and mediate the anti-angiogenic
effects of sema3s. In the present study the present inventors have
tried to evaluate the relative importance of the anti-angiogenic
effects versus the direct effects of the sema3s in the
determination of the anti-tumorigenic properties of different
sema3s. Taken together, the present examples indicate that the
expression of a given semaphorin receptor by the tumorigenic cells
is probably the most important factor which determines whether a
given class-3 semaphorin will function as an effective inhibitor of
tumor development. Thus, the development of tumors from MDA-MB-231
cells that express np1 but not np2 is strongly inhibited by the np1
agonists sema3A and sema3D but not by the np2 agonist sema3G. This
conclusion is also supported by experiments which have shown that
in the case of the np2 expressing MDA-MB-435 cells sema3A does not
inhibit tumor development and sema3D inhibits tumor development
weakly while sema3G and sema3F function in the case of these cells
as very effective inhibitors. Furthermore, neither sema3A nor
sema3F were able to inhibit the development of tumors from
MDA-MB-468 cell that do not express neuropilins.
[0233] Sema3E is unique among sema3s as it is the only semaphorin
which does not bind to neuropilins and instead activates directly
PlexD1. Both MDA-MB-231 and MDA-MB-435 cells express PlexD1
although the expression levels in MDA-MB-435 cells are
significantly lower than in MDA-MB-231 cells. Sema3E inhibited
significantly tumor development from MDA-MB-231 cells but not at
all from MDA-MB-435 cells. It is possible that the levels of
expression of PlexD1 in the MDA-MB-435 cells are below a critical
threshold that does not enable inhibition of tumor development by
sema3E. Thus, this result may perhaps also be regarded as one that
supports the above mentioned rule of thumb. PlexD1 can form
complexes with neuropilins, and at least in the case of np1 this
interaction can affect the nature of the biological response to
sema3E. It is therefore possible that in the MDA-MB-435 cells, the
effect of sema3E may be inhibited by np2 while in MDA-MB-231 cells
np1 may affect sema3E signaling differently, resulting in diverse
biological responses.
[0234] The present inventors showed for the first time that sema3D,
sema3G, sema3A and sema3E function as inhibitors of tumor
angiogenesis since their expression in tumor cells resulted in a
significant reduction in the density of tumor associated blood
vessels. These results indicate that as a rule, semaphorin
expression tends to reduce the density of blood vessels in tumors
that develop from MDA-MB-231, MDA-MB-435 or MCF-7 cells even in
cases in which tumor development is not inhibited by the given
semaphorin. However, there were a few exceptions to this rule. The
density of blood vessels in tumors derived from sema3F expressing
MCF-7 cells was not reduced in comparison with tumors derived from
control MCF-7 cells. However, the development of tumors from MCF-7
cells requires estrogen, a hormone that was recently found to
inhibit the expression of the sema3F receptor np2, which may
perhaps explain the lack of the anti-angiogenic effect in this
case. In addition it was found that expression of sema3E in
MDA-MB-231 cells also failed to reduce the density of blood vessels
in resulting tumors in spite of significant inhibition of tumor
growth.
[0235] The density of tumor associated blood vessels is determined
by a balance between the rate of tumor angiogenesis, which tends to
increase blood vessel density, and the rate of tumor cell
proliferation which tends to decrease it. It is therefore possible
that a small tumor whose expansion was strongly inhibited by an
anti-angiogenic agent will contain the same density of blood
vessels as a tumor whose expansion was much less affected by the
anti-angiogenic agent, simply because in the latter case the
reduction in the density of blood vessels did not reach that
threshold below which the expansion of the tumor mass is inhibited.
It was observed that in most cases the expression of class-3
semaphorins by the tumor cells reduces the density of blood vessels
in resulting tumors, regardless of the receptor types with which
the specific semaphorins interact and regardless of whether the
sema3s were able to inhibit the development of the tumors. For
example, sema3G decreased significantly the density of blood
vessels in tumors derived from MDA-MB-231 cells as did sema3D and
sema3F even though in contrast with these semaphorins sema3G did
not inhibit tumor development.
[0236] The present inventors reasoned that if the expression of
semaphorin receptors by the tumor cells is the primary factor that
determines whether sema3s will be able to inhibit tumor
development, than the expression of semaphorins should affect the
behavior of such tumor cells in in-vitro assays too. Class-3
semaphorins such as sema3F and sema3B inhibit the adhesion and
migration of some tumor cells. It was therefore surprising that
neither the proliferation, nor the adhesive properties of the
different tumor cells used in this study were modified the
expression of the different semaphorins. So far, the only property
of the tumor cells which was found to be affected by the expression
of semaphorins was their ability to form colonies in soft-agar. In
general, semaphorins that inhibited the formation of tumors from
either MDA-MB-231 or MDA-MB-435 cells were also able to inhibit the
anchorage independent growth of the tumor cells. Anchorage
independent growth is a hallmark of most malignant cells and these
observations indicate that the sema3s directly influence a tumor
cell characteristic that is correlated with their malignant
properties. However, a few exceptions to this rule were also
noticed. Expression of sema3E in MDA-MB-231 cells did not inhibit
the anchorage free growth of the cells even though the formation of
tumors from these cells was inhibited. Another discrepancy was
noted in the case of sema3F, which when expressed in MDA-MB-435
cells inhibited strongly tumor formation but not the anchorage free
growth of these cells. The reason for these discrepancies is still
under investigation, but nevertheless, in general there is good
agreement between the effects of the sema3s on the development of
the tumors and their ability to inhibit anchorage free growth.
[0237] In conclusion, the present inventors have found for the
first time that sema3A, sema3D, sema3E and sema3G possess
anti-tumorigenic properties similar to those displayed by the
previously identified tumor suppressors sema3F and sema3B. It was
also found that all of these semaphorins can repulse endothelial
cells with varying potencies and that all of them are able to
significantly reduce the density of blood vessels in tumors that
develop from tumor cells expressing these semaphorins. Sema3E was
an exception since even though it functioned as a potent repulsive
agent for endothelial cells in-vitro, it had no effect on the
density of blood vessels in tumors that developed from MDA-MB-231
cells and only a small effect on the density of blood vessels in
tumors that develop from MDA-MB-435 cells. In addition, a strong
correlation between the ability to inhibit tumor growth in-vivo and
the ability to inhibit anchorage independent growth in-vitro by
individual semaphorins was noted. These observations lead to the
conclusion that efficient inhibition of tumor development by
semaphorins is enabled when an individual semaphorin is able to
inhibit directly the malignant properties of the tumor cells and
when this semaphorin is also able to efficiently inhibit tumor
angiogenesis. The present results argue that semaphorins may find
use as general anti-angiogenic agents. However, for maximal
effectiveness as anti-tumorigenic agents the selection of specific
semaphorins or semaphorin combinations will have to take into
account the identity of the semaphorin receptors expressed by the
tumorigenic cells of target tumors.
[0238] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0239] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
into the specification, to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated herein by reference. In
addition, citation or identification of any reference in this
application shall not be construed as an admission that such
reference is available as prior art to the present invention. To
the extent that section headings are used, they should not be
construed as necessarily limiting.
Sequence CWU 1
1
40120DNAArtificial sequenceSingle strand DNA oligonucleotide
1ccaccttaaa atctgcaggc 20220DNAArtificial sequenceSingle strand DNA
oligonucleotide 2gtgcatgaag gacagcctct 20320DNAArtificial
sequenceSingle strand DNA oligonucleotide 3ttgcagtctc tgtcctccaa
20420DNAArtificial sequenceSingle strand DNA oligonucleotide
4gaaaaatgcg aatggctgat 20520DNAArtificial sequenceSingle strand DNA
oligonucleotide 5ccctgaggtt gcagaagaag 20620DNAArtificial
sequenceSingle strand DNA oligonucleotide 6gtcccactgg agaactgcat
20720DNAArtificial sequenceSingle strand DNA oligonucleotide
7ggtttgagag gtccaccagg 20820DNAArtificial sequenceSingle strand DNA
oligonucleotide 8ccgtggctgc ctatgactat 20920DNAArtificial
sequenceSingle strand DNA oligonucleotide 9catctcgtac tggaccccac
201020DNAArtificial sequenceSingle strand DNA oligonucleotide
10tttacaacgg ctacagcgtg 201118DNAArtificial sequenceSingle strand
DNA oligonucleotide 11accacgaagg cacggaag 181217DNAArtificial
sequenceSingle strand DNA oligonucleotide 12agccagcgga gggacag
171320DNAArtificial sequenceSingle strand DNA oligonucleotide
13agcagtgcgc tcttaaccat 201420DNAArtificial sequenceSingle strand
DNA oligonucleotide 14caaaggccag agagtggttc 201519DNAArtificial
sequenceSingle strand DNA oligonucleotide 15gagcagctcc acagtccag
191617DNAArtificial sequenceSingle strand DNA oligonucleotide
16gtgctcgaca gcgtggt 1717644PRTHomo sapiens 17Met Glu Arg Gly Leu
Pro Leu Leu Cys Ala Val Leu Ala Leu Val Leu 1 5 10 15 Ala Pro Ala
Gly Ala Phe Arg Asn Asp Lys Cys Gly Asp Thr Ile Lys 20 25 30 Ile
Glu Ser Pro Gly Tyr Leu Thr Ser Pro Gly Tyr Pro His Ser Tyr 35 40
45 His Pro Ser Glu Lys Cys Glu Trp Leu Ile Gln Ala Pro Asp Pro Tyr
50 55 60 Gln Arg Ile Met Ile Asn Phe Asn Pro His Phe Asp Leu Glu
Asp Arg 65 70 75 80 Asp Cys Lys Tyr Asp Tyr Val Glu Val Phe Asp Gly
Glu Asn Glu Asn 85 90 95 Gly His Phe Arg Gly Lys Phe Cys Gly Lys
Ile Ala Pro Pro Pro Val 100 105 110 Val Ser Ser Gly Pro Phe Leu Phe
Ile Lys Phe Val Ser Asp Tyr Glu 115 120 125 Thr His Gly Ala Gly Phe
Ser Ile Arg Tyr Glu Ile Phe Lys Arg Gly 130 135 140 Pro Glu Cys Ser
Gln Asn Tyr Thr Thr Pro Ser Gly Val Ile Lys Ser 145 150 155 160 Pro
Gly Phe Pro Glu Lys Tyr Pro Asn Ser Leu Glu Cys Thr Tyr Ile 165 170
175 Val Phe Ala Pro Lys Met Ser Glu Ile Ile Leu Glu Phe Glu Ser Phe
180 185 190 Asp Leu Glu Pro Asp Ser Asn Pro Pro Gly Gly Met Phe Cys
Arg Tyr 195 200 205 Asp Arg Leu Glu Ile Trp Asp Gly Phe Pro Asp Val
Gly Pro His Ile 210 215 220 Gly Arg Tyr Cys Gly Gln Lys Thr Pro Gly
Arg Ile Arg Ser Ser Ser 225 230 235 240 Gly Ile Leu Ser Met Val Phe
Tyr Thr Asp Ser Ala Ile Ala Lys Glu 245 250 255 Gly Phe Ser Ala Asn
Tyr Ser Val Leu Gln Ser Ser Val Ser Glu Asp 260 265 270 Phe Lys Cys
Met Glu Ala Leu Gly Met Glu Ser Gly Glu Ile His Ser 275 280 285 Asp
Gln Ile Thr Ala Ser Ser Gln Tyr Ser Thr Asn Trp Ser Ala Glu 290 295
300 Arg Ser Arg Leu Asn Tyr Pro Glu Asn Gly Trp Thr Pro Gly Glu Asp
305 310 315 320 Ser Tyr Arg Glu Trp Ile Gln Val Asp Leu Gly Leu Leu
Arg Phe Val 325 330 335 Thr Ala Val Gly Thr Gln Gly Ala Ile Ser Lys
Glu Thr Lys Lys Lys 340 345 350 Tyr Tyr Val Lys Thr Tyr Lys Ile Asp
Val Ser Ser Asn Gly Glu Asp 355 360 365 Trp Ile Thr Ile Lys Glu Gly
Asn Lys Pro Val Leu Phe Gln Gly Asn 370 375 380 Thr Asn Pro Thr Asp
Val Val Val Ala Val Phe Pro Lys Pro Leu Ile 385 390 395 400 Thr Arg
Phe Val Arg Ile Lys Pro Ala Thr Trp Glu Thr Gly Ile Ser 405 410 415
Met Arg Phe Glu Val Tyr Gly Cys Lys Ile Thr Asp Tyr Pro Cys Ser 420
425 430 Gly Met Leu Gly Met Val Ser Gly Leu Ile Ser Asp Ser Gln Ile
Thr 435 440 445 Ser Ser Asn Gln Gly Asp Arg Asn Trp Met Pro Glu Asn
Ile Arg Leu 450 455 460 Val Thr Ser Arg Ser Gly Trp Ala Leu Pro Pro
Ala Pro His Ser Tyr 465 470 475 480 Ile Asn Glu Trp Leu Gln Ile Asp
Leu Gly Glu Glu Lys Ile Val Arg 485 490 495 Gly Ile Ile Ile Gln Gly
Gly Lys His Arg Glu Asn Lys Val Phe Met 500 505 510 Arg Lys Phe Lys
Ile Gly Tyr Ser Asn Asn Gly Ser Asp Trp Lys Met 515 520 525 Ile Met
Asp Asp Ser Lys Arg Lys Ala Lys Ser Phe Glu Gly Asn Asn 530 535 540
Asn Tyr Asp Thr Pro Glu Leu Arg Thr Phe Pro Ala Leu Ser Thr Arg 545
550 555 560 Phe Ile Arg Ile Tyr Pro Glu Arg Ala Thr His Gly Gly Leu
Gly Leu 565 570 575 Arg Met Glu Leu Leu Gly Cys Glu Val Glu Ala Pro
Thr Ala Gly Pro 580 585 590 Thr Thr Pro Asn Gly Asn Leu Val Asp Glu
Cys Asp Asp Asp Gln Ala 595 600 605 Asn Cys His Ser Gly Thr Gly Asp
Asp Phe Gln Leu Thr Gly Gly Thr 610 615 620 Thr Val Leu Ala Thr Glu
Lys Pro Thr Val Ile Asp Ser Thr Ile Gln 625 630 635 640 Ser Gly Ile
Lys 18909PRTHomo sapiens 18Met Asp Met Phe Pro Leu Thr Trp Val Phe
Leu Ala Leu Tyr Phe Ser 1 5 10 15 Arg His Gln Val Arg Gly Gln Pro
Asp Pro Pro Cys Gly Gly Arg Leu 20 25 30 Asn Ser Lys Asp Ala Gly
Tyr Ile Thr Ser Pro Gly Tyr Pro Gln Asp 35 40 45 Tyr Pro Ser His
Gln Asn Cys Glu Trp Ile Val Tyr Ala Pro Glu Pro 50 55 60 Asn Gln
Lys Ile Val Leu Asn Phe Asn Pro His Phe Glu Ile Glu Lys 65 70 75 80
His Asp Cys Lys Tyr Asp Phe Ile Glu Ile Arg Asp Gly Asp Ser Glu 85
90 95 Ser Ala Asp Leu Leu Gly Lys His Cys Gly Asn Ile Ala Pro Pro
Thr 100 105 110 Ile Ile Ser Ser Gly Ser Met Leu Tyr Ile Lys Phe Thr
Ser Asp Tyr 115 120 125 Ala Arg Gln Gly Ala Gly Phe Ser Leu Arg Tyr
Glu Ile Phe Lys Thr 130 135 140 Gly Ser Glu Asp Cys Ser Lys Asn Phe
Thr Ser Pro Asn Gly Thr Ile 145 150 155 160 Glu Ser Pro Gly Phe Pro
Glu Lys Tyr Pro His Asn Leu Asp Cys Thr 165 170 175 Phe Thr Ile Leu
Ala Lys Pro Lys Met Glu Ile Ile Leu Gln Phe Leu 180 185 190 Ile Phe
Asp Leu Glu His Asp Pro Leu Gln Val Gly Glu Gly Asp Cys 195 200 205
Lys Tyr Asp Trp Leu Asp Ile Trp Asp Gly Ile Pro His Val Gly Pro 210
215 220 Leu Ile Gly Lys Tyr Cys Gly Thr Lys Thr Pro Ser Glu Leu Arg
Ser 225 230 235 240 Ser Thr Gly Ile Leu Ser Leu Thr Phe His Thr Asp
Met Ala Val Ala 245 250 255 Lys Asp Gly Phe Ser Ala Arg Tyr Tyr Leu
Val His Gln Glu Pro Leu 260 265 270 Glu Asn Phe Gln Cys Asn Val Pro
