U.S. patent application number 11/205225 was filed with the patent office on 2006-02-23 for xaf genes and polypeptides: methods and reagents for modulating apoptosis.
This patent application is currently assigned to Aegera Therapeutics Inc.. Invention is credited to Robert G. Korneluk, Peter Liston, Alexander E. MacKenzie, Katsuyuki Tamai.
Application Number | 20060040862 11/205225 |
Document ID | / |
Family ID | 27368127 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060040862 |
Kind Code |
A1 |
Korneluk; Robert G. ; et
al. |
February 23, 2006 |
XAF genes and polypeptides: methods and reagents for modulating
apoptosis
Abstract
The invention provides novel XAF nucleic acid sequences. Also
provided are XAF polypeptides, anti-XAF antibodies, and methods for
modulating apoptosis and detecting compounds which modulate
apoptosis.
Inventors: |
Korneluk; Robert G.;
(Ottawa, CA) ; Tamai; Katsuyuki; (Nagano, JP)
; Liston; Peter; (Ottawa, CA) ; MacKenzie;
Alexander E.; (Ottawa, CA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Aegera Therapeutics Inc.
Verdun
CA
|
Family ID: |
27368127 |
Appl. No.: |
11/205225 |
Filed: |
August 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10288273 |
Nov 5, 2002 |
6946544 |
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11205225 |
Aug 16, 2005 |
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09616614 |
Jul 14, 2000 |
6495339 |
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10288273 |
Nov 5, 2002 |
|
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09100391 |
Jun 19, 1998 |
6107088 |
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09616614 |
Jul 14, 2000 |
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60056338 |
Aug 18, 1997 |
|
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60054491 |
Aug 1, 1997 |
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60052402 |
Jul 14, 1997 |
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Current U.S.
Class: |
514/18.9 ;
514/19.3; 530/350 |
Current CPC
Class: |
C12Q 1/6883 20130101;
G01N 33/5008 20130101; C12Q 1/6886 20130101; G01N 2510/00 20130101;
A61K 38/1709 20130101; A01K 2217/05 20130101; G01N 33/68 20130101;
C12Q 2600/136 20130101; C12Q 2600/158 20130101; C07K 14/4703
20130101; C07K 14/4747 20130101; G01N 33/5017 20130101; C07K 16/18
20130101; G01N 33/5011 20130101 |
Class at
Publication: |
514/012 ;
530/350 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07K 14/705 20060101 C07K014/705 |
Claims
1. A method of treating a patient diagnosed as having a condition
involving insufficient apoptosis, said method comprising providing
said patient with a polypeptide, said polypeptide comprising at
least 85% amino acid sequence identity to SEQ ID NO:2, 4, 10, or
12, or a fragment thereof, said polypeptide having XAF biological
activity, said providing in an amount sufficient to increase
apoptosis in a cell of said patient.
2. The method of claim 1, wherein said patient is a human.
3. The method of claim 1, wherein said polypeptide is in a
pharmaceutically acceptable carrier.
4. The method of claim 3, wherein said carrier is a liquid solution
or suspension.
5. The method of claim 3, wherein said carrier is in the form of a
tablet or capsule.
6. The method of claim 3, wherein said carrier is suitable for
parenteral, intravenous, intra-arterial, subcutaneous,
intramuscular, intracranial, intraorbital, ophthalmic,
intraventricular, intracapsular, intraspinal, intracisternal,
intraperitoneal, intranasal, or oral administration.
7. The method of claim 1, wherein said polypeptide comprises at
least 85% amino acid sequence identity to SEQ ID NO:2, or a
fragment thereof having XAF activity.
8. The method of claim 7, wherein said polypeptide comprises the
amino acid sequence of SEQ ID NO:2.
9. The method of claim 1, wherein said polypeptide comprises at
least 85% amino acid sequence identity to SEQ ID NO:4, or a
fragment thereof having XAF activity.
10. The method of claim 9, wherein said polypeptide comprises the
amino acid sequence of SEQ ID NO:4.
11. The method of claim 1, wherein said polypeptide comprises at
least 85% amino acid sequence identity to SEQ ID NO: 10, or a
fragment thereof having XAF activity.
12. The method of claim 11, wherein said polypeptide comprises the
amino acid sequence of SEQ ID NO: 10.
13. The method of claim 1, wherein said polypeptide comprises at
least 85% amino acid sequence identity to SEQ ID NO:12, or a
fragment thereof having XAF activity.
14. The method of claim 13, wherein said polypeptide comprises the
amino acid sequence of SEQ ID NO:12.
15. The method of claim 1, wherein said condition is breast cancer,
uterine cervical carcinoma, gastric carcinoma, ovarian epithelial
cancer, pediatric medulloblastoma, lung carcinoma, or prostate
cancer.
16. The method of claim 1, wherein said providing is parenteral,
intravenous, intra-arterial, subcutaneous, intramuscular,
intracranial, intraorbital, ophthalmic, intraventricular,
intracapsular, intraspinal, intracisternal, intraperitoneal,
intranasal, by suppositories, or oral.
17. The method of claim 1, wherein said cell is a peripheral blood
leukocyte, a muscle cell, an intestinal cell, an ovarian cell, a
placental cell, or a thymus cell.
18. The method of claim 17, wherein said peripheral blood leukocyte
is a lymphocyte.
19. The method of claim 17, wherein said muscle cell is a
myocardial cell.
20. The method of claim 17, wherein said thymus cell is a
thymocyte.
21. The method of claim 1, wherein said XAF biological activity is
selected from the group consisting of the ability to bind an IAP,
the ability to bind another XAF polypeptide, the ability to cause
apoptosis when introduced into a cell, the ability to enhance the
NF-.kappa.B inducing activity of a TRAF, and the ability to bind a
XAF-1, XAF02L, or XAF-2S specific antibody.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/288,273, filed Nov. 5, 2002, which is a continuation of U.S.
application Ser. No. 09/616,614, filed Jul. 14, 2000, now U.S. Pat.
No. 6,495,339, which is a divisional of U.S. application Ser. No.
09/100,391, filed Jun. 19, 1998, now U.S. Pat. No. 6,107,088, which
claims the benefit of U.S. Provisional Application Ser. Nos.
60/056,338, filed Aug. 18, 1997, 60/054,491, filed Aug. 1, 1997,
and 60/052,402, filed Jul. 14, 1997, the disclosures of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to apoptosis, tumor necrosis
factor-.alpha. (TNF-.alpha.) mediated signalling, cell cycle and
tumor growth suppression.
[0003] Apoptosis is a morphologically distinct form of programmed
cell death that is important in the normal development and
maintenance of multicellular organisms. Dysregulation of apoptosis
can take the form of inappropriate suppression of cell death, as
occurs in the development of cancers, or in a failure to control
the extent of cell death, as is believed to occur in acquired
immunodeficiency and certain neurodegenerative disorders.
[0004] Some baculoviruses encode proteins termed "inhibitors of
apoptosis proteins" (IAPs) because they inhibit the apoptosis that
would otherwise occur when insect cells are infected by the virus.
These proteins are thought to work in a manner that is independent
of other viral proteins. The baculovirus IAP genes include
sequences encoding a ring zinc finger-like motif (RZF), which may
be involved in DNA binding, and two N-terminal domains that consist
of a 70 amino acid repeat motif termed a BIR domain (Baculovirus
IAP Repeat).
[0005] We have recently discovered a mammalian family of IAP
polypeptides. These polypeptides include the human proteins HIAP-1,
HIAP-2, and XIAP and their murine homologs. A related protein,
NAIP, has also been found. The mammalian IAP levels have been shown
to be increased both in cancer cells and cells which survive events
known to induce apoptosis (e.g., ischemia). The IAPs have also been
shown to block apoptosis triggered by diverse stimuli. These
results are consistent with a role for the mammalian IAPs as
inhibitors of apoptosis.
[0006] The IAP family is now known to include at least two
Drosophila proteins, in addition to the original four mammalian
homologues (Hay et al., Cell 83: 1253-1262, 1995). Although we and
others have established that the IAPs can suppress apoptosis in
tissue culture model systems their mechanism, of action is still
under investigation.
SUMMARY OF THE INVENTION
[0007] We have discovered a novel family of genes, the XAFs.
Members of the XAF gene family encode proteins that interact with
IAPs and are associated with apoptosis. Our discovery allows the
development of diagnostic, prognostic, and therapeutic compounds
and methods for the detection and treatment of diseases involving
apoptosis.
[0008] In a first aspect, the invention features substantially pure
nucleic acid encoding a XAF polypeptide.
[0009] In a second aspect, the invention features substantially
pure nucleic acid corresponding to at least ten nucleotides of a
nucleic acid encoding a XAF polypeptide, where the nucleic acid is
antisense nucleic acid and the antisense nucleic acid is sufficient
to decrease XAF biological activity. In various embodiments of this
aspect, the antisense nucleic acid corresponds to at least fifteen
nucleotides of a nucleic acid encoding a XAF polypeptide, at least
thirty nucleotides of a nucleic acid encoding a XAF polypeptide, or
at least 100 nucleotides of a nucleic acid encoding a XAF
polypeptide. In other embodiments, the XAF biological activity is
decreased by at least 20%, 40%, 60%, or 80%. In yet another
embodiment of this aspect of the invention, the antisense nucleic
acid is in a vector where the vector is capable of directing
expression of the antisense nucleic acid in a vector-containing
cell.
[0010] In a third aspect, the invention features a vector that
includes a substantially pure nucleic acid encoding a XAF
polypeptide, where the vector is capable of directing expression of
the polypeptide in a vector-containing cell.
[0011] In another related aspect, the invention features a cell
that contains a substantially pure nucleic acid encoding a XAF
polypeptide. In a preferred embodiment of this aspect, the nucleic
acid is expressed in the cell. In various preferred embodiments,
the cell is present in a patient having a disease that is caused by
excessive or insufficient cell death and the cell is selected from
the group that includes a fibroblast, a neuron, a glial cell, an
insect cell, an embryonic stem cell, a myocardial cell, and a
lymphocyte.
[0012] In a fifth aspect, the invention features a transgenic
animal generated from a cell genetically engineered to lack nucleic
acid encoding a XAF polypeptide, where the transgenic animal lacks
expression of the XAF polypeptide.
[0013] In a related aspect, the invention features a transgenic
animal generated from a cell that contains a substantially pure
nucleic acid that replaces DNA encoding a XAF polypeptide, where
the nucleic acid is expressed in the transgenic animal.
[0014] In various embodiments of this aspect, the XAF polypeptide
is from a mammal (e.g., a human or a rodent). In another
embodiment, the nucleic acid is genomic DNA or cDNA, and is
operably linked to regulatory sequences for expression of the
polypeptide where the regulatory sequences include a promoter
(e.g., a constitutive promoter, a promoter inducible by one or more
external agents, or a cell-type specific promoter). In other
preferred embodiments, the XAF polypeptide is selected from a group
that includes XAF-1, XAF-2 N terminus, XAF-2L and XAF-2S. In
another embodiment, the XAF-1 has the amino acid sequence of SEQ ID
NO.: 2 or the nucleic acid sequence of SEQ ID NO.: 1, and may
include a deletion of the nucleic acids encoding the carboxy
terminal amino acids 173 to 317 of XAF-1 (SEQ ID NO.: 8); or a
deletion of the nucleic acids encoding the amino terminal amino
acids 1 to 172 of XAF-1 (SEQ ID NO.: 7). In another embodiment of
this aspect of the invention, the XAF-2 N terminus polypeptide has
the amino acid sequence of SEQ ID NO.: 4 or the nucleic acid
sequence of SEQ ID NO.: 3. In another embodiment, the XAF-2L
polypeptide has the amino acid sequence of SEQ ID NO.: 10 or the
nucleic acid sequence of SEQ ID NO.: 9. In yet another embodiment,
the XAF-2S polypeptide has the amino acid sequence of SEQ ID NO.:
12 or the nucleic acid sequence of SEQ ID NO.: 11.
[0015] In a seventh aspect, the invention features a method of
identifying a compound that modulates apoptosis. The method
includes: (a) providing a cell that has a XAF gene; (b) contacting
the cell with a candidate compound; and (c) monitoring expression
of the XAF gene, where an alteration in the level of expression of
the XAF gene indicates the presence of a compound which modulates
apoptosis. In one preferred embodiment of this aspect, the
alteration that is an increase indicates the compound is increasing
apoptosis, and the alteration that is a decrease indicates the
compound is decreasing apoptosis. In various embodiments of this
aspect, the cell is transformed and the cell is not able to induce
apoptosis by expression of p53.
[0016] In a related aspect, the invention features another method
of identifying a compound that is able to modulate apoptosis that
includes: (a) providing a cell including a reporter gene operably
linked to a promoter from a XAF gene; (b) contacting the cell with
a candidate compound; and (c) measuring expression of the reporter
gene, where a change in the expression in response to the candidate
compound identifies a compound that is able to modulate apoptosis.
In one preferred embodiment of this aspect, the alteration that is
an increase indicates the compound is increasing apoptosis, and the
alteration that is a decrease indicates the compound is decreasing
apoptosis. In various embodiments of this aspect, the cell is
transformed and the cell is not able to induce apoptosis by
expression of p53.
[0017] In a ninth aspect, the invention features a method of
identifying a compound that is able to inhibit XAF-mediated
apoptosis that includes: (a) providing a cell expressing an
apoptosis-inducing amount of XAF; (b) contacting the cell with a
candidate compound; and (c) measuring the level of apoptosis in the
cell, where a decrease in the level relative to a level in a cell
not contacted with the candidate compound indicates a compound that
able to inhibit XAF-mediated apoptosis. In various embodiments of
this aspect, the cell is transformed and the cell is not able to
induce apoptosis by expression of p53.
[0018] In a tenth aspect, the invention features a method of
identifying a compound that is able to induce XAF-mediated
apoptosis that includes: (a) providing a cell expressing an
apoptosis-inducing amount of XAF; (b) contacting the cell with a
candidate compound; and (c) measuring level of apoptosis in the
cell, where an increase in the level relative to a level in a cell
not contacted with the candidate compound indicates a compound that
able to induce XAF-mediated apoptosis. In various embodiments of
this aspect, the cell is transformed and the cell is not able to
induce apoptosis by expression of p53.
[0019] In related aspects, the invention features other methods of
identifying a compound that is able to modulate apoptosis.
[0020] One such method includes: (a) providing a cell expressing a
TRAF polypeptide, a XAF polypeptide, and a reporter gene operably
linked to DNA that includes an NF-.kappa.B binding site; (b)
contacting the cell with a candidate compound; and (c) measuring
expression of the reporter gene, where a change in expression in
response to the compound indicates that the compound is able to
modulate apoptosis. In a preferred embodiment of this aspect of the
invention, the TRAF is selected from a group that includes TRAF2,
TRAF5, and TRAF6. In various embodiments of this aspect, the cell
is transformed and the cell is not able to induce apoptosis by
expression of p53.
[0021] A second such method includes: (a) providing a cell
expressing a TRAF polypeptide, a XAF polypeptide, an IAP
polypeptide, and a reporter gene operably linked to DNA that
includes an NF-.kappa.B binding site; (b) contacting the cell with
a candidate compound; and (c) measuring expression of the reporter
gene, where a change in expression in response to the compound
indicates that the compound is able to modulate apoptosis. In a
preferred embodiment of this aspect of the invention, the IAP is
XIAP. In another preferred embodiment of this aspect of the
invention, the TRAF is selected from a group that includes TRAF2,
TRAF5, and TRAF6. In various embodiments of this aspect, the cell
is transformed and the cell is not able to induce apoptosis by
expression of p53.
[0022] A third such method includes: (a) providing a cell having:
(i) a reporter gene operably linked to a DNA-binding-protein
recognition site; (ii) a first fusion gene capable of expressing a
first fusion protein, where the first fusion protein includes a XAF
polypeptide covalently bonded to a binding moiety capable of
specifically binding to the DNA-binding-protein recognition site;
(iii) a second fusion gene capable of expressing a second fusion
protein, where the second fusion protein includes a XAF polypeptide
covalently bonded to a gene activating moiety; (b) exposing the
cell to the compound; and (c) measuring reporter gene expression in
the cell, where a change in the reporter gene expression indicates
that the compound is capable of modulating apoptosis. In a
preferred embodiment of this aspect of the invention, the cell is a
yeast cell.
[0023] A fourth method for detecting a compound capable of
modulating apoptosis includes: (a) providing a cell having: (i) a
reporter gene operably linked to a DNA-binding-protein recognition
site; (ii) a first fusion gene capable of expressing a first fusion
protein, where the first fusion protein includes a XAF polypeptide
covalently bonded to a binding moiety capable of specifically
binding to the DNA-binding-protein recognition site; (iii) a second
fusion gene capable of expressing a second fusion protein, where
the second fusion protein includes an IAP polypeptide covalently
bonded to a gene activating moiety; (b) exposing the cell to the
compound; and (c) measuring reporter gene expression in the cell,
where a change in the reporter gene expression indicates that the
compound is capable of modulating apoptosis. In a preferred
embodiment of this aspect of the invention, the IAP is XIAP. In
another preferred embodiment, the cell is a yeast cell.
[0024] A fifth such method includes: (a) providing a cell having:
(i) a reporter gene operably linked to a DNA-binding-protein
recognition site; (ii) a first fusion gene capable of expressing a
first fusion protein, where the first fusion protein includes an
IAP polypeptide covalently bonded to a binding moiety capable of
specifically binding to the DNA-binding-protein recognition site;
(iii) a second fusion gene capable of expressing a second fusion
protein, where the second fusion protein includes a XAF polypeptide
covalently bonded to a gene activating moiety; (b) exposing the
cell to the compound; and (c) measuring reporter gene expression in
the cell, where a change in the reporter gene expression indicates
that the compound is capable of modulating apoptosis. In a
preferred embodiment of this aspect of the invention, the IAP is
XIAP. In another preferred embodiment, the cell is a yeast
cell.
[0025] A sixth such method includes: (a) providing a first XAF
polypeptide immobilized on a solid-phase substrate; (b) contacting
the first XAF polypeptide with a second XAF polypeptide; (c)
contacting the first XAF polypeptide and the second XAF polypeptide
with a compound; and (d) measuring amount of binding of the first
XAF polypeptide to the second XAF polypeptide, where a change in
the amount relative to an amount not contacted with the compound
indicates that the compound is capable of modulating apoptosis.
[0026] A seventh method for detecting a compound capable of
modulating apoptosis includes: (a) contacting a XAF polypeptide
immobilized on a solid-phase substrate; (b) providing the XAF
polypeptide with an IAP polypeptide; (c) contacting the XAF
polypeptide and the IAP polypeptide with a compound; and (d)
measuring amount of binding of the XAF polypeptide to the IAP
polypeptide, where a change in the amount relative to an amount not
contacted with the compound indicates that the compound is capable
of modulating apoptosis. In a preferred embodiment of this aspect
of the invention, the IAP is XIAP.
[0027] An eighth such method includes: (a) providing an IAP
polypeptide immobilized on a solid-phase substrate; (b) contacting
the IAP polypeptide with a XAF polypeptide; (c) contacting the IAP
polypeptide and the XAF polypeptide with a compound; and (d)
measuring amount of binding of the IAP polypeptide to the XAF
polypeptide, where a change in the amount relative to an amount not
contacted with the compound indicates that the compound is capable
of modulating apoptosis. In a preferred embodiment of this aspect
of the invention, the IAP is XIAP.
[0028] In various preferred embodiments of the seventh to
eighteenth method aspects of the invention, the XAF is XAF-1; the
XAF is the N-terminus of XAF-2; the XAF is XAF-2L, or the XAF is
XAF-2S. In other embodiments, the XAF is from a mammal (e.g., a
human or a rodent).
[0029] In a nineteenth aspect, the invention features a method of
increasing apoptosis in a cell by administering to the cell an
apoptosis inducing amount of XAF polypeptide or fragment
thereof.
[0030] In related aspects, the invention includes methods of
increasing apoptosis by either providing a transgene encoding a XAF
polypeptide or fragment thereof to a cell of an animal such that,
the transgene is positioned for expression in the cell; or by
administering to the cell a compound which increases XAF biological
activity in a cell (e.g., by administering a polypeptide fragment
of a XAF polypeptide, a mutant of a XAF polypeptide, or a nucleic
acid encoding a XAF polypeptide, a mutant thereof, or a polypeptide
fragment thereof).
[0031] In preferred embodiment of the nineteenth, twentieth, and
twenty-first aspects of the invention, the XAF is selected from a
group that includes XAF-1, XAF-2 N-terminus, XAF-2L, and XAF-2S. In
various preferred embodiments, the XAF is from a mammal (e.g., a
human or rodent); the cell is in a mammal (e.g., a human or
rodent); the cell is in an mammal diagnosed as having a condition
involving insufficient apoptosis, (e.g., a cancer such as breast
cancer, uterine cervical carcinoma, gastric carcinoma, ovarian
epithelial cancer, pediatric medulloblastoma, lung carcinoma,
prostate cancer); and the cell is a peripheral blood leukocyte
(e.g., a lymphocyte), a muscle cell (e.g., a myocardial cell), an
intestinal cell, an ovarian cell, a placental cell, or a thymus
cell (e.g., a thymocyte).
[0032] In a twenty-second aspect, the invention features a method
of inhibiting apoptosis in a cell, by administering to the cell an
apoptosis-inhibiting amount of XAF polypeptide or fragment
thereof.
[0033] In related aspects, the invention features a method of
inhibiting apoptosis in a cell by providing to the cell a transgene
encoding a XAF polypeptide or fragment positioned for expression in
the cell; and a method of inhibiting apoptosis by administering a
compound which decreases XAF biological activity (e.g., an antibody
which specifically binds to a XAF polypeptide (e.g., a neutralizing
antibody), a polypeptide fragment of a XAF polypeptide, a mutant
form of a XAF polypeptide, an antisense nucleic acid complementary
to the XAF coding sequence, a negative regulator of the
XAF-dependent apoptotic pathway, or a XAF antisense nucleic
acid).
[0034] In a preferred embodiment of the twenty-second,
twenty-third, and twenty-fourth aspects of the invention, the XAF
is selected from a group that includes XAF-1, XAF-2 N-terminus,
XAF-2L, and XAF-2S. In various preferred embodiments, the XAF is
from a mammal (e.g., a human or rodent); the cell is in a mammal
(e.g., a human or rodent); and the mammal bearing the cell is an
mammal diagnosed as having a condition involving excessive
apoptosis (e.g., AIDS, a neurodegenerative disease, a
myelodysplastic syndrome, or an ischemic injury (caused by, e.g., a
myocardial infarction, a stroke, or a reperfusion injury, a
toxin-induced liver disease, physical injury, renal failure, a
secondary exsaunguination or blood flow interruption resulting from
any other primary diseases)). In other preferred embodiments, the
cell is a muscle cell (e.g., a myocardial cell), a peripheral blood
leukocyte (e.g., a lymphocyte, such as a T lymphocyte (preferably,
a CD4.sup.+ T lymphocyte)), an intestinal cell, an ovarian cell, a
placental cell, a thymus cell (e.g., a thymocyte), or a breast
cell.
[0035] In the twenty-fifth and twenty-sixth aspects, the invention
features methods of diagnosing a mammal for the presence of disease
involving altered apoptosis or an increased likelihood of
developing a disease involving altered apoptosis. The methods
include isolating a sample of nucleic acid from the mammal and
determining whether the nucleic acid includes a XAF mutation, where
the presence of a mutation is an indication that the animal has an
apoptosis disease or an increased likelihood of developing a
disease involving apoptosis; or measuring XAF gene expression in a
sample from an animal to be diagnosed, where an alteration in the
expression or activity relative to a sample from an unaffected
mammal is an indication that the mammal has a disease involving
apoptosis or increased likelihood of developing such a disease. In
preferred embodiments, XAF gene expression is measured by assaying
the amount of XAF polypeptide or XAF biological activity in the
sample (e.g., the XAF polypeptide is measured by immunological
methods), or XAF gene expression is measured by assaying the amount
of XAF RNA in the sample.
[0036] In one preferred embodiment of the twenty-fifth and
twenty-sixth of the invention, the XAF is selected from a group
that includes XAF-1, XAF-2 N-terminus, XAF-2L, and XAF-2S. In
another preferred embodiment, the mammal is a human.
[0037] In a twenty-seventh aspect, the invention features a kit for
diagnosing a mammal for the presence of a disease involving altered
apoptosis or an increased likelihood of developing a disease
involving altered apoptosis that includes a substantially pure
antibody that specifically binds a XAF polypeptide.
[0038] Another such kit includes a material for measuring XAF RNA
(e.g., a probe). In a preferred embodiment, the material is a
nucleic acid probe.
[0039] A third such kit includes both a substantially pure antibody
that specifically binds a XAF polypeptide, as well as a material
for measuring XAF RNA. In a preferred embodiment, the kit also
includes a means for detecting the binding of the antibody to the
is XAF polypeptide. In another preferred embodiment, the material
is a nucleic acid probe.
[0040] In a thirtieth aspect, the invention features a method of
obtaining a XAF polypeptide, including: (a) providing a cell with
DNA encoding a XAF polypeptide, the DNA being positioned for
expression in the cell; (b) culturing the cell under conditions for
expressing the DNA; and (c) isolating the XAF polypeptide.
[0041] In preferred embodiments of this aspect of the invention,
the XAF is XAF-1, XAF-2 N terminus, XAF-2L, or XAF-2S. In another
preferred embodiment, the DNA further includes a promoter inducible
by one or more external agents.
[0042] In a thirty-first aspect, the invention features a method of
isolating a XAF gene or portion thereof having sequence identity to
human XAF-1. The method includes amplifying by polymerase chain
reaction the XAF gene or portion thereof using oligonucleotide
primers wherein the primers (a) are each greater than 13
nucleotides in length; (b) each have regions of complementarity to
opposite DNA strands in a region of the nucleotide sequence of FIG.
1; and (c) optionally contain sequences capable of producing
restriction endonuclease cut sites in the amplified product; and
isolating the XAF gene or portion thereof.
[0043] In a related aspect, the invention features a method of
isolating a XAF gene or portion thereof having sequence identity to
human XAF-2L or XAF-2S. The method includes amplifying by
polymerase chain reaction the XAF gene or portion thereof using
oligonucleotide primers wherein the primers (a) are each greater
than 13 nucleotides in length; (b) each have regions of
complementarity to opposite DNA strands in a region of the
nucleotide sequence of FIG. 37A; and (c) optionally contain
sequences capable of producing restriction endonuclease cut sites
in the amplified product; and isolating the XAF gene or portion
thereof.
