U.S. patent application number 10/139662 was filed with the patent office on 2003-02-06 for family of immunoregulators designated leukocyte immunoglobulin-like receptors (lir).
This patent application is currently assigned to Immunex Corporation. Invention is credited to Anderson, Dirk M., Borges, Luis G., Cosman, David J..
Application Number | 20030027358 10/139662 |
Document ID | / |
Family ID | 23202628 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027358 |
Kind Code |
A1 |
Cosman, David J. ; et
al. |
February 6, 2003 |
Family of immunoregulators designated leukocyte immunoglobulin-like
receptors (LIR)
Abstract
A new family of immunoreceptor molecules of the immunoglobulin
superfamily, (LIR) polypeptides is described. Disclosed are
sequences encoding LIR family members and their deduced amino acid
sequences, polypeptides encoded by DNA that hybridizes to defined
nucleotide sequences, processes for producing polypeptides of the
LIR family, and specific antibodies directed against LIR
polypeptides. LIR family members can be used to treat autoimmune
diseases and disease states associated with suppressed immune
function.
Inventors: |
Cosman, David J.;
(Bainbridge Island, WA) ; Anderson, Dirk M.;
(Seattle, WA) ; Borges, Luis G.; (Seattle,
WA) |
Correspondence
Address: |
IMMUNEX CORPORATION
LAW DEPARTMENT
51 UNIVERSITY STREET
SEATTLE
WA
98101
|
Assignee: |
Immunex Corporation
|
Family ID: |
23202628 |
Appl. No.: |
10/139662 |
Filed: |
May 2, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10139662 |
May 2, 2002 |
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09310463 |
May 12, 1999 |
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6384203 |
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09310463 |
May 12, 1999 |
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08842248 |
Apr 24, 1997 |
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Current U.S.
Class: |
436/518 ;
435/320.1; 435/326; 435/69.1; 530/388.1; 536/23.53 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/70535 20130101; A61P 37/06 20180101; A61P 37/04 20180101;
C07K 2319/00 20130101; C07K 14/70503 20130101; A61P 37/02
20180101 |
Class at
Publication: |
436/518 ;
530/388.1; 536/23.53; 435/69.1; 435/326; 435/320.1 |
International
Class: |
C12P 021/02; C07H
021/04; C12P 021/08; G01N 033/543; C07K 016/40 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule encoding an LIR polypeptide,
wherein said LIR polypeptide comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:12, SEQ ID NO:14,
SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36 and SEQ ID NO:38.
2. An isolated nucleic acid molecule of claim 1, wherein the
nucleic acid molecule has a nucleotide sequence selected from the
group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:21, SEQ
ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID
NO:37.
3. An isolated nucleic acid molecule according to claim 1, wherein
the nucleic acid molecule comprises nucleotides 171-1040 of SEQ ID
NO:11.
4. An isolated nucleic acid molecule according to claim 1, wherein
the nucleic acid molecule comprises nucleotides 183-1652 of SEQ ID
NO:13.
5. An isolated nucleic acid molecule according to claim 1, wherein
the nucleic acid molecule comprises nucleotides 67-1839 of SEQ ID
NO:21.
6. An isolated nucleic acid molecule according to claim 1, wherein
the nucleic acid molecule comprises nucleotides 69-968 of SEQ ID
NO:29.
7. An isolated nucleic acid molecule according to claim 1, wherein
the nucleic acid molecule comprises nucleotides 95-958 of SEQ ID
NO:31.
8. An isolated nucleic acid molecule according to claim 1, wherein
the nucleic acid molecule comprises nucleotides 115-912 of SEQ ID
NO:33.
9. An isolated nucleic acid molecule according to claim 1, wherein
the nucleic acid molecule comprises nucleotides 73-834 of SEQ ID
NO:35.
10. An isolated nucleic acid molecule according to claim 1, wherein
the nucleic acid molecule comprises nucleotides 1-1350 of SEQ ID
NO:37.
11. An isolated nucleic acid molecule encoding a soluble LIR
polypeptide, wherein said LIR polypeptide comprises an amino acid
sequence selected from the group consisting of: the extracellular
domain of a LIR family member, wherein the extracellular domain is
selected from the group consisting of: amino acids x.sub.5 to 261
of SEQ ID NO:12, wherein x.sub.5 is amino acid 1 or 17; amino acids
x.sub.6 to 461 of SEQ ID NO:14, wherein x.sub.6 is amino acid 1 or
17; amino acids x.sub.6 to 456 of SEQ ID NO:22.sub.10, wherein x is
amino acid 1 or 17; amino acids x.sub.11 to 262 of SEQ ID NO:30,
wherein x.sub.11 is amino acid 1 or 35; amino acids x.sub.12 to 250
of SEQ ID NO:32, wherein x.sub.12 is amino acid 1 or 36; amino
acids x.sub.13 to 265 of SEQ ID NO:34, wherein x.sub.13 is amino
acid 1 or 35;. amino acids x.sub.14 to 253 of SEQ ID NO:36, wherein
x.sub.14 is amino acid 1 or 36; and amino acids 1-393 of SEQ ID
NO:38.
12. An isolated nucleic acid molecule encoding a soluble LIR
polypeptide comprising at least one Ig-like domain, wherein said
LIR polypeptide comprises at least 85 amino acids selected from the
group consisting of: amino acids x.sub.5 to 261 of SEQ ID NO:12,
wherein x.sub.5 is amino acid 1 or 17; amino acids x.sub.6 to 461
of SEQ ID NO:14, wherein X.sub.6 is amino acid 1 or 17; amino acids
x.sub.10 to 456 of SEQ ID NO:22.sub.10, wherein x is amino acid 1
or 17; amino acids x.sub.11 to 262 of SEQ ID NO:30, wherein
x.sub.11 is amino acid 1 or 35; amino acids x.sub.12 to 250 of SEQ
ID NO:32, wherein x.sub.12 is amino acid 1 or 36; amino acids
x.sub.13 to 265 of SEQ ID NO:34, wherein x.sub.13 is amino acid 1
or 35; amino acids x.sub.14 to 253 of SEQ ID NO:36, wherein
x.sub.14 is amino acid 1 or 36; and amino acids 1 to 393 of SEQ ID
NO:38.
13. An isolated polypeptide encoded by a nucleic acid molecule of
claim 1.
14. An antibody capable of binding specifically to a polypeptide of
claim 13.
15. A nucleic acid molecule that encodes a fusion protein
comprising the Fc region of Ig and an amino acid sequence selected
from the group consisting of: amino acids x.sub.5 to 261 of SEQ ID
NO:12, wherein x.sub.5 is amino acid 1 or 17; amino acids x.sub.6
to 461 of SEQ ID NO:14, wherein x.sub.6 is amino acid 1 or 17;
amino acids x.sub.10 to 456 of SEQ ID NO:22.sub.10, wherein x is
amino acid 1 or 17; amino acids x.sub.11 to 262 of SEQ ID NO:30,
wherein x.sub.11 is amino acid 1 or 35; amino acids x.sub.12 to 250
of SEQ ID NO:32, wherein x.sub.12 is amino acid 1 or 36; amino
acids x.sub.13 to 265 of SEQ ID NO:34, wherein x.sub.3 is amino
acid 1 or 35; amino acids x.sub.14 to 253 of SEQ ID NO:36, wherein
x.sub.14 is amino acid 1 or 36; amino acids 1 to 393 of SEQ ID
NO:38.
16. A recombinant expression vector comprising a nucleic acid
molecule according to claim 1.
17. A process for preparing an LIR polypeptide, the process
comprising culturing a host cell transformed with an expression
vector of claim 16 under conditions that promote expression of said
polypeptide, and recovering said polypeptide.
18. A composition comprising a pyhsiologically acceptable carrier
and a polypeptide of claim 13.
19. A host cell transformed or transfected with an expression
vector according to claim 16.
Description
BACKGROUND OF THE INVENTION
[0001] Immune system cellular activity is controlled by a complex
network of cell surface interactions and associated signaling
processes. When a cell surface receptor is activated by its ligand
a signal is sent to the cell, depending upon the signal
transduction pathway that is engaged, the signal can be inhibitory
or activatory. For many receptor systems cellular activity is
regulated by a balance between activatory signals and inhibitory
signals. In some of these it is known that positive signals
associated with the engagement of a cell surface receptor by its
ligand are downmodulated or inhibited by negative signals sent by
the engagement of a different cell surface receptor by its
ligand.
[0002] The biochemical mechanisms of these positive and negative
signaling pathways have been studied for a number of known immune
system receptor and ligand interactions. Many receptors that
mediate positive signaling have cytoplasmic tails containing sites
of tyrosine phosphatase phosphorylation known as immunoreceptor
tyrosine-based activation motifs (ITAM). A common mechanistic
pathway for positive signaling involves the activation of tyrosine
kinases which phosphorylate sites on the cytoplasmic domains of the
receptors and on other signaling molecules. Once the receptors are
phosphorylated, binding sites for signal transduction molecules are
created which initiate the signaling pathways and activate the
cell. The inhibitory pathways involve receptors having
immunoreceptor tyrosine based inhibitory motifs (ITIM) which, like
the ITAMs, are phosphorylated by tyrosine kinases. Receptors having
these motifs are involved in inhibitory signaling because these
motifs provide binding sites for tyrosine phosphatases which block
signaling by removing tyrosine from activated receptors or signal
transduction molecules. While many of the details of the activation
and inhibitory mechanisms are unknown, it is clear that functional
balance in the immune system depends upon opposing activatory and
inhibitory signals.
[0003] One example of immune system activity that is regulated by a
balance of positive and negative signaling is B cell proliferation.
The B cell antigen receptor is a B cell surface immunoglobulin
which, when bound to antigen, mediates a positive signal leading to
B cell proliferation. However, B cells also express Fc.gamma.
RIIb1, a low affinity IgG receptor. When an antigen is part of an
immune complex with soluble immunoglobulin, the immune complex can
bind B cells by engaging both the B cell antigen receptor via the
antigen and Fc.gamma. RIIb1 via the soluble immunoglobulin.
Co-engagement of the Fc.gamma. RIIb1 with the B cell receptor
complex downmodulates the activation signal and prevents B cell
proliferation. Fc.gamma. RIIb1 receptors contain ITIM motifs which
are thought to deliver inhibitory signals to B cells via
interaction of the ITIMs with tyrosine phosphatases upon
co-engagement with B cell receptors.