Leu Gly Met Glu Ser Gly Arg Ile 275 280 285 Ala Asn Glu Gln Ile Ser
Ala Ser Ser Thr Tyr Ser Asp Gly Arg Trp 290 295 300 Thr Pro Gln Gln
Ser Arg Leu His Gly Asp Asp Asn Gly Trp Thr Pro 305 310 315 320 Asn
Leu Asp Ser Asn Lys Glu Tyr Leu Gln Val Asp Leu Arg Phe Leu 325 330
335 Thr Met Leu Thr Ala Ile Ala Thr Gln Gly Ala Ile Ser Arg Glu Thr
340 345 350 Gln Asn Gly Tyr Tyr Val Lys Ser Tyr Lys Leu Glu Val Ser
Thr Asn 355 360 365 Gly Glu Asp Trp Met Val Tyr Arg His Gly Lys Asn
His Lys Val Phe 370 375 380 Gln Ala Asn Asn Asp Ala Thr Glu Val Val
Leu Asn Lys Leu His Ala 385 390 395 400 Pro Leu Leu Thr Arg Phe Val
Arg Ile Arg Pro Gln Thr Trp His Ser 405 410 415 Gly Ile Ala Leu Arg
Leu Glu Leu Phe Gly Cys Arg Val Thr Asp Ala 420 425 430 Pro Cys Ser
Asn Met Leu Gly Met Leu Ser Gly Leu Ile Ala Asp Ser 435 440 445 Gln
Ile Ser Ala Ser Ser Thr Gln Glu Tyr Leu Trp Ser Pro Ser Ala 450 455
460 Ala Arg Leu Val Ser Ser Arg Ser Gly Trp Phe Pro Arg Ile Pro Gln
465 470 475 480 Ala Gln Pro Gly Glu Glu Trp Leu Gln Val Asp Leu Gly
Thr Pro Lys 485 490 495 Thr Val Lys Gly Val Ile Ile Gln Gly Ala Arg
Gly Gly Asp Ser Ile 500 505 510 Thr Ala Val Glu Ala Arg Ala Phe Val
Arg Lys Phe Lys Val Ser Tyr 515 520 525 Ser Leu Asn Gly Lys Asp Trp
Glu Tyr Ile Gln Asp Pro Arg Thr Gln 530 535 540 Gln Pro Lys Leu Phe
Glu Gly Asn Met His Tyr Asp Thr Pro Asp Ile 545 550 555 560 Arg Arg
Phe Asp Pro Ile Pro Ala Gln Tyr Val Arg Val Tyr Pro Glu 565 570 575
Arg Trp Ser Pro Ala Gly Ile Gly Met Arg Leu Glu Val Leu Gly Cys 580
585 590 Asp Trp Thr Asp Ser Lys Pro Thr Val Glu Thr Leu Gly Pro Thr
Val 595 600 605 Lys Ser Glu Glu Thr Thr Thr Pro Tyr Pro Thr Glu Glu
Glu Ala Thr 610 615 620 Glu Cys Gly Glu Asn Cys Ser Phe Glu Asp Asp
Lys Asp Leu Gln Leu 625 630 635 640 Pro Ser Gly Phe Asn Cys Asn Phe
Asp Phe Leu Glu Glu Pro Cys Gly 645 650 655 Trp Met Tyr Asp His Ala
Lys Trp Leu Arg Thr Thr Trp Ala Ser Ser 660 665 670 Ser Ser Pro Asn
Asp Arg Thr Phe Pro Asp Asp Arg Asn Phe Leu Arg 675 680 685 Leu Gln
Ser Asp Ser Gln Arg Glu Gly Gln Tyr Ala Arg Leu Ile Ser 690 695 700
Pro Pro Val His Leu Pro Arg Ser Pro Val Cys Met Glu Phe Gln Tyr 705
710 715 720 Gln Ala Thr Gly Gly Arg Gly Val Ala Leu Gln Val Val Arg
Glu Ala 725 730 735 Ser Gln Glu Ser Lys Leu Leu Trp Val Ile Arg Glu
Asp Gln Gly Gly 740 745 750 Glu Trp Lys His Gly Arg Ile Ile Leu Pro
Ser Tyr Asp Met Glu Tyr 755 760 765 Gln Ile Val Phe Glu Gly Val Ile
Gly Lys Gly Arg Ser Gly Glu Ile 770 775 780 Ala Ile Asp Asp Ile Arg
Ile Ser Thr Asp Val Pro Leu Glu Asn Cys 785 790 795 800 Met Glu Pro
Ile Ser Ala Phe Ala Asp Glu Tyr Glu Val Asp Trp Ser 805 810 815 Asn
Ser Ser Ser Ala Thr Ser Gly Ser Gly Ala Pro Ser Thr Asp Lys 820 825
830 Glu Lys Ser Trp Leu Tyr Thr Leu Asp Pro Ile Leu Ile Thr Ile Ile
835 840 845 Ala Met Ser Ser Leu Gly Val Leu Leu Gly Ala Thr Cys Ala
Gly Leu 850 855 860 Leu Leu Tyr Cys Thr Cys Ser Tyr Ser Gly Leu Ser
Ser Arg Ser Cys 865 870 875 880 Thr Thr Leu Glu Asn Tyr Asn Phe Glu
Leu Tyr Asp Gly Leu Lys His 885 890 895 Lys Val Lys Met Asn His Gln
Lys Cys Cys Ser Glu Ala 900 905 191873PRTHomo sapiens 19Met Trp Ala
Glu Ala Gly Leu Pro Arg Ala Gly Gly Gly Ser Gln Pro 1 5 10 15 Pro
Phe Arg Thr Phe Ser Ala Ser Asp Trp Gly Leu Thr His Leu Val 20 25
30 Val His Glu Gln Thr Gly Glu Val Tyr Val Gly Ala Val Asn Arg Ile
35 40 45 Tyr Lys Leu Ser Gly Asn Leu Thr Leu Leu Arg Ala His Val
Thr Gly 50 55 60 Pro Val Glu Asp Asn Glu Lys Cys Tyr Pro Pro Pro
Ser Val Gln Ser 65 70 75 80 Cys Pro His Gly Leu Gly Ser Thr Asp Asn
Val Asn Lys Leu Leu Leu 85 90 95 Leu Asp Tyr Ala Ala Asn Arg Leu
Leu Ala Cys Gly Ser Ala Ser Gln 100 105 110 Gly Ile Cys Gln Phe Leu
Arg Leu Asp Asp Leu Phe Lys Leu Gly Glu 115 120 125 Pro His His Arg
Lys Glu His Tyr Leu Ser Ser Val Gln Glu Ala Gly 130 135 140 Ser Met
Ala Gly Val Leu Ile Ala Gly Pro Pro Gly Gln Gly Gln Ala 145 150 155
160 Lys Leu Phe Val Gly Thr Pro Ile Asp Gly Lys Ser Glu Tyr Phe Pro
165 170 175 Thr Leu Ser Ser Arg Arg Leu Met Ala Asn Glu Glu Asp Ala
Asp Met 180 185 190 Phe Gly Phe Val Tyr Gln Asp Glu Phe Val Ser Ser
Gln Leu Lys Ile 195 200 205 Pro Ser Asp Thr Leu Ser Lys Phe Pro Ala
Phe Asp Ile Tyr Tyr Val 210 215 220 Tyr Ser Phe Arg Ser Glu Gln Phe
Val Tyr Tyr Leu Thr Leu Gln Leu 225 230 235 240 Asp Thr Gln Leu Thr
Ser Pro Asp Ala Ala Gly Glu His Phe Phe Thr 245 250 255 Ser Lys Ile
Val Arg Leu Cys Val Asp Asp Pro Lys Phe Tyr Ser Tyr 260 265 270 Val
Glu Phe Pro Ile Gly Cys Glu Gln Ala Gly Val Glu Tyr Arg Leu 275 280
285 Val Gln Asp Ala Tyr Leu Ser Arg Pro Gly Arg Ala Leu Ala His Gln
290 295 300 Leu Gly Leu Ala Glu Asp Glu Asp Val Leu Phe Thr Val Phe
Ala Gln 305 310 315 320 Gly Gln Lys Asn Arg Val Lys Pro Pro Lys Glu
Ser Ala Leu Cys Leu 325 330 335 Phe Thr Leu Arg Ala Ile Lys Glu Lys
Ile Lys Glu Arg Ile Gln Ser 340 345 350 Cys Tyr Arg Gly Glu Gly Lys
Leu Ser Leu Pro Trp Leu Leu Asn Lys 355 360 365 Glu Leu Gly Cys Ile
Asn Ser Pro Leu Gln Ile Asp Asp Asp Phe Cys 370 375 380 Gly Gln Asp
Phe Asn Gln Pro Leu Gly Gly Thr Val Thr Ile Glu Gly 385 390 395 400
Thr Pro Leu Phe Val Asp Lys Asp Asp Gly Leu Thr Ala Val Ala Ala 405
410 415 Tyr Asp Tyr Arg Gly Arg Thr Val Val Phe Ala Gly Thr Arg Ser
Gly 420 425 430 Arg Ile Arg Lys Ile Leu Val Asp Leu Ser Asn Pro Gly
Gly Arg Pro 435 440 445 Ala Leu Ala Tyr Glu Ser Val Val Ala Gln Glu
Gly
Ser Pro Ile Leu 450 455 460 Arg Asp Leu Val Leu Ser Pro Asn His Gln
Tyr Leu Tyr Ala Met Thr 465 470 475 480 Glu Lys Gln Val Thr Arg Val
Pro Val Glu Ser Cys Val Gln Tyr Thr 485 490 495 Ser Cys Glu Leu Cys
Leu Gly Ser Arg Asp Pro His Cys Gly Trp Cys 500 505 510 Val Leu His
Ser Ile Cys Ser Arg Arg Asp Ala Cys Glu Arg Ala Asp 515 520 525 Glu
Pro Gln Arg Phe Ala Ala Asp Leu Leu Gln Cys Val Gln Leu Thr 530 535
540 Val Gln Pro Arg Asn Val Ser Val Thr Met Ser Gln Val Pro Leu Val
545 550 555 560 Leu Gln Ala Trp Asn Val Pro Asp Leu Ser Ala Gly Val
Asn Cys Ser 565 570 575 Phe Glu Asp Phe Thr Glu Ser Glu Ser Val Leu
Glu Asp Gly Arg Ile 580 585 590 His Cys Arg Ser Pro Ser Ala Arg Glu
Val Ala Pro Ile Thr Arg Gly 595 600 605 Gln Gly Asp Gln Arg Val Val
Lys Leu Tyr Leu Lys Ser Lys Glu Thr 610 615 620 Gly Lys Lys Phe Ala
Ser Val Asp Phe Val Phe Tyr Asn Cys Ser Val 625 630 635 640 His Gln
Ser Cys Leu Ser Cys Val Asn Gly Ser Phe Pro Cys His Trp 645 650 655
Cys Lys Tyr Arg His Val Cys Thr His Asn Val Ala Asp Cys Ala Phe 660
665 670 Leu Glu Gly Arg Val Asn Val Ser Glu Asp Cys Pro Gln Ile Leu
Pro 675 680 685 Ser Thr Gln Ile Tyr Val Pro Val Gly Val Val Lys Pro
Ile Thr Leu 690 695 700 Ala Ala Arg Asn Leu Pro Gln Pro Gln Ser Gly
Gln Arg Gly Tyr Glu 705 710 715 720 Cys Leu Phe His Ile Pro Gly Ser
Pro Ala Arg Val Thr Ala Leu Arg 725 730 735 Phe Asn Ser Ser Ser Leu
Gln Cys Gln Asn Ser Ser Tyr Ser Tyr Glu 740 745 750 Gly Asn Asp Val
Ser Asp Leu Pro Val Asn Leu Ser Val Val Trp Asn 755 760 765 Gly Asn
Phe Val Ile Asp Asn Pro Gln Asn Ile Gln Ala His Leu Tyr 770 775 780
Lys Cys Pro Ala Leu Arg Glu Ser Cys Gly Leu Cys Leu Lys Ala Asp 785
790 795 800 Pro Arg Phe Glu Cys Gly Trp Cys Val Ala Glu Arg Arg Cys
Ser Leu 805 810 815 Arg His His Cys Ala Ala Asp Thr Pro Ala Ser Trp
Met His Ala Arg 820 825 830 His Gly Ser Ser Arg Cys Thr Asp Pro Lys
Ile Leu Lys Leu Ser Pro 835 840 845 Glu Thr Gly Pro Arg Gln Gly Gly
Thr Arg Leu Thr Ile Thr Gly Glu 850 855 860 Asn Leu Gly Leu Arg Phe
Glu Asp Val Arg Leu Gly Val Arg Val Gly 865 870 875 880 Lys Val Leu
Cys Ser Pro Val Glu Ser Glu Tyr Ile Ser Ala Glu Gln 885 890 895 Ile
Val Cys Glu Ile Gly Asp Ala Ser Ser Val Arg Ala His Asp Ala 900 905
910 Leu Val Glu Val Cys Val Arg Asp Cys Ser Pro His Tyr Arg Ala Leu
915 920 925 Ser Pro Lys Arg Phe Thr Phe Val Thr Pro Thr Phe Tyr Arg
Val Ser 930 935 940 Pro Ser Arg Gly Pro Leu Ser Gly Gly Thr Trp Ile
Gly Ile Glu Gly 945 950 955 960 Ser His Leu Asn Ala Gly Ser Asp Val
Ala Val Ser Val Gly Gly Arg 965 970 975 Pro Cys Ser Phe Ser Trp Arg
Asn Ser Arg Glu Ile Arg Cys Leu Thr 980 985 990 Pro Pro Gly Gln Ser
Pro Gly Ser Ala Pro Ile Ile Ile Asn Ile Asn 995 1000 1005 Arg Ala
Gln Leu Thr Asn Pro Glu Val Lys Tyr Asn Tyr Thr Glu 1010 1015 1020
Asp Pro Thr Ile Leu Arg Ile Asp Pro Glu Trp Ser Ile Asn Ser 1025
1030 1035 Gly Gly Thr Leu Leu Thr Val Thr Gly Thr Asn Leu Ala Thr
Val 1040 1045 1050 Arg Glu Pro Arg Ile Arg Ala Lys Tyr Gly Gly Ile
Glu Arg Glu 1055 1060 1065 Asn Gly Cys Leu Val Tyr Asn Asp Thr Thr
Met Val Cys Arg Ala 1070 1075 1080 Pro Ser Val Ala Asn Pro Val Arg
Ser Pro Pro Glu Leu Gly Glu 1085 1090 1095 Arg Pro Asp Glu Leu Gly
Phe Val Met Asp Asn Val Arg Ser Leu 1100 1105 1110 Leu Val Leu Asn
Ser Thr Ser Phe Leu Tyr Tyr Pro Asp Pro Val 1115 1120 1125 Leu Glu
Pro Leu Ser Pro Thr Gly Leu Leu Glu Leu Lys Pro Ser 1130 1135 1140
Ser Pro Leu Ile Leu Lys Gly Arg Asn Leu Leu Pro Pro Ala Pro 1145
1150 1155 Gly Asn Ser Arg Leu Asn Tyr Thr Val Leu Ile Gly Ser Thr
Pro 1160 1165 1170 Cys Thr Leu Thr Val Ser Glu Thr Gln Leu Leu Cys
Glu Ala Pro 1175 1180 1185 Asn Leu Thr Gly Gln His Lys Val Thr Val
Arg Ala Gly Gly Phe 1190 1195 1200 Glu Phe Ser Pro Gly Thr Leu Gln
Val Tyr Ser Asp Ser Leu Leu 1205 1210 1215 Thr Leu Pro Ala Ile Val
Gly Ile Gly Gly Gly Gly Gly Leu Leu 1220 1225 1230 Leu Leu Val Ile
Val Ala Val Leu Ile Ala Tyr Lys Arg Lys Ser 1235 1240 1245 Arg Asp
Ala Asp Arg Thr Leu Lys Arg Leu Gln Leu Gln Met Asp 1250 1255 1260
Asn Leu Glu Ser Arg Val Ala Leu Glu Cys Lys Glu Ala Phe Ala 1265
1270 1275 Glu Leu Gln Thr Asp Ile His Glu Leu Thr Asn Asp Leu Asp
Gly 1280 1285 1290 Ala Gly Ile Pro Phe Leu Asp Tyr Arg Thr Tyr Ala
Met Arg Val 1295 1300 1305 Leu Phe Pro Gly Ile Glu Asp His Pro Val
Leu Lys Glu Met Glu 1310 1315 1320 Val Gln Ala Asn Val Glu Lys Ser
Leu Thr Leu Phe Gly Gln Leu 1325 1330 1335 Leu Thr Lys Lys His Phe
Leu Leu Thr Phe Ile Arg Thr Leu Glu 1340 1345 1350 Ala Gln Arg Ser
Phe Ser Met Arg Asp Arg Gly Asn Val Ala Ser 1355 1360 1365 Leu Ile
Met Thr Ala Leu Gln Gly Glu Met Glu Tyr Ala Thr Gly 1370 1375 1380
Val Leu Lys Gln Leu Leu Ser Asp Leu Ile Glu Lys Asn Leu Glu 1385
1390 1395 Ser Lys Asn His Pro Lys Leu Leu Leu Arg Arg Thr Glu Ser
Val 1400 1405 1410 Ala Glu Lys Met Leu Thr Asn Trp Phe Thr Phe Leu
Leu Tyr Lys 1415 1420 1425 Phe Leu Lys Glu Cys Ala Gly Glu Pro Leu
Phe Met Leu Tyr Cys 1430 1435 1440 Ala Ile Lys Gln Gln Met Glu Lys
Gly Pro Ile Asp Ala Ile Thr 1445 1450 1455 Gly Glu Ala Arg Tyr Ser
Leu Ser Glu Asp Lys Leu Ile Arg Gln 1460 1465 1470 Gln Ile Asp Tyr
Lys Thr Leu Thr Leu Asn Cys Val Asn Pro Glu 1475 1480 1485 Asn Glu
Asn Ala Pro Glu Val Pro Val Lys Gly Leu Asp Cys Asp 1490 1495 1500
Thr Val Thr Gln Ala Lys Glu Lys Leu Leu Asp Ala Ala Tyr Lys 1505
1510 1515 Gly Val Pro Tyr Ser Gln Arg Pro Lys Ala Ala Asp Met Asp
Leu 1520 1525 1530 Glu Trp Arg Gln Gly Arg Met Ala Arg Ile Ile Leu
Gln Asp Glu 1535 1540 1545 Asp Val Thr Thr Lys Ile Asp Asn Asp Trp
Lys Arg Leu Asn Thr 1550 1555 1560 Leu Ala His Tyr Gln Val Thr Asp
Gly Ser Ser Val Ala Leu Val 1565 1570 1575 Pro Lys Gln Thr Ser Ala
Tyr Asn Ile Ser Asn Ser Ser Thr Phe 1580 1585 1590 Thr Lys Ser Leu
Ser Arg Tyr Glu Ser Met Leu Arg Thr Ala Ser 1595 1600 1605 Ser Pro
Asp Ser Leu Arg Ser Arg Thr Pro Met Ile Thr Pro Asp 1610 1615 1620
Leu Glu Ser Gly Thr Lys Leu Trp His Leu Val Lys Asn His Asp 1625
1630 1635 His Leu Asp Gln Arg Glu Gly Asp Arg Gly Ser Lys Met Val
Ser 1640 1645 1650 Glu Ile Tyr Leu Thr Arg Leu Leu Ala Thr Lys Gly
Thr Leu Gln 1655 1660 1665 Lys Phe Val Asp Asp Leu Phe Glu Thr Ile
Phe Ser Thr Ala His 1670 1675 1680 Arg Gly Ser Ala Leu Pro Leu Ala
Ile Lys Tyr Met Phe Asp Phe 1685 1690 1695 Leu Asp Glu Gln Ala Asp
Lys His Gln Ile His Asp Ala Asp Val 1700 1705 1710 Arg His Thr Trp
Lys Ser Asn Cys Leu Pro Leu Arg Phe Trp Val 1715 1720 1725 Asn Val
Ile Lys Asn Pro Gln Phe Val Phe Asp Ile His Lys Asn 1730 1735 1740
Ser Ile Thr Asp Ala Cys Leu Ser Val Val Ala Gln Thr Phe Met 1745
1750 1755 Asp Ser Cys Ser Thr Ser Glu His Lys Leu Gly Lys Asp Ser
Pro 1760 1765 1770 Ser Asn Lys Leu Leu Tyr Ala Lys Asp Ile Pro Asn
Tyr Lys Ser 1775 1780 1785 Trp Val Glu Arg Tyr Tyr Ala Asp Ile Ala
Lys Met Pro Ala Ile 1790 1795 1800 Ser Asp Gln Asp Met Ser Ala Tyr
Leu Ala Glu Gln Ser Arg Leu 1805 1810 1815 His Leu Ser Gln Phe Asn
Ser Met Ser Ala Leu His Glu Ile Tyr 1820 1825 1830 Ser Tyr Ile Thr
Lys Tyr Lys Asp Glu Ile Leu Ala Ala Leu Glu 1835 1840 1845 Lys Asp
Glu Gln Ala Arg Arg Gln Arg Leu Arg Ser Lys Leu Glu 1850 1855 1860
Gln Val Val Asp Thr Met Ala Leu Ser Ser 1865 1870 201894PRTHomo
sapiens 20Met Glu Gln Arg Arg Pro Trp Pro Arg Ala Leu Glu Val Asp
Ser Arg 1 5 10 15 Ser Val Val Leu Leu Ser Val Val Trp Val Leu Leu
Ala Pro Pro Ala 20 25 30 Ala Gly Met Pro Gln Phe Ser Thr Phe His
Ser Glu Asn Arg Asp Trp 35 40 45 Thr Phe Asn His Leu Thr Val His
Gln Gly Thr Gly Ala Val Tyr Val 50 55 60 Gly Ala Ile Asn Arg Val
Tyr Lys Leu Thr Gly Asn Leu Thr Ile Gln 65 70 75 80 Val Ala His Lys
Thr Gly Pro Glu Glu Asp Asn Lys Ser Cys Tyr Pro 85 90 95 Pro Leu
Ile Val Gln Pro Cys Ser Glu Val Leu Thr Leu Thr Asn Asn 100 105 110
Val Asn Lys Leu Leu Ile Ile Asp Tyr Ser Glu Asn Arg Leu Leu Ala 115
120 125 Cys Gly Ser Leu Tyr Gln Gly Val Cys Lys Leu Leu Arg Leu Asp
Asp 130 135 140 Leu Phe Ile Leu Val Glu Pro Ser His Lys Lys Glu His
Tyr Leu Ser 145 150 155 160 Ser Val Asn Lys Thr Gly Thr Met Tyr Gly
Val Ile Val Arg Ser Glu 165 170 175 Gly Glu Asp Gly Lys Leu Phe Ile
Gly Thr Ala Val Asp Gly Lys Gln 180 185 190 Asp Tyr Phe Pro Thr Leu
Ser Ser Arg Lys Leu Pro Arg Asp Pro Glu 195 200 205 Ser Ser Ala Met
Leu Asp Tyr Glu Leu His Ser Asp Phe Val Ser Ser 210 215 220 Leu Ile
Lys Ile Pro Ser Asp Thr Leu Ala Leu Val Ser His Phe Asp 225 230 235
240 Ile Phe Tyr Ile Tyr Gly Phe Ala Ser Gly Gly Phe Val Tyr Phe Leu
245 250 255 Thr Val Gln Pro Glu Thr Pro Glu Gly Val Ala Ile Asn Ser
Ala Gly 260 265 270 Asp Leu Phe Tyr Thr Ser Arg Ile Val Arg Leu Cys
Lys Asp Asp Pro 275 280 285 Lys Phe His Ser Tyr Val Ser Leu Pro Phe
Gly Cys Thr Arg Ala Gly 290 295 300 Val Glu Tyr Arg Leu Leu Gln Ala
Ala Tyr Leu Ala Lys Pro Gly Asp 305 310 315 320 Ser Leu Ala Gln Ala
Phe Asn Ile Thr Ser Gln Asp Asp Val Leu Phe 325 330 335 Ala Ile Phe
Ser Lys Gly Gln Lys Gln Tyr His His Pro Pro Asp Asp 340 345 350 Ser
Ala Leu Cys Ala Phe Pro Ile Arg Ala Ile Asn Leu Gln Ile Lys 355 360
365 Glu Arg Leu Gln Ser Cys Tyr Gln Gly Glu Gly Asn Leu Glu Leu Asn
370 375 380 Trp Leu Leu Gly Lys Asp Val Gln Cys Thr Lys Ala Pro Val
Pro Ile 385 390 395 400 Asp Asp Asn Phe Cys Gly Leu Asp Ile Asn Gln
Pro Leu Gly Gly Ser 405 410 415 Thr Pro Val Glu Gly Leu Thr Leu Tyr