[0044] In another related aspect, the invention features a method
of isolating a XAF gene or fragment thereof from a cell, including
the steps of: (a) providing a sample of cellular DNA; (b) providing
a pair of oligonucleotides having sequence homology to a conserved
region of a XAF gene; (c) combining the pair of oligonucleotides
with the cellular DNA sample under conditions suitable for
polymerase chain reaction-mediated DNA amplification; and (d)
isolating the amplified XAF gene or fragment thereof. In a
preferred embodiment of the above three aspects, the polymerase
chain reaction is reverse-transcription polymerase chain reaction
(e.g., RACE).
[0045] In yet another related aspect, the invention features a
method of identifying a XAF gene in a mammalian cell that includes:
(a) providing a preparation of mammalian cellular DNA; (b)
providing a detectably-labeled DNA sequence having identity to a
conserved region of a second known XAF gene; and (c) contacting the
preparation of cellular DNA with the detectably-labeled DNA
sequence under hybridization conditions that provide detection of a
gene having 50% or greater nucleotide sequence identity to the
detectably-labeled DNA sequence; and identifying the XAF gene. In
one preferred embodiment of this method for detecting a XAF gene,
the DNA sequence includes at least a portion of XAF-1. In another
preferred embodiment, the DNA sequence includes at least a portion
of XAF-2L. In another preferred embodiment, the DNA sequence
includes at least a portion of XAF-2S.
[0046] In a thirty-fifth aspect, the invention features a method
for identifying a XAF gene that includes the steps of: (a)
providing a mammalian cell sample; (b) introducing by
transformation into the cell sample a candidate XAF gene; (c)
expressing the candidate XAF gene within the cell sample; and (d)
determining whether the sample exhibits an altered level of
apoptosis, where an alteration in the level of apoptosis identifies
a XAF gene. Preferably, the alteration is an increase in apoptosis
and the cell is a leukocyte, a fibroblast, an insect cell, a glial
cell, a myocardial cell, an embryonic stem cell, or a neuron.
[0047] In other aspects, the invention features a XAF nucleic acid
for use in modulating apoptosis, a XAF polypeptide for use in
modulating apoptosis, the use of a XAF polypeptide for the
manufacture of a medicament for the modulation of apoptosis, and
the use of a XAF nucleic acid for the manufacture of a medicament
for the modulation of apoptosis. Preferably, the XAF is selected
from a group that includes XAF-1, XAF-2 N terminus, XAF-2L, and
XAF-2S.
[0048] In a fortieth aspect, the invention features a substantially
pure antibody that specifically binds a XAF polypeptide, or a
fragment or a mutant thereof. In one preferred embodiment of this
aspect, the XAF polypeptide is selected from a group that includes
XAF-1, XAF-2 N terminus XAF-2S, and XAF-2L. In other preferred
embodiments, the XAF polypeptide is from a mammal (e.g., a human or
a rodent), and the antibody is a polyclonal antibody, a monoclonal
antibody, or a neutralizing antibody.
[0049] By "XAF", "XAF protein", or "XAF polypeptide" is meant a
polypeptide, or fragment thereof, which has at least 30%, more
preferably at least 35%, and most preferably 40% amino acid
identity to either the amino-terminal 131 amino acids of the human
XAF-1 (SEQ ID NO.: 2) or the amino-terminal 135 amino acids of
human XAF-2L (SEQ ID NO.: 10) polypeptides. It is understood that
polypeptide products from splice variants of XAF gene sequences are
also included in this definition. Preferably, the XAF protein is
encoded by nucleic acid having a sequence which hybridizes to a
nucleic acid sequences present in either SEQ ID NO.: 1 or SEQ ID
NO.: 9 under stringent conditions. Even more preferably the encoded
polypeptide also has XAF biological activity. Preferably, the XAF
polypeptide has at least three zinc finger domains. More
preferably, the XAF polypeptide has at least six zinc finger
domains, at least five of which occur within 150 amino acids of the
N-terminus.
[0050] By "zinc finger" is meant a binding domain capable of
associating with zinc. A preferable zinc binding domain has the
amino acid sequence 5' C-X.sub.2-5-C-X.sub.11-18C/H-X.sub.2-5-C/H
3' (SEQ ID NO.: 6), wherein "X" may be any amino acid. A more
preferable zinc binding domain has the amino acid sequence 5'
C-X.sub.1-2C-X.sub.11-H-X.sub.3-5--C 3' (SEQ ID NO.: 7), wherein
"X" may be any amino acid. Even more preferably, a zinc binding
domain has the amino acid sequence 5'
C-X.sub.2-H-X.sub.11-H-X.sub.3-C 3' (SEQ ID NO.: 8), wherein "X"
may be any amino acid. Most preferably, a zinc binding domain is
one found in a XAF polypeptide.
[0051] By "XAF biological activity" is meant any one or more of the
biological activities described herein for XAF-1, XAF-2L, or
XAF-2S, including, without limitation, the ability to bind an IAP
(e.g., a XIAP), or another XAF polypeptide; the ability to cause
apoptosis when transfected into a cell (particularly in a HeLa
cell); the ability to enhance the NF-.kappa.B inducing activity of
a TRAF; and the ability to specifically bind a XAF-1, XAF-2L, or
XAF-2S specific antibody.
[0052] By "modulating apoptosis" or "altering apoptosis" is meant
increasing or decreasing the number of cells that undergo apoptosis
(than would otherwise be the case) in a given cell population.
Preferably, the cell population is selected from a group including
T cells, neuronal cells, fibroblasts, myocardial cells, or any
other cell line known to undergo apoptosis in a laboratory setting
(e.g., the baculovirus infected insect cells or an in vivo assay).
It will be appreciated that the degree of modulation provided by a
XAF polypeptide or a modulating compound in a given assay will
vary, but that one skilled in the art can determine the
statistically significant change or a therapeutically effective
change in the level of apoptosis which identifies a XAF polypeptide
or a compound which modulates XAF or is a XAF therapeutic.
[0053] By "high stringency conditions" is meant hybridization in
2.times.SSC at 40.degree. C. with a DNA probe length of at least 40
nucleotides. For other definitions of high stringency conditions,
see Ausubel, F. et al., 1994, Current Protocols in Molecular
Biology, John Wiley & Sons, New York, 6.3.1-6.3.6, hereby
incorporated by reference.
[0054] By "IAP" is meant an amino acid sequence which has identity
to baculovirus inhibitors of apoptosis. Mammalian IAPs include,
without limitation, NAIP, HIAP1, HIAP2, and XIAP. Preferably, such
a polypeptide has an amino acid sequence which is at least 45%,
preferably 60%, and most preferably 85% or even 95% identical to at
least one of the amino acid sequences of a baculovirus IAP.
[0055] By "inhibiting apoptosis" is meant any decrease in the
number of cells which undergo apoptosis relative to an untreated
control. Preferably, the decrease is at least 25%, more preferably
the decrease is 50%, and most preferably the decrease is at least
one-fold.
[0056] By "polypeptide" is meant any chain of more than two amino
acids, regardless of post-translational modification such as
glycosylation or phosphorylation.
[0057] By "pharmaceutically acceptable carrier" is meant a carrier
which is physiologically acceptable to the treated mammal while
retaining the therapeutic properties of the compound with which it
is administered. One exemplary pharmaceutically acceptable carrier
is physiological saline. Other physiologically acceptable carriers
and their formulations are known to one skilled in the art and
described, for example, in Remington's Pharmaceutical Sciences,
(18.sup.th edition), ed. A. Gennaro, 1990, Mack Publishing Company,
Easton, Pa.
[0058] By "substantially identical" is meant a polypeptide or
nucleic acid exhibiting at least 50%, preferably 85%, more
preferably 90%, and most preferably 95% homology to a reference
amino acid or nucleic acid sequence. For polypeptides, the length
of comparison sequences will generally be at least 16 amino acids,
preferably at least 20 amino acids, more preferably at least 25
amino acids, and most preferably 35 amino acids. For nucleic acids,
the length of comparison sequences will generally be at least 50
nucleotides, preferably at least 60 nucleotides, more preferably at
least 75 nucleotides, and most preferably 110 nucleotides.
[0059] Sequence identity is typically measured using sequence
analysis software with the default parameters specified therein
(e.g., Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). This software program
matches similar sequences by assigning degrees of homology to
various substitutions, deletions, and other modifications.
Conservative substitutions typically include substitutions within
the following groups: glycine, alanine, valine, isoleucine,
leucine; aspartic acid, glutamic acid, asparagine, glutamine;
serine, threonine; lysine, arginine; and phenylalanine,
tyrosine.
[0060] By "substantially pure polypeptide" is meant a polypeptide
that has been separated from the components that naturally
accompany it. Typically, the polypeptide is substantially pure when
it is at least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the polypeptide is a XAF polypeptide that
is at least 75%, more preferably at least 90%, and most preferably
at least 99%, by weight, pure. A substantially pure XAF polypeptide
may be obtained, for example, by extraction from a natural source
(e.g., a fibroblast, neuronal cell, or lymphocyte) by expression of
a recombinant nucleic acid encoding a XAF polypeptide, or by
chemically synthesizing the protein. Purity can be measured by any
appropriate method, e.g., by column chromatography, polyacrylamide
gel electrophoresis, or HPLC analysis.
[0061] A protein is substantially free of naturally associated
components when it is separated from those contaminants which
accompany it in its natural state. Thus, a protein which is
chemically synthesized or produced in a cellular system different
from the cell from which it naturally originates will be
substantially free from its naturally associated components.
Accordingly, substantially pure polypeptides not only includes
those derived from eukaryotic organisms but also those synthesized
in E. Coli or other prokaryotes. By "substantially pure DNA" is
meant DNA that is free of the genes which, in the
naturally-occurring genome of the organism from which the DNA of
the invention is derived, flank the gene. The term therefore
includes, for example, a recombinant DNA which is incorporated into
a vector; into an autonomously replicating plasmid or virus; or
into the genomic DNA of a prokaryote or eukaryote; or which exists
as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment
produced by PCR or restriction endonuclease digestion) independent
of other sequences. It also includes a recombinant DNA which is
part of a hybrid gene encoding additional polypeptide sequence.
[0062] By "TRAF" is meant a member of the TRAF family of proteins.
TRAF family members each possess an amino terminal RING zinc finger
and/or additional zinc fingers, a leucine zipper, and a unique,
conserved carboxy terminal coiled coil motif, the TRAF-C domain,
which defines the family. TRAF1 and TRAF2 were first identified as
components of the TNF-R2 signaling complex (Rothe et al., Cell 78:
681-692, 1994). Preferred TRAF polypeptides are TRAF2, TRAF5, and
TRAF6.
[0063] By "transgene" is meant any piece of DNA which is inserted
by artifice into a cell, and becomes part of the genome of the
organism which develops from that cell. Such a transgene may
include a gene which is partly or entirely heterologous (i.e.,
foreign) to the transgenic organism, or may represent a gene
homologous to an endogenous gene of the organism.
[0064] By "transgenic" is meant any cell which includes a DNA
sequence which is inserted by artifice into a cell and becomes part
of the genome of the organism which develops from that cell. As
used herein, the transgenic organisms are generally transgenic
mammals (e.g., rodents such as rats or mice) and the DNA
(transgene) is inserted by artifice into the nuclear genome.
[0065] By "knockout mutation" is meant an alteration in the nucleic
acid sequence that reduces the biological activity of the
polypeptide normally encoded therefrom by at least 80% relative to
the unmutated gene. The mutation may, without limitation, be an
insertion, deletion, frameshift mutation, or a missense mutation.
Preferably, the mutation is an insertion or deletion, or is a
frameshift mutation that creates a stop codon.
[0066] By "transformation" is meant any method for introducing
foreign molecules into a cell. Lipofection, calcium phosphate
precipitation, retroviral delivery, electroporation, and biolistic
transformation are just a few of the teachings which may be used.
For example, biolistic transformation is a method for introducing
foreign molecules into a cell using velocity driven
microprojectiles such as tungsten or gold particles. Such
velocity-driven methods originate from pressure bursts which
include, but are not limited to, helium-driven, air-driven, and
gunpowder-driven techniques. Biolistic transformation may be
applied to the transformation or transfection of a wide variety of
cell types and intact tissues including, without limitation,
intracellular organelles (e.g., and mitochondria and chloroplasts),
bacteria, yeast, fungi, algae, animal tissue, and cultured
cells.
[0067] By "transformed cell" is meant a cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding (as used herein) a XAF
polypeptide.
[0068] By "positioned for expression" is meant that the DNA
molecule is positioned adjacent to a DNA sequence which directs
transcription and translation of the sequence (i.e., facilitates
the production of, e.g., a XAF-1 polypeptide, a recombinant protein
or a RNA molecule).
[0069] By "reporter gene" is meant any gene which encodes a product
whose expression is detectable. A reporter gene product may have
one of the following attributes, without restriction: fluorescence
(e.g., green fluorescent protein), enzymatic activity (e.g.,
luciferase or chloramphenicol acetyl transferase), toxicity (e.g.,
ricin), or an ability to be specifically bound by a second molecule
(e.g., biotin or a detectably labeled antibody).
[0070] By "promoter" is meant a minimal sequence sufficient to
direct transcription. Also included in the invention are those
promoter elements which are sufficient to render promoter-dependent
gene expression controllable for cell type-specific,
tissue-specific or inducible by external signals or agents; such
elements may be located in the 5' or 3' or intron sequence regions
of the native gene.
[0071] By "operably linked" is meant that a gene and one or more
regulatory sequences are connected in such a way as to permit gene
expression when the appropriate molecules (e.g., transcriptional
activator proteins) are bound to the regulatory sequences.
[0072] By "conserved region" is meant any stretch of six or more
contiguous amino acids exhibiting at least 30%, preferably 50%, and
most preferably 70% amino acid sequence identity between two or
more of the XAF family members, (e.g., between human XAF-1 and
another human XAF).
[0073] By "detectably-labeled" is meant any means for marking and
identifying the presence of a molecule, e.g., an oligonucleotide
probe or primer, a gene or fragment thereof, or a cDNA molecule.
Methods for detectably-labeling a molecule are well known in the
art and include, without limitation, radioactive labeling (e.g.,
with an isotope such as .sup.32P or .sup.35S) and nonradioactive
labeling (e.g., chemiluminescent labeling, e.g., fluorescein
labeling).
[0074] By "antisense," as used herein in reference to nucleic
acids, is meant a nucleic acid sequence that is complementary to
the coding strand of a gene, preferably, a XAF gene.
[0075] By "purified antibody" is meant antibody which is at least
60%, by weight, free from proteins and naturally occurring organic
molecules with which it is naturally associated. Preferably, the
preparation is at least 75%, more preferably 90%, and most
preferably at least 99%, by weight, antibody, e.g., a XAF-1, XAF-2
N-terminus, XAF-2L, or XAF-2S specific antibody. A purified
antibody may be obtained, for example, by affinity chromatography
using recombinantly-produced protein or conserved motif peptides
and standard techniques.
[0076] By "specifically binds" is meant an antibody that recognizes
and binds a XAF polypeptide but that does not substantially
recognize and bind other non-XAF molecules in a sample, e.g., a
biological sample, that naturally includes protein. A preferred
antibody binds to the XAF-1 peptide sequence of FIG. 1 (SEQ ID NO.:
2). Another preferred antibody binds to the XAF-2 N-terminus
peptide sequence of FIG. 35 (SEQ ID NO.: 4). Yet another preferred
antibody binds to the XAF-2L peptide sequence of FIG. 37 (SEQ ID
NO.: 10). Still another preferred antibody binds to the XAF-2S
peptide sequence of FIG. 38C (SEQ ID NO.: 12). A more preferred
antibody binds to two or more of XAF-1 (SEQ ID NO.: 2), XAF-2
N-terminus (SEQ ID NO.: 4), XAF-2L (SEQ ID NO.: 10) and XAF-2S (SEQ
ID NO.: 12).
[0077] By "neutralizing antibodies" is meant antibodies that
interfere with any of the biological activities of a XAF
polypeptide, particularly the ability of a XAF to participate in
apoptosis. The neutralizing antibody may reduce the ability of a
XAF polypeptide to participate in apoptosis by, preferably 50%,
more preferably by 70%, and most preferably by 90% or more. Any
standard assay of apoptosis, including those described herein, may
be used to assess potentially neutralizing antibodies.
[0078] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0079] FIG. 1 is a listing of the cDNA (above; SEQ ID NO.: 1) and
predicted amino acid (below; SEQ ID NO.: 2) sequences of human
XAF-1.
[0080] FIG. 2 is a schematic diagram of the six predicted Zn finger
binding domains corresponding to the N-terminal 178 amino acids of
XAF-1 (SEQ ID NO.: 6).
[0081] FIG. 3 is a Northern blot analysis of XAF-1 mRNA in multiple
human tissues and various cell lines.
[0082] FIGS. 4A and 4B are a Northern dot-blot analysis of XAF-1
mRNA in multiple adult and fetal human tissues.
[0083] FIG. 5 is a genomic Southern blot analysis of XAF-1.
[0084] FIG. 6 is a Western blotting analysis of XAF-1 protein
expression level in various cell lines.
[0085] FIG. 7 are schematic diagrams of XAF-1 constructs.
[0086] FIG. 8 is a Western blotting analysis of XAF-1, XAF-1N (SEQ
ID NO.: 7) and XAF-1C (SEQ ID NO.: 8) protein expression levels
when transiently expressed in 293T cells.
[0087] FIG. 9 is a graph of showing the effect of p53 and XAF
overexpression on survival of HEL cells.
[0088] FIG. 10 shows the effect of p53 and XAF overexpression on
survival of HeLa cells.
[0089] FIGS. 11A, 11B, and 11C show photographs of HEL cells
infected with adeno-LacZ, adeno-p53 and adeno-XAF-1,
respectively.
[0090] FIGS. 12A, 12B, and 12C show photographs of HeLa cells
infected with adeno-LacZ, adeno-p53 and adeno-XAF-1,
respectively.
[0091] FIGS. 13A, 13B, 13C, and 13D are graphs showing cell cycle
profiles of HEL cells transfected with nothing, adeno-LacZ,
adeno-p53, and adeno-XAF-1, respectively.
[0092] FIGS. 14A, 14B, 14C, and 14D are graphs showing cell cycle
profiles of HeLa cells transfected with nothing, adeno-LacZ,
adeno-p53, and adeno-XAF-1, respectively.
[0093] FIGS. 15A and 15B show localization of human XAF-1 by FISH.
FIG. 15A shows metaphase spread hybridized with XAF-1 genomic
probe. FIG. 15B shows metaphase spread after G banding with Giemsa
stain. Specific fluorescent signals on 17p13.3 are indicated by
arrows.
[0094] FIGS. 16A and 16B show subcellular localization of the XAF-1
protein.
[0095] FIGS. 17A, 17B, and 17C are photographs of CHO-K1 cells
expressing green fluorescent protein (GFP)-labeled XAF-1 visualized
with a fluorescent microscope.
[0096] FIGS. 18A and 118B are photographs of 3Y1 cells expressing
GFP visualized with a fluorescent microscope.
[0097] FIGS. 19A and 19B are photographs of 3Y1 cells expressing
GFP-labeled XAF-1 visualized with a fluorescent microscope.
[0098] FIG. 20 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by expression of indicated proteins.
[0099] FIG. 21 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by co-expression of indicated
proteins.
[0100] FIG. 22 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by TRAF6 co-expressed with indicated
amounts of XAF-1 protein.
[0101] FIG. 23 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by TRAF6 co-expressed with indicated
amounts of XIAP protein.
[0102] FIG. 24 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by TRAF6 co-expressed with XIAP and XAF-1
proteins.
[0103] FIG. 25 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by TRAF2 co-expressed with XIAP and XAF-1
proteins.
[0104] FIG. 26 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by TRAF6 co-expressed with either
full-length XAF-1 protein, a fragment representing the N-terminus
of XAF-1 protein, or a fragment representing the C-terminus of
XAF-1 protein.
[0105] FIG. 27 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by either TRAF5 or TRAF6 when
co-expressed with either XAF-1 antisense DNA or Bcl-2 antisense
DNA.
[0106] FIG. 28 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by interleukin-1.beta. (IL-1.beta.) in
the presence of either XAF-1 antisense RNA or Bcl-2 antisense RNA
expression.
[0107] FIG. 29 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by interleukin-1.beta. (IL-1.beta.) in
the presence of DNA encoding for XAF-1 protein.
[0108] FIG. 30 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by TRAF2, TRAF5, or TRAF6 co-expressed
with A20 protein.
[0109] FIG. 31 is a graph of relative luciferase activity induced
by NF-.kappa.B activation by increasing amounts of A20 protein
co-expressed with TRAF6 alone, or in combination with XAF-1.
[0110] FIG. 32 is a Western blot analysis of myc-tagged proteins
from affinity-purifications with GST-control and GST-XAF-1 fusion
proteins.
[0111] FIG. 33 is an autoradiograph of an in vitro binding assay of
in vitro translated HIAP-1 and TRAF2 proteins with GST-control and
GST-XAF-1 fusion proteins.
[0112] FIG. 34 is a table listing the interaction results of a
yeast two-hybrid assay.
[0113] FIG. 35 is a listing of the cDNA (above; SEQ ID NO.: 3) and
the predicted amino acid (below; SEQ ID NO.: 4) sequences of the
N-terminus of human XAF-2. The seven zinc finger motifs are boxed
and labeled in Roman numerals.
[0114] FIG. 36 is a listing of the 3' untranslated region (UTR) DNA
sequence (SEQ ID NO.: 5) of human XAF-2 which is located about 250
base pairs C-terminally of SEQ ID NO.:3.
[0115] FIG. 37A is a listing of the full length 5' nucleotide
(above; SEQ ID NO.: 9) and amino acid (below; SEQ ID NO.: 10)
sequences of the long (XAF-2L) splice variant of XAF-2. The shorter
splice variant of XAF-2 (XAF-2S) is spliced as indicated.
[0116] FIG. 37B is an alignment comparing the nucleic acid sequence
of XAF-2L (above) with the entire nucleic acid sequence of XAF-2S
(below; SEQ ID NO.: 11).
[0117] FIGS. 38A, 38B, and 38C are the amino acid sequence listings
of XAF-1, XAF-2L, and XAF-2S (SEQ ID NO.: 12), respectively, with
the zinc finger binding domains indicated.
[0118] FIG. 39 is an alignment comparing the sequence of the first
396 amino acids of XAF-2L (above) with the entire amino acid
sequence of XAF-1 (below).
[0119] FIG. 40 is a set of two schematic drawings indicating the
alignment of the zinc finger binding domains in XAF-1 (above) and
XAF-2L (below).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0120] We have discovered a new family of proteins, the XAFs, which
interact with IAPs and are involved the TNF.alpha. signal
transduction pathway which regulates apoptosis.
[0121] The TNF receptor superfamily includes at least 13
transmembrane type I glycoproteins composed of two identical
subunits with variable numbers of a characteristic cysteine rich
extracellular repeat. Included among these members are TNF receptor
1 (TNF-R1), TNF receptor 2 (TNF-R2), CD40, Fas, and CD30. The
corresponding ligands for these receptors are typically type II
transmembrane glycoproteins expressed on the surface of interacting
cells. In some instances, notably lymphotoxin-.alpha. (also known
as TNF.beta.) and the majority of tumor necrosis factor-.alpha.
(TNF.alpha.), the ligand is secreted from the cell.
[0122] The signals generated by ligated members of the TNF receptor
superfamily can be stimulatory or inhibitory depending on the
nature and activation state of the target cell. However, there is
considerable overlap in the signal transduction pathways; for
instance, ligation of TNF-R1, TNF-R2, CD30, and CD40 (Kitson et
al., Nature 384: 372-275, 1996) all result in NF-.kappa.B
activation, a transcription factor found latent in the cytoplasm of
cells complexed to an inhibitor protein termed I-.kappa.B. Receptor
ligation induces the phosphorylation of I-.kappa.B, which renders
I-.kappa.B susceptible to ubiquitination and subsequent
degradation. I-.kappa.B degradation unveils the nuclear
translocation signal in NF-.kappa.B and allows nuclear localization
and activation of transcription from NF-.kappa.B dependent
promoters (reviewed in Grilli et al., Int. Rev. Cytol. 143: 1-60,
1993).
[0123] Tumor necrosis factor-.alpha. (TNF.alpha.), mediates its
diverse effects through both the 55-60 kDa TNF-R1 and 75-80 kDa
TNF-R2 receptors. The cytoplasmic domains of TNF-R1 and TNF-R2 are
not conserved, which is reflected in both the protein factors
associated with the cytoplasmic domains and in the consequences of
receptor stimulation. TNF-.alpha. signaling through TNF-R2 can
induce either proliferative responses (i.e., thymocyte and
mononuclear proliferation; Tartaglia et al., Proc. Natl. Acad. Sci.
USA 88: 9292-9296, 1991; Tartaglia, et al., J. Immunol. 151:
4637-4641, 1993; Gehr et al., J. Immunol. 149: 911-917, 1992), or
cytolytic responses (Heller et al., Cell 70: 47-52, 1992; Grell et
al., Lymphokine Cytokine Res. 12: 143-148, 1993) depending upon the
cell type and activation state.
[0124] Immunoprecipitation of TNF-R2 complexes and peptide sequence
analysis of the associated proteins identified HIAP-1 and HIAP-2 as
components of the unstimulated TNF-R2 signaling complex.
Protein-protein interaction analysis has established that the BIR
domains of HIAP-1 and HIAP-2 can bind interchangeably to the TRAF-N
domains of TRAF1 and TRAF2 (Rothe et al., Cell 83: 1243-1252,
1995). To date, very little is known regarding the distribution and
function of the protein components of the TNF-R2 complex following
receptor ligation. Likewise, the functional consequences of HIAP-1
and HIAP-2 in the TNF-R2 receptor complex have not been
determined.
[0125] The role of HIAP-2 in the TNF-R1 receptor signaling complex
has, in contrast, been more clearly defined.
[0126] The intracellular domain of TNF-R1 contains an approximately
80 amino acid protein-protein interaction motif termed a "death
domain", which is also found in the low affinity nerve growth
factor and Fas receptors. The cytoplasmic death domain of TNF-R1
does not appear to associate with components of the signal
transduction pathways prior to ligand binding. The primary effects
of TNF-R1 aggregation are NF-.kappa.B activation and apoptosis.
These effects are dependent upon interaction of TNF-R1 with TRADD
(INF-R1 associated death domain protein; Hsu et al., Cell 81:
495-504, 1995), through their respective death domains. TRADD
functions as an adapter molecule which can recruit a variety of
proteins to the signaling complex. The formation of alternative
signaling complexes likely determines the ultimate fate of the
cell.