[0004] The cytolytic activity of Natural Killer (NK) cells is
another example of immune system activity which is regulated by a
balance between positive signals that initiate cell function and
inhibitory signals which prevent the activity. The receptors that
activate NK cytotoxic activity are not fully understood. However,
if the target cells express cell-surface MHC class I antigens for
which the NK cell has a specific receptor, the target cell is
protected from NK killing. These specific receptors, known as
Killer Inhibitory Receptors (KIRs) send a negative signal when
engaged by their MHC ligand, downregulating NK cell cytotoxic
activity.
[0005] KIRs belong to the immunoglobulin superfamily or the C-type
lectin family (see Lanier et al., Immunology Today 17:86-91,1996).
Known human NK KIRs are members of the immunoglobulin superfamily
and display differences and similarities in their extracellular,
transmembrane and cytoplasmic regions. A cytoplasmic domain amino
acid sequence common to many of the KIRs is an ITIM motif having
the sequence YxxL/V. In some cases, it has been shown that
phosphorylated ITIMs recruit tyrosine phosphatases which
dephosphorylate molecules in the signal transduction pathway and
prevent cell activation (see Burshtyn et al., Immunity 4:77-85,
1996). The KIRs commonly have two of these motifs spaced apart by
26 amino acids [YxxL/V(x).sub.26YxxL/V]. At least two NK cell
receptors, each specific for a human leukocyte antigen (HLA) C
allele (an MHC class I molecule), exist as an inhibitory and an
activatory receptor. These receptors are highly homologous in the
extracellular portions, but have major differences in their
transmembrane and cytoplasmic portions. One of the differences is
the appearance of the ITIM motif in the inhibitory receptor and the
lack of the ITIM motif in the activating receptor (see Biassoni et
al., Journal. Exp. Med, 183:645-650, 1996).
[0006] An immunoreceptor expressed by mouse mast cells, gp49B 1,
also a member of the immunoglobulin superfamily, is known to
downregulate cell activation signals and contains a pair of ITIM
motifs. gp49B 1 shares a high degree of homology with human KIRs
(Katz et al., Cell Biology, 93: 10809-10814, 1996). Mouse NK cells
also express a family of immunoreceptors, the Ly49 family, which
contain the ITIM motif and function in a manner similar to human
KIRs. However, the Ly49 immunoreceptors have no structural homology
with human KIRs and contain an extracellular C-type lectin domain,
making them a member of the lectin superfamily of molecules (see
Lanier et al., Immunology Today 17.86-91, 1996).
[0007] Clearly, the immune system activatory and inhibitory signals
mediated by opposing kinases and phosphatases are very important
for maintaining balance in the immune system. Systems with a
predominance of activatory signals will lead to autoimmunity and
inflammation. Immune systems with a predominance of inhibitory
signals are less able to challenge infected cells or cancer cells.
Isolating new activatory or inhibitory receptors is highly
desirable for studying the biological signal(s) transduced via the
receptor. Additionally, identifying such molecules provides a means
of regulating and treating diseased states associated with
autoimmunity, inflammation and infection.
[0008] For example engaging a newly discovered cell surface
receptor having ITIM motifs with an agonistic antibody or ligand
can be used to downregulate a cell function in disease states in
which the immune system is overactive and excessive inflammation or
immunopathology is present. On the other hand, using an
antagonistic antibody specific to the receptor or a soluble form of
the receptor can be used to block the interaction of the cell
surface receptor with the receptor's ligand to activate the
specific immune function in disease states associated with
suppressed immune function. Conversely, since receptors lacking the
ITIM motif send activatory signals once engaged as described above,
the effect of antibodies and soluble receptors is the opposite of
that just described.
SUMMARY OF THE INVENTION
[0009] The present invention provides a new family of
immunoreceptor molecules of the immunoglobulin superfamily,
designated herein as the Leukocyte Immunoglobulin-Like Receptor
(LIR) polypeptides. Within the scope of the present invention are
DNA sequences encoding LIR family members and their deduced amino
acid sequences disclosed herein. Further included in the present
invention are polypeptides encoded by DNA that hybridize to
oligonucleotide probes having defined sequences or to DNA or RNA
complementary to the probes. The present invention also includes
recombinant expression vectors comprising DNA encoding LIR family
members. Also within the scope of the present invention are
nucleotide sequences which, due to the degeneracy of the genetic
code, encode polypeptides that are identical to polypeptides
encoded by the nucleic acid sequences described above, and
sequences complementary to those nucleotide sequences.
[0010] Further, the present invention includes processes for
producing polypeptides of the LIR family by culturing host cells
transformed with a recombinant expression vector that contains an
LIR family member encoding DNA sequence under conditions
appropriate for expressing an LIR polypeptide family member, then
recovering the expressed LIR polypeptide from the culture.
[0011] The invention also provides agonistic and antagonistic
antibodies to LIR family proteins.
[0012] Further still within the present invention are fusion
proteins that include a soluble portion of an LIR family member and
the Fc portion of Ig.
[0013] Certain autoimmune disorders are associated with the failure
of a negative signaling LIR to downregulate cell function. Such
disorders may be treated by administering a therapeutically
effective amount of an agonistic antibody or ligand of one or more
a LIR family member to a patient afflicted with such a disorder.
Disorders mediated by disease states associated with suppressed
immune function can be treated by administering a soluble form of
the negative signaling LIR. Conversely, disorders mediated by
diseases associated with failure of a activatory signaling LIR can
be treated by administering an agonistic antibody of the activatory
receptor. Disorders mediated by states associated with autoimmune
function can be treated by administering a soluble form of the
activatory receptor.
DETAILED DESCRIPTION OF THE INVENTION
[0014] A viral glycoprotein having a sequence similarity to MHC
class I antigens has been used to isolate and identify a new
polypeptide, designated LIR-P3G2, and several members of a new
family of cell surface polypeptides that has been designated the
LIR polypeptide family. The present invention encompasses isolated
nucleic acid molecules encoding LIR polypeptides, and further
encompasses isolated LIR polypeptides. Exemplary nucleic acids
encoding LIR polypeptides according to the present invention
include those nucleotide sequences shown in SEQ ID NOS:1, 3, 7, 9,
11, 13, 15, 17, 19, 21, 29, 31, 33, 35 and 37, and exemplary LIR
polypeptide sequences are shown in SEQ ID NOS:2, 4, 8, 10, 12, 14,
16, 18, 20, 22, 30, 32, 34, 36 and 38.
[0015] The LIR polypeptide family members possess extracellular
regions having immunoglobulin-like domains, placing the members in
a new subfamily of the immunoglobulin superfamily. While the LIR
family members are characterized as having very similar
extracellular portions, the family includes three groups of
polypeptides that are distinguishable by their transmembrane
regions and their cytoplasmic regions. One group of the LIR
polypeptides has a transmembrane region that includes a positively
charged residue and a short cytoplasmic tail and a second group has
a nonpolar transmembrane region and a long cytoplasmic tail. A
third group includes polypeptides expressed as soluble proteins
having no transmembrane region or cytoplasmic tail. One of the LIR
proteins has characteristics of both groups one and two, and may
represent a fourth group. A number of recent reports have described
nucleic acid molecules having sequences related to the LIR family
of proteins (Hillier et al., GenBank Accession Number N95687, Apr.
9, 1996; Colonna, M., GenBank Accession Nos. AF041261 and AFo41262,
Jan. 7, 1999; Lamerdin et al., GenBank Accession No. AC006293, Jan.
6, 1999; Steffans et al., GenBank Accession Nos. AH007466 and
AH007465, Mar. 4, 1999; Cosman et al., Immunity 7:273-282 (1997);
Borges et al., J. Imunol. 159:5192-96 (1997); Samaridis and
Colonna, Eur. J. Immunol 27:660-665 (1997); Colonna et al., J. Exp.
Med. 186:1809-1818 (1997); Wagtmann et al., Curr. Biol. 7:615-618
(1997); Rojo et al., J. Immunol. 158:9-12 (1997); Arm et al., J.
Immunol. 159:2342-2349(1997); Cella et al., J. Exp. Med.
185:1743-51 (1997); Torkar et al., Eur. J. Immunol. 28:3959-67
(1998); Yamashita et al., J. Biochem. 123:358-68 (1998); WO
98/31806; WO 98/24906; WO 98/09638).
[0016] The LIR polypeptides encompassed by the subject invention
contain at least one Ig-like domain in the extracellular region of
the protein, preferably contain either two or four Ig-like domains
in the extracellular region. Some LIR polypeptides may contain more
than four Ig-like domains. An Ig-like domain is a structural unit
that has been identified in a wide variety of cellular proteins.
Ig-like domains contain a common fold that forms a sandwich of two
.beta. sheets that is stabilized by a characteristic intrachain
disulfide bond. Ig-like domains are readily recognizable by
reference to a large body of knowledge concerning this structural
entity (see, e.g., Williams and Barclay, Ann. Rev. Immunol.
6:381-405 (1988)). Typically, Ig-like domains contain about 100
amino acids, although the number of amino acids may vary, e.g.,
from about 85 to 105 amino acids. Molecules that exhibit Ig-like
domains generally play a recognition role at the cell surface,
often mediating cell-cell interactions in a variety of biological
systems.
[0017] LIR-P3G2 (SEQ ID NO:2) is expressed by a variety of cells
and recognizes HLA-B44 molecules, HLA-A2 MHC molecules and the
alleles described in Example 14. Another LIR family member,
designated LIR-pbm8 (SEQ ID NO:9) is expressed by a variety of
cells and also recognizes a number of MHC class I molecules. By
analogy with known molecules, LIR-P3G2, LIR-pbm8 and LIR members
have a role in immune recognition and self/nonself
discrimination.