Thr Thr Ser Arg Asp Arg Met 420 425 430 Thr Ser Val Ala Ser Tyr Val
Tyr Asn Gly Tyr Ser Val Val Phe Val 435 440 445 Gly Thr Lys Ser Gly
Lys Leu Lys Lys Ile Arg Ala Asp Gly Pro Pro 450 455 460 His Gly Gly
Val Gln Tyr Glu Met Val Ser Val Leu Lys Asp Gly Ser 465 470 475 480
Pro Ile Leu Arg Asp Met Ala Phe Ser Ile Asp Gln Arg Tyr Leu Tyr 485
490 495 Val Met Ser Glu Arg Gln Val Thr Arg Val Pro Val Glu Ser Cys
Glu 500 505 510 Gln Tyr Thr Thr Cys Gly Glu Cys Leu Ser Ser Gly Asp
Pro His Cys 515 520 525 Gly Trp Cys Ala Leu His Asn Met Cys Ser Arg
Arg Asp Lys Cys Gln 530 535 540 Gln Ala Trp Glu Pro Asn Arg Phe Ala
Ala Ser Ile Ser Gln Cys Val 545 550 555 560 Ser Leu Ala Val His Pro
Ser Ser Ile Ser Val Ser Glu His Ser Arg 565 570 575 Leu Leu Ser Leu
Val Val Ser Asp Ala Pro Asp Leu Ser Ala Gly Ile 580 585 590 Ala Cys
Ala Phe Gly Asn Leu Thr Glu Val Glu Gly Gln Val Ser Gly 595 600 605
Ser Gln Val Ile Cys Ile Ser Pro Gly Pro Lys Asp Val Pro Val Ile 610
615 620 Pro Leu Asp Gln Asp Trp Phe Gly Leu Glu Leu Gln Leu Arg Ser
Lys 625 630 635 640 Glu Thr Gly Lys Ile Phe Val Ser Thr Glu Phe Lys
Phe Tyr Asn Cys 645 650 655 Ser Ala His Gln Leu Cys Leu Ser Cys Val
Asn Ser Ala Phe Arg Cys 660 665 670 His Trp Cys Lys Tyr Arg Asn Leu
Cys Thr His Asp Pro Thr Thr Cys 675 680 685 Ser Phe Gln Glu Gly Arg
Ile Asn Ile Ser Glu Asp Cys Pro Gln Leu 690 695 700 Val Pro Thr Glu
Glu Ile Leu Ile Pro Val Gly Glu Val Lys Pro Ile 705 710 715 720 Thr
Leu Lys Ala Arg Asn Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730
735 Tyr Glu Cys Val Leu Asn Ile Gln Gly Ala Ile His Arg Val Pro Ala
740 745 750 Leu Arg Phe Asn Ser Ser Ser Val Gln Cys Gln Asn Ser Ser
Tyr Gln 755 760 765 Tyr Asp Gly Met Asp Ile Ser Asn Leu Ala Val Asp
Phe Ala Val Val 770 775 780 Trp Asn Gly Asn Phe Ile Ile Asp Asn Pro
Gln Asp Leu Lys Val His 785 790 795 800 Leu Tyr Lys Cys Ala Ala Gln
Arg Glu Ser Cys Gly Leu Cys Leu Lys 805 810 815 Ala Asp Arg Lys Phe
Glu Cys Gly Trp Cys Ser Gly Glu Arg Arg Cys 820 825 830 Thr Leu His
Gln His Cys Thr Ser Pro Ser Ser Pro Trp Leu Asp
Trp 835 840 845 Ser Ser His Asn Val Lys Cys Ser Asn Pro Gln Ile Thr
Glu Ile Leu 850 855 860 Thr Val Ser Gly Pro Pro Glu Gly Gly Thr Arg
Val Thr Ile His Gly 865 870 875 880 Val Asn Leu Gly Leu Asp Phe Ser
Glu Ile Ala His His Val Gln Val 885 890 895 Ala Gly Val Pro Cys Thr
Pro Leu Pro Gly Glu Tyr Ile Ile Ala Glu 900 905 910 Gln Ile Val Cys
Glu Met Gly His Ala Leu Val Gly Thr Thr Ser Gly 915 920 925 Pro Val
Arg Leu Cys Ile Gly Glu Cys Lys Pro Glu Phe Met Thr Lys 930 935 940
Ser His Gln Gln Tyr Thr Phe Val Asn Pro Ser Val Leu Ser Leu Asn 945
950 955 960 Pro Ile Arg Gly Pro Glu Ser Gly Gly Thr Met Val Thr Ile
Thr Gly 965 970 975 His Tyr Leu Gly Ala Gly Ser Ser Val Ala Val Tyr
Leu Gly Asn Gln 980 985 990 Thr Cys Glu Phe Tyr Gly Arg Ser Met Ser
Glu Ile Val Cys Val Ser 995 1000 1005 Pro Pro Ser Ser Asn Gly Leu
Gly Pro Val Pro Val Ser Val Ser 1010 1015 1020 Val Asp Arg Ala His
Val Asp Ser Asn Leu Gln Phe Glu Tyr Ile 1025 1030 1035 Asp Asp Pro
Arg Val Gln Arg Ile Glu Pro Glu Trp Ser Ile Ala 1040 1045 1050 Ser
Gly His Thr Pro Leu Thr Ile Thr Gly Phe Asn Leu Asp Val 1055 1060
1065 Ile Gln Glu Pro Arg Ile Arg Val Lys Phe Asn Gly Lys Glu Ser
1070 1075 1080 Val Asn Val Cys Lys Val Val Asn Thr Thr Thr Leu Thr
Cys Leu 1085 1090 1095 Ala Pro Ser Leu Thr Thr Asp Tyr Arg Pro Gly
Leu Asp Thr Val 1100 1105 1110 Glu Arg Pro Asp Glu Phe Gly Phe Val
Phe Asn Asn Val Gln Ser 1115 1120 1125 Leu Leu Ile Tyr Asn Asp Thr
Lys Phe Ile Tyr Tyr Pro Asn Pro 1130 1135 1140 Thr Phe Glu Leu Leu
Ser Pro Thr Gly Val Leu Asp Gln Lys Pro 1145 1150 1155 Gly Ser Pro
Ile Ile Leu Lys Gly Lys Asn Leu Cys Pro Pro Ala 1160 1165 1170 Ser
Gly Gly Ala Lys Leu Asn Tyr Thr Val Leu Ile Gly Glu Thr 1175 1180
1185 Pro Cys Ala Val Thr Val Ser Glu Thr Gln Leu Leu Cys Glu Pro
1190 1195 1200 Pro Asn Leu Thr Gly Gln His Lys Val Met Val His Val
Gly Gly 1205 1210 1215 Met Val Phe Ser Pro Gly Ser Val Ser Val Ile
Ser Asp Ser Leu 1220 1225 1230 Leu Thr Leu Pro Ala Ile Val Ser Ile
Ala Ala Gly Gly Ser Leu 1235 1240 1245 Leu Leu Ile Ile Val Ile Ile
Val Leu Ile Ala Tyr Lys Arg Lys 1250 1255 1260 Ser Arg Glu Asn Asp
Leu Thr Leu Lys Arg Leu Gln Met Gln Met 1265 1270 1275 Asp Asn Leu
Glu Ser Arg Val Ala Leu Glu Cys Lys Glu Ala Phe 1280 1285 1290 Ala
Glu Leu Gln Thr Asp Ile Asn Glu Leu Thr Ser Asp Leu Asp 1295 1300
1305 Arg Ser Gly Ile Pro Tyr Leu Asp Tyr Arg Thr Tyr Ala Met Arg
1310 1315 1320 Val Leu Phe Pro Gly Ile Glu Asp His Pro Val Leu Arg
Glu Leu 1325 1330 1335 Glu Val Gln Gly Asn Gly Gln Gln His Val Glu
Lys Ala Leu Lys 1340 1345 1350 Leu Phe Ala Gln Leu Ile Asn Asn Lys
Val Phe Leu Leu Thr Phe 1355 1360 1365 Ile Arg Thr Leu Glu Leu Gln
Arg Ser Phe Ser Met Arg Asp Arg 1370 1375 1380 Gly Asn Val Ala Ser
Leu Ile Met Thr Gly Leu Gln Gly Arg Leu 1385 1390 1395 Glu Tyr Ala
Thr Asp Val Leu Lys Gln Leu Leu Ser Asp Leu Ile 1400 1405 1410 Asp
Lys Asn Leu Glu Asn Lys Asn His Pro Lys Leu Leu Leu Arg 1415 1420
1425 Arg Thr Glu Ser Val Ala Glu Lys Met Leu Thr Asn Trp Phe Ala
1430 1435 1440 Phe Leu Leu His Lys Phe Leu Lys Glu Cys Ala Gly Glu
Pro Leu 1445 1450 1455 Phe Met Leu Tyr Cys Ala Ile Lys Gln Gln Met
Glu Lys Gly Pro 1460 1465 1470 Ile Asp Ala Ile Thr Gly Glu Ala Arg
Tyr Ser Leu Ser Glu Asp 1475 1480 1485 Lys Leu Ile Arg Gln Gln Ile
Glu Tyr Lys Thr Leu Ile Leu Asn 1490 1495 1500 Cys Val Asn Pro Asp
Asn Glu Asn Ser Pro Glu Ile Pro Val Lys 1505 1510 1515 Val Leu Asn
Cys Asp Thr Ile Thr Gln Val Lys Glu Lys Ile Leu 1520 1525 1530 Asp
Ala Val Tyr Lys Asn Val Pro Tyr Ser Gln Arg Pro Arg Ala 1535 1540
1545 Val Asp Met Asp Leu Glu Trp Arg Gln Gly Arg Ile Ala Arg Val
1550 1555 1560 Val Leu Gln Asp Glu Asp Ile Thr Thr Lys Ile Glu Gly
Asp Trp 1565 1570 1575 Lys Arg Leu Asn Thr Leu Met His Tyr Gln Val
Ser Asp Arg Ser 1580 1585 1590 Val Val Ala Leu Val Pro Lys Gln Thr
Ser Ser Tyr Asn Ile Pro 1595 1600 1605 Ala Ser Ala Ser Ile Ser Arg
Thr Ser Ile Ser Arg Tyr Asp Ser 1610 1615 1620 Ser Phe Arg Tyr Thr
Gly Ser Pro Asp Ser Leu Arg Ser Arg Ala 1625 1630 1635 Pro Met Ile
Thr Pro Asp Leu Glu Ser Gly Val Lys Val Trp His 1640 1645 1650 Leu
Val Lys Asn His Asp His Gly Asp Gln Lys Glu Gly Asp Arg 1655 1660
1665 Gly Ser Lys Met Val Ser Glu Ile Tyr Leu Thr Arg Leu Leu Ala
1670 1675 1680 Thr Lys Gly Thr Leu Gln Lys Phe Val Asp Asp Leu Phe
Glu Thr 1685 1690 1695 Leu Phe Ser Thr Val His Arg Gly Ser Ala Leu
Pro Leu Ala Ile 1700 1705 1710 Lys Tyr Met Phe Asp Phe Leu Asp Glu
Gln Ala Asp Arg His Ser 1715 1720 1725 Ile His Asp Thr Asp Val Arg
His Thr Trp Lys Ser Asn Cys Leu 1730 1735 1740 Pro Leu Arg Phe Trp
Val Asn Val Ile Lys Asn Pro Gln Phe Val 1745 1750 1755 Phe Asp Ile
His Lys Gly Ser Ile Thr Asp Ala Cys Leu Ser Val 1760 1765 1770 Val
Ala Gln Thr Phe Met Asp Ser Cys Ser Thr Ser Glu His Arg 1775 1780
1785 Leu Gly Lys Asp Ser Pro Ser Asn Lys Leu Leu Tyr Ala Lys Asp
1790 1795 1800 Ile Pro Ser Tyr Lys Ser Trp Val Glu Arg Tyr Tyr Ala
Asp Ile 1805 1810 1815 Ala Lys Leu Pro Ala Ile Ser Asp Gln Asp Met
Asn Ala Tyr Leu 1820 1825 1830 Ala Glu Gln Ser Arg Leu His Ala Val
Glu Phe Asn Met Leu Ser 1835 1840 1845 Ala Leu Asn Glu Ile Tyr Ser
Tyr Val Ser Lys Tyr Ser Glu Glu 1850 1855 1860 Leu Ile Gly Ala Leu
Glu Gln Asp Glu Gln Ala Arg Arg Gln Arg 1865 1870 1875 Leu Ala Tyr
Lys Val Glu Gln Leu Ile Asn Ala Met Ser Ile Glu 1880 1885 1890 Ser
211871PRTHomo sapiens 21Met Pro Ser Val Cys Leu Leu Leu Leu Leu Phe
Leu Ala Val Gly Gly 1 5 10 15 Ala Leu Gly Asn Arg Pro Phe Arg Ala
Phe Val Val Thr Asp Thr Thr 20 25 30 Leu Thr His Leu Ala Val His
Arg Val Thr Gly Glu Val Phe Val Gly 35 40 45 Ala Val Asn Arg Val
Phe Lys Leu Ala Pro Asn Leu Thr Glu Leu Arg 50 55 60 Ala His Val
Thr Gly Pro Val Glu Asp Asn Ala Arg Cys Tyr Pro Pro 65 70 75 80 Pro
Ser Met Arg Val Cys Ala His Arg Leu Ala Pro Val Asp Asn Ile 85 90
95 Asn Lys Leu Leu Leu Ile Asp Tyr Ala Ala Arg Arg Leu Val Ala Cys
100 105 110 Gly Ser Ile Trp Gln Gly Ile Cys Gln Phe Leu Arg Leu Asp
Asp Leu 115 120 125 Phe Lys Leu Gly Glu Pro His His Arg Lys Glu His
Tyr Leu Ser Gly 130 135 140 Ala Gln Glu Pro Asp Ser Met Ala Gly Val
Ile Val Glu Gln Gly Gln 145 150 155 160 Gly Pro Ser Lys Leu Phe Val
Gly Thr Ala Val Asp Gly Lys Ser Glu 165 170 175 Tyr Phe Pro Thr Leu
Ser Ser Arg Lys Leu Ile Ser Asp Glu Asp Ser 180 185 190 Ala Asp Met
Phe Ser Leu Val Tyr Gln Asp Glu Phe Val Ser Ser Gln 195 200 205 Ile
Lys Ile Pro Ser Asp Thr Leu Ser Leu Tyr Pro Ala Phe Asp Ile 210 215
220 Tyr Tyr Ile Tyr Gly Phe Val Ser Ala Ser Phe Val Tyr Phe Leu Thr
225 230 235 240 Leu Gln Leu Asp Thr Gln Gln Thr Leu Leu Asp Thr Ala
Gly Glu Lys 245 250 255 Phe Phe Thr Ser Lys Ile Val Arg Met Cys Ala
Gly Asp Ser Glu Phe 260 265 270 Tyr Ser Tyr Val Glu Phe Pro Ile Gly
Cys Ser Trp Arg Gly Val Glu 275 280 285 Tyr Arg Leu Val Gln Ser Ala
His Leu Ala Lys Pro Gly Leu Leu Leu 290 295 300 Ala Gln Ala Leu Gly
Val Pro Ala Asp Glu Asp Val Leu Phe Thr Ile 305 310 315 320 Phe Ser
Gln Gly Gln Lys Asn Arg Ala Ser Pro Pro Arg Gln Thr Ile 325 330 335
Leu Cys Leu Phe Thr Leu Ser Asn Ile Asn Ala His Ile Arg Arg Arg 340
345 350 Ile Gln Ser Cys Tyr Arg Gly Glu Gly Thr Leu Ala Leu Pro Trp
Leu 355 360 365 Leu Asn Lys Glu Leu Pro Cys Ile Asn Thr Pro Met Gln
Ile Asn Gly 370 375 380 Asn Phe Cys Gly Leu Val Leu Asn Gln Pro Leu
Gly Gly Leu His Val 385 390 395 400 Ile Glu Gly Leu Pro Leu Leu Ala
Asp Ser Thr Asp Gly Met Ala Ser 405 410 415 Val Ala Ala Tyr Thr Tyr
Arg Gln His Ser Val Val Phe Ile Gly Thr 420 425 430 Arg Ser Gly Ser
Leu Lys Lys Val Arg Val Asp Gly Phe Gln Asp Ala 435 440 445 His Leu
Tyr Glu Thr Val Pro Val Val Asp Gly Ser Pro Ile Leu Arg 450 455 460
Asp Leu Leu Phe Ser Pro Asp His Arg His Ile Tyr Leu Leu Ser Glu 465
470 475 480 Lys Gln Val Ser Gln Leu Pro Val Glu Thr Cys Glu Gln Tyr
Gln Ser 485 490 495 Cys Ala Ala Cys Leu Gly Ser Gly Asp Pro His Cys
Gly Trp Cys Val 500 505 510 Leu Arg His Arg Cys Cys Arg Glu Gly Ala
Cys Leu Gly Ala Ser Ala 515 520 525 Pro His Gly Phe Ala Glu Glu Leu
Ser Lys Cys Val Gln Val Arg Val 530 535 540 Arg Pro Asn Asn Val Ser
Val Thr Ser Pro Gly Val Gln Leu Thr Val 545 550 555 560 Thr Leu His
Asn Val Pro Asp Leu Ser Ala Gly Val Ser Cys Ala Phe 565 570 575 Glu
Ala Ala Ala Glu Asn Glu Ala Val Leu Leu Pro Ser Gly Glu Leu 580 585
590 Leu Cys Pro Ser Pro Ser Leu Gln Glu Leu Arg Ala Leu Thr Arg Gly
595 600 605 His Gly Ala Thr Arg Thr Val Arg Leu Gln Leu Leu Ser Lys
Glu Thr 610 615 620 Gly Val Arg Phe Ala Gly Ala Asp Phe Val Phe Tyr
Asn Cys Ser Val 625 630 635 640 Leu Gln Ser Cys Met Ser Cys Val Gly
Ser Pro Tyr Pro Cys His Trp 645 650 655 Cys Lys Tyr Arg His Thr Cys
Thr Ser Arg Pro His Glu Cys Ser Phe 660 665 670 Gln Glu Gly Arg Val
His Ser Pro Glu Gly Cys Pro Glu Ile Leu Pro 675 680 685 Ser Gly Asp
Leu Leu Ile Pro Val Gly Val Met Gln Pro Leu Thr Leu 690 695 700 Arg
Ala Lys Asn Leu Pro Gln Pro Gln Ser Gly Gln Lys Asn Tyr Glu 705 710
715 720 Cys Val Val Arg Val Gln Gly Arg Gln Gln Arg Val Pro Ala Val
Arg 725 730 735 Phe Asn Ser Ser Ser Val Gln Cys Gln Asn Ala Ser Tyr
Ser Tyr Glu 740 745 750 Gly Asp Glu His Gly Asp Thr Glu Leu Asp Phe
Ser Val Val Trp Asp 755 760 765 Gly Asp Phe Pro Ile Asp Lys Pro Pro
Ser Phe Arg Ala Leu Leu Tyr 770 775 780 Lys Cys Trp Ala Gln Arg Pro
Ser Cys Gly Leu Cys Leu Lys Ala Asp 785 790 795 800 Pro Arg Phe Asn
Cys Gly Trp Cys Ile Ser Glu His Arg Cys Gln Leu 805 810 815 Arg Thr
His Cys Pro Ala Pro Lys Thr Asn Trp Met His Leu Ser Gln 820 825 830
Lys Gly Thr Arg Cys Ser His Pro Arg Ile Thr Gln Ile His Pro Leu 835
840 845 Val Gly Pro Lys Glu Gly Gly Thr Arg Val Thr Ile Val Gly Asp
Asn 850 855 860 Leu Gly Leu Leu Ser Arg Glu Val Gly Leu Arg Val Ala
Gly Val Arg 865 870 875 880 Cys Asn Ser Ile Pro Ala Glu Tyr Ile Ser
Ala Glu Arg Ile Val Cys 885 890 895 Glu Met Glu Glu Ser Leu Val Pro
Ser Pro Pro Pro Gly Pro Val Glu 900 905 910 Leu Cys Val Gly Asp Cys
Ser Ala Asp Phe Arg Thr Gln Ser Glu Gln 915 920 925 Val Tyr Ser Phe
Val Thr Pro Thr Phe Asp Gln Val Ser Pro Ser Arg 930 935 940 Gly Pro
Ala Ser Gly Gly Thr Arg Leu Thr Ile Ser Gly Ser Ser Leu 945 950 955
960 Asp Ala Gly Ser Arg Val Thr Val Thr Val Arg Asp Ser Glu Cys Gln
965 970 975 Phe Val Arg Arg Asp Ala Lys Ala Ile Val Cys Ile Ser Pro
Leu Ser 980 985 990 Thr Leu Gly Pro Ser Gln Ala Pro Ile Thr Leu Ala
Ile Asp Arg Ala 995 1000 1005 Asn Ile Ser Ser Pro Gly Leu Ile Tyr
Thr Tyr Thr Gln Asp Pro 1010 1015 1020 Thr Val Thr Arg Leu Glu Pro
Thr Trp Ser Ile Ile Asn Gly Ser 1025 1030 1035 Thr Ala Ile Thr Val
Ser Gly Thr His Leu Leu Thr Val Gln Glu 1040 1045 1050 Pro Arg Val
Arg Ala Lys Tyr Arg Gly Ile Glu Thr Thr Asn Thr 1055 1060 1065 Cys
Gln Val Ile Asn Asp Thr Ala Met Leu Cys Lys Ala Pro Gly 1070 1075
1080 Ile Phe Leu Gly Arg Pro Gln Pro Arg Ala Gln Gly Glu His Pro
1085 1090 1095 Asp Glu Phe Gly Phe Leu Leu Asp His Val Gln Thr Ala
Arg Ser 1100 1105 1110 Leu Asn Arg Ser Ser Phe Thr Tyr Tyr Pro Asp
Pro Ser Phe Glu 1115 1120 1125 Pro Leu Gly Pro Ser Gly Val Leu Asp
Val Lys Pro Gly Ser His 1130 1135 1140 Val Val Leu Lys Gly Lys Asn
Leu Ile Pro Ala Ala Ala Gly Ser 1145 1150 1155 Ser Arg Leu Asn Tyr
Thr Val Leu Ile Gly Gly Gln Pro Cys Ser 1160 1165 1170 Leu Thr Val
Ser Asp Thr Gln Leu Leu Cys Asp Ser Pro Ser Gln 1175 1180 1185 Thr
Gly Arg Gln Pro Val Met Val Leu Val Gly Gly
Leu Glu Phe 1190 1195 1200 Trp Leu Gly Thr Leu His Ile Ser Ala Glu
Arg Ala Leu Thr Leu 1205 1210 1215 Pro Ala Met Met Gly Leu Ala Ala
Gly Gly Gly Leu Leu Leu Leu 1220 1225 1230 Ala Ile Thr Ala Val Leu
Val Ala Tyr Lys Arg Lys Thr Gln Asp 1235 1240 1245 Ala Asp Arg Thr
Leu Lys Arg Leu Gln Leu Gln Met Asp Asn Leu 1250 1255 1260 Glu Ser
Arg Val Ala Leu Glu Cys Lys Glu Ala Phe Ala Glu Leu 1265 1270 1275
Gln Thr Asp Ile Asn Glu Leu Thr Asn His Met Asp Glu Val Gln 1280
1285 1290 Ile Pro Phe Leu Asp Tyr Arg Thr Tyr Ala Val Arg Val Leu
Phe 1295 1300 1305 Pro Gly Ile Glu Ala His Pro Val Leu Lys Glu Leu
Asp Thr Pro 1310 1315 1320 Pro Asn Val Glu Lys Ala Leu Arg Leu Phe
Gly Gln Leu Leu His 1325 1330 1335 Ser Arg Ala Phe Val Leu Thr Phe
Ile His Thr Leu Glu Ala Gln 1340 1345 1350 Ser Ser Phe Ser Met Arg
Asp Arg Gly Thr Val Ala Ser Leu Thr 1355 1360 1365 Met Val Ala Leu
Gln Ser Arg Leu Asp Tyr Ala Thr Gly Leu Leu 1370 1375 1380 Lys Gln
Leu Leu Ala Asp Leu Ile Glu Lys Asn Leu Glu Ser Lys 1385 1390 1395
Asn His Pro Lys Leu Leu Leu Arg Arg Thr Glu Ser Val Ala Glu 1400
1405 1410 Lys Met Leu Thr Asn Trp Phe Thr Phe Leu Leu His Lys Phe
Leu 1415 1420 1425 Lys Glu Cys Ala Gly Glu Pro Leu Phe Leu Leu Tyr
Cys Ala Ile 1430 1435 1440 Lys Gln Gln Met Glu Lys Gly Pro Ile Asp