[0127] In certain circumstances, TRADD is capable of triggering the
formation of a protein complex called the DISC (Death Inducing
Signaling Complex). DISC formation occurs when FADD is recruited to
the TNF-R1/TRADD complex, again through interaction of death
domains (Chinnaiyan et al., Cell 81: 505-512 1995; Chinnaiyan et
al., J. Biol. Chem. 271: 4961-4965, 1996). In addition to a carboxy
terminal death domain, FADD possesses an amino terminal "death
effector domain" (DED), which triggers apoptosis by recruiting
FLICE (caspase-8). FLICE possesses an unusually long amino terminal
pro-domain containing two DED homologous sequences which bind to
the FADD DED. Bringing FLICE molecules into close proximity results
in proteolytic auto-activation. The cleavage event that activates
FLICE also releases the enzyme from the DISC, at which point it
proteolytically activates other caspases and ultimately results in
apoptosis (Muzio et al., Cell 85: 817-827, 1996, Boldin et al., 85:
803-815 1996). Dominant-negative mutants of FADD block apoptosis
through either Fas or TNF-R1, indicating that the FADD component is
responsible for propagating the cell death signal generated through
either receptor (Chinnaiyan et al., J. Biol. Chem. 271: 4961-4965,
1996).
[0128] However, TNF.alpha. binding to TNF-R1 does not result in
apoptosis in all circumstances. The formation of an alternative
signaling complex contributes to the pliability of the TNF.alpha.
response. The "survival complex" that corresponds to the DISC
consists of TRADD bound to TRAF2 (TNF receptor associated factor-2)
and HIAP-2 (Hsu et al., Immunity 4: 387-389, 1996; Hsu et al., Cell
84: 299-308, 1996). HIAP-2 is complexed to TRAF2 prior to TNF-R1
stimulation (Hsu et al., Cell 84: 299-308, 1996). This protein
interaction may enhance the affinity of TRAF2 for binding to TRADD,
thereby favoring the formation of TRADD/TRAF2 complexes rather than
the TRADD/FADD/FLICE DISC. Alternatively, HIAP-2 may interact with
other components of the apoptotic pathway, such as the caspases; in
ways which suppress the apoptotic signals that would otherwise be
generated.
[0129] We have now demonstrated that XAF family members interact
with IAPs and are clearly involved in apoptotic and NF-.kappa.B
inducing signaling pathways in mammalian cells. Overexpression of
XAF-1 causes cell death in transformed cells. Interestingly,
overexpression in non-transformed cells merely leads to growth
(cell cycle) arrest. The distinct functions transformed and merely
proliferating cells is surprising and significant. Our Western and
Northern blot analyses indicate that XAF-1 is expressed in a
variety of tissues and cell types. Since apoptosis is an event
non-specific to any particular cell or tissue type, these findings
are in keeping with the involvement of the XAF-1 protein in
apoptosis in a variety of contexts.
[0130] We have also discovered a second XAF family member, XAF-2L.
XAF-2L, like XAF-1, also has seven zinc finger binding domains. A
second shorter XAF-2 splice variant, XAF-2S, has also been
discovered.
I. The XAF-1 Gene
[0131] A yeast 2-hybrid screen of a human placenta cDNA library
with XIAP as the `bait` protein identified a 37 kDa zinc finger
protein termed XAF-1 (XIAP Associated Factor 1). XAF-1 displays
significant homology to members of the TRAF family, particularly
TRAF6, but lacks the TRAF-C and TRAF-N domains.
II. Synthesis of XAF Proteins
[0132] The characteristics of the cloned XAF gene sequences may be
analyzed by introducing the sequence into various cell types or
using in vitro extracellular systems. The function of XAF proteins
may then be examined under different physiological conditions. For
example, the XAF-1-encoding DNA sequence may be manipulated in
studies to understand the expression of the XAF-1 gene and gene
product. Alternatively, cell lines may be produced which
over-express the XAF gene product allowing purification of XAF for
biochemical characterization, large-scale production, antibody
production, and patient therapy.
[0133] For protein expression, eukaryotic and prokaryotic
expression systems may be generated in which XAF gene sequences are
introduced into a plasmid or other vector which is then used to
transform living cells. Constructs in which the XAF cDNAs
containing the entire open reading frames inserted in the correct
orientation into an expression plasmid may be used for protein
expression. Alternatively, portions of the XAF gene sequences,
including wild-type or mutant XAF sequences, may be inserted.
Prokaryotic and eukaryotic expression systems allow various
important functional domains of the XAF proteins to be recovered as
fusion proteins and then used for binding, structural and
functional studies and also for the generation of appropriate
antibodies. Since XAF-1 protein expression increases apoptosis in
immortalized cells, it may be desirable to express the protein
under the control of an inducible promoter.
[0134] Typical expression vectors contain promoters that direct the
synthesis of large amounts of mRNA corresponding to the inserted
XAF nucleic acid in the plasmid bearing cells. They may also
include eukaryotic or prokaryotic origin of replication sequences
allowing for their autonomous replication within the host organism,
sequences that encode genetic traits that allow vector-containing
cells to be selected for in the presence of otherwise toxic drugs,
and sequences that increase the efficiency with which the
synthesized mRNA is translated. Stable long-term vectors may be
maintained as freely replicating entities by using regulatory
elements of, for example, viruses (e.g., the OriP sequences from
the Epstein Barr Virus genome). Cell lines may also be produced
which have integrated the vector into the genomic DNA, and in this
manner the gene product is produced on a continuous basis.
[0135] Expression of foreign sequences in bacteria such as
Escherichia coli requires the insertion of the XAF nucleic acid
sequence into a bacterial expression vector. This plasmid vector
contains several elements required for the propagation of the
plasmid in bacteria, and expression of inserted DNA of the plasmid
by the plasmid-carrying bacteria. Propagation of only
plasmid-bearing bacteria is achieved by introducing in the plasmid
selectable marker-encoding sequences that allow plasmid-bearing
bacteria to grow in the presence of otherwise toxic drugs. The
plasmid also bears a transcriptional promoter capable of producing
large amounts of mRNA from the cloned gene. Such promoters may or
may not be inducible promoters which initiate transcription upon
induction. The plasmid also preferably contains a polylinker to
simplify insertion of the gene in the correct orientation within
the vector. In a simple E. coli expression vector utilizing the lac
promoter, the expression vector plasmid contains a fragment of the
E. coli chromosome containing the lac promoter and the neighboring
lacZ gene. In the presence of the lactose analog IPTG, RNA
polymerase normally transcribes the lacZ gene producing lacZ mRNA
which is translated into the encoded protein, .beta.-galactosidase.
The lacZ gene can be cut out of the expression vector with
restriction endonucleases and replaced by a XAF gene sequence, or
fragment, fusion, or mutant thereof. When this resulting plasmid is
transfected into E. coli, addition of IPTG and subsequent
transcription from the lac promoter produces XAF mRNA, which is
translated into a XAF polypeptide.
[0136] Once the appropriate expression vectors containing a XAF
gene, or fragment, fusion, or mutant thereof, are constructed they
are introduced into an appropriate host cell by transformation
techniques including calcium phosphate transfection, DEAE-dextran
transfection; electroporation, micro-injection, protoplast fusion
and liposome-mediated transfection. The host cell which are
transfected with the vectors of this invention may be selected from
the group consisting of E. coli, pseudomonas, Bacillus subtilus, or
other bacilli, other bacteria, yeast, fungi, insect (using, for
example, baculoviral vectors for expression), mouse or other animal
or human tissue cells. Mammalian cells can also be used to express
the XAF-1 protein using a vaccinia virus expression system
described in Ausubel et al. (Current Protocols in Molecular
Biology, John Wiley & Sons, New York, N.Y., 1994).
[0137] In vitro expression of XAF proteins, fusions, polypeptide
fragments, or mutants encoded by cloned DNA is also possible using
the T7 late-promoter expression system. This system depends on the
regulated expression of T7 RNA polymerase which is an enzyme
encoded in the DNA of bacteriophage T7. The T7 RNA polymerase
transcribes DNA beginning within a specific 23-bp promoter sequence
called the T7 late promoter. Copies of the T7 late promoter are
located at several sites on the T7 genome, but none is present in
E. coli chromosomal DNA. As a result, in T7 infected cells, T7 RNA
polymerase catalyzes transcription of viral genes but not of E.
coli genes. In this expression system recombinant E. coli cells are
first engineered to carry the gene encoding T7 RNA polymerase next
to the lac promoter. In the presence of IPTG, these cells
transcribe the T7 polymerase gene at a high rate and synthesize
abundant amounts of T7 RNA polymerase. These cells are then
transformed with plasmid vectors that carry a copy of the T7 late
promoter protein. When IPTG is added to the culture medium
containing these transformed E. coli cells, large amounts of T7 RNA
polymerase are produced. The polymerase then binds to the T7 late
promoter on the plasmid expression vectors, catalyzing
transcription of the inserted cDNA at a high rate. Since each E.
coli cell contains many copies of the expression vector, large
amounts of mRNA corresponding to the cloned cDNA can be produced in
this system and the resulting protein can be radioactively labeled.
Plasmid vectors containing late promoters and the corresponding RNA
polymerases from related bacteriophages such as T3, T5, and SP6 may
also be used for in vitro production of proteins from cloned DNA.
E. coli can also be used for expression by infection with M13 Phage
mGPI-2. E. coli vectors can also be used with phage lambda
regulatory sequences, by fusion protein vectors, by maltose-binding
protein fusions, and by glutathione-S-transferase fusion
proteins.
[0138] Eukaryotic expression systems permit appropriate
post-translational modifications to expressed proteins. Transient
transfection of a eukaryotic expression plasmid allows the
transient production of a XAF polypeptide by a transfected host
cell. XAF proteins may also be produced by a stably-transfected
mammalian cell line. A number of vectors suitable for stable
transfection of mammalian cells are available to the public (e.g.,
see Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985,
Supp. 1987), as are methods for constructing such cell lines (see
e.g., Ausubel et al., supra). In one example, cDNA encoding a XAF-1
protein, fusion, mutant, or polypeptide fragment is cloned into an
expression vector that includes the dihydrofolate reductase (DHFR)
gene. Integration of the plasmid and, therefore, integration of the
XAF-1-encoding gene into the host cell chromosome is selected for
by inclusion of 0.01-300 .mu.M methotrexate in the cell culture
medium (as described, Ausubel et al., supra). This dominant
selection can be accomplished in most cell types. Recombinant
protein expression can be increased by DHFR-mediated amplification
of the transfected gene. Methods for selecting cell lines bearing
gene amplifications are described in Ausubel et al. (supra). These
methods generally involve extended culture in medium containing
gradually increasing levels of methotrexate. The most commonly used
DHFR-containing expression vectors are pCVSEII-DHFR and pAdD26SV(A)
(described in Ausubel et al., supra). The host cells described
above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO
DHFR.sup.- cells, ATCC Accession No. CRL 9096) are among those most
preferred for DHFR selection of a stably-transfected cell line or
DHFR-mediated gene amplification.
[0139] Eukaryotic cell expression of XAF proteins allows for
studies of the XAF genes and gene products including determination
of proper expression and post-translational modifications for
biological activity, identifying regulatory elements located in the
5' region of XAF genes and their roles in tissue regulation of XAF
protein expression. It also permits the production of large amounts
of normal and mutant proteins for isolation and purification, and
the use of cells expressing XAF proteins as a functional assay
system for antibodies generated against the protein. Eukaryotic
cells expressing XAF proteins may also be used to test the
effectiveness of pharmacological agents on XAF associated
apoptosis, or as means by which to study XAF proteins as components
of a signal transduction system. Expression of XAF proteins,
fusions, mutants, and polypeptide fragments in eukaryotic cells
also enables the study of the function of the normal complete
protein, specific portions of the protein, or of naturally
occurring polymorphisms and artificially produced mutated proteins.
The XAF DNA sequences can be altered using procedures known in the
art, such as restriction endonuclease digestion, DNA polymerase
fill-in, exonuclease deletion, terminal deoxynucleotide transferase
extension, ligation of synthetic or cloned DNA sequences and
site-directed sequence alteration using specific oligonucleotides
together with PCR.
[0140] Another preferred eukaryotic expression system is the
baculovirus system using, for example, the vector pBacPAK9, which
is available from Clontech (Palo Alto, Calif.). If desired, this
system may be used in conjunction with other protein expression
techniques, for example, the myc tag approach described by Evan et
al. (Mol. Cell Biol. 5:3610-3616, 1985).
[0141] Once the recombinant protein is expressed, it can be
isolated from the expressing cells by cell lysis followed by
protein purification techniques, such as affinity chromatography.
In this example, an anti-XAF antibody, which may be produced by the
methods described herein, can be attached to a column and used to
isolate the recombinant XAF proteins. Lysis and fractionation of
XAF protein-harboring cells prior to affinity chromatography may be
performed by standard methods (see e.g., Ausubel et al., supra).
Once isolated, the recombinant protein can, if desired, be purified
further by e.g., by high performance liquid chromatography (HPLC;
e.g., see Fisher, Laboratory Techniques In Biochemistry And
Molecular Biology, Work and Burdon, Eds., Elsevier, 1980).
[0142] Polypeptides of the invention, particularly short XAF-1
fragments and longer fragments of the N-terminus and C-terminus of
the XAF-1 protein, can also be produced by chemical synthesis
(e.g., by the methods described in Solid Phase Peptide Synthesis,
2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). These
general techniques of polypeptide expression and purification can
also be used to produce and isolate useful XAF-1 polypeptide
fragments or analogs, as described herein.
[0143] Those skilled in the art of molecular biology will
understand that a wide variety of expression systems may be used to
produce the recombinant XAF proteins. The precise host cell used is
not critical to the invention. The XAF proteins may be produced in
a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g.,
S. cerevisiae, insect cells such as Sf9 cells, or mammalian cells
such as COS-1, NIH 3T3, or HeLa cells). These cells are
commercially available from, for example, the American Type Culture
Collection, Rockville, Md. (see also Ausubel et al., supra). The
method of transformation and the choice of expression vehicle
(e.g., expression vector) will depend on the host system selected.
Transformation and transfection methods are described, e.g., in
Ausubel et al. (supra), and expression vehicles may be chosen from
those provided, e.g., in Pouwels et al., supra.
III. Testing for the Presence of XAF Biological Activity
[0144] Identification of XAF-1 and XAF-2 splice variants allow the
study of XAF biological activity in apoptosis-associated cellular
events. For example, administration of a XAF-1 protein, or
polypeptide fragment thereof, may have an ability to induce
apoptosis, as measured by apoptosis assays known in the art and
described herein. An apoptosis-inhibiting amount of a XAF reagent
(e.g., a compound that reduced the biological function of XAF-1,
such as a XAF-1 neutralizing antibody or antisense XAF-1 nucleic
acid) may be similarly assessed. Such assays may be carried out in
a cell which either expresses endogenous XAF-1, or a cell to which
is introduced a heterologous amount of a XAF-1 polypeptide.
Preferably, the cell is capable of undergoing apoptosis. Apoptosis
or inhibition thereof may be assessed in these XAF expressing
cells, whereby such apoptosis inducing or inhibiting activity is
evaluated based upon the level of expression of the XAF
polypeptide.
[0145] Another approach, which utilizes the activation of the
nuclear transcription factor, NF-.kappa.B (Kunkel et al., Crit.
Rev. Immunol. 9: 93-117, 1989) in TNF-mediated signal transduction.
In this system the role of a XAF in NF-.kappa.B activation may be
readily elucidated in various assays known in the art, such as the
I-.kappa.B degradation assay. Another method of rapidly measuring
NF-.kappa.B activity is through the use of a reporter gene whose
expression is directed by a NF-.kappa.B binding site containing
promoter (Zeichner et al., J. Virol. 65: 2436-2444, 1991). The
expression vector is preferably inserted by artifice into a cell
capable of undergoing apoptosis or is responsive to TNF-receptor
family-mediated signal transduction. By detecting a change in the
level of expression of the reporter gene, an NF-.kappa.B-inducing
ability of a XAF may be readily assessed. This method may also be
used to detect an NF-.kappa.B-inhibiting ability of a XAF wherein
NF-.kappa.B activation is stimulated by another component of the
TNF-receptor signalling pathway (e.g., TRAF6).
[0146] It will be understood that these analyses may be undertaken
with XAF-1 or other XAF proteins (e.g., XAF-2L).
IV. Cellular Distribution of XAF-1
[0147] We have looked at the distribution of XAF-1 mRNA expression
using radiolabeled antisense XAF-1 DNA and have found that XAF-1
mRNA is expressed in at least the following adult tissues: heart,
brain, placenta, liver, skeletal muscle, kidney, pancreas, spleen,
thymus, prostate, testis, ovary, appendix, trachea, small
intestine, submucosal lining of the colon, and peripheral blood
leukocytes. XAF-1 mRNA was further found to be expressed in fetal
tissue, including fetal brain, fetal heart, fetal kidney, fetal
liver, fetal spleen, fetal thymus, and fetal lung.
V. XAF Fragments
[0148] Polypeptide fragments which incorporate various portions of
XAF proteins are useful in identifying the domains important for
the biological activities of XAF proteins. Methods for generating
such fragments are well known in the art (see, for example, Ausubel
et al., supra) using the nucleotide sequences provided herein. For
example, a XAF protein fragment may be generated by PCR amplifying
the desired fragment using oligonucleotide primers designed based
upon the XAF-1 (SEQ ID NO.: 1) nucleic acid sequences. Preferably
the oligonucleotide primers include unique restriction enzyme site
which facilitate insertion of the fragment into the cloning site of
a mammalian expression vector. This vector may then be introduced
into a mammalian cell by artifice by the various techniques known
in the art and described herein, resulting in the production of a
XAF gene fragment.
[0149] In one approach, XAF-1 polypeptide fragments have been
useful in evaluating the portions of the protein involved in
NF-.kappa.B regulation. In particular, polypeptide fragments of the
amino- and carboxyl-termini of XAF-1 protein were used to induce or
prevent activity induction by various other components of the
TNF-receptor signalling pathway (e.g., TRAF6).
[0150] In an alternative approach, polypeptide fragments of various
portions of the XAF-1 protein are useful in modulating XAF-1
mediated apoptosis, as may be assessed in the various apoptosis
assays known in the art and described herein. XAF-1 polypeptide
fragments may be used to alter XAF-1 mediated apoptosis by
inhibiting binding of the full length XAF-1 to, for example, itself
to form XAF-1:XAF-1 homodimers, to another XAF protein (e.g.,
XAF-2) to form XAF-1:XAF-2 heterodimers, or to XIAP to form
XAF-1:XIAP heterodimers. Preferably, such fragments may include the
XAF-1:XAF-1 binding domain, the XAF-1:XAF-2 binding domain or the
XAF-1:XIAP binding domain.
VI. XAF Antibodies
[0151] In order to prepare polyclonal antibodies, XAF proteins,
fragments of XAF proteins, or fusion proteins containing defined
portions of XAF proteins can be synthesized in bacteria by
expression of corresponding DNA sequences in a suitable cloning
vehicle. Fusion proteins are commonly used as a source of antigen
for producing antibodies. Two widely used expression systems for E.
coli are lacZ fusions using the pUR series of vectors and trpE
fusions using the pATH vectors. The proteins can be purified, and
then coupled to a carrier protein and mixed with Freund's adjuvant
(to help stimulate the antigenic response by the animal of choice)
and injected into rabbits or other laboratory animals.
Alternatively, protein can be isolated from XAF expressing cultured
cells. Following booster injections at bi-weekly intervals, the
rabbits or other laboratory animals are then bled and the sera
isolated. The sera can be used directly or can be purified prior to
use, by various methods including affinity chromatography employing
reagents such as Protein A-Sepharose, Antigen Sepharose, and
Anti-mouse-Ig-Sepharose. The sera can then be used to probe protein
extracts from XAF expressing tissues run on a polyacrylamide gel to
identify XAF proteins. Alternatively, synthetic peptides can be
made that correspond to the antigenic portions of the protein and
used to innoculate the animals.
[0152] In order to generate peptide or full-length protein for use
in making, for example, XAF-1-specific antibodies, a XAF-1 coding
sequence can be expressed as a C-terminal fusion with glutathione
S-transferase (GST; Smith et al., Gene 67: 31-40, 1988). The fusion
protein can be purified on glutathione-Sepharose beads, eluted with
glutathione, and cleaved with thrombin (at the engineered cleavage
site), and purified to the degree required to successfully immunize
rabbits. Primary immunizations can be carried out with Freund's
complete adjuvant and subsequent immunizations performed with
Freund's incomplete adjuvant. Antibody titers are monitored by
Western blot and immunoprecipitation analyses using the
thrombin-cleaved XAF-1 fragment of the GST-XAF-1 fusion protein.
Immune sera are affinity purified using CNBr--Sepharose-coupled
XAF-1 protein. Antiserum specificity is determined using a panel of
unrelated GST proteins (including GSTp53, Rb, HPV-16 E6, and E6-AP)
and GST-trypsin (which was generated by PCR using known
sequences).
[0153] It is also understood by those skilled in the art that
monoclonal XAF antibodies may be produced by using as antigen XAF
protein isolated from XAF expressing cultured cells or XAF protein
isolated from tissues. The cell extracts, or recombinant protein
extracts, containing XAF protein, may for example, be injected with
Freund's adjuvant into mice. After being injected, the mice spleens
may be removed and resuspended in phosphate buffered saline (PBS).
The spleen cells serve as a source of lymphocytes, some of which
are producing antibody of the appropriate specificity. These are
then fused with a permanently growing myeloma partner cells, and
the products of the fusion are plated into a number of tissue
culture wells in the presence of a selective agent such as
hypoxanthine, aminopterine, and thymidine (HAT). The wells are then
screened by ELISA to identify those containing cells making
antibody capable of binding a XAF protein or polypeptide fragment
or mutant thereof. These are then re-plated and after a period of
growth, these wells are again screened to identify
antibody-producing cells. Several cloning procedures are carried
out until over 90% of the wells contain single clones which are
positive for antibody production. From this procedure a stable line
of clones which produce the antibody is established. The monoclonal
antibody can then be purified by affinity chromatography using
Protein A Sepharose, ion-exchange chromatography, as well as
variations and combinations of these techniques. Truncated versions
of monoclonal antibodies may also be produced by recombinant
methods in which plasmids are generated which express the desired
monoclonal antibody fragment(s) in a suitable host.
[0154] As an alternate or adjunct immunogen to GST fusion proteins,
peptides corresponding to relatively unique hydrophilic regions of,
for example, XAF-1 may be generated and coupled to keyhole limpet
hemocyanin (KLH) through an introduced C-terminal lysine. Antiserum
to each of these peptides is similarly affinity purified on
peptides conjugated to BSA, and specificity is tested by ELISA and
Western blotting using peptide conjugates, and by Western blotting
and immunoprecipitation using XAF-1 expressed as a GST fusion
protein.
[0155] Alternatively, monoclonal antibodies may be prepared using
the XAF proteins described above and standard hybridoma technology
(see, e.g., Kohler et al., Nature 256: 495, 1975; Kohler et al.,
Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:
292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell
Hybridomas, Elsevier, New York, N.Y., 1981; Ausubel et al., supra).
Once produced, monoclonal antibodies are also tested for specific
XAF protein recognition by Western blot or immunoprecipitation
analysis (by the methods described in Ausubel et al., supra).
[0156] Monoclonal and polyclonal antibodies that specifically
recognize a XAF protein (or fragments thereof), such as those
described herein containing a XAF-1 C-terminal domain, are
considered useful in the invention. They may, for example, be used
in an reporter gene assay to monitor the NF-.kappa.B inducing
effects (via TRAF6) of a XAF protein. Antibodies that inhibit a
XAF-1 described herein may be especially useful in preventing
apoptosis in cells undergoing undesirable cell death or growth
arrest.
[0157] Antibodies of the invention may be produced using XAF amino
acid sequences that do not reside within highly conserved regions,
and that appear likely to be antigenic, as analyzed by criteria
such as those provided by the Peptide Structure Program (Genetics
Computer Group Sequence Analysis Package, Program Manual for the
GCG Package, Version 7, 1991) using the algorithm of Jameson and
Wolf (CABIOS 4:181, 1988). These fragments can be generated by
standard techniques, e.g., by the PCR, and cloned into the pGEX
expression vector (Ausubel et al., supra). GST fusion proteins are
expressed in E. coli and purified using a glutathione agarose
affinity matrix as described in Ausubel et al. (supra). To generate
rabbit polyclonal antibodies, and to minimize the potential for
obtaining antisera that is non-specific, or exhibits low-affinity
binding to a XAF, two or three fusions are generated for each
protein, and each fusion is injected into at least two rabbits.
Antisera are raised by injections in series, preferably including
at least three booster injections.
[0158] In addition, antibodies of the invention may be produced
using XAF amino acid sequences that do reside within highly
conserved regions. For example, amino acid sequences from the
N-terminal 150 amino acids of either XAF-1 or XAF-2 may be used as
antigen to generate antibodies specific toward both XAF-1 and
XAF-2, and possibly specific toward other members of the XAF family
of proteins. These antibodies may be screened as described
above.
[0159] In addition to intact monoclonal and polyclonal anti-XAF-1
antibodies, the invention features various genetically engineered
antibodies, humanized antibodies, and antibody fragments, including
F(ab')2, Fab', Fab, Fv and sFv fragments. Antibodies can be
humanized by methods known in the art, e.g., monoclonal antibodies
with a desired binding specificity can be commercially humanized
(Scotgene, Scotland; Oxford Molecular, Palo Alto, Calif.). Fully
human antibodies, such as those expressed in transgenic animals,
are also features of the invention (Green et al., Nature Genetics
7: 13-21, 1994).
[0160] Ladner (U.S. Pat. Nos. 4,946,778 and 4,704,692) describes
methods for preparing single polypeptide chain antibodies. Ward et
al. (Nature 341: 544-546, 1989) describe the preparation of heavy
chain variable domains, which they term "single domain antibodies,"
which have high antigen-binding affinities. McCafferty et al.
(Nature 348: 552-554, 1990) show that complete antibody V domains
can be displayed on the surface of fd bacteriophage, that the phage
bind specifically to antigen, and that rare phage (one in a
million) can be isolated after affinity chromatography. Boss et al.
(U.S. Pat. No. 4,816,397) describe various methods for producing
immunoglobulins, and immunologically functional fragments thereof,
which include at least the variable domains of the heavy and light
chain in a single host cell. Cabilly et al. (U.S. Pat. No.
4,816,567) describe methods for preparing chimeric antibodies.
VII. Use of XAF Antibodies
[0161] Antibodies to XAF proteins may be used, as noted above, to
detect XAF proteins or to inhibit the biological activities of XAF
proteins. In addition, the antibodies may be coupled to compounds
for diagnostic and/or therapeutic uses such as radionucleotides for
imaging and therapy and liposomes for the targeting of compounds to
a specific tissue location.