[0018] Examples 1-3 below describe isolating cDNA encoding P3G2
(LIR-P3G2) and a substantially identical polypeptide designated
18A3 (LIR-18A3). Briefly, the LIR-P3G2 family member was isolated
by first expressing UL18, a Class I MHC-like molecule and using
UL18 to isolate and identify P3G2 and 18A3, which are closely
related and probably are variants of the same gene, which is
designated "LIR-1." The nucleotide sequences of the isolated P3G2
cDNA and 18A3 cDNA are presented in SEQ ID NO: 1 and SEQ ID NO:3,
respectively. The amino acid sequences encoded by the cDNA
presented in SEQ ID NO:1 and SEQ ID NO:3 are presented in SEQ ID
NO:2 and SEQ ID NO:4, respectively. The P3G2 amino acid sequence
(SEQ ID NO:2) has a predicted extracellular domain of 458 amino
acids (1-458) including a signal peptide of 16 amino acids (amino
acids 1-16); a transmembrane domain of 25 amino acids (amino acids
459-483) and, a cytoplasmic domain of 167 amino acids (amino acids
484-650). The extracellular domain includes four
immunoglobulin-like domains. Ig-like domain I includes
approximately amino acids 17-118; Ig-like domain II includes
approximately amino acids 119-220; Ig-like domain III includes
approximately amino acids 221-318; and Ig-like domain IV includes
approximately amino acids 319419. Significantly, the cytoplasmic
domain of this polypeptide includes four ITIM motifs, each having
the consensus sequence of YxxL/V. The first ITIM motif pair is
found at amino acids 533-536 and 562-565 and the second pair is
found at amino acids 614-617 and 644-647. This feature is identical
to the ITIM motifs found in KIRs except that KIRs contain only one
pair of ITIM motifs.
[0019] The 18A3 amino acid sequence (SEQ ID NO:4) has a predicted
extracellular region of 459 amino acids (1-459) including a signal
peptide of 16 amino acids (amino acids 1-16); a transmembrane
domain of 25 amino acids (amino acids 460484) and a cytoplasmic
domain of 168 amino acids (485-652). The 18A3 amino acids sequence
(SEQ ID NO:4) is substantially identical to that of P3G2 (SEQ ID
NO:2) except that 18A3 has two additional amino acids (at amino
acid 438 and 552) and 18A3 possesses an isoleucine residue at amino
acid 142 in contrast to a threonine residue for P3G2. Additionally,
18A3 has a serine residue at amino acid 155 and P3G2 has an
isoleucine at 155. Finally, the 18A3 polypeptide has a glutamic
acid at amino acid 627 and P3G2 has a lysine at 625 which is
aligned with the 627 residue of the 18A3 polypeptide. The four ITIM
motifs in the 18A3 cytoplasmic domain are at amino acids 534-537
and 564-567 and at 616-619 and 646-649. Glycosylation sites occur
at the amino acid triplet Asn-X-Y, where X is any amino acid except
Pro and Y is Ser or Thr. Thus, potential glycosylation sites on
LIR-P3G2 occur at amino acids 140-142; 281-283; 302-304; and
341-343. Sites on LIR-18A3 are at 281-283; 302-304; and 341-343.
The features of these encoded polypeptides are consistent with type
I transmembrane glycoproteins.
[0020] Examples 8-10 describe isolating and identifying eight
additional LIR polypeptide family members by probing cDNA libraries
for plasmids that hybridize to a probe obtained from DNA encoding
the extracellular region of LIR-P3G2. The nucleotide sequences
(cDNA) of the isolated LIR family members are presented in SEQ ID
NO:7 (designated pbm25, or LIR4), SEQ ID NO:9 (designated pbm8, or
LIR-2), SEQ ID NO: 11 (designated pbm36-2, or LIR-6b), SEQ ID NO:
13 (designated pbm364, or LIR-6a); SEQ ID NO: 15 (designated pbmhh,
or LIR-7); SEQ ID NO: 17 (designated pbm2, or LIR-5), SEQ ID NO:19
(designated pbml7, or LIR-3) and SEQ ID NO:21 (designated pbmnew,
or LIR-8). The amino acid sequences encoded thereby are presented
in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO:22,
respectively.
[0021] Example 15 describes the isolation of LIR-9m1 (SEQ ID
NOS:29, 30), LIR-9m2 (SEQ ID NO:31, 32), LIR-9s1 (SEQ ID NO:33,
34), and LIR-9s2 (SEQ ID NO:35, 36), which are four alternatively
spliced variants of LIR-9, another new member of the LIR family.
The first step in identifying these LIR-9 group of clones was the
isolation of a short cDNA clone that was obtained from a human
dendritic cell library and whose sequence analysis indicated that
it had significant homology with the LIR family, particularly with
the sequences shown in SEQ ID NOS: 11, 13 and 15. Using PCR primers
based on this clone, further cloning efforts yielded four
full-length cDNAs corresponding to LIR-9m1, -9m2, -9s1 and -9s2.
LIR-9m1 and LIR-9m2 are transmembrane proteins that differ by 12
amino acids that are found in the extracellular region of LIR-9m1,
but that are absent from LIR9m2. These 12 amino acids correspond to
amino acids 29-40 of SEQ ID NO:30. LIRs-9s1 and -9s2 do not contain
a transmembrane domain, thus encode soluble versions of LIR-9. The
LIR-9s 1 polypeptide (SEQ ID NO:34) includes the 12 amino acid
insert that is present in LIR-9ml. Amino acids 1-238 of LIR-9s1
(SEQ ID NO:34) and LIR-9m1 (SEQ ID NO:30) are identical, but the
remainder of the LIR-9s1 sequence is not identical to the
corresponding region of LIR-9m1. Amino acids 1-226 of LIR-9s2 (SEQ
ID NO:36) are identical to the first 226 amino acids of LIR-9m2
(SEQ ID NO:32), but the remaining amino acid sequence of LIR-9s2
diverges from that of LIR-9m2.
[0022] The same PCR primers that were used to isolate the LIR-9
clones yielded an additional cloned LIR cDNA that has been
designated LIR-10 (SEQ ID NOS:37 and 38). By comparing the
nucleotide sequence of LIR-10 with the most closely related LIRs
that were previously identified, i.e, with SEQ ID NOS: 13 and 15,
it has been determined that the LIR-10 cDNA is an incomplete clone
that lacks sequences located at the 5' end of the corresponding
mRNA, including the 5' untranslated region, and nucleotides
encoding the first 26 amino acids of the LIR-10 protein.
[0023] The identified extracellular, transmembrane and cytoplasmic
regions for the polypeptides of LIR family members shown in SEQ ID
NOS:10, 12, 14, 16, 18, 20, 22, 30, 32, 34, 36 and 38 are presented
below. The polypeptides presented in SEQ ID NOS:8, 34 and 36 are
soluble proteins having no transmembrane or cytoplasmic regions. As
will be understood by the skilled artisan, the transmembrane region
of P3G2 and 18A3 described above and those of LIR polypeptide
family members presented below are identified in accordance with
conventional criteria for identifying hydrophobic domains
associated with such regions. Accordingly, the precise boundaries
of any selected transmembrane region may vary from those presented
herein. Typically, the transmembrane domain does not vary by more
than five amino acids on either end of the domain as described
herein. Computer programs known in the art and useful for
identifying such hydrophobic regions in proteins are available.
[0024] The polypeptide presented in SEQ ID NO:8 (LIR-pbm25) has an
extracellular domain that includes the entire amino acid sequence
of amino acids 1439 and a signal peptide of amino acids 1-16. The
amino acid sequence presented in SEQ ID NO: O (LIR-pbm8) has a
predicted extracellular region of 458 amino acids (1-458) including
a 16 amino acid signal peptide (amino acids 1-16); a transmembrane
domain that includes amino acids 459-483; and a cytoplasmic domain
that includes amino acids 484-598. The extracellular domain
includes four immunoglobulin-like domains and the cytoplasmic
domain includes an ITIM motif at amino acids 533-536 and
562-565.
[0025] The amino acid sequence presented in SEQ ID NO: 12
(LIR-pbm36-2) has a predicted extracellular domain of amino acids
including a 16 amino acid signal peptide of from amino acids 1-16;
a transmembrane domain which includes amino acids 262-280 and a
cytoplasmic domain of from amino acids 281-289. The transmembrane
domain includes a charged arginine residue at 264 and the
cytoplasmic domain is short, having only a length of only 9 amino
acids.
[0026] The amino acid sequence presented in SEQ ID NO:14
(LIR-pbm36-4) has a predicted extracellular domain of amino acids
1-461 including a signal peptide from amino acids 1-16; a
transmembrane domain that includes amino acids 462-480 and
possesses a charged arginine residue at amino acid 464; and a
cytoplasmic domain that includes amino acids 481-489. SEQ ID NO:14
is nearly identical to that of SEQ ID NO: 12 except that it
possesses four immunoglobulin domains in contrast to the two
domains found in the extracellular region of SEQ ID NO:12. The
amino acid sequences presented in SEQ ID NO:12 and SEQ ID NO:14 are
likely proteins encoded by alternatively spliced transcripts from
the same gene.
[0027] The amino acid sequence presented in SEQ ID NO:16
(LIR-pbmhh) has a predicted extracellular domain that includes
amino acids 1-449 and a signal peptide from amino acids 1-16; a
transmembrane domain that includes amino acids 450-468 with a
charged arginine residue at amino acid 452; and a cytoplasmic
domain that includes amino acids 469-483. The cytoplasmic domain is
short with a length of 15 amino acids. The extracellular domain
includes four immunoglobulin-like domains.
[0028] The amino acid sequence presented in SEQ ID NO:18 (LIR-pbm2)
has a predicted extracellular region that includes amino acids
1-259 and a signal peptide of amino acids 1-16; a transmembrane
domain that includes amino acids 260-280; and a cytoplasmic domain
that includes amino acids 281-448. This LIR family member has
cytoplasmic domain which includes an ITIM motif at amino acids
412-415 and 442-445. The extracellular domain includes two
immunoglobulin-like domains.
[0029] The amino acid sequence presented in SEQ ID NO:20
(LIR-pbm17) has a predicted extracellular domain of amino acids
1-443 that includes a signal peptide of amino acids 1-16; a
transmembrane domain which includes amino acids 444-464; and a
cytoplasmic domain of amino acids 465-631. The extracellular domain
has four immunoglobulin-like domains. SEQ ID NO:20 has two pairs of
ITIM YxxL/V motifs in the cytoplasmic domain. A first pair is at
amino acids 514-517 and 543-546, and a second pair is at amino
acids 595-598 and 625-628.
