U.S. patent application number 12/628136 was filed with the patent office on 2011-01-13 for card domain containing polypeptides, encoding nucleic acids, and methods of use.
This patent application is currently assigned to Burnham Institute for Medical Research. Invention is credited to Jason S. Damiano, Adam Godzik, Hideki Hayashi, Sug Hyung Lee, Vasco A. Oliveira, Kryzysztof Pawlowski, Frederick F. Pio, John C. Reed, Christian Stehlik.
Application Number | 20110008319 12/628136 |
Document ID | / |
Family ID | 27501147 |
Filed Date | 2011-01-13 |
United States Patent
Application |
20110008319 |
Kind Code |
A1 |
Reed; John C. ; et
al. |
January 13, 2011 |
CARD DOMAIN CONTAINING POLYPEPTIDES, ENCODING NUCLEIC ACIDS, AND
METHODS OF USE
Abstract
The invention provides caspase recruitment domain
(CARD)-containing polypeptides, CARD, NB-ARC, ANGIO-R, LRR and SAM
domains therefrom, as well as encoding nucleic acid molecules and
specific antibodies. The invention also provides related screening,
diagnostic and therapeutic methods.
Inventors: |
Reed; John C.; (Rancho Santa
Fe, CA) ; Pio; Frederick F.; (Vancouver, CA) ;
Godzik; Adam; (San Diego, CA) ; Stehlik;
Christian; (San Deigo, CA) ; Damiano; Jason S.;
(La Jolla, CA) ; Lee; Sug Hyung; (San Deigo,
CA) ; Oliveira; Vasco A.; (San Deigo, CA) ;
Hayashi; Hideki; (Nagasaki City, JP) ; Pawlowski;
Kryzysztof; (Malmo, SE) |
Correspondence
Address: |
MCDERMOTT, WILL & EMERY LLP
600 13th Street, NW
Washington
DC
20005-3096
US
|
Assignee: |
Burnham Institute for Medical
Research
La Jolla
CA
|
Family ID: |
27501147 |
Appl. No.: |
12/628136 |
Filed: |
November 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11782892 |
Jul 25, 2007 |
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12628136 |
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09864921 |
May 23, 2001 |
7196182 |
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11782892 |
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60275980 |
Mar 14, 2001 |
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60367337 |
Oct 10, 2000 |
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60325756 |
May 24, 2000 |
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Current U.S.
Class: |
424/130.1 ;
435/320.1; 435/325; 530/324; 530/328; 530/350; 536/23.5 |
Current CPC
Class: |
G06F 16/958 20190101;
C07K 2319/00 20130101; A61P 35/00 20180101; C07K 14/4702 20130101;
G06F 16/335 20190101 |
Class at
Publication: |
424/130.1 ;
536/23.5; 530/350; 530/328; 530/324; 435/320.1; 435/325 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/04 20060101 C07H021/04; C07K 14/00 20060101
C07K014/00; C07K 7/06 20060101 C07K007/06; A61P 35/00 20060101
A61P035/00; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10 |
Goverment Interests
[0002] This invention was made in part with U.S. Government support
under NIH Grant No. GM61694 awarded by the National Institutes of
Health. The U.S. Government has certain rights in this invention.
Claims
1. An isolated nucleic acid molecule encoding a CARD-containing
polypeptide, or a CARD, NB-ARC, ANGIO-R, LRR or SAM domain
therefrom, selected from: (a) DNA encoding a polypeptide comprising
the amino acid sequence set forth in SEQ ID NOS: 12, 168, 188, 170,
172, 174, 176, 97, 99, 101, 103, 178, 180, 182, 184, 86 and 90; and
(b) DNA that hybridizes to the DNA of (a) under moderately
stringent conditions, wherein said DNA encodes a biologically
active polypeptide.
2. The nucleic acid molecule of claim 1, wherein the nucleotide
sequence of said nucleic acid molecule comprises any of SEQ ID
NOS:11, 167, 187, 169, 171, 173, 175, 96, 98, 100, 102, 177, 179,
181, 183, 85 and 89.
3. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is cDNA.
4. A vector containing the nucleic acid molecule of claim 1.
5. Recombinant cells containing the nucleic acid molecule of claim
1.
6. An isolated oligonucleotide comprising at least 15 contiguous
nucleotides of the nucleic acid molecule of claim 2.
7. An oligonucleotide according to claim 6, wherein said
oligonucleotide is labeled with a detectable marker.
8. A kit for detecting the presence of CARD-encoding nucleic acid
molecule comprising at least one oligonucleotide according to claim
6.
9. An isolated CARD-containing polypeptide, or a CARD, NB-ARC,
ANGIO-R, LRR or SAM domain therefrom, comprising an amino acid
sequence at least 70% identical to the amino acid sequence set
forth in any of SEQ ID NOS:12, 168, 188, 170, 172, 174, 176, 97,
99, 101, 103, 178, 180, 182, 184, 86 and 90.
10. The CARD-containing polypeptide of claim 9, wherein said
polypeptide is encoded by a nucleotide sequence set forth as any of
SEQ ID NOS:11, 167, 187, 169, 171, 173, 175, 96, 98, 100, 102, 177,
179, 181, 183, 85 and 89.
11. A peptide, comprising at least 10 contiguous amino acids of the
polypeptide of claim 9.
12-26. (canceled)
27. A composition comprising a compound selected from the group
consisting of a CARD-containing polypeptide, a functional fragment
therefrom, and an anti-CARD antibody; and a pharmaceutically
acceptable carrier.
28. (canceled)
29. A chimeric polypeptide comprising a domain of claim 9, wherein
said domain is selected from the group consisting of SEQ ID
NOS:168, 170, 172, 174, 176, 178, 180, 182 and 184.
30. (canceled)
Description
[0001] This application is a continuation of application Ser. No.
11/782,892, filed Mar. 26, 2007, which is a continuation of
application Ser. No. 09/864,921, filed May 23, 2001, which claims
the benefit of three U.S. Provisional Applications: Application
Ser. No. 60/325,756, filed May 24, 2000, which was converted from
U.S. Ser. No. 09/579,240; and Application No. 60/367,337, filed
Oct. 10, 2000, which was converted from U.S. Ser. No. 09/686,347;
and Application No. 60/275,980, filed Mar. 14, 2001, each of which
is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates generally to the fields of molecular
biology and molecular medicine and more specifically to the
identification of proteins involved in programmed cell death,
cytokine processing and receptor signal transduction, and
associations of these proteins.
[0005] 2. Background Information
[0006] Programmed cell death is a physiologic process that ensures
homeostasis is maintained between cell production and cell turnover
in essentially all self-renewing tissues. In many cases,
characteristic morphological changes, termed "apoptosis," occur in
a dying cell. Since similar changes occur in different types of
dying cells, cell death appears to proceed through a common pathway
in different cell types.
[0007] In addition to maintaining tissue homeostasis, apoptosis
also occurs in response to a variety of external stimuli, including
growth factor deprivation, alterations in calcium levels,
free-radicals, cytotoxic lymphokines, infection by some viruses,
radiation and most chemotherapeutic agents. Thus, apoptosis is an
inducible event that likely is subject to similar mechanisms of
regulation as occur, for example, in a metabolic pathway. In this
regard, dysregulation of apoptosis also can occur and is observed,
for example, in some types of cancer cells, which survive for a
longer time than corresponding normal cells, and in
neurodegenerative diseases where neurons die prematurely. In viral
infections, induction of apoptosis can figure prominently in the
pathophysiology of the disease process, because immune-based for
eradication of viral infections depend on elimination of
virus-producing host cells by immune cell attack resulting in
apoptosis.
[0008] Some of the proteins involved in programmed cell death have
been identified and associations among some of these proteins have
been described. However, additional apoptosis regulating proteins
remain to be found and the mechanisms by which these proteins
mediate their activity remains to be elucidated. The identification
of the proteins involved in cell death and an understanding of the
associations between these proteins can provide a means for
manipulating the process of apoptosis in a cell and, therefore,
selectively regulating the relative lifespan of a cell or its
relative resistance to cell death stimuli.
[0009] The principal effectors of apoptosis are a family of
intracellular proteases known as Caspases, representing an
abbreviation for Cysteine Aspartyl Proteases. Caspases are found as
inactive zymogens in essentially all animal cells. During
apoptosis, the caspases are activated by proteolytic processing at
specific aspartic acid residues, resulting in the production of
subunits that assemble into an active protease typically consisting
of a heterotetramer containing two large and two small subunits.
The phenomenon of apoptosis is produced directly or indirectly by
the activation of caspases in cells, resulting in the proteolytic
cleavage of specific substrate proteins. Moreover, in many cases,
caspases can cleave and activate themselves and each other,
creating cascades of protease activation and mechanisms for
"auto"-activation. Thus, knowledge about the proteins that interact
with and regulate caspases is important for devising strategies for
manipulating cell life and death in therapeutically useful ways. In
addition, because capsases can also participate in cytokine
activation and other processes, knowledge about the proteins that
interact with caspases can be important for manipulating immune
responses and other biochemical processes in useful ways.
[0010] One of the mechanisms for regulating caspase activation
involves protein-protein interactions mediated by a family of
protein domains known as caspase recruitment domains (CARDs). The
identification of proteins that contain CARD domains and the
elucidation of the proteins with which they interact, therefore,
can form the basis for strategies designed to alter apoptosis,
cytokine production, cytokine receptor signaling, and other
cellular processes. Thus, a need exists to identify proteins that
contain CARD domains. The present invention satisfies this need and
provides additional advantages as well.
SUMMARY OF THE INVENTION
[0011] The invention provides caspase recruitment domain
(CARD)-containing polypeptides, and CARD, NB-ARC, ANGIO-R, LRR and
SAM domains therefrom. Also provided are chimeric polypeptides
containing a CARD, NB-ARC, ANGIO-R, LRR or SAM domain of a
CARD-containing polypeptide. Methods of producing CARD-containing
polypeptides, and compositions containing CARD-containing
polypeptides and a pharmaceutically acceptable carrier, are also
provided.
[0012] The invention further provides nucleic acid molecules
encoding CARD-containing polypeptides and CARD, NB-ARC, ANGIO-R,
LRR and SAM domains therefrom. Also provided are antibodies
directed against such polypeptides.
[0013] The invention also provides methods for identifying a
nucleic acid molecule encoding a CARD-containing polypeptide, and
methods for detecting the presence of a CARD-containing polypeptide
in a sample.
[0014] Further provided are methods of identifying a
CARD-associated polypeptide (CAP), and methods of identifying an
effective agent that alters the association of a CARD-containing
polypeptide with a CAP. The invention also provides methods of
identifying an effective agent that modulates an activity of a
NB-ARC domain of a CARD-containing polypeptide.
[0015] The invention also provides methods of altering the level of
a biochemical process modulated by a CARD-containing
polypeptide.
[0016] The invention further provides methods of treating a
pathology characterized by abnormal cell proliferation, abnormal
cell death, or inflammation.
[0017] Also provided are methods of diagnosing or predicting
clinical prognosis of a pathology characterized by an increased or
decreased level of a CARD-containing polypeptide in a subject.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1A shows the genomic organization of the CLAN(CARD
4/5X) gene on chromosome 2 deduced from the BAC 164M19 sequence
from the SPG4 candidate region at 2p21-2p22 (Accession No.
AL121653) and Homo sapiens chromosome 2 working draft sequence
(Accession No. NT.sub.--005194.1). FIG. 1B shows mRNA splicing
generating CLAN A, B, C and D. FIG. 1C shows the deduced domain
structure for the splice forms of CARD4/5X (CLAN A, B, C and
D).
[0019] FIG. 2 shows an alignment of the protein sequence of the
isoforms of CLAN (designated CLAN A, B, C and D; SEQ ID NOS:97, 99,
103 and 101, respectively). Dark boxes indicate identical amino
acids, and white boxes indicate conserved amino acids.
[0020] FIG. 3 shows the amino acid sequences of the CARD-A, CARD-B
and NB-ARC domains of CARD3X (SEQ ID NOS: 170, 172 and 174,
respectively).
[0021] FIG. 4 shows an alignment of COP-1 (SEQ ID NO:86) and
caspase-1 (SEQ ID NO:87). The amino acids shaded in black are
identical.
[0022] FIG. 5 shows an alignment of COP-2 (SEQ ID NO:90) and
caspase-1 (SEQ ID NO:87), with the consensus sequence (SEQ ID
NO:91) shown above the aligned sequences. The amino acids shaded in
black are identical.
[0023] FIG. 6 shows IL-1.beta. secretion by COS7 cells transfected
with the indicated amounts of expression vectors encoding the
indicated proteins.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention provides novel polypeptides involved
in programmed cell death, or apoptosis. The principal effectors of
apoptosis are a family of intracellular cysteine aspartyl
proteases, known as caspases. Caspase activity in the cell is
regulated by protein-protein interactions. Similarly,
protein-protein interactions influence the activity of other
proteins involved in apoptosis. Several protein interaction domains
have been implicated in interactions among some
apoptosis-regulating proteins. Among these is the caspase
recruitment domain, or CARD-containing polypeptide which are so
named for the ability of the CARD-containing polypeptides to bind
caspases. In addition to their ability to bind caspases, numerous
CARD-containing polypeptides bind other proteins, particularly,
other CARD-containing polypeptides. Further, CARD-containing
polypeptides influence a variety of cellular and biochemical
processes beyond apoptosis, including cell adhesion, inflammation
and cytokine receptor signaling.
[0025] In accordance with the present invention, there are provided
isolated CARD-containing polypeptides or functional fragments
thereof, comprising substantially the same amino acid sequence as
set forth in any of SEQ ID NOS: 12, 168, 188, 170, 172, 174, 176,
97, 99, 101, 103, 178, 180, 182, 184, 86 and 90.
[0026] The sequence identifiers set forth above correspond to the
molecules described herein as set forth in Table 1
TABLE-US-00001 TABLE 1 Nucleotide Polypeptide Designation SEQ ID
NO: SEQ ID NO: CARD2X 11 12 CARD2X CARD Domain 167 168 CARD3X 187
188 and 189 CARD3X CARDA Domain 169 170 CARD3X CARDB Domain 171 172
CARD3X NB-ARC Domain 173 174 CARD3X ANGIO-R Domain 175 176 CLAN A
96 97 CLAN B 98 99 CLAN C 100 101 CLAN D 102 103 CLAN CARD 177 178
CLAN NACHT 179 180 CLAN LRR 181 182 CLAN SAM 183 184 COP1 85 86
COP2 89 90
[0027] The terms "CARD-containing protein" or "CARD-containing
polypeptide" as used herein refer to a protein or polypeptide
containing a CARD domain. As used herein, the term "CARD domain"
refers to a Caspase Recruitment Domain. A CARD domain is a well
known protein domain of approximately 80 amino acids with
characteristic sequence conservation as described, for example, in
Hofmann et al., Trends Biochem. Sci. 22:155-156 (1997). CARD
domains have been found in some members of the Caspase family of
cell death proteases. Caspases-1, 2, 4, 5, 9, and 11 contain CARD
domains near their NH2-termini. These CARD domains mediate
interactions of the zymogen inactive forms of caspases with other
proteins which can either activate or inhibit the activation of
these enzymes.
[0028] For example, the CARD domain of pro-caspase-9 binds to the
CARD domain of a caspase-activating protein called Apaf-1
(Apoptosis Protease Activating Factor-1). Similarly, the CARD
domain of pro-caspase-1 permits interactions with another CARD
protein known as Cardiac (also referred to as RIP2 and RICK), which
results in activation of the caspase-1 protease (Thome et al.,
Curr. Biol. 16:885-888 (1998)). Additionally, pro-caspase-2 binds
to the CARD protein Raidd (also know as Cradd), which permits
recruitment of pro-caspase-2 to Tumor Necrosis Factor (TNF)
Receptor complexes and which results in activation of the caspase-2
protease (Ahmad et al., Cancer Res. 57:615-619 (1997)). CARD
domains can also participate in homotypic interactions with
themselves, resulting in self-association of polypeptides that
contain these protein-interaction domains and producing dimeric or
possibly even oligomeric complexes.
[0029] CARD domains can be found in association with other types of
functional domains within a single polypeptide, thus providing a
mechanism for bringing a functional domain into close proximity or
contact with a target protein via CARD:CARD associations involving
two CARD-containing polypeptides. For example, the Caenorhabiditis
elegans cell death gene ced-4 encodes a protein that contains a
CARD domain and a ATP-binding oligomerization domain called an
NB-ARC domain (van der Biezen and Jones, Curr. Biol. 8:R226-R227).
The CARD domain of the CED-4 protein interacts with the CARD domain
of a pro-caspase called CED-3. The NB-ARC domain allows CED-4 to
self-associate, thereby forming an oligomeric complex which brings
associated pro-CED-3 molecules into close proximity to each other.
Because most pro-caspases possess at least a small amount of
protease activity even in their unprocessed form, the assembly of a
complex that brings the proforms of caspase into juxtaposition can
result in trans-processing of zymogens, producing the
proteolytically processed and active caspase. Thus, CED-4 employs a
CARD domain for binding a pro-caspase and an NB-ARC domain for
self-oligomerization, resulting in caspase clustering, proteolytic
processing and activation.
[0030] In addition to their role in caspase activation, CARD
domains have been implicated in other cellular processes. Some
CARD-containing polypeptides, for example, induce activation of the
transcription factor NF-kB. NF-kB activation is induced by many
cytokines and plays an important role in cytokine receptor signal
transduction mechanisms (DiDonato et al., Nature 388:548-554
(1997)). Moreover, CARD domains are found in some proteins that
inhibit rather than activate caspases, such as the IAP (Inhibitor
of Apoptosis Protein) family members, cIAP1 and cIAP2 (Rothe et
al., Cell 83:1243-1252 (1995)) and oncogenic mutants of the Bcl-10
protein (Willis et al., Cell 96:35-45 (1999)). Also, though caspase
activation resulting from CARD domain interactions is often
involved in inducing apoptosis, other caspases are primarily
involved in proteolytic processing and activation of inflammatory
cytokines (such as pro-IL-1b and pro-IL-18). Thus, CARD-containing
polypeptides can also be involved in cytokine receptor signaling
and cytokine production, and, therefore, can be involved in
regulation of immune and inflammatory responses.
[0031] In view of the function of the CARD domain within the
invention CARD-containing polypeptides or functional fragments
thereof, polypeptides of the invention are contemplated herein for
use in methods to alter biochemical processes such as apoptosis,
NF-kB induction, cytokine processing, cytokine receptor signaling,
caspase-mediated proteolysis, thus having modulating effects on
cell life and death (i.e., apoptosis), inflammation, cell adhesion,
and other cellular and biochemical processes.
[0032] Invention CARD-containing polypeptides or functional
fragments thereof (including CARD domains) are also contemplated in
methods to identify CARD-binding agents and CARD-associated
polypeptides (CAPs) that alter apoptosis, NF-kB induction, cytokine
processing, cytokine receptor signaling, caspase-mediated
proteolysis, thus having modulating effects on cell life and death
(i.e., apoptosis), inflammation, cell adhesion, and other cellular
and biochemical processes.
[0033] It is also contemplated herein that invention
CARD-containing polypeptides can associate with other
CARD-containing polypeptides to form invention hetero-oligomers or
homo-oligomers, such as heterodimers or homodimers. In particular,
the association of the CARD domain of invention polypeptides with
other CARD-containing polypeptides, such as Apaf-1, CED-4,
caspases-1, 2, 9, 11, cIAPs-1 and 2, CARDIAK, Raidd, Dark, CLAN,
other invention CARD-containing polypeptides, and the like,
including homo-oligomerization, is sufficiently specific such that
the bound complex can form in vivo in a cell or in vitro under
suitable conditions. Similarly therefore, an invention
CARD-containing polypeptide can associate with another
CARD-containing polypeptide by CARD:CARD form invention
hetero-oligomers or homo-oligomers, such as heterodimers or
homodimers.
[0034] In accordance with the present invention, sequences for
novel CARD-containing polypeptides have been determined. Thus, the
present invention provides novel CARD-containing polypeptides,
including the newly identified CARD-containing polypeptides
designated CARD2X, CARD3X, CLAN A, CLAN B, CLAN C, CLAN D, COP-1
and COP-2 (set forth in SEQ ID NOS: 12, 188, 97, 99, 101, 103, 86
and 90).
[0035] In addition to CARD domains, invention polypeptides can
contain one or more additional domains. The locations within the
reference sequence of the domains described herein are set forth in
Table 2.
TABLE-US-00002 TABLE 2 Domain Corresponding amino acids SEQ ID NO:
CARD2X 4-78 of SEQ ID NO: 12 167 (nt) CARD Domain 168 (aa) CARD3X
2-78 of SEQ ID NO: 107 169 (nt) CARDA Domain 170 (aa) CARD3X
105-185 of SEQ ID NO: 107 171 (nt) CARDB Domain 172 (aa) CARD3X
265-560 of SEQ ID NO: 107 173 (nt) NB-ARC Domain 174 (aa) CARD3X
437-839 of SEQ ID NO: 107 175 (nt) ANGIO-R Domain 176 (aa) CLAN
1-87 of SEQ ID NO: 97 177 (nt) CARD Domain 178 (aa) CLAN 161-457 of
SEQ ID NO: 97 179 (nt) NACHT Domain 180 (aa) CLAN 760-965 of SEQ ID
NO: 97 181 (nt) LRR Domain 182 (aa) CLAN 642-696 of SEQ ID NO: 97
183 (nt) SAM Domain 184 (aa)
[0036] CARD3X (SEQ ID NO:88) contains at least four distinct
domains: two CARD domains, designated CARD-A and CARD-B, an NB-ARC
domain and an angio-R domain. A second in-frame, open reading frame
that begins after a stop codon encodes a domain with several
leucine rich repeats (LRR) (SEQ ID NO:189) (see Example). An
invention CARD3X polypeptide can thus contain the amino acid
sequence designated SEQ ID NO:188 and the amino acid sequence
designated SEQ ID NO:189; contain SEQ ID NO:188 but not SEQ ID
NO:189; or contain SEQ ID NO:189 but not SEQ ID NO:188. A murine
CARD3X polypeptide can contain the amino acid sequence designated
SEQ ID NO:193, which is homologous to a portion of the human CARD3X
ANGIO-R domain, with or without one or more additional CARD3X
domains.
[0037] CLAN exists in four isoforms (see Example), each of which
contains a CARD domain. The longest isoform, CLAN-A, also contains
an NB-ARC(NACHT) domain, a LRR domain and a SAM domain. CLAN
represents a new member of the CED-4 related protein family.
Numerous CED-4-related proteins have recently been identified.
These proteins belong to the CED-4 family of proteins, and include
CED-4 (Yuan and Horvitz, Development 116:309-320 (1992)), Apaf-1,
(Zou et al., Cell 90:405-413 (1997)), Dark (Rodriguez et al.,
Nature Cell Biol. 1:272-279 (1999)), and CARD4/Nod1 (Bertin et al.,
J. Biol. Chem. 274:12955-12958 (1999) and Inohara et al., J. Biol.
Chem. 274:14560-14567 (1999)). As used herein, a "CED-4 family"
member or "CED-4 protein family" member, also referred to herein as
a "NAC" polypeptide, is a polypeptide that comprises a NB-ARC
domain and a CARD domain. The CED-4 homolog in humans and rodents,
referred to as Apaf-1, contains a (i) CARD domain, (ii) NB-ARC
domain, and (iii) multiple copies of a WD-repeat domain. In
contrast to CED-4 which can spontaneously oligomerize, the
mammalian Apaf-1 protein is an inactive monomer until induced to
oligomerize by binding of a co-factor protein, cytochrome c (Li et
al., Cell 91:479-489 (1997)). In Apaf-1, the WD repeat domains
prevent oligomerization of the Apaf-1 protein, until coming into
contact with cytochrome c. Thus, the WD-repeats function as a
negative-regulatory domain that maintains Apaf-1 in a latent state
until cytochrome c release from damaged mitochondria triggers the
assembly of an oligomeric Apaf-1 complex (Saleh, J. Biol. Chem.
274:17941-17945 (1999)). By binding pro-caspase-9 through its CARD
domain, Apaf-1 oligomeric complexes are thought to bring the
zymogen forms of caspase-9 into close proximity, permitting them to
cleave each other and produce the proteolytic processed and active
caspase-9 protease (Zou et al., J. Biol. Chem. 274:11549-11556
(1999)).
[0038] Another characteristic of the invention CARD-containing
polypeptides is that they can associate with pro-caspases, caspases
or with caspase-associated proteins, thereby altering caspase
proteolytic activity. Caspase proteolytic activity is associated
with apoptosis of cells, and additionally with cytokine production.
Therefore, an invention CARD-containing polypeptide can alter
apoptosis or cytokine production by altering caspase proteolytic
activity. As used herein a "caspase" is any member of the cysteine
aspartyl proteases. Typically, as caspase can associate with a
CARD-containing polypeptide of the invention such as a NAC
polypeptide. Similarly, a "pro-caspase" is an inactive or
less-active precursor form of a caspase, which is typically
converted to the more active caspase form by a proteolytic event,
and often a proteolytic event preceded by a protein:protein
interaction such as a CARD: CARD interaction, and the like.
[0039] As described in the Example, COP-1 interacts with the
prodomain of pro-caspase-1 and also with RIP2, a protein previously
demonstrated to bind the prodomain of pro-caspase-1. COP-1 competes
with RIP2 for binding to pro-caspase-1, thereby inhibiting
RIP2-mediated caspase-1 oligomerization. Consequently, COP-1
interferes with the ability of RIP2 to enhance caspase-1-induced
secretion of mature IL-1.beta.. Therefore, COP-1 is likely to play
a role in controlling IL-1.beta. generation and thereby opposing
IL-1.beta.-induced inflammation. IL-1.beta. plays a critical role
in septic shock, which currently represents the most common cause
of lethality in patients treated in the intensive care setting.
Accordingly, COP-1 likely plays a role in IL-1.beta. homeostasis to
prevent systemic inflammatory reactions when challenged with
gram-negative bacteria or other inflammatory insults.
[0040] As also described in the Example, because of their
interactions with diverse other CARD proteins, the isoforms of CLAN
(A, B, C and D) likely influence apoptosis, cytokine processing, or
NF-kB activity. Interactions of CLAN with pro-caspase-1 likely
indicates a role for CLAN as a IL-1.beta. regulator. In this
regard, different isoforms of CLAN likely have opposing effects on
pro-caspase-1 activation. The longest isoform, CLAN-A, for example,
can trigger pro-caspase-1 activation by the "induced proximity"
mechanism as a result of oligomerization mediated by its
NB-ARC(NACHT) domain. In contrast, the shorter isoforms of CLAN
lacking this self-oligomerization can operate as competitive
antagonists of pro-caspase-1 activation, analogous to ICEBERG, a
CARD-containing protein that competes with CARDIAK (RIP2/RICK) for
binding to pro-caspase-1. Interactions of CLAN with NAC also
suggest this protein can have an influence on apoptosis mediated by
Apaf-1, in as much as NAC binds Apaf-1 and enhances its ability to
activate caspase-9 in response to cytochrome c. Finally, CLAN
associations with NF-kB regulators such as Bcl-10 and Nod2 strongly
suggest that at least some of the CLAN isoforms can influence the
activity of this transcription factor.
[0041] In addition to the ability to bind caspases, invention
CARD-containing polypeptides can contain a protease domain, such as
a protease domain found in caspase, and the like. A caspase
protease domain hydrolyzes amide bonds, particularly the amide bond
of a peptide or polypeptide backbone. Typically, a caspase protease
domain contains a P20/P10 domain in the active site region of the
caspase protease domain. Thus, a caspase protease domain has
proteolytic activity.
[0042] CARD-containing polypeptides are also known to induce
activation of the transcription factor NF-kB. Thus, an invention
CARD-containing polypeptide can also alter transcription by, for
example, modulation of NF-kB activity, and the like.
[0043] The NB-ARC(NACHT) domain of invention NAC polypeptides such
as CLAN and CARD3X (see Example) associates with other
polypeptides, particularly with polypeptides comprising NB-ARC
domains. Thus, a functional NB-ARC domain associates with NB-ARC
domain-containing polypeptides by way of NB-ARC:NB-ARC association.
As used herein, the term "associate" or "association" means that
CARD-containing polypeptide such as a NAC polypeptide can bind to a
polypeptide relatively specifically and, therefore, can form a
bound complex. For example, the association of a CARD domain of an
invention CARD-containing polypeptide with another CARD-containing
polypeptide or the association of a NB-ARC domain of NAC with
another NB-ARC domain-containing polypeptides is sufficiently
specific such that the bound complex can form in vivo in a cell or
in vitro under suitable conditions.
[0044] Further, a NB-ARC domain demonstrates both
nucleotide-binding (e.g., ATP-binding) and hydrolysis activities,
which is typically required for its ability to associate with
NB-ARC domain-containing polypeptides. Thus, an NB-ARC domain of
the invention NAC comprises one or more nucleotide binding sites.
As used herein, a nucleotide binding site is a portion of a
polypeptide that specifically binds a nucleotide such as, e.g.,
ADP, ATP, and the like. Typically, the nucleotide binding site of
NB-ARC will comprise a P-loop, a kinase 2 motif, or a kinase 3a
motif of the invention NAC (these motifs are defined, for example,
in van der Biezen and Jones, supra). Preferably, the nucleotide
binding site of NB-ARC comprises a P-loop of the invention NAC. The
NB-ARC domain of the an invention CARD-containing polypeptide,
therefore, is capable of associating with other NB-ARC domains in
homo- or hetero-oligormerization. Additionally, the NB-ARC domain
is characterized by nucleotide hydrolysis activity, which can
influence the ability of an NB-ARC domain to associate with another
NB-ARC domain.
[0045] An invention NAC, therefore, is capable of CARD:CARD
association and/or NB-ARC:NB-ARC association, resulting in a
multifunctional polypeptide capable of one or more specific
associations with other polypeptides. An invention NAC can alter
cell processes such as apoptosis, cytokine production, and the
like. For example, it is contemplated herein that an invention NAC
polypeptide can increase the level of apoptosis in a cell. It is
also contemplated herein that an invention NAC can decrease the
level of apoptosis in a cell. For example, a NAC which does not
induce apoptosis may form hetero-oligomers with a NAC which is
apoptotic, thus interfering with the apoptosis-inducing activity of
NAC.
[0046] In another embodiment of the invention, a CARD-containing
polypeptide of the invention, such as CLAN (SEQ ID NOS:96, 98, 100
and 102) and an isoform of CARD3X (containing SEQ ID NO:189) also
contains Leucine-Rich Repeats (LRR) domain. LRR domains are well
known in the art and, in one embodiment, the LRR domain of an
invention CARD-containing polypeptide has substantially the same
sequence as a LRR described in another CARD-containing polypeptide
known as Nod1 (Inohara et al., J. Biol. Chem. 274:14560-14567
(1999)). The function of the LRR domain is to mediate specific
interactions with other polypeptides.
[0047] In another embodiment of the invention, there are provided
CARD-containing polypeptides that contain an NB-ARC domain and a
CARD domain. NAC polypeptide sequences disclosed herein, for
example, CARD4/5X (CLAN), modulate a variety of biochemical
processes such as apoptosis. NAC polypeptides can also have other
domains that modulate biochemical processes such as an LRR domain
or a WD domain.
[0048] Those of skill in the art will recognize that numerous
residues of the above-described sequences can be substituted with
other, chemically, sterically and/or electronically similar
residues without substantially altering the biological activity of
the resulting CARD-containing polypeptide species. In addition,
larger polypeptide sequences comprising substantially the same
sequence as amino acids set forth in SEQ ID NOS:12, 168, 188, 170,
172, 174, 176, 97, 99, 101, 103, 178, 180, 182, 184, 86 and 90,
therein are contemplated within the scope of the invention.
[0049] As employed herein, the term "substantially the same amino
acid sequence" refers to amino acid sequences having at least about
70% or 75% identity with respect to the reference amino acid
sequence, and retaining comparable functional and biological
activity characteristic of the polypeptide defined by the reference
amino acid sequence. Preferably, polypeptides having "substantially
the same amino acid sequence" will have at least about 80%, 82%,
84%, 86% or 88%, more preferably 90%, 91%, 92%, 93% or 94% amino
acid identity with respect to the reference amino acid sequence;
with greater than about 95%, 96%, 97%, 98% or 99% amino acid
sequence identity being especially preferred. It is recognized,
however, that polypeptides or nucleic acids containing less than
the described levels of sequence identity arising as splice
variants or that are modified by conservative amino acid
substitutions, or by substitution of degenerate codons are also
encompassed within the scope of the present invention.
[0050] In accordance with the invention, specifically included
within the definition of substantially the same amino acid sequence
is the predominant amino acid sequence of a particular invention
CARD-containing polypeptide or domain disclosed herein. The
predominant amino acid sequence refers to the most commonly
expressed naturally occurring amino acid sequence in a species
population. A predominant polypeptide with multiple isoforms will
have the most commonly expressed amino acid sequence for each
isoform. A predominant CARD-containing polypeptide of the invention
refers to an amino acid sequence having sequence identity to an
amino acid sequence disclosed herein that is greater than that of
any other naturally occurring protein of a particular species
(e.g., human).
[0051] Given the teachings herein of the location and nucleic acid
or amino acid sequences corresponding to the invention
CARD-containing polypeptides, one of skill in the art can readily
confirm and, if necessary, revise the nucleic acid or amino acid
sequences associated with the CARD-containing polypeptides of the
invention. For example, the sequences can be confirmed by probing a
cDNA library with a nucleic acid probe corresponding to a nucleic
acid of the invention using PCR or other known methods. Further, an
appropriate bacterial artificial chromosome containing the region
of the genome encoding an invention CARD-containing polypeptide can
be commercially obtained and probed using PCR, restriction mapping,
sequencing, and other known methods.
[0052] The term "biologically active" or "functional", when used
herein as a modifier of invention CARD-containing polypeptides, or
polypeptide fragments thereof, refers to a polypeptide that
exhibits functional characteristics similar to a CARD-containing
polypeptide of the invention. Biological activities of a
CARD-containing polypeptide include, for example, the ability to
bind, preferably in vivo, to a nucleotide, to a CARD-associated
polypeptide, to a NB-ARC-containing polypeptide, or to
homo-oligomerize, or to alter protease activation, particularly
caspase activation, or to catalyze reactions such as proteolysis or
nucleotide hydrolysis, or to alter NF-kB activity, or to alter
apoptosis, cytokine processing, cytokine receptor signaling,
inflammation, immune response, and other biological activities
described herein.
[0053] The ability of a CARD-containing polypeptide to bind another
polypeptide such as a CARD-associated polypeptide can be assayed,
for example, using the methods well known in the art such as yeast
two-hybrid assays, co-immunoprecipitation, GST fusion
co-purification, and other methods provided in standard technique
manuals such as Sambrook, supra, and Ausubel et al., supra. Another
biological activity of a CARD-containing polypeptide is the ability
to act as an immunogen for the production of polyclonal and
monoclonal antibodies that bind specifically to an invention
CARD-containing polypeptide. Thus, an invention nucleic acid
encoding a CARD-containing polypeptide can encode a polypeptide
specifically recognized by an antibody that also specifically
recognizes a CARD-containing polypeptide (preferably human)
including the amino acid set forth in SEQ ID NOS: 12, 168, 188,
170, 172, 174, 176, 97, 99, 101, 103, 178, 180, 182, 184, 86 and
90. Such immunologic activity may be assayed by any method known to
those of skill in the art. For example, a test-polypeptide can be
used to produce antibodies, which are then assayed for their
ability to bind to an invention polypeptide. If the antibody binds
to the test-polypeptide and to the reference polypeptide with
substantially the same affinity, then the polypeptide possesses the
requisite immunologic biological activity.
[0054] As used herein, the term "substantially purified" means a
polypeptide that is in a form that is relatively free from
contaminating lipids, polypeptides, nucleic acids or other cellular
material normally associated with a polypeptide in a cell. A
substantially purified CARD-containing polypeptide can be obtained
by a variety of methods well-known in the art, e.g., recombinant
expression systems described herein, chemical synthesis or
purification from native sources. Purification methods can include,
for example, precipitation, gel filtration, ion-exchange,
reverse-phase and affinity chromatography, and the like. Other
well-known methods are described in Deutscher et al., "Guide to
Protein Purification" Methods in Enzymology Vol. 182, (Academic
Press, (1990)). Alternatively, the isolated polypeptides of the
present invention can be obtained using well-known recombinant
methods as described, for example, in Sambrook et al., supra,
(1989) and Ausubel et al., supra (2000). The methods and conditions
for biochemical purification of a polypeptide of the invention can
be chosen by those skilled in the art, and purification monitored,
for example, by an immunological assay, binding assay, or a
functional assay.
[0055] In addition to the ability of invention CARD-containing
polypeptides, or functional fragments thereof, to interact with
other, heterologous proteins (e.g., CARD-containing polypeptides),
invention CARD-containing polypeptides have the ability to
self-associate to form invention homo-oligomers such as homodimers.
This self-association is possible through interactions between CARD
domains, and also through interactions between NB-ARC domains.
Further, self-association can take place as a result of
interactions between LRR domains.
[0056] In accordance with the invention, there are also provided
mutations and fragments of CARD-containing polypeptides which have
activity different than a predominant naturally occurring
CARD-containing polypeptide activity. As used herein, a "mutation"
can be any deletion, insertion, or change of one or more amino
acids in the predominant naturally occurring protein sequence
(e.g., wild-type), and a "fragment" is any truncated form, either
carboxy-terminal, amino-terminal, or both, of the predominant
naturally occurring protein. Preferably, the different activity of
the mutation or fragment is a result of the mutant polypeptide or
fragment maintaining some but not all of the activities of the
respective predominant naturally occurring CARD-containing
polypeptide.
[0057] For example, a functional fragment of an invention
polypeptide can contain or consist of one or more of the following:
a CARD domain, a NB-ARC domain, a LRR domain, a SAM domain, or an
angio-R domain. In a specific example, a fragment of a
CARD-containing polypeptide such as CLAN can contain a CARD domain
and LRR domain, but lack a functional NB-ARC domain. Such a
fragment will maintain a portion of the predominant naturally
occurring CLAN activity (e.g., CARD domain functionality), but not
all such activities (e.g., lacking an active NB-ARC domain). The
resultant fragment will therefore have an activity different than
the predominant naturally occurring CLAN activity. In another
example, the CLAN polypeptide might have only the NB-ARC domain,
allowing it to interact with other NB-ARC domain proteins in
forming homo-oligomers or hetero-oligomers. In one embodiment, the
activity of the fragment will be "dominant-negative." A
dominant-negative activity will allow the fragment to reduce or
inactivate the activity of one or more isoforms of a predominant
naturally occurring CARD-containing polypeptide. Another functional
fragment can include an angio-R domain (see Example), or any of the
domains disclosed herein (see, for example, Table 2).
[0058] Isoforms of the CARD-containing polypeptides are also
provided which arise from alternative mRNA splicing and may alter
or modify the interactions of the CARD-containing polypeptide with
other polypeptides. For example, four isoforms of CLAN and three
isoforms of CARD3X are disclosed herein. Additional isoforms of the
CARD-containing polypeptides designated SEQ ID NOS: 12, 188, 97,
99, 101, 103, 86 and 90, are contemplated herein and therefore, are
encompassed within the scope of the invention CARD-containing
polypeptides.
[0059] Methods to identify polypeptides containing a functional
fragment of a CARD-containing polypeptide of the invention are well
known in the art and are disclosed herein. For example, genomic or
cDNA libraries, including universal cDNA libraries can be probed
according to methods disclosed herein or other methods known in the
art. Full-length polypeptide encoding nucleic acids such as
full-length cDNAs can be obtained by a variety of methods
well-known in the art. For example, 5' and 3' RACE, methodology is
well known in the art and described in Ausubel et al., supra, and
the like.
[0060] In another embodiment of the invention, chimeric
polypeptides are provided comprising a CARD-containing polypeptide,
or a functional fragment thereof, fused with another protein or
functional fragment thereof. Functional fragments of a
CARD-containing polypeptide include, for example, NB-ARC(NACHT),
CARD, LRR, and ANGIO-R domains or other fragments that retain a
biological activity of an invention CARD-containing polypeptide.
Polypeptides with which the CARD-containing polypeptide or
functional fragment thereof are fused will include, for example,
glutathione-S-transferase, an antibody, or other proteins or
functional fragments thereof which facilitate recovery of the
chimera. Further, polypeptides with which a CARD-containing
polypeptide or functional fragment thereof are fused will include,
for example, luciferase, green fluorescent protein, an antibody, or
other proteins or functional fragments thereof which facilitate
identification of the chimera. Still further polypeptides with
which a CARD-containing polypeptide or functional fragment thereof
are fused will include, for example, the LexA DNA binding domain,
ricin, a-sarcin, an antibody or fragment thereof, or other
polypeptides which have therapeutic properties or other biological
activity.
[0061] Further invention chimeric polypeptides contemplated herein
are chimeric polypeptides wherein a functional fragment of a
CARD-containing polypeptide is fused with a catalytic domain or a
protein interaction domain from a heterologous polypeptide. For
example, the NB-ARC domain of CLAN, as disclosed herein, can be
replaced by the NB-ARC domain of other CARD polypeptides, such as
CARD3X, and the like. Another example of such a chimera is a
polypeptide wherein the CARD domain of CLAN is replaced by the CARD
domain from CARD2X or CARD3X, and the like. In a further example,
an NB-ARC domain can be fused with a caspase catalytic P20 domain
to form a novel chimera with caspase activity. One of skill in the
art will appreciate that a large number of chimeric polypeptides
are readily available by combining domains of two or more
CARD-containing polypeptides of the invention. Further, chimeric
polypeptides can contain a functional fragment of a CARD-containing
polypeptide of the invention fused with a domain of a protein known
in the art, such as CED-4, Apaf-1, caspase-1, and the like.
[0062] In another embodiment of the invention, polypeptides are
provided comprising 10 or more contiguous amino acids selected from
the group consisting of SEQ ID NOS:12, 188, 97, 99, 101, 103, 86
and 90.
[0063] As used herein, the term "polypeptide" when used in
reference to a CARD-containing polypeptide or fragment is intended
to refer to a peptide or polypeptide of two or more amino acids.
The term "polypeptide analog" includes any polypeptide having an
amino acid residue sequence substantially the same as a sequence
specifically described herein in which one or more residues have
been conservatively substituted with a functionally similar residue
and which displays the ability to functionally mimic a
CARD-containing polypeptide as described herein. A "modification"
of an invention polypeptide also encompasses conservative
substitutions of an invention polypeptide amino acid sequence.
Conservative substitutions of encoded amino acids include, for
example, amino acids that belong within the following groups: (1)
non-polar amino acids (Gly, Ala, Val, Leu, and Be); (2) polar
neutral amino acids (Cys, Met, Ser, Thr, Asn, and Gln); (3) polar
acidic amino acids (Asp and Glu); (4) polar basic amino acids (Lys,
Arg and His); and (5) aromatic amino acids (Phe, Trp, Tyr, and
His). Other groupings of amino acids can be found, for example in
Taylor, J. Theor. Biol. 119:205-218 (1986), which is incorporated
herein by reference. Other minor modifications are included within
invention polypeptides so long as the polypeptide retains some or
all of its function as described herein.
[0064] The amino acid length of functional fragments or polypeptide
analogs of the present invention can range from about 5 amino acids
up to the full-length protein sequence of an invention
CARD-containing polypeptide. In certain embodiments, the amino acid
lengths include, for example, at least about 10 amino acids, at
least about 15, at least about 20, at least about 25, at least
about 30, at least about 35, at least about 40, at least about 45,
at least about 50, at least about 55, at least about 60, at least
about 65, at least about 70, at least about 75, at least about 80,
at least about 85, at least about 90, at least about 95, at least
about 100, at least about 125, at least about 150, at least about
175, at least about 200, at least about 250 or more amino acids in
length up to the full-length CARD-containing polypeptide sequence.