VIII. Detection of XAF Gene Expression
[0162] As noted, the antibodies described above may be used to
monitor XAF protein expression. In addition, in situ hybridization
is a method which may be used to detect the expression of XAF
genes. In situ hybridization techniques, such as fluorescent in
situ hybridization (FISH), rely upon the hybridization of a
specifically labeled nucleic acid probe to the cellular RNA in
individual cells or tissues. Therefore, it allows the
identification of mRNA within intact tissues, such as the heart. In
this method, oligonucleotides or cloned nucleotide (RNA or DNA)
fragments corresponding to unique portions of XAF genes are used to
detect specific mRNA species, e.g., in the heart. Numerous other
gene expression detection techniques are known to those of skill in
the art and may be employed here.
IX. Identification of Compounds that Modulate XAF Protein
Expression
[0163] Based on our experimental results, we have developed a
number of screening procedures for identifying therapeutic
compounds (e.g., anti-apoptotic or apoptotic-inducing) which can be
used in human patients. In particular examples, compounds that down
regulate expression of XAF proteins are considered useful in the
invention for treatment of diseases hallmarked by an excessive
amount of apoptosis, such as neurodegenerative disorders.
Similarly, compounds that up regulate or activate XAF proteins are
also considered useful as drugs for the treatment of diseases
hallmarked by impaired apoptosis, such as cancer. In general, the
screening methods of the invention involve screening any number of
compounds for therapeutically active agents by employing any number
of in vitro or in vivo experimental systems.
[0164] The methods of the invention simplify the evaluation,
identification, and development of active agents for the treatment
and prevention of conditions involving an inappropriate amount of
apoptosis, which may be excessive or insufficient, depending upon
the condition. These screening methods provide a facile means for
selecting natural product extracts or compounds of interest from a
large population which are further evaluated and condensed to a few
active and selective materials. Constituents of this pool are then
purified and evaluated in the methods of the invention to determine
their anti-apoptotic or apoptotic-inducing activities.
[0165] In general, novel drugs for the treatment of conditions
involving an appropriate level of apoptosis are identified from
large libraries of both natural product or synthetic (or
semi-synthetic) extracts or chemical libraries according to methods
known in the art. Those skilled in the field of drug discovery and
development will understand that the precise source of test
extracts or compounds is not critical to the screening procedure(s)
of the invention. Accordingly, virtually any number of chemical
extracts or compounds can be screened using the exemplary methods
described herein. Examples of such extracts or compounds include,
but are not limited to, plant-, fungal-, prokaryotic- or
animal-based extracts, fermentation broths, and synthetic
compounds, as well as modification of existing compounds. Numerous
methods are also available for generating random or directed
synthesis (e.g., semi-synthesis or total synthesis) of any number
of chemical compounds, including, but not limited to, saccharide-,
lipid-, peptide-, and nucleic acid-based compounds. Synthetic
compound libraries are commercially available from Brandon
Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee,
Wis.). Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant, and animal extracts are commercially
available from a number of sources, including Biotics (Sussex, UK),
Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft.
Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In
addition, natural and synthetically produced libraries are
produced, if desired, according to methods known in the art, e.g.,
by standard extraction and fractionation methods. Furthermore, if
desired, any library or compound is readily modified using standard
chemical, physical, or biochemical methods.
[0166] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication
(e.g., taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their anti-apoptotic or apoptotic-inducing activities should be
employed whenever possible.
[0167] When a crude extract is found to have anti-apoptotic or
apoptotic-inducing activities or both, further fractionation of the
positive lead extract is necessary to isolate chemical constituents
responsible for the observed effect. Thus, the goal of the
extraction, fractionation, and purification process is the careful
characterization and identification of a chemical entity within the
crude extract having anti-apoptotic or apoptotic-inducing
activities. The same in vivo and in vitro assays described herein
for the detection of activities in mixtures of compounds can be
used to purify the active component and to test derivatives
thereof. Methods of fractionation and purification of such
heterogenous extracts are known in the art. If desired, compounds
shown to be useful agents for the treatment of pathogenicity are
chemically modified according to methods known in the art.
Compounds identified as being of therapeutic value are subsequently
analyzed using any standard animal model of degenerative disease or
cancer known in the art.
[0168] Below we describe screening methods for identifying and
evaluating the efficacy of a compound as an anti-apoptotic or
apoptotic-inducing agent. These methods are intended to illustrate,
not limit, the scope of the claimed invention.
[0169] 1) Screens for Compounds Affecting XAF Protein
Expression
[0170] XAF cDNAs may be used to facilitate the identification of
compounds that increase or decrease XAF protein expression. In one
approach, candidate compounds are added, in varying concentrations,
to the culture medium of cells expressing XAF mRNA. The XAF mRNA
expression is then measured, for example, by Northern blot analysis
(Ausubel et al., supra) using a XAF DNA, or cDNA or RNA fragment,
as a hybridization probe. The level of XAF mRNA expression in the
presence of the candidate compound is compared to the level of XAF
mRNA expression in the absence of the candidate compound, all other
factors (e.g., cell type and culture conditions) being equal.
[0171] The effect of candidate compounds on XAF-mediated apoptosis
may, instead, be measured at the level of translation by using the
general approach described above with standard protein detection
techniques, such as Western blotting or immunoprecipitation with a
XAF-specific antibody (for example, the XAF-1 specific antibody
described herein).
[0172] In an alternative approach to detecting compounds which
regulate XAF at the level of transcription, candidate compounds may
be tested for an ability to regulate a reporter gene whose
expression is directed by a XAF gene promoter. For example, a cell
unlikely to undergo apoptosis may be transfected with a expression
plasmid that includes a luciferase reporter gene operably linked to
the XAF-1 promoter. Candidate compounds may then be added, in
varying concentrations, to the culture medium of the cells.
Luciferase expression levels may then be measured by subjecting the
compound-treated transfected cells to standard luciferase assays
known in the art, such as the luciferase assay system kit used
herein that is commercially available from Promega, and rapidly
assessing the level of luciferase activity on a luminometer. The
level of luciferase expression in the presence of the candidate
compound is compared to the level of luciferase expression in the
absence of the candidate compound, all other factors (e.g., cell
type and culture conditions) being equal.
[0173] Compounds that modulate the level of XAF protein expression
may be purified, or substantially purified, or may be one component
of a mixture of compounds such as an extract or supernatant
obtained from cells, from mammalian serum, or from growth medium in
which mammalian cells have been cultured (Ausubel et al., supra).
In an assay of a mixture of compounds, XAF protein expression is
tested against progressively smaller subsets of the compound pool
(e.g., produced by standard purification techniques such as HPLC or
FPLC) until a single compound or minimal number of effective
compounds is demonstrated to modulate XAF protein expression.
[0174] 2) Screens for Compounds Affecting XAF Biological
Activity
[0175] Compounds may also be screened for their ability to
modulate, for example, XAF-1 apoptosis inducing activity. In this
approach, the degree of apoptosis in the presence of a candidate
compound is compared to the degree of apoptosis in its absence,
under equivalent conditions. Again, the screen may begin with a
pool of candidate compounds, from which one or more useful
modulator compounds are isolated in a step-wise fashion. Apoptosis
activity may be measured by any standard assay, for example, those
described herein.
[0176] Another method for detecting compounds that modulate the
apoptosis-inducing activity of XAF has been to screen for compounds
that interact physically with a given XAF polypeptide, e.g., XAF-1.
These compounds were detected by adapting yeast two-hybrid
expression systems known in the art. These systems detected protein
interactions using a transcriptional activation assay and are
generally described by Gyuris et al. (Cell 75:791-803, 1993) and
Field et al. (Nature 340:245-246, 1989), and are commercially
available from Clontech (Palo Alto, Calif.). In addition, PCT
Publication WO 95/28497 describes a yeast two-hybrid assay in which
proteins involved in apoptosis, by virtue of their interaction with
BCL-2, were detected. A similar method has been used to identify
proteins and other compounds that interacted with XAF-1, and is
used to identify XAF-2 splice variant interactors.
[0177] A compound that promotes an increase in the expression or
biological activity of the XAF protein, e.g., XAF-1, is considered
particularly useful in the invention; such a molecule may be used,
for example, as a therapeutic to increase cellular levels of XAF-1
and thereby exploit the ability of XAF-1 polypeptides to induce
apoptosis. This would be advantageous in the treatment of diseases
involving insufficient apoptosis (e.g., cancer).
[0178] A compound that decreases XAF-1 activity (e.g., by
decreasing XAF-1 gene expression or biological activity) may also
be used to increase cellular proliferation. This would be
advantageous in the treatment of degenerative diseases, such as
neurodegenerative diseases (e.g., Alzheimer's disease, Huntington's
disease) or other tissue-specific degenerative diseases (e.g.,
cirrhosis of the liver, T-lymphocyte depletion in AIDS, hair
loss).
[0179] Molecules that are found, by the methods described above, to
effectively modulate XAF gene expression or polypeptide activity
may be tested further in animal models. If they continue to
function successfully in an in vivo setting, they may be used as
therapeutics to either inhibit or enhance apoptosis, as
appropriate.
X. Therapies
[0180] Therapies may be designed to circumvent or overcome a XAF
gene defect or inadequate XAF gene expression, and thus modulate
and possibly alleviate conditions involving an inappropriate amount
of apoptosis. XAF-1 is expressed in the every tissue looked at thus
far. Hence, in considering various therapies, it is understood that
such therapies may be targeted at any tissues demonstrated to
express XAF-1. In particular, therapies to enhance XAF-1 gene
expression are useful in promoting apoptosis in cancerous cells.
Apoptosis-inducing XAF-1 reagents may include, without limitation,
full length or fragment XAF-1 polypeptides, XAF-1 mRNA, or any
compound which increases XAF-1 apoptosis-inducing activity.
[0181] a) Protein Therapy
[0182] Treatment or prevention of inappropriate apoptosis can be
accomplished by replacing mutant or surplus XAF protein with normal
protein, by modulating the function of mutant protein, or by
delivering normal XAF protein to the appropriate cells. It is also
be possible to modify the pathophysiologic pathway (e.g., a signal
transduction pathway) in which the protein participates in order to
correct the physiological defect.
[0183] To replace a mutant protein with normal protein, or to add
protein to cells which no longer express sufficient XAF, it is
necessary to obtain large amounts of pure XAF protein from cultured
cell systems which can express the protein. Delivery of the protein
to the affected tissues (e.g., cancerous tissues) can then be
accomplished using appropriate packaging or administrating systems.
Alternatively, small molecule analogs may be used and administered
to act as XAF agonists and in this manner produce a desired
physiological effect. Methods for finding such molecules are
provided herein.
[0184] b) Gene Therapy
[0185] Gene therapy is another potential therapeutic approach in
which normal copies of the XAF gene or nucleic acid encoding XAF
antisense RNA are introduced into selected tissues to successfully
encode for normal and abundant protein or XAF antisense RNA in
cells which inappropriately either suppress cell death (e.g.,
cancerous ovarian cells) or enhance the rate of cell death (e.g.,
neuronal cell death leading to disease), respectively. The gene
must be delivered to those cells in a form in which it can be taken
up and encode for sufficient protein to provide effective function.
Alternatively, in some mutants it may be possible to promote
apoptosis by introducing another copy of the homologous gene
bearing a second mutation in that gene or to alter the mutation, or
use another gene to block any negative effect.
[0186] Transducing retroviral vectors can be used for somatic cell
gene therapy especially because of their high efficiency of
infection and stable integration and expression. The targeted cells
however must be able to divide and the expression levels of normal
protein should be high. For example, the full length XAF-1 gene, or
portions thereof, can be cloned into a retroviral vector and driven
from its endogenous promoter or from the retroviral long terminal
repeat or from a promoter specific for the target cell type of
interest (such as neurons). Other viral vectors which can be used
include adenovirus, adeno-associated virus, vaccinia virus, bovine
papilloma virus, or a herpes virus such as Epstein-Barr Virus.
[0187] Gene transfer could also be achieved using non-viral means
requiring infection in vitro. This would include calcium phosphate,
DEAE dextran, electroporation, and protoplast fusion. Liposomes may
also be potentially beneficial for delivery of DNA into a cell.
Although these methods are available, many of these are lower
efficiency.
[0188] Transplantation of normal genes into the affected cells of a
patient can also be useful therapy. In this procedure, a normal XAF
gene is transferred into a cultivatable cell type, either
exogenously or endogenously to the patient. These cells are then
injected serotologically into the targeted tissue(s).
[0189] Retroviral vectors, adenoviral vectors,
adenovirus-associated viral vectors, or other viral vectors with
the appropriate tropism for cells likely to be involved in
apoptosis (for example, epithelial cells) may be used as a gene
transfer delivery system for a therapeutic XAF gene construct.
Numerous vectors useful for this purpose are generally known
(Miller, Human Gene Therapy 15-14, 1990; Friedman, Science
244:1275-1281, 1989; Eglitis and Anderson, BioTechniques 6:
608-614, 1988; Tolstoshev and Anderson, Curr. Opin. Biotech. 1:
55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et
al., Nucl. Acid Res and Mol. Biol. 36: 311-322, 1987; Anderson,
Science 226: 401-409, 1984; Moen, Blood Cells 17: 407-416, 1991;
Miller et al., Biotech. 7: 980-990, 1989; Le Gal La Salle et al.,
Science 259: 988-990, 1993; and Johnson, Chest 107: 77S-83S, 1995).
Retroviral vectors are particularly well developed and have been
used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:
370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). Non-viral
approaches may also be employed for the introduction of therapeutic
DNA into cells otherwise predicted to undergo apoptosis. For
example, XAF may be introduced into a neuron or a T cell by
lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413,
1987; Ono et al., Neurosci. Lett. 117: 259, 1990; Brigham et al.,
Am. J. Med. Sci. 298: 278, 1989; Staubinger et al., Meth. Enz.
101:512, 1983, asialorosonucoid-polylysine conjugation (Wu et al.,
J. Biol. Chem. 263: 14621, 1988; Wu et al., J. Biol. Chem. 264:
16985, 1989); or, less preferably, micro-injection under surgical
conditions (Wolff et al., Science 247: 1465, 1990).
[0190] In another approach that may be utilized with all of the
above methods, a therapeutic XAF DNA construct is preferably
applied to the site of the desired apoptosis event (for example, by
injection). However, it may also be applied to tissue in the
vicinity of the desired apoptosis event or to a blood vessel
supplying the cells (e.g., cancerous cells) desired to undergo
apoptosis.
[0191] In the constructs described, XAF cDNA expression can be
directed from any suitable promoter (e.g., the human
cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein
promoters), and regulated by any appropriate mammalian regulatory
element. For example, if desired, enhancers known to preferentially
direct gene expression in neural cells, lymphocytes, or muscle
cells may be used to direct XAF expression. The enhancers used
could include, without limitation, those that are characterized as
tissue- or cell-specific in their expression. Alternatively, if a
XAF genomic clone is used as a therapeutic construct (for example,
following isolation by hybridization with the XAF cDNA described
above), regulation may be mediated by the cognate regulatory
sequences or, if desired, by regulatory sequences derived from a
heterologous source, including any of the promoters or regulatory
elements described above.
[0192] Antisense based strategies have employed to explore XAF gene
function and as a basis for therapeutic drug design. The principle
is based on the hypothesis that sequence-specific suppression of
gene expression can be achieved by intracellular hybridization
between mRNA and a complementary antisense species. The formation
of a hybrid RNA duplex may then interfere with the
processing/transport/translation and/or stability of the target XAF
mRNA. Antisense strategies may use a variety of approaches
including the use of antisense oligonucleotides and injection of
antisense RNA. For our analysis of XAF-1 gene function, we employed
the method of transfection of antisense RNA expression vectors into
targeted cells. Antisense effects can be induced by control (sense)
sequences, however, the extent of phenotypic changes are highly
variable. Phenotypic effects induced by antisense effects are based
on changes in criteria such as protein levels, protein activity
measurement, and target mRNA levels.
[0193] For example, XAF-1 gene therapy may also be accomplished by
direct administration of antisense XAF-1 mRNA to a cell that is
expected to undergo undesired apoptosis. The antisense XAF-1 mRNA
may be produced and isolated by any standard technique, but is most
readily produced by in vitro transcription using an antisense XAF-1
cDNA under the control of a high efficiency promoter (e.g., the T7
promoter). Administration of antisense XAF-1 mRNA to cells can be
carried out by any of the methods for direct nucleic acid
administration described above.
[0194] Another therapeutic approach within the invention involves
administration of recombinant XAF polypeptide, either directly to
the site of a desired apoptosis event (for example, by injection)
or systemically (for example, by any conventional recombinant
protein administration technique). The dosage of XAF depends on a
number of factors, including the size and health of the individual
patient, but, generally, between 0.1 mg and 100 mg inclusive are
administered per day to an adult in any pharmaceutically acceptable
formulation.
XI. Administration of XAF Polypeptides, XAF Genes, or Modulators of
XAF Synthesis or Function
[0195] A XAF protein, gene, or modulator may be administered within
a pharmaceutically-acceptable diluent, carrier, or excipient, in
unit dosage form. Conventional pharmaceutical practice may be
employed to provide suitable formulations or compositions to
administer neutralizing XAF antibodies or XAF-inhibiting compounds
(e.g., antisense XAF-1 or a XAF-1 dominant negative mutant) to
patients suffering from a disease (e.g., a degenerative disease)
that is caused by excessive apoptosis. Administration may begin
before the patient is symptomatic. Any appropriate route of
administration may be employed, for example, administration may be
parenteral, intravenous, intra-arterial, subcutaneous,
intramuscular, intracranial, intraorbital, ophthalmic,
intraventricular, intracapsular, intraspinal, intracisternal,
intraperitoneal, intranasal, aerosol, by suppositories, or oral
administration. Therapeutic formulations may be in the form of
liquid solutions or suspensions; for oral administration,
formulations may be in the form of tablets or capsules; and for
intranasal formulations, in the form of powders, nasal drops, or
aerosols.
[0196] Methods well known in the art for making formulations are
found, for example, in Remington's Pharmaceutical Sciences,
(18.sup.th edition), ed. A. Gennaro, 1990, Mack Publishing Company,
Easton, Pa. Formulations for parenteral administration may, for
example, contain excipients, sterile water, or saline, polyalkylene
glycols such as polyethylene glycol, oils of vegetable origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for XAF modulatory compounds include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Formulations for
inhalation may contain excipients, for example, lactose, or may be
aqueous solutions containing, for example, polyoxyethylene-9-lauryl
ether, glycocholate and deoxycholate, or may be oily solutions for
administration in the form of nasal drops, or as a gel.
[0197] If desired, treatment with a XAF protein, gene, or
modulatory compound may be combined with more traditional therapies
for the disease involving excessive apoptosis, such as surgery,
steroid therapy, or chemotherapy for autoimmune disease; antiviral
therapy for AIDS; and tissue plasminogen activator (TPA) for
ischemic injury. Likewise, treatment with a XAF protein, gene, or
modulatory compound may be combined with more traditional therapies
for the disease involving insufficient apoptosis, such as surgery,
radiation therapy, and chemotherapy for cancer.
XII. Detection of Conditions Involving Altered Apoptosis
[0198] XAF polypeptides and nucleic acid sequences find diagnostic
use in the detection or monitoring of conditions involving aberrant
levels of apoptosis. For example, decreased expression of XAF-1 may
be correlated with decreased apoptosis in humans. Accordingly, a
decrease or increase in the level of XAF-1 production may provide
an indication of a deleterious condition. Levels of XAF expression
may be assayed by any standard technique. For example, XAF
expression in a biological sample (e.g., a biopsy) may be monitored
by standard Northern blot analysis or may be aided by PCR (see,
e.g., Au subel et al., supra; PCR Technology: Principles and
Applications for DNA Amplification, H. A. Ehrlich, Ed. Stockton
Press, NY; Yap et al. Nucl. Acids. Res. 19: 4294, 1991).
[0199] Alternatively, a biological sample obtained from a patient
may be analyzed for one or more mutations in XAF nucleic acid
sequences using a mismatch detection approach. Generally, these
techniques involve PCR amplification of nucleic acid from the
patient sample, followed by identification of the mutation (i.e.,
mismatch) by either altered hybridization, aberrant electrophoretic
gel migration, binding or cleavage mediated by mismatch binding
proteins, or direct nucleic acid sequencing. Any of these
techniques may be used to facilitate mutant XAF detection, and each
is well known in the art; examples of particular techniques are
described, without limitation, in Orita et al. (Proc. Natl. Acad.
Sci. USA 86: 2766-2770, 1989) and Sheffield et al. (Proc. Natl.
Acad. Sci. USA 86: 232-236, 1989).
[0200] In yet another approach, immunoassays are used to detect or
monitor XAF protein expression in a biological sample. XAF-specific
polyclonal or monoclonal antibodies (produced as described above)
may be used in any standard immunoassay format (e.g., ELISA,
Western blot, or RIA) to measure XAF polypeptide levels. These
levels would be compared to wild-type XAF levels. For example, a
decrease in XAF-1 production may indicate a condition involving
insufficient apoptosis. Examples of immunoassays are described,
e.g., in Ausubel et al., supra. Immunohistochemical techniques may
also be utilized for XAF detection. For example, a tissue sample
may be obtained from a patient, sectioned, and stained for the
presence of XAF using an anti-XAF antibody and any standard
detection system (e.g., one which includes a secondary antibody
conjugated to horseradish peroxidase). General guidance regarding
such techniques can be found in, e.g., Bancroft and Stevens (Theory
and Practice of Histological Techniques, Churchill Livingstone,
0.1982) and Ausubel et al. (supra).
[0201] In one preferred example, a combined diagnostic method may
be employed that begins with an evaluation of XAF protein
production (for example, by immunological techniques or the protein
truncation test (Hogerrorst et al., Nature Genetics 10: 208-212,
1995) and also includes a nucleic acid-based detection technique
designed to identify more subtle XAF mutations (for example, point
mutations). As described above, a number of mismatch detection
assays are available to those skilled in the art, and any preferred
technique may be used. Mutations in XAF may be detected that either
result in loss of XAF expression or loss of normal XAF biological
activity. In a variation of this combined diagnostic method, XAF-1
biological activity is measured as apoptotic-inducing activity
using any appropriate apoptosis assay system (for example, those
described herein).
[0202] Mismatch detection assays also provide an opportunity to
diagnose a XAF-mediated predisposition to diseases caused by
inappropriate apoptosis. For example, a patient heterozygous for a
XAF-1 mutation that induces a XAF-1 overexpression may show no
clinical symptoms and yet possess a higher than normal probability
of developing one or more types of neurodegenerative,
myelodysplastic or having severe sequelae to an ischemic event.
Given this diagnosis, a patient may take precautions to minimize
their exposure to adverse environmental factors (for example, UV
exposure or chemical mutagens) and to carefully monitor their
medical condition (for example, through frequent physical
examinations). This type of XAF-1 diagnostic approach may also be
used to detect XAF-1 mutations in prenatal screens. The XAF-1
diagnostic assays described above may be carried out using any
biological sample (for example, any biopsy sample or other tissue)
in which XAF-1 is normally expressed. Identification of a mutant
XAF-1 gene may also be assayed using these sources for test
samples.
[0203] Alternatively, a XAF mutation, particularly as part of a
diagnosis for predisposition to XAF-associated degenerative
disease, may be tested using a DNA sample from any cell, for
example, by mismatch detection techniques. Preferably, the DNA
sample is subjected to PCR amplification prior to analysis.
XIII. Preventative Anti-Apoptotic Therapy
[0204] In a patient diagnosed to be heterozygous for a XAF mutation
or to be susceptible to XAF mutations or aberrant XAF expression
(even if those mutations or expression patterns do not yet result
in XAF overexpression or increased XAF biological activity), or a
patient diagnosed with a degenerative disease (e.g., motor neuron
degenerative diseases such as SMA or ALS diseases), or diagnosed as
HIV positive, any of the above therapies may be administered before
the occurrence of the disease phenotype. For example, the therapies
may be provided to a patient who is HIV positive but does not yet
show a diminished T cell count or other overt signs of AIDS. In
particular, compounds shown to decrease XAF-1 expression or XAF-1
biological activity may be administered to patients diagnosed with
degenerative diseases by any standard dosage and route of
administration (see above). Alternatively, gene therapy using a
antisense XAF-1 mRNA expression construct may be undertaken to
reverse or prevent the cell defect prior to the development of the
degenerative disease.
[0205] The methods of the instant invention may be used to reduce
or diagnose the disorders described herein in any mammal, for
example, humans, domestic pets, or livestock. Where a non-human
mammal is treated or diagnosed, the XAF polypeptide, nucleic acid,
or antibody employed is preferably specific for that species.
XIV. Identification of Additional XAF Genes
[0206] Standard techniques, such as the polymerase chain reaction
(PCR) and DNA hybridization, may be used to clone additional XAF
homologues in other species. Southern blots of murine genomic DNA
hybridized at low stringency with probes specific for human XAF
reveal bands that correspond to XAF and/or related family members.
Thus, additional XAF sequences may be readily identified using low
stringency hybridization. Furthermore, murine and human
XAF-specific primers may be used to clone additional XAF related
genes by RT-PCR.
[0207] Thus far, we have identified multiple ESTs in the data base
that have significant homology to XAF-1. From the EST sequences, we
have made oligo primers and PCR cloned "XAF-2." The N terminus of
the XAF-2 protein has five of the amino-terminal zinc fingers of
XAF-1, with a unique carboxy terminus that has two additional RING
zinc fingers, so that the entire XAF-2 protein, like XAF-1, has
seven Zinc finger binding domains.
XV. Characterization of XAF Activity and Intracellular Localization
Studies
[0208] The ability of XAF proteins to modulate apoptosis can be
defined in in vitro systems in which alterations of apoptosis can
be detected. Mammalian expression constructs carrying XAF cDNAs,
which are either full-length or truncated, can be introduced into
cell lines such as CHO, NIH 3T3, HL60, Rat-1, or Jurkat cells. In
addition, SF9 insect cells may be used, in which case the XAF gene
is preferentially expressed using an insect baculovirus expression
system. Following transfection, apoptosis can be induced by
standard methods, which include serum withdrawal, or application of
staurosporine, menadione (which induces apoptosis via free radical
formation), or anti-Fas or anti-TNF-R1 antibodies. As a control,
cells are cultured under the same conditions as those induced to
undergo apoptosis, but either not transfected, or transfected with
a vector that lacks a XAF insert. The ability of each XAF construct
to induce or inhibit apoptosis upon expression can be quantified by
calculating the survival index of the cells, i.e., the ratio of
surviving transfected cells to surviving control cells. These
experiments can confirm the presence of apoptosis inducing activity
of the full length XAF-1 protein and, as discussed below, can also
be used to determine the functional region(s) of XAF-1 protein.