[0030] The amino acid sequence presented in SEQ ID NO:22
(LIR-pbmnew) has a predicted extracellular domain of amino acids
1-456 including a signal peptide of amino acids 1-16; a
transmembrane domain which includes amino acids 457-579; and a
cytoplasmic domain of amino acids 580-590. The extracellular
includes four immunoglobulin-like domains. SEQ ID NO:22 has an ITIM
motif at amino acids 554-557 and 584-587.
[0031] The LIR-9m1 protein has an extracellular domain located at
amino acids 1-262 of SEQ ID NO:30, including a signal peptide at
amino acids 1-34 of SEQ ID NO:30. Amino acids 263-284 of SEQ ID
NO:30 define the transmembrane region of LIR-9m1, and amino acids
285-299 of SEQ ID NO:30 form the cytoplasmic region. For LIR-9m2,
the extracellular region corresponds to amino acids 1-250 of SEQ ID
NO:32, including a signal sequence at amino acids 1-35 of SEQ ID
NO:32, a transmembrane region at residues 251-272 of SEQ ID NO:32,
and a cytoplasmic region at amino acids 273-287 of SEQ ID NO:32.
LIR-9s1 (SEQ ID NO:34) and LIR-9s2 (SEQ ID NO:36) consist,
respectively, of 265 and 253 amino acids, with their signal
sequences being found at amino acids 1-34 of SEQ ID NO:34, and
amino acids 1-35 of SEQ ID NO:36.
[0032] For LIR-10, amino acids 1-393 of SEQ ID NO:38 correspond to
most of the extracellular portion of the LIR-10 protein, though the
coding sequences for about 26 amino acids at the amino terminus of
this protein, including the signal peptide, are believed to be
missing from the LIR-10 cDNA clone that is described herein. The
transmembrane region of LIR-10 is defined by amino acids 394417 of
SEQ ID NO:38, and the intracellular region by amino acids 418449. A
single ITIM motif is located at amino acids 438443 of SEQ ID
NO:38.
[0033] The amino acid sequences presented in SEQ ID NO: 2, 4, 8,
10, 12, 14, 16, 18, 20, 22, 30, 32, 34, 36 and 38 reveal that the
LIR family, with the exception of LIR-10, can be categorized into
three groups of polypeptides. One group includes the polypeptides
of SEQ ID NOS: 12, 14, 16, 30 and 32, which are distinguishable by
a charged arginine residue in their transmembrane regions and their
short cytoplasmic regions. A second group includes SEQ ID NO: 2, 4,
10, 18, 20 and 22 which are distinguishable by their hydrophobic
cytoplasmic domains and the presence of one or more ITIM motifs in
their cytoplasmic regions. A third group includes the polypeptides
of SEQ ID NOS: 8, 34 and 36, which are expressed as soluble
polypeptides and have no transmembrane or cytoplasmic regions.
These soluble polypeptides may function to block the interactions
of cell surface family members with their receptors. Alternatively,
the soluble polypeptides may act as an activatory signal when bound
to the receptor. Like the members of group one, LIR-10 has a
relatively short cytoplasmic domain and a charged residue in its
transmembrane domain, though its charged residue is histidine
instead of arginine. However, LIR-10 also has an ITIM motif in its
cytoplasmic domain, like the members of group two. Thus, LIR-10 has
some of the characteristics of both groups one and two, and may
represent a fourth group of LIR proteins. The LIR polypeptides are
characterized generally by the ability of their encoding DNA to
hybridize to DNA encoding the P3G2 extracellular region.
[0034] The invention should be understood to encompass isolated
nucleic acid molecules encoding LIR polypeptides having the amino
acid sequences shown in SEQ ID NOS:2, 4, 8, 10, 12, 14, 16, 18, 20,
22, 30, 32, 34, 36 and 38. In one embodiment of the invention,
these nucleic acid molecules have the nucleic acid sequences shown
in SEQ ID NOS:1, 3, 7, 9, 11, 13, 15, 17, 19, 21, 29, 31, 33,
35and37.
[0035] The extracellular regions of the LIR family member proteins
presented in SEQ ID NO:2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 30, 32,
34, 36 and 38 have a high degree of homology, which varies from
59%-84. Several of the LIR isolates are closely related, thus must
represent allelic variants or splicing variants. For example, the
extracellular regions of SEQ ID NO: 12 and SEQ ID NO: 14 share
sequence homology which is close to 100%, thus indicating that
these polypeptides derive from the same gene. In addition, SEQ ID
NOS:2 and 4 share sequence homology that is in excess of 95%, thus
probably represent two alleles of the same gene. Moreover, as
discussed above, the extracellular regions of SEQ ID NOS:30, 32, 34
and 36 are nearly identical, thus indicting that these four
proteins derive from mRNAs that are splicing variants.
[0036] While sharing some structural similarities with other
members of the immunoglobulin superfamily, the LIR family members
have limited homology to other members of the immunoglobulin
superfamily. Molecules having the closest structural similarity to
the LIRs are the human KIRs and mouse gp49. However, LIR
extracellular regions share only a 38-42% identity with the
extracellular regions of NKAT3 and p58 Cl-39, respectively. The
extracellular regions of the LIR family members are only 35-47%
homologous with that of mouse gp49. In contrast, KIRs in general
are known to share at least a 80% amino acid identity, with NKAT3
and p58 CL-39 being 81% homologous. Additionally, none of the known
KIR molecules has four extracellular immunoglobulin domains which
is characteristic of all but two of the known LIR family members.
In view of the high sequence homology among the LIR related
polypeptides disclosed herein and their relatively low homology
with KIRs, the LIR polypeptides are members of a new family of
immunoregulators.
[0037] An analysis of the amino acid sequences of the LIR
polypeptides reveals that specific stretches of amino acids of the
LIR polypeptides are highly conserved. One conserved region is a
sequence of 46 amino acids found at amino acids 5-50 of SEQ ID
NO:2. A data base search determined that the LIR family members
differ substantially from the most structurally similar prior art
polypeptides in this LIR conserved region. The data base search and
structural analysis was performed using BLAST NB1, a local
alignment search tool for searching data bases and aligning amino
acid sequences to determine identities and variations in a given
sequence. The BLAST NB1 software is accessible on the internet at
http://www3.ncb1.nlm.nih.gov/entrez/blast. The BLAST NB 1 search
for sequences having homology to the sequence of amino acids 5 to
50 of SEQ ID NO:2 found that the most structurally similar proteins
are Fc.gamma.IIR, gp49B form 2, and gp49B form 1 having identities
with amino acids 5 to 50 of SEQ ID NO:2 of 63%, 67%, and 67%
respectively. This contrasts with an LIR family identity with amino
acids 5 to 50 of SEQ ID NO:2 which ranges from about 71% to 100%.
Specifically, LIR family members of the present invention contain
conserved regions near their amino termini having the following
identities with amino acids 5-50 of SEQ ID NO:2: SEQ ID NO:8 has a
96% identity; SEQ ID NO:10 has a 90% identity; SEQ ID NO:12 has a
96% identity; SEQ ID NO: 14 has a 91% identity; SEQ ID NO: 16 has a
97% identity; SEQ ID NO:18 has a 77% identity; SEQ ID NO:20 has an
80% identity; SEQ ID NO:22 has an 80% identity; SEQ ID NO:30 has a
78% identity; SEQ ID NO:32 has a 71% identity; SEQ ID NO:34 has a
78% identity; SEQ ID NO:36 has a 71% identity. This conserved
region appears to be present also in LIR-10 (SEQ ID NO:38), but is
incomplete due to the LIR-10 cDNA clone disclosed herein being
truncated at its 5' end.
[0038] Sequence identity as used herein is the number of aligned
amino acids which are identical, divided by the total number of
amino acids in the shorter of the two sequences being compared. A
number of computer programs are available commercially for aligning
sequences and determining sequence identities and variations. These
programs provide identity information based upon the above stated
definition of identity. One suitable computer program is the GAP
program, version 6.0, described by Devereux et al. (Nucl. Acids
Res. 12:387, 1984) and available from the University of Wisconsin
Genetics Computer Group (UWGCG). The GAP program utilizes the
alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443,
1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482,
1981). The preferred default parameters for the GAP program
include: (1) a unary comparison matrix (containing a value of 1 for
identities and 0 for non-identities) for nucleotides or amino
acids, and the weighted comparison matrix of Gribskov and Burgess,
Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and
Dayhoff, eds., Atlas of Protein Sequence and Structure, National
Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of
3.0 for each gap and an additional 0.10 penalty for each symbol in
each gap; and (3) no penalty for end gaps. Another similar program,
also available from the University of Wisconsin as part of the GCG
computer package for sequence manipulation is the BESTFIT
program.
[0039] In another aspect, the polypeptides of the present invention
have conserved regions which are uniquely characterized as having
the amino acid sequence (SEQ ID NO:28):
[0040] Leu Xaa.sub.a Leu Ser Xaa.sub.b Xaa.sub.c Pro Arg Thr
Xaa.sub.d Xaa.sub.e Gln Xaa.sub.f Gly Xaa.sub.g Xaa.sub.h Pro
Xaa.sub.i Pro Thr Leu Trp Ala Glu Pro Xaa.sub.j Ser Phe Ile
Xaa.sub.j Xaa.sub.70 Ser Asp Pro Lys Leu Xaa.sub.k Leu Val
Xaa.sub.m Thr Gly,
[0041] where Xaa.sub.a is Gly or Arg; Xaa.sub.b is Leu or Val;
Xaa.sub.c is Gly or Asp; Xaa.sub.d is His Arg or Cys; Xaa.sub.e is
Val or Met; Xaa.sub.f is Ala or Thr; Xaa.sub.g is His Pro or Thr;
Xaa.sub.h Leu Ile or Phe; Xaa.sub.i is Gly Asp or Ala; Xaa.sub.j is
Thr Ile Ser or Ala; Xaa.sub.k is Gly or Val; Xaa.sub.m is Met or
Ala; and Xaa.sub.70 is a sequence of 70 amino acids.
[0042] As mentioned above, certain LIR family members have ITIM
motifs (YxxL/V.sub.25-.sub.26YxxL/V) in their cytoplasmic domains.
It is known that many immune regulating receptors such as KIRs,
CD22, Fc.gamma.RIIb1 also have ITIMs in their cytoplasmic domain
and function to send inhibitory signals which down regulate or
inhibit cell function. It has been shown that these receptors
associate with SHP-1 phosphatase via binding to the ITIM motifs.