Ala Ile Thr Gly Glu 1445 1450 1455 Ala Arg Tyr Ser Leu Ser Glu Asp
Lys Leu Ile Arg Gln Gln Ile 1460 1465 1470 Asp Tyr Lys Thr Leu Thr
Leu His Cys Val Cys Pro Glu Asn Glu 1475 1480 1485 Gly Ser Ala Gln
Val Pro Val Lys Val Leu Asn Cys Asp Ser Ile 1490 1495 1500 Thr Gln
Ala Lys Asp Lys Leu Leu Asp Thr Val Tyr Lys Gly Ile 1505 1510 1515
Pro Tyr Ser Gln Arg Pro Lys Ala Glu Asp Met Asp Leu Glu Trp 1520
1525 1530 Arg Gln Gly Arg Met Thr Arg Ile Ile Leu Gln Asp Glu Asp
Val 1535 1540 1545 Thr Thr Lys Ile Glu Cys Asp Trp Lys Arg Leu Asn
Ser Leu Ala 1550 1555 1560 His Tyr Gln Val Thr Asp Gly Ser Leu Val
Ala Leu Val Pro Lys 1565 1570 1575 Gln Val Ser Ala Tyr Asn Met Ala
Asn Ser Phe Thr Phe Thr Arg 1580 1585 1590 Ser Leu Ser Arg Tyr Glu
Ser Leu Leu Arg Thr Ala Ser Ser Pro 1595 1600 1605 Asp Ser Leu Arg
Ser Arg Ala Pro Met Ile Thr Pro Asp Gln Glu 1610 1615 1620 Thr Gly
Thr Lys Leu Trp His Leu Val Lys Asn His Asp His Ala 1625 1630 1635
Asp His Arg Glu Gly Asp Arg Gly Ser Lys Met Val Ser Glu Ile 1640
1645 1650 Tyr Leu Thr Arg Leu Leu Ala Thr Lys Gly Thr Leu Gln Lys
Phe 1655 1660 1665 Val Asp Asp Leu Phe Glu Thr Val Phe Ser Thr Ala
His Arg Gly 1670 1675 1680 Ser Ala Leu Pro Leu Ala Ile Lys Tyr Met
Phe Asp Phe Leu Asp 1685 1690 1695 Glu Gln Ala Asp Gln Arg Gln Ile
Ser Asp Pro Asp Val Arg His 1700 1705 1710 Thr Trp Lys Ser Asn Cys
Leu Pro Leu Arg Phe Trp Val Asn Val 1715 1720 1725 Ile Lys Asn Pro
Gln Phe Val Phe Asp Ile His Lys Asn Ser Ile 1730 1735 1740 Thr Asp
Ala Cys Leu Ser Val Val Ala Gln Thr Phe Met Asp Ser 1745 1750 1755
Cys Ser Thr Ser Glu His Arg Leu Gly Lys Asp Ser Pro Ser Asn 1760
1765 1770 Lys Leu Leu Tyr Ala Lys Asp Ile Pro Asn Tyr Lys Ser Trp
Val 1775 1780 1785 Glu Arg Tyr Tyr Arg Asp Ile Ala Lys Met Ala Ser
Ile Ser Asp 1790 1795 1800 Gln Asp Met Asp Ala Tyr Leu Val Glu Gln
Ser Arg Leu His Ala 1805 1810 1815 Ser Asp Phe Ser Val Leu Ser Ala
Leu Asn Glu Leu Tyr Phe Tyr 1820 1825 1830 Val Thr Lys Tyr Arg Gln
Glu Ile Leu Thr Ala Leu Asp Arg Asp 1835 1840 1845 Ala Ser Cys Arg
Lys His Lys Leu Arg Gln Lys Leu Glu Gln Ile 1850 1855 1860 Ile Ser
Leu Val Ser Ser Asp Ser 1865 1870 221894PRTHomo sapiens 22Met Lys
Ala Met Pro Trp Asn Trp Thr Cys Leu Leu Ser His Leu Leu 1 5 10 15
Met Val Gly Met Gly Ser Ser Thr Leu Leu Thr Arg Gln Pro Ala Pro 20
25 30 Leu Ser Gln Lys Gln Arg Ser Phe Val Thr Phe Arg Gly Glu Pro
Ala 35 40 45 Glu Gly Phe Asn His Leu Val Val Asp Glu Arg Thr Gly
His Ile Tyr 50 55 60 Leu Gly Ala Val Asn Arg Ile Tyr Lys Leu Ser
Ser Asp Leu Lys Val 65 70 75 80 Leu Val Thr His Glu Thr Gly Pro Asp
Glu Asp Asn Pro Lys Cys Tyr 85 90 95 Pro Pro Arg Ile Val Gln Thr
Cys Asn Glu Pro Leu Thr Thr Thr Asn 100 105 110 Asn Val Asn Lys Met
Leu Leu Ile Asp Tyr Lys Glu Asn Arg Leu Ile 115 120 125 Ala Cys Gly
Ser Leu Tyr Gln Gly Ile Cys Lys Leu Leu Arg Leu Glu 130 135 140 Asp
Leu Phe Lys Leu Gly Glu Pro Tyr His Lys Lys Glu His Tyr Leu 145 150
155 160 Ser Gly Val Asn Glu Ser Gly Ser Val Phe Gly Val Ile Val Ser
Tyr 165 170 175 Ser Asn Leu Asp Asp Lys Leu Phe Ile Ala Thr Ala Val
Asp Gly Lys 180 185 190 Pro Glu Tyr Phe Pro Thr Ile Ser Ser Arg Lys
Leu Thr Lys Asn Ser 195 200 205 Glu Ala Asp Gly Met Phe Ala Tyr Val
Phe His Asp Glu Phe Val Ala 210 215 220 Ser Met Ile Lys Ile Pro Ser
Asp Thr Phe Thr Ile Ile Pro Asp Phe 225 230 235 240 Asp Ile Tyr Tyr
Val Tyr Gly Phe Ser Ser Gly Asn Phe Val Tyr Phe 245 250 255 Leu Thr
Leu Gln Pro Glu Met Val Ser Pro Pro Gly Ser Thr Thr Lys 260 265 270
Glu Gln Val Tyr Thr Ser Lys Leu Val Arg Leu Cys Lys Glu Asp Thr 275
280 285 Ala Phe Asn Ser Tyr Val Glu Val Pro Ile Gly Cys Glu Arg Ser
Gly 290 295 300 Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr Leu Ser Lys
Ala Gly Ala 305 310 315 320 Val Leu Gly Arg Thr Leu Gly Val His Pro
Asp Asp Asp Leu Leu Phe 325 330 335 Thr Val Phe Ser Lys Gly Gln Lys
Arg Lys Met Lys Ser Leu Asp Glu 340 345 350 Ser Ala Leu Cys Ile Phe
Ile Leu Lys Gln Ile Asn Asp Arg Ile Lys 355 360 365 Glu Arg Leu Gln
Ser Cys Tyr Arg Gly Glu Gly Thr Leu Asp Leu Ala 370 375 380 Trp Leu
Lys Val Lys Asp Ile Pro Cys Ser Ser Ala Leu Leu Thr Ile 385 390 395
400 Asp Asp Asn Phe Cys Gly Leu Asp Met Asn Ala Pro Leu Gly Val Ser
405 410 415 Asp Met Val Arg Gly Ile Pro Val Phe Thr Glu Asp Arg Asp
Arg Met 420 425 430 Thr Ser Val Ile Ala Tyr Val Tyr Lys Asn His Ser
Leu Ala Phe Val 435 440 445 Gly Thr Lys Ser Gly Lys Leu Lys Lys Ile
Arg Val Asp Gly Pro Arg 450 455 460 Gly Asn Ala Leu Gln Tyr Glu Thr
Val Gln Val Val Asp Pro Gly Pro 465 470 475 480 Val Leu Arg Asp Met
Ala Phe Ser Lys Asp His Glu Gln Leu Tyr Ile 485 490 495 Met Ser Glu
Arg Gln Leu Thr Arg Val Pro Val Glu Ser Cys Gly Gln 500 505 510 Tyr
Gln Ser Cys Gly Glu Cys Leu Gly Ser Gly Asp Pro His Cys Gly 515 520
525 Trp Cys Val Leu His Asn Thr Cys Thr Arg Lys Glu Arg Cys Glu Arg
530 535 540 Ser Lys Glu Pro Arg Arg Phe Ala Ser Glu Met Lys Gln Cys
Val Arg 545 550 555 560 Leu Thr Val His Pro Asn Asn Ile Ser Val Ser
Gln Tyr Asn Val Leu 565 570 575 Leu Val Leu Glu Thr Tyr Asn Val Pro
Glu Leu Ser Ala Gly Val Asn 580 585 590 Cys Thr Phe Glu Asp Leu Ser
Glu Met Asp Gly Leu Val Val Gly Asn 595 600 605 Gln Ile Gln Cys Tyr
Ser Pro Ala Ala Lys Glu Val Pro Arg Ile Ile 610 615 620 Thr Glu Asn
Gly Asp His His Val Val Gln Leu Gln Leu Lys Ser Lys 625 630 635 640
Glu Thr Gly Met Thr Phe Ala Ser Thr Ser Phe Val Phe Tyr Asn Cys 645
650 655 Ser Val His Asn Ser Cys Leu Ser Cys Val Glu Ser Pro Tyr Arg
Cys 660 665 670 His Trp Cys Lys Tyr Arg His Val Cys Thr His Asp Pro
Lys Thr Cys 675 680 685 Ser Phe Gln Glu Gly Arg Val Lys Leu Pro Glu
Asp Cys Pro Gln Leu 690 695 700 Leu Arg Val Asp Lys Ile Leu Val Pro
Val Glu Val Ile Lys Pro Ile 705 710 715 720 Thr Leu Lys Ala Lys Asn
Leu Pro Gln Pro Gln Ser Gly Gln Arg Gly 725 730 735 Tyr Glu Cys Ile
Leu Asn Ile Gln Gly Ser Glu Gln Arg Val Pro Ala 740 745 750 Leu Arg
Phe Asn Ser Ser Ser Val Gln Cys Gln Asn Thr Ser Tyr Ser 755 760 765
Tyr Glu Gly Met Glu Ile Asn Asn Leu Pro Val Glu Leu Thr Val Val 770
775 780 Trp Asn Gly His Phe Asn Ile Asp Asn Pro Ala Gln Asn Lys Val
His 785 790 795 800 Leu Tyr Lys Cys Gly Ala Met Arg Glu Ser Cys Gly
Leu Cys Leu Lys 805 810 815 Ala Asp Pro Asp Phe Ala Cys Gly Trp Cys
Gln Gly Pro Gly Gln Cys 820 825 830 Thr Leu Arg Gln His Cys Pro Ala
Gln Glu Ser Gln Trp Leu Glu Leu 835 840 845 Ser Gly Ala Lys Ser Lys
Cys Thr Asn Pro Arg Ile Thr Glu Ile Ile 850 855 860 Pro Val Thr Gly
Pro Arg Glu Gly Gly Thr Lys Val Thr Ile Arg Gly 865 870 875 880 Glu
Asn Leu Gly Leu Glu Phe Arg Asp Ile Ala Ser His Val Lys Val 885 890
895 Ala Gly Val Glu Cys Ser Pro Leu Val Asp Gly Tyr Ile Pro Ala Glu
900 905 910 Gln Ile Val Cys Glu Met Gly Glu Ala Lys Pro Ser Gln His
Ala Gly 915 920 925 Phe Val Glu Ile Cys Val Ala Val Cys Arg Pro Glu
Phe Met Ala Arg 930 935 940 Ser Ser Gln Leu Tyr Tyr Phe Met Thr Leu
Thr Leu Ser Asp Leu Lys 945 950 955 960 Pro Ser Arg Gly Pro Met Ser
Gly Gly Thr Gln Val Thr Ile Thr Gly 965 970 975 Thr Asn Leu Asn Ala
Gly Ser Asn Val Val Val Met Phe Gly Lys Gln 980 985 990 Pro Cys Leu
Phe His Arg Arg Ser Pro Ser Tyr Ile Val Cys Asn Thr 995 1000 1005
Thr Ser Ser Asp Glu Val Leu Glu Met Lys Val Ser Val Gln Val 1010
1015 1020 Asp Arg Ala Lys Ile His Gln Asp Leu Val Phe Gln Tyr Val
Glu 1025 1030 1035 Asp Pro Thr Ile Val Arg Ile Glu Pro Glu Trp Ser
Ile Val Ser 1040 1045 1050 Gly Asn Thr Pro Ile Ala Val Trp Gly Thr
His Leu Asp Leu Ile 1055 1060 1065 Gln Asn Pro Gln Ile Arg Ala Lys
His Gly Gly Lys Glu His Ile 1070 1075 1080 Asn Ile Cys Glu Val Leu
Asn Ala Thr Glu Met Thr Cys Gln Ala 1085 1090 1095 Pro Ala Leu Ala
Leu Gly Pro Asp His Gln Ser Asp Leu Thr Glu 1100 1105 1110 Arg Pro
Glu Glu Phe Gly Phe Ile Leu Asp Asn Val Gln Ser Leu 1115 1120 1125
Leu Ile Leu Asn Lys Thr Asn Phe Thr Tyr Tyr Pro Asn Pro Val 1130
1135 1140 Phe Glu Ala Phe Gly Pro Ser Gly Ile Leu Glu Leu Lys Pro
Gly 1145 1150 1155 Thr Pro Ile Ile Leu Lys Gly Lys Asn Leu Ile Pro
Pro Val Ala 1160 1165 1170 Gly Gly Asn Val Lys Leu Asn Tyr Thr Val
Leu Val Gly Glu Lys 1175 1180 1185 Pro Cys Thr Val Thr Val Ser Asp
Val Gln Leu Leu Cys Glu Ser 1190 1195 1200 Pro Asn Leu Ile Gly Arg
His Lys Val Met Ala Arg Val Gly Gly 1205 1210 1215 Met Glu Tyr Ser
Pro Gly Met Val Tyr Ile Ala Pro Asp Ser Pro 1220 1225 1230 Leu Ser
Leu Pro Ala Ile Val Ser Ile Ala Val Ala Gly Gly Leu 1235 1240 1245
Leu Ile Ile Phe Ile Val Ala Val Leu Ile Ala Tyr Lys Arg Lys 1250
1255 1260 Ser Arg Glu Ser Asp Leu Thr Leu Lys Arg Leu Gln Met Gln
Met 1265 1270 1275 Asp Asn Leu Glu Ser Arg Val Ala Leu Glu Cys Lys
Glu Ala Phe 1280 1285 1290 Ala Glu Leu Gln Thr Asp Ile His Glu Leu
Thr Ser Asp Leu Asp 1295 1300 1305 Gly Ala Gly Ile Pro Phe Leu Asp
Tyr Arg Thr Tyr Thr Met Arg 1310 1315 1320 Val Leu Phe Pro Gly Ile
Glu Asp His Pro Val Leu Arg Asp Leu 1325 1330 1335 Glu Val Pro Gly
Tyr Arg Gln Glu Arg Val Glu Lys Gly Leu Lys 1340 1345 1350 Leu Phe
Ala Gln Leu Ile Asn Asn Lys Val Phe Leu Leu Ser Phe 1355 1360 1365
Ile Arg Thr Leu Glu Ser Gln Arg Ser Phe Ser Met Arg Asp Arg 1370
1375 1380 Gly Asn Val Ala Ser Leu Ile Met Thr Val Leu Gln Ser Lys
Leu 1385 1390 1395 Glu Tyr Ala Thr Asp Val Leu Lys Gln Leu Leu Ala
Asp Leu Ile 1400 1405 1410 Asp Lys Asn Leu Glu Ser Lys Asn His Pro
Lys Leu Leu Leu Arg 1415 1420 1425 Arg Thr Glu Ser Val Ala Glu Lys
Met Leu Thr Asn Trp Phe Thr 1430 1435 1440 Phe Leu Leu Tyr Lys Phe
Leu Lys Glu Cys Ala Gly Glu Pro Leu 1445 1450 1455 Phe Ser Leu Phe
Cys Ala Ile Lys Gln Gln Met Glu Lys Gly Pro 1460 1465 1470 Ile Asp
Ala Ile Thr Gly Glu Ala Arg Tyr Ser Leu Ser Glu Asp 1475 1480 1485
Lys Leu Ile Arg Gln Gln Ile Asp Tyr Lys Thr Leu Val Leu Ser 1490
1495 1500 Cys Val Ser Pro Asp Asn Ala Asn Ser Pro Glu Val Pro Val
Lys 1505 1510 1515 Ile Leu Asn Cys Asp Thr Ile Thr Gln Val Lys Glu
Lys Ile Leu 1520 1525 1530 Asp Ala Ile Phe Lys Asn Val Pro Cys Ser
His Arg Pro Lys Ala 1535 1540 1545 Ala Asp Met Asp Leu Glu Trp Arg
Gln Gly Ser Gly Ala Arg Met 1550 1555 1560 Ile Leu
Gln Asp Glu Asp Ile Thr Thr Lys Ile Glu Asn Asp Trp 1565 1570 1575
Lys Arg Leu Asn Thr Leu Ala His Tyr Gln Val Pro Asp Gly Ser 1580
1585 1590 Val Val Ala Leu Val Ser Lys Gln Val Thr Ala Tyr Asn Ala
Val 1595 1600 1605 Asn Asn Ser Thr Val Ser Arg Thr Ser Ala Ser Lys
Tyr Glu Asn 1610 1615 1620 Met Ile Arg Tyr Thr Gly Ser Pro Asp Ser
Leu Arg Ser Arg Thr 1625 1630 1635 Pro Met Ile Thr Pro Asp Leu Glu
Ser Gly Val Lys Met Trp His 1640 1645 1650 Leu Val Lys Asn His Glu
His Gly Asp Gln Lys Glu Gly Asp Arg 1655 1660 1665 Gly Ser Lys Met
Val Ser Glu Ile Tyr Leu Thr Arg Leu Leu Ala 1670 1675 1680 Thr Lys
Gly Thr Leu Gln Lys Phe Val Asp Asp Leu Phe Glu Thr 1685 1690 1695
Ile Phe Ser Thr Ala His Arg Gly Ser Ala Leu Pro Leu Ala Ile 1700
1705 1710 Lys Tyr Met Phe Asp Phe Leu Asp Glu Gln Ala Asp Lys His
Gly 1715 1720 1725 Ile His Asp Pro His Val Arg His Thr Trp Lys Ser
Asn Cys Leu 1730 1735 1740 Pro Leu Arg Phe Trp Val Asn Met Ile Lys
Asn Pro Gln Phe Val 1745 1750 1755 Phe Asp Ile His Lys Asn Ser Ile
Thr Asp Ala Cys Leu Ser Val 1760 1765 1770 Val Ala Gln Thr Phe Met
Asp Ser Cys Ser Thr Ser Glu His Arg 1775 1780 1785 Leu Gly Lys Asp
Ser Pro Ser Asn Lys Leu Leu Tyr Ala Lys Asp 1790 1795 1800 Ile Pro
Ser Tyr Lys Asn Trp Val Glu Arg Tyr Tyr Ser Asp Ile 1805 1810 1815
Gly Lys Met Pro Ala Ile Ser Asp Gln Asp Met Asn Ala Tyr Leu 1820
1825 1830 Ala Glu Gln Ser Arg Met His Met Asn Glu Phe Asn Thr Met
Ser 1835 1840 1845 Ala Leu Ser Glu Ile Phe Ser Tyr Val Gly Lys Tyr
Ser Glu Glu 1850 1855 1860 Ile Leu Gly Pro Leu Asp His Asp Asp Gln
Cys Gly Lys Gln Lys 1865 1870 1875 Leu Ala Tyr Lys Leu Glu Gln Val
Ile Thr Leu Met Ser Leu Asp 1880 1885 1890 Ser 23492PRTHomo sapiens
23Met Lys Ala Met Pro Trp Asn Trp Thr Cys Leu Leu Ser His Leu Leu 1
5 10 15 Met Val Gly Met Gly Ser Ser Thr Leu Leu Thr Arg Gln Pro Ala
Pro 20 25 30 Leu Ser Gln Lys Gln Arg Ser Phe Val Thr Phe Arg Gly
Glu Pro Ala 35 40 45 Glu Gly Phe Asn His Leu Val Val Asp Glu Arg
Thr Gly His Ile Tyr 50 55 60 Leu Gly Ala Val Asn Arg Ile Tyr Lys
Leu Ser Ser Asp Leu Lys Val 65 70 75 80 Leu Val Thr His Glu Thr Gly
Pro Asp Glu Asp Asn Pro Lys Cys Tyr 85 90 95 Pro Pro Arg Ile Val
Gln Thr Cys Asn Glu Pro Leu Thr Thr Thr Asn 100 105 110 Asn Val Asn
Lys Met Leu Leu Ile Asp Tyr Lys Glu Asn Arg Leu Ile 115 120 125 Ala
Cys Gly Ser Leu Tyr Gln Gly Ile Cys Lys Leu Leu Arg Leu Glu 130 135
140 Asp Leu Phe Lys Leu Gly Glu Pro Tyr His Lys Lys Glu His Tyr Leu
145 150 155 160 Ser Gly Val Asn Glu Ser Gly Ser Val Phe Gly Val Ile
Val Ser Tyr 165 170 175 Ser Asn Leu Asp Asp Lys Leu Phe Ile Ala Thr
Ala Val Asp Gly Lys 180 185 190 Pro Glu Tyr Phe Pro Thr Ile Ser Ser
Arg Lys Leu Thr Lys Asn Ser 195 200 205 Glu Ala Asp Gly Met Phe Ala
Tyr Val Phe His Asp Glu Phe Val Ala 210 215 220 Ser Met Ile Lys Ile
Pro Ser Asp Thr Phe Thr Ile Ile Pro Asp Phe 225 230 235 240 Asp Ile
Tyr Tyr Val Tyr Gly Phe Ser Ser Gly Asn Phe Val Tyr Phe 245 250 255
Leu Thr Leu Gln Pro Glu Met Val Ser Pro Pro Gly Ser Thr Thr Lys 260
265 270 Glu Gln Val Tyr Thr Ser Lys Leu Val Arg Leu Cys Lys Glu Asp
Thr 275 280 285 Ala Phe Asn Ser Tyr Val Glu Val Pro Ile Gly Cys Glu
Arg Ser Gly 290 295 300 Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr Leu
Ser Lys Ala Gly Ala 305 310 315 320 Val Leu Gly Arg Thr Leu Gly Val
His Pro Asp Asp Asp Leu Leu Phe 325 330 335 Thr Val Phe Ser Lys Gly
Gln Lys Arg Lys Met Lys Ser Leu Asp Glu 340 345 350 Ser Ala Leu Cys
Ile Phe Ile Leu Lys Gln Ile Asn Asp Arg Ile Lys 355 360 365 Glu Arg
Leu Gln Ser Cys Tyr Arg Gly Glu Gly Thr Leu Asp Leu Ala 370 375 380
Trp Leu Lys Val Lys Asp Ile Pro Cys Ser Ser Ala Leu Leu Thr Ile 385
390 395 400 Asp Asp Asn Phe Cys Gly Leu Asp Met Asn Ala Pro Leu Gly
Val Ser 405 410 415 Asp Met Val Arg Gly Ile Pro Val Phe Thr Glu Asp
Arg Asp Arg Met 420 425 430 Thr Ser Val Ile Ala Tyr Val Tyr Lys Asn
His Ser Leu Ala Phe Val 435 440 445 Gly Thr Lys Ser Gly Lys Leu Lys
Lys Met Pro Gly Thr Ser Leu Cys 450 455 460 Pro Thr Leu Glu Leu Gln
Thr Gly Pro Arg Ser His Arg Ala Thr Val 465 470 475 480 Thr Leu Glu
Leu Leu Phe Ser Ser Cys Ser Ser Asn 485 490 24522PRTHomo sapiens
24Met Lys Ala Met Pro Trp Asn Trp Thr Cys Leu Leu Ser His Leu Leu 1
5 10 15 Met Val Gly Met Gly Ser Ser Thr Leu Leu Thr Arg Gln Pro Ala
Pro 20 25 30 Leu Ser Gln Lys Gln Arg Ser Phe Val Thr Phe Arg Gly
Glu Pro Ala 35 40 45 Glu Gly Phe Asn His Leu Val Val Asp Glu Arg
Thr Gly His Ile Tyr 50 55 60 Leu Gly Ala Val Asn Arg Ile Tyr Lys
Leu Ser Ser Asp Leu Lys Val 65 70 75 80 Leu Val Thr His Glu Thr Gly
Pro Asp Glu Asp Asn Pro Lys Cys Tyr 85 90 95 Pro Pro Arg Ile Val
Gln Thr Cys Asn Glu Pro Leu Thr Thr Thr Asn 100 105 110 Asn Val Asn