These assays may also be performed in combination with the
application of additional compounds in order to identify compounds
that modulate apoptosis via XAF expression.
XVI. Examples of Additional Apoptosis Assays
[0209] Specific examples of apoptosis assays are also provided in
the following references. Assays for apoptosis in lymphocytes are
disclosed by: Li et al., "Induction of apoptosis in uninfected
lymphocytes by HIV-1 Tat protein", Science 268: 429-431, 1995;
Gibellini et al., "Tat-expressing Jurkat cells show an increased
resistance to different apoptotic stimuli, including acute human
immunodeficiency virus-type 1 (HIV-1) infection", Br. J. Haematol.
89: 24-33, 1995; Martin et al., "HIV-1 infection of human CD4.sup.+
T cells in vitro. Differential induction of apoptosis in these
cells." J. Immunol. 152:330-342, 1994; Terai et al., "Apoptosis as
a mechanism of cell death in cultured T lymphoblasts acutely
infected with HIV-1", J. Clin. Invest. 87: 1710-1715, 1991; Dhein
et al., "Autocrine T-cell suicide mediated by APO-1/(Fas/CD95)",
Nature 373: 438-441, 1995; Katsikis et al., "Fas antigen
stimulation induces marked apoptosis of T lymphocytes in human
immunodeficiency virus-infected individuals", J. Exp. Med.
1815:2029-2036, 1995; Westendorp et al., "Sensitization of T cells
to CD95-mediated apoptosis by HIV-1 Tat and gp12O", Nature 375:497,
1995; DeRossi et al., Virology 198:234-244, 1994.
[0210] Assays for apoptosis in fibroblasts are disclosed by:
Vossbeck et al., "Direct transforming activity of TGF-beta on rat
fibroblasts", Int. J. Cancer 61:92-97, 1995; Goruppi et al.,
"Dissection of c-myc domains involved in S phase induction of
NIH3T3 fibroblasts", Oncogene 9:1537-44, 1994; Fernandez et al.,
"Differential sensitivity of normal and Ha-ras transformed C3H
mouse embryo fibroblasts to tumor necrosis factor: induction of
bcl-2, c-myc, and manganese superoxide dismutase in resistant
cells", Oncogene 9:2009-2017, 1994; Harrington et al.,
"c-Myc-induced apoptosis in fibroblasts is inhibited by specific
cytokines", EMBO J. 13:3286-3295, 1994; Itoh et al., "A novel
protein domain required for apoptosis. Mutational analysis of human
Fas antigen", J. Biol. Chem. 268:10932-10937, 1993.
[0211] Assays for apoptosis in neuronal cells are disclosed by:
Melino et al., "Tissue transglutaminase and apoptosis: sense and
antisense transfection studies with human neuroblastoma cells",
Mol. Cell Biol. 14:6584-6596, 1994; Rosenbaum et al., "Evidence for
hypoxia-induced, programmed cell death of cultured neurons", Ann.
Neurol. 36:864-870, 1994; Sato et al., "Neuronal differentiation of
PC12 cells as a result of prevention of cell death by bcl-2", J.
Neurobiol. 25:1227-1234, 1994; Ferrari et al., "N-acetylcysteine D-
and L-stereoisomers prevents apoptotic death of neuronal cells", J.
Neurosci. 1516:2857-2866, 1995; Talley et al., "Tumor necrosis
factor alpha-induced apoptosis in human neuronal cells: protection
by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA",
Mol. Cell Biol. 1585:2359-2366, 1995; Talley et al., "Tumor
Necrosis Factor Alpha-Induced Apoptosis in Human Neuronal Cells:
Protection by the Antioxidant N-Acetylcysteine and the Genes bcl-2
and crmA", Mol. Cell. Biol. 15:2359-2366, 1995; Walkinshaw et al.,
"Induction of apoptosis in catecholaminergic PC12 cells by L-DOPA.
Implications for the treatment of Parkinson's disease", J. Clin.
Invest. 95:2458-2464, 1995.
[0212] Assays for apoptosis in insect cells are disclosed by: Clem
et al., "Prevention of apoptosis by a baculovirus gene during
infection of insect cells", Science 254:1388-1390, 1991; Crook et
al., "An apoptosis-inhibiting baculovirus gene with a zinc
finger-like motif", J. Virol. 67:2168-2174, 1993; Rabizadeh et al.,
"Expression of the baculovirus p35 gene inhibits mammalian neural
cell death", J. Neurochem. 61:2318-2321, 1993; Birnbaum et al., "An
apoptosis inhibiting gene from a nuclear polyhedrosis virus
encoding a polypeptide with Cys/His sequence motifs", J. Virol.
68:2521-2528, 1994; Clem et al., Mol. Cell. Biol. 14:5212-5222,
1994.
XVII. Construction of a Transgenic Animal
[0213] Characterization of XAF genes provides information that is
necessary for XAF knockout animal models to be developed by
homologous recombination. Preferably, the model is a mammalian
animal, most preferably a mouse. Similarly, an animal model of XAF
overproduction may be generated by integrating one or more XAF
sequences into the genome, according to standard transgenic
techniques.
[0214] A replacement-type targeting vector, which would be used to
create a knockout model, can be constructed using an isogenic
genomic clone, for example, from a mouse strain such as 129/Sv
(Stratagene Inc., LaJolla, Calif.). The targeting vector will be
introduced into a suitably-derived line of embryonic stem (ES)
cells by electroporation to generate ES cell lines that carry a
profoundly truncated form of a XAF gene. To generate chimeric
founder mice, the targeted cell lines will be injected into a mouse
blastula stage embryo. Heterozygous offspring will be interbred to
homozygosity. Knockout mice would provide the means, in vivo, to
screen for therapeutic compounds that modulate apoptosis via a
XAF-dependent pathway. Making such mice may require use of loxP
sites if there are multiple copies of XAF genes (i.e., genes
encoding XAF-1 and another XAF polypeptide) on the chromosome (see
Sauer and Henderson, Nucleic Aids Res. 17: 147-61, 1989).
[0215] The following examples are to illustrate the invention. They
are not meant to limit the invention in any way.
EXAMPLE I
cDNA and Predicted Amino Acid Sequences of Cloned Human XAF-1
[0216] Yeast 2-hybrid analysis (see U.S. Ser. No. 08/511,485 and
related applications) with XIAP as the "bait" protein identified a
37 kDa, RING zinc finger protein termed XAF-1 (XIAP associated
factor 1).
Methods
[0217] The plasmid pAS2-XIAP, which encodes the GAL4 DNA-binding
domain fused to full-length XIAP, was constructed by inserting the
coding region of full length XIAP into the pAS2 plasmid which is
commercially available from Clontech. PAS2-XIAP was then used as
bait (DNA-binding domain hybrid) in yeast two-hybrid screens of the
human placenta cDNA library commercially available from Clontech.
The yeast two-hybrid assay and isolation of positive clones and
subsequent interaction analyses were carried out as described (PCT
Publication WO 95/28497). DNA sequence was performed on an Applied
Biosytems model 373A automated DNA sequencer.
Results
[0218] Shown in FIG. 1 is the complete nucleotide sequence of XAF-1
cDNA determined for the coding strand (SEQ ID NO: 1; EMBL accession
number X99699) and is shown with its encoded protein below in
single letter code (SEQ ID NO.: 2). The asterisk indicates the stop
codon. The entire XAF-1 protein is predicted to have seven Zinc
finger binding domains, six of which are located in the N-terminal
178 amino acids. XAF-1 displays significant homology to members of
the TRAF family, particularly TRAF6, but lacks the TRAF-C and
TRAF-N domains.
EXAMPLE II
Predicted Zinc fingers of XAF-1 Amino-Terminus
Results
[0219] Shown on FIG. 2 is a schematic of the six predicted Zinc
finger binding domains corresponding to the N-terminal 178 amino
acids of XAF-1 (SEQ ID NO.: 6).
EXAMPLE III
Northern Blot Analysis of XAF-1 mRNA in Multiple Human Tissues
Methods
[0220] Using methods described in the art (see, for example,
Ausubel, et al., supra), mRNA was collected from tissues from
heart, brain, placenta, lunch, liver, skeletal muscle, kidney,
pancreas, spleen, thymus, prostate, testis, ovary, small intestine,
mucosal lining of the colon, and peripheral blood leukocytes. mRNA
was also collected from the following cell lines: [0221] HL-60. a
promyelocytic leukemia; [0222] HeLa/S3, a cervix epitheliod
carcinoma; [0223] K-562, a chronic myelogenous leukemia; [0224]
MOLT-4, a lymphobastic leukemia; [0225] Raji, a Burkitt's lymphoma;
[0226] SW480, a colorectal adenocarcinoma; [0227] A549, a lung
carcinoma; and [0228] G361, a melanoma.
[0229] The mRNA samples were electrophoretically resolved and
transferred to a nitrocellulose membrane, which was then subjected
to Northern blot analysis for the presence and expression levels of
XAF-1 mRNA using radioisotope labeled XAF-1 cDNA as a probe (as
described in Ausubel, et al., supra).
[0230] Additional mRNA was also collected from lung, trachea, and
placenta, as well as various subunits of the brain, heart, testis,
kidney, and fetal tissue. RNA from yeast and E. Coli bacteria was
also collected. This RNA, as well as DNA collected from human, E.
Coli bacteria, and yeast, was dot-blotted on a dot-blot apparatus,
electrophoretically transferred to a nitrocellulose membrane, and
probed with radioisotope labeled XAF-1 cDNA for the presence and
expression levels of XAF-1 mRNA.
Results
[0231] mRNA encoding XAF-1 is clearly expressed in normal cells in
various tissues. FIG. 3 shows a Northern blotting analysis reveals
XAF-1 mRNA to be widely distributed among the various tissues
tested, with expression levels highest in the heart, placenta,
spleen, thymus, ovary, small intestine, mucosal lining of the
colon, and peripheral blood leukocytes. XAF-1 mRNA is also present
in K-562 and MOLT-4 leukemic cell lines.
[0232] The dot-blot analysis of various tissues shown in FIG. 4
reveals that XAF-1 mRNA is widely distributed among the various
indicated regions of the brain, heart, testes, kidney, lung,
trachea, placenta, and fetal tissue. XAF-1 mRNA is not found,
however, in yeast or the E. coli strain of bacteria.
EXAMPLE IV
Genomic Southern Blot Analysis of XAF-1
Methods
[0233] Genomic DNA was prepared from HEC38-0 human endometrial
adenocarcinoma cells available from the ATCC (Bethesda, Md.) and
Raji cells, digested with BamH1, EcoR1 and HindIII restriction
endonucleases, electrophoretically resolved and transferred to a
nitrocellulose membrane. Membrane bound DNA was subjected to
Southern blot analysis using radioisotope labeled XAF-1 cDNA as a
probe.
Results
[0234] As shown in FIG. 5, the gene encoding XAF-1 appears to be
limited in copy number in the human genome and is the same in both
HEC38-0 and Raji cells, indicating that there is most likely only
one gene encoding XAF-1, and that this gene is the same in the two
cell lines assayed.
EXAMPLE V
Western Blot Analysis of XAF-1 Protein in Various Cell Lines
Methods
[0235] A number of transformed, immortalized and a primary cell
line were tested by Western blot analysis for the presence and
expression levels of XAF-1 protein using mouse polyclonal
anti-XAF-1 antisera, which were obtained by providing GST-fusion
proteins of XAF-1 and XIAP to the MBL Co., Ltd. (Japan) for use as
immunogens. Cells were lysed, and lysates SDS-PAGE resolved,
electrophoretically transferred to a nylon membrane, and
immunoblotted with anti-XAF-1 polyclonal antisera. The
membrane-bound proteins were then blotted with commercially
available horseradish peroxidase conjugated anti-mouse secondary
antibody and visualized with a chemiluminescent substrate.
[0236] The cell lines used in Western blotting analysis were:
[0237] HeLa: Epitheliod carcinoma, cervix, human; [0238] A431:
Epidermoid carcinoma, human; [0239] SUDHL6: Hodgkin's lymphoma,
human; [0240] P19: Embryonal carcinoma, mouse; [0241] cos-7: Kidney
fibroblast, SV40 transformed, African green monkey; [0242] 293T:
Adenovirus type 5 transformed primary embryonal kidney, human;
[0243] CHO: Chinese hamster ovary;
[0244] For use as a positive control for Western blotting analysis,
293Tcells transiently expressing a myc-tagged XAF-1 protein were
generated by the following method:
[0245] 293T cells (2.times.10.sup.5) were transfected with 4 .mu.g
of plasmid DNA encoding XAF-1 by standard lipofection methods using
Trans-IT lipofection reagent commercially available from Mirus.
Results
[0246] Shown in FIG. 6 is the Western blotting analysis of the
various cell lines for XAF-1 expression. By this type of analysis,
XAF-1 expression appears to be ubiquitous, with low levels seen in
a number of transformed cell lines.
EXAMPLE VI
XAF-1 Constructs and Expression
Methods
[0247] Mammalian expression vectors encoding full length XAF-1, the
N-terminal 173 amino acids of XAF-1 containing six potential zinc
fingers, including the region with significant homology to TRAF4
and TRAF6 (XAF-1N; SEQ ID NO.: 7), the C-terminal 173-317 amino
acids of XAF-1 containing a single potential zinc finger domain
(XAF-1C; SEQ ID NO.: 8) were constructed by insertion of each
coding region into the pcDNA3-myc expression vector which contains
an N-terminal c-myc epitope sequence (similar vectors are
commercially available from Invitrogen). To generate the XAF-1
antisense construct, a 720 bp fragment of XAF-1 corresponding to
723-1 nucleotides (non-coding orientation) was cloned into the
pcDNA3 expression vector (Invitrogen).
[0248] 293T cells (2.times.10.sup.5) were transiently transfected
with 4 .mu.g of plasmid DNA encoding XAF-1, XAF-1N, or XAF-1C by
standard lipofection methods using Trans-IT lipofection reagent
commercially available from Mirus. About 48 hours following
transfection, the cells were lysed, and 10.sup.6 cell equivalents
were resolved by SDS-PAGE and electrophoretically transferred to a
nylon membrane. The membrane-bound proteins were then immunoblotted
with an anti-myc monoclonal antibody (9E10) (commercially available
from Amersham Life Sciences), followed by a commercially available
horseradish peroxidase conjugated secondary anti-mouse antibody.
Immunoreactive proteins were visualized by chemiluminescence
following addition of substrate.
Results
[0249] Shown in FIG. 7 are schematic diagrams of the polypeptides
encoded for by the various XAF-1 constructs. Although XAF-1
antisense is shown here in the "coding" orientation, in the vector,
it inserted and expressed in the "non-coding" orientation.
[0250] Shown in FIG. 8 is the Western blot analysis of 293T cells
transiently transfected with XAF-1, XAF-12N and XAF-1C probed with
anti-c-myc antibody. The expressed proteins show correct
electrophoretic mobility predicted from the amino acid
sequences.
EXAMPLE VII
Effect of XAF-1 Overexpression on Cell Survival
Methods
[0251] Recombinant adenoviruses were constructed that overexpress
either the LacZ protein (negative control), p53 (positive control
for cell cycle arrest), or the XAF-1 protein. HeLa (cervical
carcinoma, available from the ATCC, Bethesda, Md.) and HEL (human
embryonic in lung epithelial cells, available from the ATCC,
Bethesda, Md.) were infected with recombinant adenovirus at a
multiplicity of infection (MOI) of 10. Triplicate samples of
infected cells were harvested at t=0, 24, 48, 72, and 96 hours post
infection. Cell viability was assessed using standard MTT assays.
Briefly, the media was removed from the well and replaced with 1/10
volume of MTT (3-[4,5-dimethylthiazol-2-,
yl]-2,5-diphenyltetrazoleum bromide, available from Sigma) in
phosphate buffered saline and incubated at 37.degree. C. for 4
hours. Converted dye was then extracted using acidic isopropanol
(0.1 N HCl in 100% isopropanol) and absorbance determined at 570 nm
in a spectrophotometer. Conversion of the substrate to the 570 nm
absorbing dye is carried out by mitochondrial enzymes active in
living, but not dead cells.
[0252] The methods are further described in: Carmichael, J. et al.,
(1987) Cancer Res. 47:936-942 and Miyake, S et al., (1996) Proc.
Natl. Acad. Sci. USA 93:1320-1324.
Results
[0253] As seen in FIGS. 9 and 10, adenovirus-LacZ had no effect on
cell viability (compare to the control, CON, which were not
infected). In contrast, p53 induced a profound decrease in the
number of viable cells when primary HEL cells are used (FIG. 9),
but not in the HeLa cancer cell line (FIG. 10). The XAF-1
expressing adenovirus resulted in a similar decrease in the number
of viable cells in both HEL and HeLa cell lines. The decrease in
viability in the HeLa cell lines would therefore seem to be p53a
independent. Photographs of adeno-LacZ infected, adeno-p53 infected
and adeno-XAF-1 infected HEL (FIGS. 11A, 11B, 11C) and HeLa cells
(FIGS. 12A, 12B, 12C) are included. The morphology of the XAF-1
overexpressing HEL cells is consistent with cell cycle arrest. In
contrast, the XAF-1 overexpressing HeLa cells demonstrate classical
features of apoptosis, including pyknotic nuclei and extensive
blebbing. Photographs were taken four days post-infection using a
standard phase-contrast, inverted tissue culture microscope.
EXAMPLE VIII
Cell Cycle Analysis on XAF-1 Overexpressing HEL and HeLa Cells
Methods
[0254] 1.times.10.sup.5 HeLa or HEL cells were infected at an MOI
of 10 with recombinant adenoviruses expressing either LacZ
(negative control), p53 (positive control for cell cycle arrest) or
XAF-1. Cell were harvested at 96 hours post-infection, rinsed with
PBS and fixed with 100% ethanol. Fixed cells were centrifuged 5 min
at 1000 RPM, the ethanol removed, and the cells resuspended in 1 ml
PBS. 100 .mu.l of 0.1 mg/ml RNAse was added and the cells incubated
at 37.degree. C. for 30 minutes. 100 .mu.l of 1 mg/ml propidium
iodide was added to stain for DNA content. Cells were then analyzed
on a FACS machine and cell cycle effects examined.
Results
[0255] In HEL cells, adeno-LacZ infection had no effect on the cell
cycle profiles (compare FIG. 13A [uninfected] with FIG. 13B [LacZ
infected]). In contrast, both p53 (FIG. 13C) and XAF-1 (FIG. 13D)
expressing adenoviruses caused a virtually complete cessation of
cell cycle and a G1 arrest (note absence of S phase cells and
accumulation of G1 arrested cells). The effects of p53 and XAF-1
were identical. Infection of HeLa cells with the LacZ virus had no
effect, as seen in FIGS. 14A and 14B). In contrast to the HEL
cells, HeLa cells did not arrest when infected with the adeno-p53
virus (FIG. 14C). With the adeno-XAF-1 virus, HeLa cells did not
arrest in G1, but instead underwent apoptosis (FIG. 14C). (Note:
the changing scales on the FACS outputs give the impression of a G2
arrest [i.e., cell with 2n DNA]. In fact, the numbers of cells in S
and G2 did not change significantly). There is a loss of G1 cells
and an increase in the number of cells with less than 1n DNA
content, indicating apoptosis.
EXAMPLE IX
Chromosomal Localization of the XAF-1 Gene by Fluorescent In Situ
Hybridization (FISH)
Methods
[0256] FISH was performed on freshly isolated mouse spleen
lymphocytes cultured in RPMI 1640 media containing 15% fetal calf
serum, 3 .mu.g/ml concanavalin A, 10 .mu.g/ml lipopolysaccharide,
and 50 nM mercaptoethanol. Lymphocytes were synchronized with 180
.mu.g/ml BrdU for 14 hours followed by 4 hr growth in .alpha.-MEM
containing 2.5 .mu.g/ml thymidine. Chromosome spreads were prepared
on slides using hypotonic lysis, after which the chromosomes were
fixed and air dried. 1 .mu.g of DNA probe derived from a XAF-1
specific genomic phage clone was labeled with biotinylated dATP
using the BRL BioNick labeling kit at 15.degree. C. for 1 hr (Gibco
BRL). Slides were baked at 55.degree. C. for 1 hr, RNAse A treated,
and the chromosomes denatured in 70% formamide in 2.times.SSC for 2
min at 70.degree. C., followed by ethanol dehydration. Probe
hybridization to the denatured chromosomes was performed overnight
in 50% formamide, 10% dextran sulphate, 1 .mu.g/ml mouse cot I DNA.
Slides were washed with 2.times.SSC/50% formamide followed by
2.times.SSC at 42.degree. C. Biotin labeled DNA was amplified and
detected using fluorescein isothiocyanate conjugated avidin and
anti-avidin antibodies (FIG. 15A). Chromosomes were counterstained
with Giemsa and photographed (FIG. 15B).
Results
[0257] The XAF-1 gene was found to map to the extreme end of
chromosome 17, in the p13.3 region. This region is known to encode
an as yet unidentified tumor suppressor gene(s). This tumor
suppressor gene is believed to be involved in a large number of
tumor types, including uterine cervical carcinoma (Park et al.,
Cancer Genet. Cytogenet. 79: 74-78, 1995), breast tumors (Cornelis
et al., Cancer Res. 54: 4200-4206, 1994, Merlo et al., Cancer
Genet. Cytogenet. 76: 106-111, 1994), gastric carcinoma (Kim et
al., Lab. Invest. 72: 232-236, 1995), ovarian epithelial cancer
(Wertheim et al., Oncogene 12: 2147-2153, 1996), pediatric
medulloblastoma (McDonald et al., Genomics 23: 229-232, 1994,
reviewed in Cogan and McDonald, J. of Neuro-Oncology 29: 103-112,
1996) and lung carcinoma (White et al., Br. J. Cancer 74: 863-870,
1996). Thus XAF-1 maybe a tumor suppressor and therapies designed
to over-express XAF-1 in cancer cells may be effective (i.e., gene
therapy, compounds that up-regulate endogenous XAF-1 or compounds
that activate the XAF-1 pathway). Furthermore, the X4F-1 gene may
provide an important staging/prognostic indicator in cancer
diagnostics through the development of a LOH type assay using PCR
based detection of microsatellites in the XAF-1 locus.
EXAMPLE X
Sub-Cellular Localization of the XAF-1 Protein
Methods
[0258] Triplicate plates of HeLa cells (ATCC, Bethesda, Md.) were
infected with a recombinant adenovirus expressing the XAF-1 open
reading frame under the control of the chicken .beta.-actin
promoter at a multiplicity of infection 10. At 48 hrs post
infection, the cells were harvested in 5 ml of phosphate buffered
saline, pelleted by low speed centrifugation (5 min, 1000 rpm in a
Beckman JA-10 rotor at 4.degree. C.), and cell extracts prepared as
follows: [0259] cells were washed with isotonic Tris buffered
saline (pH 7.0) [0260] cells were lysed by freeze/thawing 5 times
in Cell Extraction Buffer (50 mM PIPES, 50 mM KCl, 5 mM EGTA, 2 mM
MgCl.sub.2, 1 mM DTT, and 20 .mu.M cytochalasin B) [0261] nuclei
were pelleted by centrifugation at 5000 RPM in a JA-17 rotor for 5
minutes. Nuclear pellet was resuspended in isotonic Tris pH 7.0,
and frozen at -80.degree. C. [0262] cytoplasmic extract was further
processed by centrifugation at 60,000 RPM in a TA 100.3 rotor for
30 minutes. Supernatant (cytoplasmic extract) was frozen at
-80.degree. C. Pelleted material (membrane fraction) was
resuspended in isotonic Tris pH 7.0, and frozen. [0263] nuclear,
membrane, and cytoplasmic fractions were electrophoresed on a 12.5%
SDS polyacrylamide gel, and electroblotted onto PVDF membranes.
[0264] Western blotting was first performed using rabbit polyclonal
anti-XAF-1 antibody at a concentration of 1:1,500 in Tris buffered
saline containing 0.5% NP-40 and 3% skim milk powder. The secondary
antibody was a horse radish peroxidase coupled goat anti-rabbit IgG
(Amersham) used at 1:2000 dilution in the same buffer system.
Chemiluminescent detection of bound antibody was performed using
Amersham's ECL kit according to the manufacturer's directions. The
membrane was then re-probed with polyclonal anti-XIAP antibody at
1:2000 dilution and processed as above. Results
[0265] FIG. 16A demonstrates that the vast majority of the
adenovirus expressed XAF-1 protein fractionates in the nuclear
compartment. A very small fraction of the protein was observed in
the membrane fraction, likely as a result of incomplete separation
of the nuclear and membrane fractions. None of the protein was
observed in the cytoplasmic fraction. FIG. 16B demonstrates that
overexpression of the XAF-1 protein resulted in a re-distribution
of >1/2 of the endogenous XIAP protein from the cytoplasmic
fraction to the nuclear fraction. One explanation for this is that
the function of XAF-1 is to relocate the XIAP protein to its "real"
site of action, in the nucleus. Alternatively, XIAP may be
interfering with the function of XAF-1 in the nucleus.
EXAMPLE XI
XAF-1 Protein is Found in the Nucleus by GFP Staining
Methods
[0266] An expression vector called pGFP-XAF-1 was constructed that
generates a fusion protein between green fluorescent protein (GFP)
and XAF-1 (Clontech). The coding region of GFP was fused to the
amino terminus of the full length XAF-1 coding region. CHO-K1 cells
or 3Y1 primary rat embryo fibroblast cells from Fischer rat fetus
(available from the Riken gene bank, Tsukuba, Japan) were
transiently transfected by standard lipofection methods using the
Trans-IT lipofection reagent commercially available from Mirus with
pGFP or pGFP-XAF-1. 24 hours following transfection, the cells were
visualized on a fluorescent microscope with a blue filter.
[0267] All cells were counter stained with evans blue.
Results
[0268] FIGS. 17A, 17B, and 17C are photographs of transfected
CHO-K1 cells. FIGS. 17A and 17B shows that in CHO-K1 cells
transiently transfected with pGFP-XAF-1, the GFP-labeled XAF-1
protein was localized to the nucleus. This is in contrast to the
GFP homogenously distributed throughout the cytoplasm and nucleus
in the CHO-K1 cells transiently transfected with pGFP shown on FIG.
17C.
[0269] FIGS. 18A and 18B shows the GFP homogenously distributed
throughout the cytoplasm and nucleus in 3Y1 cells transiently
transfected with pGFP.
[0270] FIGS. 19A and 19B shows the GFP-labeled XAF-1 protein
localized to the nucleus in 3Y1 cells transiently transfected with
pGFP-XAF-1.
[0271] We have furthermore found that XAF-1 expression resulted in
a re-distribution of XIAP protein from the cytoplasm to the
nucleus.