The functional fragments can be contiguous amino acid sequences of
an invention polypeptide, including contiguous amino acid sequences
of SEQ ID NOS: 12, 188, 97, 99, 101, 103, 86 and 90. A peptide of
at least about 10 amino acids can be used, for example, as an
immungen to raise antibodies specific for an invention
CARD-containing polypeptide.
[0065] A modification of a polypeptide can also include
derivatives, analogues and functional mimetics thereof, provided
that such polypeptide displays a CARD-containing polypeptide
biological activity. For example, derivatives can include chemical
modifications of the polypeptide such as alkylation, acylation,
carbamylation, iodination, or any modification that derivatizes the
polypeptide. Such derivatized molecules include, for example, those
molecules in which free amino groups have been derivatized to form
amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl
groups. Free carboxyl groups can be derivatized to form salts,
methyl and ethyl esters or other types of esters or hydrazides.
Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl
derivatives. The imidazole nitrogen of histidine can be derivatized
to form N-im-benzylhistidine. Also included as derivatives or
analogues are those peptides which contain one or more naturally
occurring amino acid derivatives of the twenty standard amino
acids, for example, 4-hydroxyproline, 5-hydroxylysine,
3-methylhistidine, homoserine, ornithine or carboxyglutamate, and
can include amino acids that are not linked by peptide bonds.
Polypeptides of the present invention also include any polypeptide
having one or more additions and/or deletions of residues, relative
to the sequence of a polypeptide whose sequence is shown herein, so
long as CARD-containing polypeptide activity is maintained.
[0066] A modification of an invention polypeptide includes
functional mimetics thereof. Mimetics encompass chemicals
containing chemical moieties that mimic the function of the
polypeptide. For example, if a polypeptide contains two charged
chemical moieties having functional activity, a mimetic places two
charged chemical moieties in a spatial orientation and constrained
structure so that the charged chemical function is maintained in
three-dimensional space. Thus, a mimetic, which orients functional
groups that provide a function of a CARD-containing polypeptide,
are included within the meaning of a CARD-containing polypeptide
derivative. All of these modifications are included within the term
"polypeptide" so long as the invention polypeptide or functional
fragment retains its function. Exemplary mimetics are
peptidomimetics, peptoids, or other peptide-like polymers such as
poly(b-amino acids), and also non-polymeric compounds upon which
functional groups that mimic a peptide are positioned.
[0067] Another embodiment of the invention provides a
CARD-containing polypeptide, or a functional fragment thereof,
fused with a moiety to form a conjugate. As used herein, a "moiety"
can be a physical, chemical or biological entity which contributes
functionality to a CARD-containing polypeptide or a functional
fragment thereof. Functionalities contributed by a moiety include
therapeutic or other biological activity, or the ability to
facilitate identification or recovery of a CARD-containing
polypeptide. Therefore, a moiety will include molecules known in
the art to be useful for detection of the conjugate by, for
example, by fluorescence, magnetic imaging, detection of
radioactive emission. A moiety may also be useful for recovery of
the conjugate, for example a His tag or other known tags used for
protein isolation and/or purification, or a physical substance such
as a bead. A moiety can be a therapeutic compound, for example, a
cytotoxic drug which can be useful to effect a biological change in
cells to which the conjugate localizes.
[0068] An example of the means for preparing the invention
polypeptide(s) is to express nucleic acids encoding a
CARD-containing polypeptide in a suitable host cell, such as a
bacterial cell, a yeast cell, an amphibian cell such as an oocyte,
or a mammalian cell, using methods well known in the art, and
recovering the expressed polypeptide, again using well-known
purification methods. Invention polypeptides can be isolated
directly from cells that have been transformed with expression
vectors as known in the art. Recombinantly expressed polypeptides
of the invention can also be expressed as fusion proteins with
appropriate affinity tags, such as glutathione S transferase (GST)
or poly His, and affinity purified. The invention polypeptide,
biologically functional fragments, and functional equivalents
thereof can also be produced by in vitro transcription/translation
methods known in the art, such as using reticulocyte lysates, as
used for example, in the TNT system (Promega). The invention
polypeptide, biologically functional fragments, and functional
equivalents thereof can also be produced by chemical synthesis. For
example, synthetic polypeptides can be produced using Applied
Biosystems, Inc. Model 430A or 431A automatic peptide synthesizer
(Foster City, Calif.) employing the chemistry provided by the
manufacturer.
[0069] In accordance with another embodiment of the invention,
there are provided isolated nucleic acids encoding a
CARD-containing polypeptide or functional fragment thereof. The
isolated nucleic acids can be selected from: [0070] (a) DNA
encoding a polypeptide containing the amino acid sequence set forth
in SEQ ID NOs: 12, 168, 188, 170, 172, 174, 176, 97, 99, 101, 103,
178, 180, 182, 184, 86 and 90, or [0071] (b) DNA that hybridizes to
the DNA of (a) under moderately stringent conditions, where the DNA
encodes biologically active CARD-containing polypeptide, or [0072]
(c) DNA degenerate with respect to (b), where the DNA encodes
biologically active CARD-containing polypeptide.
[0073] The nucleic acid molecules described herein are useful for
producing invention polypeptides, when such nucleic acids are
incorporated into a variety of protein expression systems known to
those of skill in the art. In addition, such nucleic acid molecules
or fragments thereof can be labeled with a readily detectable
substituent and used as hybridization probes for assaying for the
presence and/or amount of an invention CARD-encoding gene or mRNA
transcript in a given sample. The nucleic acid molecules described
herein, and fragments thereof, are also useful as primers and/or
templates in a PCR reaction for amplifying genes encoding invention
polypeptides described herein.
[0074] The term "nucleic acid" (also referred to as
polynucleotides) encompasses ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA), probes, oligonucleotides, and primers
and can be single stranded or double stranded. DNA can be either
complementary DNA (cDNA) or genomic DNA, e.g. a CARD-encoding gene,
and can represent the sense strand, the anti-sense strand, or both.
Examples of nucleic acids are RNA, cDNA, or isolated genomic DNA
encoding a CARD-containing polypeptide. One means of isolating a
CARD-encoding nucleic acid is to probe a mammalian genomic or cDNA
library with a natural or artificially designed DNA probe using
methods well known in the art. DNA probes derived from the
CARD-encoding gene are particularly useful for this purpose. DNA
and cDNA molecules that encode CARD-containing polypeptides can be
used to obtain complementary genomic DNA, cDNA or RNA from
mammalian (e.g., human, mouse, rat, rabbit, pig, and the like), or
other animal sources, or to isolate related cDNA or genomic clones
by screening cDNA or genomic libraries, using methods described in
more detail below. Such nucleic acids include, but are not limited
to, nucleic acids comprising substantially the same nucleotide
sequence as set forth in SEQ ID NOS: 11, 167, 187, 169, 171, 173,
175, 96, 98, 100, 102, 177, 179, 181, 183, 85 and 89. In general, a
genomic sequence of the invention includes regulatory regions such
as promoters, enhancers, and introns that are outside of the exons
encoding a CARD-containing polypeptide but does not include
proximal genes that do not encode a CARD-containing
polypeptide.
[0075] Thus a CARD-encoding nucleic acid as used herein refers to a
nucleic acid encoding a CARD-containing polypeptide of the
invention, or a functional fragment thereof.
[0076] Use of the terms "isolated" and/or "purified" and/or
"substantially purified" in the present specification and claims as
a modifier of DNA, RNA, polypeptides or proteins means that the
DNA, RNA, polypeptides or proteins so designated have been produced
in such form by the hand of man, and thus are separated from their
native in vivo cellular environment, and are substantially free of
any other species of nucleic acid or protein. As a result of this
human intervention, the recombinant DNAs, RNAs, polypeptides and
proteins of the invention are useful in ways described herein that
the DNAs, RNAs, polypeptides or proteins as they naturally occur
are not.
[0077] Invention nucleic acids encoding CARD-containing
polypeptides and invention CARD-containing polypeptides can be
obtained from any species of organism, such as prokaryotes,
eukaryotes, plants, fungi, vertebrates, invertebrates, and the
like. A particular species can be mammalian, As used herein,
"mammalian" refers to a subset of species from which an invention
CARD-encoding nucleic acid is derived, e.g., human, rat, mouse,
rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline, and
the like. A preferred CARD-encoding nucleic acid herein, is human
CARD-encoding nucleic acid.
[0078] In one embodiment of the present invention, cDNAs encoding
the invention CARD-containing polypeptides disclosed herein
comprise substantially the same nucleotide sequence as the coding
region set forth in any of SEQ ID NOS: 11, 167, 187, 169, 171, 173,
175, 96, 98, 100, 102, 177, 179, 181, 183, 85 and 89.
[0079] As employed herein, the term "substantially the same
nucleotide sequence" refers to a nucleic acid molecule (DNA or RNA)
having sufficient identity to the reference polynucleotide, such
that it will hybridize to the reference nucleotide under moderately
or highly stringent hybridization conditions. In one embodiment, a
nucleic acid molecule having substantially the same nucleotide
sequence as the reference nucleotide sequence encodes substantially
the same amino acid sequence as that set forth in any of SEQ ID
NOS: 12, 168, 188, 170, 172, 174, 176, 97, 99, 101, 103, 178, 180,
182, 184, 86 and 90. In another embodiment, a nucleic acid molecule
having "substantially the same nucleotide sequence" as the
reference nucleotide sequence has at least 60%, or at least 65%
identity with respect to the reference nucleotide sequence, such as
at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the
reference nucleotide sequence.
[0080] In accordance with the invention, specifically included
within the definition of substantially the same nucleotide sequence
is the predominant nucleotide sequence of a particular invention
CARD-containing polypeptide described herein. The predominant
nucleotide sequence refers to the most commonly present naturally
occurring nucleotide sequence in a species population. A
predominant CARD-encoding nucleic acid of the invention refers to a
nucleotide sequence having sequence identity to a nucleotide
sequence disclosed herein that is greater than that of any other
naturally occurring nucleotide sequence of a particular species
(e.g., human).
[0081] In one embodiment, a nucleic acid molecule that has
substantially the same nucleotide sequence as a reference sequence
is a modification of the reference sequence. As used herein, a
"modification" of a nucleic acid can include one or several
nucleotide additions, deletions, or substitutions with respect to a
reference sequence. A modification of a nucleic acid can include
substitutions that do not change the encoded amino acid sequence
due to the degeneracy of the genetic code. Such modifications can
correspond to variations that are made deliberately, or which occur
as mutations during nucleic acid replication.
[0082] Exemplary modifications of the recited nucleotide sequences
include sequences that correspond to homologs of other species,
including mammalian species such as mouse, primates, including
monkey and baboon, rat, rabbit, bovine, porcine, ovine, canine,
feline, or other animal species. The corresponding nucleotide
sequences of non-human species can be determined by methods known
in the art, such as by PCR or by screening genomic, cDNA or
expression libraries.
[0083] Another exemplary modification of the invention
CARD-encoding nucleic acid or CARD-containing polypeptide can
correspond to splice variant forms of the CARD-encoding nucleotide
sequence. Additionally, a modification of a nucleotide sequence can
include one or more non-native nucleotides, having, for example,
modifications to the base, the sugar, or the phosphate portion, or
having a modified phosphodiester linkage. Such modifications can be
advantageous in increasing the stability of the nucleic acid
molecule.
[0084] Furthermore, a modification of a nucleotide sequence can
include, for example, a detectable moiety, such as a radiolabel, a
fluorochrome, a ferromagnetic substance, a luminescent tag or a
detectable binding agent such as biotin. Such modifications can be
advantageous in applications where detection of a CARD-encoding
nucleic acid molecule is desired.
[0085] In another embodiment, a nucleic acid molecule that has
substantially the same nucleotide sequence as a reference sequence
is a functionally equivalent nucleic acid, which indicates that it
is phenotypically similar to the reference nucleic acid. As used
herein, the phrase "functionally equivalent nucleic acids"
encompasses nucleic acids characterized by slight and
non-consequential sequence variations that will function in
substantially the same manner to produce the same polypeptide
product(s) as the nucleic acids disclosed herein. In particular,
functionally equivalent nucleic acids encode polypeptides that are
the same as those encoded by the nucleic acids disclosed herein or
that have conservative amino acid variations, as described above.
These variations include those recognized by skilled artisans as
those that do not substantially alter the tertiary structure of the
protein.
[0086] Further provided are nucleic acids encoding CARD-containing
polypeptides that, by virtue of the degeneracy of the genetic code,
do not necessarily hybridize to the invention nucleic acids under
specified hybridization conditions. Preferred nucleic acids
encoding the invention CARD-containing polypeptides are comprised
of nucleotides that encode substantially the same amino acid
sequence as set forth in SEQ ID NOS:12, 168, 188, 170, 172, 174,
176, 97, 99, 101, 103, 178, 180, 182, 184, 86 and 90.
[0087] Hybridization refers to the binding of complementary strands
of nucleic acid (i.e., sense:antisense strands or probe:target-DNA)
to each other through hydrogen bonds, similar to the bonds that
naturally occur in chromosomal DNA. Stringency levels used to
hybridize a given probe with target-DNA can be readily varied by
those of skill in the art.
[0088] The phrase "stringent hybridization" is used herein to refer
to conditions under which polynucleic acid hybrids are stable. As
known to those of skill in the art, the stability of hybrids is
reflected in the melting temperature (Tm) of the hybrids. In
general, the stability of a hybrid is a function of sodium ion
concentration and temperature. Typically, the hybridization
reaction is performed under conditions of lower stringency,
followed by washes of varying, but higher, stringency. Reference to
hybridization stringency relates to such washing conditions.
[0089] As used herein, the phrase "moderately stringent
hybridization" refers to conditions that permit target-nucleic acid
to bind a complementary nucleic acid. The hybridized nucleic acids
will generally have at least about 60% identity, at least about 75%
identity, such as at least about 85% identity; or at least about
90% identity. Moderately stringent conditions are conditions
equivalent to hybridization in 50% formamide, 5.times.Denhart's
solution, 5.times.SSPE, 0.2% SDS at 42.degree. C., followed by
washing in 0.2.times.SSPE, 0.2% SDS, at 42.degree. C.
[0090] The phrase "high stringency hybridization" refers to
conditions that permit hybridization of only those nucleic acid
sequences that form stable hybrids in 0.018M NaCl at 65.degree. C.,
for example, if a hybrid is not stable in 0.018M NaCl at 65.degree.
C., it will not be stable under high stringency conditions, as
contemplated herein. High stringency conditions can be provided,
for example, by hybridization in 50% formamide, 5.times.Denhart's
solution, 5.times.SSPE, 0.2% SDS at 42.degree. C., followed by
washing in 0.1.times.SSPE, and 0.1% SDS at 65.degree. C.
[0091] The phrase "low stringency hybridization" refers to
conditions equivalent to hybridization in 10% formamide,
5.times.Denhart's solution, 6.times.SSPE, 0.2% SDS at 22.degree.
C., followed by washing in 1.times.SSPE, 0.2% SDS, at 37.degree. C.
Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and
1% bovine serum albumin (BSA). 20.times.SSPE (sodium chloride,
sodium phosphate, ethylene diamide tetraacetic acid (EDTA))
contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M
(EDTA). Other suitable moderate stringency and high stringency
hybridization buffers and conditions are well known to those of
skill in the art and are described, for example, in Sambrook et
al., supra (1989); and Ausubel et al., supra, 2000). Nucleic acids
encoding polypeptides hybridize under moderately stringent or high
stringency conditions to substantially the entire sequence, or
substantial portions, for example, typically at least 15-30
nucleotides of the nucleic acid sequence set forth in SEQ ID
NOS:11, 167, 187, 169, 171, 173, 175, 96, 98, 100, 102, 177, 179,
181, 183, 85 and 89.
[0092] As used herein, the term "degenerate" refers to codons that
differ in at least one nucleotide from a reference nucleic acid,
e.g., SEQ ID NOS:11, 167, 187, 169, 171, 173, 175, 96, 98, 100,
102, 177, 179, 181, 183, 85 and 89, but encode the same amino acids
as the reference nucleic acid. For example, codons specified by the
triplets "UCU", "UCC", "UCA", and "UCG" are degenerate with respect
to each other since all four of these codons encode the amino acid
serine.
[0093] The invention also provides a modification of a nucleotide
sequence that hybridizes to a CARD-encoding nucleic acid molecule,
for example, a nucleic acid molecule referenced as any of SEQ ID
NOS:11, 167, 187, 169, 171, 173, 175, 96, 98, 100, 102, 177, 179,
181, 183, 85 and 89 under moderately stringent conditions.
Modifications of nucleotide sequences, where the modification has
at least 60% identity to a CARD-encoding nucleotide sequence, are
also provided. The invention also provides modification of a
CARD-encoding nucleotide sequence having at least 65% identity, at
least 70% identity, at least 72% identity, at least 74% identity,
at least 76% identity, at least 78% identity, at least 80%
identity, at least 82% identity, at least 84% identity, at least
86% identity, at least 88% identity, at least 90% identity, at
least 91% identity, at least 92% identity, at least 93% identity,
at least 94% identity, at least 95% identity, at least 96%
identity, at least 97% identity, at least 98% identity or at least
99% identity.
[0094] Identity of any two nucleic acid or amino acid sequences can
be determined by those skilled in the art based, for example, on a
BLAST 2.0 computer alignment, using default parameters. BLAST 2.0
searching is known in the art and is publicly available, for
example, at http://www.ncbi.nlm.nih.gov/BLAST/, as described by
Tatiana et al., FEMS Microbiol Lett. 174:247-250 (1999); Altschul
et al., Nucleic Acids Res., 25:3389-3402 (1997).
[0095] One means of isolating a nucleic acid encoding a
CARD-containing polypeptide is to probe a cDNA library or genomic
library with a natural or artificially designed nucleic acid probe
using methods well known in the art. Nucleic acid probes derived
from a CARD-encoding gene are particularly useful for this purpose.
DNA and cDNA molecules that encode CARD-containing polypeptides can
be used to obtain complementary genomic DNA, cDNA or RNA from
mammals, for example, human, mouse, rat, rabbit, pig, and the like,
or other animal sources, or to isolate related cDNA or genomic
clones by the screening of cDNA or genomic libraries, by methods
well known in the art (see, for example, the Examples set forth
hereinafter; and Sambrook et al., supra, 1989; Ausubel et al.,
supra, 2000).
[0096] Another useful method for producing a CARD-encoding nucleic
acid molecule of the invention involves amplification of the
nucleic acid molecule using PCR and invention oligonucleotides and,
optionally, purification of the resulting product by gel
electrophoresis. Either PCR or RT-PCR can be used to produce a
CARD-encoding nucleic acid molecule having any desired nucleotide
boundaries as described in the Examples. Desired modifications to
the nucleic acid sequence can also be introduced by choosing an
appropriate oligonucleotide primer with one or more additions,
deletions or substitutions. Such nucleic acid molecules can be
amplified exponentially starting from as little as a single gene or
mRNA copy, from any cell, tissue or species of interest.
[0097] The invention additionally provides a nucleic acid that
hybridizes under high stringency conditions to the CARD coding
portion of any of SEQ ID NOS:11, 187, 96, 98, 100, 102, 85 and 89,
such as to any of SEQ ID NOS: 168, 170, 172 and 178. The invention
also provides a nucleic acid having a nucleotide sequence
substantially the same as set that forth in any of SEQ ID 11, 167,
187, 169, 171, 173, 175, 96, 98, 100, 102, 177, 179, 181, 183, 85
and 89.
[0098] The invention also provides a method for identifying nucleic
acids encoding a mammalian CARD-containing polypeptide by
contacting a sample containing nucleic acids with one or more
invention nucleic acid molecules or oligonucleotides, wherein the
contacting is effected under high stringency hybridization
conditions, and identifying a nucleic acid that hybridizes to the
oligonucleotide. The invention additionally provides a method of
detecting a CARD-encoding nucleic acid molecule in a sample by
contacting the sample with two or more invention oligonucleotides,
amplifying a nucleic acid molecule, and detecting the
amplification. The amplification can be performed, for example,
using PCR. The invention further provides oligonucleotides that
function as single stranded nucleic acid primers for amplification
of a CARD-encoding nucleic acid, wherein the primers comprise a
nucleic acid sequence derived from the nucleic acid sequences set
forth as SEQ ID NOS:11, 187, 96, 98, 100, 102, 85 and 89.
[0099] In accordance with a further embodiment of the present
invention, optionally labeled CARD-encoding cDNAs, or fragments
thereof, can be employed to probe library(ies) such as cDNA,
genomic, BAC, and the like for predominant nucleic acid sequences
or additional nucleic acid sequences encoding novel CARD-containing
polypeptides. Construction and screening of suitable mammalian cDNA
libraries, including human cDNA libraries, is well-known in the
art, as demonstrated, for example, in Ausubel et al., supra.
Screening of such a cDNA library is initially carried out under
low-stringency conditions, which comprise a temperature of less
than about 42.degree. C., a formamide concentration of less than
about 50%, and a moderate to low salt concentration.
[0100] Probe-based screening conditions can comprise a temperature
of about 37.degree. C., a formamide concentration of about 20%, and
a salt concentration of about 5.times. standard saline citrate
(SSC; 20.times.SSC contains 3M sodium chloride, 0.3M sodium
citrate, pH 7.0). Such conditions will allow the identification of
sequences which have a substantial degree of similarity with the
probe sequence, without requiring perfect homology. The phrase
"substantial similarity" refers to sequences which share at least
50% homology. Hybridization conditions are selected which allow the
identification of sequences having at least 70% homology with the
probe, while discriminating against sequences which have a lower
degree of homology with the probe. As a result, nucleic acids
having substantially the same nucleotide sequence as any of SEQ ID
NOS:11, 167, 187, 169, 171, 173, 175, 96, 98, 100, 102, 177, 179,
181, 183, 85 and 89 are obtained.
[0101] As used herein, a nucleic acid "probe" is single-stranded
nucleic acid, or analog thereof, that has a sequence of nucleotides
that includes at least 15, at least 20, at least 50, at least 100,
at least 200, at least 300, at least 400, or at least 500
contiguous bases that are substantially the same as, or the
complement of, any contiguous bases set forth in any of SEQ ID
NOS:11, 187, 96, 98, 100, 102, 85 and 89. In addition, the entire
cDNA encoding region of an invention CARD-containing polypeptide,
or an entire sequence substantially the same as SEQ ID NOS:11, 187,
96, 98, 100, 102, 85 and 89 can be used as a probe. Probes can be
labeled by methods well-known in the art, as described hereinafter,
and used in various diagnostic kits.
[0102] The invention additionally provides an oligonucleotide
comprising between 15 and 300 contiguous nucleotides of any of SEQ
ID NOS:11, 187, 96, 98, 100, 102, 85 and 89 or the anti-sense
strand thereof. As used herein, the term "oligonucleotide" refers
to a nucleic acid molecule that includes at least 15 contiguous
nucleotides from a reference nucleotide sequence, can include at
least 16, 17, 18, 19, 20 or at least 25 contiguous nucleotides, and
often includes at least 30, 40, 50, 60, 70, 80, 90, 100, 125, 150,
175, 200, 225, 250, 275, 300, 325, up to 350 contiguous nucleotides
from the reference nucleotide sequence. The reference nucleotide
sequence can be the sense strand or the anti-sense strand.
[0103] The oligonucleotides of the invention that contain at least
15 contiguous nucleotides of a reference CARD-encoding nucleotide
sequence are able to hybridize to CARD-encoding nucleotide
sequences under moderately stringent hybridization conditions and
thus can be advantageously used, for example, as probes to detect
CARD-encoding DNA or RNA in a sample, and to detect splice variants
thereof; as sequencing or PCR primers; as antisense reagents to
block transcription of CARD-encoding RNA in cells; or in other
applications known to those skilled in the art in which
hybridization to a CARD-encoding nucleic acid molecule is
desirable.
[0104] In accordance with another embodiment of the invention, a
method is provided for identifying nucleic acids encoding a
CARD-containing polypeptide. The method comprises contacting a
sample containing nucleic acids with an invention probe or an
invention oligonucleotide, wherein said contacting is effected
under high stringency hybridization conditions, and identifying
nucleic acids which hybridize thereto. Methods for identification
of nucleic acids encoding a CARD-containing polypeptide are
disclosed herein and exemplified in the Examples.
[0105] Also provided in accordance with present invention is a
method for identifying a CARD-encoding nucleotide sequence
comprising the steps of using a CARD-encoding nucleotide sequence
selected from SEQ ID NOS:11, 167, 187, 169, 171, 173, 175, 96, 98,
100, 102, 177, 179, 181, 183, 85 and 89 to identify a candidate
CARD-encoding nucleotide sequence and verifying the candidate
CARD-encoding nucleotide sequence by aligning the candidate
sequence with known CARD-encoding nucleotide sequences, where a
conserved CARD domain sequence or a predicted three dimensional
polypeptide structure similar to a known CARD domain three
dimensional structure confirms the candidate sequence as a
CARD-encoding sequence. Methods for identifying CARD-encoding
sequences are provided herein (See Examples).
[0106] It is understood that a CARD-encoding nucleic acid molecule
of the invention, as used herein, specifically excludes previously
known nucleic acid molecules consisting of nucleotide sequences
having identity with the CARD-encoding nucleotide sequence (SEQ ID
NOS:11, 167, 187, 169, 171, 173, 175, 96, 98, 100, 102, 177, 179,
181, 183, 85 and 89), such as Expressed Sequence Tags (ESTs),
Sequence Tagged Sites (STSs) and genomic fragments, deposited in
public databases such as the nr, dbest, dbsts, gss and htgs
databases, which are available for searching at
http://www.ncbi.nlm.nih.gov/blast/.
[0107] In particular, an invention CARD-encoding nucleic acid
molecule excludes the exact, specific and complete nucleic acid
molecule sequence corresponding to any of the nucleotide sequences
having the Genbank (gb), EMBL (emb) or DDBJ (dbj) accession numbers
described below. Accession numbers specifically excluded include
GI:6165147 (Phase-1), AC007728 (Phase-1), NT-002476 (Phase-1),
AC010968 (Phase-1), AP001153, AC022468 (Phase-1), GI:6253000
(Phase-1), AC0097959 (Phase-1), GI:6497652 (Phase-1)
(contig:23086:40635), GI:6497652 (Phase-1) (contig:41136:57024),
AC023068 (Phase-1), W58453, AA257158, AA046000, AW085161, AI189838,
AA418021, AA046105, W58488, AA418193, AA257066, AI217611, AW295205,
A1023795, AL389934, AA070591, AA070591, AC027011, AP002787,
AQ889169, AV719179, AI263294, AV656315, AW337918, BF207840,
AW418826, BK903662, A1023795, H25984, AL121653 and
NT.sub.--005194.1. The human contig referenced as GenBank accession
No. AC007608 is also specifically excluded from a CARD encoding
nucleic acid molecule. The genomic contigs referenced as GenBank
accession numbers GI 5001450, GI 8575872 and GI 9795562 are also
specifically excluded from invention nucleic acid molecules. Since
one of skill in the art will realize that the above-recited
excluded sequences may be revised at a later date, the skilled
artisan will recognize that the above-recited sequences are
excluded as they stand on the priority date of this
application.
[0108] The isolated nucleic acid molecules of the invention can be
used in a variety of diagnostic and therapeutic applications. For
example, the isolated nucleic acid molecules of the invention can
be used as probes, as described above; as templates for the
recombinant expression of CARD-containing polypeptides; or in
screening assays such as two-hybrid assays to identify cellular
molecules that bind CARD-containing polypeptides.
[0109] The invention thus provides methods for detecting a
CARD-encoding nucleic acid in a sample. The methods of detecting a
CARD-encoding nucleic acid in a sample can be either qualitative or
quantitative, as desired. For example, the presence, abundance,
integrity or structure of a CARD-encoding nucleic acid can be
determined, as desired, depending on the assay format and the probe
used for hybridization or primer pair chosen for application.
[0110] Useful assays for detecting a CARD-containing nucleic acid
based on specific hybridization with an isolated invention
oligonucleotide are well known in the art and include, for example,
in situ hybridization, which can be used to detect altered
chromosomal location of the nucleic acid molecule, altered gene
copy number, and RNA abundance, depending on the assay format used.
Other hybridization assays include, for example, Northern blots and
RNase protection assays, which can be used to determine the
abundance and integrity of different RNA splice variants, and
Southern blots, which can be used to determine the copy number and
integrity of DNA. A hybridization probe can be labeled with any
suitable detectable moiety, such as a radioisotope, fluorochrome,
chemiluminescent marker, biotin, or other detectable moiety known
in the art that is detectable by analytical methods.
[0111] As used herein, the terms "label" and "indicating means" in
their various grammatical forms refer to single atoms and molecules
that are either directly or indirectly involved in the production
of a detectable signal. Any label or indicating means can be linked
to invention nucleic acid probes, expressed proteins, polypeptide
fragments, or antibody molecules. These atoms or molecules can be
used alone or in conjunction with additional reagents. Such labels
are themselves well-known in clinical diagnostic chemistry.
[0112] Useful assays for detecting a CARD-encoding nucleic acid in
a sample based on amplifying a CARD-encoding nucleic acid with two
or more invention oligonucleotides are also well known in the art,
and include, for example, qualitative or quantitative polymerase
chain reaction (PCR); reverse-transcription PCR(RT-PCR); single
strand conformational polymorphism (SSCP) analysis, which can
readily identify a single point mutation in DNA based on
differences in the secondary structure of single-strand DNA that
produce an altered electrophoretic mobility upon non-denaturing gel
electrophoresis; and coupled PCR, transcription and translation
assays, such as a protein truncation test, in which a mutation in
DNA is determined by an altered protein product on an
electrophoresis gel. Additionally, the amplified CARD-encoding
nucleic acid can be sequenced to detect mutations and mutational
hot-spots, and specific assays for large-scale screening of samples
to identify such mutations can be developed.
[0113] Also provided are antisense-nucleic acids having a sequence
capable of binding specifically with full-length or any portion of
an mRNA that encodes CARD-containing polypeptides so as to prevent
translation of the mRNA. The antisense-nucleic acid can have a
sequence capable of binding specifically with any portion of the
sequence of the cDNA encoding CARD-containing polypeptides. As used
herein, the phrase "binding specifically" encompasses the ability
of a nucleic acid sequence to recognize a complementary nucleic
acid sequence and to form double-helical segments therewith via the
formation of hydrogen bonds between the complementary base pairs.
An example of an antisense-nucleic acid is an antisense-nucleic
acid comprising chemical analogs of nucleotides.
[0114] The present invention provides means to alter levels of
expression of CARD-containing polypeptides by recombinantly
expressing CARD-containing anti-sense nucleic acids or employing
synthetic anti-sense nucleic acid compositions (hereinafter SANC)
that inhibit translation of mRNA encoding these polypeptides.
Synthetic oligonucleotides, or other antisense-nucleic acid
chemical structures designed to recognize and selectively bind to
mRNA are constructed to be complementary to full-length or portions
of a CARD-encoding strand, including nucleotide sequences
substantially the same as SEQ ID NOS:11, 187, 96, 98, 100, 102, 85
and 89.
[0115] The SANC is designed to be stable in the blood stream for
administration to a subject by injection, or in laboratory cell
culture conditions. The SANC is designed to be capable of passing
through the cell membrane in order to enter the cytoplasm of the
cell by virtue of physical and chemical properties of the SANC,
which render it capable of passing through cell membranes, for
example, by designing small, hydrophobic SANC chemical structures,
or by virtue of specific transport systems in the cell which
recognize and transport the SANC into the cell. In addition, the
SANC can be designed for administration only to certain selected
cell populations by targeting the SANC to be recognized by specific
cellular uptake mechanisms which bind and take up the SANC only
within select cell populations. In a particular embodiment the SANC
is an antisense oligonucleotide.
[0116] For example, the SANC may be designed to bind to a receptor
found only in a certain cell type, as discussed above. The SANC is
also designed to recognize and selectively bind to target mRNA
sequence, which can correspond to a sequence contained within the
sequences shown in SEQ ID NOS:11, 187, 96, 98, 100, 102, 85 and 89.
The SANC is designed to inactivate target mRNA sequence by either
binding thereto and inducing degradation of the mRNA by, for
example, RNase I digestion, or inhibiting translation of mRNA
target sequence by interfering with the binding of
translation-regulating factors or ribosomes, or inclusion of other
chemical structures, such as ribozyme sequences or reactive
chemical groups which either degrade or chemically modify the
target mRNA. SANCs have been shown to be capable of such properties
when directed against mRNA targets (see Cohen et al., TIPS, 10:435
(1989) and Weintraub, Sci. American, January (1990), pp. 40).
[0117] The invention further provides a method of altering the
level of a biochemical process modulated by a CARD-containing
polypeptide by introducing an antisense nucleotide sequence into
the cell, wherein the antisense nucleotide sequence specifically
hybridizes to a CARD-encoding nucleic acid molecule, wherein the
hybridization reduces or inhibits the expression of the
CARD-containing polypeptide in the cell. The use of anti-sense
nucleic acids, including recombinant anti-sense nucleic acids or
SANCs, can be advantageously used to inhibit cell death.
[0118] Compositions comprising an amount of the antisense-nucleic
acid of the invention, effective to reduce expression of
CARD-containing polypeptides by entering a cell and binding
specifically to CARD-encoding mRNA so as to prevent translation and
an acceptable hydrophobic carrier capable of passing through a cell
membrane are also provided herein. Suitable hydrophobic carriers
are described, for example, in U.S. Pat. Nos. 5,334,761; 4,889,953;
4,897,355, and the like. The acceptable hydrophobic carrier capable
of passing through cell membranes may also comprise a structure
which binds to a receptor specific for a selected cell type and is
thereby taken up by cells of the selected cell type. For example,
the structure can be part of a protein known to bind to a cell-type
specific receptor such as a tumor.
[0119] Antisense-nucleic acid compositions are useful to inhibit
translation of mRNA encoding invention polypeptides. Synthetic
oligonucleotides, or other antisense chemical structures are
designed to bind to CARD-encoding mRNA and inhibit translation of
mRNA and are useful as compositions to inhibit expression of
CARD-encoding genes or CARD-associated polypeptide genes in a
tissue sample or in a subject.
[0120] The invention also provides vectors containing the
CARD-encoding nucleic acids of the invention. Suitable expression
vectors are well-known in the art and include vectors capable of
expressing nucleic acid operatively linked to a regulatory sequence
or element such as a promoter region or enhancer region that is
capable of regulating expression of such nucleic acid. Appropriate
expression vectors include those that are replicable in eukaryotic
cells and/or prokaryotic cells and those that remain episomal or
those which integrate into the host cell genome.
[0121] Promoters or enhancers, depending upon the nature of the
regulation, can be constitutive or regulated. The regulatory
sequences or regulatory elements are operatively linked to a
nucleic acid of the invention such that the physical and functional
relationship between the nucleic acid and the regulatory sequence
allows transcription of the nucleic acid.
[0122] Suitable vectors for expression in prokaryotic or eukaryotic
cells are well known to those skilled in the art (see, for example,
Ausubel et al., supra, 2000). Vectors useful for expression in
eukaryotic cells can include, for example, regulatory elements
including the SV40 early promoter, the cytomegalovirus (CMV)
promoter, the mouse mammary tumor virus (MMTV) steroid-inducible
promoter, Moloney murine leukemia virus (MMLV) promoter, and the
like. The vectors of the invention are useful for subcloning and
amplifying a CARD-encoding nucleic acid molecule and for
recombinantly expressing a CARD-containing polypeptide. A vector of
the invention can include, for example, viral vectors such as a
bacteriophage, a baculovirus or a retrovirus; cosmids or plasmids;
and, particularly for cloning large nucleic acid molecules,
bacterial artificial chromosome vectors (BACs) and yeast artificial
chromosome vectors (YACs). Such vectors are commercially available,
and their uses are well known in the art. One skilled in the art
will know or can readily determine an appropriate promoter for
expression in a particular host cell.
[0123] The invention additionally provides recombinant cells
containing CARD-encoding nucleic acids of the invention. The
recombinant cells are generated by introducing into a host cell a
vector containing a CARD-encoding nucleic acid molecule. The
recombinant cells are transducted, transfected or otherwise
genetically modified. Exemplary host cells that can be used to
express recombinant CARD molecules include mammalian primary cells;
established mammalian cell lines, such as COS, CHO, HeLa, NIH3T3,
HEK 293 and PC12 cells; amphibian cells, such as Xenopus embryos
and oocytes and other vertebrate cells. Exemplary host cells also
include insect cells such as Drosophila, yeast cells such as
Saccharomyces cerevisiae, Saccharomyces pombe, or Pichia pastoris,
and prokaryotic cells such as Escherichia coli. Additional host
cells can be obtained, for example, from ATCC (Manassas, Va.).
[0124] In one embodiment, CARD-encoding nucleic acids can be
delivered into mammalian cells, either in vivo or in vitro using
suitable vectors well-known in the art. Suitable vectors for
delivering a CARD-containing polypeptide, or a functional fragment
thereof to a mammalian cell, include viral vectors such as
retroviral vectors, adenovirus, adeno-associated virus, lentivirus,
herpesvirus, as well as non-viral vectors such as plasmid vectors.
Such vectors are useful for providing therapeutic amounts of a
CARD-containing polypeptide (see, for example, U.S. Pat. No.
5,399,346, issued Mar. 21, 1995). Delivery of CARD polypeptides or
nucleic acids therapeutically can be particularly useful when
targeted to a tumor cell, thereby inducing apoptosis in tumor
cells. In addition, where it is desirable to limit or reduce the in
vivo expression of a CARD-containing polypeptide, the introduction
of the antisense strand of the invention nucleic acid is
contemplated.
[0125] Viral based systems provide the advantage of being able to
introduce relatively high levels of the heterologous nucleic acid
into a variety of cells. Suitable viral vectors for introducing an
invention CARD-encoding nucleic acid into mammalian cells are well
known in the art. These viral vectors include, for example, Herpes
simplex virus vectors (Geller et al., Science, 241:1667-1669
(1988)); vaccinia virus vectors (Piccini et al., Meth. Enzymology,
153:545-563 (1987)); cytomegalovirus vectors (Mocarski et al., in
Viral Vectors, Y. Gluzman and S. H. Hughes, Eds., Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp. 78-84));
Moloney murine leukemia virus vectors (Danos et al., Proc. Natl.
Acad. Sci. USA, 85:6460-6464 (1988); Blaese et al., Science,
270:475-479 (1995); Onodera et al., J. Virol., 72:1769-1774
(1998)); adenovirus vectors (Berkner, Biotechniques, 6:616-626
(1988); Cotten et al., Proc. Natl. Acad. Sci. USA, 89:6094-6098
(1992); Graham et al., Meth. Mol. Biol., 7:109-127 (1991); Li et
al., Human Gene Therapy, 4:403-409 (1993); Zabner et al., Nature
Genetics, 6:75-83 (1994)); adeno-associated virus vectors (Goldman
et al., Human Gene Therapy, 10:2261-2268 (1997); Greelish et al.,
Nature Med., 5:439-443 (1999); Wang et al., Proc. Natl. Acad. Sci.
USA, 96:3906-3910 (1999); Snyder et al., Nature Med., 5:64-70
(1999); Herzog et al., Nature Med., 5:56-63 (1999)); retrovirus
vectors (Donahue et al., Nature Med., 4:181-186 (1998); Shackleford
et al., Proc. Natl. Acad. Sci. USA, 85:9655-9659 (1988); U.S. Pat.
Nos. 4,405,712, 4,650,764 and 5,252,479, and WIPO publications WO
92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829;
and lentivirus vectors (Kafri et al., Nature Genetics, 17:314-317
(1997)).
[0126] For example, in one embodiment of the present invention,
adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes
(Wagner et al., Proc. Natl. Acad. Sci., USA, 89:6099-6103 (1992);
Curiel et al., Hum. Gene Ther., 3:147-154 (1992); Gao et al., Hum.
Gene Ther., 4:14-24 (1993)) are employed to transduce mammalian
cells with heterologous CARD-encoding nucleic acid. Any of the
plasmid expression vectors described herein may be employed in a
TfAdpl-DNA complex.
[0127] Vectors useful for therapeutic administration of a
CARD-encoding nucleic acid can contain a regulatory element that
provides tissue specific or inducible expression of an operatively
linked nucleic acid. One skilled in the art can readily determine
an appropriate tissue-specific promoter or enhancer that allows
expression of a CARD polypeptide or nucleic acid in a desired
tissue. Any of a variety of inducible promoters or enhancers can
also be included in the vector for regulatable expression of a CARD
polypeptide or nucleic acid. Such inducible systems, include, for
example, tetracycline inducible system (Gossen & Bizard, Proc.
Natl. Acad. Sci. USA, 89:5547-5551 (1992); Gossen et al., Science,
268:1766-1769 (1995); Clontech, Palo Alto, Calif.); metalothionein
promoter induced by heavy metals; insect steroid hormone responsive
to ecdysone or related steroids such as muristerone (No et al.,
Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996); Yao et al.,
Nature, 366:476-479 (1993); Invitrogen, Carlsbad, Calif.); mouse
mammory tumor virus (MMTV) induced by steroids such as
glucocortocoid and estrogen (Lee et al., Nature, 294:228-232
(1981); and heat shock promoters inducible by temperature
changes.
[0128] An inducible system particularly useful for therapeutic
administration utilizes an inducible promoter that can be regulated
to deliver a level of therapeutic product in response to a given
level of drug administered to an individual and to have little or
no expression of the therapeutic product in the absence of the
drug. One such system utilizes a Ga14 fusion that is inducible by
an antiprogestin such as mifepristone in a modified adenovirus
vector (Burien et al., Proc. Natl. Acad. Sci. USA, 96:355-360
(1999). Another such inducible system utilizes the drug rapamycin
to induce reconstitution of a transcriptional activator containing
rapamycin binding domains of FKBP12 and FRAP in an adeno-associated
virus vector (Ye et al., Science, 283:88-91 (1999)). It is
understood that any combination of an inducible system can be
combined in any suitable vector, including those disclosed herein.
Such a regulatable inducible system is advantageous because the
level of expression of the therapeutic product can be controlled by
the amount of drug administered to the individual or, if desired,
expression of the therapeutic product can be terminated by stopping
administration of the drug.
[0129] The invention also provides a method for expression of a
CARD-containing polypeptide by culturing cells containing a
CARD-encoding nucleic acid under conditions suitable for expression
of a CARD-containing polypeptide. Thus, there is provided a method
for the recombinant production of a CARD-containing polypeptide of
the invention by expressing the CARD-encoding nucleic acid
sequences in suitable host cells. Recombinant DNA expression
systems that are suitable to produce a CARD-containing polypeptide
described herein are well-known in the art (see, for example,
Ausubel et al., supra, 2000). For example, the above-described
nucleotide sequences can be incorporated into vectors for further
manipulation. As used herein, vector refers to a recombinant DNA or
RNA plasmid or virus containing discrete elements that are used to
introduce heterologous DNA into cells for either expression or
replication thereof.
[0130] The invention additionally provides an isolated anti-CARD
antibody having specific reactivity with a invention
CARD-containing polypeptide. The anti-CARD antibody can be a
monoclonal antibody or a polyclonal antibody. The invention further
provides cell lines producing monoclonal antibodies having specific
reactivity with an invention CARD-containing protien.
[0131] The invention thus provides antibodies that specifically
bind a CARD-containing polypeptide. As used herein, the term
"antibody" is used in its broadest sense to include polyclonal and
monoclonal antibodies, as well as antigen binding fragments of such
antibodies. With regard to an anti-CARD antibody of the invention,
the term "antigen" means a native or synthesized CARD-containing
polypeptide or fragment thereof. An anti-CARD antibody, or antigen
binding fragment of such an antibody, is characterized by having
specific binding activity for a CARD polypeptide or a peptide
portion thereof of at least about 1.times.10.sup.5 M.sup.-1. Thus,
Fab, F(ab').sub.2, Fd and Fv fragments of an anti-CARD antibody,
which retain specific binding activity for a CARD-containing
polypeptide, are included within the definition of an antibody.