Lys Met Leu Leu Ile Asp Tyr Lys Glu Asn Arg Leu Ile 115 120 125 Ala
Cys Gly Ser Leu Tyr Gln Gly Ile Cys Lys Leu Leu Arg Leu Glu 130 135
140 Asp Leu Phe Lys Leu Gly Glu Pro Tyr His Lys Lys Glu His Tyr Leu
145 150 155 160 Ser Gly Val Asn Glu Ser Gly Ser Val Phe Gly Val Ile
Val Ser Tyr 165 170 175 Ser Asn Leu Asp Asp Lys Leu Phe Ile Ala Thr
Ala Val Asp Gly Lys 180 185 190 Pro Glu Tyr Phe Pro Thr Ile Ser Ser
Arg Lys Leu Thr Lys Asn Ser 195 200 205 Glu Ala Asp Gly Met Phe Ala
Tyr Val Phe His Asp Glu Phe Val Ala 210 215 220 Ser Met Ile Lys Ile
Pro Ser Asp Thr Phe Thr Ile Ile Pro Asp Phe 225 230 235 240 Asp Ile
Tyr Tyr Val Tyr Gly Phe Ser Ser Gly Asn Phe Val Tyr Phe 245 250 255
Leu Thr Leu Gln Pro Glu Met Val Ser Pro Pro Gly Ser Thr Thr Lys 260
265 270 Glu Gln Val Tyr Thr Ser Lys Leu Val Arg Leu Cys Lys Glu Asp
Thr 275 280 285 Ala Phe Asn Ser Tyr Val Glu Val Pro Ile Gly Cys Glu
Arg Ser Gly 290 295 300 Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr Leu
Ser Lys Ala Gly Ala 305 310 315 320 Val Leu Gly Arg Thr Leu Gly Val
His Pro Asp Asp Asp Leu Leu Phe 325 330 335 Thr Val Phe Ser Lys Gly
Gln Lys Arg Lys Met Lys Ser Leu Asp Glu 340 345 350 Ser Ala Leu Cys
Ile Phe Ile Leu Lys Gln Ile Asn Asp Arg Ile Lys 355 360 365 Glu Arg
Leu Gln Ser Cys Tyr Arg Gly Glu Gly Thr Leu Asp Leu Ala 370 375 380
Trp Leu Lys Val Lys Asp Ile Pro Cys Ser Ser Ala Leu Leu Thr Ile 385
390 395 400 Asp Asp Asn Phe Cys Gly Leu Asp Met Asn Ala Pro Leu Gly
Val Ser 405 410 415 Asp Met Val Arg Gly Ile Pro Val Phe Thr Glu Asp
Arg Asp Arg Met 420 425 430 Thr Ser Val Ile Ala Tyr Val Tyr Lys Asn
His Ser Leu Ala Phe Val 435 440 445 Gly Thr Lys Ser Gly Lys Leu Lys
Lys Ser Phe Gly Thr Gly Pro Gln 450 455 460 Gly Gly Ile Thr Gln Glu
Trp Ile Gly Val Glu Gly Asp Pro Pro Gly 465 470 475 480 Ala Asn Ile
Ala Ser Gln Glu Gln Met Leu Cys Val Tyr Leu Gln Cys 485 490 495 Ser
Ser His Lys Ala Ile Ser Asp Gln Arg Val Gln Pro Leu Leu Cys 500 505
510 Cys Phe Leu Asn Val Pro Gly Asn Ser Ser 515 520 251925PRTHomo
sapiens 25Met Ala Pro Arg Ala Ala Gly Gly Ala Pro Leu Ser Ala Arg
Ala Ala 1 5 10 15 Ala Ala Ser Pro Pro Pro Phe Gln Thr Pro Pro Arg
Cys Pro Val Pro 20 25 30 Leu Leu Leu Leu Leu Leu Leu Gly Ala Ala
Arg Ala Gly Ala Leu Glu 35 40 45 Ile Gln Arg Arg Phe Pro Ser Pro
Thr Pro Thr Asn Asn Phe Ala Leu 50 55 60 Asp Gly Ala Ala Gly Thr
Val Tyr Leu Ala Ala Val Asn Arg Leu Tyr 65 70 75 80 Gln Leu Ser Gly
Ala Asn Leu Ser Leu Glu Ala Glu Ala Ala Val Gly 85 90 95 Pro Val
Pro Asp Ser Pro Leu Cys His Ala Pro Gln Leu Pro Gln Ala 100 105 110
Ser Cys Glu His Pro Arg Arg Leu Thr Asp Asn Tyr Asn Lys Ile Leu 115
120 125 Gln Leu Asp Pro Gly Gln Gly Leu Val Val Val Cys Gly Ser Ile
Tyr 130 135 140 Gln Gly Phe Cys Gln Leu Arg Arg Arg Gly Asn Ile Ser
Ala Val Ala 145 150 155 160 Val Arg Phe Pro Pro Ala Ala Pro Pro Ala
Glu Pro Val Thr Val Phe 165 170 175 Pro Ser Met Leu Asn Val Ala Ala
Asn His Pro Asn Ala Ser Thr Val 180 185 190 Gly Leu Val Leu Pro Pro
Ala Ala Gly Ala Gly Gly Ser Arg Leu Leu 195 200 205 Val Gly Ala Thr
Tyr Thr Gly Tyr Gly Ser Ser Phe Phe Pro Arg Asn 210 215 220 Arg Ser
Leu Glu Asp His Arg Phe Glu Asn Thr Pro Glu Ile Ala Ile 225 230 235
240 Arg Ser Leu Asp Thr Arg Gly Asp Leu Ala Lys Leu Phe Thr Phe Asp
245 250 255 Leu Asn Pro Ser Asp Asp Asn Ile Leu Lys Ile Lys Gln Gly
Ala Lys 260 265 270 Glu Gln His Lys Leu Gly Phe Val Ser Ala Phe Leu
His Pro Ser Asp 275 280 285 Pro Pro Pro Gly Ala Gln Ser Tyr Ala Tyr
Leu Ala Leu Asn Ser Glu 290 295 300 Ala Arg Ala Gly Asp Lys Glu Ser
Gln Ala Arg Ser Leu Leu Ala Arg 305 310 315 320 Ile Cys Leu Pro His
Gly Ala Gly Gly Asp Ala Lys Lys Leu Thr Glu 325 330 335 Ser Tyr Ile
Gln Leu Gly Leu Gln Cys Ala Gly Gly Ala Gly Arg Gly 340 345 350 Asp
Leu Tyr Ser Arg Leu Val Ser Val Phe Pro Ala Arg Glu Arg Leu 355 360
365 Phe Ala Val Phe Glu Arg Pro Gln Gly Ser Pro Ala Ala Arg Ala Ala
370 375 380 Pro Ala Ala Leu Cys Ala Phe Arg Phe Ala Asp Val Arg Ala
Ala Ile 385 390 395 400 Arg Ala Ala Arg Thr Ala Cys Phe Val Glu Pro
Ala Pro Asp Val Val 405 410 415 Ala Val Leu Asp Ser Val Val Gln Gly
Thr Gly Pro Ala Cys Glu Arg 420 425 430 Lys Leu Asn Ile Gln Leu Gln
Pro Glu Gln Leu Asp Cys Gly Ala Ala 435 440 445 His Leu Gln His Pro
Leu Ser Ile Leu Gln Pro Leu Lys Ala Thr Pro 450 455 460 Val Phe Arg
Ala Pro Gly Leu Thr Ser Val Ala Val Ala Ser Val Asn 465 470 475 480
Asn Tyr Thr Ala Val Phe Leu Gly Thr Val Asn Gly Arg Leu Leu Lys 485
490 495 Ile Asn Leu Asn Glu Ser Met Gln Val Val Ser Arg Arg Val Val
Thr 500 505 510 Val Ala Tyr Gly Glu Pro Val His His Val Met Gln Phe
Asp Pro Ala 515 520 525 Asp Ser Gly Tyr Leu Tyr Leu Met Thr Ser His
Gln Met Ala Arg Val 530 535 540 Lys Val Ala Ala Cys Asn Val His Ser
Thr Cys Gly Asp Cys Val Gly 545 550 555 560 Ala Ala Asp Ala Tyr Cys
Gly Trp Cys Ala Leu Glu Thr Arg Cys Thr 565 570 575 Leu Gln Gln Asp
Cys Thr Asn Ser Ser Gln Gln His Phe Trp Thr Ser 580 585 590 Ala Ser
Glu Gly Pro Ser Arg Cys Pro Ala Met Thr Val Leu Pro Ser 595 600 605
Glu Ile Asp Val Arg Gln Glu Tyr Pro Gly Met Ile Leu Gln Ile Ser 610
615 620 Gly Ser Leu Pro Ser Leu Ser Gly Met Glu Met Ala Cys Asp Tyr
Gly 625 630 635 640 Asn Asn Ile Arg Thr Val Ala Arg Val Pro Gly Pro
Ala Phe Gly His 645 650 655 Gln Ile Ala Tyr Cys Asn Leu Leu Pro Arg
Asp Gln Phe Pro Pro Phe 660 665 670 Pro Pro Asn Gln Asp His Val Thr
Val Glu Met Ser Val Arg Val Asn 675 680 685 Gly Arg Asn Ile Val Lys
Ala Asn Phe Thr Ile Tyr Asp Cys Ser Arg 690 695 700 Thr Ala Gln Val
Tyr Pro His Thr Ala Cys Thr Ser Cys Leu Ser Ala 705 710 715 720 Gln
Trp Pro Cys Phe Trp Cys Ser Gln Gln His Ser Cys Val Ser Asn 725 730
735 Gln Ser Arg Cys Glu Ala Ser Pro Asn Pro Thr Ser Pro Gln Asp Cys
740 745 750 Pro Arg Thr Leu Leu Ser Pro Leu Ala Pro Val Pro Thr Gly
Gly Ser 755 760 765 Gln Asn Ile Leu Val Pro Leu Ala Asn Thr Ala Phe
Phe Gln Gly Ala 770 775 780 Ala Leu Glu Cys Ser Phe Gly Leu Glu Glu
Ile Phe Glu Ala Val Trp 785 790 795 800 Val Asn Glu Ser Val Val Arg
Cys Asp Gln Val Val Leu His Thr Thr 805 810 815 Arg Lys Ser Gln Val
Phe Pro Leu Ser Leu Gln Leu Lys Gly Arg Pro 820 825 830 Ala Arg Phe
Leu Asp Ser Pro Glu Pro Met Thr Val Met Val Tyr Asn 835 840 845 Cys
Ala Met Gly Ser Pro Asp Cys Ser Gln Cys Leu Gly Arg Glu Asp 850 855
860 Leu Gly His Leu Cys Met Trp Ser Asp Gly Cys Arg Leu Arg Gly Pro
865 870 875 880 Leu Gln Pro Met Ala Gly Thr Cys Pro Ala Pro Glu Ile
Arg Ala Ile 885 890 895 Glu Pro Leu Ser Gly Pro Leu Asp Gly Gly Thr
Leu Leu Thr Ile Arg 900 905 910 Gly Arg Asn Leu Gly Arg Arg Leu Ser
Asp Val Ala His Gly Val Trp 915 920 925 Ile Gly Gly Val Ala Cys Glu
Pro Leu Pro Asp Arg Tyr Thr Val Ser 930
935 940 Glu Glu Ile Val Cys Val Thr Gly Pro Ala Pro Gly Pro Leu Ser
Gly 945 950 955 960 Val Val Thr Val Asn Ala Ser Lys Glu Gly Lys Ser
Arg Asp Arg Phe 965 970 975 Ser Tyr Val Leu Pro Leu Val His Ser Leu
Glu Pro Thr Met Gly Pro 980 985 990 Lys Ala Gly Gly Thr Arg Ile Thr
Ile His Gly Asn Asp Leu His Val 995 1000 1005 Gly Ser Glu Leu Gln
Val Leu Val Asn Asp Thr Asp Pro Cys Thr 1010 1015 1020 Glu Leu Met
Arg Thr Asp Thr Ser Ile Ala Cys Thr Met Pro Glu 1025 1030 1035 Gly
Ala Leu Pro Ala Pro Val Pro Val Cys Val Arg Phe Glu Arg 1040 1045
1050 Arg Gly Cys Val His Gly Asn Leu Thr Phe Trp Tyr Met Gln Asn
1055 1060 1065 Pro Val Ile Thr Ala Ile Ser Pro Arg Arg Ser Pro Val
Ser Gly 1070 1075 1080 Gly Arg Thr Ile Thr Val Ala Gly Glu Arg Phe
His Met Val Gln 1085 1090 1095 Asn Val Ser Met Ala Val His His Ile
Gly Arg Glu Pro Thr Leu 1100 1105 1110 Cys Lys Val Leu Asn Ser Thr
Leu Ile Thr Cys Pro Ser Pro Gly 1115 1120 1125 Ala Leu Ser Asn Ala
Ser Ala Pro Val Asp Phe Phe Ile Asn Gly 1130 1135 1140 Arg Ala Tyr
Ala Asp Glu Val Ala Val Ala Glu Glu Leu Leu Asp 1145 1150 1155 Pro
Glu Glu Ala Gln Arg Gly Ser Arg Phe Arg Leu Asp Tyr Leu 1160 1165
1170 Pro Asn Pro Gln Phe Ser Thr Ala Lys Arg Glu Lys Trp Ile Lys
1175 1180 1185 His His Pro Gly Glu Pro Leu Thr Leu Val Ile His Lys
Glu Gln 1190 1195 1200 Asp Ser Leu Gly Leu Gln Ser His Glu Tyr Arg
Val Lys Ile Gly 1205 1210 1215 Gln Val Ser Cys Asp Ile Gln Ile Val
Ser Asp Arg Ile Ile His 1220 1225 1230 Cys Ser Val Asn Glu Ser Leu
Gly Ala Ala Val Gly Gln Leu Pro 1235 1240 1245 Ile Thr Ile Gln Val
Gly Asn Phe Asn Gln Thr Ile Ala Thr Leu 1250 1255 1260 Gln Leu Gly
Gly Ser Glu Thr Ala Ile Ile Val Ser Ile Val Ile 1265 1270 1275 Cys
Ser Val Leu Leu Leu Leu Ser Val Val Ala Leu Phe Val Phe 1280 1285
1290 Cys Thr Lys Ser Arg Arg Ala Glu Arg Tyr Trp Gln Lys Thr Leu
1295 1300 1305 Leu Gln Met Glu Glu Met Glu Ser Gln Ile Arg Glu Glu
Ile Arg 1310 1315 1320 Lys Gly Phe Ala Glu Leu Gln Thr Asp Met Thr
Asp Leu Thr Lys 1325 1330 1335 Glu Leu Asn Arg Ser Gln Gly Ile Pro
Phe Leu Glu Tyr Lys His 1340 1345 1350 Phe Val Thr Arg Thr Phe Phe
Pro Lys Cys Ser Ser Leu Tyr Glu 1355 1360 1365 Glu Arg Tyr Val Leu
Pro Ser Gln Thr Leu Asn Ser Gln Gly Ser 1370 1375 1380 Ser Gln Ala
Gln Glu Thr His Pro Leu Leu Gly Glu Trp Lys Ile 1385 1390 1395 Pro
Glu Ser Cys Arg Pro Asn Met Glu Glu Gly Ile Ser Leu Phe 1400 1405
1410 Ser Ser Leu Leu Asn Asn Lys His Phe Leu Ile Val Phe Val His
1415 1420 1425 Ala Leu Glu Gln Gln Lys Asp Phe Ala Val Arg Asp Arg
Cys Ser 1430 1435 1440 Leu Ala Ser Leu Leu Thr Ile Ala Leu His Gly
Lys Leu Glu Tyr 1445 1450 1455 Tyr Thr Ser Ile Met Lys Glu Leu Leu
Val Asp Leu Ile Asp Ala 1460 1465 1470 Ser Ala Ala Lys Asn Pro Lys
Leu Met Leu Arg Arg Thr Glu Ser 1475 1480 1485 Val Val Glu Lys Met
Leu Thr Asn Trp Met Ser Ile Cys Met Tyr 1490 1495 1500 Ser Cys Leu
Arg Glu Thr Val Gly Glu Pro Phe Phe Leu Leu Leu 1505 1510 1515 Cys
Ala Ile Lys Gln Gln Ile Asn Lys Gly Ser Ile Asp Ala Ile 1520 1525
1530 Thr Gly Lys Ala Arg Tyr Thr Leu Asn Glu Glu Trp Leu Leu Arg
1535 1540 1545 Glu Asn Ile Glu Ala Lys Pro Arg Asn Leu Asn Val Ser
Phe Gln 1550 1555 1560 Gly Cys Gly Met Asp Ser Leu Ser Val Arg Ala
Met Asp Thr Asp 1565 1570 1575 Thr Leu Thr Gln Val Lys Glu Lys Ile
Leu Glu Ala Phe Cys Lys 1580 1585 1590 Asn Val Pro Tyr Ser Gln Trp
Pro Arg Ala Glu Asp Val Asp Leu 1595 1600 1605 Glu Trp Phe Ala Ser
Ser Thr Gln Ser Tyr Ile Leu Arg Asp Leu 1610 1615 1620 Asp Asp Thr
Ser Val Val Glu Asp Gly Arg Lys Lys Leu Asn Thr 1625 1630 1635 Leu
Ala His Tyr Lys Ile Pro Glu Gly Ala Ser Leu Ala Met Ser 1640 1645
1650 Leu Ile Asp Lys Lys Asp Asn Thr Leu Gly Arg Val Lys Asp Leu
1655 1660 1665 Asp Thr Glu Lys Tyr Phe His Leu Val Leu Pro Thr Asp
Glu Leu 1670 1675 1680 Ala Glu Pro Lys Lys Ser His Arg Gln Ser His
Arg Lys Lys Val 1685 1690 1695 Leu Pro Glu Ile Tyr Leu Thr Arg Leu
Leu Ser Thr Lys Gly Thr 1700 1705 1710 Leu Gln Lys Phe Leu Asp Asp
Leu Phe Lys Ala Ile Leu Ser Ile 1715 1720 1725 Arg Glu Asp Lys Pro
Pro Leu Ala Val Lys Tyr Phe Phe Asp Phe 1730 1735 1740 Leu Glu Glu
Gln Ala Glu Lys Arg Gly Ile Ser Asp Pro Asp Thr 1745 1750 1755 Leu
His Ile Trp Lys Thr Asn Ser Leu Pro Leu Arg Phe Trp Val 1760 1765
1770 Asn Ile Leu Lys Asn Pro Gln Phe Val Phe Asp Ile Asp Lys Thr
1775 1780 1785 Asp His Ile Asp Ala Cys Leu Ser Val Ile Ala Gln Ala
Phe Ile 1790 1795 1800 Asp Ala Cys Ser Ile Ser Asp Leu Gln Leu Gly
Lys Asp Ser Pro 1805 1810 1815 Thr Asn Lys Leu Leu Tyr Ala Lys Glu
Ile Pro Glu Tyr Arg Lys 1820 1825 1830 Ile Val Gln Arg Tyr Tyr Lys
Gln Ile Gln Asp Met Thr Pro Leu 1835 1840 1845 Ser Glu Gln Glu Met
Asn Ala His Leu Ala Glu Glu Ser Arg Lys 1850 1855 1860 Tyr Gln Asn
Glu Phe Asn Thr Asn Val Ala Met Ala Glu Ile Tyr 1865 1870 1875 Lys
Tyr Ala Lys Arg Tyr Arg Pro Gln Ile Met Ala Ala Leu Glu 1880 1885
1890 Ala Asn Pro Thr Ala Arg Arg Thr Gln Leu Gln His Lys Phe Glu
1895 1900 1905 Gln Val Val Ala Leu Met Glu Asp Asn Ile Tyr Glu Cys
Tyr Ser 1910 1915 1920 Glu Ala 1925 26771PRTHomo sapiens 26Met Gly
Trp Leu Thr Arg Ile Val Cys Leu Phe Trp Gly Val Leu Leu 1 5 10 15
Thr Ala Arg Ala Asn Tyr Gln Asn Gly Lys Asn Asn Val Pro Arg Leu 20
25 30 Lys Leu Ser Tyr Lys Glu Met Leu Glu Ser Asn Asn Val Ile Thr
Phe 35 40 45 Asn Gly Leu Ala Asn Ser Ser Ser Tyr His Thr Phe Leu
Leu Asp Glu 50 55 60 Glu Arg Ser Arg Leu Tyr Val Gly Ala Lys Asp
His Ile Phe Ser Phe 65 70 75 80 Asp Leu Val Asn Ile Lys Asp Phe Gln
Lys Ile Val Trp Pro Val Ser 85 90 95 Tyr Thr Arg Arg Asp Glu Cys
Lys Trp Ala Gly Lys Asp Ile Leu Lys 100 105 110 Glu Cys Ala Asn Phe
Ile Lys Val Leu Lys Ala Tyr Asn Gln Thr His 115 120 125 Leu Tyr Ala
Cys Gly Thr Gly Ala Phe His Pro Ile Cys Thr Tyr Ile 130 135 140 Glu
Ile Gly His His Pro Glu Asp Asn Ile Phe Lys Leu Glu Asn Ser 145 150
155 160 His Phe Glu Asn Gly Arg Gly Lys Ser Pro Tyr Asp Pro Lys Leu
Leu 165 170 175 Thr Ala Ser Leu Leu Ile Asp Gly Glu Leu Tyr Ser Gly
Thr Ala Ala 180 185 190 Asp Phe Met Gly Arg Asp Phe Ala Ile Phe Arg
Thr Leu Gly His His 195 200 205 His Pro Ile Arg Thr Glu Gln His Asp
Ser Arg Trp Leu Asn Asp Pro 210 215 220 Lys Phe Ile Ser Ala His Leu
Ile Ser Glu Ser Asp Asn Pro Glu Asp 225 230 235 240 Asp Lys Val Tyr
Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu His 245 250 255 Ser Gly
Lys Ala Thr His Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp 260 265 270
Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys 275
280 285 Ala Arg Leu Ile Cys Ser Val Pro Gly Pro Asn Gly Ile Asp Thr
His 290 295 300 Phe Asp Glu Leu Gln Asp Val Phe Leu Met Asn Phe Lys
Asp Pro Lys 305 310 315 320 Asn Pro Val Val Tyr Gly Val Phe Thr Thr
Ser Ser Asn Ile Phe Lys 325 330 335 Gly Ser Ala Val Cys Met Tyr Ser
Met Ser Asp Val Arg Arg Val Phe 340 345 350 Leu Gly Pro Tyr Ala His
Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro 355 360 365 Tyr Gln Gly Arg
Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys 370 375 380 Thr Phe
Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro Asp Asp Val Ile 385 390 395
400 Thr Phe Ala Arg Ser His Pro Ala Met Tyr Asn Pro Val Phe Pro Met
405 410 415 Asn Asn Arg Pro Ile Val Ile Lys Thr Asp Val Asn Tyr Gln
Phe Thr 420 425 430 Gln Ile Val Val Asp Arg Val Asp Ala Glu Asp Gly
Gln Tyr Asp Val 435 440 445 Met Phe Ile Gly Thr Asp Val Gly Thr Val
Leu Lys Val Val Ser Ile 450 455 460 Pro Lys Glu Thr Trp Tyr Asp Leu
Glu Glu Val Leu Leu Glu Glu Met 465 470 475 480 Thr Val Phe Arg Glu
Pro Thr Ala Ile Ser Ala Met Glu Leu Ser Thr 485 490 495 Lys Gln Gln
Gln Leu Tyr Ile Gly Ser Thr Ala Gly Val Ala Gln Leu 500 505 510 Pro
Leu His Arg Cys Asp Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys 515 520
525 Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ser Ala Cys Ser Arg
530 535 540 Tyr Phe Pro Thr Ala Lys Arg Arg Thr Arg Arg Gln Asp Ile
Arg Asn 545 550 555 560 Gly Asp Pro Leu Thr His Cys Ser Asp Leu His
His Asp Asn His His 565 570 575 Gly His Ser Pro Glu Glu Arg Ile Ile