EXAMPLE XII
Neither XAF-1 Nor Mammalian IAPs Over-expression can induce
NF-.kappa.B Activation in 293 T Cells
[0272] The members of the growing family of TRAF proteins each
possesses an amino terminal RING zinc finger and/or additional zinc
fingers, a leucine zipper, and a unique, conserved carboxy terminal
coiled coil motif, the TRAF-C domain, which defines the family.
TRAF1 and TRAF2 were first identified as components of the TNF-R2
signaling complex (Rothe et al., Cell 78: 681-692, 1994). The
interaction of the TRAF proteins are complex, reflecting their
putative role as adapter molecules that exhibit no apparent
enzymatic activity themselves.
Methods
[0273] Mammalian expression vectors encoding XAF-1, HIAP-1, HIAP-2,
XIAP, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, RIP, and TRADD were
constructed by insertion of each coding region into the pcDNA3-myc
expression vector which contains an N-terminal c-myc epitope
sequence (similar vectors are commercially available from
Invitrogen). The NF-.kappa.B firefly luciferase reporter plasmid
pELAM-Lu was constructed by insertion of PCR-amplified E-selectin
promoter sequences from position -730 to position 52 into the
pGL3-Basic vector which is commercially available from Promega.
[0274] 293T cells were seeded into collagen-coated six-well plates
at 2.times.10.sup.5 cells per well 24 hrs before transfection.
Cells were then transfected with 0.5 .mu.g of pELAM-Lu reporter
plasmid, 0.05 .mu.g of pRL-CMV, 1 .mu.g of indicated expression
plasmid and enough pCMV-myc control plasmid to give 4 .mu.g of
total DNA by standard lipofection methods using Trans-IT
lipofection reagent commercially available from Mirus. Twenty-four
hours after transfection, cells were washed with PBS and lysed in
400 .mu.l of Passive Lysis Buffer commercially available from
Promega. Lysate (20 .mu.l) from each samples was used to measure
firefly luciferase activity. Firefly luciferase activity was
determined and normalized on the basis of Renilla luciferase
expression level. Luciferase activity was measured in a model
TD20/20 luminometer using the Dual luciferase assay system
according to the manufacturer's protocol (Promega). Values shown
are averages for an experiment in which each transfection was
performed in duplicate.
Results
[0275] XAF-1, HIAP-1, HIAP-2, and XIAP do not induced NF-.kappa.B
activation in 293 T cells. As shown in FIG. 20, when expressed
singly in 293T cells, none of the IAPs or XAF-1 resulted in
measurable activation of NF-.kappa.B, as measured by luciferase
activity. TRAF2, TRAF5, TRAF6, RIP, and TRADD expression plasmids,
however, all strongly transactivated the reporter gene. TRAF1,
TRAF3, and TRAF4 failed to transactivate the reporter.
[0276] We have also obtained data showing that XIAP can activate
NF-.kappa.B in HeLa cells.
EXAMPLE XIII
Co-Expression of XAF-1 and Mammalian IAPs do not Induce NF-.kappa.B
Activation in 293 T Cells
Methods
[0277] 293T cells were seeded into collagen-coated six-well plates
at 2.times.10.sup.5 cells per well 24 hrs before transfection.
Cells were then transfected with 0.5 .mu.g of pELAM-Lu reporter
plasmid, 0.05 .mu.g of pRL-CMV, 4 .mu.g of indicated expression
plasmid(s) and enough pCMV-myc control plasmid to give 5 .mu.g of
total DNA by standard lipofection methods using Trans-IT
lipofection reagent commercially available from Mirus. Twenty-four
hours after transfection, cells were washed with PBS and lysed in
400 .mu.l of Passive Lysis Buffer commercially available from
Promega. Lysate (20 .mu.l) from each samples was used to measure
firefly luciferase activity. Firefly luciferase activity was
determined and normalized on the basis of Renilla luciferase
expression level. Luciferase activity was measured in a model
TD20/20 luminometer using the Dual luciferase assay system
according to the manufacture's protocol (Promega). Values shown are
averages for an experiment in which each transfection was performed
in duplicate.
Results
[0278] As shown in FIG. 21, none of the IAPs, alone, or in
combination with XAF-1, resulted in measurable activation of
NF-.kappa.B when expressed in 293T cells. Expression of TRAF6,
shown here as a positive control, did induce NF-.kappa.B
activation.
EXAMPLE XIV
Dose Response Effect of XAF-1 Expression on TRAF6-Mediated
NF-.kappa.B Activation
Methods
[0279] 293T cells were seeded into collagen-coated six-well plates
at 2.times.10.sup.5 cells per well 24 hrs before transfection.
Cells were then transfected with 0.5 .mu.g of pELAM-Lu reporter
plasmid, 0.1 .mu.g of pRL-CMV, 0.5 .mu.g of pCMV-TRAF6, indicated
amounts of pCMV-XAF-1 and enough pCMV-myc control plasmid to give 4
.mu.g of total DNA by standard lipofection methods using Trans-IT
lipofection reagent commercially available from Mirus. Twenty-four
hours after transfection, cells were washed with PBS and lysed in
400 .mu.l of Passive Lysis Buffer commercially available from
Promega. Lysate (20 .mu.l) from each samples was used to measure
firefly luciferase activity. Firefly luciferase activity was
determined and normalized on the basis of Renilla luciferase
expression level. Luciferase activity was measured in a model
TD20/20 luminometer using the Dual luciferase assay system
according to the manufacture's protocol (Promega). Values shown are
averages for an experiment in which each transfection was performed
in duplicate.
Results
[0280] As the results shown in FIG. 22 demonstrate, although
expression of TRAF6 was by itself capable of inducing NF-.kappa.B
activity, co-expression of TRAF6 with XAF-1 resulted in an
increased level of NF-.kappa.B activation which increased as the
amount of XAF-1 expression increased. Hence, XAF-1 was able to
enhance the NF-.kappa.B inducing abilities of TRAF6.
EXAMPLE XV
Dose Response Effect of XIAP Expression on TRAF6-Mediated
NF-.kappa.B Activation
Methods
[0281] 293T cells were seeded into collagen-coated six-well plates
at 2.times.10.sup.5 cells per well 24 hrs before transfection.
Cells were then transfected with 0.5 .mu.g of pELAM-Lu reporter
plasmid, 0.1 .mu.g of pRL-CMV, 0.5 .mu.g of pCMV-TRAF6, indicated
amounts of pCMV-XIAP and enough pCMV-myc control plasmid to give 4
.mu.g of total DNA by standard lipofection methods using Trans-IT
lipofection reagent commercially available from Mirus. Twenty-four
hours after transfection, cells were washed with PBS and lysed in
400 .mu.l of Passive Lysis Buffer commercially available from
Promega. Lysate (20 .mu.l) from each sample was used to measure
firefly luciferase activity. Firefly luciferase activity was
determined and normalized on the basis of Renilla luciferase
expression level. Luciferase activity was measured in a model
TD20/20 luminometer using the Dual luciferase assay system
according to the manufacturer's protocol (Promega). Values shown
are averages for an experiment in which each transfection was
performed in duplicate.
Results
[0282] The results shown in FIG. 23 demonstrate that although
expression of TRAF6 was by itself capable of inducing NF-.kappa.B
activity, co-expression of TRAF6 with XIAP resulted in an increased
level of NF-.kappa.B activation which increased as the amount of
XIAP expression increased. Hence, XIAP was able to enhance the
NF-.kappa.B inducing abilities of TRAF6.
EXAMPLE XVI
Synergistic Effect of XAF-1 and XIAP Expression on TRAF6- and
TRAF2-Mediated NF-.kappa.B Activation
Methods
[0283] 293T cells were seeded into collagen-coated six-well plates
at 2.times.10.sup.5 cells per well 24 hrs before transfection.
Cells were then transfected with 0.5 .mu.g of pELAM-Lu
(pGL3-E-selectin promoter) and 0.05 .mu.g of pRL-CMV, 1 .mu.g of
pCMV-TRAF6 or 1 .mu.g of pCMV-TRAF2, 1 .mu.g of pCMV-XAF-1 and/or
pCMV-XIAP, and enough pCMV-myc control plasmid to give 4 .mu.g of
total DNA by standard lipofection methods using Trans-IT
lipofection reagent (Mirus). Twenty-four hours after transfection,
cells were washed with PBS and lysed in 400 .mu.l of Passive Lysis
Buffer (Promega). Lysate (20 .mu.l) from each samples was used to
measure firefly luciferase activity. Firefly luciferase activity
was determined and normalized on the basis of Renilla luciferase
expression level. Luciferase activity was measured in a model
TD20/20 luminometer (Promega) using Dual luciferase assay system
according to the manufacture's protocol (Promega). Values shown are
averages for an experiment in which each transfection was performed
in duplicate.
Results
[0284] XIAP and XAF-1 were additive in their effects on TRAF6
mediated NF-.kappa.B transactivation, as shown on FIG. 24. FIG. 25
indicates that XIAP and XAF-1 were also able to assist in TRAF2
mediated NF-.kappa.B transactivation, although to a lesser extent
than their assistance in TRAF6 mediated NF-.kappa.B
transactivation. Hence, XIAP and XAF-1 work synergistically in
their signal transducing capabilities.
EXAMPLE XVII
C-terminus of XAF-1 Enhances TRAF6-Mediated NF-.kappa.B
Activation
Methods
[0285] Expression plasmids that express either the amino terminal
domain of XAF-1 containing six potential zinc fingers, including
the region with significant homology to TRAF4 and TRAF6 (XAF-1N) or
the carboxy terminus containing a single potential zinc finger
domain (XAF-1C) were tested for their capacity to augment TRAF6
mediated NF-.kappa.B activity
[0286] 293T cells (2.times.10.sup.5) were transfected with 0.5
.mu.g of pELAM-Lu reporter plasmid, 0.1 .mu.g of pRL-TK
commercially available from Promega, 0.5 .mu.g of pCMV-TRAF6, 1
.mu.g of indicated expression plasmid and enough pCMV-myc control
plasmid to give 4 .mu.g of total DNA. Firefly luciferase activity
were determined 24 hrs after transfection and normalized on the
basis of Renilla luciferase expression level. Values shown are
averages for an experiment in which each transfection was performed
in duplicate.
Results
[0287] As FIG. 26 demonstrates, we have found that the carboxy
terminus of XAF-1 protein mediates the additive effect of XAF-1 on
TRAF6 induction of NF-.kappa.B. XAF-1N expression did not augment
the ability to TRAF6 to induce NF-.kappa.B, whereas XAF-1C
augmented NF-.kappa.B induction by TRAF6 substantially. Full length
XAF-1, as we showed previously in FIG. 21, clearly enhanced TRAF6
induction of NF-.kappa.B.
EXAMPLE XVIII
Inhibitory Effect of Antisense XAF-1 Expression on TRAF5-and
TRAF6-Mediated NF-.kappa.B Activation in 293 T Cells
Methods
[0288] To generate the bcl-2 antisense construct, a 1.5 kb EcoRI
fragment of bcl-2 was cloned in a non-coding orientation into the
pcDNA3 plasmid commercially available from Invitrogen.
[0289] 293T cells (2.times.10.sup.5) were transfected with 0.5
.mu.g of pELAM-Lu reporter plasmid, 0.1 .mu.g of pRL-TK
commercially available from Promega, 0.5 .mu.g of pCMV-TRAF5 or
pCMV-TRAF6, 3 .mu.g of indicated antisense plasmid: antisense XAF-1
(240-1) or antisense bcl-2 (450-23), and enough pCMV-myc control
plasmid to give 5 .mu.g of total DNA. Firefly luciferase activity
were determined 24 hrs after transfection and normalized on the
basis of Renilla luciferase expression level. Values shown are
averages for an experiment in which each transfection was performed
in duplicate.
Results
[0290] FIG. 27 demonstrates that expression of antisense XAF-1
significantly inhibited TRAF6 induced activation of NF-.kappa.B
and, to a lesser extent, TRAF5 induced activation of NF-.kappa.B.
This inhibition was specific to XAF-1 since antisense bcl-2 did not
have the same effect.
EXAMPLE XIX
Inhibitory Effect of Antisense XAF-1 Expression on
IL-1.beta.-Induced NF-.kappa.B Activation
Methods
[0291] 293T cells (2.times.10.sup.5) were transfected with 0.5
.mu.g of pELAM-Lu reporter plasmid, 0.1 .mu.g of pRL-TK
commercially available from Promega, indicated amounts of antisense
plasmid: antisense XAF-1 (240-1) or antisense bcl-2 (1486-23), and
enough pCMV-myc control plasmid to give 5 .mu.g of total DNA. 24
hrs after transfection, cells were treated for 6 hrs with 20 ng/ml
of interleukin-1 (IL-1.beta.). Firefly luciferase activity were
determined after IL-1.beta. treatment and normalized on the basis
of Renilla luciferase expression level. Values shown are averages
for an experiment in which each transfection was performed in
duplicate.
Results
[0292] As shown on FIG. 28, expression of antisense XAF-1 inhibited
interleukin-1.beta. induced activation of NF-.kappa.B. This
inhibition was specific to XAF-1 since antisense bcl-2 does not
have the same effect.
EXAMPLE XX
Dose Response Effect of XAF-1 Expression on IL-1.beta.-Induced
NF-.kappa.B Activation
Methods
[0293] 293T cells (2.times.10.sup.5) were transfected with 0.5
.mu.g of pELAM-Lu reporter plasmid, 0.1 .mu.g of pRL-TK
commercially available from Promega, indicated amounts of
pCMV-XAF-1 and enough pCMV-myc control plasmid to give 5 .mu.g of
total DNA. 24 hrs after transfection, cells were treated for 6 hrs
with 20 ng/ml of interleukin-1.beta. (IL-1.beta.). Firefly
luciferase activity were determined after IL-1.beta. treatment and
normalized on the basis of Renilla luciferase expression level.
Values shown are averages for an experiment in which each
transfection was performed in duplicate.
Results
[0294] Expression of full length XAF-1 augmented
interleukin-1.beta. mediated induction of NF-.kappa.B in a
dose-dependent manner, as is demonstrated in FIG. 29.
EXAMPLE XXI
Inhibitory Effect of A20 Expression on TRAF2-, TRAF5- and
TRAF6-Mediated NF-.kappa.B Activation
[0295] The A20 protein is induced by NF-.kappa.B and binds to both
TRAF1 and TRAF2, again via the TRAF-C domain. Binding of A20 to
TRAF2 interferes with NF-.kappa.B activation in a negative
feed-back loop (Song et al., Proc. Natl. Acad. Sci. USA 93:
6721-6725, 1996). It has previously been established that
over-expression of A20 can render cells resistant to the apoptotic
effects of TNF.alpha. (Opipari et al., J. Biol. Chem. 267:
12424-12427, 1992), and may also participate in rendering B cells
resistant to apoptosis following CD40 signaling (Sarma et al., 270:
12353-12346, 1995).
Methods
[0296] 293T cells (2.times.10.sup.5) were transfected with 0.5
.mu.g of pELAM-Lu reporter plasmid, 0.1 .mu.g of pRL-TK
commercially available from Promega, 0.5 .mu.g of pCMV-TRAF2,
pCMV-TRAF5 or pCMV-TRAF6, 0.3 .mu.g of pCMV-A20 and enough pCMV-myc
control plasmid to give 4 .mu.g of total DNA. Firefly luciferase
activity were determined 24 hrs after transfection and normalized
on the basis of Renilla luciferase expression level. Values shown
are averages for an experiment in which each transfection was
performed in duplicate
Results
[0297] In the experiments shown on FIG. 30, co-transfection of an
A20 expression vector with either TRAF2, TRAF5 or TRAF6 resulted in
virtually complete inhibition of NF-.kappa.B transactivation.
EXAMPLE XXII
XAF-1 Counters the Effect of A20 Expression on TRAF6 Mediated
Induction of NF-.kappa.B
Methods
[0298] 293T cells (2.times.10.sup.5) were transfected with 0.5
.mu.g of pELAM-Lu reporter plasmid, 0.1 .mu.g of pRL-TK
commercially available from Promega, 0.5 .mu.g of pCMV-TRAF6, 2
.mu.g of pCMV-XAF-1, indicated amounts of pCMV-A20 and enough
pCMV-myc control plasmid to give 5 .mu.g of total DNA. Firefly
luciferase activity were determined 24 hrs after transfection and
normalized on the basis of Renilla luciferase expression level.
Values shown are averages for an experiment in which each
transfection was performed in duplicate.
Results
[0299] As shown in FIG. 31, XAF-1 expression had a partial
neutralizing effect on the A20-mediated inhibitory function of
TRAF6-mediated NF-.kappa.B activation.
EXAMPLE XXIII
Interaction of XAF-1 with the Various TRAFs and Mammalian IAPs
Methods
[0300] XIAP and XAF-1 coding regions were cloned in frame into the
pGEX-4T-1 expression vector which is commercially available from
Pharmacia. Expression and purification of GST-fusion proteins were
performed essentially according to the manufacturer's protocol
(Pharmacia).
[0301] 293T cells were transiently transfected with myc-epitope
tagged TRAFs and mammalian IAPs expression vectors (5 .mu.g). After
36 hrs, cells were lysed and cell lysates were incubated with
GST-XAF-1 fusion protein or GST-control protein
(Glutathione-s-transferase from Schistosoma Japonicum) immobilized
on 10 .mu.l of glutathione beads. Protein adsorbed to beads were
analyzed by SDS-PAGE, followed by Western blotting using anti-c-myc
monoclonal antibody (9E10). Lanes were loaded as follows: [0302]
lane 1: HIAP-2, [0303] lane 2: TRAF1, [0304] lane 3: TRAF2, [0305]
lane 4: TRAF3, [0306] lane 5: A20. Proteins in A lanes were
affinity-purified with the GST-XAF-1 fusion protein. Proteins in B
lanes were affinity-purified with the GST-control protein.
Results
[0307] GST interaction analysis indicated that XAF-1 can form
complexes with a variety of cellular proteins, including HIAP-2,
TRAF1, TRAF2, and A20, as is shown on FIG. 32. In this type of
analysis, indirect interactions cannot be distinguished from direct
binding. For instance, XAF-1 may bind TRAF2 directly (as shown by
two-hybrid analysis) which in turn can interact with either TRAF1
or A20.
EXAMPLE XXIV
In Vitro Translated TRAF2 and HIAP-1 Bind XAF-1
Methods
[0308] .sup.35S-labeled in vitro translated proteins were generated
by using the various TRAF2 and HIAP-1 expression constructs in
pCDNA3-myc with the TNT T7 Coupled Reticulocyte Lysate System,
according to the manufacturer's descriptions (Promega) and .sup.35S
labeled methionine, commercially available from DuPont/NEN.
[0309] .sup.35S-labeled in vitro translated proteins were incubated
with GST-XAF-1 fusion protein or GST-control protein immobilized on
10 .mu.l of glutathione beads. Protein adsorbed to beads were
analyzed by SDS-PAGE. The protein bearing gel was then dried, and
adsorbed proteins were detected by autoradiograph of the gel. The
lanes were loaded as follows: [0310] lane 1: HIAP1, [0311] lane 2:
TRAF2. Proteins in A lanes were affinity-purified with the
GST-XAF-1 fusion protein. Proteins in B lanes were
affinity-purified with the GST-control protein. Results
[0312] As shown on FIG. 33, both in vitro translated HIAP-1 and
TRAF2 bound the GST-XAF-1 fusion protein, but do not bind the GST
control protein. Since this experiment was done in a cell-free
system, we have demonstrated that the HIAP-1:XAF-1 and the
TRAF2:XAF-1 interactions are direct.
EXAMPLE XXV
XAF-1 Directly Interacts with XIAP, HIAP-1, HIAP-2, and TRAF2
Methods
[0313] The plasmids pAS2-XIAP, pAS2-HIAP-1, pAS2-HIAP-2,
pAS2-TRAF2, pAS2-TRAF4, pAS2-XAF-1, and pAS2 (vector only) which
encode the GAL4 DNA-binding domains fused to indicated full-length
proteins, were used as baits (DNA-binding domain hybrids) in
two-hybrid screens of pGAD GH plasmids (commercially available from
Clontech) encoding XIAP, HIAP-1, HIAP-2, TRAF2, TRAF4, and XAF-1 as
preys (activation domain hybrids). The yeast two-hybrid assay and
isolation of positive clones and subsequent interaction analyses
were carried out as described elsewhere (PCT Publication WO
95/28497). DNA sequence was performed on an Applied Biosytems model
373A automated DNA sequencer.
Results
[0314] Shown in FIG. 34 is a listing of the XAF-1 interactions with
mammalian IAPs and TRAFS found in the yeast two-hybrid assay. Our
results indicated that XAF-1 directly interacts with XIAP, HIAP-1,
HIAP-2, and TRAF2 (but not TRAF4). As has been established in the
literature, TRAF2 can interact with TRAF1 or A20. Since we have
shown here in yeast two-hybrid analysis that XAF-1 binds TRAF2
directly, it may be through this interaction that XAF-1 is able to
form a complex with TRAF1 and A20, as we showed in FIG. 32.
EXAMPLE XXVI
Identification and Cloning of Human XAF-2
Methods
[0315] We screened the database for ESTs that have significant
homology to XAF-1. A number of such ESTs were identified. From the
EST sequences, we have made oligonucleotide primers and PCR cloned
a cDNA encoding a protein which we have named "XAF-2".
Results
[0316] FIG. 35 shows the partial 5' nucleic acid (SEQ ID NO.: 3)
and N-terminal amino acid (SEQ ID NO.: 4) sequences of the long
splice variant of XAF-2. The N-terminus of XAF-2 protein has five
zinc fingers in the N-terminal 150 amino acids which show 38% amino
acid identity to XAF-1 (SEQ ID NO.: 2). XAF-2 also has a unique
C-terminus that has two RING zinc fingers, so that the entire XAF-2
protein, like XAF-1, has seven zinc finger binding domains. FIG. 36
shows sequence of the 3' untranslated region (UTR) located
approximately 250 nucleic acid residues C-terminally to the nucleic
acid sequence of FIG. 35. There are at least two splice variants of
XAF-2. FIG. 37A shows the full length 5' nucleotide (above; SEQ ID
NO.: 9) and amino acid (below; SEQ ID NO.: 10) sequences of the
long (XAF-2L) splice variant of XAF-2. The shorter splice form of
XAF-2 (XAF-2S) is spliced as indicated in FIG. 37A, with the
nucleic acid encoding XAF-2S indicated in FIG. 37B, lower sequence
(SEQ ID NO.: 11). FIGS. 38A, 38B, and 38C show the indicated zinc
finger binding domains in the amino acid sequence listings of
XAF-1, XAF-2L, and XAF-2S, respectively. XAF-2L and XAF-1 shown an
overall amino acid sequence identity of 27%, although the first 135
amino acids of XAF-2L and the first 131 amino acids of XAF-1 share
a 40% amino acid sequence identity (FIG. 39). As indicated in FIG.
40, the alignment of the zinc finger binding domains in XAF-1 and
XAF-2L is not equivalent: the sixth zinc domain of XAF-2L aligns
with the seventh zinc domain of XAF-1. However, the two XAF
molecules both have seven zinc finger binding domains overall.
EXAMPLE XXVII
A Screen for Candidate Compounds which Modulate XAF-1
Expression
[0317] Compounds are screened for an ability to modulate XAF-1
expression by looking at the ability of the compounds to modulate
the expression of a luciferase reporter gene operably linked to the
XAF-1 promoter.
Methods
[0318] The XAF-1 promoter firefly luciferase reporter plasmid
pXAF-1 prom-Lu is constructed by insertion of PCR-amplified XAF-1
promoter sequences into a vector such as the pGL3-Basic vector
which is commercially available from Promega.
[0319] COS cells are seeded into six-well plates at
2.times.10.sup.5 cells per well 24 hrs before transfection. Cells
are then transfected with 1.0 .mu.g of pXAF-1prom-Lu reporter
plasmid, and 3.0 .mu.g pCMV-myc control plasmid by standard
lipofection methods using Trans-IT lipofection reagent commercially
available from Mirus. Twenty-four hours after transfection, varying
concentrations of different compounds are added to the culture
supernatant of transfected cells, such that there is one compound,
or combination thereof, per well. Twelve hours following treatment
with the compound, the cells are washed with PBS and lysed in 400
.mu.l of Passive Lysis Buffer commercially available from Promega.
Lysate (20 .mu.l) from each samples is used to measure firefly
luciferase activity. Firefly luciferase activity is determined and
normalized on the basis of Renilla luciferase expression level.
Luciferase activity is measured in a model TD20/20 luminometer
using the Dual luciferase assay system according to the
manufacture's protocol (Promega).
Results
[0320] Compound-treated cells which show an increased firefly
luciferase activity as compared to untreated control cells indicate
a compound with an ability to increase XAF-1 activity.
Compound-treated cells which show a decreased firefly luciferase
activity as compared to untreated control cells indicate a compound
with an ability to decrease XAF-1 activity.
Other Embodiments
[0321] In other embodiments, the invention includes any protein
which is substantially identical to a mammalian XAF polypeptide
provided in FIG. 1 (SEQ. ID NO.: 2), FIG. 35 (SEQ ID NO.: 4), FIG.
37A (SEQ ID NO.: 10) and FIG. 38C (SEQ ID NO.: 12); such homologues
include other substantially pure naturally-occurring mammalian XAF
proteins as well as splice variants, allelic variants; natural
mutants; induced mutants; DNA sequences which encode proteins and
also hybridize to the XAF DNA sequences of FIG. 1 (SEQ ID NO.: 1),
FIG. 35 (SEQ ID NO.: 3), FIG. 37A (SEQ ID NO.: 9) and FIG. 37B (SEQ
ID NO.: 11) under high stringency conditions (e.g., hybridizing at
2.times.SSC at 40.degree. C. with a probe length of at least 40
nucleotides) or, less preferably, under low stringency conditions
(e.g., hybridizing at 5.times.SSC at 25.degree. C. with a probe
length of at least 80 nucleotides); and proteins specifically bound
by antisera directed to a XAF polypeptide. The term also includes
chimeric polypeptides that include a portion derived from a XAF
polypeptide.
[0322] The invention further includes analogs of any
naturally-occurring XAF polypeptides. Analogs can differ from the
naturally-occurring XAF proteins by amino acid sequence
differences, by post-translational modifications, or by both.