Recruitment of the SHP-1 phosphatase by the receptor appears to be
required for intracellular signaling pathways that regulate the
inhibitory function of the receptors. The experiment described in
Example 11 demonstrates that LIR-P3G2 and LIR-pbm8 polypeptides
associate with SHP-1 phosphatase upon phosphorylation and generate
inhibitory signals through monocyte activation pathways. It is
known that many immune regulating receptors such as KIRs, CD22,
Fc.gamma.RIIIb1 have ITIMs in their cytoplasmic domain and function
to send inhibitory signals which down regulate or inhibit cell
function. Thus, by analogy with KIRs, CD22 and Fc.gamma.RIIb1, LIR
family members presented in SEQ ID NO:2, 4, 10, 18, 20, 22 and 38
that have ITIM motifs deliver an inhibitory signal via the
interaction of its ITIM with SHP-1 tyrosine phosphatase, or other
tyrosine phosphatases, when the LIR is coligated with an
appropriate receptor. Also by analogy with immunoregulatory
receptors possessing ITIMs, LIR family members have a regulatory
influence on humoral, inflammatory and allergic responses.
[0043] The LIR family members presented in SEQ ID NO: 12, 14, 16,
30 and 32 have relatively short cytoplasmic domains, have
transmembrane regions possessing at least one charged residue, and
do not possess the ITIM motif. By analogy with membrane proteins
that lack ITIM motifs and have charged transmembrane regions, these
family members mediate stimulatory or activatory signals to cells.
For example, membrane bound proteins containing a charged residue
in the transmembrane regions are known to associate with other
membrane-bound proteins that possess cytoplasmic tails having
motifs known as immunoreceptor tyrosine-based activation motifs
(ITAM). Upon association, the ITAMs become phosphorylated and
propagate an activation signal.
[0044] The LIR polypeptide designated LIR-P3G2 is expressed on the
surface of transfected or normal cells. This is evidenced by the
results of the experiments described in Example 3 and Example 5 in
which flow cytometry and precipitation techniques demonstrate that
LIR-P3G2 is found on monocytes, a subpopulation of NK cells, and B
cells. P3G2 was detected on small subset of T cells. P3G2 is
expressed as a 110-120 kDa glycoprotein. Since P3G2 has four
potential glycosylation sites, the molecular size of this protein
will vary with the degree of its glycosylation. Glycosylation sites
occur at the amino acid triplet Asn-X-Y, where X is any amino acid
except Pro and Y is Ser or Thr. Potential glycosylation sites on
P3G2 occur at amino acids 139-141; 280-282; 302-304; and
340-342.
[0045] P3G2-LIR isolated as described in Example 3 was tested for
its ability to bind to cell surface ligands distinct from UL18. As
demonstrated by the experimental results detailed in Example 7,
P3G2 binds HLA-B 44 and HLA-A2, class I MHC antigens. Similarly, as
demonstrated in Example 14, LIR-P3G2 and LIR-pbm8 bind to a variety
of HLA-A, -B, and -C alleles and recognize a broad spectrum of MHC
class I specificities. Since Class I MHC molecules play a central
role in immune surveillance, self/non-self discrimination, the
immune response to infection etc., the LIR-P3G2 and LIR-pbm8
polypeptides have a role in regulation of immune responses. It is
known that NK cytolytic activity for killing tumor cells and cells
infected with a virus is regulated by a delicate modulation of
activatory and inhibitory signals. It has been shown that receptors
specific for the same HLA class I molecules to which LIR-P3G2 and
LIR-pbm8 bind may be activatory or inhibitory in their triggering
mechanism. By analogy, LIR-P3G2 and LIR-pbm8, which bind MHC class
I molecules, play a role in balancing immune system cell activity
and are useful in treating disease states in which the immune
system balance is disrupted.
[0046] Within the scope of the present invention are polypeptides
which include amino acid sequences encoded by DNA that hybridizes
to LIR-P3G2 extracellular DNA probes under moderate to highly
stringent conditions as taught herein. Probes that hybridize to DNA
that encode polypeptides of the present invention include probes
which encompass nucleotides 310-1684 of SEQ ID NO: 1 or fragments
thereof. Fragments of SEQ ID NO: 1 utilized as hybridization probes
are preferably greater than 17 nucleotides in length, and more
typically are greater than 20 nucleotides in length, and may
include nucleotides 358-1684; nucleotides 322-459 (encoding LIR
conserved sequence); or DNA or RNA sequences complementary to SEQ
ID NOS:5, 6, 23, 24, 27 and 1 or fragments thereof. Fragments of
SEQ ID NOS:5, 6, 23, 24 and 27 include these sequences without the
restriction sites. The nucleotide sequences described herein also
can be used to design PCR primers, for which a convenient length is
about 17-30 nucleotides.
[0047] Conditions for hybridization may be moderately stringent
conditions described in, for example, in Sambrook et al, Molecular
Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory Press, 1989, which is hereby incorporated by reference
(see, e.g., Vol. 1, pp 1.101-104). Conditions of moderate
stringency, as defined by Sambrook et al., include, for example,
the use of a prewashing solution containing 5.times.SSC, 0.5% SDS,
1.0 mM EDTA (pH 8.0) and hybridization conditions of about
55.degree. C. in 5.times.SSC, incubated overnight. Highly stringent
conditions include higher temperatures of hybridization and
washing. The skilled artisan will recognize that a given degree of
stringency may be maintained while varying the hybridization or
wash temperature or composition of the hybridization buffer in
accord with formulae known to those in the art (e.g., see Sambrook
et al., 9.50-9.51 and 11.45-11.47). Such formulae take into account
factors such as the length of the probe, the G+C content of the
probe, salt concentration of the hybridization buffer. If desired,
formamide may be added to the hybridization buffer, which permits
the use of lower hybridization temperatures (e.g., see Sambrook et
al., 9.50-9.51).
[0048] Preferred embodiments include amino acid sequences encoded
by DNA that hybridizes to probes of the extracellular region of
LIR-P3G2 having at least 17 nucleotides. Preferred hybridizing
conditions include an incubation temperature of 63.degree. C for 16
hours in a solution of Denhart's solution, 0.05 M TRIS at pH 7.5,
0.9 M NaCl, 0.1% sodium pyrophosphate, 1% SDS and 200 .mu.g/mL
salmon sperm DNA, followed by washing with 2.times.SSC at
63.degree. C. for one hour and then washing with 1.times.SSC at
63.degree. C. for one hour. However, as explained above, one
skilled in the art can devise other hybridization conditions that
produce the same degree of stringency. Generally, stringent
hybridization conditions involve a combination of buffer and
incubation temperature that supports the formation of specific,
i.e., well-matched duplexes while still allowing the formation of
stable duplexes at an acceptable rate. Conditions of reduced
stringency permit the formation of stable duplexes containing a
higher degree of mismatched base pairs than can form under more
stringent conditions.
[0049] Stringent hybridization conditions for PCR primers can be
achieved, for example, by hybridizing labeled probes to
filter-bound target nucleic acid overnight at 50-55.degree. C. in
aqueous buffer containing 5.times.SSC or 6.times.SSC (1
.times.SSC=0.15 M NaCl, 0.015 M sodium citrate), followed by washes
in 6.times.SSC at 50-55.degree. C. However, the skilled artisan
will recognize that stringent hybridization conditions for
oligonucleotide probes will vary, depending on the length, base
composition and sequence of the probe (e.g., see Sambrook et al.,
11.45-11.49).
[0050] The present invention includes polypeptides having amino
acid sequences that differ from, but are highly homologous to,
those presented in SEQ ID NOS:2, 4, 8, 10, 12, 14, 16, 18, 20, 22,
30, 32, 34, 36 and 38. Examples include, but are not limited to,
homologs derived from other mammalian species, variants (both
naturally occurring variants and those generated by recombinant DNA
technology), and LIR P3G2 and LIR family member fragments that
retain a desired biological activity. Preferably, such polypeptides
exhibit a biological activity associated with the LIR polypeptides
described in SEQ ID NOS:2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 30,
32, 34, 36 and 38, and comprise an amino acid sequence that is at
least 80% identical to any of the amino acid sequences of the
signal peptide and extracellular domains of the polypeptides
presented in SEQ ID NOS:2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 30,
32, 34, 36 and 38. Preferably such polypeptides are at least 90%
identical to any of the amino acid sequences of the signal peptide
and extracellular domains of the polypeptides presented in SEQ ID
NOS: 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 30, 32, 34, 36 and 38.
Determining the degree of identity between polypeptides can be
achieved using any algorithms or computer programs designed for
analyzing protein sequences. The commercially available GAP program
described below is one such program. Other programs include the
BESTFIT and GCG programs which are also commercially available.
[0051] Within the scope of the present invention are LIR
polypeptide fragments that retain a desired biological property of
an LIR polypeptide family member such as binding to MHC class I or
other ligand. In one such embodiment, LIR polypeptide fragments are
soluble LIR polypeptides comprising all or part of the
extracellular domain, but lacking the transmembrane region that
would cause retention of the polypeptide on a cell membrane.
Soluble LIR polypeptides are capable of being secreted from the
cells in which they are expressed. Advantageously, a heterologous
signal peptide is fused to the N-terminus such that the soluble LIR
is secreted upon expression. Soluble LIR polypeptides include
extracellular domains incorporating the signal peptide and those in
which the signal peptide is cleaved signal peptide.
[0052] The use of soluble forms of a LIR family member is
advantageous for certain applications. One such advantage is the
ease of purifying soluble forms from recombinant host cells. Since
the soluble proteins are secreted from the cells, the protein need
not be extracted from cells during the recovery process.
Additionally, soluble proteins are generally more suitable for
intravenous administration and can be used to block the interaction
of cell surface LIR family members with their ligands in order to
mediate a desirable immune function.