Specific binding activity of a CARD-containing polypeptide can be
readily determined by one skilled in the art, for example, by
comparing the binding activity of an anti-CARD antibody to a
CARD-containing polypeptide versus a reference polypeptide that is
not a CARD-containing polypeptide. Methods of preparing polyclonal
or monoclonal antibodies are well known to those skilled in the art
(see, for example, Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press (1988)).
[0132] In addition, the term "antibody" as used herein includes
naturally occurring antibodies as well as non-naturally occurring
antibodies, including, for example, single chain antibodies,
chimeric, bifunctional and humanized antibodies, as well as
antigen-binding fragments thereof. Such non-naturally occurring
antibodies can be constructed using solid phase peptide synthesis,
can be produced recombinantly or can be obtained, for example, by
screening combinatorial libraries consisting of variable heavy
chains and variable light chains as described by Huse et al.,
Science 246:1275-1281 (1989)). These and other methods of making,
for example, chimeric, humanized, CDR-grafted, single chain, and
bifunctional antibodies are well known to those skilled in the art
(Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al.,
Nature 341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard
et al., Protein Engineering: A practical approach (IRL Press 1992);
Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press
1995)).
[0133] Anti-CARD antibodies can be raised using a CARD immunogen
such as an isolated CARD-containing polypeptide having
substantially the same amino acid sequence as SEQ ID NOS:12, 188,
97, 99, 101, 103, 86 and 90, or a fragment thereof, which can be
prepared from natural sources or produced recombinantly, or a
peptide portion of the CARD-containing polypeptide. Such peptide
portions of a CARD-containing polypeptide are functional antigenic
fragments if the antigenic peptides can be used to generate a
CARD-specific antibody. A non-immunogenic or weakly immunogenic
CARD-containing polypeptide or portion thereof can be made
immunogenic by coupling the hapten to a carrier molecule such as
bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
Various other carrier molecules and methods for coupling a hapten
to a carrier molecule are well known in the art (see, for example,
Harlow and Lane, supra, 1988). An immunogenic CARD-containing
polypeptide fragment can also be generated by expressing the
peptide as a fusion protein, for example, to glutathione S
transferase (GST), polyHis or the like. Methods for expressing
peptide fusions are well known to those skilled in the art (Ausubel
et al., supra, (2000)).
[0134] The invention further provides a method for detecting the
presence of a human CARD-containing polypeptide in a sample by
contacting a sample with a CARD-specific antibody, and detecting
the presence of specific binding of the antibody to the sample,
thereby detecting the presence of a human CARD-containing
polypeptide in the sample. CARD-specific antibodies can be used in
diagnostic methods and systems to detect the level of
CARD-containing polypeptide present in a sample. As used herein,
the term "sample" is intended to mean any biological fluid, cell,
tissue, organ or portion thereof, that includes or potentially
includes CARD nucleic acids or polypeptides. The term includes
samples present in an individual as well as samples obtained or
derived from the individual. For example, a sample can be a
histologic section of a specimen obtained by biopsy, or cells that
are placed in or adapted to tissue culture. A sample further can be
a subcellular fraction or extract, or a crude or substantially pure
nucleic acid or polypeptide preparation.
[0135] CARD-specific antibodies can also be used for the
immunoaffinity or affinity chromatography purification of an
invention CARD-containing polypeptide. In addition, methods are
contemplated herein for detecting the presence of an invention
CARD-containing polypeptide in a cell, comprising contacting the
cell with an antibody that specifically binds to CARD-containing
polypeptides under conditions permitting binding of the antibody to
the CARD-containing polypeptides, detecting the presence of the
antibody bound to the CARD-containing polypeptide, and thereby
detecting the presence of invention polypeptides in a cell. With
respect to the detection of such polypeptides, the antibodies can
be used for in vitro diagnostic or in vivo imaging methods.
[0136] Immunological procedures useful for in vitro detection of
target CARD-containing polypeptides in a sample include
immunoassays that employ a detectable antibody. Such immunoassays
include, for example, immunohistochemistry, immunofluorescence,
ELISA assays, radioimmunoassay, FACS analysis, immunoprecipitation,
immunoblot analysis, Pandex microfluorimetric assay, agglutination
assays, flow cytometry and serum diagnostic assays, which are well
known in the art (Harlow and Lane, supra, 1988; Harlow and Lane,
Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press
(1999)).
[0137] An antibody can be made detectable by various means well
known in the art. For example, a detectable marker can be directly
attached to the antibody or indirectly attached using, for example,
a secondary agent that recognizes the CARD specific antibody.
Useful markers include, for example, radionucleotides, enzymes,
binding proteins such as biotin, fluorogens, chromogens and
chemiluminescent labels.
[0138] An antibody can also be detectable by, for example, a
fluorescent labeling agent that chemically binds to antibodies or
antigens without denaturation to form a fluorochrome (dye) that is
a useful immunofluorescent tracer. A description of
immunofluorescent analytic techniques is found in DeLuca,
"Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis
et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982),
which is incorporated herein by reference.
[0139] In one embodiment, the indicating group is an enzyme, such
as horseradish peroxidase (HRP), glucose oxidase, and the like. In
another embodiment, radioactive elements are employed labeling
agents. The linking of a label to a substrate, i.e., labeling of
nucleic acid probes, antibodies, polypeptides, and proteins, is
well known in the art. For instance, an invention antibody can be
labeled by metabolic incorporation of radiolabeled amino acids
provided in the culture medium. See, for example, Galfre et al.,
Meth. Enzymol., 73:3-46 (1981). Conventional means of protein
conjugation or coupling by activated functional groups are
particularly applicable. See, for example, Aurameas et al., Scand.
J. Immunol., Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al.,
Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.
[0140] In addition to detecting the presence of a CARD-containing
polypeptide, invention anti-CARD antibodies are contemplated for
use herein to alter the activity of the CARD-containing polypeptide
in living animals, in humans, or in biological tissues or fluids
isolated therefrom. The term "alter" refers to the ability of a
compound such as a CARD-containing polypeptide, a CARD-encoding
nucleic acid, an agent or other compound to increase or decrease
biological activity which is modulated by the compound, by
functioning as an agonist or antagonist of the compound.
Accordingly, compositions comprising a carrier and an amount of an
antibody having specificity for CARD-containing polypeptides
effective to block naturally occurring ligands or other
CARD-associated polypeptides from binding to invention
CARD-containing polypeptides are contemplated herein. For example,
a monoclonal antibody directed to an epitope of an invention
CARD-containing polypeptide, including an amino acid sequence
substantially the same as SEQ ID 12, 188, 97, 99, 101, 103, 86 and
90, can be useful for this purpose.
[0141] The present invention further provides transgenic non-human
mammals that are capable of expressing exogenous nucleic acids
encoding CARD-containing polypeptides. As employed herein, the
phrase "exogenous nucleic acid" refers to nucleic acid sequence
which is not native to the host, or which is present in the host in
other than its native environment, for example, as part of a
genetically engineered DNA construct. In addition to naturally
occurring CARD-containing polypeptide levels, a CARD-containing
polypeptide of the invention can either be overexpressed or
underexpressed in transgenic mammals, for example, underexpressed
in a knock-out animal.
[0142] Also provided are transgenic non-human mammals capable of
expressing CARD-encoding nucleic acids so mutated as to be
incapable of normal activity. Therefore, the transgenic non-human
mammals do not express native CARD-containing polypeptide or have
reduced expression of native CARD-containing polypeptide. The
present invention also provides transgenic non-human mammals having
a genome comprising antisense nucleic acids complementary to
CARD-encoding nucleic acids, placed so as to be transcribed into
antisense mRNA complementary to CARD-encoding mRNA, which
hybridizes to the mRNA and, thereby, reduces the translation
thereof. The nucleic acid can additionally comprise an inducible
promoter and/or tissue specific regulatory elements, so that
expression can be induced, or restricted to specific cell
types.
[0143] Animal model systems useful for elucidating the
physiological and behavioral roles of CARD-containing polypeptides
are also provided, and are produced by creating transgenic animals
in which the expression of the CARD-containing polypeptide is
altered using a variety of techniques. Examples of such techniques
include the insertion of normal or mutant versions of nucleic acids
encoding a CARD-containing polypeptide by microinjection,
retroviral infection or other means well known to those skilled in
the art, into appropriate fertilized embryos to produce a
transgenic animal, see, for example, Hogan et al., Manipulating the
Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory,
(1986)). Transgenic animal model systems are useful for in vivo
screening of compounds for identification of specific ligands, such
as agonists or antagonists, which activate or inhibit a biological
activity.
[0144] Also contemplated herein, is the use of homologous
recombination of mutant or normal versions of CARD-encoding genes
with the native gene locus in transgenic animals, to alter the
regulation of expression or the structure of CARD-containing
polypeptides by replacing the endogeneous gene with a recombinant
or mutated CARD-encoding gene. Methods for producing a transgenic
non-human mammal including a gene knock-out non-human mammal, are
well known to those skilled in the art (see, Capecchi et al.,
Science 244:1288 (1989); Zimmer et al., Nature 338:150 (1989);
Shastry, Experentia, 51:1028-1039 (1995); Shastry, Mol. Cell.
Biochem., 181:163-179 (1998); and U.S. Pat. No. 5,616,491, issued
Apr. 1, 1997, No. 5,750,826, issued May 12, 1998, and No.
5,981,830, issued Nov. 9, 1999).
[0145] In addition to homologous recombination, additional methods
such as microinjection can be used which add genes to the host
genome without removing host genes. Microinjection can produce a
transgenic animal that is capable of expressing both endogenous and
exogenous CARD-containing polypeptides. Inducible promoters can be
linked to the coding region of nucleic acids to provide a means to
regulate expression of the transgene. Tissue specific regulatory
elements can be linked to the coding region to permit
tissue-specific expression of the transgene. Transgenic animal
model systems are useful for in vivo screening of compounds for
identification of specific ligands, i.e., agonists and antagonists,
which activate or inhibit CARD-containing polypeptide
responses.
[0146] In accordance with another embodiment of the invention, a
method is provided for identifying a CARD-associated polypeptide
(CAP). The method is carried out by contacting an invention
CARD-containing polypeptide with a candidate CAP and detecting
association of the CARD-containing polypeptide with the CAP.
[0147] As used herein, the term "CARD-associated polypeptide" or
"CAP" means a polypeptide that can specifically bind to the
CARD-containing polypeptides of the invention, or to any functional
fragment of a CARD-containing polypeptide of the invention. Because
CARD-containing polypeptides of the invention contain domains which
can self-associate, CARD-containing polypeptides are encompassed by
the term CAP. An exemplary CAP is a protein or a polypeptide
portion of a protein that can bind an NB-ARC(NACHT), CARD, LRR or
ANGIO-R domain of an invention CARD-containing polypeptide. A CAP
can be identified, for example, using in vitro protein binding
assays similar to those described in, for example, Ausubel et al.,
supra, 2000, and by in vivo methods including methods such as yeast
two-hybrid assays, or other protein-interaction assays and methods
known in the art.
[0148] Normal association of CARD-containing polypeptide and a CAP
polypeptide in a cell can be altered due, for example, to the
expression in the cell of a variant CAP or CARD-containing
polypeptide, respectively, either of which can compete with the
normal binding function of a CARD-containing polypeptide and,
therefore, can decrease the association of CAP and CARD-containing
polypeptides in a cell. The term "variant" is used generally herein
to mean a polypeptide that is different from the CAP or
CARD-containing polypeptide that normally is found in a particular
cell type. Thus, a variant can include a mutated protein or a
naturally occurring protein, such as an isoform, that is not
normally found in a particular cell type.
[0149] CARD-containing polypeptides and CARD-associated
polypeptides of the invention can be characterized, for example,
using in vitro binding assays or the yeast two hybrid system. An in
vivo transcription activation assay such as the yeast two hybrid
system is particularly useful for identifying and manipulating the
association of proteins. In addition, the results observed in such
an assay likely mirror the events that naturally occur in a cell.
Thus, the results obtained in such an in vivo assay can be
predictive of results that can occur in a cell in a subject such as
a human subject.
[0150] A transcription activation assay such as the yeast two
hybrid system is based on the modular nature of transcription
factors, which consist of functionally separable DNA-binding and
trans-activation domains. When expressed as separate proteins,
these two domains fail to mediate gene transcription. However,
transcription activation activity can be restored if the
DNA-binding domain and the trans-activation domain are bridged
together due, for example, to the association of two proteins. The
DNA-binding domain and trans-activation domain can be bridged, for
example, by expressing the DNA-binding domain and trans-activation
domain as fusion proteins (hybrids), provided that the proteins
that are fused to the domains can associate with each other. The
non-covalent bridging of the two hybrids brings the DNA-binding and
trans-activation domains together and creates a transcriptionally
competent complex. The association of the proteins is determined by
observing transcriptional activation of a reporter gene.
[0151] The yeast two hybrid systems exemplified herein use various
strains of S. cerevisiae as host cells for vectors that express the
hybrid proteins. A transcription activation assay also can be
performed using, for example, mammalian cells. However, the yeast
two hybrid system is particularly useful due to the ease of working
with yeast and the speed with which the assay can be performed. For
example, yeast host cells containing a lacZ reporter gene linked to
a LexA operator sequence can be used to demonstrate that a CARD
domain of an invention CARD-containing polypeptide can interact
with itself or other CARD-containing polypeptides. For example, the
DNA-binding domain can consist of the LexA DNA-binding domain,
which binds the LexA promoter, fused to the CARD domain of a
CARD-containing polypeptide of the invention and the
trans-activation domain can consist of the B42 acidic region
separately fused to several cDNA sequences which encode known
CARD-containing polypeptides. When the LexA domain is
non-covalently bridged to a trans-activation domain fused to a
CARD-containing polypeptide, the association can activate
transcription of the reporter gene.
[0152] A CAP, for example, a CARD-containing polypeptide, an
NB-ARC-containing polypeptide or a LRR-containing polypeptide, also
can be identified using well known in vitro assays, for example, an
assay utilizing a glutathione-S-transferase (GST) fusion protein.
Such an in vitro assay provides a simple, rapid and inexpensive
method for identifying and isolating a CAP. Such an in vitro assay
is particularly useful in confirming results obtained in vivo and
can be used to characterize specific binding domains of a CAP. For
example, a GST can be fused to a CARD-containing polypeptide of the
invention, and expressed and purified by binding to an affinity
matrix containing immobilized glutathione. If desired, a sample
that can contain a CAP or active fragments of a CAP can be passed
over an affinity column containing bound GST/CARD and a CAP that
binds to a CARD-containing polypeptide can be obtained. In
addition, GST/CARD can be used to screen a cDNA expression library,
wherein binding of the GST/CARD fusion protein to a clone indicates
that the clone contains a cDNA encoding a CAP.
[0153] Thus, one of skill in the art will recognize that using the
CARD-containing polypeptides described herein, a variety of
methods, such as protein purification, protein interaction cloning,
or protein mass-spectrometry, can be used to identify a CAP.
[0154] Although the term "CAP" is used generally, it should be
recognized that a CAP that is identified using the novel
polypeptides described herein can be a fragment of a protein. Thus,
as used herein, a CAP also includes a polypeptide that specifically
associates to a portion of an invention CARD-containing polypeptide
that does not include a CARD domain. For example, a CAP can
associate with the NB-ARC domain of CLAN or CARD3X. As used herein,
a "candidate CAP" refers to a polypeptide containing a polypeptide
sequence know or suspected of binding one or more CARD-containing
polypeptides of the invention. Thus, a CAP can represent a
full-length protein or a CARD-associating fragment thereof. Since a
CAP polypeptide can be a full-length protein or a CARD-associating
fragment thereof, one of skill in the art will recognize that a
CAP-encoding nucleic acid, such as the genomic sequence, an mRNA
sequence or a cDNA sequence need not encode the full-length
protein. Thus, a cDNA can encode a polypeptide that is a fragment
of a full-length CAP which, nevertheless, binds one or more
invention CARD-containing polypeptides. It is also within the scope
of the invention that a full-length CAP can assume a conformation
that does not, absent some post-translational modification, bind a
CARD-containing polypeptide of the invention, due, for example, to
steric blocking of the binding site. Thus, a CAP can be a protein
or a polypeptide portion of a protein that can bind one of the
CARD-containing polypeptides of the invention. Also, it should be
recognized that a CAP can be identified by using a minimal
polypeptide derived from the sequences of the CARD-containing
polypeptides of the invention, and does not necessarily require
that the full-length molecules be employed for identifying such
CAPs.
[0155] Since CARD-containing polypeptides can be involved in
apoptosis, the association of a CAP with a CARD-containing
polypeptide can affect the sensitivity or resistance of a cell to
apoptosis or can induce or block apoptosis induced by external or
internal stimuli. The identification of various CAPs by use of
known methods can be used to determine the function of these CAPs
in cell death or signal transduction pathways controlled by
CARD-containing polypeptides, allowing for the development of
assays that are useful for identifying agents that effectively
alter the association of a CAP with a CARD-containing polypeptide.
Such agents can be useful for providing effective therapy for
conditions caused, at least in part, by insufficient apoptosis,
such as a cancer, autoimmune disease or certain viral infections.
Such agents can also be useful for providing an effective therapy
for diseases where excessive apoptosis is known to occur, such as
stroke, heart failure, or AIDS.
[0156] Assays of the invention can be used for identification of
agents that alter the self-association of the CARD-containing
polypeptides of the invention. Thus, the methods of the invention
can be used to identify agents that alter the self-association of
CARD2X, CARD3X, CLAN A, CLAN B, CLAN C, CLAN D, COP-1 and COP-2
(set forth in SEQ ID NOS: 12, 188, 97, 99, 101, 103, 86 and 90) via
their CARD domains, NB-ARC domains, LRR domains, or other domains
within these polypeptides.
[0157] The ATP-binding and hydrolysis of the NB-ARC domains can be
critical for function of a NAC polypeptide, for example, by
altering the oligomerization of the NAC. Thus, agents that
interfere with or enhance ATP or nucleotide binding and/or
hydrolysis by the NB-ARC domain of a NAC polypeptide of the
invention, such as CLAN (SEQ ID NOS:97, 99, 101 or 103) can also be
useful for altering the activity of these polypeptides in
cells.
[0158] A further embodiment of the invention provides a method to
identify agents that can effectively alter CARD-containing
polypeptide activity, for example the ability of CARD-containing
polypeptides to associate with one or more heterologous proteins.
Thus, the present invention provides a screening assay useful for
identifying an effective agent, which can alter the association of
a CARD-containing polypeptide with a CARD-associated polypeptide
(CAP), such as a heterologous CARD-containing polypeptide. Since
CARD-containing polypeptides are involved in biochemical processes
such as apoptosis, the identification of such effective agents can
be useful for altering the level of a biochemical process such as
apoptosis in a cell, for example in a cell of a subject having a
pathology characterized by an increased or decreased level of
apoptosis.
[0159] Further, effective agents can be useful for alteration of
other biochemical process modulated by a CARD-containing
polypeptide of the invention. Additional biochemical processes
modulated by CARD-containing polypeptide include, for example,
NF-kB induction, cytokine processing, cytokine receptor signaling,
cJUN N-terminal kinase induction, and caspase-mediated proteolysis
activation/inhibition, transcription, inflammation and cell
adhesion.
[0160] As used herein, the term "agent" means a chemical or
biological molecule such as a simple or complex organic molecule, a
peptide, a peptido-mimetic, a polypeptide, a protein or an
oligonucleotide that has the potential for altering the association
of a CARD-containing polypeptide with a heterologous protein or
altering the ability of a CARD-containing polypeptide to
self-associate or altering the ligand binding or catalytic activity
of a CARD-containing polypeptide. An exemplary ligand binding
activity is nucleotide binding activity, such as ADP or ATP binding
activity; and exemplary catalytic activities are nucleotide
hydrolytic activity and proteolytic activity. In addition, the term
"effective agent" is used herein to mean an agent that is confirmed
as capable of altering the association of a CARD-containing
polypeptide with a heterologous protein or altering the ability of
a CARD-containing polypeptide to self-associate or altering the
ligand binding or catalytic activity of a CARD-containing
polypeptide. For example, an effective agent may be an anti-CARD
antibody, a CARD-associated polypeptide, a caspase inhibitor, and
the like.
[0161] As used herein, the term "alter the association" means that
the association between two specifically interacting polypeptides
either is increased or decreased due to the presence of an
effective agent. As a result of an altered association of
CARD-containing polypeptide with another polypeptide in a cell, the
activity of the CARD-containing polypeptide or the CAP can be
increased or decreased, thereby altering a biochemical process, for
example, the level of apoptosis in the cell. As used herein, the
term "alter the activity" means that the agent can increase or
decrease the activity of a CARD-containing polypeptide in a cell,
thereby modulating a biochemical process in a cell, for example,
the level of apoptosis in the cell. Similarly, the term "alter the
level" of a biological process modulated by a CARD-containing
polypeptide refers to an increase or decrease a biochemical process
which occurs upon altering the activity of a CARD-containing
polypeptide. For example, an effective agent can increase or
decrease the CARD:CARD-associating activity of a CARD-containing
polypeptide, which can result in decreased apoptosis. In another
example, alteration of the ATP hydrolysis activity can modulate the
ability of the NB-ARC domain of a CARD-containing polypeptide to
associate with other NB-ARC-containing polypeptides, such as
Apaf-1, thereby altering any process effected by such association
between a CARD-containing polypeptide and an NB-ARC-containing
polypeptide.
[0162] An effective agent can act by interfering with the ability
of a CARD-containing polypeptide to associate with another
polypeptide, or can act by causing the dissociation of a
CARD-containing polypeptide from a complex with a CARD-associated
polypeptide, wherein the ratio of bound CARD-containing polypeptide
to free CARD-containing polypeptide is related to the level of a
biochemical process, such as, apoptosis, in a cell. For example,
binding of a ligand to a CAP can allow the CAP, in turn, to bind a
specific CARD-containing polypeptide such that all of the specific
CARD-containing polypeptide is bound to a CAP, and can result in
decreased apoptosis. The association, for example, of a
CARD-containing polypeptide and a CARD-containing polypeptide can
result in activation or inhibition of the NB-ARC:NB-ARC-associating
activity of a CARD-containing polypeptide. In the presence of an
effective agent, the association of a CARD-containing polypeptide
and a CAP can be altered, which can, for example, alter the
activation of caspases in the cell. As a result of the altered
caspase activation, the level of apoptosis in a cell can be
increased or decreased. Thus, the identification of an effective
agent that alters the association of a CARD-containing polypeptide
with another polypeptide can allow for the use of the effective
agent to increase or decrease the level of a biological process
such as apoptosis.
[0163] An effective agent can be useful, for example, to increase
the level of apoptosis in a cell such as a cancer cell, which is
characterized by having a decreased level of apoptosis as compared
to its normal cell counterpart. An effective agent also can be
useful, for example, to decrease the level of apoptosis in a cell
such as a T lymphocyte in a subject having a viral disease such as
acquired immunodeficiency syndrome, which is characterized by an
increased level of apoptosis in an infected T cell as compared to a
normal T cell. Thus, an effective agent can be useful as a
medicament for altering the level of apoptosis in a subject having
a pathology characterized by increased or decreased apoptosis. In
addition, an effective agent can be used, for example, to decrease
the level of apoptosis and, therefore, increase the survival time
of a cell such as a hybridoma cell in culture. The use of an
effective agent to prolong the survival of a cell in vitro can
significantly improve bioproduction yields in industrial tissue
culture applications.
[0164] A CARD-containing polypeptide that lacks the ability to bind
the NB-ARC domain or LRR domain of another polypeptide but retains
the ability to self-associate via its CARD domain or to bind to
other CARD-containing polypeptides is an example of an effective
agent, since the expression of a non-NB-ARC-associating or
non-catalytically active CARD-containing polypeptide in a cell can
alter the association of a the endogenous CARD-containing
polypeptide with itself or with CAPs.
[0165] Thus, it should be recognized that a mutation of a
CARD-containing polypeptide can be an effective agent, depending,
for example, on the normal levels of CARD-containing polypeptide
and CARD-associated polypeptide that occur in a particular cell
type. In addition, an active fragment of a CARD-containing
polypeptide can be an effective agent, provided the active fragment
can alter the association of a CARD-containing polypeptide and
another polypeptide in a cell. Such active fragments, which can be
peptides as small as about five amino acids, can be identified, for
example, by screening a peptide library (see, for example, Ladner
et al., U.S. Pat. No. 5,223,409) to identify peptides that can bind
a CARD-associated polypeptide.
[0166] Similarly, a fragment of a CARD-associated polypeptide also
can be an effective agent. A fragment of CARD-associated
polypeptide can be useful, for example, for decreasing the
association of a CARD-containing polypeptide with a CAP in a cell
by competing for binding to the CARD-containing polypeptide. A
non-naturally occurring peptido-mimetic also can be useful as an
effective agent. Such a peptido-mimetic can include, for example, a
peptoid, which is peptide-like sequence containing N-substituted
glycines, or an oligocarbamate. A peptido-mimetic can be
particularly useful as an effective agent due, for example, to
having an increased stability to enzymatic degradation in vivo.
[0167] In accordance with another embodiment of the present
invention, there is provided a method of identifying an effective
agent that alters the association of an invention CARD-containing
polypeptide with a CARD-associated polypeptide (CAP), by the steps
of: [0168] (a) contacting a CARD-containing polypeptide and a CAP
polypeptide, under conditions that allow the CARD-containing
polypeptide and CAP polypeptide to associate, with an agent
suspected of being able to alter the association of the
CARD-containing polypeptide and CAP polypeptides; and [0169] (b)
detecting the altered association of the CARD-containing
polypeptide and CAP polypeptide, where the altered association
identifies an effective agent.
[0170] Methods well-known in the art for detecting the altered
association of the CARD-containing polypeptide and CAP
polypeptides, for example, measuring protein:protein binding,
protein degradation or apoptotic activity can be employed in
bioassays described herein to identify agents as agonists or
antagonists of CARD-containing polypeptides. As described herein,
CARD-containing polypeptides have the ability to self-associate.
Thus, methods for identifying effective agents that alter the
association of a CARD-containing polypeptide with a CAP are useful
for identifying effective agents that alter the ability of a
CARD-containing polypeptide to self-associate.
[0171] As used herein, "conditions that allow said CARD-containing
polypeptide and CAP polypeptide to associate" refers to
environmental conditions in which a CARD-containing polypeptide and
CAP specifically associate. Such conditions will typically be
aqueous conditions, with a pH between 3.0 and 11.0, and temperature
below 100.degree. C. Preferably, the conditions will be aqueous
conditions with salt concentrations below the equivalent of 1 M
NaCl, and pH between 5.0 and 9.0, and temperatures between
0.degree. C. and 50.degree. C. Most preferably, the conditions will
range from physiological conditions of normal yeast or mammalian
cells, or conditions favorable for carrying out in vitro assays
such as immunoprecipitation and GST protein:protein association
assays, and the like.
[0172] In another embodiment of the invention, a method is provided
for identifying agents that modulate a ligand binding or catalytic
activity of an invention CARD-containing polypeptide. The method
contains the steps of contacting an invention CARD-containing
polypeptide with an agent suspected of modulating a ligand binding
or catalytic activity of the CARD-containing polypeptide and
measuring a ligand binding or catalytic activity of the
CARD-containing polypeptide, where modulated ligand binding or
catalytic activity identifies the agent as an agent that alters the
ligand binding or catalytic activity of a CARD-containing
polypeptide.
[0173] As used herein in regard to ligand binding or catalytic
activity, "modulate" refers to an increase or decrease in ligand
binding or catalytic activity. Thus, modulation encompasses
inhibition of ligand binding or catalytic activity as well as
activation or enhancement of ligand binding or catalytic activity.
Exemplary ligand binding activities include nucleotide binding
activity. Exemplary catalytic binding activities include nucleotide
hydrolysis and proteolysis activities.
[0174] Methods for measuring ligand binding or catalytic activities
are well known in the art, as disclosed herein. For example, an
agent known or suspected of modulating ligand binding or catalytic
activity can be contacted with an invention CARD-containing
polypeptide in vivo or in vitro, and the ligand binding or
catalytic activity can be measured using known methods. For
example, enzymatic activity can be measured using a cleavable
reporter, where the cleavable reporter generates or alters a
measurable signal such as absorption, fluorescence or radioactive
decay. Exemplary agents that can modulate ligand binding or
catalytic activity include peptides, peptidomimetics and other
peptide analogs, non-peptide organic molecules such as naturally
occurring protease inhibitors and derviatives thereof, nucleotides
and nucleotide analogs, and the like. Such inhibitors can be either
reversible or irreversible, as is well known in the art.
[0175] Agents that modulate the ligand binding or catalytic
activity of a CARD-containing polypeptide identified using the
invention methods can be used to modulate the activity of a
CARD-containing polypeptide. For example, and agent can modulate
the nucleotide binding or nucleotide hydrolytic activity of an
NB-ARC domain of a CARD-containing polypeptide. In another example,
an agent can modulate the catalytic activity of a protease domain
such as a caspase domain. Methods of modulating the ligand binding
or catalytic activities of invention CARD-containing proteins can
be used in methods of altering biochemical processes modulated by
CARD-containing proteins, such as the biochemical processes
disclosed herein.
[0176] In yet another embodiment of the present invention, there
are provided methods for altering ligand binding or catalytic
activity of a CARD-containing polypeptide of the invention, the
method comprising: [0177] contacting an CARD-containing polypeptide
with an effective amount of an agent identified by the
herein-described bioassays.
[0178] The present invention also provides in vitro screening
assays. Such screening assays are particularly useful in that they
can be automated, which allows for high through-put screening, for
example, of randomly or rationally designed agents such as drugs,
peptidomimetics or peptides in order to identify those agents that
effectively alter the association of a CARD-containing polypeptide
and a CAP or the catalytic or ligand binding activity of a
CARD-containing polypeptide and, thereby, alter a biochemical
process modulated by a CARD-containing polypeptide such as
apoptosis. An in vitro screening assay can utilize, for example, a
CARD-containing polypeptide including a CARD-containing fusion
protein such as a CARD-glutathione-S-transferase fusion protein.
For use in the in vitro screening assay, the CARD-containing
polypeptide should have an affinity for a solid substrate as well
as the ability to associate with a CARD-associated polypeptide. For
example, when a CARD-containing polypeptide is used in the assay,
the solid substrate can contain a covalently attached anti-CARD
antibody. Alternatively, a GST/CARD fusion protein can be used in
the assay and the solid substrate can contain covalently attached
glutathione, which is bound by the GST component of the GST/CARD
fusion protein. Similarly, a CARD-associated polypeptide, or
GST/NB-ARC-containing polypeptide fusion protein can be used in any
of a variety of in vitro enzymatic or in vitro binding assays known
in the art and described in texts such as Ausubel et al., supra,
2000.
[0179] An in vitro screening assay can be performed by allowing a
CARD-containing polypeptide, for example, to bind to the solid
support, then adding a CARD-associated polypeptide and an agent to
be tested. Reference reactions, which do not contain an agent, can
be performed in parallel. Following incubation under suitable
conditions, which include, for example, an appropriate buffer
concentration and pH and time and temperature that permit binding
of the particular CARD-containing polypeptide and CARD-associated
polypeptide, the amount of protein that has associated in the
absence of an agent and in the presence of an agent can be
determined. The association of a CARD-associated polypeptide with a
CARD-containing polypeptide can be detected, for example, by
attaching a detectable moiety such as a radionuclide or a
fluorescent label to a CARD-associated polypeptide and measuring
the amount of label that is associated with the solid support,
wherein the amount of label detected indicates the amount of
association of the CARD-associated polypeptide with a
CARD-containing polypeptide. An effective agent is determined by
comparing the amount of specific binding in the presence of an
agent as compared to a reference level of binding, wherein an
effective agent alters the association of CARD-containing
polypeptide with the CARD-associated polypeptide. Such an assay is
particularly useful for screening a panel of agents such as a
peptide library in order to detect an effective agent.
[0180] Various binding assays to identify cellular proteins that
interact with protein binding domains are known in the art and
include, for example, yeast two-hybrid screening assays (see, for
example, U.S. Pat. Nos. 5,283,173, 5,468,614 and 5,667,973; Ausubel
et al., supra, 2000; Luban et al., Curr. Opin. Biotechnol. 6:59-64
(1995)) and affinity column chromatography methods using cellular
extracts. By synthesizing or expressing polypeptide fragments
containing various CARD-associating sequences or deletions, the
CARD binding interface can be readily identified.
[0181] Another assay for screening of agents that alter the
activity of a CARD-containing polypeptide is based on altering the
phenotype of yeast by expressing a CARD-containing polypeptide. In
one embodiment, expression of a CARD-containing polypeptide can be
inducible (Tao et al., J. Biol. Chem. 273:23704-23708 (1998), and
the compounds can be screened when CARD-containing polypeptide
expression is induced. CARD-containing polypeptides of the
invention can also be co-expressed in yeast with CAP polypeptides
used to screen for compounds that antagonize the activity of the
CARD-containing polypeptide.
[0182] Also provided with the present invention are assays to
identify agents that alter CARD-containing polypeptide expression.
Methods to determine CARD-containing polypeptide expression can
involve detecting a change in CARD-containing polypeptide abundance
in response to contacting the cell with an agent that modulates
CARD-containing polypeptide expression. Assays for detecting
changes in polypeptide expression include, for example,
immunoassays with CARD-specific antibodies, such as immunoblotting,
immunofluorescence, immunohistochemistry and immunoprecipitation
assays, as described herein.
[0183] As understood by those of skill in the art, assay methods
for identifying agents that alter CARD-containing polypeptide
activity generally require comparison to a reference. One type of a
"reference" is a cell or culture that is treated substantially the
same as the test cell or test culture exposed to the agent, with
the distinction that the "reference" cell or culture is not exposed
to the agent. Another type of "reference" cell or culture can be a
cell or culture that is identical to the test cells, with the
exception that the "reference" cells or culture do not express a
CARD-containing polypeptide. Accordingly, the response of the
transfected cell to an agent is compared to the response, or lack
thereof, of the "reference" cell or culture to the same agent under
the same reaction conditions.
[0184] Methods for producing pluralities of agents to use in
screening for compounds that alter the activity of a
CARD-containing polypeptide, including chemical or biological
molecules such as simple or complex organic molecules,
metal-containing compounds, carbohydrates, peptides, proteins,
peptidomimetics, glycoproteins, lipoproteins, nucleic acids,
antibodies, and the like, are well known in the art and are
described, for example, in Huse, U.S. Pat. No. 5,264,563; Francis
et al., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al.,
Curr. Biol., 2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998);
Eichler et al., Med. Res. Rev. 15:481-496 (1995); and the like.
Libraries containing large numbers of natural and synthetic agents
also can be obtained from commercial sources. Combinatorial
libraries of molecules can be prepared using well known
combinatorial chemistry methods (Gordon et al., J. Med. Chem. 37:
1233-1251 (1994); Gordon et al., J. Med. Chem. 37: 1385-1401
(1994); Gordon et al., Acc. Chem. Res. 29:144-154 (1996); Wilson
and Czarnik, eds., Combinatorial Chemistry: Synthesis and
Application, John Wiley & Sons, New York (1997)).
[0185] The invention further provides a method of diagnosing or
predicting clinical prognosis of a pathology characterized by an
increased or decreased level of a CARD-containing polypeptide in a
subject. The method includes the steps of (a) obtaining a test
sample from the subject; (b) contacting the sample with an agent
that can bind a CARD-containing polypeptide of the invention under
suitable conditions, wherein the conditions allow specific binding
of the agent to the CARD-containing polypeptide; and (c) comparing
the amount of the specific binding in the test sample with the
amount of specific binding in a reference sample, wherein an
increased or decreased amount of the specific binding in the test
sample as compared to the reference sample is diagnostic of, or
predictive of the clinical prognosis of, a pathology. The agent can
be, for example, an anti-CARD antibody, a
CARD-associated-polypeptide (CAP), or a CARD-encoding nucleic
acid.
[0186] Exemplary pathologies for diagnosis or the prediction of
clinical prognosis include any of the pathologies described herein,
such as neoplastic pathologies (e.g. cancer), autoimmune diseases,
and other pathologies related to abnormal cell proliferation or
abnormal cell death (e.g. apoptosis), as disclosed herein.
[0187] The invention also provides a method of diagnosing cancer or
monitoring cancer therapy by contacting a test sample from a
patient with a CARD-specific antibody. The invention additionally
provides a method of assessing prognosis (e.g., predicting the
clinical prognosis) of patients with cancer comprising contacting a
test sample from a patient with a CARD-specific antibody.
[0188] The invention additionally provides a method of diagnosing
cancer or monitoring cancer therapy by contacting a test sample
from a patient with a oligonucleotide that selectively hybridizes
to a CARD-encoding nucleic acid molecule. The invention further
provides a method of assessing prognosis (e.g., predicting the
clinical prognosis) of patients with cancer by contacting a test
sample from a patient with a oligonucleotide that selectively
hybridizes to a CARD-encoding nucleic acid molecule.
[0189] The methods of the invention for diagnosing cancer or
monitoring cancer therapy using a CARD-specific antibody or
oligonucleotide or nucleic acid that selectively hybridizes to a
CARD-encoding nucleic acid molecule can be used, for example, to
segregate patients into a high risk group or a low risk group for
diagnosing cancer or predicting risk of metastasis or risk of
failure to respond to therapy. Therefore, the methods of the
invention can be advantageously used to determine, for example, the
risk of metastasis in a cancer patient, or the risk of an
autoimmune disease of a patient, or as a prognostic indicator of
survival or disease progression in a cancer patient or patient with
an autoimmune disease. One of ordinary skill in the art would
appreciate that the prognostic indicators of survival for cancer
patients suffering from stage I cancer can be different from those
for cancer patients suffering from stage IV cancer. For example,
prognosis for stage I cancer patients can be oriented toward the
likelihood of continued growth and/or metastasis of the cancer,
whereas prognosis for stage IV cancer patients can be oriented
toward the likely effectiveness of therapeutic methods for treating
the cancer. Accordingly, the methods of the invention directed to
measuring the level of or determining the presence of a
CARD-containing polypeptide or CARD-encoding nucleic acid can be
used advantageously as a prognostic indicator for the presence or
progression of a cancer or response to therapy.
[0190] The invention further provides methods for introducing a
CARD-encoding nucleic acid into a cell in a subject, for example,
for gene therapy. Viruses are specialized infectious agents that
can elude host defense mechanisms and can infect and propagate in
specific cell types. Viral based systems provide the advantage of
being able to introduce relatively high levels of the heterologous
nucleic acid into a variety of cells. Suitable viral vectors for
introducing an invention CARD-encoding nucleic acid into mammalian
cells (e.g., vascular tissue segments) are well known in the art.
These viral vectors include, for example, Herpes simplex virus
vectors (e.g., Geller et al., Science, 241:1667-1669 (1988)),
Vaccinia virus vectors (e.g., Piccini et al., Meth. in Enzymology,
153:545-563 (1987); Cytomegalovirus vectors (Mocarski et al., in
Viral Vectors, Y. Gluzman and S. H. Hughes, Eds., Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp. 78-84),
Moloney murine leukemia virus vectors (Danos et al., Proc. Natl.
Acad. Sci., USA, 85:6469 (1980)), adenovirus vectors (e.g., Logan
et al., Proc. Natl. Acad. Sci., USA, 81:3655-3659 (1984); Jones et
al., Cell, 17:683-689 (1979); Berkner, Biotechniques, 6:616-626
(1988); Cotten et al., Proc. Natl. Acad. Sci., USA, 89:6094-6098
(1992); Graham et al., Meth.
Mol. Biol., 7:109-127 (1991)), adeno-associated virus vectors,
retrovirus vectors (see, e.g., U.S. Pat. Nos. 4,405,712 and
4,650,764), and the like. Especially preferred viral vectors are
the adenovirus and retroviral vectors.
[0191] Suitable retroviral vectors for use herein are described,
for example, in U.S. Pat. No. 5,252,479, and in WIPO publications
WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829,
incorporated herein by reference, which provide a description of
methods for efficiently introducing nucleic acids into human cells
using such retroviral vectors. Other retroviral vectors include,
for example, the mouse mammary tumor virus vectors (e.g.,
Shackleford et al., Proc. Natl. Acad. Sci. USA, 85:9655-9659
(1988)), and the like.
[0192] In particular, the specificity of viral vectors for
particular cell types can be utilized to target predetermined cell
types. Thus, the selection of a viral vector will depend, in part,
on the cell type to be targeted. For example, if a
neurodegenerative disease is to be treated by increasing the level
of a CARD-containing polypeptide in neuronal cells affected by the
disease, then a viral vector that targets neuronal cells can be
used. A vector derived from a herpes simplex virus is an example of
a viral vector that targets neuronal cells (Battleman et al., J.
Neurosci. 13:941-951 (1993), which is incorporated herein by
reference). Similarly, if a disease or pathological condition of
the hematopoietic system is to be treated, then a viral vector that
is specific for a particular blood cell or its precursor cell can
be used. A vector based on a human immunodeficiency virus is an
example of such a viral vector (Carroll et al., J. Cell. Biochem.
17E:241 (1993), which is incorporated herein by reference). In
addition, a viral vector or other vector can be constructed to
express a CARD-encoding nucleic acid in a tissue specific manner by
incorporating a tissue-specific promoter or enhancer into the
vector (Dai et al., Proc. Natl. Acad. Sci. USA 89:10892-10895
(1992), which is incorporated herein by reference).
[0193] For gene therapy, a vector containing a CARD-encoding
nucleic acid or an antisense nucleotide sequence can be
administered to a subject by various methods. For example, if viral
vectors are used, administration can take advantage of the target
specificity of the vectors. In such cases, there in no need to
administer the vector locally at the diseased site. However, local
administration can be a particularly effective method of
administering a CARD-encoding nucleic acid. In addition,
administration can be via intravenous or subcutaneous injection
into the subject. Following injection, the viral vectors will
circulate until they recognize host cells with the appropriate
target specificity for infection. Injection of viral vectors into
the spinal fluid also can be an effective mode of administration,
for example, in treating a neurodegenerative disease.
[0194] Receptor-mediated DNA delivery approaches also can be used
to deliver a CARD-encoding nucleic acid molecule into cells in a
tissue-specific manner using a tissue-specific ligand or an
antibody that is non-covalently complexed with the nucleic acid
molecule via a bridging molecule (Curiel et al., Hum. Gene Ther.
3:147-154 (1992); Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987),
each of which is incorporated herein by reference). Direct
injection of a naked or a nucleic acid molecule encapsulated, for
example, in cationic liposomes also can be used for stable gene
transfer into non-dividing or dividing cells in vivo (Ulmer et al.,
Science 259:1745-1748 (1993), which is incorporated herein by
reference). In addition, a CARD-encoding nucleic acid molecule can
be transferred into a variety of tissues using the particle
bombardment method (Williams et al., Proc. Natl. Acad. Sci. USA
88:2726-2730 (1991), which is incorporated herein by reference).
Such nucleic acid molecules can be linked to the appropriate
nucleotide sequences required for transcription and
translation.
[0195] A particularly useful mode of administration of a
CARD-encoding nucleic acid is by direct inoculation locally at the
site of the disease or pathological condition. Local administration
can be advantageous because there is no dilution effect and,
therefore, the likelihood that a majority of the targeted cells
will be contacted with the nucleic acid molecule is increased.
Thus, local inoculation can alleviate the targeting requirement
necessary with other forms of administration and, if desired, a
vector that infects all cell types in the inoculated area can be
used. If expression is desired in only a specific subset of cells
within the inoculated area, then a promoter, an enhancer or other
expression element specific for the desired subset of cells can be
linked to the nucleic acid molecule. Vectors containing such
nucleic acid molecules and regulatory elements can be viral
vectors, viral genomes, plasmids, phagemids and the like.
Transfection vehicles such as liposomes also can be used to
introduce a non-viral vector into recipient cells. Such vehicles
are well known in the art.
[0196] The present invention also provides therapeutic compositions
useful for practicing the therapeutic methods described herein.