Tyr Gly Val Glu Asn Ser Ser 580 585 590 Thr Phe Leu Glu Cys Ser Pro
Lys Ser Gln Arg Ala Leu Val Tyr Trp 595 600 605 Gln Phe Gln Arg Arg
Asn Glu Glu Arg Lys Glu Glu Ile Arg Val Asp 610 615 620 Asp His Ile
Ile Arg Thr Asp Gln Gly Leu Leu Leu Arg Ser Leu Gln 625 630 635 640
Gln Lys Asp Ser Gly Asn Tyr Leu Cys His Ala Val Glu His Gly Phe 645
650 655 Ile Gln Thr Leu Leu Lys Val Thr Leu Glu Val Ile Asp Thr Glu
His 660 665 670 Leu Glu Glu Leu Leu His Lys Asp Asp Asp Gly Asp Gly
Ser Lys Thr 675 680 685 Lys Glu Met Ser Asn Ser Met Thr Pro Ser Gln
Lys Val Trp Tyr Arg 690 695 700 Asp Phe Met Gln Leu Ile Asn His Pro
Asn Leu Asn Thr Met Asp Glu 705 710 715 720 Phe Cys Glu Gln Val Trp
Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg 725 730 735 Pro Gly His Thr
Pro Gly Asn Ser Asn Lys Trp Lys His Leu Gln Glu 740 745 750 Asn Lys
Lys Gly Arg Asn Arg Arg Thr His Glu Phe Glu Arg Ala Pro 755 760 765
Arg Ser Val 770 27748PRTHomo sapiens 27Met Gly Arg Ala Gly Ala Ala
Ala Val Ile Pro Gly Leu Ala Leu Leu 1 5 10 15 Trp Ala Val Gly Leu
Gly Ser Ala Ala Pro Ser Pro Pro Arg Leu Arg 20 25 30 Leu Ser Phe
Gln Glu Leu Gln Ala Trp His Gly Leu Gln Thr Phe Ser 35 40 45 Leu
Glu Arg Thr Cys Cys Tyr Gln Ala Leu Leu Val Asp Glu Glu Arg 50 55
60 Gly Arg Leu Phe Val Gly Ala Glu Asn His Val Ala Ser Leu Asn Leu
65 70 75 80 Asp Asn Ile Ser Lys Arg Ala Lys Lys Leu Ala Trp Pro Ala
Pro Val 85 90 95 Glu Trp Arg Glu Glu Cys Asn Trp Ala Gly Lys Asp
Ile Gly Thr Glu 100 105 110 Cys Met Asn Phe Val Lys Leu Leu His Ala
Tyr Asn Arg Thr His Leu 115 120 125 Leu Ala Cys Gly Thr Gly Ala Phe
His Pro Thr Cys Ala Phe Val Glu 130 135 140 Val Gly His Arg Ala Glu
Glu Pro Val Leu Arg Leu Asp Pro Gly Arg 145 150 155 160 Ile Glu Asp
Gly Lys Gly Lys Ser Pro Tyr Asp Pro Arg His Arg Ala 165 170 175 Ala
Ser Val Leu Val Gly Glu Glu Leu Tyr Ser Gly Val Ala Ala Asp 180 185
190 Leu Met Gly Arg Asp Phe Thr Ile Phe Arg Ser Leu Gly Gln Arg Pro
195 200 205 Ser Leu Arg Thr Glu Pro His Asp Ser Arg Trp Leu Asn Glu
Pro Lys 210 215 220 Phe Val Lys Val Phe Trp Ile Pro Glu Ser Glu Asn
Pro Asp Asp Asp 225 230 235 240 Lys Ile Tyr Phe Phe Phe Arg Glu Thr
Ala Val Glu Ala Ala Pro Ala 245 250 255 Leu Gly Arg Leu Ser Val Ser
Arg Val Gly Gln Ile Cys Arg Asn Asp 260 265 270 Val Gly Gly Gln Arg
Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys 275 280 285 Ala Arg Leu
Val Cys Ser Val Pro Gly Val Glu Gly Asp Thr His Phe 290 295 300 Asp
Gln Leu Gln Asp Val Phe Leu Leu Ser Ser Arg Asp His Arg Thr 305 310
315 320 Pro Leu Leu Tyr Ala Val Phe Ser Thr Ser Ser Ile Phe Gln Gly
Ser 325 330 335 Ala Val Cys Val Tyr Ser Met Asn Asp Val Arg Arg Ala
Phe Leu Gly 340 345 350 Pro Phe Ala His Lys Glu Gly Pro Met His Gln
Trp Val Ser Tyr Gln 355 360 365 Gly Arg Val Pro Tyr Pro Arg Pro Gly
Met Cys Pro Ser Lys Thr Phe 370 375 380 Gly Thr Phe Ser Ser Thr Lys
Asp Phe Pro Asp Asp Val Ile Gln Phe 385 390 395 400 Ala Arg Asn His
Pro Leu Met Tyr Asn Ser Val Leu Pro Thr Gly Gly 405 410 415 Arg Pro
Leu Phe Leu Gln Val Gly Ala Asn Tyr Thr Phe Thr Gln Ile 420 425 430
Ala Ala Asp Arg Val Ala Ala Ala Asp Gly His Tyr Asp Val Leu Phe 435
440 445 Ile Gly Thr Asp Val Gly Thr Val Leu Lys Val Ile Ser Val Pro
Lys 450 455 460 Gly Ser Arg Pro Ser Ala Glu Gly Leu Leu Leu Glu Glu
Leu His Val 465 470 475 480 Phe Glu Asp Ser Ala Ala Val Thr Ser Met
Gln Ile Ser Ser Lys Arg 485 490
495 His Gln Leu Tyr Val Ala Ser Arg Ser Ala Val Ala Gln Ile Ala Leu
500 505 510 His Arg Cys Ala Ala His Gly Arg Val Cys Thr Glu Cys Cys
Leu Ala 515 520 525 Arg Asp Pro Tyr Cys Ala Trp Asp Gly Val Ala Cys
Thr Arg Phe Gln 530 535 540 Pro Ser Ala Lys Arg Arg Phe Arg Arg Gln
Asp Val Arg Asn Gly Asp 545 550 555 560 Pro Ser Thr Leu Cys Ser Gly
Asp Ser Ser Arg Pro Ala Leu Leu Glu 565 570 575 His Lys Val Phe Gly
Val Glu Gly Ser Ser Ala Phe Leu Glu Cys Glu 580 585 590 Pro Arg Ser
Leu Gln Ala Arg Val Glu Trp Thr Phe Gln Arg Ala Gly 595 600 605 Val
Thr Ala His Thr Gln Val Leu Ala Glu Glu Arg Thr Glu Arg Thr 610 615
620 Ala Arg Gly Leu Leu Leu Arg Arg Leu Arg Arg Arg Asp Ser Gly Val
625 630 635 640 Tyr Leu Cys Ala Ala Val Glu Gln Gly Phe Thr Gln Pro
Leu Arg Arg 645 650 655 Leu Ser Leu His Val Leu Ser Ala Thr Gln Ala
Glu Arg Leu Ala Arg 660 665 670 Ala Glu Glu Ala Ala Pro Ala Ala Pro
Pro Gly Pro Lys Leu Trp Tyr 675 680 685 Arg Asp Phe Leu Gln Leu Val
Glu Pro Gly Gly Gly Gly Ser Ala Asn 690 695 700 Ser Leu Arg Met Cys
Arg Pro Gln Pro Ala Leu Gln Ser Leu Pro Leu 705 710 715 720 Glu Ser
Arg Arg Lys Gly Arg Asn Arg Arg Thr His Ala Pro Glu Pro 725 730 735
Arg Ala Glu Arg Gly Pro Arg Ser Ala Thr His Trp 740 745
28751PRTHomo sapiens 28Met Ala Phe Arg Thr Ile Cys Val Leu Val Gly
Val Phe Ile Cys Ser 1 5 10 15 Ile Cys Val Lys Gly Ser Ser Gln Pro
Gln Ala Arg Val Tyr Leu Thr 20 25 30 Phe Asp Glu Leu Arg Glu Thr
Lys Thr Ser Glu Tyr Phe Ser Leu Ser 35 40 45 His His Pro Leu Asp
Tyr Arg Ile Leu Leu Met Asp Glu Asp Gln Asp 50 55 60 Arg Ile Tyr
Val Gly Ser Lys Asp His Ile Leu Ser Leu Asn Ile Asn 65 70 75 80 Asn
Ile Ser Gln Glu Ala Leu Ser Val Phe Trp Pro Ala Ser Thr Ile 85 90
95 Lys Val Glu Glu Cys Lys Met Ala Gly Lys Asp Pro Thr His Gly Cys
100 105 110 Gly Asn Phe Val Arg Val Ile Gln Thr Phe Asn Arg Thr His
Leu Tyr 115 120 125 Val Cys Gly Ser Gly Ala Phe Ser Pro Val Cys Thr
Tyr Leu Asn Arg 130 135 140 Gly Arg Arg Ser Glu Asp Gln Val Phe Met
Ile Asp Ser Lys Cys Glu 145 150 155 160 Ser Gly Lys Gly Arg Cys Ser
Phe Asn Pro Asn Val Asn Thr Val Ser 165 170 175 Val Met Ile Asn Glu
Glu Leu Phe Ser Gly Met Tyr Ile Asp Phe Met 180 185 190 Gly Thr Asp
Ala Ala Ile Phe Arg Ser Leu Thr Lys Arg Asn Ala Val 195 200 205 Arg
Thr Asp Gln His Asn Ser Lys Trp Leu Ser Glu Pro Met Phe Val 210 215
220 Asp Ala His Val Ile Pro Asp Gly Thr Asp Pro Asn Asp Ala Lys Val
225 230 235 240 Tyr Phe Phe Phe Lys Glu Lys Leu Thr Asp Asn Asn Arg
Ser Thr Lys 245 250 255 Gln Ile His Ser Met Ile Ala Arg Ile Cys Pro
Asn Asp Thr Gly Gly 260 265 270 Leu Arg Ser Leu Val Asn Lys Trp Thr
Thr Phe Leu Lys Ala Arg Leu 275 280 285 Val Cys Ser Val Thr Asp Glu
Asp Gly Pro Glu Thr His Phe Asp Glu 290 295 300 Leu Glu Asp Val Phe
Leu Leu Glu Thr Asp Asn Pro Arg Thr Thr Leu 305 310 315 320 Val Tyr
Gly Ile Phe Thr Thr Ser Ser Ser Val Phe Lys Gly Ser Ala 325 330 335
Val Cys Val Tyr His Leu Ser Asp Ile Gln Thr Val Phe Asn Gly Pro 340
345 350 Phe Ala His Lys Glu Gly Pro Asn His Gln Leu Ile Ser Tyr Gln
Gly 355 360 365 Arg Ile Pro Tyr Pro Arg Pro Gly Thr Cys Pro Gly Gly
Ala Phe Thr 370 375 380 Pro Asn Met Arg Thr Thr Lys Glu Phe Pro Asp
Asp Val Val Thr Phe 385 390 395 400 Ile Arg Asn His Pro Leu Met Tyr
Asn Ser Ile Tyr Pro Ile His Lys 405 410 415 Arg Pro Leu Ile Val Arg
Ile Gly Thr Asp Tyr Lys Tyr Thr Lys Ile 420 425 430 Ala Val Asp Arg
Val Asn Ala Ala Asp Gly Arg Tyr His Val Leu Phe 435 440 445 Leu Gly
Thr Asp Arg Gly Thr Val Gln Lys Val Val Val Leu Pro Thr 450 455 460
Asn Asn Ser Val Ser Gly Glu Leu Ile Leu Glu Glu Leu Glu Val Phe 465
470 475 480 Lys Asn His Ala Pro Ile Thr Thr Met Lys Ile Ser Ser Lys
Lys Gln 485 490 495 Gln Leu Tyr Val Ser Ser Asn Glu Gly Val Ser Gln
Val Ser Leu His 500 505 510 Arg Cys His Ile Tyr Gly Thr Ala Cys Ala
Asp Cys Cys Leu Ala Arg 515 520 525 Asp Pro Tyr Cys Ala Trp Asp Gly
His Ser Cys Ser Arg Phe Tyr Pro 530 535 540 Thr Gly Lys Arg Arg Ser
Arg Arg Gln Asp Val Arg His Gly Asn Pro 545 550 555 560 Leu Thr Gln
Cys Arg Gly Phe Asn Leu Lys Ala Tyr Arg Asn Ala Ala 565 570 575 Glu
Ile Val Gln Tyr Gly Val Lys Asn Asn Thr Thr Phe Leu Glu Cys 580 585
590 Ala Pro Lys Ser Pro Gln Ala Ser Ile Lys Trp Leu Leu Gln Lys Asp
595 600 605 Lys Asp Arg Arg Lys Glu Val Lys Leu Asn Glu Arg Ile Ile
Ala Thr 610 615 620 Ser Gln Gly Leu Leu Ile Arg Ser Val Gln Gly Ser
Asp Gln Gly Leu 625 630 635 640 Tyr His Cys Ile Ala Thr Glu Asn Ser
Phe Lys Gln Thr Ile Ala Lys 645 650 655 Ile Asn Phe Lys Val Leu Asp
Ser Glu Met Val Ala Val Val Thr Asp 660 665 670 Lys Trp Ser Pro Trp
Thr Trp Ala Ser Ser Val Arg Ala Leu Pro Phe 675 680 685 His Pro Lys
Asp Ile Met Gly Ala Phe Ser His Ser Glu Met Gln Met 690 695 700 Ile
Asn Gln Tyr Cys Lys Asp Thr Arg Gln Gln His Gln Gln Gly Asp 705 710
715 720 Glu Ser Gln Lys Met Arg Gly Asp Tyr Gly Lys Leu Lys Ala Leu
Ile 725 730 735 Asn Ser Arg Lys Ser Arg Asn Arg Arg Asn Gln Leu Pro
Glu Ser 740 745 750 29777PRTHomo sapiens 29Met Asn Ala Asn Lys Asp
Glu Arg Leu Lys Ala Arg Ser Gln Asp Phe 1 5 10 15 His Leu Phe Pro
Ala Leu Met Met Leu Ser Met Thr Met Leu Phe Leu 20 25 30 Pro Val
Thr Gly Thr Leu Lys Gln Asn Ile Pro Arg Leu Lys Leu Thr 35 40 45
Tyr Lys Asp Leu Leu Leu Ser Asn Ser Cys Ile Pro Phe Leu Gly Ser 50
55 60 Ser Glu Gly Leu Asp Phe Gln Thr Leu Leu Leu Asp Glu Glu Arg
Gly 65 70 75 80 Arg Leu Leu Leu Gly Ala Lys Asp His Ile Phe Leu Leu
Ser Leu Val 85 90 95 Asp Leu Asn Lys Asn Phe Lys Lys Ile Tyr Trp
Pro Ala Ala Lys Glu 100 105 110 Arg Val Glu Leu Cys Lys Leu Ala Gly
Lys Asp Ala Asn Thr Glu Cys 115 120 125 Ala Asn Phe Ile Arg Val Leu
Gln Pro Tyr Asn Lys Thr His Ile Tyr 130 135 140 Val Cys Gly Thr Gly
Ala Phe His Pro Ile Cys Gly Tyr Ile Asp Leu 145 150 155 160 Gly Val
Tyr Lys Glu Asp Ile Ile Phe Lys Leu Asp Thr His Asn Leu 165 170 175
Glu Ser Gly Arg Leu Lys Cys Pro Phe Asp Pro Gln Gln Pro Phe Ala 180
185 190 Ser Val Met Thr Asp Glu Tyr Leu Tyr Ser Gly Thr Ala Ser Asp
Phe 195 200 205 Leu Gly Lys Asp Thr Ala Phe Thr Arg Ser Leu Gly Pro
Thr His Asp 210 215 220 His His Tyr Ile Arg Thr Asp Ile Ser Glu His
Tyr Trp Leu Asn Gly 225 230 235 240 Ala Lys Phe Ile Gly Thr Phe Phe
Ile Pro Asp Thr Tyr Asn Pro Asp 245 250 255 Asp Asp Lys Ile Tyr Phe
Phe Phe Arg Glu Ser Ser Gln Glu Gly Ser 260 265 270 Thr Ser Asp Lys
Thr Ile Leu Ser Arg Val Gly Arg Val Cys Lys Asn 275 280 285 Asp Val
Gly Gly Gln Arg Ser Leu Ile Asn Lys Trp Thr Thr Phe Leu 290 295 300
Lys Ala Arg Leu Ile Cys Ser Ile Pro Gly Ser Asp Gly Ala Asp Thr 305
310 315 320 Tyr Phe Asp Glu Leu Gln Asp Ile Tyr Leu Leu Pro Thr Arg
Asp Glu 325 330 335 Arg Asn Pro Val Val Tyr Gly Val Phe Thr Thr Thr
Ser Ser Ile Phe 340 345 350 Lys Gly Ser Ala Val Cys Val Tyr Ser Met
Ala Asp Ile Arg Ala Val 355 360 365 Phe Asn Gly Pro Tyr Ala His Lys
Glu Ser Ala Asp His Arg Trp Val 370 375 380 Gln Tyr Asp Gly Arg Ile
Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser 385 390 395 400 Lys Thr Tyr
Asp Pro Leu Ile Lys Ser Thr Arg Asp Phe Pro Asp Asp 405 410 415 Val
Ile Ser Phe Ile Lys Arg His Ser Val Met Tyr Lys Ser Val Tyr 420 425
430 Pro Val Ala Gly Gly Pro Thr Phe Lys Arg Ile Asn Val Asp Tyr Arg
435 440 445 Leu Thr Gln Ile Val Val Asp His Val Ile Ala Glu Asp Gly
Gln Tyr 450 455 460 Asp Val Met Phe Leu Gly Thr Asp Ile Gly Thr Val
Leu Lys Val Val 465 470 475 480 Ser Ile Ser Lys Glu Lys Trp Asn Met
Glu Glu Val Val Leu Glu Glu 485 490 495 Leu Gln Ile Phe Lys His Ser
Ser Ile Ile Leu Asn Met Glu Leu Ser 500 505 510 Leu Lys Gln Gln Gln
Leu Tyr Ile Gly Ser Arg Asp Gly Leu Val Gln 515 520 525 Leu Ser Leu
His Arg Cys Asp Thr Tyr Gly Lys Ala Cys Ala Asp Cys 530 535 540 Cys
Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Asn Ala Cys Ser 545 550
555 560 Arg Tyr Ala Pro Thr Ser Lys Arg Arg Ala Arg Arg Gln Asp Val
Lys 565 570 575 Tyr Gly Asp Pro Ile Thr Gln Cys Trp Asp Ile Glu Asp
Ser Ile Ser 580 585 590 His Glu Thr Ala Asp Glu Lys Val Ile Phe Gly
Ile Glu Phe Asn Ser 595 600 605 Thr Phe Leu Glu Cys Ile Pro Lys Ser
Gln Gln Ala Thr Ile Lys Trp 610 615 620 Tyr Ile Gln Arg Ser Gly Asp
Glu His Arg Glu Glu Leu Lys Pro Asp 625 630 635 640 Glu Arg Ile Ile
Lys Thr Glu Tyr Gly Leu Leu Ile Arg Ser Leu Gln 645 650 655 Lys Lys
Asp Ser Gly Met Tyr Tyr Cys Lys Ala Gln Glu His Thr Phe 660 665 670
Ile His Thr Ile Val Lys Leu Thr Leu Asn Val Ile Glu Asn Glu Gln 675
680 685 Met Glu Asn Thr Gln Arg Ala Glu His Glu Glu Gly Lys Val Lys
Asp 690 695 700 Leu Leu Ala Glu Ser Arg Leu Arg Tyr Lys Asp Tyr Ile
Gln Ile Leu 705 710 715 720 Ser Ser Pro Asn Phe Ser Leu Asp Gln Tyr
Cys Glu Gln Met Trp His 725 730 735 Arg Glu Lys Arg Arg Gln Arg Asn
Lys Gly Gly Pro Lys Trp Lys His 740 745 750 Met Gln Glu Met Lys Lys
Lys Arg Asn Arg Arg His His Arg Asp Leu 755 760 765 Asp Glu Leu Pro
Arg Ala Val Ala Thr 770 775 30775PRTHomo sapiens 30Met Ala Ser Ala
Gly His Ile Ile Thr Leu Leu Leu Trp Gly Tyr Leu 1 5 10 15 Leu Glu
Leu Trp Thr Gly Gly His Thr Ala Asp Thr Thr His Pro Arg 20 25 30
Leu Arg Leu Ser His Lys Glu Leu Leu Asn Leu Asn Arg Thr Ser Ile 35
40 45 Phe His Ser Pro Phe Gly Phe Leu Asp Leu His Thr Met Leu Leu
Asp 50 55 60 Glu Tyr Gln Glu Arg Leu Phe Val Gly Gly Arg Asp Leu
Val Tyr Ser 65 70 75 80 Leu Ser Leu Glu Arg Ile Ser Asp Gly Tyr Lys
Glu Ile His Trp Pro 85 90 95 Ser Thr Ala Leu Lys Met Glu Glu Cys
Ile Met Lys Gly Lys Asp Ala 100 105 110 Gly Glu Cys Ala Asn Tyr Val
Arg Val Leu His His Tyr Asn Arg Thr 115 120 125 His Leu Leu Thr Cys
Gly Thr Gly Ala Phe Asp Pro Val Cys Ala Phe 130 135 140 Ile Arg Val
Gly Tyr His Leu Glu Asp Pro Leu Phe His Leu Glu Ser 145 150 155 160
Pro Arg Ser Glu Arg Gly Arg Gly Arg Cys Pro Phe Asp Pro Ser Ser 165
170 175 Ser Phe Ile Ser Thr Leu Ile Gly Ser Glu Leu Phe Ala Gly Leu
Tyr 180 185 190 Ser Asp Tyr Trp Ser Arg Asp Ala Ala Ile Phe Arg Ser
Met Gly Arg 195 200 205 Leu Ala His Ile Arg Thr Glu His Asp Asp Glu
Arg Leu Leu Lys Glu 210 215 220 Pro Lys Phe Val Gly Ser Tyr Met Ile
Pro Asp Asn Glu Asp Arg Asp 225 230 235 240 Asp Asn Lys Val Tyr Phe
Phe Phe Thr Glu Lys Ala Leu Glu Ala Glu 245 250 255 Asn Asn Ala His
Ala Ile Tyr Thr Arg Val Gly Arg Leu Cys Val Asn 260 265 270 Asp Val
Gly Gly Gln Arg Ile Leu Val Asn Lys Trp Ser Thr Phe Leu 275 280 285
Lys Ala Arg Leu Val Cys Ser Val Pro Gly Met Asn Gly Ile Asp Thr 290
295 300 Tyr Phe Asp Glu Leu Glu Asp Val Phe Leu Leu Pro Thr Arg Asp
His 305 310 315 320 Lys Asn Pro Val Ile Phe Gly Leu Phe Asn Thr Thr
Ser Asn Ile Phe 325 330 335 Arg Gly His Ala Ile Cys Val Tyr His Met
Ser Ser Ile Arg Ala Ala 340 345 350 Phe Asn Gly Pro Tyr Ala His Lys
Glu Gly Pro Glu Tyr His Trp Ser 355 360 365 Val Tyr Glu Gly Lys Val
Pro Tyr Pro Arg Pro Gly Ser Cys Ala Ser 370 375 380 Lys Val Asn Gly
Gly Arg Tyr Gly Thr Thr Lys Asp Tyr Pro Asp Asp 385 390 395 400 Ala
Ile Arg Phe Ala Arg Ser His Pro Leu Met Tyr Gln Ala Ile Lys 405 410
415 Pro Ala His Lys Lys Pro Ile Leu Val Lys Thr Asp Gly Lys Tyr Asn
420 425 430 Leu Lys Gln Ile Ala Val Asp Arg Val Glu Ala Glu Asp Gly
Gln Tyr 435 440 445 Asp Val Leu Phe Ile Gly Thr Asp Asn Gly Ile Val
Leu Lys Val Ile 450 455 460 Thr Ile Tyr Asn Gln Glu Met Glu Ser Met
Glu Glu Val Ile Leu Glu 465 470 475 480 Glu Leu Gln Ile Phe Lys Asp
Pro Val Pro Ile Ile Ser Met Glu Ile 485 490 495 Ser Ser Lys Arg Gln