[0053] Further encompassed within the present invention are soluble
LIR polypeptides, which may include the entire extracellular domain
or any desirable fragment thereof, including extracellular domains
that exclude signal peptides. Thus, for example, soluble LIR
polypeptides include amino acids x,458 of SEQ ID NO:2, where
x.sub.1 is amino acids 1 or 17; amino acids x.sub.2-459 of SEQ ID
NO:4, where x.sub.2 is amino acid 1 or 17; amino acids x.sub.3-439
of SEQ ID NO:8, where x.sub.3 is amino acid 1 or 17; amino acids
x.sub.4-458 of SEQ ID NO:10, where x.sub.4 is amino acid 1 or 17;
amino acids x.sub.5-241 of SEQ ID NO:12, where amino acid x.sub.5
is amino acid 1 or 17; amino acids x.sub.6-461 of SEQ ID NO: 14,
where x.sub.6 is amino acid 1 or 17; amino acids x.sub.7-449 of SEQ
ID NO: 16, where x.sub.7 is amino acid 1 or 17; amino acids
x.sub.8-259 of SEQ ID NO:18, where x.sub.8 is amino acid 1 or 17;
amino acids x.sub.9-443 of SEQ ID NO:20, where x.sub.9 is amino
acid 1 or 17; amino acids x.sub.10-456 of SEQ ID NO:22, where
x.sub.10 is amino acid 1 or 17; amino acids x.sub.11-262 of SEQ ID
NO:30, where x.sub.11 is amino acid 1 or 35; amino acids
x.sub.12-250 of SEQ ID NO:32, where x.sub.12 is amino acid 1 or 36;
amino acids x13of SEQ ID NO:34, where x.sub.13 is amino acid 1 or
35; amino acids x.sub.14of SEQ ID NO:36, where x.sub.14 is amino
acid 1 or 36; and amino acids 1-393 of SEQ ID NO:38. The above
identified soluble LIR polypeptides include LIR extracellular
regions that include and exclude signal peptides. Also encompassed
herein are LIRs that lack a transmembrane and cytoplasmic region,
such as SEQ ID NOS:8, 34 and 36. Additional soluble LIR
polypeptides include fragments of the extracellular domains of
family members that retain a desired biological activity, such as
binding to ligands that include MHC class I molecules.
[0054] LIR family member fragments, including soluble polypeptides,
may be prepared by any of a number of conventional techniques. A
DNA sequence encoding a desired LIR polypeptide encoding fragment
may be subcloned into an expression vector for production of the
LIR polypeptide fragment. The selected encoding DNA sequence
advantageously is fused to a sequence encoding a suitable leader or
signal peptide. The desired LIR member encoding DNA fragment may be
chemically synthesized using known DNA synthesis techniques. DNA
fragments also may be produced by restriction endonuclease
digestion of a full length cloned DNA sequence, and isolated by
electrophoresis on an appropriate gel. If necessary,
oligonucleotides that reconstruct the 5' or 3' terminus to a
desired point may be ligated to a DNA fragment generated by
restriction enzyme digestion. Such oligonucleotides may
additionally contain a restriction endonuclease cleavage site
upstream of the desired coding sequence, and position an initiation
codon (ATG) at the N-terminus of the coding sequence.
[0055] Another technique useful for obtaining a DNA sequence
encoding a desired protein fragment is the well-known polymerase
chain reaction (PCR) procedure. Oligonucleotides which define the
termini of the desired DNA are used as primers to synthesize
additional DNA from a desired DNA template. The oligonucleotides
may also contain recognition sites for restriction endonucleases,
to facilitate inserting the amplified DNA fragment into an
expression vector. PCR techniques are described, for example, in
Saiki et al., Science 239:487(1988): Recombinant DNA Methodology,
Wu et al., eds., Academic Press, Inc., San Diego (1989), pp.
189-196; and PCR Protocols: A Guide to Methods and Applications,
Innis et al., eds., Academic Press, Inc. (1990).
[0056] The LIR nucleic acid molecules of the present invention
include isolated cDNA, chemically synthesized DNA, DNA isolated by
PCR, cloned genomic DNA, and combinations thereof. Genomic LIR
family DNA may be isolated by hybridization to the LIR family cDNA
disclosed herein using standard techniques. Isolated RNA
transcribed from LIR family DNA molecules is also encompassed by
the present invention.
[0057] Within the scope of the present invention are DNA fragments
such as LIR polypeptide coding regions and DNA fragments that
encode soluble polypeptides. Examples of DNA fragments that encode
soluble polypeptides include DNA that encodes entire extracellular
regions of LIR family members and DNA that encodes extracellular
region fragments such as regions lacking the signal peptide. More
specifically, the present invention includes nucleotides 310-2262
of SEQ ID NO:1 (P3G2 coding region); nucleotides x.sub.1-1683 of
SEQ ID NO:1, where x.sub.1 is 310 or 358 (encoding the P3G2
extracellular domain); nucleotides 168-2126 of SEQ ID NO:3 (the
18A3 coding region) and nucleotides x.sub.2-1544 of SEQ ID NO:3,
where x.sub.2 is 168 or 216 (the 18A3 extracellular domain coding
region); nucleotides x3 -1412 of SEQ ID NO:7, where x.sub.3 is 93
or 141 (the pbm25 coding region and extracellular region);
nucleotides 184-1980 of SEQ ID NO:9, (the pbm8 coding region) and
nucleotides x.sub.4-1557 of SEQ ID NO:9, where x.sub.3 is 184 or
232 (the pmb8 extracellular domain coding region); nucleotides
171-1040 of SEQ ID NO:11 (pbm36-2 coding region) and nucleotides
x.sub.5-878 of SEQ ID NO:11, where x.sub.5 is 171 or 219 (encoding
the pbm36-2 extracellular domain); nucleotides 183-1652 of SEQ ID
NO:13 (coding region for pbm36-4) and nucleotides x.sub.6-1565 of
SEQ ID NO:13, where x.sub.6 is 183 or 231 (encoding the pbm3-64
extracellular domain); nucleotides 40-1491 of SEQ ID NO:15 (the
pbmhh coding region) and nucleotides x.sub.7-1386 of SEQ ID NO:15,
where x.sub.7 is 40 or 88 (encoding the pbmhh extracellular
domain); nucleotides 30-1376 of SEQ ID NO:17 (the pbm2 coding
region) and nucleotides x.sub.8-806 of SEQ ID NO:17, where x.sub.8
is 30 or 78 (encoding the pbm2 extracellular region); nucleotides
66-1961 of SEQ ID NO:19 (the pbm17 coding region) and nucleotides
x.sub.9-1394 of SEQ ID NO:19, where x.sub.9 is 66 or 114 (encoding
the pbm17 extracellular domain); nucleotides 67-1839 of SEQ ID
NO:21 (the pbmnew coding region) and nucleotides x.sub.10-1434 of
SEQ ID NO:21, where x.sub.10 is 67 or 115 (encoding the pbmnew
extracellular domain); nucleotides 69-968 of SEQ ID NO:29 (the
coding region of LIR-9m1) and nucleotides x.sub.11-854 of SEQ ID
NO:29, where x.sub.11 is 69 or 170 (encoding the LIR-9m1
extracellular domain); nucleotides 95-958 of SEQ ID NO:31 (the
LIR-9m2 coding region) and nucleotides x.sub.12-844 of SEQ ID
NO:31, where x.sub.12 is 95 or 200 (encoding the LIR-9m2
extracellular domain); nucleotides x.sub.13-912 of SEQ ID NO:33,
where x.sub.13 is 115 or 216 (the LIR-9s1 coding region and
extracellular region); nucleotides x.sub.14-834 of SEQ ID NO:35,
where x.sub.14 is 73 or 178 (the LIR-9s2 coding region and
extracellular region); nucleotides 1-1350 of SEQ ID NO:37 (the
LIR-10 coding region) and nucleotides 1-1179 of SEQ ID NO:37
(encoding all but a few amino-terminal amino acids of the LIR-10
extracellular domain).
[0058] Included in the present invention are DNAs encoding
biologically active fragments of the LIR proteins whose amino acid
sequences are presented in SEQ ID NOS:2, 4, 8, 10, 12, 14, 16, 18,
20, 22, 30, 32, 34, 36 and 38.
[0059] The present invention encompasses nucleotide sequences
which, due to the degeneracy of the genetic code, encode
polypeptides that are identical to polypeptides encoded by the
nucleic acid sequences described above, and sequences complementary
to them. Accordingly, within the present invention are DNA encoding
biologically active LIR family members that include the coding
region of a native human LIR family member cDNA, or fragments
thereof, and DNA that is degenerate as a result of the genetic code
to the native LIR polypeptide DNA sequence or the DNA of native LIR
family members described herein.
[0060] In another aspect, the present invention includes LIR
variants and derivatives as well as variants and derivatives of LIR
family polypeptides, both recombinant and non-recombinant, that
retain a desired biological activity. An LIR variant, as referred
to herein, is a polypeptide substantially homologous to a native
LIR polypeptide, as described herein, except the variant amino acid
sequence differs from that of the native polypeptide because of one
or more deletions, insertions or substitutions.
[0061] LIR family variants may be obtained from mutations of native
LIR nucleotide sequences. Within the present invention are such DNA
mutations or variants that include nucleotide sequences having one
or more nucleotide additions, nucleotide deletions, or nucleotide
substitutions compared to native DNA of LIR family members and that
encode variant LIR polypeptides or variant LIR family members
having a desired biological activity. Preferably the biological
activity is substantially the same as that of the native LIR
polypeptide.
[0062] Variant amino acid sequences and variant nucleotide
sequences of the present invention preferably are at least 80%
identical to that of a native LIR family member sequence. One
method for determining the degree of homology or identity between a
native amino acid or nucleotide sequence and a variant amino acid
or nucleotide sequence is to compare the sequences using computer
programs available for such purposes. One suitable computer program
is the GAP program, version 6.0, described by Devereux et al.
(Nucl. Acids Res. 12:387, 1984) and available from the University
of Wisconsin Genetics Computer Group (UWGCG). The GAP program
utilizes the alignment method of Needleman and Wunsch (J. Mol.
Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl.
Math 2:482, 1981). Briefly, the GAP program defines identity as the
number of aligned symbols (i.e., nucleotides or amino acids) which
are identical, divided by the total number of symbols in the
shorter of the two sequences being compared. The preferred default
parameters for the GAP program include: (1) a unary comparison
matrix (containing a value of 1 for identities and 0 for
non-identities) for nucleotides, and the weighted comparison matrix
of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as
described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence
and Structure, National Biomedical Research Foundation, pp.
353-358, 1979; (2) a penalty of 3.0 for each gap and an additional
0.10 penalty for each symbol in each gap; and (3) no penalty for
end gaps.