Analogs of the invention will generally exhibit at least 85%, more
preferably 90%, and most preferably 95% or even 99% identity with
all or part of a naturally occurring XAF-1, XAF-2 N-terminus,
XAF-2L, or XAF-2S amino acid sequence. The length of sequence
comparison is at least 15 amino acid residues, preferably at least
25 amino acid residues, and more preferably more than 35 amino acid
residues. Modifications include in vivo and in vitro chemical
derivatization of polypeptides, e.g., acetylation, carboxylation,
phosphorylation, or glycosylation; such modifications may occur
during polypeptide synthesis or processing or following treatment
with isolated modifying enzymes. Analogs can also differ from the
naturally-occurring XAF-1, XAF-2 N-terminus, XAF-2L or XAF-2S
polypeptide by alterations in primary sequence. These include
genetic variants, both natural and induced (for example, resulting
from random mutagenesis by irradiation or exposure to
ethanemethylsulfate or by site-specific mutagenesis as described in
Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory
Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also
included are cyclized peptides, molecules, and analogs which
contain residues other than L-amino acids, e.g., D-amino acids or
non-naturally occurring or synthetic amino acids, e.g., B or Y
amino acids. In addition to full-length polypeptides, the invention
also includes XAF-1, XAF-2 N-terminus, XAF-2L and XAF-2S
polypeptide fragments. As used herein, the term "fragment," means
at least 20 contiguous amino acids, preferably at least 30
contiguous amino acids, more preferably at least 50 contiguous
amino acids, and most preferably at least 60 to 80 or more
contiguous amino acids. Fragments of XAF-1, XAF-2 N-terminus,
XAF-2L and XAF-2S polypeptides can be generated by methods known to
those skilled in the art or may result from normal protein
processing (e.g., removal of amino acids from the nascent
polypeptide that are not required for biological activity or
removal of amino acids by alternative mRNA splicing or alternative
protein processing events).
[0323] Preferable fragments or analogs according to the invention
are those which facilitate specific detection of a XAF-1, XAF-2 N
terminus, XAF-2L or XAF-2S nucleic acid or amino acid sequence in a
sample to be diagnosed. Particularly useful XAF-1 fragments for
this purpose include, without limitation, the amino acid fragments
shown in FIG. 7.
[0324] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each independent publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0325] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure come within
known or customary practice, within the art to which the invention
pertains and may be applied to the essential features hereinbefore
set forth.
Sequence CWU 1
1
143 1 1326 DNA Homo sapiens 1 atggaaggag acttctcggt gtgcaggaac
tgtaaaagac atgtagtctc tgccaacttc 60 accctccatg aggcttactg
cctgcggttc ctggtcctgt gtccggagtg tgaggagcct 120 gtccccaagg
aaaccatgga ggagcactgc aagcttgagc accagcaggt tgggtgtacg 180
atgtgtcagc agagcatgca gaagtcctcg ctggagtttc ataaggccaa tgagtgccag
240 gagcgccctg ttgagtgtaa gttctgcaaa ctggacatgc agctcagcaa
gctggagctc 300 cacgagtcct actgtggcag ccggacagag ctctgccaag
gctgtggcca gttcatcatg 360 caccgcatgc tcgcccagca cagagatgtc
tgtcggagtg aacaggccca gctcgggaaa 420 ggggaaagaa tttcagctcc
tgaaagggaa atctactgtc attattgcaa ccaaatgatt 480 ccagaaaata
agtatttcca ccatatgggt aaatgttgtc cagactcaga gtttaagaaa 540
cactttcctg ttggaaatcc agaaattctt ccttcatctc ttccaagtca agctgctgaa
600 aatcaaactt ccacgatgga gaaagatgtt cgtccaaaga caagaagtat
aaacagattt 660 cctcttcatt ctgaaagttc atcaaagaaa gcaccaagaa
gcaaaaacaa aaccttggat 720 ccacttttga tgtcagagcc caagcccagg
accagctccc ctagaggaga taaagcagcc 780 tatgacattc tgaggagatg
ttctcagtgt ggcatcctgc ttcccctgcc gatcctaaat 840 caacatcagg
agaaatgccg gtggttagct tcatcaaaaa ggaaaacaag tgagaaattt 900
cagctagatt tggaaaagga aaggtactac aaattcaaaa gatttcactt ttaacactgg
960 cattcctgcc tacttgctgt ggtggtcttg tgaaaggtga tgggttttat
tcgttgggct 1020 ttaaaagaaa aggtttggca gaactaaaaa caaaactcac
gtatcatctc aatagataca 1080 gaaaaggctt ttgataaaat tcaacttgac
ttcatgttaa aaaccctcaa caaaccaggc 1140 gtcgaaggaa catacctcaa
aataataaga gccatctatg acaaaaccac agccaacatc 1200 atactgaatg
agcaaaagct ggagcattac tcttgagaag tagaacaagg cacttcagtc 1260
ctattcaaca tagtactgga agtctcgcca cagcaatcag gcaagagaaa gaagtaaaag
1320 gcaccc 1326 2 317 PRT Homo sapiens 2 Met Glu Gly Asp Phe Ser
Val Cys Arg Asn Cys Lys Arg His Val Val 1 5 10 15 Ser Ala Asn Phe
Thr Leu His Glu Ala Tyr Cys Leu Arg Phe Leu Val 20 25 30 Leu Cys
Pro Glu Cys Glu Glu Pro Val Pro Lys Glu Thr Met Glu Glu 35 40 45
His Cys Lys Leu Glu His Gln Gln Val Gly Cys Thr Met Cys Gln Gln 50
55 60 Ser Met Gln Lys Ser Ser Leu Glu Phe His Lys Ala Asn Glu Cys
Gln 65 70 75 80 Glu Arg Pro Val Glu Cys Lys Phe Cys Lys Leu Asp Met
Gln Leu Ser 85 90 95 Lys Leu Glu Leu His Glu Ser Tyr Cys Gly Ser
Arg Thr Glu Leu Cys 100 105 110 Gln Gly Cys Gly Gln Phe Ile Met His
Arg Met Leu Ala Gln His Arg 115 120 125 Asp Val Cys Arg Ser Glu Gln
Ala Gln Leu Gly Lys Gly Glu Arg Ile 130 135 140 Ser Ala Pro Glu Arg
Glu Ile Tyr Cys His Tyr Cys Asn Gln Met Ile 145 150 155 160 Pro Glu
Asn Lys Tyr Phe His His Met Gly Lys Cys Cys Pro Asp Ser 165 170 175
Glu Phe Lys Lys His Phe Pro Val Gly Asn Pro Glu Ile Leu Pro Ser 180
185 190 Ser Leu Pro Ser Gln Ala Ala Glu Asn Gln Thr Ser Thr Met Glu
Lys 195 200 205 Asp Val Arg Pro Lys Thr Arg Ser Ile Asn Arg Phe Pro
Leu His Ser 210 215 220 Glu Ser Ser Ser Lys Lys Ala Pro Arg Ser Lys
Asn Lys Thr Leu Asp 225 230 235 240 Pro Leu Leu Met Ser Glu Pro Lys
Pro Arg Thr Ser Ser Pro Arg Gly 245 250 255 Asp Lys Ala Ala Tyr Asp
Ile Leu Arg Arg Cys Ser Gln Cys Gly Ile 260 265 270 Leu Leu Pro Leu
Pro Ile Leu Asn Gln His Gln Glu Lys Cys Arg Trp 275 280 285 Leu Ala
Ser Ser Lys Arg Lys Thr Ser Glu Lys Phe Gln Leu Asp Leu 290 295 300
Glu Lys Glu Arg Tyr Tyr Lys Phe Lys Arg Phe His Phe 305 310 315 3
1311 DNA Homo sapiens 3 gcagctagtg tgtcatttca gcgtttctcc tctcgtccct
ggaagagcta aagatggctg 60 aatttctaga tgaccaggaa actcgactgt
gtgacaactg caaaaaagaa attcctgtgt 120 ttaactttac catccatgag
atccactgtc aaaggaacat tggtatgtgt cctacctgta 180 aggaaccatt
tcccaaatct gacatggaga ctcacatggc tgcagaacac tgtcaggtga 240
cctgcaaatg taacaagaag ttggagaaga ggctgttaaa gaagcatgag gagactgagt
300 gccctttgcg gcttgctgtc tgccagcact gtgatttaga actttccatt
ctcaaactga 360 aggaacatga agattattgt ggtgcccgga cggaactatg
tggcaactgt ggtcgcaatg 420 tccttgtgaa agatctgaag actcaccctg
aagtttgtgg gagagagggg gaggaaaaga 480 gaaatgaggt tgccatacct
cctaatgcat atgatgaatc ttggggtcag gatggaatct 540 ggattgcatc
ccaactcctc agacaaattg aggctctgga cccacccatg aggctgccgc 600
gaaggcccct gagagccttt gaatcagatg ttttccacaa tagaactacc aaccaaagga
660 acattacagc ccaggtttca attcagaata atctgtttga agaacaagag
aggcaggaaa 720 ggaatagagg ccaacagccc cccaaagagg gtggtgaaga
gagtgcaaac ttggacttca 780 tgttggccct aagtctgcaa aatgaaggcc
aagcctccag tgtggcagag caggacttct 840 ggagggccgt atgtgaggcc
gaccagtctc atggcggtcc caggtctctc agtgacataa 900 agggtgcagc
tgacgagatc atgttgcctt gtgaattttg tgaggagctc tacccagagg 960
aactgctgat tgaccatcag acaagctgta acccttcacg tgccttacct tcactcaata
1020 ctggcagctc ttcccccaga ggggtggagg aacctgatgt catcttccag
aactccttgc 1080 aacaggctgc aagtaaccag ttagactctt tgatgggcct
gagcaattca caccctgtgg 1140 aggagagcat cattatccca tgtgaattct
gtggggtaca gctggaagag gaggtgctgt 1200 tccatcacca ggaccagtgt
gaccaacgcc cagccactgc aaccaaccat gtgacagagg 1260 ggattcctag
actggattcc cagcctcaag agccccttcc ccttgttttt a 1311 4 419 PRT Homo
sapiens 4 Met Ala Glu Phe Leu Asp Asp Gln Glu Thr Arg Leu Cys Asp
Asn Cys 1 5 10 15 Lys Lys Glu Ile Pro Val Phe Asn Phe Thr Ile His
Glu Ile His Cys 20 25 30 Gln Arg Asn Ile Gly Met Cys Pro Thr Cys
Lys Glu Pro Phe Pro Lys 35 40 45 Ser Asp Met Glu Thr His Met Ala
Ala Glu His Cys Gln Val Thr Cys 50 55 60 Lys Cys Asn Lys Lys Leu
Glu Lys Arg Leu Leu Lys Lys His Glu Glu 65 70 75 80 Thr Glu Cys Pro
Leu Arg Leu Ala Val Cys Gln His Cys Asp Leu Glu 85 90 95 Leu Ser
Ile Leu Lys Leu Lys Glu His Glu Asp Tyr Cys Gly Ala Arg 100 105 110
Thr Glu Leu Cys Gly Asn Cys Gly Arg Asn Val Leu Val Lys Asp Leu 115
120 125 Lys Thr His Pro Glu Val Cys Gly Arg Glu Gly Glu Glu Lys Arg
Asn 130 135 140 Glu Val Ala Ile Pro Pro Asn Ala Tyr Asp Glu Ser Trp
Gly Gln Asp 145 150 155 160 Gly Ile Trp Ile Ala Ser Gln Leu Leu Arg
Gln Ile Glu Ala Leu Asp 165 170 175 Pro Pro Met Arg Leu Pro Arg Arg
Pro Leu Arg Ala Phe Glu Ser Asp 180 185 190 Val Phe His Asn Arg Thr
Thr Asn Gln Arg Asn Ile Thr Ala Gln Val 195 200 205 Ser Ile Gln Asn
Asn Leu Phe Glu Glu Gln Glu Arg Gln Glu Arg Asn 210 215 220 Arg Gly
Gln Gln Pro Pro Lys Glu Gly Gly Glu Glu Ser Ala Asn Leu 225 230 235
240 Asp Phe Met Leu Ala Leu Ser Leu Gln Asn Glu Gly Gln Ala Ser Ser
245 250 255 Val Ala Glu Gln Asp Phe Trp Arg Ala Val Cys Glu Ala Asp
Gln Ser 260 265 270 His Gly Gly Pro Arg Ser Leu Ser Asp Ile Lys Gly
Ala Ala Asp Glu 275 280 285 Ile Met Leu Pro Cys Glu Phe Cys Glu Glu
Leu Tyr Pro Glu Glu Leu 290 295 300 Leu Ile Asp His Gln Thr Ser Cys
Asn Pro Ser Arg Ala Leu Pro Ser 305 310 315 320 Leu Asn Thr Gly Ser
Ser Ser Pro Arg Gly Val Glu Glu Pro Asp Val 325 330 335 Ile Phe Gln
Asn Ser Leu Gln Gln Ala Ala Ser Asn Gln Leu Asp Ser 340 345 350 Leu
Met Gly Leu Ser Asn Ser His Pro Val Glu Glu Ser Ile Ile Ile 355 360
365 Pro Cys Glu Phe Cys Gly Val Gln Leu Glu Glu Glu Val Leu Phe His
370 375 380 His Gln Asp Gln Cys Asp Gln Arg Pro Ala Thr Ala Thr Asn
His Val 385 390 395 400 Thr Glu Gly Ile Pro Arg Leu Asp Ser Gln Pro
Gln Glu Pro Leu Pro 405 410 415 Leu Val Phe 5 1169 DNA Homo sapiens
5 tgggtgccag cccagctctc cttgtgtgcc gaagctcagc aactcagaca gccaggacat
60 ccaggggcgg aatcgagaca gccagaatgg ggccatagcc cctgggcacg
tttcagtgat 120 tcgccctcct caaaatctct acccagaaaa cattgtgccc
tctttctccc gtgggccttc 180 agggagatac ggagctagtg gtaggagtga
aggtggcagg aattcccggg tcacccctgc 240 agctgccaac taccgcagca
gaactgcaaa ggcaaagcct tccaagcaac agggagctgg 300 ggatgcagaa
gaggaagagg aggagtaatg gtgtctccag agactttaca tcggttcctg 360
tcttctgtgc acagcagcac ttgccgctgt gcaggcccac ctctttggct ctttgggtgg
420 gagagttttt ccagatttta gatttttcta ggttatggcc attttgtgtc
ttttgaggtt 480 gtgctgtggg ggtttgggtt tgagggaagg gagcagggtg
gcggttgagg aacgcttcag 540 ccttagctgc tacctttcgg cagcagtgaa
atacaagctg cagcctcggc tgccagggct 600 cccttttgac ttattgtcgc
cactgcccct tggtgctgtg tggtcccagt ggaaggaggg 660 gaagattttg
gaaacctggt agccaccagt aaggtgattc tctgccctgt tggggcctaa 720
atttgggggc ttttgggcaa cctctccgtg tactgcgtct gtccacactc gattgggccc
780 caggtgtgta tgaggcgctc tggtaaggtg ctcaggccag ttgcaatgtc
tgtcagtaac 840 gaggcttttg atgtgttgag ctggaggtga gtggaccggg
ggctgtgttt taagctgctt 900 ccttggcatt tggcatcact gccttctgtt
cccgggggag catggatctt ttgtcctcac 960 tgctttctaa tggggagggc
tgagggctcc ctgtccccac agcaggtatg gttgctctgc 1020 cccagcccca
cacttgctct gaaaaccaag tgtcagagcc ccttcccctt gtttttattt 1080
tactgttata ataattatta acttccttgt aatagaaata aagtttgtac ttggaaaaaa
1140 aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1169 6 178 PRT Homo sapiens 6
Met Glu Gly Asp Phe Ser Val Cys Arg Asn Cys Lys Arg His Val Val 1 5
10 15 Ser Ala Asn Phe Thr Leu His Glu Ala Tyr Cys Leu Arg Phe Leu
Val 20 25 30 Leu Cys Pro Glu Cys Glu Glu Pro Val Pro Lys Glu Thr
Met Glu Glu 35 40 45 His Cys Lys Leu Glu His Gln Gln Val Gly Cys
Thr Met Cys Gln Gln 50 55 60 Ser Met Gln Lys Ser Ser Leu Glu Phe
His Lys Ala Asn Glu Cys Gln 65 70 75 80 Glu Arg Pro Val Glu Cys Lys
Phe Cys Lys Leu Asp Met Gln Leu Ser 85 90 95 Lys Leu Glu Leu His
Glu Ser Tyr Cys Gly Ser Arg Thr Glu Leu Cys 100 105 110 Gln Gly Cys
Gly Gln Phe Ile Met His Arg Met Leu Ala Gln His Arg 115 120 125 Asp
Val Cys Arg Ser Glu Gln Ala Gln Leu Gly Lys Gly Glu Arg Ile 130 135
140 Ser Ala Pro Glu Arg Glu Ile Tyr Cys His Tyr Cys Asn Gln Met Ile
145 150 155 160 Pro Glu Asn Lys Tyr Phe His His Met Gly Lys Cys Cys
Pro Asp Ser 165 170 175 Glu Phe 7 172 PRT Homo sapiens 7 Met Glu
Gly Asp Phe Ser Val Cys Arg Asn Cys Lys Arg His Val Val 1 5 10 15
Ser Ala Asn Phe Thr Leu His Glu Ala Tyr Cys Leu Arg Phe Leu Val 20
25 30 Leu Cys Pro Glu Cys Glu Glu Pro Val Pro Lys Glu Thr Met Glu
Glu 35 40 45 His Cys Lys Leu Glu His Gln Gln Val Gly Cys Thr Met
Cys Gln Gln 50 55 60 Ser Met Gln Lys Ser Ser Leu Glu Phe His Lys
Ala Asn Glu Cys Gln 65 70 75 80 Glu Arg Pro Val Glu Cys Lys Phe Cys
Lys Leu Asp Met Gln Leu Ser 85 90 95 Lys Leu Glu Leu His Glu Ser
Tyr Cys Gly Ser Arg Thr Glu Leu Cys 100 105 110 Gln Gly Cys Gly Gln
Phe Ile Met His Arg Met Leu Ala Gln His Arg 115 120 125 Asp Val Cys
Arg Ser Glu Gln Ala Gln Leu Gly Lys Gly Glu Arg Ile 130 135 140 Ser
Ala Pro Glu Arg Glu Ile Tyr Cys His Tyr Cys Asn Gln Met Ile 145 150
155 160 Pro Glu Asn Lys Tyr Phe His His Met Gly Lys Cys 165 170 8
145 PRT Homo sapiens 8 Cys Pro Asp Ser Glu Phe Lys Lys His Phe Pro
Val Gly Asn Pro Glu 1 5 10 15 Ile Leu Pro Ser Ser Leu Pro Ser Gln
Ala Ala Glu Asn Gln Thr Ser 20 25 30 Thr Met Glu Lys Asp Val Arg
Pro Lys Thr Arg Ser Ile Asn Arg Phe 35 40 45 Pro Leu His Ser Glu
Ser Ser Ser Lys Lys Ala Pro Arg Ser Lys Asn 50 55 60 Lys Thr Leu
Asp Pro Leu Leu Met Ser Glu Pro Lys Pro Arg Thr Ser 65 70 75 80 Ser
Pro Arg Gly Asp Lys Ala Ala Tyr Asp Ile Leu Arg Arg Cys Ser 85 90
95 Gln Cys Gly Ile Leu Leu Pro Leu Pro Ile Leu Asn Gln His Gln Glu
100 105 110 Lys Cys Arg Trp Leu Ala Ser Ser Lys Arg Lys Thr Ser Glu
Lys Phe 115 120 125 Gln Leu Asp Leu Glu Lys Glu Arg Tyr Tyr Lys Phe
Lys Arg Phe His 130 135 140 Phe 145 9 2643 DNA Homo sapiens 9
gcagctagtg tgtcatttca gcgtttctcc tctcgtccct ggaagagcta aagatggctg
60 aatttctaga tgaccaggaa actcgactgt gtgacaactg caaaaaagaa
attcctgtgt 120 ttaactttac catccatgag atccactgtc aaaggaacat
tggtatgtgt cctacctgta 180 aggaaccatt tcccaaatct gacatggaga
ctcacatggc tgcagaacac tgtcaggtga 240 cctgcaaatg taacaagaag
ttggagaaga ggctgttaaa gaagcatgag gagactgagt 300 gccctttgcg
gcttgctgtc tgccagcact gtgatttaga actttccatt ctcaaactga 360
aggaacatga agattattgt ggtgcccgga cggaactatg tggcaactgt ggtcgcaatg
420 tccttgtgaa agatctgaag actcaccctg aagtttgtgg gagagagggg
gaggaaaaga 480 gaaatgaggt tgccatacct cctaatgcat atgatgaatc
ttggggtcag gatggaatct 540 ggattgcatc ccaactcctc agacaaattg
aggctctgga cccacccatg aggctgccgc 600 gaaggcccct gagagccttt
gaatcagatg ttttccacaa tagaactacc aaccaaagga 660 acattacagc
ccaggtttca attcagaata atctgtttga agaacaagag aggcaggaaa 720
ggaatagagg ccaacagccc cccaaagagg gtggtgaaga gagtgcaaac ttggacttca
780 tgttggccct aagtctgcaa aatgaaggcc aagcctccag tgtggcagag
caggacttct 840 ggagggccgt atgtgaggcc gaccagtctc atggcggtcc
caggtctctc agtgacataa 900 agggtgcagc tgacgagatc atgttgcctt
gtgaattttg tgaggagctc tacccagagg 960 aactgctgat tgaccatcag
acaagctgta acccttcacg tgccttacct tcactcaata 1020 ctggcagctc
ttcccccaga ggggtggagg aacctgatgt catcttccag aacttcttgc 1080
aacaggctgc aagtaaccag ttagactctt tgatgggcct gagcaattca caccctgtgg
1140 aggagagcat cattatccca tgtgaattct gtggggtaca gctggaagag
gaggtgctgt 1200 tccatcacca ggaccagtgt gaccaacgcc cagccactgc
aaccaaccat gtgacagagg 1260 ggattcctag actggattcc cagcctcaag
agaccccacc agagctgccc aggaggcgtg 1320 tcagacacca gggagacctg
tcttctggtt acctggatga tactaagcag gaaacagcta 1380 atgggcccac
ctcctgtctg cctcccagcc gacccattaa caatatgaca gctacctata 1440
accagctatc gagatcaaca tcaggcccca gacctgggtg ccagcccagc tctccttgtg
1500 tgccgaagct cagcaactca gacagccagg acatccaggg gcggaatcga
gacagccaga 1560 atggggccat agcccctggg cacgtttcag tgattcgccc
tcctcaaaat ctctacccag 1620 aaaacattgt gccctctttc tcccctgggc
cttcagggag atacggagct agtggtagga 1680 gtgaaggtgg caggaattcc
cgggtcaccc ctgcagctgc caactaccgc agcagaactg 1740 caaaggcaaa
gccttccaag caacagggag ctggggatgc agaagaggaa gaggaggagt 1800
aatggtgtct ccagagactt tacatcggtt cctgtcttct gtgcacagca gcacttgccg
1860 ctgtgcaggc ccacctcttt ggctctttgg gtgggagagt ttttccagat
tttagatttt 1920 tctaggttat ggccattttg tgtcttttga ggttgtgctg
tgggggtttg ggtttgaggg 1980 aagggagcag ggtggcggtt gaggaacgct
tcagccttag ctgctacctt tcggcagcag 2040 tgaaatacaa gctgcagcct
cggctgccag ggctcccttt tgacttattg tcgccactgc 2100 cccttggtgc
tgtgtggtcc cagtggaagg aggggaagat tttggaaacc tggtagccac 2160
cagtaaggtg attctctgcc ctgttggggc ctaaatttgg gggcttttgg gcaacctctc
2220 cgtgtactgc gtctgtccac actcgattgg gccccaggtg tgtatgaggc
gctctggtaa 2280 ggtgctcagg ccagttgcaa tgtctgtcag taacgaggct
tttgatgtgt tgagctggag 2340 gtgagtggac cgggggctgt gttttaagct
gcttccttgg catttggcat cactgccttc 2400 tgttcccggg ggagcatgga
tcttttgtcc tcactgcttt ctaatgggga gggctgaggg 2460 ctccctgtcc
ccacagcagg tatggttgct ctgccccagc cccacacttg ctctgaaaac 2520
caagtgtcag agccccttcc ccttgttttt attttactgt tataataatt attaacttcc
2580 ttgtaataga aataaagttt gtacttggaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2640 aaa 2643 10 582 PRT Homo sapiens 10 Met Ala Glu Phe
Leu Asp Asp Gln Glu Thr Arg Leu Cys Asp Asn Cys 1 5 10 15 Lys Lys
Glu Ile Pro Val Phe Asn Phe Thr Ile His Glu Ile His Cys 20 25 30
Gln Arg Asn Ile Gly Met Cys Pro Thr Cys Lys Glu Pro Phe Pro Lys 35
40 45 Ser Asp Met Glu Thr His Met Ala Ala Glu His Cys Gln Val Thr
Cys 50 55 60 Lys Cys Asn Lys Lys Leu Glu Lys Arg Leu Leu Lys Lys
His Glu Glu 65 70 75 80 Thr Glu Cys Pro Leu Arg Leu Ala Val Cys Gln
His Cys Asp Leu Glu 85 90 95 Leu Ser Ile Leu Lys Leu Lys Glu His
Glu Asp Tyr Cys Gly Ala Arg 100 105 110 Thr Glu Leu Cys
Gly Asn Cys Gly Arg Asn Val Leu Val Lys Asp Leu 115 120 125 Lys Thr
His Pro Glu Val Cys Gly Arg Glu Gly Glu Glu Lys Arg Asn 130 135 140
Glu Val Ala Ile Pro Pro Asn Ala Tyr Asp Glu Ser Trp Gly Gln Asp 145
150 155 160 Gly Ile Trp Ile Ala Ser Gln Leu Leu Arg Gln Ile Glu Ala
Leu Asp 165 170 175 Pro Pro Met Arg Leu Pro Arg Arg Pro Leu Arg Ala
Phe Glu Ser Asp 180 185 190 Val Phe His Asn Arg Thr Thr Asn Gln Arg
Asn Ile Thr Ala Gln Val 195 200 205 Ser Ile Gln Asn Asn Leu Phe Glu
Glu Gln Glu Arg Gln Glu Arg Asn 210 215 220 Arg Gly Gln Gln Pro Pro
Lys Glu Gly Gly Glu Glu Ser Ala Asn Leu 225 230 235 240 Asp Phe Met
Leu Ala Leu Ser Leu Gln Asn Glu Gly Gln Ala Ser Ser 245 250 255 Val
Ala Glu Gln Asp Phe Trp Arg Ala Val Cys Glu Ala Asp Gln Ser 260 265
270 His Gly Gly Pro Arg Ser Leu Ser Asp Ile Lys Gly Ala Ala Asp Glu
275 280 285 Ile Met Leu Pro Cys Glu Phe Cys Glu Glu Leu Tyr Pro Glu
Glu Leu 290 295 300 Leu Ile Asp His Gln Thr Ser Cys Asn Pro Ser Arg
Ala Leu Pro Ser 305 310 315 320 Leu Asn Thr Gly Ser Ser Ser Pro Arg
Gly Val Glu Glu Pro Asp Val 325 330 335 Ile Phe Gln Asn Phe Leu Gln
Gln Ala Ala Ser Asn Gln Leu Asp Ser 340 345 350 Leu Met Gly Leu Ser
Asn Ser His Pro Val Glu Glu Ser Ile Ile Ile 355 360 365 Pro Cys Glu
Phe Cys Gly Val Gln Leu Glu Glu Glu Val Leu Phe His 370 375 380 His
Gln Asp Gln Cys Asp Gln Arg Pro Ala Thr Ala Thr Asn His Val 385 390
395 400 Thr Glu Gly Ile Pro Arg Leu Asp Ser Gln Pro Gln Glu Thr Pro
Pro 405 410 415 Glu Leu Pro Arg Arg Arg Val Arg His Gln Gly Asp Leu
Ser Ser Gly 420 425 430 Tyr Leu Asp Asp Thr Lys Gln Glu Thr Ala Asn
Gly Pro Thr Ser Cys 435 440 445 Leu Pro Pro Ser Arg Pro Ile Asn Asn
Met Thr Ala Thr Tyr Asn Gln 450 455 460 Leu Ser Arg Ser Thr Ser Gly
Pro Arg Pro Gly Cys Gln Pro Ser Ser 465 470 475 480 Pro Cys Val Pro
Lys Leu Ser Asn Ser Asp Ser Gln Asp Ile Gln Gly 485 490 495 Arg Asn
Arg Asp Ser Gln Asn Gly Ala Ile Ala Pro Gly His Val Ser 500 505 510
Val Ile Arg Pro Pro Gln Asn Leu Tyr Pro Glu Asn Ile Val Pro Ser 515
520 525 Phe Ser Pro Gly Pro Ser Gly Arg Tyr Gly Ala Ser Gly Arg Ser
Glu 530 535 540 Gly Gly Arg Asn Ser Arg Val Thr Pro Ala Ala Ala Asn
Tyr Arg Ser 545 550 555 560 Arg Thr Ala Lys Ala Lys Pro Ser Lys Gln
Gln Gly Ala Gly Asp Ala 565 570 575 Glu Glu Glu Glu Glu Glu 580 11
1302 DNA Homo sapiens 11 gcagctagtg tgtcatttca gcgtttctcc
tctcgtccct ggaagagcta aagatggctg 60 aatttctaga tgaccaggaa
actcgactgt gtgacaactg caaaaaagaa attcctgtgt 120 ttaactttac
catccatgag atccactgtc aaaggaacat tggtatgtgt cctacctgta 180
aggaaccatt tcccaaatct gacatggaga ctcacatggc tgcagaacac tgtcaggtga
240 cctgcaaatg taacaagaag ttggagaaga ggctgttaaa gaagcatgag
gagactgagt 300 gccctttgcg gcttgctgtc tgccagcact gtgatttaga
actttccatt ctcaaactga 360 aggtcacccc tgcagctgcc aactaccgca
gcagaactgc aaaggcaaag ccttccaagc 420 aacagggagc tggggatgca
gaagaggaag aggaggagta atggtgtctc cagagacttt 480 acatcggttc
ctgtcttctg tgcacagcag cacttgccgc tgtgcaggcc cacctctttg 540
gctctttggg tgggagagtt tttccagatt ttagattttt ctaggttatg gccattttgt
600 gtcttttgag gttgtgctgt gggggtttgg gtttgaggga agggagcagg
gtggcggttg 660 aggaacgctt cagccttagc tgctaccttt cggcagcagt
gaaatacaag ctgcagcctc 720 ggctgccagg gctccctttt gacttattgt
cgccactgcc ccttggtgct gtgtggtccc 780 agtggaagga ggggaagatt
ttggaaacct ggtagccacc agtaaggtga ttctctgccc 840 tgttggggcc
taaatttggg ggcttttggg caacctctcc gtgtactgcg tctgtccaca 900
ctcgattggg ccccaggtgt gtatgaggcg ctctggtaag gtgctcaggc cagttgcaat
960 gtctgtcagt aacgaggctt ttgatgtgtt gagctggagg tgagtggacc
gggggctgtg 1020 ttttaagctg cttccttggc atttggcatc actgccttct
gttcccgggg gagcatggat 1080 cttttgtcct cactgctttc taatggggag
ggctgagggc tccctgtccc cacagcaggt 1140 atggttgctc tgccccagcc
ccacacttgc tctgaaaacc aagtgtcaga gccccttccc 1200 cttgttttta
ttttactgtt ataataatta ttaacttcct tgtaatagaa ataaagtttg 1260
tacttggaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 1302 12 135 PRT Homo
sapiens 12 Met Ala Glu Phe Leu Asp Asp Gln Glu Thr Arg Leu Cys Asp