Therapeutic compositions of the present invention, such as
pharmaceutical compositions, contain a physiologically compatible
carrier together with an invention CARD-containing polypeptide (or
functional fragment thereof), an invention CARD-encoding nucleic
acid, an agent that alters CARD activity or expression identified
by the methods described herein, or an anti-CARD antibody, as
described herein, dissolved or dispersed therein as an active
ingredient. In a preferred embodiment, the therapeutic composition
is not immunogenic when administered to a mammal or human patient
for therapeutic purposes.
[0197] As used herein, the terms "pharmaceutically acceptable",
"physiologically compatible" and grammatical variations thereof, as
they refer to compositions, carriers, diluents and reagents, are
used interchangeably and represent that the materials are capable
of administration to a mammal without the production of undesirable
physiological effects.
[0198] The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein is well
known in the art. Typically such compositions are prepared as
injectibles either as liquid solutions or suspensions; however,
solid forms suitable for solution, or suspension, in liquid prior
to use can also be prepared. The preparation can also be
emulsified.
[0199] The active ingredient can be mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient in amounts suitable for use in the therapeutic methods
described herein. Suitable excipients are, for example, water,
saline, dextrose, glycerol, ethanol, or the like, as well as
combinations of any two or more thereof. In addition, if desired,
the composition can contain minor amounts of auxiliary substances
such as wetting or emulsifying agents, pH buffering agents, and the
like, which enhance the effectiveness of the active ingredient.
[0200] The therapeutic composition of the present invention can
include pharmaceutically acceptable salts of the components
therein. Pharmaceutically acceptable nontoxic salts include the
acid addition salts (formed with the free amino groups of the
polypeptide) that are formed with inorganic acids such as, for
example, hydrochloric acid, hydrobromic acid, perchloric acid,
nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid,
acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic
acid, oxalic acid, malonic acid, succinic acid, maleic acid,
fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic
acid, sulfanilic acid, and the like.
[0201] Salts formed with the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium
hydroxide, ammonium hydroxide, potassium hydroxide, and the like;
and organic bases such as mono-, di-, and tri-alkyl and -aryl
amines (e.g., triethylamine, diisopropyl amine, methyl amine,
dimethyl amine, and the like) and optionally substituted
ethanolamines (e.g., ethanolamine, diethanolamine, and the
like).
[0202] Physiologically tolerable carriers are well known in the
art. Exemplary liquid carriers are sterile aqueous solutions that
contain no materials in addition to the active ingredients and
water, or contain a buffer such as sodium phosphate at
physiological pH, physiological saline or both, such as
phosphate-buffered saline. Still further, aqueous carriers can
contain more than one buffer salt, as well as salts such as sodium
and potassium chlorides, dextrose, polyethylene glycol and other
solutes.
[0203] Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary additional
liquid phases include glycerin, vegetable oils such as cottonseed
oil, and water-oil emulsions.
[0204] As described herein, an "effective amount" is a
predetermined amount calculated to achieve the desired therapeutic
effect, i.e., to alter the protein binding activity of a
CARD-containing polypeptide or the catalytic activity of a
CARD-containing polypeptide, resulting in altered biochemical
process modulated by a CARD-containing polypeptide. The required
dosage will vary with the particular treatment and with the
duration of desired treatment; however, it is anticipated that
dosages between about 10 micrograms and about 1 milligram per
kilogram of body weight per day will be used for therapeutic
treatment. It may be particularly advantageous to administer such
agents in depot or long-lasting form as discussed herein. A
therapeutically effective amount is typically an amount of an agent
identified herein that, when administered in a physiologically
acceptable composition, is sufficient to achieve a plasma
concentration of from about 0.1 .mu.g/ml to about 100 .mu.g/ml,
preferably from about 1.0 .mu.g/ml to about 50 .mu.g/ml, more
preferably at least about 2 .mu.g/ml and usually 5 to 10 .mu.g/ml.
Therapeutic invention anti-CARD antibodies can be administered in
proportionately appropriate amounts in accordance with known
practices in this art.
[0205] Also provided herein are methods of treating pathologies
characterized by abnormal cell proliferation, abnormal cell death,
or inflammation said method comprising administering an effective
amount of an invention therapeutic composition. Such compositions
are typically administered in a physiologically compatible
composition.
[0206] Exemplary abnormal cell proliferation diseases associated
with CARD-containing polypeptides contemplated herein for treatment
according to the present invention include cancer pathologies,
keratinocyte hyperplasia, neoplasia, keloid, benign prostatic
hypertrophy, inflammatory hyperplasia, fibrosis, smooth muscle cell
proliferation in arteries following balloon angioplasty
(restenosis), and the like. Exemplary cancer pathologies
contemplated herein for treatment include, gliomas, carcinomas,
adenocarcinomas, sarcomas, melanomas, hamartomas, leukemias,
lymphomas, and the like. Further diseases associated with
CARD-containing polypeptides contemplated herein for treatment
according to the present invention include inflammatory diseases
and diseases of cell loss. Such diseases include allergies,
inflammatory diseases including arthritis, lupus, Schrogen's
syndrome, Crohn's disease, ulcerative colitis, as well as allograft
rejection, such as graft-versus-host disease, and the like.
CARD-containing polypeptides can also be useful in design of
strategies for preventing diseases related to abnormal cell death
in conditions such as stroke, myocardial infarction, heart failure,
neurodegenerative diseases such as Parkinson's and Alzheimer's
diseases, and for immunodeficiency associated diseases such as HIV
infection, HIV-related disease, and the like.
[0207] Methods of treating pathologies can include methods of
modulating the activity of one or more oncogenic proteins, wherein
the oncogenic proteins specifically interact with a CARD-containing
polypeptide of the invention. Methods of modulating the activity of
such oncogenic proteins will include contacting the oncogenic
protein with a substantially pure CARD-containing polypeptide or an
active fragment (i.e., oncogenic protein-binding fragment) thereof.
This contacting will alter the activity of the oncogenic protein,
thereby providing a method of treating a pathology caused by the
oncogenic protein. Further methods of modulating the activity of
oncogenic proteins will include contacting the oncogenic protein
with an agent, wherein the agent alters interaction between a
CARD-containing polypeptide and an oncogenic protein.
[0208] Also contemplated herein, are therapeutic methods using
invention pharmaceutical compositions for the treatment of
pathological disorders in which there is too little cell division,
such as, for example, bone marrow aplasias, immunodeficiencies due
to a decreased number of lymphocytes, and the like. Methods of
treating a variety of inflammatory diseases with invention
therapeutic compositions are also contemplated herein, such as
treatment of sepsis, fibrosis (e.g., scarring), arthritis, graft
versus host disease, and the like.
[0209] The present invention also provides methods for diagnosing a
pathology that is characterized by an increased or decreased level
of a biochemical process to determine whether the increased or
decreased level of the biochemical process is due, for example, to
increased or decreased expression of a CARD-containing polypeptide
or to expression of a variant CARD-containing polypeptide. As
disclosed herein, such biochemical processes include apoptosis,
NF-kB induction, cytokine processing, caspase-mediated proteolysis,
transcription, inflammation, cell adhesion, and the like. The
identification of such a pathology, which can be due to altered
association of a CARD-containing polypeptide with a CARD-associated
polypeptide in a cell, or altered ligand binding or catalytic
activity of a CARD-containing polypeptide, can allow for
intervention therapy using an effective agent or a nucleic acid
molecule or an antisense nucleotide sequence as described herein.
In general, a test sample can be obtained from a subject having a
pathology characterized by having or suspected of having increased
or decreased apoptosis and can be compared to a reference sample
from a normal subject to determine whether a cell in the test
sample has, for example, increased or decreased expression of a
CARD-encoding gene. The level of a CARD-containing polypeptide in a
cell can be determined by contacting a sample with a reagent such
as an anti-CARD antibody or a CARD-associated polypeptide, either
of which can specifically bind a CARD-containing polypeptide. For
example, the level of a CARD-containing polypeptide in a cell can
determined by well known immunoassay or immunohistochemical methods
using an anti-CARD antibody (see, for example, Reed et al., Anal.
Biochem. 205:70-76 (1992); see, also, Harlow and Lane, supra,
(1988)). As used herein, the term "reagent" means a chemical or
biological molecule that can specifically bind to a CARD-containing
polypeptide or to a bound CARD/CARD-associated polypeptide complex.
For example, either an anti-CARD antibody or a CARD-associated
polypeptide can be a reagent for a CARD-containing polypeptide,
whereas either an anti-CARD antibody or an anti-CARD-associated
polypeptide antibody can be a reagent for a CARD/CARD-associated
polypeptide complex.
[0210] As used herein, the term "test sample" means a cell or
tissue specimen that is obtained from a subject and is to be
examined for expression of a CARD-encoding gene in a cell in the
sample. A test sample can be obtained, for example, during surgery
or by needle biopsy and can be examined using the methods described
herein to diagnose a pathology characterized by increased or
decreased apoptosis. Increased or decreased expression of a
CARD-encoding gene in a cell in a test sample can be determined,
for example, by comparison to an expected normal level of
CARD-containing polypeptide or CARD-encoding mRNA in a particular
cell type. A normal range of CARD-containing polypeptide or
CARD-encoding mRNA levels in various cell types can be determined
by sampling a statistically significant number of normal subjects.
In addition, a reference sample can be evaluated in parallel with a
test sample in order to determine whether a pathology characterized
by increased or decreased apoptosis is due to increased or
decreased expression of a CARD-encoding gene. The test sample can
be examined using, for example, immunohistochemical methods as
described above or the sample can be further processed and
examined. For example, an extract of a test sample can be prepared
and examined to determine whether a CARD-containing polypeptide in
the sample can associate with a CARD-associated polypeptide in the
same manner as a CARD-containing polypeptide from a reference cell
or whether, instead, a variant CARD-containing polypeptide is
expressed in the cell.
[0211] In accordance with another embodiment of the present
invention, there are provided diagnostic systems, preferably in kit
form, comprising at least one invention CARD-encoding nucleic acid,
CARD-containing polypeptide, and/or anti-CARD antibody described
herein, in a suitable packaging material. In one embodiment, for
example, the diagnostic nucleic acids are derived from any of SEQ
ID NOS:11, 187, 96, 98, 100, 102, 85 and 89. Invention diagnostic
systems are useful for assaying for the presence or absence of
CARD-encoding nucleic acid in either genomic DNA or in transcribed
CARD-encoding nucleic acid, such as mRNA or cDNA.
[0212] A suitable diagnostic system includes at least one invention
CARD-encoding nucleic acid, CARD-containing polypeptide, and/or
anti-CARD antibody, preferably two or more invention nucleic acids,
proteins and/or antibodies, as a separately packaged chemical
reagent(s) in an amount sufficient for at least one assay.
Instructions for use of the packaged reagent are also typically
included. Those of skill in the art can readily incorporate
invention nucleic acid probes and/or primers into kit form in
combination with appropriate buffers and solutions for the practice
of the invention methods as described herein.
[0213] As employed herein, the phrase "packaging material" refers
to one or more physical structures used to house the contents of
the kit, such as invention nucleic acid probes or primers, and the
like. The packaging material is constructed by well known methods,
preferably to provide a sterile, contaminant-free environment. The
packaging material has a label which indicates that the invention
nucleic acids can be used for detecting a particular CARD-encoding
sequence including the nucleotide sequences set forth in SEQ ID
NOS:11, 187, 96, 98, 100, 102, 85 and 89 or mutations or deletions
therein, thereby diagnosing the presence of, or a predisposition
for a pathology such as cancer or an autoimmune disease. In
addition, the packaging material contains instructions indicating
how the materials within the kit are employed both to detect a
particular sequence and diagnose the presence of, or a
predisposition for a pathology such as cancer or an autoimmune
disease.
[0214] The packaging materials employed herein in relation to
diagnostic systems are those customarily utilized in nucleic
acid-based diagnostic systems. As used herein, the term "package"
refers to a solid matrix or material such as glass, plastic, paper,
foil, and the like, capable of holding within fixed limits an
isolated nucleic acid, oligonucleotide, or primer of the present
invention. Thus, for example, a package can be a glass vial used to
contain milligram quantities of a contemplated nucleic acid,
oligonucleotide or primer, or it can be a microtiter plate well to
which microgram quantities of a contemplated nucleic acid probe
have been operatively affixed.
[0215] "Instructions for use" typically include a tangible
expression describing the reagent concentration or at least one
assay method parameter, such as the relative amounts of reagent and
sample to be admixed, maintenance time periods for reagent/sample
admixtures, temperature, buffer conditions, and the like.
[0216] A diagnostic assay should include a simple method for
detecting the amount of a CARD-containing polypeptide or
CARD-encoding nucleic acid in a sample that is bound to the
reagent. Detection can be performed by labeling the reagent and
detecting the presence of the label using well known methods (see,
for example, Harlow and Lane, supra, 1988; chap. 9, for labeling an
antibody). A reagent can be labeled with various detectable
moieties including a radiolabel, an enzyme, biotin or a
fluorochrome. Materials for labeling the reagent can be included in
the diagnostic kit or can be purchased separately from a commercial
source. Following contact of a labeled reagent with a test sample
and, if desired, a control sample, specifically bound reagent can
be identified by detecting the particular moiety.
[0217] A labeled antibody that can specifically bind the reagent
also can be used to identify specific binding of an unlabeled
reagent. For example, if the reagent is an anti-CARD antibody, a
second antibody can be used to detect specific binding of the
anti-CARD antibody. A second antibody generally will be specific
for the particular class of the first antibody. For example, if an
anti-CARD antibody is of the IgG class, a second antibody will be
an anti-IgG antibody. Such second antibodies are readily available
from commercial sources. The second antibody can be labeled using a
detectable moiety as described above. When a sample is labeled
using a second antibody, the sample is first contacted with a first
antibody, then the sample is contacted with the labeled second
antibody, which specifically binds to the first antibody and
results in a labeled sample.
[0218] In accordance with another embodiment of the invention,
there are provided methods for determining a prognosis of disease
free or overall survival in a patient suffering from cancer. For
example, it is contemplated herein that abnormal levels of
CARD-containing polypeptides (either higher or lower) in primary
tumor tissue show a high correlation with either increased or
decreased tumor recurrence or spread, and therefore indicates the
likelihood of disease free or overall survival. Thus, the present
invention advantageously provides a significant advancement in
cancer management because early identification of patients at risk
for tumor recurrence or spread will permit aggressive early
treatment with significantly enhanced potential for survival. Also
provided are methods for predicting the risk of tumor recurrence or
spread in an individual having a cancer tumor; methods for
screening a cancer patient to determine the risk of tumor
metastasis; and methods for determining the proper course of
treatment for a patient suffering from cancer. These methods are
carried out by collecting a sample from a patient and comparing the
level of CARD-encoding gene expression in the patient to the level
of expression in a control or to a reference level of CARD-encoding
gene expression as defined by patient population sampling, tissue
culture analysis, or any other method known for determining
reference levels for determination of disease prognosis. The level
of CARD-encoding gene expression in the patient is then classified
as higher than the reference level or lower than the reference
level, wherein the prognosis of survival or tumor recurrence is
different for patients with higher levels than the prognosis for
patients with lower levels.
[0219] All U.S. patents and all publications mentioned herein are
incorporated in their entirety by reference thereto. The invention
will now be described in greater detail by reference to the
following non-limiting examples.
EXAMPLES
1.0 Identification of CARD-Containing Polypeptides
[0220] The process of gene identification and assembling include
the following steps:
[0221] A) Identification of new candidate CARD containing
polypeptides. A database search was performed using the TBLASTN
program with the CARD domain of caspase-1 and caspase-12 as the
query in the following NCBI databases: high throughput genome
sequence (HTGS), genomic survey sequence (GSS) and expressed
sequence tag (EST) databases.
[0222] B) Verification that the new candidate CARD containing
polypeptide is novel. Using PSI-BLAST, each new candidate CARD gene
was queried in the annotated non-redundant (NR) database at NCBI.
When the new candidate gene showed significant but not identical
homology with other known CARD containing polypeptides during this
search, the CARD containing polypeptide candidate was kept for
further analysis.
[0223] C) 3-D-Model Building of new candidate CARD polypeptide:
When the sequence homology was low (<25% identity),
three-dimensional criteria was added to characterization of new
CARD-containing polypeptides. The candidate CARD fragment was
analyzed by a profile-profile sequence comparison method which
aligns the candidate CARD domain with a database of sequences of
known three-dimensional structure. From this analysis, a sequence
alignment was produced and a three-dimensional model was built
according to the known structure of CARD domain of IAP-1. In most
cases, the best score was produced using CARD domain sequences
having known three-dimensional structures. The quality of the
three-dimensional model obtained from the alignments confirmed that
novel CARD-domain containing polypeptides had been identified.
[0224] D) Identification of additional domains in the full length
protein. Full length protein sequences were obtained using the
closest full-length caspase homolog of the new CARD identified in
step B as query. TBLASTN searches of the sequences containing the
newly identified CARD domains were performed. Longer aligned
fragments or multiple aligned fragments in the accession number
corresponding to the newly identified CARD containing polypeptides
indicated a longer protein.
[0225] E) These additional domains were assembled using the
following gene building procedure: [0226] Genomic DNA fragments
were identified by T-BLAST-N analysis using mouse caspase-12 and
human caspase-1 full length protein as query and scanning HTGS
database from NCBI of incomplete DNA genomics sequences. New
fragments homologous to caspase-12 and caspase-1 were further
confirmed by psi-blast analysis using the TBLASTN genomic DNA
homolog fragment as query and scanning NR database. The boundary of
each fragment was identified according to the following criteria:
[0227] Disruption of sequence similarity between the protein
alignment of the target fragment and the query. [0228] Extension of
the protein sequence alignment between query and target using ORF
finder. [0229] Protein sequence overlap between two contiguous
fragments in sequence relative to the query. [0230] Conservation of
exon-intron junction between DNA sequence of the target and query.
[0231] Orientation of the ORF of the different genomic DNA
fragment. [0232] Presence of contiguous fragments, based on
sequence alignment with the query, on the same contig. [0233]
Finally, the reconstituted sequences were aligned by CLUSTALW with
the query and exon-intron junctions further refined by repeating
the above process.
[0234] 2.0 Identification of CARD2X, CARD3X and CLAN. Nucleic acids
encoding CARD containing proteins CARD2X, CARD3X and CLAN were
identified from different CARD queries using tblastn and
systematically scanning gss, htgs, and all EST databases at NCBI.
Further analysis using translated genomic fragment containing CARD
domains larger than the CARD domain itself as query were performed
to identify additional domains. Genomic DNA were translated in all
reading frames and examined for additional domains using psi-blast
and nr database.
[0235] 3.0 Cloning and sequencing of large cDNA. For cDNA larger
than 1500 bp, cloning is accomplished by amplification of multiple
fragments of the cDNA. Jurkat total RNA is reverse-transcribed to
complementary DNAs using MMLV reverse transcriptase (Stratagene)
and random hexanucleotide primers. Overlapping cDNA fragments of a
CARD-containing polypeptide are amplified from the Jurkat
complementary DNAs with Turbo Pfu DNA polymerase (Stratagene) using
an oligonucleotide primer set for every 1500 by of cDNA, where the
amplified cDNA fragment contains a unique restriction site near the
end that is to be ligated with an adjacent amplified cDNA
fragment.
[0236] The resultant cDNA fragments are ligated into mammalian
expression vector pcDNA-myc (Invitrogen, modified as described in
Roy et al., EMBO J. 16:6914-6925 (1997)) and assembled to
full-length cDNA by consecutively ligating adjacent fragments at
the unique endonuclease sites form the full-length cDNA. Sequencing
analysis of the assembled full-length cDNA is carried out, and
splice isoforms of CARD-containing polypeptides can be
identified.
[0237] 4.0 Plasmid Constructions. Complementary DNA encoding a
CARD-containing polypeptide, or a functional fragment thereof is
amplified from Jurkat cDNAs with Turbo Pfu DNA polymerase
(Stratagene) and desired primers, such as those described above.
The resultant PCR fragments are digested with restriction enzymes
such as EcoRI and Xho I and ligated into pGEX-4T1 (Pharmacia) and
pcDNA-myc vectors.
[0238] 5.0 In vitro Protein Binding Assays. CARD-containing or
fragments thereof encoded in pGEX-4T1 are expressed in XL-1 blue E.
coli cells (Stratagene), and affinity-purified using glutathione
(GSH)-sepharose according to known methods, such as those in
Current Protocols in Molecular Biology, Ausubel et al. eds., John
Wiley and Sons (1999). For GST pull-down assays, purified CARD-GST
fusion proteins and GST alone (0.1-0.5 .mu.g immobilized on 10-15
.mu.l GSH-sepharose beads) are incubated with 1 mg/ml of BSA in 100
.mu.l Co-IP buffer (142.4 mM KCl, 5 mM M.sub.gCl.sub.2, 10 mM HEPES
(pH 7.4), 0.5 mM EGTA, 0.2% NP-40, 1 mM DTT, and 1 mM PMSF) for 30
min. at room temperature. The beads are then incubated with 1 .mu.l
of rat reticulocyte lysates (TnT-lysate; Promega, Inc.) containing
.sup.35S-labeled, in vitro translated CARD-containing or control
protein Skp-1 in 100 .mu.l Co-IP buffer supplemented with 0.5 mg/ml
BSA for overnight at 4.degree. C. The beads are washed four times
in 500 .mu.l Co-IP buffer, followed by boiling in 20 .mu.l
Laemmli-SDS sample buffer. The eluted proteins are analyzed by
SDS-PAGE. The bands of SDS-PAGE gels are detected by
fluorography.
[0239] The resultant oligomerization pattern will reveal that
CARD:CARD and other protein:protein interactions occur with
CARD-containing polypeptides or fragments thereof.
[0240] In vitro translated candidate CARD-associated polypeptides
such as Apaf-1 (lacking its WD domain), CED4, and control Skp-1 are
subjected to GST pull-down assay using GSH-sepharose beads
conjugated with GST and GST-CARD-containing polypeptides as
described above. Lanes containing GST-CARD yield significant
signals when incubated with a CARD-associated polypeptide whereas,
the controls GST alone and Skp-1 yield negligible signals.
[0241] 6.0 Protein Interaction Studies in Yeast. EGY48 yeast cells
(Saccharomyces cerevisiae: MAT.alpha., trpl, ura3, his,
leu2::plexApo6-leu2) are transformed with pGilda-CARD plasmids (his
marker) encoding the LexA DNA binding domain fused to:
CARD-containing polypeptides, fragments thereof, or CARD-associated
polypeptides. EGY48 are also transformed with a LexA-LacZ reporter
plasmid pSH1840 (ura3 marker), as previously described (Durfee et
al., 1993; Sato et al., 1995). Sources for cells and plasmids are
described previously in U.S. Pat. No. 5,632,994, and in Zervous et
al., Cell 72:223-232 (1993); Gyuris et al., Cell 75:791-803 (1993);
Golemis et al., In Current Protocols in Molecular Biology (ed.
Ausubel et al.; Green Publ.; NY 1994), each of which is
incorporated herein by reference. Transformants are replica-plated
on Burkholder's minimal medium (BMM) plates supplemented with
leucine and 2% glucose as previously described (Sato et al., Gene
140:291-292 (1994)). Protein-protein interactions are scored by
growth of transformants on leucine deficient BMM plates containing
2% galactose and 1% raffinose.
[0242] Protein-protein interactions are also evaluated using
.beta.-galactosidase activity assays. Colonies grown on
BMM/Leu/Glucose plates are filter-lifted onto nitrocellulose
membranes, and incubated over-night on BMM/Leu/galactose plates.
Yeast cells are lysed by soaking filters in liquid nitrogen and
thawing at room temperature. .beta.-galactosidase activity is
measured by incubating the filter in 3.2 ml Z buffer (60 mM,
Na.sub.2HPO.sub.4, 40 mM Na.sub.2HPO.sub.4, 10 mM KCl, 1 mM
MgSO.sub.4) supplemented with 50 .mu.l X-gal solution (20 mg/ml).
Levels of .beta.-galactosidase activity are scaled according to the
intensity of blue color generated for each transformant.
[0243] The results of this experiment will show colonies on leucine
deficient plates for yeast containing CARD/LexA fusions together
with CARD-associated polypeptide/B42. In addition, the
CARD/LexA:CARD-associated polypeptide/B42 cells will have
significant amounts of LacZ activity.
[0244] 7.0 Self-Association of NB-ARC domain of CARD-containing
polypeptides. In vitro translated, .sup.35S-labeled rat
reticulocyte lysates (1 .mu.l) containing NB-ARC or Skp-1 (used as
a control) are incubated with GSH-sepharose beads conjugated with
purified GST-NB-ARC or GST alone for GST pull-down assay, resolved
on SDS-PAGE and visualized by fluorography as described above. One
tenth of input is loaded for NB-ARC or Skp-1 as controls.
[0245] 8.0 Protein-Protein Interactions of CARD-containing
polypeptides. Transient transfection of 293T, a human embryonic
kidney fibroblast cell line, are conducted using SuperFect reagents
(Qiagen) according to manufacturer's instructions. The cDNA
fragments encoding full-length CED4 and the truncated form of
Apaf-1 (Apaf-1.DELTA.WD) comprising amino acids 1-420 of the human
Apaf-1 protein are amplified by PCR and subcloned into pcDNA3HA at
EcoRI and Xho I sites. Expression plasmids encoding catalytically
inactive forms of caspases such as pro-Casp8 (pro-Casp8 (C/A)) are
prepared by replacing Cys 377 with an Ala using site-directed
mutagenesis and pro-Casp9 (pro-Casp9 (C/A)) has been described
previously, Cardone et al., Science 282:1318-1321 (1998)). 293T
cells are transiently transfected with an expression plasmid (2
.mu.g) encoding HA-tagged human Apaf-1.DELTA.WD, CED4, pro-Casp8
(C/A) or C-Terminal Flag-tagged pro-Casp9 (C/A) in the presence or
absence of a plasmid (2 .mu.g) encoding myc-tagged CARD-containing
polypeptide. After 24 hr growth in culture, transfected cells are
collected and lysed in Co-IP buffer (142.4 mM KCl, 5 mM MgCl.sub.2,
10 mM HEPES (pH 7.4), 0.5 mM EGTA, 0.1% NP-40, and 1 mM DTT)
supplemented with 12.5 mM .beta.-glycerolphosphate, 2 mM NaF, 1 mM
Na.sub.3VO.sub.4, 1 mM PMSF, and 1.times. protenase inhibitor mix
(Boehringer Mannheim). Cell lysates are clarified by
microcentrifugation and subjected to immunoprecipitation using
either a mouse monoclonal antibody to myc (Santa Cruz
Biotechnologies, Inc) or a control mouse IgG. Proteins from the
immune complexes are resolved by SDS-PAGE, transferred to
nitrocellulose membranes, and subjected to immunoblot analysis
using anti-HA antibodies followed by anti-myc antibodies using a
standard Western blotting procedure and ECL reagents from
Amersham-Pharmacia Biotechnologies, Inc. (Krajewski et al., Proc.
Natl. Acad. Sci. USA 96:5752-5757 (1999)).
[0246] 9.0 Cloning and characterization of CARD2X. CARD2X-encoding
cDNA was obtained by PCR using primers CGGAATTCATGGCTACCGAGAGTACTCC
(SEQ ID NO:76) and GTAAAACGACGGCCAGT (SEQ ID NO:77) to amplify a
0.9 kb cDNA molecule from a human skeletal muscle cDNA library
(Clontech). The PCR products was then purified by agarose gel
electrophoresis and the purified products subcloned into
pBluescript II SK vector (Stratagene). Using the forward primers,
the PCR fragments were directly sequenced using the ABI PRISM Big
Dye Terminal Cycle sequencing kit, according to manufacturer's
instructions (Perkin Elmer). Based on the sequence obtained, a
third CARD2X-specific primer was generated having the sequence
GCAGAAGCCACTGTGGAAGAGGAGGTT (SEQ ID NO:78). In identifying the 3'
end of the CARD2X-encoding cDNA, this third CARD2X-specific primer
was used in conjunction with a phage-specific primer having the
sequence ATACGACTCACTATAGGGCGAATTGGCC (SEQ ID NO:79) to amplify a
0.3 kb cDNA molecule using methods described above. The 0.3 kb cDNA
molecule was cloned and sequenced as described above, and the
sequences of the 0.3 and 0.9 kb cDNA molecules were merged to
produce a 1.0 kb cDNA sequence.
[0247] The sequence of CARD2X was confirmed. Additional 5'
untranslated sequence was identified (nucleotide sequence of CARD2X
including 5' untranslated sequence, SEQ ID NO:84). The CARD domain
extends from amino acids 4 to 78 of SEQ ID NO:12.
[0248] The association between CARD2X and other CARD-containing
proteins was determined. HEK 293T cells in 6-well plates were
transfected using SuperFect (Qiagen) with pairwise combinations of
Myc-tagged or FLAG-tagged CARD2X, CARDIAK or NOD1 (total DNA 2
.mu.g). After 24 hours, cells were collected in 400 .mu.l of lysis
buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, and 1 mM EDTA
supplemented with 1.times. protease inhibitors mix
(Roche/Boehringer Mannheim)). Cell lysates were clarified by
centrifugation and subjected to immunoprecipitation using
Agarose-beads conjugated with anti-FLAG M2 antibody (Sigma).
Immune-complexes were washed three times with wash buffer (20 mM
Tris, pH 7.4, 100 mM NaCl, 0.05% NP-40, and 1 mM EDTA), and
resolved on SDS-PAGE gels. Proteins in the gels were transferred to
nitrocellulose membranes, immunoblotted with anti-Myc antibodies,
and detected with ECL (Amersham-Pharmacia Biotech).
Epitope-specific antibodies for myc, FLAG, or HA tag were obtained
from Santa Cruz Biotech, Roche/Boehringer Mannheim, and Sigma. The
results of these co-immunoprecipitation assays demonstrated that
CARD2X specifically associates with both NOD1 and with CARDIAK.
[0249] The effect of CARDIAK on CARD2X phosphorylation was next
determined. HEK 293T cells transiently expressing FLAG-CARDIAK were
lysed and immunoprecipitated with Agarose-beads conjugated with
anti-FLAG M2 antibody. In vitro phosphorylation was performed in
the immune complex with or without purified Myc-CARD-2.times. as a
substrate. The kinase reaction was initiated by adding 1 .mu.M of
[.gamma.-.sup.32P]ATP in 10 .mu.l of kinase buffer (50 mM Tris,
pH7.4, 100 mM NaCl, 6 mM MgCl.sub.2, 1 mM MnCl, and 1 mM EDTA).
After 20 min at 37.degree. C., the reaction was stopped by adding
10 .mu.l of 2.times.SDS sample buffer, and subjected to SDS-PAGE
and autoradiography. The results of these assays indicated that
CARD2X is not phosphorylated directly by CARDIAK.
[0250] Phosphatase assays were also performed to examine
phosphorylation of CARD2X. HEK 293 cells were transfected with
plasmids encoding Myc-CARD-2.times. with or without FLAG-CARDIAK or
FLAG-CARDIAK (K47M), which is a kinase deficient mutant of CARDIAK.
The cleared lysates were diluted 1:20 with 20 .mu.l of reaction
buffer (25 mM Tris, pH8.0, 50 mM NaCl, 5 mM MgCl.sub.2), and
optionally treated with 2 units of calf intestine alkaline
phosphatase (Gibco BRL) for 30 min at 37.degree. C. The reaction
was terminated by adding 7 .mu.l 4.times.SDS sample buffer, and
subjected to SDS-PAGE and immunoblot. The phosphorylated form of
CARD2X migrates more slowly that CARD2X, and is not observed after
phosphatase treatment. The results of these assays indicated that
CARD2X is phosphorylated in vivo in the presence of either CARDIAK
or kinase-deficient CARDIAK, but not in their absence. Taken
together with the in vitro phosphorylation results above, these
results indicate that CARDIAK is indirectly involved in CARD2X
phosphorylation.
[0251] The 30-35 residues at the carboxy terminus of CARD2X have
homology to human Alu family sequences and RhoGAP. Thus, this
region can have activity similar to that observed in human Alu
family sequences and RhoGAP.
[0252] 10.0 Cloning and characterization of CLAN. CLAN encoding
cDNA was obtained by polymerase chain reaction (PCR) using primers
CXF1: TACTTACTTTGTCCCTTCA (SEQ ID NO:74) and CXR2:
TATTTGTCCCCATCTCGTC (SEQ ID NO:75) to amplify cDNA from a human
genomic library. Thirty cycles of PCR were carried out using Turbo
Pfu DNA polymerase (Stratagene) at annealing temperature 47.degree.
C. and extension temperature 72.degree. C. The PCR product was then
purified by agarose gel electrophoresis and the purified product
subcloned into pGEM-T vector (Promega).
[0253] The HTSG database of human genomic DNA sequence data was
searched for regions capable of encoding CARDs using the CARD
amino-acid sequence of cIAP-1 as a query with the TBLASTn method.
This search revealed strong homology with a human genomic clone
(Accession number: AQ889169) that mapped to human chromosome
2p21-22. This locus was not recognized in the human genomic
database and was not previously annotated. In initial studies, two
genes encoding CARD domain containing polypeptides, designated
CARD4X and CARD5X, were identified. Upon further characterization,
it was determined that CARD4X (also known as NAC-X or NAC-4) and
CARD5X were actually encoded by the same gene, which is therefore
referenced as CARD4/5X. CARD4/5X was subsequently designated CLAN,
which stands for "CARD, LRR and NACHT-containing protein," because
at least one of the proteins encoded by it contains CARD, Leucine
Rich Repeat (LRR) and NACHT (NB-ARC) domains, as described
below.
[0254] The CLAN gene locus lies in close proximity to the gene
encoding Spastin (on chromosome 2p21-22), a AAA protein which is
frequently mutated in autosomal dominant hereditary spastic
paraplegia (AD-HSP). The CLAN locus is found on the strand opposite
the SPG4 (SPAST) locus but with no overlapping regions. This result
suggests that mutations in the CLAN gene potentially occur in
patients with this neurodegenerative disorder.
[0255] Using GENESCAN for exon prediction, additional regions
potentially encoding a NACHT (NB-ARC) domain and regions
corresponding to Leucine-Rich Repeat (LRR) domains were also
recognized 3' to the potential CARD-encoding sequences, suggesting
the presence of a CED4-like gene.
[0256] 10.1 Cloning of CLAN cDNAs. CLAN-specific primers
corresponding to sequences within the putative CARD and NACHT
(NB-ARC) regions (as determined from genomic DNA sequence data)
were used in conjunction with 2 universal primers to isolate CLAN
cDNAs from first-strand liver and lung cDNA by nested PCR according
to the manufacturer's protocol (SMART RACE, Clontech). Primers used
for amplification are 5' RACE primers
(5'-CATGTGAATGATCCCTCTAGCAG-3' (SEQ ID NO:153); nested
5'-GGGCTCGGCTATCGTGCTCTA-3' (SEQ ID NO:154)) and 3' RACE primers
(5'-ACGATAGCCGAGCCCTTATTC-3' (SEQ ID NO:155); nested
5'-GTATGGAATGTTCTGAATCGC-3' (SEQ ID NO;156)). Amplification
products were purified from agarose gels, ligated into the TA
cloning vector (Promega), and sequenced. Four open reading frames
were deduced and multiple clones of each isoform were sequenced to
ensure fidelity of PCR products.
[0257] The longest transcript, termed CLAN-A, was 3.370
kilobasepairs (kbp) in length (SEQ ID NO:96) with an open reading
frame (ORF) coding for a 1024 amino-acid protein (SEQ ID NO:97)
containing a CARD, NACHT (NB-ARC), and LRR-domains, as well as a
predicted SAM domain. A second transcript, termed CLAN-B, was 1.374
kbp in length (SEQ ID NO:98), with an ORF coding for a 359
amino-acid protein (SEQ ID NO:99) containing an identical CARD
directly spliced to the LRRs. CLAN-C, the third transcript
isolated, was 0.768 kbp in length (SEQ ID NO:102) and encoded a 156
amino acid protein (SEQ ID NO:103) containing the CARD and an
additional region lacking homology to recognizable domains.
Finally, the shortest transcript found, CLAN-D, was 0.578 kbp in
length (SEQ ID NO:100) and contained an ORF encoding a 92
amino-acid protein (SEQ ID NO:101) encompassing only the CARD
followed by 9 amino acids.
[0258] Comparisons of these cDNA sequence data with the genomic DNA
sequence data found in the HTSG database suggested that the CLAN
gene consists of 12 exons, spanning 41.3 kbp on chromosome 2p21-22
(FIG. 1A). Six differences were found between the sequence of the
CLAN cDNA and the sequence within the public database.
Additionally, nucleotide regions 1-12 and 3372-3396 do not have
equivalent fragments in the public database.
[0259] Southern blot analysis was also performed. For Southern blot
analysis, 10 .mu.g of restriction endonuclease (EcoRI or PstI)
digested genomic DNA was loaded per lane and hybridized with the
CARD domain of CLAN as a probe. The probe was derived from the CLAN
A-isoform (see FIGS. 1 and 2), nucleotides 276 to 507 plus an
additional 20 upstream nucleotides, which are not present in the
cDNA but are present in the genomic DNA. CLAN was found to be a
single copy gene.
[0260] Two different transcriptional start sites are utilized
(corresponding to the beginning of either exon 1 or 2); however
both are spliced to exon 3 at the beginning of the CARD. Exons 6
and 7 contain additional internal splice donor sites which are
utilized to generate CLAN-G. FIG. 1B shows the pattern of mRNA
splicing events predicted to give rise to the CLAN-A, CLAN-B,
CLAN-C, and CLAN-D transcripts and encoded proteins. All the
exon/intron splice junctions follow the conserved GT/AG consensus
rule.
[0261] As predicted by SMART (EMBL, Heidelberg, Germany), CLAN
contains a CARD (amino acids 1-87 of SEQ ID NO:97). A .psi.-BLAST
search of the non-redundant database using the CLAN CARD as query
identified several homologous CARDs including those from cIAP1 and
2 (58%), caspase-1 and ICEBERG (50%), Nod1, Nod2, and Card8
(.about.38%) and caspase-13, Ced3, caspase-9, Bc110 (CIPER) and
CARKIAK/RIP2 (.about.30%).
[0262] Following the CARD, a domain containing consensus sequences
for Walker A and B boxes is present (Walker et al., EMBO J.
8:945-951 (1982)) as well as additional characteristics of the
family of NTPases termed the NACHT family (Koonin et al., Trends.
Biochem. Sci. 25:2230224 (2000)). By .psi.-BLAST search the NACHT
domain of CLAN ("NB" in FIG. 1, amino acids 161-457 of SEQ ID
NO:97) shows highest similarity to the NACHT domain of NAIP (60%),
followed by Nod1 (49%) and Nod2 (47%).
[0263] Leucine Rich Repeat (LRR) domains are also found near the
C-terminus of the A and .beta. isoforms of the protein. The
C-terminal end consists of four repeated LRRs, each containing a
predicted .beta. sheet and .alpha. helical structure, which is in
agreement with the prototypical horseshoe-shaped structure of LRRs
(Kobe et al., Cum Opin. Struct. Biol. 5:409-416 (1999). LRR 1
(amino acids 760-791 of SEQ ID NO:97) represents a non-Kobe and
Deisenhofer (non-K/D) LRR, whereas LRRs 2, 3, and 4 (amino acids
817-848; 845-876; and 934-965 of SEQ ID NO:97, respectively) are in
accordance with Kobe and Deisenhofer (K/D) LRR. LRR 2 also shares
sequence homology to a prototypical Ribonuclease Inhibitor type A
(R1 type A). By .psi.-BLAST searches the LRRs show 49% sequence
identity to the placental ribonuclease/angiogenin inhibitor
(RAI).
[0264] Sequences located between the NACHT (NB-ARC) and LRR domains
show some similarity to the sterile alpha motif (SAM) (amino acids
642-696 of SEQ ID NO:97), a domain built of five alpha helices
originally found in proteins involved in numerous developmental
processes. The SAM domain has been shown to function as a
protein-protein interaction domain, with ability to homo- as well
as hetero-oligomerize with other SAMs (Stapleton et al., Nat.
Struct. Biol. 6:44-49 (1999)).
[0265] 10.2 In vivo expression of CLAN. In order to determine which
of the various splice variants of CLAN are expressed in adult human
tissues, Northern blot analysis was performed. Hybridization probes
corresponding to the common CARD domain of all 4 CLAN isoforms or
the NACHT and LRR regions were radiolabeled by random priming with
hexanucleotides (Roche) and .alpha.-.sup.32P-dCTP, or
Digoxigenin-labeled with a commercially available kit (Roche),
incubated with blots containing human poly(A)+ RNA derived from
various human tissues (Origene), washed at high stringency, and
exposed to X-ray film. Positive signals were detected by
autoradiography or by immunoblotting with HRP-conjugated anti-DIG
antibody and an enhanced chemiluminescence method (ECL)
(Amersham).
[0266] Northern blot analysis with CARD of CLAN revealed expression
of an approximately 1.5 kbp transcript corresponding to CLAN-B in
nearly all tissues examined, with highest expression in lung and
spleen. Northern blot analysis using the NACHT and LRR of CLAN-A as
a probe revealed expression of an approximately 3.5 kbp mRNA
corresponding to CLAN-A primarily in the lung.
[0267] To further explore the tissue-specific patterns of
expression of CLAN splicing variants, RT-PCR assays were devised
specific for the A, B, C, and D isoforms. A panel of cDNA specimens
derived from various human tissues was utilized (Clontech), as well
as blood cells, prepared as followed. Peripheral blood leukocytes
were obtained from heparinized venous blood by Ficoll-Paque
(Amersham) density-gradient centrifugation. Red blood cells were
removed from granulocytes by short incubation in hypotonic lysis
buffer. Monocytes were separated from lymphocytes by adherence to
plastic dishes. Total RNA was isolated from cells using TRIZOL
reagent (BRL) and 2 .mu.g was used to generate cDNA in a reverse
transcription reaction with Superscript II (BRL).
[0268] PCR was carried out on the cDNA samples in an Eppendorf
thermal cycler using Taq polymerase (BRL) and the following
isoform-specific primer pairs: CLAN-A
5'-GGTGGAGCAGGATGCTGCTAGAGG-3' (SEQ ID NO:159),
5'-CACAGTGGTCCAGGCTCCGAATGAAGTCA-3' (SEQ ID NO:160); CLAN-B
5'-CATCATTTGCTGCGAGAAGGTGGAG-3' (SEQ ID NO:161),
5'-TTAACTTGGATAACACTTGGCTAAG-3' (SEQ ID NO:162);
CLAN-C5'-GTAAACATCATTTGCTGCGAGAA-3' (SEQ ID NO:163),
5'-CCCGGGCAGGTAGAAGATGCTAT-3' (SEQ ID NO:164); CLAN-D
5'AATTTCATAAAGGACAATAGCCGAG-3' (SEQ ID NO:165),
5'-TGTCTACTGTACTTTCTAAGCTGTT-3' (SEQ ID NO:166).
[0269] RT-PCR analysis showed that CLAN-B was present throughout
human tissues (brain, heart, kidney, liver, lung, pancreas,
placenta, skeletal muscle, colon, ovary, leukocytes, prostate,
small intestine, spleen, testis, thymus), consistent with the
Northern blot analysis. In contrast, CLAN-A was restricted to lung,
colon, brain, prostate, spleen and leukocytes, but not other
tissues. Further analysis of leukocyte sub-populations revealed
expression of the CLAN-A isoform predominantly in the monocyte cell
fraction, with lower expression found in granulocytes and no
expression in lymphocytes. Expression of CLAN-C was absent in all
normal tissues tested, however, expression was evident in the cell
line HEK293T, suggesting this transcript can be produced under some
circumstances. CLAN-D transcripts were detected only in brain by
RT-PCR.
[0270] RT-PCR was also performed on cell lines. RT-PCR was
performed using the same CLAN primers as used for RT-PCR in normal
tissues, as described above. RT-PCR was performed in various tumor
derived cell lines: M2, OVCAR3, HEY, HaCaT, 293T, SKOV-3, Jurkat,
BG-1, 697, HL-60, PC3, DU145, MDA-MB-231, MCF-7, MDA-MB-4, HS578T,
8T-549, and T-47D. Beta-actin primers were used as a control. In
contrast to normal tissue, the transcript for CLAN was mostly
absent in the cell lines tested. Weak expression was found in the
cell lines 697, MDA-MB-231, MVF-7, MDA-MB-4, HS578T, and T-47D.