[0063] Alterations of native LIR amino acid sequences may be
provided by using any of a number of known techniques. As described
above, mutations can be introduced at selected sequence sites by
synthesizing oligonucleotides containing a mutant coding sequence,
flanked by restriction sites enabling its ligation to fragments of
the native sequence. After ligating the synthesized
oligonucleotides to the native sequence fragments, the resulting
reconstructed nucleotide sequence will encode an analog or variant
polypeptide having the desired amino acid insertion, substitution,
or deletion. Another procedure suitable for preparing variant
polypeptides is oligonucleotide-directed site-specific mutagenesis
procedures which provide genes having specific codons altered in
accordance with the desired substitution, deletion, or insertion.
Techniques for making such alterations include those disclosed in
the following references: Walder et al. Gene, 42:133, 1986; Bauer
et al.,Gene 37:73, 1985; Craik, BioTechniques, 12-19 January, 1985;
Smith et al. Genetic Engineering: Principles and Methods, Plenum
Press, 1981; and U.S. Pat. Nos. 4,518,584 and 4,737,462, all of
which are incorporated herein by reference.
[0064] Variant polypeptides of the present invention may have amino
acid sequences which are conservatively substituted, meaning that
one or more amino acid residues of a native LIR polypeptide family
member is replaced by different residues, such that the variant
polypeptide retains a desired biological activity that is
essentially equivalent to that of a native LIR family member. In
general, a number of approaches to conservative substitutions are
well known in the art and can be applied in preparing variant of
the present invention. For example, amino acids of the native
polypeptide sequence may be substituted for amino acids which do
not alter the secondary and/or tertiary structure of the LIR
polypeptide. Other suitable substitutions include those which
involve amino acids outside of the ligand-binding domain of
interest. One approach to conservative amino acid substitutions
involves replacing one or amino acids with those having similar
physiochemical characteristics, e.g. substituting one aliphatic
residue for another such as Ile, Val, Leu, or Ala for one another);
substituting one polar residue for another (such as between Lys and
Arg; Glu and Asp; or Gln and Asn); or substituting entire regions
having similar hydrophobicity or hydrophilic characteristics.
[0065] LIR polypeptide variants can be tested for binding to cells
as described in Examples 5 and 6 and for phosphatase binding
activity as described in Example 11 to confirm biological activity.
Other LIR variants within the present invention include
polypeptides which are altered by changing the nucleotide sequence
encoding the polypeptide so that selected polypeptide Cys residues
are deleted or replaced with one or more alternative amino acids.
These LIR variants will not form intramolecular disulfide bridges
upon renaturation. Naturally occurring LIR polypeptides selected
for alteration by deleting or altering Cys residues preferably do
not have biological activities which depend upon disulfide bridges
formed by the Cys residue. Other possible variants are prepared by
techniques which cause the modification of adjacent dibasic amino
acid residues to enhance expression in yeast systems in which KEX2
protease activity is present. EP 212,914 discloses site-specific
mutagenesis techniques for inactivating KEX2 protease processing
sites in a protein. KEX2 protease processing sites are inactivated
by deleting, adding or substituting residues to alter Arg-Arg,
Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these
adjacent basic residues. Lys-Lys and pairings are considerably less
susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg
to Lys-Lys represents a conservative and preferred approach to
inactivating KEX2 sites.
[0066] Naturally occurring LIR variants are also encompassed by the
present invention. Examples of such variants are proteins that
result from alternative MRNA splicing events or from proteolytic
cleavage of an LIR polypeptide. Alternative splicing of mRNA may
yield a truncated but biologically active LIR polypeptide such as a
naturally occurring soluble form of the protein. Variations
attributable to proteolysis include difference in the N- or C-
termini upon expression in different types of host cells, due to
proteolytic removal of one or more terminal amino acids from the
LIR polypeptide. In addition, proteolytic cleavage may release a
soluble form of LIR from a membrane-bound form of the polypeptide.
Other naturally occurring LIR variations are those in which
differences from the amino acid sequence of SEQ ID Nos:2, 4, 8, 10,
12, 14, 16, 18, 20, 22, 30, 32, 34, 36 and 38 are attributable to
genetic polymorphism, the allelic variation among individuals.
[0067] Within the scope of the present invention are derivative LIR
family polypeptides which include native or variant LIR
polypeptides modified to form conjugates with selected chemical
moieties. The conjugates can be formed by covalently linking
another moiety to a native or variant LIR or by non-covalently
linking another moiety to a native or variant LIR. Suitable
chemical moieties include but are not limited to glycosyl groups,
lipids, phosphates, acetyl groups, and other proteins or fragments
thereof. Techniques for covalently linking chemical moieties to
proteins are well known in the art and are generally suitable for
preparing derivative LIR polypeptides. For example, active or
activated functional groups on amino acid side chains can be used
as reaction sites for covalently linking a chemical moiety to a LIR
polypeptide. Similarly, the N-terminus or C-terminus can provide a
reaction site for a chemical moiety. LIR polypeptides or fragments
conjugated with other proteins or protein fragments can be prepared
in recombinant culture as N-terminal or C-terminal fusion products.
For example, the conjugate or fusion portions may include a signal
or leader sequence attached to an LIR molecule at its N-terminus.
The signal or leader peptide co-translationally or
post-translationally directs transfer of the conjugate from its
site of synthesis to a site inside or outside of the cell
membrane.
[0068] One useful LIR polypeptide conjugate is one incorporating a
poly-His or the antigenic identification peptides described in U.S.
Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1124, 1988.
For example, the FLAG .RTM. peptide,
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:39) is highly antigenic
and provides an epitope reversibly bound by a specific monoclonal
antibody, thus enabling rapid assay and facile purification of
expressed recombinant protein. This sequence is specifically
cleaved by bovine mucosal enterokinase at the residue immediately
following the Asp-Lys pairing. Fusion proteins capped with this
peptide may be resistant to intracellular degradation in E. coli.
Murine hybridoma designated 4E11 produced a monoclonal antibody
that binds the peptide of SEQ ID NO:39 in the presence of certain
divalent metal cations, and has been deposited with the American
Type Culture Collection under accession no HB 9259. Expression
systems useful for producing recombinant proteins fused to the
FLAG.RTM. peptide, and monoclonal antibodies that bind the peptide
and are useful in purifying the recombinant proteins, are available
from Eastman Kodak Company, Scientific Imaging Systems, New Haven,
Conn.
[0069] Particularly suitable LIR fusion proteins are those in which
an LIR polypeptide is in the form of an oligomer. Oligomers may be
formed by disulfide bonds between cysteine residues on more than
one LIR polypeptide, or by noncovalent interactions between LIR
polypeptide chains. In another approach, LIR oligomers can be
formed by joining LIR polypeptides or fragment thereof via covalent
or noncovalent interactions between peptide moieties fused to the
LIR polypeptide. Suitable peptide moieties include peptide linkers
or spacers, or peptides that have the property of promoting
oligomerization. Leucine zippers and certain polypeptides derived
from antibodies are among the peptides that can promote
oligomerization of LIR polypeptides attached thereto.
[0070] Other LIR fusion proteins which promote oligomer formation
are fusion proteins having heterologous polypeptides fused to
various portions of antibody-derived polypeptides (including the Fc
domain). Procedures for preparing such fusion proteins are
described in Ashkenazi et al. PNAS USA 88:10535, 1991; Byrne et al.
Nature 344:667, 1990, and Hollenbaugh and Aruffo Current Protocols
in Immunology, Supplement 4, pages 10.19.1-10.19.11, 1992; all of
which are incorporated herein by reference. Example 1 and Example 5
below describe methods for preparing UL18:Fc and P3G2:Fc fusion
proteins, respectively, by fusing P3G2 and UL18 to an Fc region
polypeptide derived from an antibody. This is accomplished by
inserting into an expression vector a gene fusion encoding the
P3G2:Fc fusion protein and expressing the P3G2:Fc fusion protein.
The fusion proteins are allowed to assemble much like antibody
molecules, whereupon interchain disulfide bonds form between the Fc
polypeptides, yielding divalent P3G2 polypeptide. In a similar
approach, P3G2 or any LIR polypeptide may be substituted for the
variable portion of an antibody heavy or light chain. If fusion
proteins are made with heavy and light chains of an antibody, it is
possible to form a LIR oligomer with as many as four LIR
regions.
[0071] Thus, the invention encompasses nucleic acids that encode
fusion proteins that include the Fc region of Ig and an amino acid
sequence including the extracellular region of any of the LIR
family member proteins. Such extracellular regions include, e.g.,
amino acids x.sub.1-458 of SEQ ID NO:2, where x.sub.1 is amino
acids I or 17; amino acids x.sub.2-459 of SEQ ID NO:4, where
x.sub.2 is amino acid 1 or 17; amino acids x.sub.3-439 of SEQ ID
NO:8, where x.sub.3 is amino acid 1 or 17; amino acids x.sub.4458
of SEQ ID NO: 10, where x.sub.4 is amino acid 1 or 17; amino acids
x.sub.5 to 261 of SEQ ID NO: 12, wherein x.sub.5 is amino acid 1 or
17; amino acids x.sub.6 to 461 of SEQ ID NO:14, wherein x.sub.6 is
amino acid 1 or 17; amino acids x.sub.7-449 of SEQ ID NO:16, where
x.sub.7 is amino acid 1 or 17; amino acids x.sub.8-259 of SEQID
NO:18, where x.sub.8 is amino acid 1 or 17; amino acids x.sub.9-443
of SEQ ID NO:20, where x.sub.9 is amino acid 1 or 17; amino acids
x.sub.10 to 456 of SEQ ID NO:22, wherein x.sub.10 is amino acid 1
or 17; amino acids x.sub.11 to 262 of SEQ ID NO:30, wherein
x.sub.11 is amino acid 1 or 35; amino acids x.sub.12 to 250 of SEQ
ID NO:32, wherein x.sub.12 is amino acid 1 or 36; amino acids
x.sub.13 to 265 of SEQ ID NO:34, wherein x.sub.13 is amino acid 1
or 35; and amino acids x.sub.14 to 253 of SEQ ID NO:36, wherein
x.sub.14 is amino acid 1 or 36; and amino acids 1-393 of SEQ ID
NO:38.