Gln Leu Tyr Ile Gly Ser Ala Ser Ala Val Ala 500 505 510 Gln Val Arg
Phe His His Cys
Asp Met Tyr Gly Ser Ala Cys Ala Asp 515 520 525 Cys Cys Leu Ala Arg
Asp Pro Tyr Cys Ala Trp Asp Gly Ile Ser Cys 530 535 540 Ser Arg Tyr
Tyr Pro Thr Gly Thr His Ala Lys Arg Arg Phe Arg Arg 545 550 555 560
Gln Asp Val Arg His Gly Asn Ala Ala Gln Gln Cys Phe Gly Gln Gln 565
570 575 Phe Val Gly Asp Ala Leu Asp Lys Thr Glu Glu His Leu Ala Tyr
Gly 580 585 590 Ile Glu Asn Asn Ser Thr Leu Leu Glu Cys Thr Pro Arg
Ser Leu Gln 595 600 605 Ala Lys Val Ile Trp Phe Val Gln Lys Gly Arg
Glu Thr Arg Lys Glu 610 615 620 Glu Val Lys Thr Asp Asp Arg Val Val
Lys Met Asp Leu Gly Leu Leu 625 630 635 640 Phe Leu Arg Leu His Lys
Ser Asp Ala Gly Thr Tyr Phe Cys Gln Thr 645 650 655 Val Glu His Ser
Phe Val His Thr Val Arg Lys Ile Thr Leu Glu Val 660 665 670 Val Glu
Glu Glu Lys Val Glu Asp Met Phe Asn Lys Asp Asp Glu Glu 675 680 685
Asp Arg His His Arg Met Pro Cys Pro Ala Gln Ser Ser Ile Ser Gln 690
695 700 Gly Ala Lys Pro Trp Tyr Lys Glu Phe Leu Gln Leu Ile Gly Tyr
Ser 705 710 715 720 Asn Phe Gln Arg Val Glu Glu Tyr Cys Glu Lys Val
Trp Cys Thr Asp 725 730 735 Arg Lys Arg Lys Lys Leu Lys Met Ser Pro
Ser Lys Trp Lys Tyr Ala 740 745 750 Asn Pro Gln Glu Lys Lys Leu Arg
Ser Lys Pro Glu His Tyr Arg Leu 755 760 765 Pro Arg His Thr Leu Asp
Ser 770 775 31785PRTHomo sapiens 31Met Leu Val Ala Gly Leu Leu Leu
Trp Ala Ser Leu Leu Thr Gly Ala 1 5 10 15 Trp Pro Ser Phe Pro Thr
Gln Asp His Leu Pro Ala Thr Pro Arg Val 20 25 30 Arg Leu Ser Phe
Lys Glu Leu Lys Ala Thr Gly Thr Ala His Phe Phe 35 40 45 Asn Phe
Leu Leu Asn Thr Thr Asp Tyr Arg Ile Leu Leu Lys Asp Glu 50 55 60
Asp His Asp Arg Met Tyr Val Gly Ser Lys Asp Tyr Val Leu Ser Leu 65
70 75 80 Asp Leu His Asp Ile Asn Arg Glu Pro Leu Ile Ile His Trp
Ala Ala 85 90 95 Ser Pro Gln Arg Ile Glu Glu Cys Val Leu Ser Gly
Lys Asp Val Asn 100 105 110 Gly Glu Cys Gly Asn Phe Val Arg Leu Ile
Gln Pro Trp Asn Arg Thr 115 120 125 His Leu Tyr Val Cys Gly Thr Gly
Ala Tyr Asn Pro Met Cys Thr Tyr 130 135 140 Val Asn Arg Gly Arg Arg
Ala Gln Ala Thr Pro Trp Thr Gln Thr Gln 145 150 155 160 Ala Val Arg
Gly Arg Gly Ser Arg Ala Thr Asp Gly Ala Leu Arg Pro 165 170 175 Met
Pro Thr Ala Pro Arg Gln Asp Tyr Ile Phe Tyr Leu Glu Pro Glu 180 185
190 Arg Leu Glu Ser Gly Lys Gly Lys Cys Pro Tyr Asp Pro Lys Leu Asp
195 200 205 Thr Ala Ser Ala Leu Ile Asn Glu Glu Leu Tyr Ala Gly Val
Tyr Ile 210 215 220 Asp Phe Met Gly Thr Asp Ala Ala Ile Phe Arg Thr
Leu Gly Lys Gln 225 230 235 240 Thr Ala Met Arg Thr Asp Gln Tyr Asn
Ser Arg Trp Leu Asn Asp Pro 245 250 255 Ser Phe Ile His Ala Glu Leu
Ile Pro Asp Ser Ala Glu Arg Asn Asp 260 265 270 Asp Lys Leu Tyr Phe
Phe Phe Arg Glu Arg Ser Ala Glu Ala Pro Gln 275 280 285 Ser Pro Ala
Val Tyr Ala Arg Ile Gly Arg Ile Cys Leu Asn Asp Asp 290 295 300 Gly
Gly His Cys Cys Leu Val Asn Lys Trp Ser Thr Phe Leu Lys Ala 305 310
315 320 Arg Leu Val Cys Ser Val Pro Gly Glu Asp Gly Ile Glu Thr His
Phe 325 330 335 Asp Glu Leu Gln Asp Val Phe Val Gln Gln Thr Gln Asp
Val Arg Asn 340 345 350 Pro Val Ile Tyr Ala Val Phe Thr Ser Ser Gly
Ser Val Phe Arg Gly 355 360 365 Ser Ala Val Cys Val Tyr Ser Met Ala
Asp Ile Arg Met Val Phe Asn 370 375 380 Gly Pro Phe Ala His Lys Glu
Gly Pro Asn Tyr Gln Trp Met Pro Phe 385 390 395 400 Ser Gly Lys Met
Pro Tyr Pro Arg Pro Gly Thr Cys Pro Gly Gly Thr 405 410 415 Phe Thr
Pro Ser Met Lys Ser Thr Lys Asp Tyr Pro Asp Glu Val Ile 420 425 430
Asn Phe Met Arg Ser His Pro Leu Met Tyr Gln Ala Val Tyr Pro Leu 435
440 445 Gln Arg Arg Pro Leu Val Val Arg Thr Gly Ala Pro Tyr Arg Leu
Thr 450 455 460 Thr Ile Ala Val Asp Gln Val Asp Ala Ala Asp Gly Arg
Tyr Glu Val 465 470 475 480 Leu Phe Leu Gly Thr Asp Arg Gly Thr Val
Gln Lys Val Ile Val Leu 485 490 495 Pro Lys Asp Asp Gln Glu Leu Glu
Glu Leu Met Leu Glu Glu Val Glu 500 505 510 Val Phe Lys Asp Pro Ala
Pro Val Lys Thr Met Thr Ile Ser Ser Lys 515 520 525 Arg Gln Gln Leu
Tyr Val Ala Ser Ala Val Gly Val Thr His Leu Ser 530 535 540 Leu His
Arg Cys Gln Ala Tyr Gly Ala Ala Cys Ala Asp Cys Cys Leu 545 550 555
560 Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Gln Ala Cys Ser Arg Tyr
565 570 575 Thr Ala Ser Ser Lys Arg Arg Ser Arg Arg Gln Asp Val Arg
His Gly 580 585 590 Asn Pro Ile Arg Gln Cys Arg Gly Phe Asn Ser Asn
Ala Asn Lys Asn 595 600 605 Ala Val Glu Ser Val Gln Tyr Gly Val Ala
Gly Ser Ala Ala Phe Leu 610 615 620 Glu Cys Gln Pro Arg Ser Pro Gln
Ala Thr Val Lys Trp Leu Phe Gln 625 630 635 640 Arg Asp Pro Gly Asp
Arg Arg Arg Glu Ile Arg Ala Glu Asp Arg Phe 645 650 655 Leu Arg Thr
Glu Gln Gly Leu Leu Leu Arg Ala Leu Gln Leu Ser Asp 660 665 670 Arg
Gly Leu Tyr Ser Cys Thr Ala Thr Glu Asn Asn Phe Lys His Val 675 680
685 Val Thr Arg Val Gln Leu His Val Leu Gly Arg Asp Ala Val His Ala
690 695 700 Ala Leu Phe Pro Pro Leu Ser Met Ser Ala Pro Pro Pro Pro
Gly Ala 705 710 715 720 Gly Pro Pro Thr Pro Pro Tyr Gln Glu Leu Ala
Gln Leu Leu Ala Gln 725 730 735 Pro Glu Val Gly Leu Ile His Gln Tyr
Cys Gln Gly Tyr Trp Arg His 740 745 750 Val Pro Pro Ser Pro Arg Glu
Ala Pro Gly Ala Pro Arg Ser Pro Glu 755 760 765 Pro Gln Asp Gln Lys
Lys Pro Arg Asn Arg Arg His His Pro Pro Asp 770 775 780 Thr 785
32782PRTHomo sapiens 32Met Ala Pro Ser Ala Trp Ala Ile Cys Trp Leu
Leu Gly Gly Leu Leu 1 5 10 15 Leu His Gly Gly Ser Ser Gly Pro Ser
Pro Gly Pro Ser Val Pro Arg 20 25 30 Leu Arg Leu Ser Tyr Arg Asp
Leu Leu Ser Ala Asn Arg Ser Ala Ile 35 40 45 Phe Leu Gly Pro Gln
Gly Ser Leu Asn Leu Gln Ala Met Tyr Leu Asp 50 55 60 Glu Tyr Arg
Asp Arg Leu Phe Leu Gly Gly Leu Asp Ala Leu Tyr Ser 65 70 75 80 Leu
Arg Leu Asp Gln Ala Trp Pro Asp Pro Arg Glu Val Leu Trp Pro 85 90
95 Pro Gln Pro Gly Gln Arg Glu Glu Cys Val Arg Lys Gly Arg Asp Pro
100 105 110 Leu Thr Glu Cys Ala Asn Phe Val Arg Val Leu Gln Pro His
Asn Arg 115 120 125 Thr His Leu Leu Ala Cys Gly Thr Gly Ala Phe Gln
Pro Thr Cys Ala 130 135 140 Leu Ile Thr Val Gly His Arg Gly Glu His
Val Leu His Leu Glu Pro 145 150 155 160 Gly Ser Val Glu Ser Gly Arg
Gly Arg Cys Pro His Glu Pro Ser Arg 165 170 175 Pro Phe Ala Ser Thr
Phe Ile Asp Gly Glu Leu Tyr Thr Gly Leu Thr 180 185 190 Ala Asp Phe
Leu Gly Arg Glu Ala Met Ile Phe Arg Ser Gly Gly Pro 195 200 205 Arg
Pro Ala Leu Arg Ser Asp Ser Asp Gln Ser Leu Leu His Asp Pro 210 215
220 Arg Phe Val Met Ala Ala Arg Ile Pro Glu Asn Ser Asp Gln Asp Asn
225 230 235 240 Asp Lys Val Tyr Phe Phe Phe Ser Glu Thr Val Pro Ser
Pro Asp Gly 245 250 255 Gly Ser Asn His Val Thr Val Ser Arg Val Gly
Arg Val Cys Val Asn 260 265 270 Asp Ala Gly Gly Gln Arg Val Leu Val
Asn Lys Trp Ser Thr Phe Leu 275 280 285 Lys Ala Arg Leu Val Cys Ser
Val Pro Gly Pro Gly Gly Ala Glu Thr 290 295 300 His Phe Asp Gln Leu
Glu Asp Val Phe Leu Leu Trp Pro Lys Ala Gly 305 310 315 320 Lys Ser
Leu Glu Val Tyr Ala Leu Phe Ser Thr Val Ser Ala Val Phe 325 330 335
Gln Gly Phe Ala Val Cys Val Tyr His Met Ala Asp Ile Trp Glu Val 340
345 350 Phe Asn Gly Pro Phe Ala His Arg Asp Gly Pro Gln His Gln Trp
Gly 355 360 365 Pro Tyr Gly Gly Lys Val Pro Phe Pro Arg Pro Gly Val
Cys Pro Ser 370 375 380 Lys Met Thr Ala Gln Pro Gly Arg Pro Phe Gly
Ser Thr Lys Asp Tyr 385 390 395 400 Pro Asp Glu Val Leu Gln Phe Ala
Arg Ala His Pro Leu Met Phe Trp 405 410 415 Pro Val Arg Pro Arg His
Gly Arg Pro Val Leu Val Lys Thr His Leu 420 425 430 Ala Gln Gln Leu
His Gln Ile Val Val Asp Arg Val Glu Ala Glu Asp 435 440 445 Gly Thr
Tyr Asp Val Ile Phe Leu Gly Thr Asp Ser Gly Ser Val Leu 450 455 460
Lys Val Ile Ala Leu Gln Ala Gly Gly Ser Ala Glu Pro Glu Glu Val 465
470 475 480 Val Leu Glu Glu Leu Gln Val Phe Lys Val Pro Thr Pro Ile
Thr Glu 485 490 495 Met Glu Ile Ser Val Lys Arg Gln Met Leu Tyr Val
Gly Ser Arg Leu 500 505 510 Gly Val Ala Gln Leu Arg Leu His Gln Cys
Glu Thr Tyr Gly Thr Ala 515 520 525 Cys Ala Glu Cys Cys Leu Ala Arg
Asp Pro Tyr Cys Ala Trp Asp Gly 530 535 540 Ala Ser Cys Thr His Tyr
Arg Pro Ser Leu Gly Lys Arg Arg Phe Arg 545 550 555 560 Arg Gln Asp
Ile Arg His Gly Asn Pro Ala Leu Gln Cys Leu Gly Gln 565 570 575 Ser
Gln Glu Glu Glu Ala Val Gly Leu Val Ala Ala Thr Met Val Tyr 580 585
590 Gly Thr Glu His Asn Ser Thr Phe Leu Glu Cys Leu Pro Lys Ser Pro
595 600 605 Gln Ala Ala Val Arg Trp Leu Leu Gln Arg Pro Gly Asp Glu
Gly Pro 610 615 620 Asp Gln Val Lys Thr Asp Glu Arg Val Leu His Thr
Glu Arg Gly Leu 625 630 635 640 Leu Phe Arg Arg Leu Ser Arg Phe Asp
Ala Gly Thr Tyr Thr Cys Thr 645 650 655 Thr Leu Glu His Gly Phe Ser
Gln Thr Val Val Arg Leu Ala Leu Val 660 665 670 Val Ile Val Ala Ser
Gln Leu Asp Asn Leu Phe Pro Pro Glu Pro Lys 675 680 685 Pro Glu Glu
Pro Pro Ala Arg Gly Gly Leu Ala Ser Thr Pro Pro Lys 690 695 700 Ala
Trp Tyr Lys Asp Ile Leu Gln Leu Ile Gly Phe Ala Asn Leu Pro 705 710
715 720 Arg Val Asp Glu Tyr Cys Glu Arg Val Trp Cys Arg Gly Thr Thr
Glu 725 730 735 Cys Ser Gly Cys Phe Arg Ser Arg Ser Arg Gly Lys Gln
Ala Arg Gly 740 745 750 Lys Ser Trp Ala Gly Leu Glu Leu Gly Lys Lys
Met Lys Ser Arg Val 755 760 765 His Ala Glu His Asn Arg Thr Pro Arg
Glu Val Glu Ala Thr 770 775 780 332334DNAHomo sapiens 33atgaatgcta
ataaagatga aagacttaaa gccagaagcc aagattttca cctttttcct 60gctttgatga
tgctaagcat gaccatgttg tttcttccag tcactggcac tttgaagcaa
120aatattccaa gactcaagct aacctacaaa gacttgctgc tttcaaatag
ctgtattccc 180tttttgggtt catcagaagg actggatttt caaactcttc
tcttagatga ggaaagaggc 240aggctgctct tgggagccaa agaccacatc
tttctactca gtctggttga cttaaacaaa 300aattttaaga agatttattg
gcctgctgca aaggaacggg tggaattatg taaattagct 360gggaaagatg
ccaatacaga atgtgcaaat ttcatcagag tacttcagcc ctataacaaa
420actcacatat atgtgtgtgg aactggagca tttcatccaa tatgtgggta
tattgatctt 480ggagtctaca aggaggatat tatattcaaa ctagacacac
ataatttgga gtctggcaga 540ctgaaatgtc ctttcgatcc tcagcagcct
tttgcttcag taatgacaga tgagtacctc 600tactctggaa cagcttctga
tttccttggc aaagatactg cattcactcg atcccttggg 660cctactcatg
accaccacta catcagaact gacatttcag agcactactg gctcaatgga
720gcaaaattta ttggaacttt cttcatacca gacacctaca atccagatga
tgataaaata 780tatttcttct ttcgtgaatc atctcaagaa ggcagtacct
ccgataaaac catcctttct 840cgagttggaa gagtttgtaa gaatgatgta
ggaggacaac gcagcctgat aaacaagtgg 900acgacttttc ttaaggccag
actgatttgc tcaattcctg gaagtgatgg ggcagatact 960tactttgatg
agcttcaaga tatttattta ctccccacaa gagatgaaag aaatcctgta
1020gtatatggag tctttactac aaccagctcc atcttcaaag gctctgctgt
ttgtgtgtat 1080agcatggctg acatcagagc agtttttaat ggtccatatg
ctcataagga aagtgcagac 1140catcgttggg tgcagtatga tgggagaatt
ccttatccac ggcctggtac atgtccaagc 1200aaaacctatg acccactgat
taagtccacc cgagattttc cagatgatgt catcagtttc 1260ataaagcggc
actctgtgat gtataagtcc gtatacccag ttgcaggagg accaacgttc
1320aagagaatca atgtggatta cagactgaca cagatagtgg tggatcatgt
cattgcagaa 1380gatggccagt acgatgtaat gtttcttgga acagacattg
gaactgtcct caaagttgtc 1440agcatttcaa aggaaaagtg gaatatggaa
gaggtagtgc tggaggagtt gcagatattc 1500aagcactcat caatcatctt
gaacatggaa ttgtctctga agcagcaaca attgtacatt 1560ggttcccgag
atggattggt tcagctctcc ttgcacagat gcgacactta tgggaaagct
1620tgcgcagact gttgtcttgc cagagacccc tactgtgcct gggatggaaa
tgcatgctct 1680cgatatgctc ctacttctaa aaggaaagct aagaaacaag
atgtaaaata tggcgaccca 1740atcacccagt gctgggacat cgaagacagc
attagtcatg aaactgctga tgaaaaggtg 1800atttttggca ttgaatttaa
ctcaaccttt ctggaatgta tacctaaatc ccaacaagca 1860actattaaat
ggtatatcca gaggtcaggg gatgagcatc gagaggagtt gaagcccgat
1920gaaagaatca tcaaaacgga atatgggcta ctgattcgaa gtttgcagaa
gaaggattct 1980gggatgtatt actgcaaagc ccaggagcac actttcatcc
acaccatagt gaagctgact 2040ttgaatgtca ttgagaatga acagatggaa
aatacccaga gggcagagca tgaggagggg 2100aaggtcaagg atctattggc
tgagtcacgg ttgagataca aagactacat ccaaatcctt 2160agcagcccaa
acttcagcct cgaccagtac tgcgaacaga tgtggcacag ggagaagcgg
2220agacagagaa acaagggggg cccaaagtgg aagcacatgc aggaaatgaa
gaagaaacga 2280aatcgaagac atcacagaga cctggatgag ctccctagag
ctgtagccac gtag 2334342328DNAHomo sapiens 34atggcatccg cggggcacat
tatcaccttg ctcctgtggg gttacttact ggagctttgg 60acaggaggtc atacagctga
tactacccac ccccggttac gcctgtcaca taaagagctc 120ttgaatctga
acagaacatc aatatttcat agcccttttg gatttcttga tctccataca
180atgctgctgg atgaatatca agagaggctc ttcgtgggag gcagggacct
tgtatattcc 240ctcagcttgg agagaatcag tgacggctat aaagagatac
actggccgag tacagctcta 300aaaatggaag aatgcataat gaagggaaaa
gatgcgggtg aatgtgcaaa ttatgttcgg 360gttttgcatc actataacag
gacacacctt ctgacctgtg gtactggagc ttttgatcca 420gtttgtgcct
tcatcagagt tggatatcat ttggaggatc ctctgtttca cctggaatca
480cccagatctg agagaggaag gggcagatgt ccttttgacc ccagctcctc
cttcatctcc 540actttaattg gtagtgaatt gtttgctgga ctctacagtg
actactggag cagagacgct 600gcgatcttcc gcagcatggg gcgactggcc
catatccgca ctgagcatga cgatgagcgt 660ctgttgaaag aaccaaaatt
tgtaggttca tacatgattc
ctgacaatga agacagagat 720gacaacaaag tatatttctt ttttactgag
aaggcactgg aggcagaaaa caatgctcac 780gcaatttaca ccagggtcgg
gcgactctgt gtgaatgatg taggagggca gagaatactg 840gtgaataagt
ggagcacttt cctaaaagcg agactcgttt gctcagtacc aggaatgaat
900ggaattgaca catattttga tgaattagag gacgtttttt tgctacctac
cagagatcat 960aagaatccag tgatatttgg actctttaac actaccagta
atatttttcg agggcatgct 1020atatgtgtct atcacatgtc tagcattcgg
gcagccttca acggaccata tgcacataag 1080gaaggacctg aataccactg
gtcagtctat gaaggaaaag tcccttatcc aaggcctggt 1140tcttgtgcca
gcaaagtaaa tggagggaga tacggaacca ccaaggacta tcctgatgat
1200gccatccgat ttgcaagaag tcatccacta atgtaccagg ccataaaacc
tgcccataaa 1260aaaccaatat tggtaaaaac agatggaaaa tataacctga
aacaaatagc agtagatcga 1320gtggaagctg aggatggcca atatgacgtc
ttgtttattg ggacagataa tggaattgtg 1380ctgaaagtaa tcacaattta
caaccaagaa atggaatcaa tggaagaagt aattctagaa 1440gaacttcaga
tattcaagga tccagttcct attatttcta tggagatttc ttcaaaacgg
1500caacagctgt atattggatc tgcttctgct gtggctcaag tcagattcca
tcactgtgac 1560atgtatggaa gtgcttgtgc tgactgctgc ctggctcgag
acccttactg tgcctgggat 1620ggcatatcct gctcccggta ttacccaaca
ggcacacatg caaaaaggaa gttcaagaaa 1680caagatgttc gacatggaaa
tgcagctcag cagtgctttg gacaacagtt tgttggggat 1740gctttggata
agactgaaga acatctggct tatggcatag agaacaacag tactttgctg
1800gaatgtaccc cacgatcttt acaagcgaaa gttatctggt ttgtacagaa
aggacgtgag 1860acaagaaaag aggaggtgaa gacagatgac agagtggtta
agatggacct tggtttactc 1920ttcctaaggt tacacaaatc agatgctggg
acctattttt gccagacagt agagcatagc 1980tttgtccata cggtccgtaa
aatcaccttg gaggtagtgg aagaggagaa agtcgaggat 2040atgtttaaca
aggacgatga ggaggacagg catcacagga tgccttgtcc tgctcagagt
2100agcatctcgc agggagcaaa accatggtac aaggaattct tgcagctgat
cggttatagc 2160aacttccaga gagtggaaga atactgcgag aaagtatggt
gcacagatag aaagaggaaa 2220aagcttaaaa tgtcaccctc caagtggaag
tatgccaacc ctcaggaaaa gaagctccgt 2280tccaaacctg agcattaccg