[0271] 10.3 CLAN protein interactions. Interactions between the
CARD of CLAN and known CARD domains were tested in vitro and in
vivo.
[0272] To test CLAN interactions with other molecules, an in vitro
binding assay was performed. CLAN was in vitro translated in the
absence of label (i.e., cold). Other cellular proteins were labeled
in vitro with .sup.35S-Met: CLAN, caspase1, caspase2, caspase8,
caspase9, caspase10, Apaf1, Apaf1-CARD, NACa, NAC-CARD, Bc110, ASC,
cIAP1, cIAP2, XIAP, Nod1, Ced4, RAIDD, and CARDIAK. The in vitro
translated proteins were mixed separately with unlabeled CLAN and
co-immunoprecipitated using an antibody against an epitope tag
fused to CARD5X, either myc or hemaglutinin (HA). CLAN associated
proteins were eluted by boiling in Laemmli denaturing buffer and
separated by 12% SDS-PAGE. The radioactive bands were visualized by
fluorography.
[0273] Weak binding to CLAN was observed with caspase2 and cIAP1,
with stronger binding to Nod1 and Cardiak. The strongest binding
was observed with Ced4. Caspase8 binding is possibly due to its
stickiness. There was no association detected between CLAN and
itself.
[0274] To prepare appropriate expression vectors for in vivo
interaction studies, a cDNA encoding the CLAN CARD domain was
amplified using PFU polymerase and specific primers
(5'-CCCGGATCCATGAATTTCATAAAGGACAATAGC-3' (SEQ ID NO:153);
5'-CCCTTCGAACAAGTCCTGAAATAGAGGATA-3' (SEQ ID NO:154)) containing
BamHI and HindIII sites. The resulting PCR product was ligated into
pcDNA3.1(-)/Myc-His.sub.6 A (Invitrogen) which places the
myc-His.sub.6 tag at the C-terminus of expressed proteins.
pcDNA3/HA-CLAN (CARD) was created using a similar strategy.
Authenticity of all vectors was confirmed by DNA sequencing.
[0275] The CARD of CLAN was expressed as an epitope-tagged protein
in HEK293T cells in co-transfections with a variety of other
epitope-tagged CARD-containing proteins, and lysates derived from
these cells were used for co-immunoprecipitation assays. Briefly,
HEK293T cells were seeded onto six-well plates (35 mm wells) and
transfected with 0.2-2 mg plasmid DNA using Superfect (Qiagen) 24
hr later. After culturing for a day, cells were collected and lysed
in isotonic lysis buffer (142.4 mM KCl, 5 mM MgCl.sub.2, 10 mM
HEPES (pH 7.4), 0.5 mM EGTA, 0.2% NP-40, 12.5 mM
b-glycerophosphate, 2 mM NaF, 1 mM Na.sub.3VO.sub.4, 1 mM PMSF, and
1.times. protease inhibitor mix (Roche)). Lysates were clarified by
centrifugation and subjected to immunoprecipitation using
agarose-conjugated anti-c-myc antibodies (Santa Cruz), or
non-specific control antibodies and Protein G-agarose for 2-24 hr
at 4.degree. C. Immune-complexes were washed four times with lysis
buffer, boiled in Laemmli buffer, and separated by 12-15% PAGE.
Immune-complexes were then transferred to PVDF membranes and
immunoblotted with anti-c-myc (Santa Cruz), anti-HA (Roche), or
anti-flag (Sigma) antibodies. Membranes were washed, incubated with
HRP-conjugated secondary antibodies, and reactive proteins were
detected using ECL.
[0276] Co-immunoprecipitation analysis indicated that the CARD of
CLAN bound readily to full-length pro-caspase-1 but did not
significantly bind another CARD-containing caspase, caspase-9.
Among the other CED-4 family members which contain a CARD in
conjunction with a nucleotide-binding domain, CLAN interacted with
the CARDs of Nod2 and NAC, but not with Apaf-1 or Nod-1. Finally,
the CLAN CARD was found to associate with Bcl-10, but not with
another adapter protein, RAIDD.
[0277] 11.0 Cloning and characterization of CARD3X Based on an
analysis of the overlapping genomic contigs GI 8575872 and GI
5001450, a cDNA sequence for CARD3X was predicted (SEQ ID NO:82),
that encoded amino acid sequences designated SEQ ID NOS:83 and
107.
[0278] For identification of novel domains in CARD3X, the sequence
of the CARD domain of polypeptide CARD3X was used as a query for a
tblastn search in the HTGS database, and two overlapping genomic
contigs were found (GI numbers 5001450 and 8575872). This contig
was analyzed using the GenScan server
(http://ccr-081.mit.edu/GENSCAN.html) for the presence of exons.
(Burge and Karlin, J. Mol. Biol. 268:78-94 (1997)). The predicted
protein sequences coded by the exons were analyzed by comparison
with the NCBI nr protein sequence database using PSI-BLAST. The
predicted protein sequences coded by the exons were analyzed also
by comparison with a database of proteins with known
three-dimensional structures and apoptosis related domains using
the profile-profile comparison server at
http://bioinformatics.burnham-instorg/FFAS_apoptosis (Rychlewski,
et al., Protein Science 9:232-241 (2000)).
[0279] CARD3X contains two CARD domains, a CARD-A and CARD-B domain
(see FIG. 3). An NB-ARC domain was also observed (see FIG. 3). The
NB-ARC is similar to both the CLAN and APAF-1 NB-ARC domains and to
NB-ARC domains from several plant disease resistance proteins
(Aravind et al., Trends Biochem. Sci. 24:47-53 (1999); Young, Curr.
Opin. Plant Biol. 4:285-289 (2000)).
[0280] An angio-R domain was also identified at amino acids 457-839
of SEQ ID NO:107. An "angio-R" is a new domain that can be defined
as a region of a polypeptide chain that bears substantial
similarity (e.g. 25, 30, 40% sequence identity) to the 514-reside
long protein "angiotensin II/vasopressin receptor" (described in
Ruiz-Opazo et al., Nature Med. 1:1074-1081 (1995)). The "angio-R"
domain has not been previously described in any protein.
[0281] To confirm the predicted sequences, cDNAs were cloned and
sequenced. The CARD3X cDNA was cloned using a Rapid-Screen.TM.
Arrayed Placenta cDNA Library Panel from Origene Technologies, Inc.
The library cDNAs had been pre-selected for long clones,
unidirectionally cloned into the vector pCMV6-XL4, and arrayed in a
96-well format. An initial Master Plate containing 500,000 cDNA
clones was screened by PCR, using the forward primer
5'-GAAATGTGCTCGCAGGAGG-3' (SEQ ID NO:185) and the reverse primer
5'-GATGAGCTTCTGACAGGCCC-3' (SEQ ID NO:186). A set of 5000 clones
that were initially positive by PCR were screened again with the
same set of primers. Positive clones were plated on LB/Amp plates,
and a further round of single colony PCRs was performed in order to
obtain the desired clone.
[0282] Three independent clones were sequenced, each of which
corresponded to the nucleotide sequence SEQ ID NO:187. The cDNA
sequence differed at both the N- and C-terminal ends from the
CARD3X sequence predicted from analysis of genomic exons. SEQ ID
NO:187 encodes a polypeptide of 795 amino acids (SEQ ID NO:188),
followed by a stop codon. A second open reading frame begins after
the stop codon, and in the same reading frame, and encodes a
polypeptide of 180 amino acids (SEQ ID NO:189). SEQ ID NO:189
contains several leucine rich repeats.
[0283] Subsequent to the identification of the two polypeptides
encoded by SEQ ID NO:187, a publication reported the cloning of a
gene designated Nod2 cloned (Ogura et al., J. Biol. Chem.
276:4812-4818 (2001)). The published Nod2 sequence has additional
N-terminal amino acids relative to SEQ ID NO:188 and, instead of
the stop codon between the residues that encode SEQ ID NO:188 and
SEQ ID NO:189, additional coding sequence is present, which encodes
several additional leucine rich repeats. The published Nod2
sequence is 1040 amino acids.
[0284] It is proposed that SEQ ID NO:188 is a splice variant form
of CARD3X/Nod2 that does not contain an LRR domain. The LRR of Nod2
has been shown to interfere with the ability of the protein to
activate NF.kappa.B (Ogura et al., supra (2001)). Therefore, SEQ ID
NO:188 is likely expressed under physiological conditions in which
activation of NF.kappa.B is required.
[0285] Human CARD3X cDNA sequences were used as a query for BLAST
searches of several mouse databases. A genomic sequence, SEQ ID
NO:190, was identified. Nucleotides 191-614 of SEQ ID NO:190 are
homologous to the ANGIO-R coding region of human CARD3X.
Nucleotides 193-612 of SEQ ID NO:191 were predicted to encode SEQ
ID NO:191, which is highly homologous to amino acids 214-341 of the
ANGIO-R domain of human CARD3X (SEQ ID NO:176).
[0286] PCR was then performed on mouse genomic DNA obtained from
C57B6 and NIH3T3 cell lines, using the following primers: Forward
primer: 5'-CTGCAGAAGGCTGAGCCACACAACCT-3' (SEQ ID NO:194), Reverse
primer: 5'-ACAGAGTTGTAATCCAGCTGTAGGGCCACA-3' (SEQ ID NO:195). The
PCR product so obtained was sequenced (SEQ ID NO:192), and shown to
have several nucleotide differences as compared to the
corresponding region of SEQ ID NO:190. The predicted amino acid
sequence encoded by SEQ ID NO:192 (designated SEQ ID NO:193) had a
single amino acid difference in comparison with SEQ ID NO:191.
[0287] Both the CARD-A and CARD-B domains are independently cloned
into pcDNA3 with epitope tags such as myc or HA, as described
above, and binding of the CARD domains is tested with
co-immunoprecipitation to test binding of CARD3X CARD domains with
other known CARD domains, as described above.
[0288] The NB-ARC domain is cloned into a yeast two-hybrid vector
and into pcDNA3 with two alternative epitope tags (e.g., myc and
Flag) to determine whether the NB-ARC domain self-associates in an
ATP-dependent manner/P-loop mutation. The P-loop, which binds the
gamma phosphate of ATP in the NB-ARC domain, is mutated to remove a
conserved Lys in the consensus P-loop sequence G-S/T-K, where Lys
is generally mutated to Met. The NB-ARC domain is also tested for
binding to the NB-domains of other CED-4 like proteins (e.g.,
apafl, nodi, nac).
[0289] 12.0 Characterization of COP-1. Using the amino-acid
sequence of the caspase-1 prodomain as a query for BLASTn searches
of the public databases, a human EST clone (GenBank accession
number AA070591) was identified containing an ORF encoding a 97
amino-acid protein (SEQ ID NO:86) predicted to share 92% sequence
identity with the CARD of pro-caspase-1 (SEQ ID NO:87). The
predicted protein contains a CARD (residues 1-91), which is
followed by 6 amino-acids and then a stop-codon. The CARD region of
COP-1 showed 97% identity to the CARD of pro-caspase-1.
[0290] To confirm the predicted sequences, cDNAs were amplified
from various adult human tissues and sequenced. The sequenced COP-1
cDNA (SEQ ID NO:85) had the same nucleotide sequence as the
original EST.
[0291] The start codon initiating the ORF in the COP-1 cDNA clones
resides in a favorable context for translation, and is preceded by
an in-frame stop codon. The 3'-untranslated region contains TAAA
and TATA motifs, typical of short-lived mRNAs which are subject to
post-transcriptional regulation, and a candidate polyadenylation
signal sequence (AATAAA). Thus, this protein contains essentially
only a CARD, prompting the moniker CARD Only Protein (COP-1).
[0292] To determine the genomic organization of the COP-1 gene, the
COP-1 cDNA nucleotide sequence was employed for searches of the
High Throughput Genomic Sequence (HTGS) database, resulting in
identification of three genomic clones containing the COP-1 gene
(GenBank accessions numbers AC027011, AP001153 and AP002787).
Comparison of the COP-1 cDNA and genomic DNA sequences suggests a
three exon structure, in which only the first two amino-acids are
encoded in exon 1 and only the last 5 residues are encoded in exon
3, such that most of the coding regions (including the entire CARD)
are derived from exon 2. The introns separating exons 1, 2, and 3
are 631 and 844 by in length, respectively, containing consensus
dinucleotide splice donor (GT) and splice acceptor (AG) motifs.
[0293] The COP-1 genomic clones identified in the HTSG database
have been mapped to human chromosome 11q22, which is the same
chromosomal region where the pro-caspase-1 gene resides, as well as
pro-caspase-4, pro-caspase-5, and ICEBERG. To address the genomic
localization of COP, pro-caspase-4, pro-caspase-5, and ICEBERG
genes in chromosome 11, the public database of Human Genome Project
Working Draft (www.genome.cse.ucsc.edu) was searched, and the order
of these genes from centromere to telomere was determined to be
pro-caspase-4, pro-caspase-5, pro-caspase-1, COP, and ICEBERG. This
result suggests that COP-1 is a separate gene, presumably arising
from duplication of other homologous genes in this locus.
[0294] 14.1 COP-1 expression. To study the expression of COP-1,
Northern blot analysis was performed using RNA derived from several
adult human tissues and a .sup.32P-labeled COP-1 cDNA probe. Blots
containing polyA-selected mRNA from various adult tissues
(Clontech, Palo Alto, Calif.) were hybridized using a
.sup.32P-labeled COP-1 cDNA probe. The probe represented a 570 by
length cDNA containing portions of the 5'-untranslated region, the
complete ORF, and portions of the 3'-untranslated region of COP.
The COP-1 probe (from the EST clone corresponding to AA070591
obtained from the I.M.A.G.E. Consortium (Washington University
School of Medicine, St. Louis, Mo.)) was excised from the plasmid
by restriction digestion with EcoRI and XhoI, gel-purified, and
radiolabeled by the random priming method using [.alpha.-.sup.32P]
dCTP and a kit from Ambion (Austin, Tex.). After hybridization,
heat-denatured probe was annealed for 1 hr at 68.degree. C. with
QuickHyb Hybridization Solution (Stratagene, La Jolla, Calif.) and
then blots were washed with solutions containing 2.times.SSC, 0.1%
(w/v) SDS (twice each for 15 min at 25.degree. C.) followed by
0.1.times.SSC, 0.1% (w/v) SDS (twice for 10 min at 40.degree. C.).
Bands were visualized by autoradiography.
[0295] Hybridizing bands of approximately 0.6 kbp, 1.5 kbp and 2.6
kbp were identified, with the 0.6 kbp band representing the most
abundant of these transcripts and presumably corresponding to the
fully-spliced COP-1 mRNA. The less abundant larger 1.5 kbp and 2.6
kbp transcripts could represent unspliced precursors.
Alternatively, the 2.6 kbp mRNA could represent pro-caspase-1 mRNA,
resulting from probe cross-hybridization. The 0.6 kbp COP-1 mRNA
was most abundant in spleen, followed by liver, placenta, and
peripheral blood leukocytes (PBL). However, most tissues (including
heart, muscle, colon, kidney, intestine and lung) were shown to
contain at least some detectable 0.6 kbp COP-1 mRNA.
[0296] To corroborate the Northern blot analysis, COP-1 mRNA
expression in adult human tissues was also examined using RT-PCR
and COP-specific primers. cDNA samples derived from multiple human
adult tissues (Clontech, Palo Alto, Calif.) were amplified using a
set of COP-specific primers (a forward primer
5'-GAAGACAGTTACCTGGCAGA-3' (SEQ ID NO:147) and a reverse primer
5'-TTGTATTCTGAACATGGCACC-3' (SEQ ID NO:148)). The resulting PCR
products were size-fractionated by electrophoresis in 1.5% agarose
gels, then stained with ethidium bromide for UV-photography. In
some cases, bands were excised from gels, purified, and sequenced,
thus verifying amplification of the correct product by the RT-PCR
assay.
[0297] RT-PCR analysis showed that COP-1 mRNA was expressed in all
tissues analyzed (brain, heart, muscle, colon, spleen, kidney,
liver, intestine, placenta, lung and PBL), except thymus. Parallel
RT-PCR analysis of .beta.-actin mRNA served as a control. In
general, the relative levels of COP-1 mRNA detected by RT-PCR were
in agreement with the Northern blot data.
[0298] 14.2 COP-1 interactions. The prodomain of pro-caspase-1 is
required for dimerization and activation of this zymogen. Since the
prodomain of COP-1 shares a high-degree of amino-acid sequence
identity with the prodomain of caspase-1, the possibility that
COP-1 interacts with pro-caspase-1 in co-immunoprecipitation assays
was tested. Interactions with several other CARD-containing
proteins were also tested, including COP-1 itself, RIP2, Bcl-10,
cIAP1, cIAP2 and pro-caspase-9.
[0299] For these experiments, the entire open reading frame (ORF)
of COP-1 was amplified by PCR using the primers
(5'-CCAGAATTCATGGCCGACAAGGTCCTGAAG-3' (SEQ ID NO:145) (forward) and
5'-CCACTCGAGCTAATTTCCAGGTATCGGACC-3' (SEQ ID NO:146) (reverse). The
COP-1 PCR product was digested with EcoR11XhoI and ligated into
mammalian expression vectors pcDNA3-Myc, pcDNA3-HA and pcDNA3-Flag
at the EcoR11XhoI cloning sites. Plasmids encoding wild-type
pro-caspase-1, RIP2, and pro-IL-1.beta. were as described in Thome
et al., Curr, Biol. 8:885-888 (1998); Nett-Fiordalisi et al., J.
Leukoc. Biol. 58:717-724 (1995); and Wang et al., J. Biol. Chem.
271:20580-20587 (1996).
[0300] A pro-caspase-1 Cys 285 Ala mutant was made from wild-type
caspase-1 plasmid by site-directed mutagenesis, using a
commercially available kit (Stratagene, La Jolla, Calif.) and the
primers 5'-GATCATCATCCAGGCCGCCCGTGGTGACAGCCCTGG-3' (SEQ ID NO:149)
and 5'-CCAGGGCTGTCACCACGGGCGGCCTGGATGATGATC-3' (SEQ ID NO:150). A
truncation mutant of pro-caspase-1 in which a stop codon was
introduced downstream of the CARD was created by PCR using primers
5'-CGGAATTCATGGCCGACAAGGTCCTG-3' (SEQ ID NO:151) and
CGCTCGAGTTAGTCTTGCATATTAAGGTAATTTCCAGA-3' (SEQ ID NO:152).
[0301] Human embryonic kidney 293T cells were cultured at
37.degree. C. in 5% CO.sub.2 in Dulbecco's Modified Eagle's Medium
(DMEM) with 10% heat-inactivated fetal bovine serum (FBS). Cells in
log phase were transfected in 60 mm diameter dishes with expression
plasmids (5 .mu.g total DNA) using Superfect Transfection Reagent
(Qiagen, Valencia, Calif.) according to the manufacturer's
recommendations. Cells were harvested 2 days later and lysed in
ice-cold NP-40 lysis buffer (10 mM HEPES [pH 7.4], 142.5 mM KCl,
0.2% NP-40, 5 mM EGTA), supplemented with 1 mM DTT, 12.5 mM
.beta.-glycerophosphate, 1 .mu.M Na.sub.3V0.sub.4, 1 mM PMSF, and
1.times. protease inhibitor mix (Roche, Indianapolis, Ind.). Cell
lysates (0.5 ml) were clarified by centrifugation at 16,000.times.g
for 5 minutes, and subjected to immunoprecipitation using specific
antibodies, including anti-Myc antibodies (Santa Cruz
Biotechnology, Santa Cruz, Calif.), and anti-Flag antibodies
(Sigma, St. Louis, Mo.), in combination with 15 .mu.l Protein A- or
G-Sepharose (Zymed, South San Francisco, Calif.).
[0302] Immune-complexes were fractionated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and
transferred to nitrocellulose membranes. The resulting blots were
incubated with various antibodies, including anti-HA antibodies
(1:1000 v/v; Roche, Indianapolis, Ind.), anti-Myc antibodies (1:100
v/v; Santa Cruz Biotechnology, Santa Cruz, Calif.) and anti-Flag
antibodies (1:1000 v/v; Sigma, St. Louis, Mo.), followed by
horseradish peroxidase-conjugated secondary antibodies, and
detection by an enhanced chemiluminescence (ECL) method
(Amersham-Pharmacia, Piscataway, N.J.). Alternatively, lysates were
analyzed directly by immunoblotting after normalization for total
protein content.
[0303] The co-immunoprecipitation results showed that HA-COP-1
co-immunoprecipitated with Myc-COP, indicating that this protein
can self-associate. In addition, HA-COP-1 co-immunoprecipitated
with Myc-tagged pro-caspase-1 (C285A mutant) as well as with a
fragment of pro-caspase-1 containing only its CARD-carrying
prodomain. Thus, COP-1 binds pro-caspase-1 through its CARD domain.
For these co-immunoprecipitation experiments, the active site
cysteine of pro-caspase-1 was mutated to avoid induction of
apoptosis, which can occur when over-expressing this protease.
Additionally, Myc-COP-1 co-immunoprecipitated with Flag-RIP2. In
contrast, COP-1 did not co-immunoprecipitate with the
CARD-containing proteins Bcl-10, cIAP1, cIAP2, or pro-caspase-9,
thus demonstrating the specificity of these results.
[0304] RIP2 has been shown to bind and activate caspase-1 through
the interaction of their CARDS, resulting in oligomerization of
pro-caspase-1 and its activation via the "induced proximity"
mechanism. The data demonstrating that COP-1 binds to both
pro-caspase-1 and RIP2 therefore suggested that COP-1 might
function as a modulator of RIP2-induced pro-caspase-1
oligomerization.
[0305] To test this hypothesis, experiments were performed in which
293T cells were transiently transfected with expression plasmids
encoding Myc-tagged pro-caspase-1 (C285A mutant) and HA-tagged
pro-caspase-1 (C285A mutant), with or without Flag-tagged RIP2 and
COP, after which Myc-pro-caspase-1 and HA-pro-caspase-1 association
was monitored by co-immunoprecipitation assays.
[0306] As determined by this co-immunoprecipitation assay,
pro-caspase-1 self-associated and this was enhanced by
co-expression of RIP2. However, when COP-1 was also co-expressed,
this RIP2-mediated effect on pro-caspase-1 self-association was
negated. These findings suggested the possibility of a competitive
mechanism, in which COP-1 competes with RIP2 for binding to
pro-caspase-1. To test this hypothesis, therefore, transfection
experiments were preformed in which Flag-RIP2 and Myc-tagged
pro-caspase-1 (C285A mutant) were expressed in 293T cells in the
presence of increasing amounts of HA-tagged COP-1. The effects of
COP-1 on association of RIP2 with pro-caspase-1 were then evaulated
by co-immunoprecipitation assays in which immunoprecipitations were
performed using anti-Flag antibody to recover Flag-RIP2 protein and
the resulting immune-complexes were analyzed by
SDS-PAGE/immunoblotting using anti-Myc antibody to detect
associated Myc-pro-caspase-1.
[0307] The results from these experiments indicated that COP-1
inhibited association of pro-caspase-1 with RIP2 in a
dose-dependent manner. Immunoblot analysis of lysates from these
same cells demonstrated that COP-1 did not affect the total levels
of pro-caspase-1 or RIP2, but rather just their association. These
results therefore confirm that COP-1 can interfere with binding of
pro-caspase-1 to RIP2.
[0308] 14.3 COP-1 inhibition of caspase-1-mediated activation of
pro-IL-1.beta.. Active caspase-1 cleaves pro-IL-1.beta., resulting
in the generation of bioactive IL-1.beta. which is secreted from
cells. It was hypothesized that COP-1 could suppress
caspase-1-induced pro-IL-1.beta. processing and thus reduce
secretion of IL-1.beta..
[0309] To test this hypothesis, COS-7, 293T, or 293HEK cells were
co-transfected in 12 well (22 mm in diameter) plates using
Lipofectamine Plus Reagent (GIBCO BRL, Grand Island, N.Y.) with
plasmids encoding mouse pro-IL-1.beta., human caspase-1, RIP2, or
COP-1, in various amounts (total DNA=2.0 .mu.g). At 1 day after
transfection, supernatants were collected and stored at -80.degree.
C. or used immediately to quantify secretion of mature murine
IL-1.beta. into the culture medium by an ELISA assay, according to
the manufacturer's protocol (R&D systems, Minneapolis,
Minn.).
[0310] Co-expression of pro-caspase-1 and pro-IL-1.beta. in COS-7
cells resulted in secretion of mature IL-1.beta. ranging from 80
.mu.g/ml to 250 .mu.g/ml, which was proportional to the amount of
pro-caspase-1 plasmid used (FIG. 17). This IL-1.beta. secretion was
enhanced by co-expression of RIP2 plasmid. In contrast, expression
of COP-1 together with pro-caspase-1, pro-IL-1.beta., and RIP2
resulted in a dose-dependent decrease in the amount of mature
IL-1.beta. secretion, proportional to the amount of COP-1-encoding
plasmid used (FIG. 6). Similar results were obtained using 293T or
293HEK cells. These results indicate that COP-1 is capable of
suppressing the caspase-1-mediated secretion of IL-1.beta..
[0311] 15.0 Identification of COP-2. A human CARD-containing
proteins, designated COP-2, for CARD-only protein 2, was identified
and the gene and cDNA cloned. The predicted protein of COP-2 has
high sequence similarity to the CARD-domain of human caspase-1. For
COP-2, two primers based on the caspase-15 genomic sequence were
designed, one in the middle of the CARD domain
(5'-aagaagagacggctgcttatcaat-3'; SEQ ID NO:104) and the other in
the catalytic domain (5'-ccacagcaggcctcgaagatgatc-3'; SEQ ID
NO:105). RT-RTR was performed, and a single band was observed,
although the band size was smaller than expected for caspase-15.
The PCR product was sequenced, and it was found that two exons were
deleted and the catalytic domain was directly connected to the CARD
domain. However, due to a frameshift, a stop codon occurs just
after the CARD domain, resulting in truncated protein and no
translation of the catalytic domain.
[0312] To clone the N-terminal region, a primer
(5'-atgatcctcctgaagaagag-3'; SEQ ID NO:106) was designed with the
genomic sequence in the most N-terminal portion of the CARD domain
including ATG. RT-PCR was performed, and the PCR product was
sequenced and found to be the same as in the genomic DNA. A merged
construct containing both the N-terminal fragment and the CARD
domain sequence was made by PCR.
[0313] The COP-2 cDNA sequence identified contained 321 nucleotides
(SEQ ID NO:89), and the deduced amino acid sequence (SEQ ID NO:90)
had a high level of identity with caspase-1. An alignment of COP-2
(SEQ ID NO:90) and caspase-1 (SEQ ID NO:87) is shown in FIG. 5,
with the consensus sequence (SEQ ID NO:91) shown above the aligned
sequences. The amino acids shaded in black are identical. The
stipled shading represents a match within 3 distance units. COP-2
is encoded by the caspase-15 gene (FIG. 3), but COP-2 is a CARD
only protein that lacks the caspase catalytic domain.
[0314] COP-2 cDNA encodes a polypeptide with downstream termination
codons, which result in shorter proteins containing a CARD domain
without associated catalytic protease domains. COP-2 is therefore
expected to function as trans-dominant inhibitor that likely
prevents caspase activation by binding to the CARD-domains
(pro-domains) in pro-enzymes such as pro-caspase-1.
[0315] COP-2 polypeptide is expected to function as A regulator of
caspase-1 activation by enhancing or suppressing the activation of
caspase-1. COP-2 binding activity is tested, for example, by making
epitope tagged fusions with COP-2 and caspase-1 and
co-immunoprecipitating to determine binding interactions with
caspase-1. Antibodies specific for COP-2 are also made.
[0316] The effect of COP-2 on caspase-1 proteolytic activity is
also tested. Methods for measuring caspase activity are well known
(see, for example, Thornberry, Nature 356:768-774 (1992);
Thornberry and Molineaux, Protein Science 4:3-12 (1995); Rano et
al., Chem. Biol. 4:149-155 (1997); Fletcher et al., J. Interferon
Cytokine Res. 15:243-248 (1995)), and are also described above.
[0317] Although the invention has been described with reference to
the examples above, it should be understood that various
modifications can be made without departing from the spirit of the
invention.
[0318] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0319] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention.
Sequence CWU 1
1
1951 10002 20003 30004 40005 50006 60007 70008 80009 900010
10000111038DNAHomo sapienCDS(1)...(930) 11atg gct acc gag agt act
ccc tca gag atc ata gaa aga gaa aga aaa 48Met Ala Thr Glu Ser Thr
Pro Ser Glu Ile Ile Glu Arg Glu Arg Lys1 5 10 15aag ttg ctt gaa atc
ctt caa cat gat cct gat tct atc tta gac acg 96Lys Leu Leu Glu Ile
Leu Gln His Asp Pro Asp Ser Ile Leu Asp Thr 20 25 30tta act tct cgg
agg ctg att tct gag gaa gag tat gag act ctg gag 144Leu Thr Ser Arg
Arg Leu Ile Ser Glu Glu Glu Tyr Glu Thr Leu Glu 35 40 45aat gtt aca
gat ctc ctg aag aaa agt cgg aag ctg tta att ttg gta 192Asn Val Thr
Asp Leu Leu Lys Lys Ser Arg Lys Leu Leu Ile Leu Val 50 55 60cag aaa
aag gga gag gcg acc tgt cag cat ttt ctc aag tgt tta ttt 240Gln Lys
Lys Gly Glu Ala Thr Cys Gln His Phe Leu Lys Cys Leu Phe65 70 75
80agt act ttt cca cag tca gct gcc att tgc ggc tta agg cat gaa gtt
288Ser Thr Phe Pro Gln Ser Ala Ala Ile Cys Gly Leu Arg His Glu Val
85 90 95tta aaa cat gag aat aca gta cct cct caa tct atg ggg gca agc
agt 336Leu Lys His Glu Asn Thr Val Pro Pro Gln Ser Met Gly Ala Ser
Ser 100 105 110aat tca gaa gat gct ttt tct cct gga ata aaa cag cct
gaa gcc cct 384Asn Ser Glu Asp Ala Phe Ser Pro Gly Ile Lys Gln Pro
Glu Ala Pro 115 120 125gag atc aca gtg ttc ttc agt gag aag gaa cac
ttg gat ttg gaa acc 432Glu Ile Thr Val Phe Phe Ser Glu Lys Glu His
Leu Asp Leu Glu Thr 130 135 140tct gag ttt ttc agg gac aag aaa act
agt tat agg gaa aca gct ttg 480Ser Glu Phe Phe Arg Asp Lys Lys Thr
Ser Tyr Arg Glu Thr Ala Leu145 150 155 160tct gcc agg aag aat gag
aag gaa tat gac aca cca gaa gtc aca tta 528Ser Ala Arg Lys Asn Glu
Lys Glu Tyr Asp Thr Pro Glu Val Thr Leu 165 170 175tca tat tca gtt
gag aaa gtt gga tgt gaa gtt cca gca act att aca 576Ser Tyr Ser Val
Glu Lys Val Gly Cys Glu Val Pro Ala Thr Ile Thr 180 185 190tat ata
aaa gat gga cag aga tat gag gag cta gat gat tct tta tac 624Tyr Ile
Lys Asp Gly Gln Arg Tyr Glu Glu Leu Asp Asp Ser Leu Tyr 195 200
205tta gga aaa gag gaa tat cta gga tct gtt gac acc cct gaa gat gca
672Leu Gly Lys Glu Glu Tyr Leu Gly Ser Val Asp Thr Pro Glu Asp Ala
210 215 220gaa gcc act gtg gaa gag gag gtt tat gat gac cca gag cac
gtt gga 720Glu Ala Thr Val Glu Glu Glu Val Tyr Asp Asp Pro Glu His
Val Gly225 230 235 240tat gat ggt gaa gag gac ttc gag aat tca gaa
acc aca gag ttc tct 768Tyr Asp Gly Glu Glu Asp Phe Glu Asn Ser Glu
Thr Thr Glu Phe Ser 245 250 255ggt gaa gaa cca agt tat gag gga tca
gaa acc agc ctt tca ttg gag 816Gly Glu Glu Pro Ser Tyr Glu Gly Ser
Glu Thr Ser Leu Ser Leu Glu 260 265 270gag gaa cag gag aaa agt ata