Asn Cys 1 5 10 15 Lys Lys Glu Ile Pro Val Phe Asn Phe Thr Ile His
Glu Ile His Cys 20 25 30 Gln Arg Asn Ile Gly Met Cys Pro Thr Cys
Lys Glu Pro Phe Pro Lys 35 40 45 Ser Asp Met Glu Thr His Met Ala
Ala Glu His Cys Gln Val Thr Cys 50 55 60 Lys Cys Asn Lys Lys Leu
Glu Lys Arg Leu Leu Lys Lys His Glu Glu 65 70 75 80 Thr Glu Cys Pro
Leu Arg Leu Ala Val Cys Gln His Cys Asp Leu Glu 85 90 95 Leu Ser
Ile Leu Lys Leu Lys Val Thr Pro Ala Ala Ala Asn Tyr Arg 100 105 110
Ser Arg Thr Ala Lys Ala Lys Pro Ser Lys Gln Gln Gly Ala Gly Asp 115
120 125 Ala Glu Glu Glu Glu Glu Glu 130 135 13 19 PRT Homo sapiens
VARIANT (1)...(19) Xaa = Any Amino Acid; Xaa at 16 and 19 is Cys or
His 13 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 15 Xaa Xaa Xaa 14 20 PRT Homo sapiens VARIANT (1)...(20)
Xaa = Any Amino Acid; Xaa at 17 and 20 is Cys or His 14 Cys Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa 20 15 21 PRT Homo sapiens VARIANT (1)...(21) Xaa = Any
Amino Acid; Xaa at 18 and 21 is Cys or His 15 Cys Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa 20 16 22 PRT Homo sapiens VARIANT (1)...(22) Xaa = Any
Amino Acid; Xaa at 19 and 22 is Cys or His 16 Cys Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa 20 17 20 PRT Homo sapiens VARIANT (1)...(20) Xaa = Any
Amino Acid; Xaa at 17 and 20 is Cys or His 17 Cys Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa 20 18 21 PRT Homo sapiens VARIANT (1)...(21) Xaa = Any Amino
Acid; Xaa at 19 and 21 is Cys or His 18 Cys Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
20 19 22 PRT Homo sapiens VARIANT (1)...(22) Xaa = Any Amino Acid;
Xaa at 19 and 22 is Cys or His 19 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa
20 20 23 PRT Homo sapiens VARIANT (1)...(23) Xaa = Any Amino Acid;
Xaa at 20 and 23 is Cys or His 20 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 21 24 PRT Homo sapiens VARIANT (1)...(24) Xaa = Any Amino
Acid; Xaa at 21 and 24 is Cys or His 21 Cys Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 22 25 PRT Homo sapiens VARIANT (1)...(25) Xaa = Any
Amino Acid; Xaa at 22 and 25 is Cys or His 22 Cys Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 23 26 PRT Homo sapiens VARIANT
(1)...(26) Xaa = Any Amino Acid; Xaa at 23 and 26 is Cys or His 23
Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 24 21 PRT Homo
sapiens VARIANT (1)...(21) Xaa = Any Amino Acid; Xaa at 19 and 21
is Cys or His 24 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 25 22 PRT Homo
sapiens VARIANT (1)...(22) Xaa = Any Amino Acid; Xaa at 19 and 22
is Cys or His 25 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa 20 26 23 PRT Homo
sapiens VARIANT (1)...(23) Xaa = Any Amino Acid; Xaa at 20 and 23
is Cys or His 26 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 27 24 PRT
Homo sapiens VARIANT (1)...(24) Xaa = Any Amino Acid; Xaa at 21 and
24 is Cys or His 27 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 28 25
PRT Homo sapiens VARIANT (1)...(25) Xaa = Any Amino Acid; Xaa at 22
and 25 is Cys or His 28 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 29 26 PRT Homo sapiens VARIANT (1)...(26) Xaa = Any Amino
Acid; Xaa at 23 and 26 is Cys or His 29 Cys Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 30 27 PRT Homo sapiens VARIANT (1)...(27)
Xaa = Any Amino Acid; Xaa at 24 and 27 is Cys or His 30 Cys Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 31 22 PRT Homo
sapiens VARIANT (1)...(22) Xaa = Any Amino Acid; Xaa at 19 and 22
is Cys or His 31 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa 20 32 23 PRT Homo
sapiens VARIANT (1)...(23) Xaa = Any Amino Acid; Xaa at 20 and 23
is Cys or His 32 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 33 24 PRT
Homo sapiens VARIANT (1)...(24) Xaa = Any Amino Acid Xaa at 21 and
24 is Cys or His 33 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 34 25
PRT Homo sapiens VARIANT (1)...(25) Xaa = Any Amino Acid; Xaa at 22
and 25 is Cys or His 34 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 35 26 PRT Homo sapiens VARIANT (1)...(26) Xaa = Any Amino
Acid; Xaa at 23 and 26 is Cys or His 35 Cys Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 36 27 PRT Homo sapiens VARIANT (1)...(27)
Xaa = Any Amino Acid; Xaa at 24 and 27 is Cys or His 36 Cys Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 37 28 PRT Homo
sapiens VARIANT (1)...(28) Xaa = Any Amino Acid; Xaa at 25 and 28
is Cys or His 37 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 38 23 PRT Homo sapiens VARIANT (1)...(23) Xaa = Any
Amino Acid; Xaa at 20 and 23 is Cys or His 38 Cys Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 39 24 PRT Homo sapiens VARIANT (1)...(24) Xaa =
Any Amino Acid; Xaa at 21 and 24 is Cys or His 39 Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 40 25 PRT Homo sapiens VARIANT
(1)...(25) Xaa = Any Amino Acid; Xaa at 22 and 25 is Cys or His 40
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 41 26 PRT Homo
sapiens VARIANT (1)...(26) Xaa = Any Amino Acid; Xaa at 23 and 26
is Cys or His 41 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 42 27 PRT Homo sapiens VARIANT (1)...(27) Xaa = Any Amino
Acid; Xaa at 24 and 27 is Cys or His 42 Cys Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 43 28 PRT Homo sapiens VARIANT
(1)...(28) Xaa = Any Amino Acid; Xaa at 25 and 28 is Cys or His 43
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 44 29
PRT Homo sapiens VARIANT (1)...(29) Xaa = Any Amino Acid; Xaa at 26
and 29 is Cys or His 44 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 45 20 PRT Homo sapiens VARIANT (1)...(20) Xaa
= Any Amino Acid; Xaa at 16 and 20 is Cys or His 45 Cys Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa 20 46 21 PRT Homo sapiens VARIANT (1)...(21) Xaa = Any
Amino Acid; Xaa at 17 and 21 is Cys or His 46 Cys Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa 20 47 22 PRT Homo sapiens VARIANT (1)...(22) Xaa = Any
Amino Acid; Xaa at 17 and 22 is Cys or His 47 Cys Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa 20 48 23 PRT Homo sapiens VARIANT (1)...(23) Xaa = Any
Amino Acid; Xaa at 19 and 23 is Cys or His 48 Cys Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 49 21 PRT Homo sapiens VARIANT (1)...(21) Xaa =
Any Amino Acid; Xaa at 17 and 21 is Cys or His 49 Cys Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa 20 50 22 PRT Homo sapiens VARIANT (1)...(22) Xaa = Any
Amino Acid; Xaa at 18 and 22 is Cys or His 50 Cys Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa 20 51 23 PRT Homo sapiens VARIANT (1)...(23) Xaa = Any
Amino Acid; Xaa at 19 and 23 is Cys or His 51 Cys Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 52 24 PRT Homo sapiens VARIANT (1)...(24) Xaa =
Any Amino Acid; Xaa at 20 and 24 is Cys or His 52 Cys Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 53 25 PRT Homo sapiens VARIANT
(1)...(25) Xaa = Any Amino Acid; Xaa at 21 and 25 is Cys or His 53
Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 54 26 PRT Homo
sapiens VARIANT (1)...(26) Xaa = Any Amino Acid; Xaa at 22 and 26
is Cys or His 54 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 55 27 PRT Homo sapiens VARIANT (1)...(27) Xaa = Any Amino
Acid; Xaa at 23 and 27 is Cys or His 55 Cys Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 56 22 PRT
Homo sapiens VARIANT (1)...(22) Xaa = Any Amino Acid; Xaa at 18 and
22 is Cys or His 56 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa 20 57 23 PRT Homo
sapiens VARIANT (1)...(23) Xaa = Any Amino Acid; Xaa at 19 and 23
is Cys or His 57 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 58 24 PRT
Homo sapiens VARIANT (1)...(24) Xaa = Any Amino Acid; Xaa at 20 and
24 is Cys or His 58 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 59 25
PRT Homo sapiens VARIANT (1)...(25) Xaa = Any Amino Acid; Xaa at 21
and 24 is Cys or His 59 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 60 26 PRT Homo sapiens VARIANT (1)...(26) Xaa = Any Amino
Acid; Xaa at 22 and 26 is Cys or His 60 Cys Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 61 27 PRT Homo sapiens VARIANT (1)...(27)
Xaa = Any Amino Acid; Xaa at 23 and 27 is Cys or His 61 Cys Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 62 28 PRT Homo
sapiens VARIANT (1)...(28) Xaa = Any Amino Acid; Xaa at 24 and 28
is Cys or His 62 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 63 23 PRT Homo sapiens VARIANT (1)...(23) Xaa = Any
Amino Acid; Xaa at 19 and 23 is Cys or His 63 Cys Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 64 24 PRT Homo sapiens VARIANT (1)...(24) Xaa =
Any Amino Acid; Xaa at 20 and 24 is Cys or His 64 Cys Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 65 25 PRT Homo sapiens VARIANT
(1)...(25) Xaa = Any Amino Acid; Xaa at 21 and 25 is Cys or His 65
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 66 26 PRT Homo
sapiens VARIANT (1)...(26) Xaa = Any Amino Acid; Xaa at 22 and 26
is Cys or His 66 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 67 27 PRT Homo sapiens VARIANT (1)...(27) Xaa = Any Amino
Acid; Xaa at 23 and 27 is Cys or His 67 Cys Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 68 28 PRT Homo sapiens VARIANT
(1)...(28) Xaa = Any Amino Acid; Xaa at 24 and 28 is Cys or His 68
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 69 29
PRT Homo sapiens VARIANT (1)...(29) Xaa = Any Amino Acid; Xaa at 25
and 29 is Cys or His 69 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 70 24 PRT Homo sapiens VARIANT (1)...(24) Xaa
= Any Amino Acid; Xaa at 20 and 24 is Cys or His 70 Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 71 25 PRT Homo sapiens VARIANT
(1)...(25) Xaa = Any Amino Acid; Xaa at 21 and 25 is Cys or His 71
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 72 26 PRT Homo
sapiens VARIANT (1)...(26) Xaa = Any Amino Acid; Xaa at 22 and 26
is Cys or His 72 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 73 27 PRT Homo sapiens VARIANT (1)...(27) Xaa = Any Amino
Acid; Xaa at 23 and 27 is Cys or His 73 Cys Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 74 28 PRT Homo sapiens VARIANT
(1)...(28) Xaa = Any Amino Acid; Xaa at 24 and 28 is Cys or His 74
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 75 29
PRT Homo sapiens VARIANT (1)...(29) Xaa = Any Amino Acid; Xaa at 25
and 29 is Cys or His 75 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 76 30 PRT Homo sapiens VARIANT (1)...(30) Xaa
= Any Amino Acid; Xaa at 26 and 30 is Cys or His 76 Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 77 22 PRT
Homo sapiens VARIANT (1)...(21) Xaa = Any Amino Acid; Xaa at 16 and
21 is Cys or His 77 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa 20 78 22 PRT Homo
sapiens VARIANT (1)...(22) Xaa = Any Amino Acid; Xaa at 17 and 22
is Cys or His 78 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa 20 79 23 PRT Homo
sapiens VARIANT (1)...(23) Xaa = Any Amino Acid; Xaa at 18 and 23
is Cys or His 79 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 80 24 PRT
Homo sapiens VARIANT (1)...(24) Xaa = Any Amino Acid; Xaa at 19 and
24 is Cys or His 80 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 81 22
PRT Homo sapiens VARIANT (1)...(22) Xaa = Any Amino Acid; Xaa at 17
and 22 is Cys or His 81 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa 20 82 23 PRT
Homo sapiens VARIANT (1)...(23) Xaa = Any Amino Acid; Xaa at 18 and
23 is Cys or His 82 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 83 24 PRT
Homo sapiens VARIANT (1)...(24) Xaa = Any Amino Acid; Xaa at 19 and
24 is Cys or His 83 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 84 25
PRT Homo sapiens VARIANT (1)...(25) Xaa = Any Amino Acid; Xaa at 20
and 25 is Cys or His 84 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 85 26 PRT Homo sapiens VARIANT (1)...(26) Xaa = Any Amino
Acid; Xaa at 21 and 26 is Cys or His 85 Cys Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 86 27 PRT Homo sapiens VARIANT (1)...(27)
Xaa = Any Amino Acid; Xaa at 22 and 27 is Cys or His 86 Cys Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 87 28 PRT Homo
sapiens VARIANT (1)...(28) Xaa = Any Amino Acid; Xaa at 23 and 28
is Cys or His 87 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 88 23 PRT Homo sapiens VARIANT (1)...(22) Xaa = Any
Amino Acid; Xaa at 16 and 22 is Cys or His 88 Cys Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 89 24 PRT Homo sapiens VARIANT (1)...(24) Xaa =
Any Amino Acid; Xaa at 19 and 24 is Cys or His 89 Cys Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 90 25 PRT Homo sapiens VARIANT
(1)...(25) Xaa = Any Amino Acid; Xaa at 20 and 25 is Cys or His 90
Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 91 26 PRT Homo
sapiens VARIANT (1)...(26) Xaa = Any Amino Acid; Xaa at 21 and 26
is Cys or His 91 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 92 27 PRT Homo sapiens VARIANT (1)...(27) Xaa = Any Amino
Acid; Xaa at 22 and 27 is Cys or His 92 Cys Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 93 28 PRT Homo sapiens VARIANT
(1)...(28) Xaa = Any Amino Acid; Xaa at 23 and 28 is Cys or His 93
Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 94 29
PRT Homo sapiens VARIANT (1)...(29) Xaa = Any Amino Acid; Xaa at 24
and 29 is Cys or His 94 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 95 24 PRT Homo sapiens VARIANT (1)...(24) Xaa
= Any Amino Acid; Xaa at 19 and 24 is Cys or His 95 Cys Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 96 25 PRT Homo sapiens VARIANT
(1)...(25) Xaa = Any Amino Acid; Xaa at 20 and 25 is Cys or His 96
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 97 26 PRT Homo
sapiens VARIANT (1)...(26) Xaa = Any Amino Acid; Xaa at 21 and 26
is Cys or His 97 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 98 27 PRT Homo sapiens VARIANT (1)...(27) Xaa = Any Amino
Acid; Xaa at 22 and 27 is Cys or His 98 Cys Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 99 28 PRT Homo sapiens VARIANT
(1)...(28) Xaa = Any Amino Acid; Xaa at 23 and 28 is Cys or His 99
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 100 29
PRT Homo sapiens VARIANT (1)...(29) Xaa = Any Amino Acid; Xaa at 24
and 29 is Cys or His 100 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 20 25 101 30 PRT Homo sapiens VARIANT
(1)...(30) Xaa = Any Amino Acid; Xaa at 25 and 30 is Cys or His 101
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30 102 25 PRT Homo sapiens VARIANT (1)...(25) Xaa = Any Amino Acid;
Xaa at 20 and 25 is Cys or His 102 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 103 26 PRT Homo sapiens VARIANT (1)...(26) Xaa =
Any Amino Acid; Xaa at 21 and 26 is Cys or His 103 Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 104 27 PRT Homo sapiens
VARIANT (1)...(27) Xaa = Any Amino Acid; Xaa at 22 and 27 is Cys or
His 104 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 105
28 PRT Homo sapiens VARIANT (1)...(28) Xaa = Any Amino Acid; Xaa at
23 and 28is Cys or His 105 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 106 29 PRT Homo sapiens VARIANT (1)...(29)
Xaa = Any Amino Acid; Xaa at 24 and 29 is Cys or His 106 Cys Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 107 30
PRT Homo sapiens VARIANT (1)...(30) Xaa = Any Amino Acid; Xaa at 25
and 30 is Cys or His 107 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 108 31 PRT Homo sapiens VARIANT
(1)...(31) Xaa = Any Amino Acid; Xaa at 26 and 31 is Cys or His 108
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30 109 22 PRT Homo sapiens VARIANT (1)...(22) Xaa = Any Amino
Acid; Xaa at 17 and 22 is Cys or His 109 Cys Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa
Xaa Xaa 20 110 23 PRT Homo sapiens VARIANT (1)...(23) Xaa = Any
Amino Acid; Xaa at 18 and 23 is Cys or His 110 Cys Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 111 24 PRT Homo sapiens VARIANT (1)...(24) Xaa =
Any Amino Acid; Xaa at 19 and 24 is Cys or His 111 Cys Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 112 25 PRT Homo sapiens VARIANT
(1)...(25) Xaa = Any Amino Acid; Xaa at 20 and 25 is Cys or His 112
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 113 23 PRT Homo
sapiens VARIANT (1)...(23) Xaa = Any Amino Acid; Xaa at 18 and 23
is Cys or His 113 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 114 24 PRT
Homo sapiens VARIANT (1)...(24) Xaa = Any Amino Acid; Xaa at 19 and
24 is Cys or His 114 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20
115 25 PRT Homo sapiens VARIANT (1)...(25) Xaa = Any Amino Acid;
Xaa at 20 and 25 is Cys or His 115 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 116 26 PRT Homo sapiens VARIANT (1)...(26) Xaa =
Any Amino Acid; Xaa at 21 and 25 is Cys or His 116 Cys Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 117 27 PRT Homo sapiens VARIANT (1)...(27) Xaa =
Any Amino Acid; Xaa at 22 and 27 is Cys or His 117 Cys Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 118 28 PRT Homo sapiens
VARIANT (1)...(28) Xaa = Any Amino Acid; Xaa at 23 and 28 is Cys or
His 118 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
119 29 PRT Homo sapiens VARIANT (1)...(29) Xaa = Any Amino Acid;
Xaa at 24 and 29 is Cys or His 119 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 120 24 PRT Homo sapiens VARIANT
(1)...(24) Xaa = Any Amino Acid; Xaa at 19 and 24 is Cys or His 120
Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 121 25 PRT Homo sapiens
VARIANT (1)...(25) Xaa = Any Amino Acid; Xaa at 20 and 25 is Cys or
His 121 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 122 26 PRT
Homo sapiens VARIANT (1)...(26) Xaa = Any Amino Acid; Xaa at 21 and
26 is Cys or His 122 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 123 27 PRT Homo sapiens VARIANT (1)...(27) Xaa = Any
Amino Acid; Xaa at 22 and 27 is Cys or His 123 Cys Xaa Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 124 28 PRT Homo sapiens
VARIANT (1)...(28) Xaa = Any Amino Acid; Xaa at 23 and 28 is Cys or
His 124 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
125 29 PRT Homo sapiens VARIANT (1)...(29) Xaa = Any Amino Acid;
Xaa at 24 and 29 is Cys or His 125 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 126 30 PRT Homo sapiens VARIANT
(1)...(30) Xaa = Any Amino Acid; Xaa at 25 and 30 is Cys or His 126
Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30 127 25 PRT Homo sapiens VARIANT (1)...(25) Xaa = Any Amino Acid;
Xaa at 20 and 25 is Cys or His 127 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 128 26 PRT Homo sapiens VARIANT (1)...(26) Xaa =
Any Amino Acid; Xaa at 21 and 26 is Cys or His 128 Cys Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 129 27 PRT Homo sapiens
VARIANT (1)...(27) Xaa = Any Amino Acid; Xaa at 22 and 27 is Cys or
His 129 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 130
28 PRT Homo sapiens VARIANT (1)...(28) Xaa = Any Amino Acid; Xaa at
23 and 28 is Cys or His 130 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 131 29 PRT Homo sapiens VARIANT (1)...(29)
Xaa = Any Amino Acid; Xaa at 24 and 29 is Cys or His 131 Cys Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 132 30
PRT Homo sapiens VARIANT (1)...(30) Xaa = Any Amino Acid; Xaa at 25
and 30 is Cys or His 132 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 133 31 PRT Homo sapiens VARIANT
(1)...(31) Xaa = Any Amino Acid; Xaa at 26 and 31 is Cys or His 133
Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30 134 26 PRT Homo sapiens VARIANT (1)...(26) Xaa = Any Amino
Acid; Xaa at 21 and 26 is Cys or His 134 Cys Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 135 27 PRT Homo sapiens VARIANT
(1)...(27) Xaa = Any Amino Acid; Xaa at 22 and 27 is Cys or His 135
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 136 28 PRT
Homo sapiens VARIANT (1)...(28) Xaa = Any Amino Acid; Xaa at 22 and
28 is Cys or His 136 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 137 29 PRT Homo sapiens VARIANT (1)...(29) Xaa =
Any Amino Acid; Xaa at 25 and 29 is Cys or His 137 Cys Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 138 30 PRT Homo
sapiens VARIANT (1)...(30) Xaa = Any Amino Acid; Xaa at 25 and 30
is Cys or His 138 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 30 139 31 PRT Homo sapiens VARIANT (1)...(31)
Xaa = Any Amino Acid; Xaa at 26 and 31 is Cys or His 139 Cys Xaa
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30 140 32 PRT Homo sapiens VARIANT (1)...(32) Xaa = Any Amino Acid;
Xaa at 27 and 32 is Cys or His 140 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 141 19 PRT Homo
sapiens VARIANT (1)...(19) Xaa = Any Amino Acid 141 Cys Xaa Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Xaa 1 5 10 15 Xaa Xaa
Cys 142 20 PRT Homo sapiens VARIANT (1)...(20) Xaa = Any Amino Acid
142 Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Xaa
1 5 10 15 Xaa Xaa Xaa Cys 20 143 21 PRT Homo sapiens VARIANT
(1)...(21) Xaa = Any Amino Acid 143 Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa His Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys
20
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