cctgcccagg cacacgctgg actcctga 2328352349DNAHomo sapiens
35atggccccct cggcctgggc catttgctgg ctgctagggg gcctcctgct ccatgggggt
60agctctggcc ccagccccgg ccccagtgtg ccccgcctgc ggctctccta ccgagacctc
120ctgtctgcca accgctctgc catctttctg ggcccccagg gctccctgaa
cctccaggcc 180atgtacctag atgagtaccg agaccgcctc tttctgggtg
gcctggacgc cctctactct 240ctgcggctgg accaggcatg gccagatccc
cgggaggtcc tgtggccacc gcagccagga 300cagagggagg agtgtgttcg
aaagggaaga gatcctttga cagagtgcgc caacttcgtg 360cgggtgctac
agcctcacaa ccggacccac ctgctagcct gtggcactgg ggccttccag
420cccacctgtg ccctcatcac agttggccac cgtggggagc atgtgctcca
cctggagcct 480ggcagtgtgg aaagtggccg ggggcggtgc cctcacgagc
ccagccgtcc ctttgccagc 540accttcatag acggggagct gtacacgggt
ctcactgctg acttcctggg gcgagaggcc 600atgatcttcc gaagtggagg
tcctcggcca gctctgcgtt ccgactctga ccagagtctc 660ttgcacgacc
cccggtttgt gatggccgcc cggatccctg agaactctga ccaggacaat
720gacaaggtgt acttcttctt ctcggagacg gtcccctcgc ccgatggtgg
ctcgaaccat 780gtcactgtca gccgcgtggg ccgcgtctgc gtgaatgatg
ctgggggcca gcgggtgctg 840gtgaacaaat ggagcacttt cctcaaggcc
aggctggtct gctcggtgcc cggccctggt 900ggtgccgaga cccactttga
ccagctagag gatgtgttcc tgctgtggcc caaggccggg 960aagagcctcg
aggtgtacgc gctgttcagc accgtcagtg ccgtgttcca gggcttcgcc
1020gtctgtgtgt accacatggc agacatctgg gaggttttca acgggccctt
tgcccaccga 1080gatgggcctc agcaccagtg ggggccctat gggggcaagg
tgcccttccc tcgccctggc 1140gtgtgcccca gcaagatgac cgcacagcca
ggacggcctt ttggcagcac caaggactac 1200ccagatgagg tgctgcagtt
tgcccgagcc caccccctca tgttctggcc tgtgcggcct 1260cgacatggcc
gccctgtcct tgtcaagacc cacctggccc agcagctaca ccagatcgtg
1320gtggaccgcg tggaggcaga ggatgggacc tacgatgtca ttttcctggg
gactgactca 1380gggtctgtgc tcaaagtcat cgctctccag gcagggggct
cagctgaacc tgaggaagtg 1440gttctggagg agctccaggt gtttaaggtg
ccaacaccta tcaccgaaat ggagatctct 1500gtcaaaaggc aaatgctata
cgtgggctct cggctgggtg tggcccagct gcggctgcac 1560caatgtgaga
cttacggcac tgcctgtgca gagtgctgcc tggcccggga cccatactgt
1620gcctgggatg gtgcctcctg tacccactac cgccccagcc ttggcaagcg
caaattcaaa 1680aagcaggaca tccggcacgg caaccctgcc ctgcagtgcc
tgggccagag ccaggaagaa 1740gaggcagtgg gacttgtggc agccaccatg
gtctacggca cggagcacaa tagcaccttc 1800ctggagtgcc tgcccaagtc
tccccaggct gctgtgcgct ggctcttgca gaggccaggg 1860gatgaggggc
ctgaccaggt gaagacggac gagcgagtct tgcacacgga gcgggggctg
1920ctgttccgca ggcttagccg tttcgatgcg ggcacctaca cctgcaccac
tctggagcat 1980ggcttctccc agactgtggt ccgcctggct ctggtggtga
ttgtggcctc acagctggac 2040aacctgttcc ctccggagcc aaagccagag
gagcccccag cccggggagg cctggcttcc 2100accccaccca aggcctggta
caaggacatc ctgcagctca ttggcttcgc caacctgccc 2160cgggtggatg
agtactgtga gcgcgtgtgg tgcaggggca ccacggaatg ctcaggctgc
2220ttccggagcc ggagccgggg caagcaggcc aggggcaaga gctgggcagg
gctggagcta 2280ggcaagaaga tgaagagccg ggtgcatgcc gagcacaatc
ggacgccccg ggaggtggag 2340gccacgtag 234936777PRTHomo sapiens 36Met
Asn Ala Asn Lys Asp Glu Arg Leu Lys Ala Arg Ser Gln Asp Phe 1 5 10
15 His Leu Phe Pro Ala Leu Met Met Leu Ser Met Thr Met Leu Phe Leu
20 25 30 Pro Val Thr Gly Thr Leu Lys Gln Asn Ile Pro Arg Leu Lys
Leu Thr 35 40 45 Tyr Lys Asp Leu Leu Leu Ser Asn Ser Cys Ile Pro
Phe Leu Gly Ser 50 55 60 Ser Glu Gly Leu Asp Phe Gln Thr Leu Leu
Leu Asp Glu Glu Arg Gly 65 70 75 80 Arg Leu Leu Leu Gly Ala Lys Asp
His Ile Phe Leu Leu Ser Leu Val 85 90 95 Asp Leu Asn Lys Asn Phe
Lys Lys Ile Tyr Trp Pro Ala Ala Lys Glu 100 105 110 Arg Val Glu Leu
Cys Lys Leu Ala Gly Lys Asp Ala Asn Thr Glu Cys 115 120 125 Ala Asn
Phe Ile Arg Val Leu Gln Pro Tyr Asn Lys Thr His Ile Tyr 130 135 140
Val Cys Gly Thr Gly Ala Phe His Pro Ile Cys Gly Tyr Ile Asp Leu 145
150 155 160 Gly Val Tyr Lys Glu Asp Ile Ile Phe Lys Leu Asp Thr His
Asn Leu 165 170 175 Glu Ser Gly Arg Leu Lys Cys Pro Phe Asp Pro Gln
Gln Pro Phe Ala 180 185 190 Ser Val Met Thr Asp Glu Tyr Leu Tyr Ser
Gly Thr Ala Ser Asp Phe 195 200 205 Leu Gly Lys Asp Thr Ala Phe Thr
Arg Ser Leu Gly Pro Thr His Asp 210 215 220 His His Tyr Ile Arg Thr
Asp Ile Ser Glu His Tyr Trp Leu Asn Gly 225 230 235 240 Ala Lys Phe
Ile Gly Thr Phe Phe Ile Pro Asp Thr Tyr Asn Pro Asp 245 250 255 Asp
Asp Lys Ile Tyr Phe Phe Phe Arg Glu Ser Ser Gln Glu Gly Ser 260 265
270 Thr Ser Asp Lys Thr Ile Leu Ser Arg Val Gly Arg Val Cys Lys Asn
275 280 285 Asp Val Gly Gly Gln Arg Ser Leu Ile Asn Lys Trp Thr Thr
Phe Leu 290 295 300 Lys Ala Arg Leu Ile Cys Ser Ile Pro Gly Ser Asp
Gly Ala Asp Thr 305 310 315 320 Tyr Phe Asp Glu Leu Gln Asp Ile Tyr
Leu Leu Pro Thr Arg Asp Glu 325 330 335 Arg Asn Pro Val Val Tyr Gly
Val Phe Thr Thr Thr Ser Ser Ile Phe 340 345 350 Lys Gly Ser Ala Val
Cys Val Tyr Ser Met Ala Asp Ile Arg Ala Val 355 360 365 Phe Asn Gly
Pro Tyr Ala His Lys Glu Ser Ala Asp His Arg Trp Val 370 375 380 Gln
Tyr Asp Gly Arg Ile Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser 385 390
395 400 Lys Thr Tyr Asp Pro Leu Ile Lys Ser Thr Arg Asp Phe Pro Asp
Asp 405 410 415 Val Ile Ser Phe Ile Lys Arg His Ser Val Met Tyr Lys
Ser Val Tyr 420 425 430 Pro Val Ala Gly Gly Pro Thr Phe Lys Arg Ile
Asn Val Asp Tyr Arg 435 440 445 Leu Thr Gln Ile Val Val Asp His Val
Ile Ala Glu Asp Gly Gln Tyr 450 455 460 Asp Val Met Phe Leu Gly Thr
Asp Ile Gly Thr Val Leu Lys Val Val 465 470 475 480 Ser Ile Ser Lys
Glu Lys Trp Asn Met Glu Glu Val Val Leu Glu Glu 485 490 495 Leu Gln
Ile Phe Lys His Ser Ser Ile Ile Leu Asn Met Glu Leu Ser 500 505 510
Leu Lys Gln Gln Gln Leu Tyr Ile Gly Ser Arg Asp Gly Leu Val Gln 515
520 525 Leu Ser Leu His Arg Cys Asp Thr Tyr Gly Lys Ala Cys Ala Asp
Cys 530 535 540 Cys Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Asn
Ala Cys Ser 545 550 555 560 Arg Tyr Ala Pro Thr Ser Lys Arg Lys Ala
Lys Lys Gln Asp Val Lys 565 570 575 Tyr Gly Asp Pro Ile Thr Gln Cys
Trp Asp Ile Glu Asp Ser Ile Ser 580 585 590 His Glu Thr Ala Asp Glu
Lys Val Ile Phe Gly Ile Glu Phe Asn Ser 595 600 605 Thr Phe Leu Glu
Cys Ile Pro Lys Ser Gln Gln Ala Thr Ile Lys Trp 610 615 620 Tyr Ile
Gln Arg Ser Gly Asp Glu His Arg Glu Glu Leu Lys Pro Asp 625 630 635
640 Glu Arg Ile Ile Lys Thr Glu Tyr Gly Leu Leu Ile Arg Ser Leu Gln
645 650 655 Lys Lys Asp Ser Gly Met Tyr Tyr Cys Lys Ala Gln Glu His
Thr Phe 660 665 670 Ile His Thr Ile Val Lys Leu Thr Leu Asn Val Ile
Glu Asn Glu Gln 675 680 685 Met Glu Asn Thr Gln Arg Ala Glu His Glu
Glu Gly Lys Val Lys Asp 690 695 700 Leu Leu Ala Glu Ser Arg Leu Arg
Tyr Lys Asp Tyr Ile Gln Ile Leu 705 710 715 720 Ser Ser Pro Asn Phe
Ser Leu Asp Gln Tyr Cys Glu Gln Met Trp His 725 730 735 Arg Glu Lys
Arg Arg Gln Arg Asn Lys Gly Gly Pro Lys Trp Lys His 740 745 750 Met
Gln Glu Met Lys Lys Lys Arg Asn Arg Arg His His Arg Asp Leu 755 760
765 Asp Glu Leu Pro Arg Ala Val Ala Thr 770 775 37775PRTHomo
sapiens 37Met Ala Ser Ala Gly His Ile Ile Thr Leu Leu Leu Trp Gly
Tyr Leu 1 5 10 15 Leu Glu Leu Trp Thr Gly Gly His Thr Ala Asp Thr
Thr His Pro Arg 20 25 30 Leu Arg Leu Ser His Lys Glu Leu Leu Asn
Leu Asn Arg Thr Ser Ile 35 40 45 Phe His Ser Pro Phe Gly Phe Leu
Asp Leu His Thr Met Leu Leu Asp 50 55 60 Glu Tyr Gln Glu Arg Leu
Phe Val Gly Gly Arg Asp Leu Val Tyr Ser 65 70 75 80 Leu Ser Leu Glu
Arg Ile Ser Asp Gly Tyr Lys Glu Ile His Trp Pro 85 90 95 Ser Thr
Ala Leu Lys Met Glu Glu Cys Ile Met Lys Gly Lys Asp Ala 100 105 110
Gly Glu Cys Ala Asn Tyr Val Arg Val Leu His His Tyr Asn Arg Thr 115
120 125 His Leu Leu Thr Cys Gly Thr Gly Ala Phe Asp Pro Val Cys Ala
Phe 130 135 140 Ile Arg Val Gly Tyr His Leu Glu Asp Pro Leu Phe His
Leu Glu Ser 145 150 155 160 Pro Arg Ser Glu Arg Gly Arg Gly Arg Cys
Pro Phe Asp Pro Ser Ser 165 170 175 Ser Phe Ile Ser Thr Leu Ile Gly
Ser Glu Leu Phe Ala Gly Leu Tyr 180 185 190 Ser Asp Tyr Trp Ser Arg
Asp Ala Ala Ile Phe Arg Ser Met Gly Arg 195 200 205 Leu Ala His Ile
Arg Thr Glu His Asp Asp Glu Arg Leu Leu Lys Glu 210 215 220 Pro Lys
Phe Val Gly Ser Tyr Met Ile Pro Asp Asn Glu Asp Arg Asp 225 230 235
240 Asp Asn Lys Val Tyr Phe Phe Phe Thr Glu Lys Ala Leu Glu Ala Glu
245 250 255 Asn Asn Ala His Ala Ile Tyr Thr Arg Val Gly Arg Leu Cys
Val Asn 260 265 270 Asp Val Gly Gly Gln Arg Ile Leu Val Asn Lys Trp
Ser Thr Phe Leu 275 280 285 Lys Ala Arg Leu Val Cys Ser Val Pro Gly
Met Asn Gly Ile Asp Thr 290 295 300 Tyr Phe Asp Glu Leu Glu Asp Val
Phe Leu Leu Pro Thr Arg Asp His 305 310 315 320 Lys Asn Pro Val Ile
Phe Gly Leu Phe Asn Thr Thr Ser Asn Ile Phe 325 330 335 Arg Gly His
Ala Ile Cys Val Tyr His Met Ser Ser Ile Arg Ala Ala 340 345 350 Phe
Asn Gly Pro Tyr Ala His Lys Glu Gly Pro Glu Tyr His Trp Ser 355 360
365 Val Tyr Glu Gly Lys Val Pro Tyr Pro Arg Pro Gly Ser Cys Ala Ser
370 375 380 Lys Val Asn Gly Gly Arg Tyr Gly Thr Thr Lys Asp Tyr Pro
Asp Asp 385 390 395 400 Ala Ile Arg Phe Ala Arg Ser His Pro Leu Met
Tyr Gln Ala Ile Lys 405 410 415 Pro Ala His Lys Lys Pro Ile Leu Val
Lys Thr Asp Gly Lys Tyr Asn 420 425 430 Leu Lys Gln Ile Ala Val Asp
Arg Val Glu Ala Glu Asp Gly Gln Tyr 435 440 445 Asp Val Leu Phe Ile
Gly Thr Asp Asn Gly Ile Val Leu Lys Val Ile 450 455 460 Thr Ile Tyr
Asn Gln Glu Met Glu Ser Met Glu Glu Val Ile Leu Glu 465 470 475 480
Glu Leu Gln Ile Phe Lys Asp Pro Val Pro Ile Ile Ser Met Glu Ile 485
490 495 Ser Ser Lys Arg Gln Gln Leu Tyr Ile Gly Ser Ala Ser Ala Val
Ala 500 505 510 Gln Val Arg Phe His His Cys Asp Met Tyr Gly Ser Ala
Cys Ala Asp 515 520 525 Cys Cys Leu Ala Arg Asp Pro Tyr Cys Ala Trp
Asp Gly Ile Ser Cys 530 535 540 Ser Arg Tyr Tyr Pro Thr Gly Thr His
Ala Lys Arg Lys Phe Lys Lys 545 550 555 560 Gln Asp Val Arg His Gly
Asn Ala Ala Gln Gln Cys Phe Gly Gln Gln 565 570 575 Phe Val Gly Asp
Ala Leu Asp Lys Thr Glu Glu His Leu Ala Tyr Gly 580 585 590 Ile Glu
Asn Asn Ser Thr Leu Leu Glu Cys Thr Pro Arg Ser Leu Gln 595 600 605
Ala Lys Val Ile Trp Phe Val Gln Lys Gly Arg Glu Thr Arg Lys Glu 610
615 620 Glu Val Lys Thr Asp Asp Arg Val Val Lys Met Asp Leu Gly Leu
Leu 625 630 635 640 Phe Leu Arg Leu His Lys Ser Asp Ala Gly Thr Tyr
Phe Cys Gln Thr 645 650 655 Val Glu His Ser Phe Val His Thr Val Arg
Lys Ile Thr Leu Glu Val 660 665 670 Val Glu Glu Glu Lys Val Glu Asp
Met Phe Asn Lys Asp Asp Glu Glu 675 680 685 Asp Arg His His Arg Met
Pro Cys Pro Ala Gln Ser Ser Ile Ser Gln 690 695 700 Gly Ala Lys Pro
Trp Tyr Lys Glu Phe Leu Gln Leu Ile Gly Tyr Ser 705 710 715 720 Asn
Phe Gln Arg Val Glu Glu Tyr Cys Glu Lys Val Trp Cys Thr Asp 725 730
735 Arg Lys Arg Lys Lys Leu Lys Met Ser Pro Ser Lys Trp Lys Tyr Ala
740 745 750 Asn Pro Gln Glu Lys Lys Leu Arg Ser Lys Pro Glu His Tyr
Arg Leu 755 760 765 Pro Arg His Thr Leu Asp Ser 770 775
38782PRTHomo sapiens 38Met Ala Pro Ser Ala Trp Ala Ile Cys Trp Leu
Leu Gly Gly Leu Leu 1 5 10 15 Leu His Gly Gly Ser Ser Gly Pro Ser
Pro Gly Pro Ser Val Pro Arg 20 25 30 Leu Arg Leu Ser Tyr Arg Asp
Leu Leu Ser Ala Asn Arg Ser Ala Ile 35 40 45 Phe Leu Gly Pro Gln
Gly Ser Leu Asn Leu Gln Ala Met Tyr Leu Asp 50 55 60 Glu Tyr Arg
Asp Arg Leu Phe Leu Gly Gly Leu Asp Ala Leu Tyr Ser 65 70 75 80 Leu
Arg Leu Asp Gln Ala Trp Pro Asp Pro Arg Glu Val Leu Trp Pro 85 90
95 Pro Gln Pro Gly Gln Arg Glu Glu Cys Val Arg Lys Gly Arg Asp Pro
100 105 110 Leu Thr Glu Cys Ala Asn Phe Val Arg Val Leu Gln Pro His
Asn Arg 115
120 125 Thr His Leu Leu Ala Cys Gly Thr Gly Ala Phe Gln Pro Thr Cys
Ala 130 135 140 Leu Ile Thr Val Gly His Arg Gly Glu His Val Leu His
Leu Glu Pro 145 150 155 160 Gly Ser Val Glu Ser Gly Arg Gly Arg Cys
Pro His Glu Pro Ser Arg 165 170 175 Pro Phe Ala Ser Thr Phe Ile Asp
Gly Glu Leu Tyr Thr Gly Leu Thr 180 185 190 Ala Asp Phe Leu Gly Arg
Glu Ala Met Ile Phe Arg Ser Gly Gly Pro 195 200 205 Arg Pro Ala Leu
Arg Ser Asp Ser Asp Gln Ser Leu Leu His Asp Pro 210 215 220 Arg Phe
Val Met Ala Ala Arg Ile Pro Glu Asn Ser Asp Gln Asp Asn 225 230 235
240 Asp Lys Val Tyr Phe Phe Phe Ser Glu Thr Val Pro Ser Pro Asp Gly
245 250 255 Gly Ser Asn His Val Thr Val Ser Arg Val Gly Arg Val Cys
Val Asn 260 265 270 Asp Ala Gly Gly Gln Arg Val Leu Val Asn Lys Trp
Ser Thr Phe Leu 275 280 285 Lys Ala Arg Leu Val Cys Ser Val Pro Gly
Pro Gly Gly Ala Glu Thr 290 295 300 His Phe Asp Gln Leu Glu Asp Val
Phe Leu Leu Trp Pro Lys Ala Gly 305 310 315 320 Lys Ser Leu Glu Val
Tyr Ala Leu Phe Ser Thr Val Ser Ala Val Phe 325 330 335 Gln Gly Phe
Ala Val Cys Val Tyr His Met Ala Asp Ile Trp Glu Val 340 345 350 Phe
Asn Gly Pro Phe Ala His Arg Asp Gly Pro Gln His Gln Trp Gly 355 360
365 Pro Tyr Gly Gly Lys Val Pro Phe Pro Arg Pro Gly Val Cys Pro Ser
370 375 380 Lys Met Thr Ala Gln Pro Gly Arg Pro Phe Gly Ser Thr Lys
Asp Tyr 385 390 395 400 Pro Asp Glu Val Leu Gln Phe Ala Arg Ala His
Pro Leu Met Phe Trp 405 410 415 Pro Val Arg Pro Arg His Gly Arg Pro
Val Leu Val Lys Thr His Leu 420 425 430 Ala Gln Gln Leu His Gln Ile
Val Val Asp Arg Val Glu Ala Glu Asp 435 440 445 Gly Thr Tyr Asp Val
Ile Phe Leu Gly Thr Asp Ser Gly Ser Val Leu 450 455 460 Lys Val Ile
Ala Leu Gln Ala Gly Gly Ser Ala Glu Pro Glu Glu Val 465 470 475 480
Val Leu Glu Glu Leu Gln Val Phe Lys Val Pro Thr Pro Ile Thr Glu 485
490 495 Met Glu Ile Ser Val Lys Arg Gln Met Leu Tyr Val Gly Ser Arg
Leu 500 505 510 Gly Val Ala Gln Leu Arg Leu His Gln Cys Glu Thr Tyr
Gly Thr Ala 515 520 525 Cys Ala Glu Cys Cys Leu Ala Arg Asp Pro Tyr
Cys Ala Trp Asp Gly 530 535 540 Ala Ser Cys Thr His Tyr Arg Pro Ser
Leu Gly Lys Arg Lys Phe Lys 545 550 555 560 Lys Gln Asp Ile Arg His
Gly Asn Pro Ala Leu Gln Cys Leu Gly Gln 565 570 575 Ser Gln Glu Glu
Glu Ala Val Gly Leu Val Ala Ala Thr Met Val Tyr 580 585 590 Gly Thr
Glu His Asn Ser Thr Phe Leu Glu Cys Leu Pro Lys Ser Pro 595 600 605
Gln Ala Ala Val Arg Trp Leu Leu Gln Arg Pro Gly Asp Glu Gly Pro 610
615 620 Asp Gln Val Lys Thr Asp Glu Arg Val Leu His Thr Glu Arg Gly
Leu 625 630 635 640 Leu Phe Arg Arg Leu Ser Arg Phe Asp Ala Gly Thr
Tyr Thr Cys Thr 645 650 655 Thr Leu Glu His Gly Phe Ser Gln Thr Val
Val Arg Leu Ala Leu Val 660 665 670 Val Ile Val Ala Ser Gln Leu Asp
Asn Leu Phe Pro Pro Glu Pro Lys 675 680 685 Pro Glu Glu Pro Pro Ala
Arg Gly Gly Leu Ala Ser Thr Pro Pro Lys 690 695 700 Ala Trp Tyr Lys
Asp Ile Leu Gln Leu Ile Gly Phe Ala Asn Leu Pro 705 710 715 720 Arg
Val Asp Glu Tyr Cys Glu Arg Val Trp Cys Arg Gly Thr Thr Glu 725 730
735 Cys Ser Gly Cys Phe Arg Ser Arg Ser Arg Gly Lys Gln Ala Arg Gly
740 745 750 Lys Ser Trp Ala Gly Leu Glu Leu Gly Lys Lys Met Lys Ser
Arg Val 755 760 765 His Ala Glu His Asn Arg Thr Pro Arg Glu Val Glu
Ala Thr 770 775 780 394PRTArtificial sequencePro-protein convertase
recognition sequence 39Arg Phe Arg Arg 1 404PRTArtificial
sequenceMutated pro-protein convertase recognition sequence 40Lys
Phe Lys Lys 1
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