gaa ggc tgg tct cga act cat ggg ctt 864Glu Glu Gln Glu Lys Ser Ile
Glu Gly Trp Ser Arg Thr His Gly Leu 275 280 285aag cga tcc tcc cac
gtt ggc ctc cca aag tgc tgg gat tac agg cgt 912Lys Arg Ser Ser His
Val Gly Leu Pro Lys Cys Trp Asp Tyr Arg Arg 290 295 300gag cca ccc
tgc ctg gcc tgaaaattct gcctcaaaca tctcaaacat 960Glu Pro Pro Cys Leu
Ala305 310ccatttatat tttgtacaag aaagtaaata aaatttttct ttttaacatt
aaaaaaaaaa 1020aaaaaaaaaa aatctaga 103812310PRTHomo sapien 12Met
Ala Thr Glu Ser Thr Pro Ser Glu Ile Ile Glu Arg Glu Arg Lys 1 5 10
15Lys Leu Leu Glu Ile Leu Gln His Asp Pro Asp Ser Ile Leu Asp Thr
20 25 30Leu Thr Ser Arg Arg Leu Ile Ser Glu Glu Glu Tyr Glu Thr Leu
Glu 35 40 45Asn Val Thr Asp Leu Leu Lys Lys Ser Arg Lys Leu Leu Ile
Leu Val 50 55 60Gln Lys Lys Gly Glu Ala Thr Cys Gln His Phe Leu Lys
Cys Leu Phe65 70 75 80Ser Thr Phe Pro Gln Ser Ala Ala Ile Cys Gly
Leu Arg His Glu Val 85 90 95Leu Lys His Glu Asn Thr Val Pro Pro Gln
Ser Met Gly Ala Ser Ser 100 105 110Asn Ser Glu Asp Ala Phe Ser Pro
Gly Ile Lys Gln Pro Glu Ala Pro 115 120 125Glu Ile Thr Val Phe Phe
Ser Glu Lys Glu His Leu Asp Leu Glu Thr 130 135 140Ser Glu Phe Phe
Arg Asp Lys Lys Thr Ser Tyr Arg Glu Thr Ala Leu145 150 155 160Ser
Ala Arg Lys Asn Glu Lys Glu Tyr Asp Thr Pro Glu Val Thr Leu 165 170
175Ser Tyr Ser Val Glu Lys Val Gly Cys Glu Val Pro Ala Thr Ile Thr
180 185 190Tyr Ile Lys Asp Gly Gln Arg Tyr Glu Glu Leu Asp Asp Ser
Leu Tyr 195 200 205Leu Gly Lys Glu Glu Tyr Leu Gly Ser Val Asp Thr
Pro Glu Asp Ala 210 215 220Glu Ala Thr Val Glu Glu Glu Val Tyr Asp
Asp Pro Glu His Val Gly225 230 235 240Tyr Asp Gly Glu Glu Asp Phe
Glu Asn Ser Glu Thr Thr Glu Phe Ser 245 250 255Gly Glu Glu Pro Ser
Tyr Glu Gly Ser Glu Thr Ser Leu Ser Leu Glu 260 265 270Glu Glu Gln
Glu Lys Ser Ile Glu Gly Trp Ser Arg Thr His Gly Leu 275 280 285Lys
Arg Ser Ser His Val Gly Leu Pro Lys Cys Trp Asp Tyr Arg Arg 290 295
300Glu Pro Pro Cys Leu Ala305 31013 1300014 1400015 1500016 1600017
1700018 1800019 1900020 2000021 2100022 2200023 2300024 2400025
2500026 2600027 2700028 2800029 2900030 3000031 3100032 3200033
3300034 3400035 3500036 3600037 3700038 3800039 3900040 4000041
4100042 4200043 4300044 4400045 4500046 4600047 4700048 4800049
4900050 5000051 5100052 5200053 5300054 5400055 5500056 5600057
5700058 5800059 5900060 6000061 6100062 6200063 6300064 6400065
6500066 6600067 6700068 6800069 6900070 7000071 7100072 7200073
730007419DNAArtificial SequencePrimer 74tacttacttt gtcccttca
197519DNAArtificial SequencePrimer 75tatttgtccc catctcgtc
197628DNAArtificial SequencePrimer 76cggaattcat ggctaccgag agtactcc
287717DNAArtificial SequencePrimer 77gtaaaacgac ggccagt
177827DNAArtificial SequencePrimer 78gcagaagcca ctgtggaaga ggaggtt
277928DNAArtificial SequencePrimer 79atacgactca ctatagggcg aattggcc
2880Primer 8000081Primer 81000823030DNAHomo sapienCDS(1)...(1680)
82tgt gaa atg tgc tcg cag gag gct ttt cag gca cag agg agc cag ctg
48Cys Glu Met Cys Ser Gln Glu Ala Phe Gln Ala Gln Arg Ser Gln Leu1
5 10 15gtc gag ctg ctg gtc tca ggg tcc ctg gaa ggc ttc gag agt gtc
ctg 96Val Glu Leu Leu Val Ser Gly Ser Leu Glu Gly Phe Glu Ser Val
Leu 20 25 30gac tgg ctg ctg tcc tgg gag gtc ctc tcc tgg gag gac tac
gag ggc 144Asp Trp Leu Leu Ser Trp Glu Val Leu Ser Trp Glu Asp Tyr
Glu Gly 35 40 45ttc cac ctc ctg ggc cag cct ctc tcc cac ttg gcc agg
cgc ctt ctg 192Phe His Leu Leu Gly Gln Pro Leu Ser His Leu Ala Arg
Arg Leu Leu 50 55 60gac acc gtc tgg aat aag ggt act tgg gcc tgt cag
aag ctc atc gcg 240Asp Thr Val Trp Asn Lys Gly Thr Trp Ala Cys Gln
Lys Leu Ile Ala65 70 75 80gct gcc caa gaa gcc cag gcc gac agc cag
tcc ccc aag ctg cat ggc 288Ala Ala Gln Glu Ala Gln Ala Asp Ser Gln
Ser Pro Lys Leu His Gly 85 90 95tgc tgg gac ccc cac tcg ctc cac cca
gcc cga gac ctg cag agt cac 336Cys Trp Asp Pro His Ser Leu His Pro
Ala Arg Asp Leu Gln Ser His 100 105 110cgg cca gcc att gtc agg agg
ctc cac agc cat gtg gag aac atg ctg 384Arg Pro Ala Ile Val Arg Arg
Leu His Ser His Val Glu Asn Met Leu 115 120 125gac ctg gca tgg gag
cgg ggt ttc gtc agc cag tat gaa tgt gat gaa 432Asp Leu Ala Trp Glu
Arg Gly Phe Val Ser Gln Tyr Glu Cys Asp Glu 130 135 140atc agg ttg
ccg atc ttc aca ccg tcc cag agg gca aga agg ctg ctt 480Ile Arg Leu
Pro Ile Phe Thr Pro Ser Gln Arg Ala Arg Arg Leu Leu145 150 155
160gat ctt gcc acg gtg aaa gcg aat gga ttg gct gcc ttc ctt cta caa
528Asp Leu Ala Thr Val Lys Ala Asn Gly Leu Ala Ala Phe Leu Leu Gln
165 170 175cat gtt cag gaa tta cca gtc cca ttg gcc ctg cct ttg gaa
gct gcc 576His Val Gln Glu Leu Pro Val Pro Leu Ala Leu Pro Leu Glu
Ala Ala 180 185 190aca tgc aag aag tat atg gcc aag ctg agg acc acg
gtg tct gct cag 624Thr Cys Lys Lys Tyr Met Ala Lys Leu Arg Thr Thr
Val Ser Ala Gln 195 200 205tct cgc ttc ctc agt acc tat gat gga gca
gag acg ctc tgc ctg gag 672Ser Arg Phe Leu Ser Thr Tyr Asp Gly Ala
Glu Thr Leu Cys Leu Glu 210 215 220gac ata tac aca gag aat gtc ctg
gag gtc tgg gca gat gtg ggc atg 720Asp Ile Tyr Thr Glu Asn Val Leu
Glu Val Trp Ala Asp Val Gly Met225 230 235 240gct gga ccc ccg cag
aag agc cca gcc acc ctg ggc ctg gag gag ctc 768Ala Gly Pro Pro Gln
Lys Ser Pro Ala Thr Leu Gly Leu Glu Glu Leu 245 250 255ttc agc acc
cct ggc cac ctc aat gac gat gcg gac act gtg ctg gtg 816Phe Ser Thr
Pro Gly His Leu Asn Asp Asp Ala Asp Thr Val Leu Val 260 265 270gtg
ggt gag gcg ggc agt ggc aag agc acg ctc ctg cag cgg ctg cac 864Val
Gly Glu Ala Gly Ser Gly Lys Ser Thr Leu Leu Gln Arg Leu His 275 280
285ttg ctg tgg gct gca ggg caa gac ttc cag gaa ttt ctc ttt gtc ttc
912Leu Leu Trp Ala Ala Gly Gln Asp Phe Gln Glu Phe Leu Phe Val Phe
290 295 300cca ttc agc tgc cgg cag ctg cag tgc atg gcc aaa cca ctc
tct gtg 960Pro Phe Ser Cys Arg Gln Leu Gln Cys Met Ala Lys Pro Leu
Ser Val305 310 315 320cgg act cta ctc ttt gag cac tgc tgt tgg cct
gat gtt ggt caa gaa 1008Arg Thr Leu Leu Phe Glu His Cys Cys Trp Pro
Asp Val Gly Gln Glu 325 330 335gac atc ttc cag tta ctc ctt gac cac
cct gac cgt gtc ctg tta acc 1056Asp Ile Phe Gln Leu Leu Leu Asp His
Pro Asp Arg Val Leu Leu Thr 340 345 350ttt gat ggc ttt gac gag ttc
aag ttc agg ttc acg gat cgt gaa cgc 1104Phe Asp Gly Phe Asp Glu Phe
Lys Phe Arg Phe Thr Asp Arg Glu Arg 355 360 365cac tgc tcc ccg acc
gac ccc acc tct gtc cag acc ctg ctc ttc aac 1152His Cys Ser Pro Thr
Asp Pro Thr Ser Val Gln Thr Leu Leu Phe Asn 370 375 380ctt ctg cag
ggc aac ctg ctg aag aat gcc cgc aag gtg gtg acc agc 1200Leu Leu Gln
Gly Asn Leu Leu Lys Asn Ala Arg Lys Val Val Thr Ser385 390 395
400cgt ccg gcc gct gtg tcg gcg ttc ctc agg aag tac atc cgc acc gag
1248Arg Pro Ala Ala Val Ser Ala Phe Leu Arg Lys Tyr Ile Arg Thr Glu
405 410 415ttc aac ctc aag ggc ttc tct gaa cag ggc atc gag ctg tac
ctg agg 1296Phe Asn Leu Lys Gly Phe Ser Glu Gln Gly Ile Glu Leu Tyr
Leu Arg 420 425 430aag cgc cat cat gag ccc ggg gtg gcg gac cgc ctc
atc cgc ctg ctc 1344Lys Arg His His Glu Pro Gly Val Ala Asp Arg Leu
Ile Arg Leu Leu 435 440 445caa gag acc tca gcc ctg cac ggt ttg tgc
cac ctg cct gtc ttc tca 1392Gln Glu Thr Ser Ala Leu His Gly Leu Cys
His Leu Pro Val Phe Ser 450 455 460tgg atg gtg tcc aaa tgc cac cag
gaa ctg ttg ctg cag gag ggg ggg 1440Trp Met Val Ser Lys Cys His Gln
Glu Leu Leu Leu Gln Glu Gly Gly465 470 475 480tcc cca aag acc act
aca gat atg tac ctg ctg att ctg cag cat ttt 1488Ser Pro Lys Thr Thr
Thr Asp Met Tyr Leu Leu Ile Leu Gln His Phe 485 490 495ctg ctg cat
gcc acc ccc cca gac tca gct tcc caa ggt ctg gga ccc 1536Leu Leu His
Ala Thr Pro Pro Asp Ser Ala Ser Gln Gly Leu Gly Pro 500 505 510agt
ctt ctt cgg ggc cgc ctc ccc acc ctc ctg cac ctg ggc aga ctg 1584Ser
Leu Leu Arg Gly Arg Leu Pro Thr Leu Leu His Leu Gly Arg Leu 515 520
525gct ctg tgg ggc ctg ggc atg tgc tgc tac gtg ttc tca gcc cag cag
1632Ala Leu Trp Gly Leu Gly Met Cys Cys Tyr Val Phe Ser Ala Gln Gln
530 535 540ctc cag gca gca cag gtc agc cct gat gac att tct ctt ggc
ttc ctg 1680Leu Gln Ala Ala Gln Val Ser Pro Asp Asp Ile Ser Leu Gly
Phe Leu545 550 555 560gtgcgtgcca aaggtgtcgt gccagggagt acggcgcccc
tggaattcct tcacatcact 1740ttccagtgct tctttgccgc gttctacctg
gcactcagtg ctgatgtgcc accagctttg 1800ctcagacacc tcttcaattg
tggcaggcca ggcaactcac caatggccag gctcctgccc 1860acgatgtgca
tccaggcctc ggagggaaag gacagcagcg tggcagcttt gctgcagaag
1920gccgagccgc acaaccttca gatcacagca gccttcctgg cagggctgtt
gtcccgggag 1980cactggggcc tgctggctga gtgccagaca tctgagaagg
ccctgctccg gcgccaggcc 2040tgtgcccgct ggtgtctggc ccgcagcctc
cgcaagcact tccactccat cccgccagct 2100gcaccgggtg aggccaagag
cgtgcatgcc atgcccgggt tcatctggct catccggagc 2160ctgtacgaga
tgcaggagga gcggctggct cggaaggctg cacgtggcct gaatgttggg
2220cacctcaagt tgacattttg cagtgtgggc cccactgagt gtgctgccct
ggcctttgtg 2280ctgcagcacc tccggcggcc cgtggccctg cagctggact
acaactctgt gggtgacatt 2340ggcgtggagc agctgctgcc ttgccttggt
gtctgcaagg ctctgtattt gcgcgataac 2400aatatctcag accgaggcat
ctgcaagctc attgaatgtg ctcttcactg cgagcaattg 2460cagaagttag
cgctggggaa taactacatc actgccgcgg gagcccaagt gctggccgag
2520gggctccgag gcaacacctc cttgcagttc ctgggattct ggggcaacag
agtgggtgac 2580gagggggccc aggccctggc tgaagccttg ggtgatcacc
agagcttgag gtggctcagc 2640ctggtgggga acaacattgg cagtgtgggt
gcccaagcct tggcactgat gctggcaaag 2700aacgtcatgc tagaagaact
ctgcctggag gagaaccatc tccaggatga aggtgtatgt 2760tctctcgcag
aaggactgaa gaaaaattca agtttgaaaa tcctgaacat aaaaattcat
2820gcttcgggat tcaacaaact cttggaaagc attttctgca tcctcctggt
tgtggaagca 2880tttttcctgc agaaagttgt caagattctt gaagaaatgg
tagtcagttg gctagaggtc 2940aggttgtcca ataactgcat cacctaccta
ggggcagaag ccctcctgca ggcccttgaa 3000aggaatgaca ccatcctgga
agtctggtaa 303083560PRTHomo sapien 83Cys Glu Met Cys Ser Gln Glu
Ala Phe Gln Ala Gln Arg Ser Gln Leu1 5 10 15Val Glu Leu Leu Val Ser
Gly Ser Leu Glu Gly Phe Glu Ser Val Leu 20 25 30Asp Trp Leu Leu Ser
Trp Glu Val Leu Ser Trp Glu Asp Tyr Glu Gly 35 40 45Phe His Leu Leu
Gly Gln Pro Leu Ser His Leu Ala Arg Arg Leu Leu 50 55 60Asp Thr Val
Trp Asn Lys Gly Thr Trp Ala Cys Gln Lys Leu Ile Ala65 70 75 80Ala
Ala Gln Glu Ala Gln Ala Asp Ser Gln Ser Pro Lys Leu His Gly 85 90
95Cys Trp Asp Pro His Ser Leu His Pro Ala Arg Asp Leu Gln Ser His
100 105 110Arg Pro Ala Ile Val Arg Arg Leu His Ser His Val Glu Asn
Met Leu 115 120 125Asp Leu Ala Trp Glu Arg Gly Phe Val Ser Gln Tyr
Glu Cys Asp Glu 130 135 140Ile Arg Leu Pro Ile Phe Thr Pro Ser Gln
Arg Ala Arg Arg Leu Leu145 150 155 160Asp Leu Ala Thr Val Lys Ala
Asn Gly Leu Ala Ala Phe Leu Leu Gln 165 170 175His Val Gln Glu Leu
Pro Val Pro Leu Ala Leu Pro Leu Glu Ala Ala 180 185 190Thr Cys Lys
Lys Tyr Met Ala Lys Leu Arg Thr Thr Val Ser Ala Gln 195 200 205Ser
Arg Phe Leu Ser Thr Tyr Asp Gly Ala Glu Thr Leu Cys Leu Glu 210 215
220Asp Ile Tyr Thr Glu Asn Val Leu Glu Val Trp Ala Asp Val Gly
Met225 230 235 240Ala Gly Pro Pro Gln Lys Ser Pro Ala Thr Leu Gly
Leu Glu Glu Leu 245 250 255Phe Ser Thr Pro Gly His Leu Asn Asp Asp
Ala Asp Thr Val Leu Val 260 265 270Val Gly Glu Ala Gly Ser Gly Lys
Ser Thr Leu Leu Gln Arg Leu His 275 280 285Leu Leu Trp Ala Ala Gly
Gln Asp Phe Gln Glu Phe Leu Phe Val Phe 290 295 300Pro Phe Ser Cys
Arg Gln Leu Gln Cys Met Ala Lys Pro Leu Ser Val305 310 315 320Arg
Thr Leu Leu Phe Glu His Cys Cys Trp Pro Asp Val Gly Gln Glu 325
330 335Asp Ile Phe Gln Leu Leu Leu Asp His Pro Asp Arg Val Leu Leu
Thr 340 345 350Phe Asp Gly Phe Asp Glu Phe Lys Phe Arg Phe Thr Asp
Arg Glu Arg 355 360 365His Cys Ser Pro Thr Asp Pro Thr Ser Val Gln
Thr Leu Leu Phe Asn 370 375 380Leu Leu Gln Gly Asn Leu Leu Lys Asn
Ala Arg Lys Val Val Thr Ser385 390 395 400Arg Pro Ala Ala Val Ser
Ala Phe Leu Arg Lys Tyr Ile Arg Thr Glu 405 410 415Phe Asn Leu Lys
Gly Phe Ser Glu Gln Gly Ile Glu Leu Tyr Leu Arg 420 425 430Lys Arg
His His Glu Pro Gly Val Ala Asp Arg Leu Ile Arg Leu Leu 435 440
445Gln Glu Thr Ser Ala Leu His Gly Leu Cys His Leu Pro Val Phe Ser
450 455 460Trp Met Val Ser Lys Cys His Gln Glu Leu Leu Leu Gln Glu
Gly Gly465 470 475 480Ser Pro Lys Thr Thr Thr Asp Met Tyr Leu Leu
Ile Leu Gln His Phe 485 490 495Leu Leu His Ala Thr Pro Pro Asp Ser
Ala Ser Gln Gly Leu Gly Pro 500 505 510Ser Leu Leu Arg Gly Arg Leu
Pro Thr Leu Leu His Leu Gly Arg Leu 515 520 525Ala Leu Trp Gly Leu
Gly Met Cys Cys Tyr Val Phe Ser Ala Gln Gln 530 535 540Leu Gln Ala
Ala Gln Val Ser Pro Asp Asp Ile Ser Leu Gly Phe Leu545 550 555
560841107DNAHomo sapien 84attctttttt taacttttac ttattcatta
ggatgatttc ataatatatt tcctggttta 60gaggaaacag gaacaatggc taccgagagt
actccctcag agatcataga aagagaaaga 120aaaaagttgc ttgaaatcct
tcaacatgat cctgattcta tcttagacac gttaacttct 180cggaggctga
tttctgagga agagtatgag actctggaga atgttacaga tctcctgaag
240aaaagtcgga agctgttaat tttggtacag aaaaagggag aggcgacctg
tcagcatttt 300ctcaagtgtt tatttagtac ttttccacag tcagctgcca
tttgcggctt aaggcatgaa 360gttttaaaac atgagaatac agtacctcct
caatctatgg gggcaagcag taattcagaa 420gatgcttttt ctcctggaat
aaaacagcct gaagcccctg agatcacagt gttcttcagt 480gagaaggaac
acttggattt ggaaacctct gagtttttca gggacaagaa aactagttat
540agggaaacag ctttgtctgc caggaagaat gagaaggaat atgacacacc
agaagtcaca 600ttatcatatt cagttgagaa agttggatgt gaagttccag
caactattac atatataaaa 660gatggacaga gatatgagga gctagatgat
tctttatact taggaaaaga ggaatatcta 720ggatctgttg acacccctga
agatgcagaa gccactgtgg aagaggaggt ttatgatgac 780ccagagcacg
ttggatatga tggtgaagag gacttcgaga attcagaaac cacagagttc
840tctggtgaag aaccaagtta tgagggatca gaaaccagcc tttcattgga
ggaggaacag 900gagaaaagta tagaaggctg gtctcgaact catgggctta
agcgatcctc ccacgttggc 960ctcccaaagt gctgggatta caggcgtgag
ccaccctgcc tggcctgaaa attctgcctc 1020aaacatctca aacatccatt
tatattttgt acaagaaagt aaataaaatt tttcttttta 1080acattaaaaa
aaaaaaaaaa aaaaaaa 110785510DNAHomo sapienCDS(15)...(305)
85aggagagaaa agcc atg gcc gac aag gtc ctg aag gag aag aga aag ctg
50 Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu 1 5 10ttt atc
cat tcc atg ggt gaa ggt aca ata aat ggc tta ctg gat gaa 98Phe Ile
His Ser Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu 15 20 25tta
tta cag aca agg gtg ctg aac cag gaa gag atg gag aaa gta aaa 146Leu
Leu Gln Thr Arg Val Leu Asn Gln Glu Glu Met Glu Lys Val Lys 30 35
40cgt gaa aat gct aca gtt atg gat aag acc cga gct ttg att gac tcc
194Arg Glu Asn Ala Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp
Ser45 50 55 60gtt att ccg aaa ggg gca cag gca tgc caa att tgc atc
aca tac att 242Val Ile Pro Lys Gly Ala Gln Ala Cys Gln Ile Cys Ile
Thr Tyr Ile 65 70 75tgt gaa gaa gac agt tac ctg gca gag acg ctg gga
ctc tca gca ggt 290Cys Glu Glu Asp Ser Tyr Leu Ala Glu Thr Leu Gly
Leu Ser Ala Gly 80 85 90ccg ata cct gga aat tagcttagct tagtacacaa
gactcccaat tactattttc 345Pro Ile Pro Gly Asn 95ttccttccca
gctcttcagg cagtgcagga caacccagct atgcccacat gctcaagccc
405agaaggcaga atcaagcttt gctttctaga agacgctcaa aggatatgga
aacaaaagtt 465gcagaggtgc catgttcaga atacaataat aaagtggagt aaaga
5108697PRTHomo sapien 86Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys
Leu Phe Ile His Ser1 5 10 15Met Gly Glu Gly Thr Ile Asn Gly Leu Leu
Asp Glu Leu Leu Gln Thr 20 25 30Arg Val Leu Asn Gln Glu Glu Met Glu
Lys Val Lys Arg Glu Asn Ala 35 40 45Thr Val Met Asp Lys Thr Arg Ala
Leu Ile Asp Ser Val Ile Pro Lys 50 55 60Gly Ala Gln Ala Cys Gln Ile
Cys Ile Thr Tyr Ile Cys Glu Glu Asp65 70 75 80Ser Tyr Leu Ala Glu
Thr Leu Gly Leu Ser Ala Gly Pro Ile Pro Gly 85 90 95Asn8797PRTHomo
sapien 87Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile
Arg Ser1 5 10 15Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu
Leu Gln Thr 20 25 30Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val Lys
Arg Glu Asn Ala 35 40 45Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp
Ser Val Ile Pro Lys 50 55 60Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr
Tyr Ile Cys Glu Glu Asp65 70 75 80Ser Tyr Leu Ala Gly Thr Leu Gly
Leu Ser Ala Asp Gln Thr Ser Gly 85 90 95Asn88 8800089321DNAHomo
sapienCDS(1)...(318) 89atg atc ctc ctg aag aag aga cgg ctg ctt atc
aat tca ttg ggt gaa 48Met Ile Leu Leu Lys Lys Arg Arg Leu Leu Ile
Asn Ser Leu Gly Glu1 5 10 15ggt aca ata aat ggc tta ctg gat gaa tta
ttg gag aca aat gtg ctg 96Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu
Leu Glu Thr Asn Val Leu 20 25 30agc cag gaa gac aca gag ata gta aaa
tgt gaa aat gtt aca gtt atc 144Ser Gln Glu Asp Thr Glu Ile Val Lys
Cys Glu Asn Val Thr Val Ile 35 40 45gat aag gcc cga gat ttg ctt gac
tct gtt att cgg aaa ggg gca ggg 192Asp Lys Ala Arg Asp Leu Leu Asp
Ser Val Ile Arg Lys Gly Ala Gly 50 55 60gca tgt gaa att tgc atc aca
tac att tgt gaa gaa gac agg tac ctg 240Ala Cys Glu Ile Cys Ile Thr
Tyr Ile Cys Glu Glu Asp Arg Tyr Leu65 70 75 80gca ggg acg ctg gga
ctc tca gca gga aat gac tac aga gct gga ggc 288Ala Gly Thr Leu Gly
Leu Ser Ala Gly Asn Asp Tyr Arg Ala Gly Gly 85 90 95att tgc tca ccg
ccc aga gca caa gac ctc tga 321Ile Cys Ser Pro Pro Arg Ala Gln Asp
Leu 100 10590106PRTHomo sapien 90Met Ile Leu Leu Lys Lys Arg Arg
Leu Leu Ile Asn Ser Leu Gly Glu1 5 10 15Gly Thr Ile Asn Gly Leu Leu
Asp Glu Leu Leu Glu Thr Asn Val Leu 20 25 30Ser Gln Glu Asp Thr Glu
Ile Val Lys Cys Glu Asn Val Thr Val Ile 35 40 45Asp Lys Ala Arg Asp
Leu Leu Asp Ser Val Ile Arg Lys Gly Ala Gly 50 55 60Ala Cys Glu Ile
Cys Ile Thr Tyr Ile Cys Glu Glu Asp Arg Tyr Leu65 70 75 80Ala Gly
Thr Leu Gly Leu Ser Ala Gly Asn Asp Tyr Arg Ala Gly Gly 85 90 95Ile
Cys Ser Pro Pro Arg Ala Gln Asp Leu 100 10591108PRTHomo sapien
91Met Ala Asp Lys Val Leu Leu Glu Lys Arg Lys Leu Leu Ile Asn Ser1
5 10 15Leu Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Glu
Thr 20 25 30Asn Val Leu Ser Gln Glu Asp Glu Ile Val Lys Arg Glu Asn
Ala Thr 35 40 45Val Ile Asp Lys Ala Arg Ala Leu Leu Asp Ser Val Ile
Arg Lys Gly 50 55 60Ala Gly Ala Cys Glu Ile Cys Ile Thr Tyr Ile Cys
Glu Glu Asp Ser65 70 75 80Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala
Gly Asn Ala Val Gln Ala 85 90 95Gly Gly Ala Cys Ser Thr Ser Ser Gly
Gln Asp Leu 100 10592 9200093 9300094 9400095 95000963396DNAHomo
sapienCDS(277)...(3348) 96cgcccgggca ggtgtttata ctccggaggg
tgtccccgtg cgtcatcggt ggagtggacc 60aaaactggtg atctgtttgc cctgtgtgac
cttgcccaga accctgctga ctgagagaac 120acatctgctg gaagtcctct
gggattcaag gtacagggaa tgaagagtag ttttacagaa 180aaaagaggac
aatattggga tcacctttga cctttccatt tggaaataat attttctatt
240gtgttataga aaggtgggaa gctttcatcc agaaca atg aat ttc ata aag gac
294 Met Asn Phe Ile Lys Asp 1 5aat agc cga gcc ctt att caa aga atg
gga atg act gtt ata aag caa 342Asn Ser Arg Ala Leu Ile Gln Arg Met
Gly Met Thr Val Ile Lys Gln 10 15 20atc aca gat gac cta ttt gta tgg
aat gtt ctg aat cgc gaa gaa gta 390Ile Thr Asp Asp Leu Phe Val Trp
Asn Val Leu Asn Arg Glu Glu Val 25 30 35aac atc att tgc tgc gag aag
gtg gag cag gat gct gct aga ggg atc 438Asn Ile Ile Cys Cys Glu Lys
Val Glu Gln Asp Ala Ala Arg Gly Ile 40 45 50att cac atg att ttg aaa
aag ggt tca gag tcc tgt aac ctc ttt ctt 486Ile His Met Ile Leu Lys
Lys Gly Ser Glu Ser Cys Asn Leu Phe Leu55 60 65 70aaa tcc ctt aag
gag tgg aac tat cct cta ttt cag gac ttg aat gga 534Lys Ser Leu Lys
Glu Trp Asn Tyr Pro Leu Phe Gln Asp Leu Asn Gly 75 80 85caa agt ctt
ttt cat cag aca tca gaa gga gac ttg gac gat ttg gct 582Gln Ser Leu
Phe His Gln Thr Ser Glu Gly Asp Leu Asp Asp Leu Ala 90 95 100cag
gat tta aag gac ttg tac cat acc cca tct ttt ctg aac ttt tat 630Gln
Asp Leu Lys Asp Leu Tyr His Thr Pro Ser Phe Leu Asn Phe Tyr 105 110
115ccc ctt ggt gaa gat att gac att att ttt aac ttg aaa agc acc ttc
678Pro Leu Gly Glu Asp Ile Asp Ile Ile Phe Asn Leu Lys Ser Thr Phe
120 125 130aca gaa cct atc ctg tgg agg aag gac caa cac cat cac cgc
gtg gag 726Thr Glu Pro Ile Leu Trp Arg Lys Asp Gln His His His Arg
Val Glu135 140 145 150cag ctg acc ctg aat ggc ctc ctg cag gct ctt
cag agc ccc tgc atc 774Gln Leu Thr Leu Asn Gly Leu Leu Gln Ala Leu
Gln Ser Pro Cys Ile 155 160 165att gaa ggg gaa tct ggc aaa ggc aag
tcc act ctg ctg cag cgc att 822Ile Glu Gly Glu Ser Gly Lys Gly Lys
Ser Thr Leu Leu Gln Arg Ile 170 175 180gcc atg ctc tgg ggc tcc gga
aag tgc aag gct ctg acc aag ttc aaa 870Ala Met Leu Trp Gly Ser Gly
Lys Cys Lys Ala Leu Thr Lys Phe Lys 185 190 195ttc gtc ttc ttc ctc
cgt ctc agc agg gcc cag ggt gga ctt ttt gaa 918Phe Val Phe Phe Leu
Arg Leu Ser Arg Ala Gln Gly Gly Leu Phe Glu 200 205 210acc ctc tgt
gat caa ctc ctg gat ata cct ggc aca atc agg aag cag 966Thr Leu Cys
Asp Gln Leu Leu Asp Ile Pro Gly Thr Ile Arg Lys Gln215 220 225
230aca ttc atg gcc atg ctg ctg aag ctg cgg cag agg gtt ctt ttc ctt
1014Thr Phe Met Ala Met Leu Leu Lys Leu Arg Gln Arg Val Leu Phe Leu
235 240 245ctt gat ggc tac aat gaa ttc aag ccc cag aac tgc cca gaa
atc gaa 1062Leu Asp Gly Tyr Asn Glu Phe Lys Pro Gln Asn Cys Pro Glu
Ile Glu 250 255 260gcc ctg ata aag gaa aac cac cgc ttc aag aac atg
gtc atc gtc acc 1110Ala Leu Ile Lys Glu Asn His Arg Phe Lys Asn Met
Val Ile Val Thr 265 270 275act acc act gag tgc ctg agg cac ata cgg
cag ttt ggt gcc ctg act 1158Thr Thr Thr Glu Cys Leu Arg His Ile Arg
Gln Phe Gly Ala Leu Thr 280 285 290gct gag gtg ggg gat atg aca gaa
gac agc gcc cag gct ctc atc cga 1206Ala Glu Val Gly Asp Met Thr Glu
Asp Ser Ala Gln Ala Leu Ile Arg295 300 305 310gaa gtg ctg atc aag
gag ctt gct gaa ggc ttg ttg ctc caa att cag 1254Glu Val Leu Ile Lys
Glu Leu Ala Glu Gly Leu Leu Leu Gln Ile Gln 315 320 325aaa tcc agg
tgc ttg agg aat ctc atg aag acc cct ctc ttt gtg gtc 1302Lys Ser Arg
Cys Leu Arg Asn Leu Met Lys Thr Pro Leu Phe Val Val 330 335 340atc
act tgt gca atc cag atg ggt gaa agt gag ttc cac tct cac aca 1350Ile
Thr Cys Ala Ile Gln Met Gly Glu Ser Glu Phe His Ser His Thr 345 350
355caa aca acg ctg ttc cat acc ttc tat gat ctg ttg ata cag aaa aac
1398Gln Thr Thr Leu Phe His Thr Phe Tyr Asp Leu Leu Ile Gln Lys Asn
360 365 370aaa cac aaa cat aaa ggt gtg gct gca agt gac ttc att cgg
agc ctg 1446Lys His Lys His Lys Gly Val Ala Ala Ser Asp Phe Ile Arg
Ser Leu375 380 385 390gac cac cgt gga gac cta gct ctg gag ggt gtg
ttc tcc cac aag ttt 1494Asp His Arg Gly Asp Leu Ala Leu Glu Gly Val
Phe Ser His Lys Phe 395 400 405gat ttc gaa ctg cag gat gtg tcc agc
gtg aat gag gat gtc ctg ctg 1542Asp Phe Glu Leu Gln Asp Val Ser Ser
Val Asn Glu Asp Val Leu Leu 410 415 420aca act ggg ctc ctc tgt aaa
tat aca gct caa agg ttc aag cca aag 1590Thr Thr Gly Leu Leu Cys Lys
Tyr Thr Ala Gln Arg Phe Lys Pro Lys 425 430 435tat aaa ttc ttt cac
aag tca ttc cag gag tac aca gca gga cga aga 1638Tyr Lys Phe Phe His
Lys Ser Phe Gln Glu Tyr Thr Ala Gly Arg Arg 440 445 450ctc agc agt
tta ttg acg tct cat gag cca gag gag gtg acc aag ggg 1686Leu Ser Ser
Leu Leu Thr Ser His Glu Pro Glu Glu Val Thr Lys Gly455 460 465
470aat ggt tac ttg cag aaa atg gtt tcc att tcg gac att aca tcc act
1734Asn Gly Tyr Leu Gln Lys Met Val Ser Ile Ser Asp Ile Thr Ser Thr
475 480 485tat agc agc ctg ctc cgg tac acc tgt ggg tca tct gtg gaa
gcc acc 1782Tyr Ser Ser Leu Leu Arg Tyr Thr Cys Gly Ser Ser Val Glu
Ala Thr 490 495 500agg gct gtt atg aag cac ctc gca gca gtg tat caa
cac ggc tgc ctt 1830Arg Ala Val Met Lys His Leu Ala Ala Val Tyr Gln
His Gly Cys Leu 505 510 515ctc gga ctt tcc atc gcc aag agg cct ctc
tgg aga cag gaa tct ttg 1878Leu Gly Leu Ser Ile Ala Lys Arg Pro Leu
Trp Arg Gln Glu Ser Leu 520 525 530caa agt gtg aaa aac acc act gag
caa gaa att ctg aaa gcc ata aac 1926Gln Ser Val Lys Asn Thr Thr Glu
Gln Glu Ile Leu Lys Ala Ile Asn535 540 545 550atc aat tcc ttt gta
gag tgt ggc atc cat tta tat caa gag agt aca 1974Ile Asn Ser Phe Val
Glu Cys Gly Ile His Leu Tyr Gln Glu Ser Thr 555 560 565tcc aaa tca
gcc ctg agc caa gaa ttt gaa gct ttc ttt caa ggt aaa 2022Ser Lys Ser
Ala Leu Ser Gln Glu Phe Glu Ala Phe Phe Gln Gly Lys 570 575 580agc
tta tat atc aac tca ggg aac atc ccc gat tac tta ttt gac ttc 2070Ser
Leu Tyr Ile Asn Ser Gly Asn Ile Pro Asp Tyr Leu Phe Asp Phe 585 590
595ttt gaa cat ttg ccc aat tgt gca agt gcc ctg gac ttc att aaa ctg
2118Phe Glu His Leu Pro Asn Cys Ala Ser Ala Leu Asp Phe Ile Lys Leu
600 605 610gac ttt tat ggg gga gct atg gct tca tgg gaa aag gct gca
gaa gac 2166Asp Phe Tyr Gly Gly Ala Met Ala Ser Trp Glu Lys Ala Ala
Glu Asp615 620 625 630aca ggt gga atc cac atg gaa gag gcc cca gaa
acc tac att ccc agc 2214Thr Gly Gly Ile His Met Glu Glu Ala Pro Glu
Thr Tyr Ile Pro Ser 635 640 645agg gct gta tct ttg ttc ttc aac tgg
aag cag gaa ttc agg act ctg 2262Arg Ala Val Ser Leu Phe Phe Asn Trp
Lys Gln Glu Phe Arg Thr Leu 650 655 660gag gtc aca ctc cgg gat ttc
agc aag ttg aat aag caa gat atc aga 2310Glu Val Thr Leu Arg Asp Phe
Ser Lys Leu Asn Lys Gln Asp Ile Arg 665 670 675tat ctg ggg aaa ata
ttc agc tct gcc aca agc ctc agg ctg caa ata 2358Tyr Leu Gly Lys Ile
Phe Ser Ser Ala Thr Ser Leu Arg Leu Gln Ile 680 685 690aag aga tgt
gct ggt gtg gct gga agc ctc agt ttg gtc ctc agc acc 2406Lys Arg Cys
Ala Gly Val Ala Gly Ser Leu Ser Leu Val Leu Ser Thr695 700 705
710tgt aag aac att tat tct ctc atg gtg gaa gcc agt ccc ctc acc ata
2454Cys Lys Asn Ile Tyr Ser Leu Met Val Glu Ala Ser Pro Leu Thr Ile
715 720 725gaa gat gag agg cac atc aca tct gta aca aac ctg aaa acc
ttg agt 2502Glu Asp Glu Arg His Ile Thr Ser Val Thr Asn Leu Lys Thr
Leu Ser 730 735
740att cat gac cta cag aat caa cgg ctg ccg ggt ggt ctg act gac agc
2550Ile His Asp Leu Gln Asn Gln Arg Leu Pro Gly Gly Leu Thr Asp Ser
745 750 755ttg ggt aac ttg aag aac ctt aca aag ctc ata atg gat aac
ata aag 2598Leu Gly Asn Leu Lys Asn Leu Thr Lys Leu Ile Met Asp Asn
Ile Lys 760 765 770atg aat gaa gaa gat gct ata aaa cta gct gaa ggc
ctg aaa aac ctg 2646Met Asn Glu Glu Asp Ala Ile Lys Leu Ala Glu Gly
Leu Lys Asn Leu775 780 785 790aag aag atg tgt tta ttt cat ttg acc
cac ttg tct gac att gga gag 2694Lys Lys Met Cys Leu Phe His Leu Thr
His Leu Ser Asp Ile Gly Glu 795 800 805gga atg gat tac ata gtc aag
tct ctg tca agt gaa ccc tgt gac ctt 2742Gly Met Asp Tyr Ile Val Lys
Ser Leu Ser Ser Glu Pro Cys Asp Leu 810 815 820gaa gaa att caa tta
gtc tcc tgc tgc ttg tct gca aat gca gtg aaa 2790Glu Glu Ile Gln Leu
Val Ser Cys Cys Leu Ser Ala Asn Ala Val Lys 825 830 835atc cta gct
cag aat ctt cac aat ttg gtc aaa ctg agc att ctt gat 2838Ile Leu Ala
Gln Asn Leu His Asn Leu Val Lys Leu Ser Ile Leu Asp 840 845 850tta
tca gaa aat tac ctg gaa aaa gat gga aat gaa gct ctt cat gaa 2886Leu
Ser Glu Asn Tyr Leu Glu Lys Asp Gly Asn Glu Ala Leu His Glu855 860
865 870ctg atc gac agg atg aac gtg cta gaa cag ctc acc gca ctg atg
ctg 2934Leu Ile Asp Arg Met Asn Val Leu Glu Gln Leu Thr Ala Leu Met
Leu 875 880 885ccc tgg ggc tgt gac gtg caa ggc agc ctg agc agc ctg
ttg aaa cat 2982Pro Trp Gly Cys Asp Val Gln Gly Ser Leu Ser Ser Leu
Leu Lys His 890 895 900ttg gag gag gtc cca caa ctc gtc aag ctt ggg
ttg aaa aac tgg aga 3030Leu Glu Glu Val Pro Gln Leu Val Lys Leu Gly
Leu Lys Asn Trp Arg 905 910 915ctc aca gat aca gag att aga att tta
ggt gca ttt ttt gga aag aac 3078Leu Thr Asp Thr Glu Ile Arg Ile Leu
Gly Ala Phe Phe Gly Lys Asn 920 925 930cct ctg aaa aac ttc cag cag
ttg aat ttg gcg gga aat cgt gtg agc 3126Pro Leu Lys Asn Phe Gln Gln
Leu Asn Leu Ala Gly Asn Arg Val Ser935 940 945 950agt gat gga tgg
ctt gcc ttc atg ggt gta ttt gag aat ctt aag caa 3174Ser Asp Gly Trp
Leu Ala Phe Met Gly Val Phe Glu Asn Leu Lys Gln 955 960 965tta gtg
ttt ttt gac ttt agt act aaa gaa ttt cta cct gat cca gca 3222Leu Val
Phe Phe Asp Phe Ser Thr Lys Glu Phe Leu Pro Asp Pro Ala 970 975
980tta gtc aga aaa ctt agc caa gtg tta tcc aag tta act ttt ctg caa
3270Leu Val Arg Lys Leu Ser Gln Val Leu Ser Lys Leu Thr Phe Leu Gln
985 990 995gaa gct agg ctt gtt ggg tgg caa ttt gat gat gat gat ctc
agt gtt 3318Glu Ala Arg Leu Val Gly Trp Gln Phe Asp Asp Asp Asp Leu
Ser Val 1000 1005 1010att aca ggt gct ttt aaa cta gta act gct
taaataaagt gtactcgaag 3368Ile Thr Gly Ala Phe Lys Leu Val Thr
Ala1015 1020ccaaaaaaaa aaaaaaaaaa aaaaaaaa 3396971024PRTHomo sapien
97Met Asn Phe Ile Lys Asp Asn Ser Arg Ala Leu Ile Gln Arg Met Gly1
5 10 15Met Thr Val Ile Lys Gln Ile Thr Asp Asp Leu Phe Val Trp Asn
Val 20 25 30Leu Asn Arg Glu Glu Val Asn Ile Ile Cys Cys Glu Lys Val
Glu Gln 35 40 45Asp Ala Ala Arg Gly Ile Ile His Met Ile Leu Lys Lys
Gly Ser Glu 50 55 60Ser Cys Asn Leu Phe Leu Lys Ser Leu Lys Glu Trp
Asn Tyr Pro Leu65 70 75 80Phe Gln Asp Leu Asn Gly Gln Ser Leu Phe
His Gln Thr Ser Glu Gly 85 90 95Asp Leu Asp Asp Leu Ala Gln Asp Leu
Lys Asp Leu Tyr His Thr Pro 100 105 110Ser Phe Leu Asn Phe Tyr Pro
Leu Gly Glu Asp Ile Asp Ile Ile Phe 115 120 125Asn Leu Lys Ser Thr
Phe Thr Glu Pro Ile Leu Trp Arg Lys Asp Gln 130 135 140His His His
Arg Val Glu Gln Leu Thr Leu Asn Gly Leu Leu Gln Ala145 150 155
160Leu Gln Ser Pro Cys Ile Ile Glu Gly Glu Ser Gly Lys Gly Lys Ser
165 170 175Thr Leu Leu Gln Arg Ile Ala Met Leu Trp Gly Ser Gly Lys
Cys Lys 180 185 190Ala Leu Thr Lys Phe Lys Phe Val Phe Phe Leu Arg
Leu Ser Arg Ala 195 200 205Gln Gly Gly Leu Phe Glu Thr Leu Cys Asp
Gln Leu Leu Asp Ile Pro 210 215 220Gly Thr Ile Arg Lys Gln Thr Phe
Met Ala Met Leu Leu Lys Leu Arg225 230 235 240Gln Arg Val Leu Phe
Leu Leu Asp Gly Tyr Asn Glu Phe Lys Pro Gln 245 250 255Asn Cys Pro
Glu Ile Glu Ala Leu Ile Lys Glu Asn His Arg Phe Lys 260 265 270Asn
Met Val Ile Val Thr Thr Thr Thr Glu Cys Leu Arg His Ile Arg 275 280
285Gln Phe Gly Ala Leu Thr Ala Glu Val Gly Asp Met Thr Glu Asp Ser
290 295 300Ala Gln Ala Leu Ile Arg Glu Val Leu Ile Lys Glu Leu Ala
Glu Gly305 310 315 320Leu Leu Leu Gln Ile Gln Lys Ser Arg Cys Leu
Arg Asn Leu Met Lys 325 330 335Thr Pro Leu Phe Val Val Ile Thr Cys
Ala Ile Gln Met Gly Glu Ser 340 345 350Glu Phe His Ser His Thr Gln
Thr Thr Leu Phe His Thr Phe Tyr Asp 355 360 365Leu Leu Ile Gln Lys
Asn Lys His Lys His Lys Gly Val Ala Ala Ser 370 375 380Asp Phe Ile
Arg Ser Leu Asp His Arg Gly Asp Leu Ala Leu Glu Gly385 390 395
400Val Phe Ser His Lys Phe Asp Phe Glu Leu Gln Asp Val Ser Ser Val
405 410 415Asn Glu Asp Val Leu Leu Thr Thr Gly Leu Leu Cys Lys Tyr
Thr Ala 420 425 430Gln Arg Phe Lys Pro Lys Tyr Lys Phe Phe His Lys
Ser Phe Gln Glu 435 440 445Tyr Thr Ala Gly Arg Arg Leu Ser Ser Leu
Leu Thr Ser His Glu Pro 450 455 460Glu Glu Val Thr Lys Gly Asn Gly
Tyr Leu Gln Lys Met Val Ser Ile465 470 475 480Ser Asp Ile Thr Ser
Thr Tyr Ser Ser Leu Leu Arg Tyr Thr Cys Gly 485 490 495Ser Ser Val
Glu Ala Thr Arg Ala Val Met Lys His Leu Ala Ala Val 500 505 510Tyr
Gln His Gly Cys Leu Leu Gly Leu Ser Ile Ala Lys Arg Pro Leu 515 520
525Trp Arg Gln Glu Ser Leu Gln Ser Val Lys Asn Thr Thr Glu Gln Glu
530 535 540Ile Leu Lys Ala Ile Asn Ile Asn Ser Phe Val Glu Cys Gly
Ile His545 550 555 560Leu Tyr Gln Glu Ser Thr Ser Lys Ser Ala Leu
Ser Gln Glu Phe Glu 565 570 575Ala Phe Phe Gln Gly Lys Ser Leu Tyr
Ile Asn Ser Gly Asn Ile Pro 580 585 590Asp Tyr Leu Phe Asp Phe Phe
Glu His Leu Pro Asn Cys Ala Ser Ala 595 600 605Leu Asp Phe Ile Lys
Leu Asp Phe Tyr Gly Gly Ala Met Ala Ser Trp 610 615 620Glu Lys Ala
Ala Glu Asp Thr Gly Gly Ile His Met Glu Glu Ala Pro625 630 635
640Glu Thr Tyr Ile Pro Ser Arg Ala Val Ser Leu Phe Phe Asn Trp Lys
645 650 655Gln Glu Phe Arg Thr Leu Glu Val Thr Leu Arg Asp Phe Ser
Lys Leu 660 665 670Asn Lys Gln Asp Ile Arg Tyr Leu Gly Lys Ile Phe
Ser Ser Ala Thr 675 680 685Ser Leu Arg Leu Gln Ile Lys Arg Cys Ala
Gly Val Ala Gly Ser Leu 690 695 700Ser Leu Val Leu Ser Thr Cys Lys
Asn Ile Tyr Ser Leu Met Val Glu705 710 715 720Ala Ser Pro Leu Thr
Ile Glu Asp Glu Arg His Ile Thr Ser Val Thr 725 730 735Asn Leu Lys
Thr Leu Ser Ile His Asp Leu Gln Asn Gln Arg Leu Pro 740 745 750Gly
Gly Leu Thr Asp Ser Leu Gly Asn Leu Lys Asn Leu Thr Lys Leu 755 760
765Ile Met Asp Asn Ile Lys Met Asn Glu Glu Asp Ala Ile Lys Leu Ala
770 775 780Glu Gly Leu Lys Asn Leu Lys Lys Met Cys Leu Phe His Leu
Thr His785 790 795 800Leu Ser Asp Ile Gly Glu Gly Met Asp Tyr Ile
Val Lys Ser Leu Ser 805 810 815Ser Glu Pro Cys Asp Leu Glu Glu Ile
Gln Leu Val Ser Cys Cys Leu 820 825 830Ser Ala Asn Ala Val Lys Ile
Leu Ala Gln Asn Leu His Asn Leu Val 835 840 845Lys Leu Ser Ile Leu
Asp Leu Ser Glu Asn Tyr Leu Glu Lys Asp Gly 850 855 860Asn Glu Ala
Leu His Glu Leu Ile Asp Arg Met Asn Val Leu Glu Gln865 870 875
880Leu Thr Ala Leu Met Leu Pro Trp Gly Cys Asp Val Gln Gly Ser Leu
885 890 895Ser Ser Leu Leu Lys His Leu Glu Glu Val Pro Gln Leu Val
Lys Leu 900 905 910Gly Leu Lys Asn Trp Arg Leu Thr Asp Thr Glu Ile
Arg Ile Leu Gly 915 920 925Ala Phe Phe Gly Lys Asn Pro Leu Lys Asn
Phe Gln Gln Leu Asn Leu 930 935 940Ala Gly Asn Arg Val Ser Ser Asp
Gly Trp Leu Ala Phe Met Gly Val945 950 955 960Phe Glu Asn Leu Lys
Gln Leu Val Phe Phe Asp Phe Ser Thr Lys Glu 965 970 975Phe Leu Pro
Asp Pro Ala Leu Val Arg Lys Leu Ser Gln Val Leu Ser 980 985 990Lys
Leu Thr Phe Leu Gln Glu Ala Arg Leu Val Gly Trp Gln Phe Asp 995
1000 1005Asp Asp Asp Leu Ser Val Ile Thr Gly Ala Phe Lys Leu Val
Thr Ala 1010 1015 1020981395DNAHomo sapienCDS(277)...(1353)
98cgcccgggca ggtgtttata ctccggaggg tgtccccgtg cgtcatcggt ggagtggacc
60aaaactggtg atctgtttgc cctgtgtgac cttgcccaga accctgctga ctgagagaac
120acatctgctg gaagtcctct gggattcaag gtacagggaa tgaagagtag
ttttacagaa 180aaaagaggac aatattggga tcacctttga cctttccatt
tggaaataat attttctatt 240gtgttataga aaggtgggaa gctttcatcc agaaca
atg aat ttc ata aag gac 294 Met Asn Phe Ile Lys Asp 1 5aat agc cga
gcc ctt att caa aga atg gga atg act gtt ata aag caa 342Asn Ser Arg
Ala Leu Ile Gln Arg Met Gly Met Thr Val Ile Lys Gln 10 15 20atc aca
gat gac cta ttt gta tgg aat gtt ctg aat cgc gaa gaa gta 390Ile Thr
Asp Asp Leu Phe Val Trp Asn Val Leu Asn Arg Glu Glu Val 25 30 35aac
atc att tgc tgc gag aag gtg gag cag gat gct gct aga ggg atc 438Asn
Ile Ile Cys Cys Glu Lys Val Glu Gln Asp Ala Ala Arg Gly Ile 40 45
50att cac atg att ttg aaa aag ggt tca gag tcc tgt aac ctc ttt ctt
486Ile His Met Ile Leu Lys Lys Gly Ser Glu Ser Cys Asn Leu Phe
Leu55 60 65 70aaa tcc ctt aag gag tgg aac tat cct cta ttt cag gac
ttg aat gga 534Lys Ser Leu Lys Glu Trp Asn Tyr Pro Leu Phe Gln Asp
Leu Asn Gly 75 80 85caa agt ggt ctg act gac agc ttg ggt aac ttg aag
aac ctt aca aag 582Gln Ser Gly Leu Thr Asp Ser Leu Gly Asn Leu Lys
Asn Leu Thr Lys 90 95 100ctc ata atg gat aac ata aag atg aat gaa
gaa gat gct ata aaa cta 630Leu Ile Met Asp Asn Ile Lys Met Asn Glu
Glu Asp Ala Ile Lys Leu 105 110 115gct gaa ggc ctg aaa aac ctg aag
aag atg tgt tta ttt cat ttg acc 678Ala Glu Gly Leu Lys Asn Leu Lys
Lys Met Cys Leu Phe His Leu Thr 120 125 130cac ttg tct gac att gga
gag gga atg gat tac ata gtc aag tct ctg 726His Leu Ser Asp Ile Gly
Glu Gly Met Asp Tyr Ile Val Lys Ser Leu135 140 145 150tca agt gaa
ccc tgt gac ctt gaa gaa att caa tta gtc tcc tgc tgc 774Ser Ser Glu
Pro Cys Asp Leu Glu Glu Ile Gln Leu Val Ser Cys Cys 155 160 165ttg
tct gca aat gca gtg aaa atc cta gct cag aat ctt cac aat ttg 822Leu
Ser Ala Asn Ala Val Lys Ile Leu Ala Gln Asn Leu His Asn Leu 170 175
180gtc aaa ctg agc att ctt gat tta tca gaa aat tac ctg gaa aaa gat
870Val Lys Leu Ser Ile Leu Asp Leu Ser Glu Asn Tyr Leu Glu Lys Asp
185 190 195gga aat gaa gct ctt cat gaa ctg atc gac agg atg aac gtg
cta gaa 918Gly Asn Glu Ala Leu His Glu Leu Ile Asp Arg Met Asn Val
Leu Glu 200 205 210cag ctc acc gca ctg atg ctg ccc tgg ggc tgt gac
gtg caa ggc agc 966Gln Leu Thr Ala Leu Met Leu Pro Trp Gly Cys Asp
Val Gln Gly Ser215 220 225 230ctg agc agc ctg ttg aaa cat ttg gag
gag gtc cca caa ctc gtc aag 1014Leu Ser Ser Leu Leu Lys His Leu Glu
Glu Val Pro Gln Leu Val Lys 235 240 245ctt ggg ttg aaa aac tgg aga
ctc aca gat aca gag att aga att tta 1062Leu Gly Leu Lys Asn Trp Arg
Leu Thr Asp Thr Glu Ile Arg Ile Leu 250 255 260ggt gca ttt ttt gga
aag aac cct ctg aaa aac ttc cag cag ttg aat 1110Gly Ala Phe Phe Gly
Lys Asn Pro Leu Lys Asn Phe Gln Gln Leu Asn 265 270 275ttg gcg gga
aat cgt gtg agc agt gat gga tgg ctt gcc ttc atg ggt 1158Leu Ala Gly
Asn Arg Val Ser Ser Asp Gly Trp Leu Ala Phe Met Gly 280 285 290gta
ttt gag aat ctt aag caa tta gtg ttt ttt gac ttt agt act aaa 1206Val
Phe Glu Asn Leu Lys Gln Leu Val Phe Phe Asp Phe Ser Thr Lys295 300
305 310gaa ttt cta cct gat cca gca tta gtc aga aaa ctt agc caa gtg
tta 1254Glu Phe Leu Pro Asp Pro Ala Leu Val Arg Lys Leu Ser Gln Val
Leu 315 320 325tcc aag tta act ttt ctg caa gaa gct agg ctt gtt ggg
tgg caa ttt 1302Ser Lys Leu Thr Phe Leu Gln Glu Ala Arg Leu Val Gly
Trp Gln Phe 330 335 340gat gat gat gat ctc agt gtt att aca ggt gct
ttt aaa cta gta act 1350Asp Asp Asp Asp Leu Ser Val Ile Thr Gly Ala
Phe Lys Leu Val Thr 345 350 355gct taaataaagt gtactcgaag caaaaaaaaa
aaaaaaaaaa aa 1395Ala99359PRTHomo sapien 99Met Asn Phe Ile Lys Asp
Asn Ser Arg Ala Leu Ile Gln Arg Met Gly1 5 10 15Met Thr Val Ile Lys
Gln Ile Thr Asp Asp Leu Phe Val Trp Asn Val 20 25 30Leu Asn Arg Glu
Glu Val Asn Ile Ile Cys Cys Glu Lys Val Glu Gln 35 40 45Asp Ala Ala
Arg Gly Ile Ile His Met Ile Leu Lys Lys Gly Ser Glu 50 55 60Ser Cys
Asn Leu Phe Leu Lys Ser Leu Lys Glu Trp Asn Tyr Pro Leu65 70 75
80Phe Gln Asp Leu Asn Gly Gln Ser Gly Leu Thr Asp Ser Leu Gly Asn
85 90 95Leu Lys Asn Leu Thr Lys Leu Ile Met Asp Asn Ile Lys Met Asn
Glu 100 105 110Glu Asp Ala Ile Lys Leu Ala Glu Gly Leu Lys Asn Leu
Lys Lys Met 115 120 125Cys Leu Phe His Leu Thr His Leu Ser Asp Ile
Gly Glu Gly Met Asp 130 135 140Tyr Ile Val Lys Ser Leu Ser Ser Glu
Pro Cys Asp Leu Glu Glu Ile145 150 155 160Gln Leu Val Ser Cys Cys
Leu Ser Ala Asn Ala Val Lys Ile Leu Ala 165 170 175Gln Asn Leu His
Asn Leu Val Lys Leu Ser Ile Leu Asp Leu Ser Glu 180 185 190Asn Tyr
Leu Glu Lys Asp Gly Asn Glu Ala Leu His Glu Leu Ile Asp 195 200
205Arg Met Asn Val Leu Glu Gln Leu Thr Ala Leu Met Leu Pro Trp Gly
210 215 220Cys Asp Val Gln Gly Ser Leu Ser Ser Leu Leu Lys His Leu
Glu Glu225 230 235 240Val Pro Gln Leu Val Lys Leu Gly Leu Lys Asn
Trp Arg Leu Thr Asp 245 250 255Thr Glu Ile Arg Ile Leu Gly Ala Phe
Phe Gly Lys Asn Pro Leu Lys 260 265 270Asn Phe Gln Gln Leu Asn Leu
Ala Gly Asn Arg Val Ser Ser Asp Gly 275 280 285Trp Leu Ala Phe Met
Gly Val Phe Glu Asn Leu Lys Gln Leu Val Phe 290 295 300Phe Asp Phe
Ser Thr Lys Glu Phe Leu Pro Asp Pro Ala Leu Val Arg305 310 315
320Lys Leu Ser Gln Val Leu Ser Lys Leu Thr Phe Leu Gln Glu Ala Arg
325 330
335Leu Val Gly Trp Gln Phe Asp Asp Asp Asp Leu Ser Val Ile Thr Gly
340 345 350Ala Phe Lys Leu Val Thr Ala 355100578DNAHomo
sapienCDS(277)...(552) 100cgcccgggca ggtgtttata ctccggaggg
tgtccccgtg cgtcatcggt ggagtggacc 60aaaactggtg atctgtttgc cctgtgtgac
cttgcccaga accctgctga ctgagagaac 120acatctgctg gaagtcctct
gggattcaag gtacagggaa tgaagagtag ttttacagaa 180aaaagaggac
aatattggga tcacctttga cctttccatt tggaaataat attttctatt
240gtgttataga aaggtgggaa gctttcatcc agaaca atg aat ttc ata aag gac
294 Met Asn Phe Ile Lys Asp 1 5aat agc cga gcc ctt att caa aga atg
gga atg act gtt ata aag caa 342Asn Ser Arg Ala Leu Ile Gln Arg Met
Gly Met Thr Val Ile Lys Gln 10 15 20atc aca gat gac cta ttt gta tgg
aat gtt ctg aat cgc gaa gaa gta 390Ile Thr Asp Asp Leu Phe Val Trp
Asn Val Leu Asn Arg Glu Glu Val 25 30 35aac atc att tgc tgc gag aag
gtg gag cag gat gct gct aga ggg atc 438Asn Ile Ile Cys Cys Glu Lys
Val Glu Gln Asp Ala Ala Arg Gly Ile 40 45 50att cac atg att ttg aaa
aag ggt tca gag tcc tgt aac ctc ttt ctt 486Ile His Met Ile Leu Lys
Lys Gly Ser Glu Ser Cys Asn Leu Phe Leu55 60 65 70aaa tcc ctt aag
gag tgg aac tat cct cta ttt cag gac ttg aat gga 534Lys Ser Leu Lys
Glu Trp Asn Tyr Pro Leu Phe Gln Asp Leu Asn Gly 75 80 85caa agt ctt
tta aca gct tagaaagtac agtagacata ctgggg 578Gln Ser Leu Leu Thr Ala
9010192PRTHomo sapien 101Met Asn Phe Ile Lys Asp Asn Ser Arg Ala
Leu Ile Gln Arg Met Gly1 5 10 15Met Thr Val Ile Lys Gln Ile Thr Asp
Asp Leu Phe Val Trp Asn Val 20 25 30Leu Asn Arg Glu Glu Val Asn Ile
Ile Cys Cys Glu Lys Val Glu Gln 35 40 45Asp Ala Ala Arg Gly Ile Ile
His Met Ile Leu Lys Lys Gly Ser Glu 50 55 60Ser Cys Asn Leu Phe Leu
Lys Ser Leu Lys Glu Trp Asn Tyr Pro Leu65 70 75 80Phe Gln Asp Leu
Asn Gly Gln Ser Leu Leu Thr Ala 85 90102768DNAHomo
sapienCDS(277)...(744) 102cgcccgggca ggtgtttata ctccggaggg
tgtccccgtg cgtcatcggt ggagtggacc 60aaaactggtg atctgtttgc cctgtgtgac
cttgcccaga accctgctga ctgagagaac 120acatctgctg gaagtcctct
gggattcaag gtacagggaa tgaagagtag ttttacagaa 180aaaagaggac
aatattggga tcacctttga cctttccatt tggaaataat attttctatt
240gtgttataga aaggtgggaa gctttcatcc agaaca atg aat ttc ata aag gac
294 Met Asn Phe Ile Lys Asp 1 5aat agc cga gcc ctt att caa aga atg
gga atg act gtt ata aag caa 342Asn Ser Arg Ala Leu Ile Gln Arg Met
Gly Met Thr Val Ile Lys Gln 10 15 20atc aca gat gac cta ttt gta tgg
aat gtt ctg aat cgc gaa gaa gta 390Ile Thr Asp Asp Leu Phe Val Trp
Asn Val Leu Asn Arg Glu Glu Val 25 30 35aac atc att tgc tgc gag aag
gtg gag cag gat gct gct aga ggg atc 438Asn Ile Ile Cys Cys Glu Lys
Val Glu Gln Asp Ala Ala Arg Gly Ile 40 45 50att cac atg att ttg aaa
aag ggt tca gag tcc tgt aac ctc ttt ctt 486Ile His Met Ile Leu Lys
Lys Gly Ser Glu Ser Cys Asn Leu Phe Leu55 60 65 70aaa tcc ctt aag
gag tgg aac tat cct cta ttt cag gac ttg aat gga 534Lys Ser Leu Lys
Glu Trp Asn Tyr Pro Leu Phe Gln Asp Leu Asn Gly 75 80 85caa agt ctt
ttt cat cag aca tca gaa gga gac ttg gac gat ttg gct 582Gln Ser Leu
Phe His Gln Thr Ser Glu Gly Asp Leu Asp Asp Leu Ala 90 95 100cag
gat tta aag gac ttg tac cat acc cca tct ttt ctg aac ttt tat 630Gln
Asp Leu Lys Asp Leu Tyr His Thr Pro Ser Phe Leu Asn Phe Tyr 105 110
115ccc ctt ggt gaa gat att gac att att ttt aac ttg aaa agc acc ttc
678Pro Leu Gly Glu Asp Ile Asp Ile Ile Phe Asn Leu Lys Ser Thr Phe
120 125 130aca gaa cct gtc ctg tgg agg aag gac caa cac cat cac cgc
gtg gag 726Thr Glu Pro Val Leu Trp Arg Lys Asp Gln His His His Arg
Val Glu135 140 145 150cag ctg acc cta gtt tta tagcatcttc tacctgcccg
ggcg 768Gln Leu Thr Leu Val Leu 155103156PRTHomo sapien 103Met Asn
Phe Ile Lys Asp Asn Ser Arg Ala Leu Ile Gln Arg Met Gly1 5 10 15Met
Thr Val Ile Lys Gln Ile Thr Asp Asp Leu Phe Val Trp Asn Val 20 25
30Leu Asn Arg Glu Glu Val Asn Ile Ile Cys Cys Glu Lys Val Glu Gln
35 40 45Asp Ala Ala Arg Gly Ile Ile His Met Ile Leu Lys Lys Gly Ser
Glu 50 55 60Ser Cys Asn Leu Phe Leu Lys Ser Leu Lys Glu Trp Asn Tyr
Pro Leu65 70 75 80Phe Gln Asp Leu Asn Gly Gln Ser Leu Phe His Gln
Thr Ser Glu Gly 85 90 95Asp Leu Asp Asp Leu Ala Gln Asp Leu Lys Asp
Leu Tyr His Thr Pro 100 105 110Ser Phe Leu Asn Phe Tyr Pro Leu Gly
Glu Asp Ile Asp Ile Ile Phe 115 120 125Asn Leu Lys Ser Thr Phe Thr
Glu Pro Val Leu Trp Arg Lys Asp Gln 130 135 140His His His Arg Val
Glu Gln Leu Thr Leu Val Leu145 150 15510424DNAArtificial
SequencePrimer 104aagaagagac ggctgcttat caat 2410524DNAArtificial
SequencePrimer 105ccacagcagg cctcgaagat gatc 2410620DNAArtificial
SequencePrimer 106atgatcctcc tgaagaagag 201071009PRTHomo sapien
107Cys Glu Met Cys Ser Gln Glu Ala Phe Gln Ala Gln Arg Ser Gln Leu1
5 10 15Val Glu Leu Leu Val Ser Gly Ser Leu Glu Gly Phe Glu Ser Val
Leu 20 25 30Asp Trp Leu Leu Ser Trp Glu Val Leu Ser Trp Glu Asp Tyr
Glu Gly 35 40 45Phe His Leu Leu Gly Gln Pro Leu Ser His Leu Ala Arg
Arg Leu Leu 50 55 60Asp Thr Val Trp Asn Lys Gly Thr Trp Ala Cys Gln
Lys Leu Ile Ala65 70 75 80Ala Ala Gln Glu Ala Gln Ala Asp Ser Gln
Ser Pro Lys Leu His Gly 85 90 95Cys Trp Asp Pro His Ser Leu His Pro
Ala Arg Asp Leu Gln Ser His 100 105 110Arg Pro Ala Ile Val Arg Arg
Leu His Ser His Val Glu Asn Met Leu 115 120 125Asp Leu Ala Trp Glu
Arg Gly Phe Val Ser Gln Tyr Glu Cys Asp Glu 130 135 140Ile Arg Leu
Pro Ile Phe Thr Pro Ser Gln Arg Ala Arg Arg Leu Leu145 150 155
160Asp Leu Ala Thr Val Lys Ala Asn Gly Leu Ala Ala Phe Leu Leu Gln
165 170 175His Val Gln Glu Leu Pro Val Pro Leu Ala Leu Pro Leu Glu
Ala Ala 180 185 190Thr Cys Lys Lys Tyr Met Ala Lys Leu Arg Thr Thr
Val Ser Ala Gln 195 200 205Ser Arg Phe Leu Ser Thr Tyr Asp Gly Ala
Glu Thr Leu Cys Leu Glu 210 215 220Asp Ile Tyr Thr Glu Asn Val Leu
Glu Val Trp Ala Asp Val Gly Met225 230 235 240Ala Gly Pro Pro Gln
Lys Ser Pro Ala Thr Leu Gly Leu Glu Glu Leu 245 250 255Phe Ser Thr
Pro Gly His Leu Asn Asp Asp Ala Asp Thr Val Leu Val 260 265 270Val
Gly Glu Ala Gly Ser Gly Lys Ser Thr Leu Leu Gln Arg Leu His 275 280
285Leu Leu Trp Ala Ala Gly Gln Asp Phe Gln Glu Phe Leu Phe Val Phe
290 295 300Pro Phe Ser Cys Arg Gln Leu Gln Cys Met Ala Lys Pro Leu
Ser Val305 310 315 320Arg Thr Leu Leu Phe Glu His Cys Cys Trp Pro
Asp Val Gly Gln Glu 325 330 335Asp Ile Phe Gln Leu Leu Leu Asp His
Pro Asp Arg Val Leu Leu Thr 340 345 350Phe Asp Gly Phe Asp Glu Phe
Lys Phe Arg Phe Thr Asp Arg Glu Arg 355 360 365His Cys Ser Pro Thr
Asp Pro Thr Ser Val Gln Thr Leu Leu Phe Asn 370 375 380Leu Leu Gln
Gly Asn Leu Leu Lys Asn Ala Arg Lys Val Val Thr Ser385 390 395
400Arg Pro Ala Ala Val Ser Ala Phe Leu Arg Lys Tyr Ile Arg Thr Glu
405 410 415Phe Asn Leu Lys Gly Phe Ser Glu Gln Gly Ile Glu Leu Tyr
Leu Arg 420 425 430Lys Arg His His Glu Pro Gly Val Ala Asp Arg Leu
Ile Arg Leu Leu 435 440 445Gln Glu Thr Ser Ala Leu His Gly Leu Cys
His Leu Pro Val Phe Ser 450 455 460Trp Met Val Ser Lys Cys His Gln
Glu Leu Leu Leu Gln Glu Gly Gly465 470 475 480Ser Pro Lys Thr Thr
Thr Asp Met Tyr Leu Leu Ile Leu Gln His Phe 485 490 495Leu Leu His
Ala Thr Pro Pro Asp Ser Ala Ser Gln Gly Leu Gly Pro 500 505 510Ser
Leu Leu Arg Gly Arg Leu Pro Thr Leu Leu His Leu Gly Arg Leu 515 520
525Ala Leu Trp Gly Leu Gly Met Cys Cys Tyr Val Phe Ser Ala Gln Gln
530 535 540Leu Gln Ala Ala Gln Val Ser Pro Asp Asp Ile Ser Leu Gly
Phe Leu545 550 555 560Val Arg Ala Lys Gly Val Val Pro Gly Ser Thr
Ala Pro Leu Glu Phe 565 570 575Leu His Ile Thr Phe Gln Cys Phe Phe
Ala Ala Phe Tyr Leu Ala Leu 580 585 590Ser Ala Asp Val Pro Pro Ala
Leu Leu Arg His Leu Phe Asn Cys Gly 595 600 605Arg Pro Gly Asn Ser
Pro Met Ala Arg Leu Leu Pro Thr Met Cys Ile 610 615 620Gln Ala Ser
Glu Gly Lys Asp Ser Ser Val Ala Ala Leu Leu Gln Lys625 630 635
640Ala Glu Pro His Asn Leu Gln Ile Thr Ala Ala Phe Leu Ala Gly Leu
645 650 655Leu Ser Arg Glu His Trp Gly Leu Leu Ala Glu Cys Gln Thr
Ser Glu 660 665 670Lys Ala Leu Leu Arg Arg Gln Ala Cys Ala Arg Trp
Cys Leu Ala Arg 675 680 685Ser Leu Arg Lys His Phe His Ser Ile Pro
Pro Ala Ala Pro Gly Glu 690 695 700Ala Lys Ser Val His Ala Met Pro
Gly Phe Ile Trp Leu Ile Arg Ser705 710 715 720Leu Tyr Glu Met Gln
Glu Glu Arg Leu Ala Arg Lys Ala Ala Arg Gly 725 730 735Leu Asn Val
Gly His Leu Lys Leu Thr Phe Cys Ser Val Gly Pro Thr 740 745 750Glu
Cys Ala Ala Leu Ala Phe Val Leu Gln His Leu Arg Arg Pro Val 755 760
765Ala Leu Gln Leu Asp Tyr Asn Ser Val Gly Asp Ile Gly Val Glu Gln
770 775 780Leu Leu Pro Cys Leu Gly Val Cys Lys Ala Leu Tyr Leu Arg
Asp Asn785 790 795 800Asn Ile Ser Asp Arg Gly Ile Cys Lys Leu Ile
Glu Cys Ala Leu His 805 810 815Cys Glu Gln Leu Gln Lys Leu Ala Leu
Gly Asn Asn Tyr Ile Thr Ala 820 825 830Ala Gly Ala Gln Val Leu Ala
Glu Gly Leu Arg Gly Asn Thr Ser Leu 835 840 845Gln Phe Leu Gly Phe
Trp Gly Asn Arg Val Gly Asp Glu Gly Ala Gln 850 855 860Ala Leu Ala
Glu Ala Leu Gly Asp His Gln Ser Leu Arg Trp Leu Ser865 870 875
880Leu Val Gly Asn Asn Ile Gly Ser Val Gly Ala Gln Ala Leu Ala Leu
885 890 895Met Leu Ala Lys Asn Val Met Leu Glu Glu Leu Cys Leu Glu
Glu Asn 900 905 910His Leu Gln Asp Glu Gly Val Cys Ser Leu Ala Glu
Gly Leu Lys Lys 915 920 925Asn Ser Ser Leu Lys Ile Leu Asn Ile Lys
Ile His Ala Ser Gly Phe 930 935 940Asn Lys Leu Leu Glu Ser Ile Phe
Cys Ile Leu Leu Val Val Glu Ala945 950 955 960Phe Phe Leu Gln Lys
Val Val Lys Ile Leu Glu Glu Met Val Val Ser 965 970 975Trp Leu Glu
Val Arg Leu Ser Asn Asn Cys Ile Thr Tyr Leu Gly Ala 980 985 990Glu
Ala Leu Leu Gln Ala Leu Glu Arg Asn Asp Thr Ile Leu Glu Val 995
1000 1005Trp108 108000109 109000110 110000111
111000112VARIANT(1)...(87)Xaa = Any Amino Acid 112000113 113000114
114000115 115000116 116000117 117000118 118000119 119000120
120000121 121000122 122000123 123000124 124000125 125000126
126000127 127000128 128000129 129000130 130000131 131000132
132000133 133000134 134000135 135000136 136000137 137000138
138000139 139000140 140000141 141000142 142000143 143000144
14400014530DNAArtificial Sequenceprimer 145ccagaattca tggccgacaa
ggtcctgaag 3014630DNAArtificial Sequenceprimer 146ccactcgagc
taatttccag gtatcggacc 3014720DNAArtificial Sequenceprimer
147gaagacagtt acctggcaga 2014821DNAArtificial Sequenceprimer
148ttgtattctg aacatggcac c 2114936DNAArtificial Sequenceprimer
149gatcatcatc caggccgccc gtggtgacag ccctgg 3615036DNAArtificial
Sequenceprimer 150ccagggctgt caccacgggc ggcctggatg atgatc
3615126DNAArtificial Sequenceprimer 151cggaattcat ggccgacaag gtcctg
2615238DNAArtificial Sequenceprimer 152cgctcgagtt agtcttgcat
attaaggtaa tttccaga 3815323DNAArtificial Sequenceprimer
153catgtgaatg atccctctag cag 2315421DNAArtificial Sequenceprimer
154gggctcggct atcgtgctct a 2115521DNAArtificial Sequenceprimer
155acgatagccg agcccttatt c 2115621DNAArtificial Sequenceprimer
156gtatggaatg ttctgaatcg c 2115733DNAArtificial Sequenceprimer
157cccggatcca tgaatttcat aaaggacaat agc 3315830DNAArtificial
Sequenceprimer 158cccttcgaac aagtcctgaa atagaggata
3015924DNAArtificial Sequenceprimer 159ggtggagcag gatgctgcta gagg
2416029DNAArtificial Sequenceprimer 160cacagtggtc caggctccga
atgaagtca 2916125DNAArtificial Sequenceprimer 161catcatttgc
tgcgagaagg tggag 2516225DNAArtificial Sequenceprimer 162ttaacttgga
taacacttgg ctaag 2516323DNAArtificial Sequenceprimer 163gtaaacatca
tttgctgcga gaa 2316423DNAArtificial Sequenceprimer 164cccgggcagg
tagaagatgc tat 2316525DNAArtificial Sequenceprimer 165aatttcataa
aggacaatag ccgag 2516625DNAArtificial Sequenceprimer 166tgtctactgt
actttctaag ctgtt 25167225DNAHomo sapiensCDS(1)...(225) 167gag agt
act ccc tca gag atc ata gaa aga gaa aga aaa aag ttg ctt 48Glu Ser
Thr Pro Ser Glu Ile Ile Glu Arg Glu Arg Lys Lys Leu Leu1 5 10 15gaa
atc ctt caa cat gat cct gat tct atc tta gac acg tta act tct 96Glu
Ile Leu Gln His Asp Pro Asp Ser Ile Leu Asp Thr Leu Thr Ser 20 25
30cgg agg ctg att tct gag gaa gag tat gag act ctg gag aat gtt aca
144Arg Arg Leu Ile Ser Glu Glu Glu Tyr Glu Thr Leu Glu Asn Val Thr
35 40 45gat ctc ctg aag aaa agt cgg aag ctg tta att ttg gta cag aaa
aag 192Asp Leu Leu Lys Lys Ser Arg Lys Leu Leu Ile Leu Val Gln Lys
Lys 50 55 60gga gag gcg acc tgt cag cat ttt ctc aag tgt 225Gly Glu
Ala Thr Cys Gln His Phe Leu Lys Cys65 70 7516875PRTHomo sapiens
168Glu Ser Thr Pro Ser Glu Ile Ile Glu Arg Glu Arg Lys Lys Leu Leu1
5 10 15Glu Ile Leu Gln His Asp Pro Asp Ser Ile Leu Asp Thr Leu Thr
Ser 20 25 30Arg Arg Leu Ile Ser Glu Glu Glu Tyr Glu Thr Leu Glu Asn
Val Thr 35 40 45Asp Leu Leu Lys Lys Ser Arg Lys Leu Leu Ile Leu Val
Gln Lys Lys 50 55 60Gly Glu Ala Thr Cys Gln His Phe Leu Lys Cys65
70 75169228DNAHomo sapiensCDS(1)...(228) 169atg tgc tcg cag gag gct
ttt cag gca cag agg agc cag ctg gtc gag 48Met Cys Ser Gln Glu
Ala Phe Gln Ala Gln Arg Ser Gln Leu Val Glu1 5 10 15ctg ctg gtc tca
ggg tcc ctg gaa ggc ttc gag agt gtc ctg gac tgg 96Leu Leu Val Ser
Gly Ser Leu Glu Gly Phe Glu Ser Val Leu Asp Trp 20 25 30ctg ctg tcc
tgg gag gtc ctc tcc tgg gag gac tac gag ggc ttc cac 144Leu Leu Ser
Trp Glu Val Leu Ser Trp Glu Asp Tyr Glu Gly Phe His 35 40 45ctc ctg
ggc cag cct ctc tcc cac ttg gcc agg cgc ctt ctg gac acc 192Leu Leu
Gly Gln Pro Leu Ser His Leu Ala Arg Arg Leu Leu Asp Thr 50 55 60gtc
tgg aat aag ggt act tgg gcc tgt cag aag ctc 228Val Trp Asn Lys Gly
Thr Trp Ala Cys Gln Lys Leu65 70 7517076PRTHomo sapiens 170Met Cys
Ser Gln Glu Ala Phe Gln Ala Gln Arg Ser Gln Leu Val Glu 1 5 10
15Leu Leu Val Ser Gly Ser Leu Glu Gly Phe Glu Ser Val Leu Asp Trp
20 25 30Leu Leu Ser Trp Glu Val Leu Ser Trp Glu Asp Tyr Glu Gly Phe
His 35 40 45Leu Leu Gly Gln Pro Leu Ser His Leu Ala Arg Arg Leu Leu
Asp Thr 50 55 60Val Trp Asn Lys Gly Thr Trp Ala Cys Gln Lys Leu65
70 75171243DNAHomo sapiensCDS(1)...(243) 171cca gcc cga gac ctg cag
agt cac cgg cca gcc att gtc agg agg ctc 48Pro Ala Arg Asp Leu Gln
Ser His Arg Pro Ala Ile Val Arg Arg Leu1 5 10 15cac agc cat gtg gag
aac atg ctg gac ctg gca tgg gag cgg ggt ttc 96His Ser His Val Glu
Asn Met Leu Asp Leu Ala Trp Glu Arg Gly Phe 20 25 30gtc agc cag tat
gaa tgt gat gaa atc agg ttg ccg atc ttc aca ccg 144Val Ser Gln Tyr
Glu Cys Asp Glu Ile Arg Leu Pro Ile Phe Thr Pro 35 40 45tcc cag agg
gca aga agg ctg ctt gat ctt gcc acg gtg aaa gcg aat 192Ser Gln Arg
Ala Arg Arg Leu Leu Asp Leu Ala Thr Val Lys Ala Asn 50 55 60gga ttg
gct gcc ttc ctt cta caa cat gtt cag gaa tta cca gtc cca 240Gly Leu
Ala Ala Phe Leu Leu Gln His Val Gln Glu Leu Pro Val Pro65 70 75
80ttg 243Leu17281PRTHomo sapiens 172Pro Ala Arg Asp Leu Gln Ser His
Arg Pro Ala Ile Val Arg Arg Leu1 5 10 15His Ser His Val Glu Asn Met
Leu Asp Leu Ala Trp Glu Arg Gly Phe 20 25 30Val Ser Gln Tyr Glu Cys
Asp Glu Ile Arg Leu Pro Ile Phe Thr Pro 35 40 45Ser Gln Arg Ala Arg
Arg Leu Leu Asp Leu Ala Thr Val Lys Ala Asn 50 55 60Gly Leu Ala Ala
Phe Leu Leu Gln His Val Gln Glu Leu Pro Val Pro65 70 75
80Leu173888DNAHomo sapiensCDS(1)...(888) 173gac gat gcg gac act gtg
ctg gtg gtg ggt gag gcg ggc agt ggc aag 48Asp Asp Ala Asp Thr Val
Leu Val Val Gly Glu Ala Gly Ser Gly Lys1 5 10 15agc acg ctc ctg cag
cgg ctg cac ttg ctg tgg gct gca ggg caa gac 96Ser Thr Leu Leu Gln
Arg Leu His Leu Leu Trp Ala Ala Gly Gln Asp 20 25 30ttc cag gaa ttt
ctc ttt gtc ttc cca ttc agc tgc cgg cag ctg cag 144Phe Gln Glu Phe
Leu Phe Val Phe Pro Phe Ser Cys Arg Gln Leu Gln 35 40 45tgc atg gcc
aaa cca ctc tct gtg cgg act cta ctc ttt gag cac tgc 192Cys Met Ala
Lys Pro Leu Ser Val Arg Thr Leu Leu Phe Glu His Cys 50 55 60tgt tgg
cct gat gtt ggt caa gaa gac atc ttc cag tta ctc ctt gac 240Cys Trp
Pro Asp Val Gly Gln Glu Asp Ile Phe Gln Leu Leu Leu Asp65 70 75
80cac cct gac cgt gtc ctg tta acc ttt gat ggc ttt gac gag ttc aag
288His Pro Asp Arg Val Leu Leu Thr Phe Asp Gly Phe Asp Glu Phe Lys
85 90 95ttc agg ttc acg gat cgt gaa cgc cac tgc tcc ccg acc gac ccc
acc 336Phe Arg Phe Thr Asp Arg Glu Arg His Cys Ser Pro Thr Asp Pro
Thr 100 105 110tct gtc cag acc ctg ctc ttc aac ctt ctg cag ggc aac
ctg ctg aag 384Ser Val Gln Thr Leu Leu Phe Asn Leu Leu Gln Gly Asn
Leu Leu Lys 115 120 125aat gcc cgc aag gtg gtg acc agc cgt ccg gcc
gct gtg tcg gcg ttc 432Asn Ala Arg Lys Val Val Thr Ser Arg Pro Ala
Ala Val Ser Ala Phe 130 135 140ctc agg aag tac atc cgc acc gag ttc
aac ctc aag ggc ttc tct gaa 480Leu Arg Lys Tyr Ile Arg Thr Glu Phe
Asn Leu Lys Gly Phe Ser Glu145 150 155 160cag ggc atc gag ctg tac
ctg agg aag cgc cat cat gag ccc ggg gtg 528Gln Gly Ile Glu Leu Tyr
Leu Arg Lys Arg His His Glu Pro Gly Val 165 170 175gcg gac cgc ctc
atc cgc ctg ctc caa gag acc tca gcc ctg cac ggt 576Ala Asp Arg Leu
Ile Arg Leu Leu Gln Glu Thr Ser Ala Leu His Gly 180 185 190ttg tgc
cac ctg cct gtc ttc tca tgg atg gtg tcc aaa tgc cac cag 624Leu Cys
His Leu Pro Val Phe Ser Trp Met Val Ser Lys Cys His Gln 195 200
205gaa ctg ttg ctg cag gag ggg ggg tcc cca aag acc act aca gat atg
672Glu Leu Leu Leu Gln Glu Gly Gly Ser Pro Lys Thr Thr Thr Asp Met
210 215 220tac ctg ctg att ctg cag cat ttt ctg ctg cat gcc acc ccc
cca gac 720Tyr Leu Leu Ile Leu Gln His Phe Leu Leu His Ala Thr Pro
Pro Asp225 230 235 240tca gct tcc caa ggt ctg gga ccc agt ctt ctt
cgg ggc cgc ctc ccc 768Ser Ala Ser Gln Gly Leu Gly Pro Ser Leu Leu
Arg Gly Arg Leu Pro 245 250 255acc ctc ctg cac ctg ggc aga ctg gct
ctg tgg ggc ctg ggc atg tgc 816Thr Leu Leu His Leu Gly Arg Leu Ala
Leu Trp Gly Leu Gly Met Cys 260 265 270tgc tac gtg ttc tca gcc cag
cag ctc cag gca gca cag gtc agc cct 864Cys Tyr Val Phe Ser Ala Gln
Gln Leu Gln Ala Ala Gln Val Ser Pro 275 280 285gat gac att tct ctt
ggc ttc ctg 888Asp Asp Ile Ser Leu Gly Phe Leu 290 295174296PRTHomo
sapiens 174Asp Asp Ala Asp Thr Val Leu Val Val Gly Glu Ala Gly Ser
Gly Lys1 5 10 15Ser Thr Leu Leu Gln Arg Leu His Leu Leu Trp Ala Ala
Gly Gln Asp 20 25 30Phe Gln Glu Phe Leu Phe Val Phe Pro Phe Ser Cys
Arg Gln Leu Gln 35 40 45Cys Met Ala Lys Pro Leu Ser Val Arg Thr Leu
Leu Phe Glu His Cys 50 55 60Cys Trp Pro Asp Val Gly Gln Glu Asp Ile
Phe Gln Leu Leu Leu Asp65 70 75 80His Pro Asp Arg Val Leu Leu Thr
Phe Asp Gly Phe Asp Glu Phe Lys 85 90 95Phe Arg Phe Thr Asp Arg Glu
Arg His Cys Ser Pro Thr Asp Pro Thr 100 105 110Ser Val Gln Thr Leu
Leu Phe Asn Leu Leu Gln Gly Asn Leu Leu Lys 115 120 125Asn Ala Arg
Lys Val Val Thr Ser Arg Pro Ala Ala Val Ser Ala Phe 130 135 140Leu
Arg Lys Tyr Ile Arg Thr Glu Phe Asn Leu Lys Gly Phe Ser Glu145 150
155 160Gln Gly Ile Glu Leu Tyr Leu Arg Lys Arg His His Glu Pro Gly
Val 165 170 175Ala Asp Arg Leu Ile Arg Leu Leu Gln Glu Thr Ser Ala
Leu His Gly 180 185 190Leu Cys His Leu Pro Val Phe Ser Trp Met Val
Ser Lys Cys His Gln 195 200 205Glu Leu Leu Leu Gln Glu Gly Gly Ser
Pro Lys Thr Thr Thr Asp Met 210 215 220Tyr Leu Leu Ile Leu Gln His
Phe Leu Leu His Ala Thr Pro Pro Asp225 230 235 240Ser Ala Ser Gln
Gly Leu Gly Pro Ser Leu Leu Arg Gly Arg Leu Pro 245 250 255Thr Leu
Leu His Leu Gly Arg Leu Ala Leu Trp Gly Leu Gly Met Cys 260 265
270Cys Tyr Val Phe Ser Ala Gln Gln Leu Gln Ala Ala Gln Val Ser Pro
275 280 285Asp Asp Ile Ser Leu Gly Phe Leu 290 2951751209DNAHomo
sapiensCDS(1)...(1209) 175gag ccc ggg gtg gcg gac cgc ctc atc cgc
ctg ctc caa gag acc tca 48Glu Pro Gly Val Ala Asp Arg Leu Ile Arg
Leu Leu Gln Glu Thr Ser1 5 10 15gcc ctg cac ggt ttg tgc cac ctg cct
gtc ttc tca tgg atg gtg tcc 96Ala Leu His Gly Leu Cys His Leu Pro
Val Phe Ser Trp Met Val Ser 20 25 30aaa tgc cac cag gaa ctg ttg ctg
cag gag ggg ggg tcc cca aag acc 144Lys Cys His Gln Glu Leu Leu Leu
Gln Glu Gly Gly Ser Pro Lys Thr 35 40 45act aca gat atg tac ctg ctg
att ctg cag cat ttt ctg ctg cat gcc 192Thr Thr Asp Met Tyr Leu Leu
Ile Leu Gln His Phe Leu Leu His Ala 50 55 60acc ccc cca gac tca gct
tcc caa ggt ctg gga ccc agt ctt ctt cgg 240Thr Pro Pro Asp Ser Ala
Ser Gln Gly Leu Gly Pro Ser Leu Leu Arg65 70 75 80ggc cgc ctc ccc
acc ctc ctg cac ctg ggc aga ctg gct ctg tgg ggc 288Gly Arg Leu Pro
Thr Leu Leu His Leu Gly Arg Leu Ala Leu Trp Gly 85 90 95ctg ggc atg
tgc tgc tac gtg ttc tca gcc cag cag ctc cag gca gca 336Leu Gly Met
Cys Cys Tyr Val Phe Ser Ala Gln Gln Leu Gln Ala Ala 100 105 110cag
gtc agc cct gat gac att tct ctt ggc ttc ctg gtg cgt gcc aaa 384Gln
Val Ser Pro Asp Asp Ile Ser Leu Gly Phe Leu Val Arg Ala Lys 115 120
125ggt gtc gtg cca ggg agt acg gcg ccc ctg gaa ttc ctt cac atc act
432Gly Val Val Pro Gly Ser Thr Ala Pro Leu Glu Phe Leu His Ile Thr
130 135 140ttc cag tgc ttc ttt gcc gcg ttc tac ctg gca ctc agt gct
gat gtg 480Phe Gln Cys Phe Phe Ala Ala Phe Tyr Leu Ala Leu Ser Ala
Asp Val145 150 155 160cca cca gct ttg ctc aga cac ctc ttc aat tgt
ggc agg cca ggc aac 528Pro Pro Ala Leu Leu Arg His Leu Phe Asn Cys
Gly Arg Pro Gly Asn 165 170 175tca cca atg gcc agg ctc ctg ccc acg
atg tgc atc cag gcc tcg gag 576Ser Pro Met Ala Arg Leu Leu Pro Thr
Met Cys Ile Gln Ala Ser Glu 180 185 190gga aag gac agc agc gtg gca
gct ttg ctg cag aag gcc gag ccg cac 624Gly Lys Asp Ser Ser Val Ala
Ala Leu Leu Gln Lys Ala Glu Pro His 195 200 205aac ctt cag atc aca
gca gcc ttc ctg gca ggg ctg ttg tcc cgg gag 672Asn Leu Gln Ile Thr
Ala Ala Phe Leu Ala Gly Leu Leu Ser Arg Glu 210 215 220cac tgg ggc
ctg ctg gct gag tgc cag aca tct gag aag gcc ctg ctc 720His Trp Gly
Leu Leu Ala Glu Cys Gln Thr Ser Glu Lys Ala Leu Leu225 230 235
240cgg cgc cag gcc tgt gcc cgc tgg tgt ctg gcc cgc agc ctc cgc aag
768Arg Arg Gln Ala Cys Ala Arg Trp Cys Leu Ala Arg Ser Leu Arg Lys
245 250 255cac ttc cac tcc atc ccg cca gct gca ccg ggt gag gcc aag
agc gtg 816His Phe His Ser Ile Pro Pro Ala Ala Pro Gly Glu Ala Lys
Ser Val 260 265 270cat gcc atg ccc ggg ttc atc tgg ctc atc cgg agc
ctg tac gag atg 864His Ala Met Pro Gly Phe Ile Trp Leu Ile Arg Ser
Leu Tyr Glu Met 275 280 285cag gag gag cgg ctg gct cgg aag gct gca
cgt ggc ctg aat gtt ggg 912Gln Glu Glu Arg Leu Ala Arg Lys Ala Ala
Arg Gly Leu Asn Val Gly 290 295 300cac ctc aag ttg aca ttt tgc agt
gtg ggc ccc act gag tgt gct gcc 960His Leu Lys Leu Thr Phe Cys Ser
Val Gly Pro Thr Glu Cys Ala Ala305 310 315 320ctg gcc ttt gtg ctg
cag cac ctc cgg cgg ccc gtg gcc ctg cag ctg 1008Leu Ala Phe Val Leu
Gln His Leu Arg Arg Pro Val Ala Leu Gln Leu 325 330 335gac tac aac
tct gtg ggt gac att ggc gtg gag cag ctg ctg cct tgc 1056Asp Tyr Asn
Ser Val Gly Asp Ile Gly Val Glu Gln Leu Leu Pro Cys 340 345 350ctt
ggt gtc tgc aag gct ctg tat ttg cgc gat aac aat atc tca gac 1104Leu
Gly Val Cys Lys Ala Leu Tyr Leu Arg Asp Asn Asn Ile Ser Asp 355 360
365cga ggc atc tgc aag ctc att gaa tgt gct ctt cac tgc gag caa ttg
1152Arg Gly Ile Cys Lys Leu Ile Glu Cys Ala Leu His Cys Glu Gln Leu
370 375 380cag aag tta gcg ctg ggg aat aac tac atc act gcc gcg gga
gcc caa 1200Gln Lys Leu Ala Leu Gly Asn Asn Tyr Ile Thr Ala Ala Gly
Ala Gln385 390 395 400gtg ctg gcc 1209Val Leu Ala176403PRTHomo
sapiens 176Glu Pro Gly Val Ala Asp Arg Leu Ile Arg Leu Leu Gln Glu
Thr Ser1 5 10 15Ala Leu His Gly Leu Cys His Leu Pro Val Phe Ser Trp
Met Val Ser 20 25 30Lys Cys His Gln Glu Leu Leu Leu Gln Glu Gly Gly
Ser Pro Lys Thr 35 40 45Thr Thr Asp Met Tyr Leu Leu Ile Leu Gln His
Phe Leu Leu His Ala 50 55 60Thr Pro Pro Asp Ser Ala Ser Gln Gly Leu
Gly Pro Ser Leu Leu Arg65 70 75 80Gly Arg Leu Pro Thr Leu Leu His
Leu Gly Arg Leu Ala Leu Trp Gly 85 90 95Leu Gly Met Cys Cys Tyr Val
Phe Ser Ala Gln Gln Leu Gln Ala Ala 100 105 110Gln Val Ser Pro Asp
Asp Ile Ser Leu Gly Phe Leu Val Arg Ala Lys 115 120 125Gly Val Val
Pro Gly Ser Thr Ala Pro Leu Glu Phe Leu His Ile Thr 130 135 140Phe
Gln Cys Phe Phe Ala Ala Phe Tyr Leu Ala Leu Ser Ala Asp Val145 150
155 160Pro Pro Ala Leu Leu Arg His Leu Phe Asn Cys Gly Arg Pro Gly
Asn 165 170 175Ser Pro Met Ala Arg Leu Leu Pro Thr Met Cys Ile Gln
Ala Ser Glu 180 185 190Gly Lys Asp Ser Ser Val Ala Ala Leu Leu Gln
Lys Ala Glu Pro His 195 200 205Asn Leu Gln Ile Thr Ala Ala Phe Leu
Ala Gly Leu Leu Ser Arg Glu 210 215 220His Trp Gly Leu Leu Ala Glu
Cys Gln Thr Ser Glu Lys Ala Leu Leu225 230 235 240Arg Arg Gln Ala
Cys Ala Arg Trp Cys Leu Ala Arg Ser Leu Arg Lys 245 250 255His Phe
His Ser Ile Pro Pro Ala Ala Pro Gly Glu Ala Lys Ser Val 260 265
270His Ala Met Pro Gly Phe Ile Trp Leu Ile Arg Ser Leu Tyr Glu Met
275 280 285Gln Glu Glu Arg Leu Ala Arg Lys Ala Ala Arg Gly Leu Asn
Val Gly 290 295 300His Leu Lys Leu Thr Phe Cys Ser Val Gly Pro Thr
Glu Cys Ala Ala305 310 315 320Leu Ala Phe Val Leu Gln His Leu Arg
Arg Pro Val Ala Leu Gln Leu 325 330 335Asp Tyr Asn Ser Val Gly Asp
Ile Gly Val Glu Gln Leu Leu Pro Cys 340 345 350Leu Gly Val Cys Lys
Ala Leu Tyr Leu Arg Asp Asn Asn Ile Ser Asp 355 360 365Arg Gly Ile
Cys Lys Leu Ile Glu Cys Ala Leu His Cys Glu Gln Leu 370 375 380Gln
Lys Leu Ala Leu Gly Asn Asn Tyr Ile Thr Ala Ala Gly Ala Gln385 390
395 400Val Leu Ala177261DNAHomo sapiensCDS(1)...(261) 177atg aat
ttc ata aag gac aat agc cga gcc ctt att caa aga atg gga 48Met Asn
Phe Ile Lys Asp Asn Ser Arg Ala Leu Ile Gln Arg Met Gly1 5 10 15atg
act gtt ata aag caa atc aca gat gac cta ttt gta tgg aat gtt 96Met
Thr Val Ile Lys Gln Ile Thr Asp Asp Leu Phe Val Trp Asn Val 20 25
30ctg aat cgc gaa gaa gta aac atc att tgc tgc gag aag gtg gag cag
144Leu Asn Arg Glu Glu Val Asn Ile Ile Cys Cys Glu Lys Val Glu Gln
35 40 45gat gct gct aga ggg atc att cac atg att ttg aaa aag ggt tca
gag 192Asp Ala Ala Arg Gly Ile Ile His Met Ile Leu Lys Lys Gly Ser
Glu 50 55 60tcc tgt aac ctc ttt ctt aaa tcc ctt aag gag tgg aac tat
cct cta 240Ser Cys Asn Leu Phe Leu Lys Ser Leu Lys Glu Trp Asn Tyr
Pro Leu65 70 75 80ttt cag gac ttg aat gga caa 261Phe Gln Asp Leu
Asn Gly Gln 8517887PRTHomo sapiens 178Met Asn Phe Ile Lys Asp Asn
Ser Arg Ala Leu Ile Gln Arg Met Gly1 5 10 15Met Thr Val Ile Lys Gln
Ile Thr Asp Asp Leu Phe Val Trp Asn Val 20 25 30Leu Asn Arg Glu Glu
Val Asn Ile Ile Cys Cys Glu Lys Val Glu Gln 35 40 45Asp Ala Ala Arg
Gly Ile Ile His Met Ile Leu Lys Lys Gly Ser Glu 50
55 60Ser Cys Asn Leu Phe Leu Lys Ser Leu Lys Glu Trp Asn Tyr Pro
Leu65 70 75 80Phe Gln Asp Leu Asn Gly Gln 85179891DNAHomo
sapiensCDS(1)...(891) 179ctt cag agc ccc tgc atc att gaa ggg gaa
tct ggc aaa ggc aag tcc 48Leu Gln Ser Pro Cys Ile Ile Glu Gly Glu
Ser Gly Lys Gly Lys Ser1 5 10 15act ctg ctg cag cgc att gcc atg ctc
tgg ggc tcc gga aag tgc aag 96Thr Leu Leu Gln Arg Ile Ala Met Leu
Trp Gly Ser Gly Lys Cys Lys 20 25 30gct ctg acc aag ttc aaa ttc gtc
ttc ttc ctc cgt ctc agc agg gcc 144Ala Leu Thr Lys Phe Lys Phe Val
Phe Phe Leu Arg Leu Ser Arg Ala 35 40 45cag ggt gga ctt ttt gaa acc
ctc tgt gat caa ctc ctg gat ata cct 192Gln Gly Gly Leu Phe Glu Thr
Leu Cys Asp Gln Leu Leu Asp Ile Pro 50 55 60ggc aca atc agg aag cag
aca ttc atg gcc atg ctg ctg aag ctg cgg 240Gly Thr Ile Arg Lys Gln
Thr Phe Met Ala Met Leu Leu Lys Leu Arg65 70 75 80cag agg gtt ctt
ttc ctt ctt gat ggc tac aat gaa ttc aag ccc cag 288Gln Arg Val Leu
Phe Leu Leu Asp Gly Tyr Asn Glu Phe Lys Pro Gln 85 90 95aac tgc cca
gaa atc gaa gcc ctg ata aag gaa aac cac cgc ttc aag 336Asn Cys Pro
Glu Ile Glu Ala Leu Ile Lys Glu Asn His Arg Phe Lys 100 105 110aac
atg gtc atc gtc acc act acc act gag tgc ctg agg cac ata cgg 384Asn
Met Val Ile Val Thr Thr Thr Thr Glu Cys Leu Arg His Ile Arg 115 120
125cag ttt ggt gcc ctg act gct gag gtg ggg gat atg aca gaa gac agc
432Gln Phe Gly Ala Leu Thr Ala Glu Val Gly Asp Met Thr Glu Asp Ser
130 135 140gcc cag gct ctc atc cga gaa gtg ctg atc aag gag ctt gct
gaa ggc 480Ala Gln Ala Leu Ile Arg Glu Val Leu Ile Lys Glu Leu Ala
Glu Gly145 150 155 160ttg ttg ctc caa att cag aaa tcc agg tgc ttg
agg aat ctc atg aag 528Leu Leu Leu Gln Ile Gln Lys Ser Arg Cys Leu
Arg Asn Leu Met Lys 165 170 175acc cct ctc ttt gtg gtc atc act tgt
gca atc cag atg ggt gaa agt 576Thr Pro Leu Phe Val Val Ile Thr Cys
Ala Ile Gln Met Gly Glu Ser 180 185 190gag ttc cac tct cac aca caa
aca acg ctg ttc cat acc ttc tat gat 624Glu Phe His Ser His Thr Gln
Thr Thr Leu Phe His Thr Phe Tyr Asp 195 200 205ctg ttg ata cag aaa
aac aaa cac aaa cat aaa ggt gtg gct gca agt 672Leu Leu Ile Gln Lys
Asn Lys His Lys His Lys Gly Val Ala Ala Ser 210 215 220gac ttc att
cgg agc ctg gac cac cgt gga gac cta gct ctg gag ggt 720Asp Phe Ile
Arg Ser Leu Asp His Arg Gly Asp Leu Ala Leu Glu Gly225 230 235
240gtg ttc tcc cac aag ttt gat ttc gaa ctg cag gat gtg tcc agc gtg
768Val Phe Ser His Lys Phe Asp Phe Glu Leu Gln Asp Val Ser Ser Val
245 250 255aat gag gat gtc ctg ctg aca act ggg ctc ctc tgt aaa tat
aca gct 816Asn Glu Asp Val Leu Leu Thr Thr Gly Leu Leu Cys Lys Tyr
Thr Ala 260 265 270caa agg ttc aag cca aag tat aaa ttc ttt cac aag
tca ttc cag gag 864Gln Arg Phe Lys Pro Lys Tyr Lys Phe Phe His Lys
Ser Phe Gln Glu 275 280 285tac aca gca gga cga aga ctc agc agt
891Tyr Thr Ala Gly Arg Arg Leu Ser Ser 290 295180297PRTHomo sapiens
180Leu Gln Ser Pro Cys Ile Ile Glu Gly Glu Ser Gly Lys Gly Lys Ser1
5 10 15Thr Leu Leu Gln Arg Ile Ala Met Leu Trp Gly Ser Gly Lys Cys
Lys 20 25 30Ala Leu Thr Lys Phe Lys Phe Val Phe Phe Leu Arg Leu Ser
Arg Ala 35 40 45Gln Gly Gly Leu Phe Glu Thr Leu Cys Asp Gln Leu Leu
Asp Ile Pro 50 55 60Gly Thr Ile Arg Lys Gln Thr Phe Met Ala Met Leu
Leu Lys Leu Arg65 70 75 80Gln Arg Val Leu Phe Leu Leu Asp Gly Tyr
Asn Glu Phe Lys Pro Gln 85 90 95Asn Cys Pro Glu Ile Glu Ala Leu Ile
Lys Glu Asn His Arg Phe Lys 100 105 110Asn Met Val Ile Val Thr Thr
Thr Thr Glu Cys Leu Arg His Ile Arg 115 120 125Gln Phe Gly Ala Leu
Thr Ala Glu Val Gly Asp Met Thr Glu Asp Ser 130 135 140Ala Gln Ala
Leu Ile Arg Glu Val Leu Ile Lys Glu Leu Ala Glu Gly145 150 155
160Leu Leu Leu Gln Ile Gln Lys Ser Arg Cys Leu Arg Asn Leu Met Lys
165 170 175Thr Pro Leu Phe Val Val Ile Thr Cys Ala Ile Gln Met Gly
Glu Ser 180 185 190Glu Phe His Ser His Thr Gln Thr Thr Leu Phe His
Thr Phe Tyr Asp 195 200 205Leu Leu Ile Gln Lys Asn Lys His Lys His
Lys Gly Val Ala Ala Ser 210 215 220Asp Phe Ile Arg Ser Leu Asp His
Arg Gly Asp Leu Ala Leu Glu Gly225 230 235 240Val Phe Ser His Lys
Phe Asp Phe Glu Leu Gln Asp Val Ser Ser Val 245 250 255Asn Glu Asp
Val Leu Leu Thr Thr Gly Leu Leu Cys Lys Tyr Thr Ala 260 265 270Gln
Arg Phe Lys Pro Lys Tyr Lys Phe Phe His Lys Ser Phe Gln Glu 275 280
285Tyr Thr Ala Gly Arg Arg Leu Ser Ser 290 295181618DNAHomo
sapiensCDS(1)...(618) 181ggt aac ttg aag aac ctt aca aag ctc ata
atg gat aac ata aag atg 48Gly Asn Leu Lys Asn Leu Thr Lys Leu Ile
Met Asp Asn Ile Lys Met1 5 10 15aat gaa gaa gat gct ata aaa cta gct
gaa ggc ctg aaa aac ctg aag 96Asn Glu Glu Asp Ala Ile Lys Leu Ala
Glu Gly Leu Lys Asn Leu Lys 20 25 30aag atg tgt tta ttt cat ttg acc
cac ttg tct gac att gga gag gga 144Lys Met Cys Leu Phe His Leu Thr
His Leu Ser Asp Ile Gly Glu Gly 35 40 45atg gat tac ata gtc aag tct
ctg tca agt gaa ccc tgt gac ctt gaa 192Met Asp Tyr Ile Val Lys Ser
Leu Ser Ser Glu Pro Cys Asp Leu Glu 50 55 60gaa att caa tta gtc tcc
tgc tgc ttg tct gca aat gca gtg aaa atc 240Glu Ile Gln Leu Val Ser
Cys Cys Leu Ser Ala Asn Ala Val Lys Ile65 70 75 80cta gct cag aat
ctt cac aat ttg gtc aaa ctg agc att ctt gat tta 288Leu Ala Gln Asn
Leu His Asn Leu Val Lys Leu Ser Ile Leu Asp Leu 85 90 95tca gaa aat
tac ctg gaa aaa gat gga aat gaa gct ctt cat gaa ctg 336Ser Glu Asn
Tyr Leu Glu Lys Asp Gly Asn Glu Ala Leu His Glu Leu 100 105 110atc
gac agg atg aac gtg cta gaa cag ctc acc gca ctg atg ctg ccc 384Ile
Asp Arg Met Asn Val Leu Glu Gln Leu Thr Ala Leu Met Leu Pro 115 120
125tgg ggc tgt gac gtg caa ggc agc ctg agc agc ctg ttg aaa cat ttg
432Trp Gly Cys Asp Val Gln Gly Ser Leu Ser Ser Leu Leu Lys His Leu
130 135 140gag gag gtc cca caa ctc gtc aag ctt ggg ttg aaa aac tgg
aga ctc 480Glu Glu Val Pro Gln Leu Val Lys Leu Gly Leu Lys Asn Trp
Arg Leu145 150 155 160aca gat aca gag att aga att tta ggt gca ttt
ttt gga aag aac cct 528Thr Asp Thr Glu Ile Arg Ile Leu Gly Ala Phe
Phe Gly Lys Asn Pro 165 170 175ctg aaa aac ttc cag cag ttg aat ttg
gcg gga aat cgt gtg agc agt 576Leu Lys Asn Phe Gln Gln Leu Asn Leu
Ala Gly Asn Arg Val Ser Ser 180 185 190gat gga tgg ctt gcc ttc atg
ggt gta ttt gag aat ctt aag 618Asp Gly Trp Leu Ala Phe Met Gly Val
Phe Glu Asn Leu Lys 195 200 205182206PRTHomo sapiens 182Gly Asn Leu
Lys Asn Leu Thr Lys Leu Ile Met Asp Asn Ile Lys Met1 5 10 15Asn Glu
Glu Asp Ala Ile Lys Leu Ala Glu Gly Leu Lys Asn Leu Lys 20 25 30Lys
Met Cys Leu Phe His Leu Thr His Leu Ser Asp Ile Gly Glu Gly 35 40
45Met Asp Tyr Ile Val Lys Ser Leu Ser Ser Glu Pro Cys Asp Leu Glu
50 55 60Glu Ile Gln Leu Val Ser Cys Cys Leu Ser Ala Asn Ala Val Lys
Ile65 70 75 80Leu Ala Gln Asn Leu His Asn Leu Val Lys Leu Ser Ile
Leu Asp Leu 85 90 95Ser Glu Asn Tyr Leu Glu Lys Asp Gly Asn Glu Ala
Leu His Glu Leu 100 105 110Ile Asp Arg Met Asn Val Leu Glu Gln Leu
Thr Ala Leu Met Leu Pro 115 120 125Trp Gly Cys Asp Val Gln Gly Ser
Leu Ser Ser Leu Leu Lys His Leu 130 135 140Glu Glu Val Pro Gln Leu
Val Lys Leu Gly Leu Lys Asn Trp Arg Leu145 150 155 160Thr Asp Thr
Glu Ile Arg Ile Leu Gly Ala Phe Phe Gly Lys Asn Pro 165 170 175Leu
Lys Asn Phe Gln Gln Leu Asn Leu Ala Gly Asn Arg Val Ser Ser 180 185
190Asp Gly Trp Leu Ala Phe Met Gly Val Phe Glu Asn Leu Lys 195 200
205183165DNAHomo sapiensCDS(1)...(165) 183acc tac att ccc agc agg
gct gta tct ttg ttc ttc aac tgg aag cag 48Thr Tyr Ile Pro Ser Arg
Ala Val Ser Leu Phe Phe Asn Trp Lys Gln1 5 10 15gaa ttc agg act ctg
gag gtc aca ctc cgg gat ttc agc aag ttg aat 96Glu Phe Arg Thr Leu
Glu Val Thr Leu Arg Asp Phe Ser Lys Leu Asn 20 25 30aag caa gat atc
aga tat ctg ggg aaa ata ttc agc tct gcc aca agc 144Lys Gln Asp Ile
Arg Tyr Leu Gly Lys Ile Phe Ser Ser Ala Thr Ser 35 40 45ctc agg ctg
caa ata aag aga 165Leu Arg Leu Gln Ile Lys Arg 50 5518455PRTHomo
sapiens 184Thr Tyr Ile Pro Ser Arg Ala Val Ser Leu Phe Phe Asn Trp
Lys Gln 1 5 10 15Glu Phe Arg Thr Leu Glu Val Thr Leu Arg Asp Phe
Ser Lys Leu Asn 20 25 30Lys Gln Asp Ile Arg Tyr Leu Gly Lys Ile Phe
Ser Ser Ala Thr Ser 35 40 45Leu Arg Leu Gln Ile Lys Arg 50
5518519DNAArtificial Sequenceprimer 185gaaatgtgct cgcaggagg
1918620DNAArtificial Sequenceprimer 186gatgagcttc tgacaggccc
201873063DNAHomo sapiensCDS(1)...(2385)CDS(2389)...(2928) 187tgt
gaa atg tgc tcg cag gag gct ttt cag gca cag agg agc cag ctg 48Cys
Glu Met Cys Ser Gln Glu Ala Phe Gln Ala Gln Arg Ser Gln Leu1 5 10
15gtc gag ctg ctg gtc tca ggg tcc ctg gaa ggc ttc gag agt gtc ctg
96Val Glu Leu Leu Val Ser Gly Ser Leu Glu Gly Phe Glu Ser Val Leu
20 25 30gac tgg ctg ctg tcc tgg gag gtc ctc tcc tgg gag gac tac gag
ggc 144Asp Trp Leu Leu Ser Trp Glu Val Leu Ser Trp Glu Asp Tyr Glu
Gly 35 40 45ttc cac ctc ctg ggc cag cct ctc tcc cac ttg gcc agg cgc
ctt ctg 192Phe His Leu Leu Gly Gln Pro Leu Ser His Leu Ala Arg Arg
Leu Leu 50 55 60gac acc gtc tgg aat aag ggt act tgg gcc tgt cag aag
ctc atc gcg 240Asp Thr Val Trp Asn Lys Gly Thr Trp Ala Cys Gln Lys
Leu Ile Ala65 70 75 80gct gcc caa gaa gcc cag gcc gac agc cag tcc
ccc aag ctg cat ggc 288Ala Ala Gln Glu Ala Gln Ala Asp Ser Gln Ser
Pro Lys Leu His Gly 85 90 95tgc tgg gac ccc cac tcg ctc cac cca gcc
cga gac ctg cag agt cac 336Cys Trp Asp Pro His Ser Leu His Pro Ala
Arg Asp Leu Gln Ser His 100 105 110cgg cca gcc att gtc agg agg ctc
cac agc cat gtg gag aac atg ctg 384Arg Pro Ala Ile Val Arg Arg Leu
His Ser His Val Glu Asn Met Leu 115 120 125gac ctg gca tgg gag cgg
ggt ttc gtc agc cag tat gaa tgt gat gaa 432Asp Leu Ala Trp Glu Arg
Gly Phe Val Ser Gln Tyr Glu Cys Asp Glu 130 135 140atc agg ttg ccg
atc ttc aca ccg tcc cag agg gca aga agg ctg ctt 480Ile Arg Leu Pro
Ile Phe Thr Pro Ser Gln Arg Ala Arg Arg Leu Leu145 150 155 160gat
ctt gcc acg gtg aaa gcg aat gga ttg gct gcc ttc ctt cta caa 528Asp
Leu Ala Thr Val Lys Ala Asn Gly Leu Ala Ala Phe Leu Leu Gln 165 170
175cat gtt cag gaa tta cca gtc cca ttg gcc ctg cct ttg gaa gct gcc
576His Val Gln Glu Leu Pro Val Pro Leu Ala Leu Pro Leu Glu Ala Ala
180 185 190aca tgc aag aag tat atg gcc aag ctg agg acc acg gtg tct
gct cag 624Thr Cys Lys Lys Tyr Met Ala Lys Leu Arg Thr Thr Val Ser
Ala Gln 195 200 205tct cgc ttc ctc agt acc tat gat gga gca gag acg
ctc tgc ctg gag 672Ser Arg Phe Leu Ser Thr Tyr Asp Gly Ala Glu Thr
Leu Cys Leu Glu 210 215 220gac ata tac aca gag aat gtc ctg gag gtc
tgg gca gat gtg ggc atg 720Asp Ile Tyr Thr Glu Asn Val Leu Glu Val
Trp Ala Asp Val Gly Met225 230 235 240gct gga ccc ccg cag aag agc
cca gcc acc ctg ggc ctg gag gag ctc 768Ala Gly Pro Pro Gln Lys Ser
Pro Ala Thr Leu Gly Leu Glu Glu Leu 245 250 255ttc agc acc cct ggc
cac ctc aat gac gat gcg gac act gtg ctg gtg 816Phe Ser Thr Pro Gly
His Leu Asn Asp Asp Ala Asp Thr Val Leu Val 260 265 270gtg ggt gag
gcg ggc agt ggc aag agc acg ctc ctg cag cgg ctg cac 864Val Gly Glu
Ala Gly Ser Gly Lys Ser Thr Leu Leu Gln Arg Leu His 275 280 285ttg
ctg tgg gct gca ggg caa gac ttc cag gaa ttt ctc ttt gtc ttc 912Leu
Leu Trp Ala Ala Gly Gln Asp Phe Gln Glu Phe Leu Phe Val Phe 290 295
300cca ttc agc tgc cgg cag ctg cag tgc atg gcc aaa cca ctc tct gtg
960Pro Phe Ser Cys Arg Gln Leu Gln Cys Met Ala Lys Pro Leu Ser
Val305 310 315 320cgg act cta ctc ttt gag cac tgc tgt tgg cct gat
gtt ggt caa gaa 1008Arg Thr Leu Leu Phe Glu His Cys Cys Trp Pro Asp
Val Gly Gln Glu 325 330 335gac atc ttc cag tta ctc ctt gac cac cct
gac cgt gtc ctg tta acc 1056Asp Ile Phe Gln Leu Leu Leu Asp His Pro
Asp Arg Val Leu Leu Thr 340 345 350ttt gat ggc ttt gac gag ttc aag
ttc agg ttc acg gat cgt gaa cgc 1104Phe Asp Gly Phe Asp Glu Phe Lys
Phe Arg Phe Thr Asp Arg Glu Arg 355 360 365cac tgc tcc ccg acc gac
ccc acc tct gtc cag acc ctg ctc ttc aac 1152His Cys Ser Pro Thr Asp
Pro Thr Ser Val Gln Thr Leu Leu Phe Asn 370 375 380ctt ctg cag ggc
aac ctg ctg aag aat gcc cgc aag gtg gtg acc agc 1200Leu Leu Gln Gly
Asn Leu Leu Lys Asn Ala Arg Lys Val Val Thr Ser385 390 395 400cgt
ccg gcc gct gtg tcg gcg ttc ctc agg aag tac atc cgc acc gag 1248Arg
Pro Ala Ala Val Ser Ala Phe Leu Arg Lys Tyr Ile Arg Thr Glu 405 410
415ttc aac ctc aag ggc ttc tct gaa cag ggc atc gag ctg tac ctg agg
1296Phe Asn Leu Lys Gly Phe Ser Glu Gln Gly Ile Glu Leu Tyr Leu Arg
420 425 430aag cgc cat cat gag ccc ggg gtg gcg gac cgc ctc atc cgc
ctg ctc 1344Lys Arg His His Glu Pro Gly Val Ala Asp Arg Leu Ile Arg
Leu Leu 435 440 445caa gag acc tca gcc ctg cac ggt ttg tgc cac ctg
cct gtc ttc tca 1392Gln Glu Thr Ser Ala Leu His Gly Leu Cys His Leu
Pro Val Phe Ser 450 455 460tgg atg gtg tcc aaa tgc cac cag gaa ctg
ttg ctg cag gag ggg ggg 1440Trp Met Val Ser Lys Cys His Gln Glu Leu
Leu Leu Gln Glu Gly Gly465 470 475 480tcc cca aag acc act aca gat
atg tac ctg ctg att ctg cag cat ttt 1488Ser Pro Lys Thr Thr Thr Asp
Met Tyr Leu Leu Ile Leu Gln His Phe 485 490 495ctg ctg cat gcc acc
ccc cca gac tca gct tcc caa ggt ctg gga ccc 1536Leu Leu His Ala Thr
Pro Pro Asp Ser Ala Ser Gln Gly Leu Gly Pro 500 505 510agt ctt ctt
cgg ggc cgc ctc ccc acc ctc ctg cac ctg ggc aga ctg 1584Ser Leu Leu
Arg Gly Arg Leu Pro Thr Leu Leu His Leu Gly Arg Leu 515 520 525gct
ctg tgg ggc ctg ggc atg tgc tgc tac gtg ttc tca gcc cag cag 1632Ala
Leu Trp Gly Leu Gly Met Cys Cys Tyr Val Phe Ser Ala Gln Gln 530 535
540ctc cag gca gca cag gtc agc cct gat gac att tct ctt ggc ttc ctg
1680Leu Gln Ala Ala Gln Val Ser Pro Asp Asp Ile Ser Leu Gly Phe
Leu545 550 555 560gtg cgt gcc aaa ggt gtc gtg cca ggg agt acg gcg
ccc ctg gaa ttc 1728Val Arg Ala Lys Gly Val Val Pro Gly Ser Thr Ala
Pro Leu Glu Phe 565
570 575ctt cac atc act ttc cag tgc ttc ttt gcc gcg ttc tac ctg gca
ctc 1776Leu His Ile Thr Phe Gln Cys Phe Phe Ala Ala Phe Tyr Leu Ala
Leu 580 585 590agt gct gat gtg cca cca gct ttg ctc aga cac ctc ttc
aat tgt ggc 1824Ser Ala Asp Val Pro Pro Ala Leu Leu Arg His Leu Phe
Asn Cys Gly 595 600 605agg cca ggc aac tca cca atg gcc agg ctc ctg
ccc acg atg tgc atc 1872Arg Pro Gly Asn Ser Pro Met Ala Arg Leu Leu
Pro Thr Met Cys Ile 610 615 620cag gcc tcg gag gga aag gac agc agc
gtg gca gct ttg ctg cag aag 1920Gln Ala Ser Glu Gly Lys Asp Ser Ser
Val Ala Ala Leu Leu Gln Lys625 630 635 640gcc gag ccg cac aac ctt
cag atc aca gca gcc ttc ctg gca ggg ctg 1968Ala Glu Pro His Asn Leu
Gln Ile Thr Ala Ala Phe Leu Ala Gly Leu 645 650 655ttg tcc cgg gag
cac tgg ggc ctg ctg gct gag tgc cag aca tct gag 2016Leu Ser Arg Glu
His Trp Gly Leu Leu Ala Glu Cys Gln Thr Ser Glu 660 665 670aag gcc
ctg ctc cgg cgc cag gcc tgt gcc cgc tgg tgt ctg gcc cgc 2064Lys Ala
Leu Leu Arg Arg Gln Ala Cys Ala Arg Trp Cys Leu Ala Arg 675 680
685agc ctc cgc aag cac ttc cac tcc atc ccg cca gct gca ccg ggt gag
2112Ser Leu Arg Lys His Phe His Ser Ile Pro Pro Ala Ala Pro Gly Glu
690 695 700gcc aag agc gtg cat gcc atg ccc ggg ttc atc tgg ctc atc
cgg agc 2160Ala Lys Ser Val His Ala Met Pro Gly Phe Ile Trp Leu Ile
Arg Ser705 710 715 720ctg tac gag atg cag gag gag cgg ctg gct cgg
aag gct gca cgt ggc 2208Leu Tyr Glu Met Gln Glu Glu Arg Leu Ala Arg
Lys Ala Ala Arg Gly 725 730 735ctg aat gtt ggg cac ctc aag ttg aca
ttt tgc agt gtg ggc ccc act 2256Leu Asn Val Gly His Leu Lys Leu Thr
Phe Cys Ser Val Gly Pro Thr 740 745 750gag tgt gct gcc ctg gcc ttt
gtg ctg cag cac ctc cgg cgg ccc gtg 2304Glu Cys Ala Ala Leu Ala Phe
Val Leu Gln His Leu Arg Arg Pro Val 755 760 765gcc ctg cag ctg gac
tac aac tct gtg ggt gac att ggc gtg gag cag 2352Ala Leu Gln Leu Asp
Tyr Asn Ser Val Gly Asp Ile Gly Val Glu Gln 770 775 780ctg ctg cct
tgc ctt ggt gtc tgc aag gct ctg taa ttc tgg ggc aac 2400Leu Leu Pro
Cys Leu Gly Val Cys Lys Ala Leu Phe Trp Gly Asn785 790 795aga gtg
ggt gac gag ggg gcc cag gcc ctg gct gaa gcc ttg ggt gat 2448Arg Val
Gly Asp Glu Gly Ala Gln Ala Leu Ala Glu Ala Leu Gly Asp800 805 810
815cac cag agc ttg agg tgg ctc agc ctg gtg ggg aac aac att ggc agt
2496His Gln Ser Leu Arg Trp Leu Ser Leu Val Gly Asn Asn Ile Gly Ser
820 825 830gtg ggt gcc caa gcc ttg gca ctg atg ctg gca aag aac gtc
atg cta 2544Val Gly Ala Gln Ala Leu Ala Leu Met Leu Ala Lys Asn Val
Met Leu 835 840 845gaa gaa ctc tgc ctg gag gag aac cat ctc cag gat
gaa ggt gta tgt 2592Glu Glu Leu Cys Leu Glu Glu Asn His Leu Gln Asp
Glu Gly Val Cys 850 855 860tct ctc gca gaa gga ctg aag aaa aat tca
agt ttg aaa atc ctg aac 2640Ser Leu Ala Glu Gly Leu Lys Lys Asn Ser
Ser Leu Lys Ile Leu Asn 865 870 875ata aaa att cat gct tcg gga ttc
aac aaa ctc ttg gaa agc att ttc 2688Ile Lys Ile His Ala Ser Gly Phe
Asn Lys Leu Leu Glu Ser Ile Phe880 885 890 895tgc atc ctc ctg gtt
gtg gaa gca ttt ttc ctg cag aaa gtt gtc aag 2736Cys Ile Leu Leu Val
Val Glu Ala Phe Phe Leu Gln Lys Val Val Lys 900 905 910att ctt gaa
gaa atg gta gtc agt tgg cta gag gtc agg ttg tcc aat 2784Ile Leu Glu
Glu Met Val Val Ser Trp Leu Glu Val Arg Leu Ser Asn 915 920 925aac
tgc atc acc tac cta ggg gca gaa gcc ctc ctg cag gcc ctt gaa 2832Asn
Cys Ile Thr Tyr Leu Gly Ala Glu Ala Leu Leu Gln Ala Leu Glu 930 935
940agg aat gac acc atc ctg gaa gtc tgg ctc cga ggg aac act ttc tct
2880Arg Asn Asp Thr Ile Leu Glu Val Trp Leu Arg Gly Asn Thr Phe Ser
945 950 955cta gag gag gtt gac aag ctc ggc tgc agg gac acc aga ctc
ttg ctt 2928Leu Glu Glu Val Asp Lys Leu Gly Cys Arg Asp Thr Arg Leu
Leu Leu960 965 970 975tgaagtctcc gggaggatgt tcgtctcagt ttgtttgtga
gcaggctgtg agtttgggcc 2988ccagaggctg ggtgacatgt gttggcagcc
tcttcaaaat gagccctgtc ctgcctaagg 3048ctgaacttgt tttct
3063188795PRTHomo sapiens 188Cys Glu Met Cys Ser Gln Glu Ala Phe
Gln Ala Gln Arg Ser Gln Leu1 5 10 15Val Glu Leu Leu Val Ser Gly Ser
Leu Glu Gly Phe Glu Ser Val Leu 20 25 30Asp Trp Leu Leu Ser Trp Glu
Val Leu Ser Trp Glu Asp Tyr Glu Gly 35 40 45Phe His Leu Leu Gly Gln
Pro Leu Ser His Leu Ala Arg Arg Leu Leu 50 55 60Asp Thr Val Trp Asn
Lys Gly Thr Trp Ala Cys Gln Lys Leu Ile Ala65 70 75 80Ala Ala Gln
Glu Ala Gln Ala Asp Ser Gln Ser Pro Lys Leu His Gly 85 90 95Cys Trp
Asp Pro His Ser Leu His Pro Ala Arg Asp Leu Gln Ser His 100 105
110Arg Pro Ala Ile Val Arg Arg Leu His Ser His Val Glu Asn Met Leu
115 120 125Asp Leu Ala Trp Glu Arg Gly Phe Val Ser Gln Tyr Glu Cys
Asp Glu 130 135 140Ile Arg Leu Pro Ile Phe Thr Pro Ser Gln Arg Ala
Arg Arg Leu Leu145 150 155 160Asp Leu Ala Thr Val Lys Ala Asn Gly
Leu Ala Ala Phe Leu Leu Gln 165 170 175His Val Gln Glu Leu Pro Val
Pro Leu Ala Leu Pro Leu Glu Ala Ala 180 185 190Thr Cys Lys Lys Tyr
Met Ala Lys Leu Arg Thr Thr Val Ser Ala Gln 195 200 205Ser Arg Phe
Leu Ser Thr Tyr Asp Gly Ala Glu Thr Leu Cys Leu Glu 210 215 220Asp
Ile Tyr Thr Glu Asn Val Leu Glu Val Trp Ala Asp Val Gly Met225 230
235 240Ala Gly Pro Pro Gln Lys Ser Pro Ala Thr Leu Gly Leu Glu Glu
Leu 245 250 255Phe Ser Thr Pro Gly His Leu Asn Asp Asp Ala Asp Thr
Val Leu Val 260 265 270Val Gly Glu Ala Gly Ser Gly Lys Ser Thr Leu
Leu Gln Arg Leu His 275 280 285Leu Leu Trp Ala Ala Gly Gln Asp Phe
Gln Glu Phe Leu Phe Val Phe 290 295 300Pro Phe Ser Cys Arg Gln Leu
Gln Cys Met Ala Lys Pro Leu Ser Val305 310 315 320Arg Thr Leu Leu
Phe Glu His Cys Cys Trp Pro Asp Val Gly Gln Glu 325 330 335Asp Ile
Phe Gln Leu Leu Leu Asp His Pro Asp Arg Val Leu Leu Thr 340 345
350Phe Asp Gly Phe Asp Glu Phe Lys Phe Arg Phe Thr Asp Arg Glu Arg
355 360 365His Cys Ser Pro Thr Asp Pro Thr Ser Val Gln Thr Leu Leu
Phe Asn 370 375 380Leu Leu Gln Gly Asn Leu Leu Lys Asn Ala Arg Lys
Val Val Thr Ser385 390 395 400Arg Pro Ala Ala Val Ser Ala Phe Leu
Arg Lys Tyr Ile Arg Thr Glu 405 410 415Phe Asn Leu Lys Gly Phe Ser
Glu Gln Gly Ile Glu Leu Tyr Leu Arg 420 425 430Lys Arg His His Glu
Pro Gly Val Ala Asp Arg Leu Ile Arg Leu Leu 435 440 445Gln Glu Thr
Ser Ala Leu His Gly Leu Cys His Leu Pro Val Phe Ser 450 455 460Trp
Met Val Ser Lys Cys His Gln Glu Leu Leu Leu Gln Glu Gly Gly465 470
475 480Ser Pro Lys Thr Thr Thr Asp Met Tyr Leu Leu Ile Leu Gln His
Phe 485 490 495Leu Leu His Ala Thr Pro Pro Asp Ser Ala Ser Gln Gly
Leu Gly Pro 500 505 510Ser Leu Leu Arg Gly Arg Leu Pro Thr Leu Leu
His Leu Gly Arg Leu 515 520 525Ala Leu Trp Gly Leu Gly Met Cys Cys
Tyr Val Phe Ser Ala Gln Gln 530 535 540Leu Gln Ala Ala Gln Val Ser
Pro Asp Asp Ile Ser Leu Gly Phe Leu545 550 555 560Val Arg Ala Lys
Gly Val Val Pro Gly Ser Thr Ala Pro Leu Glu Phe 565 570 575Leu His
Ile Thr Phe Gln Cys Phe Phe Ala Ala Phe Tyr Leu Ala Leu 580 585
590Ser Ala Asp Val Pro Pro Ala Leu Leu Arg His Leu Phe Asn Cys Gly
595 600 605Arg Pro Gly Asn Ser Pro Met Ala Arg Leu Leu Pro Thr Met
Cys Ile 610 615 620Gln Ala Ser Glu Gly Lys Asp Ser Ser Val Ala Ala
Leu Leu Gln Lys625 630 635 640Ala Glu Pro His Asn Leu Gln Ile Thr
Ala Ala Phe Leu Ala Gly Leu 645 650 655Leu Ser Arg Glu His Trp Gly
Leu Leu Ala Glu Cys Gln Thr Ser Glu 660 665 670Lys Ala Leu Leu Arg
Arg Gln Ala Cys Ala Arg Trp Cys Leu Ala Arg 675 680 685Ser Leu Arg
Lys His Phe His Ser Ile Pro Pro Ala Ala Pro Gly Glu 690 695 700Ala
Lys Ser Val His Ala Met Pro Gly Phe Ile Trp Leu Ile Arg Ser705 710
715 720Leu Tyr Glu Met Gln Glu Glu Arg Leu Ala Arg Lys Ala Ala Arg
Gly 725 730 735Leu Asn Val Gly His Leu Lys Leu Thr Phe Cys Ser Val
Gly Pro Thr 740 745 750Glu Cys Ala Ala Leu Ala Phe Val Leu Gln His
Leu Arg Arg Pro Val 755 760 765Ala Leu Gln Leu Asp Tyr Asn Ser Val
Gly Asp Ile Gly Val Glu Gln 770 775 780Leu Leu Pro Cys Leu Gly Val
Cys Lys Ala Leu785 790 795189180PRTHomo sapiens 189Phe Trp Gly Asn
Arg Val Gly Asp Glu Gly Ala Gln Ala Leu Ala Glu1 5 10 15Ala Leu Gly
Asp His Gln Ser Leu Arg Trp Leu Ser Leu Val Gly Asn 20 25 30Asn Ile
Gly Ser Val Gly Ala Gln Ala Leu Ala Leu Met Leu Ala Lys 35 40 45Asn
Val Met Leu Glu Glu Leu Cys Leu Glu Glu Asn His Leu Gln Asp 50 55
60Glu Gly Val Cys Ser Leu Ala Glu Gly Leu Lys Lys Asn Ser Ser Leu65
70 75 80Lys Ile Leu Asn Ile Lys Ile His Ala Ser Gly Phe Asn Lys Leu
Leu 85 90 95Glu Ser Ile Phe Cys Ile Leu Leu Val Val Glu Ala Phe Phe
Leu Gln 100 105 110Lys Val Val Lys Ile Leu Glu Glu Met Val Val Ser
Trp Leu Glu Val 115 120 125Arg Leu Ser Asn Asn Cys Ile Thr Tyr Leu
Gly Ala Glu Ala Leu Leu 130 135 140Gln Ala Leu Glu Arg Asn Asp Thr
Ile Leu Glu Val Trp Leu Arg Gly145 150 155 160Asn Thr Phe Ser Leu
Glu Glu Val Asp Lys Leu Gly Cys Arg Asp Thr 165 170 175Arg Leu Leu
Leu 180190721DNAMus musculusCDS(193)...(612) 190cctggggttc
ctgcacatta ccttccgtgc ttttttgccg ctttctactt ggctgtcagt 60gctgacacat
cggtggcctc tctcaagcac cttttcagct gtggccggct gggcagctca
120ctgctgggaa ggctgctgcc caacctgtgt atccagggct ccagagtcaa
gaagggcagc 180gaagcagccc tg ctg cag aag gct gag cca cac aac ctg caa
atc aca gca 231 Leu Gln Lys Ala Glu Pro His Asn Leu Gln Ile Thr Ala
1 5 10gcc ttc cta gca ggt ctg ttg tcc cag cag cat cgg gac ctg ttg
gct 279Ala Phe Leu Ala Gly Leu Leu Ser Gln Gln His Arg Asp Leu Leu
Ala 15 20 25gca tgc cag gtc tcc gag agg gta ctg ctc cag cgt cag gca
cgt gcc 327Ala Cys Gln Val Ser Glu Arg Val Leu Leu Gln Arg Gln Ala
Arg Ala30 35 40 45cgc tcg tgt ctg gcc cac agc ctc cgc gag cac ttc
cat tcc atc ccg 375Arg Ser Cys Leu Ala His Ser Leu Arg Glu His Phe
His Ser Ile Pro 50 55 60cct gcc gtg ccc ggt gag acc aag agc atg cat
gct atg ccg ggc ttc 423Pro Ala Val Pro Gly Glu Thr Lys Ser Met His
Ala Met Pro Gly Phe 65 70 75att tgg ctc atc cgt agc ctg tac gag atg
cag gag gag cag ttg gcc 471Ile Trp Leu Ile Arg Ser Leu Tyr Glu Met
Gln Glu Glu Gln Leu Ala 80 85 90cag gag gct gtc cgt cgc ttg gac atc
ggg cac ctg aag ttg aca ttt 519Gln Glu Ala Val Arg Arg Leu Asp Ile
Gly His Leu Lys Leu Thr Phe 95 100 105tgc aga gtg ggc cct gca gag
tgt gct gca ctg gcc ttt gta ctg caa 567Cys Arg Val Gly Pro Ala Glu
Cys Ala Ala Leu Ala Phe Val Leu Gln110 115 120 125cat ctc cag cgg
cct gtg gcc cta cag ctg gat tac aac tct gtg 612His Leu Gln Arg Pro
Val Ala Leu Gln Leu Asp Tyr Asn Ser Val 130 135 140ggagatgttg
ggagtggaac agctgcgacc gtgcctttgg ggtctgcaca gctctgtagt
672gagtgtgaca aggtcttgcc gattgggcct gtggcaaatg ctactgtca
721191140PRTMus musculus 191Leu Gln Lys Ala Glu Pro His Asn Leu Gln
Ile Thr Ala Ala Phe Leu1 5 10 15Ala Gly Leu Leu Ser Gln Gln His Arg
Asp Leu Leu Ala Ala Cys Gln 20 25 30Val Ser Glu Arg Val Leu Leu Gln
Arg Gln Ala Arg Ala Arg Ser Cys 35 40 45Leu Ala His Ser Leu Arg Glu
His Phe His Ser Ile Pro Pro Ala Val 50 55 60Pro Gly Glu Thr Lys Ser
Met His Ala Met Pro Gly Phe Ile Trp Leu65 70 75 80Ile Arg Ser Leu
Tyr Glu Met Gln Glu Glu Gln Leu Ala Gln Glu Ala 85 90 95Val Arg Arg
Leu Asp Ile Gly His Leu Lys Leu Thr Phe Cys Arg Val 100 105 110Gly
Pro Ala Glu Cys Ala Ala Leu Ala Phe Val Leu Gln His Leu Gln 115 120
125Arg Pro Val Ala Leu Gln Leu Asp Tyr Asn Ser Val 130 135
140192419DNAMus musculusCDS(1)...(417) 192ctg cag aag gct gag cca
cac aac ctg cag atc aca gca gcc ttc cta 48Leu Gln Lys Ala Glu Pro
His Asn Leu Gln Ile Thr Ala Ala Phe Leu1 5 10 15gca ggt ctg ttg tcc
cag cag cat cgg gac ctg ttg gct gca tgc cag 96Ala Gly Leu Leu Ser
Gln Gln His Arg Asp Leu Leu Ala Ala Cys Gln 20 25 30atc tcc gag agg
gtg ctg ctc cag cgt cag gca cgt gcc cgc tcg tgt 144Ile Ser Glu Arg
Val Leu Leu Gln Arg Gln Ala Arg Ala Arg Ser Cys 35 40 45ctg gcc cac
agc ctc cgc gag cac ttc cat tcc atc ccg cct gcc gtg 192Leu Ala His
Ser Leu Arg Glu His Phe His Ser Ile Pro Pro Ala Val 50 55 60ccc ggt
gag acc aag agc atg cat gct atg ccg ggc ttt att tgg ctc 240Pro Gly
Glu Thr Lys Ser Met His Ala Met Pro Gly Phe Ile Trp Leu65 70 75
80atc cgg agc ctg tac gag atg cag gag gag cag ttg gcc cag gag gct
288Ile Arg Ser Leu Tyr Glu Met Gln Glu Glu Gln Leu Ala Gln Glu Ala
85 90 95gtc cgt cgc ttg gac atc ggg cac ctg aag ttg aca ttt tgc aga
gtg 336Val Arg Arg Leu Asp Ile Gly His Leu Lys Leu Thr Phe Cys Arg
Val 100 105 110ggc cct gca gag tgt gct gcg ctg gcc ttt gta ctg caa
cat ctc cag 384Gly Pro Ala Glu Cys Ala Ala Leu Ala Phe Val Leu Gln
His Leu Gln 115 120 125cgg cct gtg gcc cta cag ctg gat tac aac tct
gt 419Arg Pro Val Ala Leu Gln Leu Asp Tyr Asn Ser 130
135193139PRTMus musculus 193Leu Gln Lys Ala Glu Pro His Asn Leu Gln
Ile Thr Ala Ala Phe Leu1 5 10 15Ala Gly Leu Leu Ser Gln Gln His Arg
Asp Leu Leu Ala Ala Cys Gln 20 25 30Ile Ser Glu Arg Val Leu Leu Gln
Arg Gln Ala Arg Ala Arg Ser Cys 35 40 45Leu Ala His Ser Leu Arg Glu
His Phe His Ser Ile Pro Pro Ala Val 50 55 60Pro Gly Glu Thr Lys Ser
Met His Ala Met Pro Gly Phe Ile Trp Leu65 70 75 80Ile Arg Ser Leu
Tyr Glu Met Gln Glu Glu Gln Leu Ala Gln Glu Ala 85 90 95Val Arg Arg
Leu Asp Ile Gly His Leu Lys Leu Thr Phe Cys Arg Val 100 105 110Gly
Pro Ala Glu Cys Ala Ala Leu Ala Phe Val Leu Gln His Leu Gln 115 120
125Arg Pro Val Ala Leu Gln Leu Asp Tyr Asn Ser 130
13519426DNAArtificial Sequenceprimer 194ctgcagaagg ctgagccaca
caacct
2619530DNAArtificial Sequenceprimer 195acagagttgt aatccagctg
tagggccaca 30
* * * * *
References