[0072] As used herein, a Fc polypeptide includes native and mutein
forms, as well as truncated Fc polypeptides containing the hinge
region that promotes dimerization. One suitable Fc polypeptide is
the native Fc region polypeptide derived from a human IgG1, which
is described in PCT application WO 93/10151, hereby incorporated
herein by reference. Another useful Fc polypeptide is the Fc mutein
described in U.S. Pat. 5,457,035. The amino acid sequence of the
mutein is identical to that of the native Fc sequence presented in
WO 93/1015 1, except that amino acid 19 has been changed from Leu
to Ala, amino acid 20 has been changed from Leu to Glu, and amino
acid 22 has been changed from Gly to Ala. This mutein Fc exhibits
reduced affinity for immunoglobulin receptors.
[0073] Alternatively, oligomeric LIR polypeptide variants may
include two or more LIR peptides joined through peptide linkers.
Examples include those peptide linkers described in U.S. Pat. No.
5,073,627, incorporated herein by reference. Fusion proteins which
include multiple LIR polypeptides separated by peptide linkers may
be produced conventional recombinant DNA technology.
[0074] Another method for preparing oligomeric LIR polypeptide
variants involves use of a leucine zipper. Leucine zipper domains
are peptides that promote oligomerization of the proteins in which
they are found. Leucine zippers were first identified in several
DNA-binding proteins (Landschulz et al. Science 240:1759, 1988).
Among the known leucine zippers are naturally occurring peptides
and peptide derivatives that dimerize or trimerize. Examples of
leucine zipper domains suitable for producing soluble oligomeric
LIR polypeptides or oligomeric polypeptides of the LIR family are
those described in PCT application WO 94/10308, incorporated herein
by reference. Recombinant fusion proteins having a soluble LIR
polypeptide fused to a peptide that dimerizes or trimerizes in
solution may be expressed in suitable host cells, and the resulting
soluble oligomeric LIR polypeptide recovered from the culture
supernatant.
[0075] Numerous reagents useful for cross-linking one protein
molecule to another are known. Heterobifunctional and
homobifunctional linkers are available for this purpose from Pierce
Chemical Company, Rockford, Ill., for example. Such linkers contain
two functional groups (e.g., esters and/or maleimides) that will
react with certain functional groups on amino acid side chains,
thus linking one polypeptide to another.
[0076] One type of peptide linker that may be employed in the
present invention separates polypeptide domains by a distance
sufficient to ensure that each domain properly folds into the
secondary and tertiary structures necessary for the desired
biological activity. The linker also should allow the extracellular
portion to assume the proper spatial orientation to form the
binding sites for ligands.
[0077] Suitable peptide linkers are known in the art, and may be
employed according to conventional techniques. Among the suitable
peptide linkers are those described in U.S. Pat. No. 4,751,180 and
4,935,233, which are hereby incorporated by reference. A peptide
linker may be attached to LIR polypeptides by any of the
conventional procedures used to attach one polypeptide to another.
The cross-linking reagents available from Pierce Chemical Company
as described above are among those that may be employed. Amino
acids having side chains reactive with such reagents may be
included in the peptide linker, e.g., at the termini thereof.
Preferably, a fusion proteins formed via a peptide linker are
prepared by recombinant DNA technology.
[0078] The fusion proteins of the present invention include
constructs in which the C-terminal portion of one protein is fused
to the linker which is fused to the N-terminal portion of another
protein. Peptides linked in such a manner produce a single protein
which retains the desired biological activities. The components of
the fusion protein are listed in their order of occurrence (i.e.,
the N-terminal polypeptide is listed first, followed by the linker
and then the C-terminal polypeptide).
[0079] A DNA sequence encoding a fusion protein is constructed
using recombinant DNA techniques to insert separate DNA fragments
encoding the desired proteins into an appropriate expression
vector. The 3' end of a DNA fragment encoding one protein is
ligated (via the linker) to the 5' end of the DNA fragment encoding
another protein with the reading frames of the sequences in phase
to permit translation of the MRNA into a single biologically active
fusion protein. A DNA sequence encoding an N-terminal signal
sequence may be retained on the DNA sequence encoding the
N-terminal polypeptide, while stop codons, which would prevent
read-through to the second (C-terminal) DNA sequence, are
eliminated. Conversely, a stop codon required to end translation is
retained on the second DNA sequence. DNA encoding a signal sequence
is preferably removed from the DNA sequence encoding the C-terminal
polypeptide.
[0080] A DNA sequence encoding a desired polypeptide linker may be
inserted between, and in the same reading frame as, the DNA
sequences encoding the two proteins using any suitable conventional
technique. For example, a chemically synthesized oligonucleotide
encoding the linker and containing appropriate restriction
endonuclease cleavage sites may be ligated between the sequences
encoding Fc and a P3G2 polypeptide.
[0081] Within the scope of the present invention are recombinant
expression vectors for expressing polypeptides of the LIR family,
and host cells transformed with the expression vectors. Expression
vectors of the invention include DNA that encodes a LIR family
member operably linked to suitable transcriptional or translational
regulatory nucleotide sequences, such as those derived from a
mammalian, microbial, viral, or insect gene. Examples of regulatory
sequences include transcriptional promoters, operators, or
enhancers, an MRNA ribosomal binding site, and appropriate
sequences which control transcription and translation initiation
and termination. Nucleotide sequences are operably linked when the
regulatory sequence functionally relates to the LIR DNA sequence.
Thus, a promoter nucleotide sequence is operably linked to a LIR
DNA sequence if the promoter nucleotide sequence controls the
transcription of the LIR DNA sequence. An origin of replication
that confers the ability to replicate in the desired host cells,
and a selection gene by which transformants are identified, are
generally incorporated in the expression vector.
[0082] In addition, a sequence encoding an appropriate signal
peptide can be incorporated into expression vectors. A DNA sequence
for a signal peptide (secretory leader) may be fused in frame to
the LIR sequence so that the LIR is initially translated as a
fusion protein comprising the signal peptide. A signal peptide that
is functional in the intended host cells promotes extracellular
secretion of the LIR polypeptide. The signal peptide is cleaved
from the LIR polypeptide upon secretion of the LIR polypeptide from
the cell.
[0083] The recombinant expression vectors of the present invention
may include any DNA encoding a LIR polypeptide. Exemplary DNAs for
inclusion in such expression vectors include the nucleic acid
molecules whose sequences are shown in SEQ ID NOS: 1, 3, 7, 9, 11,
13, 15, 17, 19, 21, 29, 31, 33, 35 and 37.
[0084] Suitable host cells for expression of LIR polypeptides
include prokaryotes, yeast or higher eukaryotic cells. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described, for example, in
Pouwels et al. Coning Vectors: A Laboratory Manual, Elsevier, N.Y.,
(1985). Cell-free translation systems could also be employed to
produce P3G2 polypeptides using RNAs derived from DNA constructs
disclosed herein.
[0085] Prokaryote host cells suitable in the practice of the
present invention include gram negative or gram positive organisms,
for example, E. coli or Bacilli.. Suitable prokaryotic host cells
for transformation include, for example, E. coli, Bacillus
subtilis, Salmonella typhimurium, and various other species such as
Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic
host cell, such as E. coli, a P3G2 polypeptide may include an
N-terminal methionine residue to facilitate expression of the
recombinant polypeptide. The N-terminal Met may be cleaved from the
expressed recombinant LIR polypeptide.
[0086] Expression vectors for use in prokaryotic host cells
generally include one or more phenotypic selectable marker genes. A
phenotypic selectable marker gene is, for example, a gene encoding
a protein that confers antibiotic resistance or that supplies an
autotrophic requirement. Examples of useful expression vectors for
prokarytoic host cells include those derived from commercially
available plasmids such as the cloning vector pBR322 (ATCC 37017).
pBR322 contains genes for ampicillin and tetracycline resistance
and thus provides simple means for identifying transformed cells.
An appropriate promoter and a LIR family DNA may be inserted into
the pBR322 vector. Other commercially available vectors include,
for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden)
and pGEM1 (Promega Biotec, Madison, Wis., USA).
[0087] Promoter sequences commonly used for recombinant prokaryotic
host cell expression vectors include .beta.-lactamase
(penicillinase), lactose promoter system (Chang et al. Nature
75:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan
(trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057,
1980); and EP-A-36776) and tac promoter (Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.
412, 1982). A particularly useful prokaryotic host cell expression
system employs a phase .lambda.P.sub.L promoter and a cI857ts
thermolabile repressor sequence. Plasmid vectors available from the
American Type Culture Collection which incorporate derivatives of
the .lambda.P.sub.L promoter include plastid pHUB2 (resident in E.
coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1,
ATCC 53082).
[0088] Alternatively, LIR polypeptides may be expressed in yeast
host cells, preferably from the Saccharomyces genus (e.g., S.
cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces
may also be employed. Yeast vectors will often contain an origin of
replication sequence from a 2.mu. yeast plasmid, an autonomously
replicating sequence (ARS), a promoter region, sequences for
polyadenylation, sequences for transcription termination, and a
selectable marker gene. Suitable promoter sequences for yeast
vectors include, among others, promoters for metallothionein,
3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem 255:2073,
1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.
7:149, 1968); and Holland et al., Biochem. 17:4900, 1978), such as
enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,
pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phospho-glucose isomerase, and
glucokinase. Other suitable vectors and promoters for use in yeast
expression are further described in Hitzeman, EPA-73,675. Another
alternative is the glucose-repressible ADH2 promoter described by
Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al.
(Nature 300:724, 1982). Shuttle vectors replicable in both yeast
and E. coli may be constructed by inserting DNA from pBR322 for
selection and replication in E. coli (Amp.sup.r gene and origin of
replication) into the above-described yeast vectors.
[0089] The yeast .alpha.-factor leader sequence may be employed to
direct secretion of the LIR polypeptide. The .alpha.-factor leader
sequence is often inserted between the promoter sequence and the
structural gene sequence. See, e.g., Kurjan et al., Cell
30:933,1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,
1984. Other leader sequences suitable for facilitating secretion of
recombinant polypeptides from yeast hosts are known to those of
skill in the art. A leader sequence may be modified near its 3' end
to contain one or more restriction sites. This will facilitate
fusion of the leader sequence to the structural gene.
[0090] Yeast transformation protocols are known to those of skill
in the art. One such protocol is described by Hinnen et al., Proc.
Natl. Acad. Sci. USA 75:1929, 1978. The Hinnen et al. protocol
selects for Trp.sup.+ transformants in a selective medium, wherein
the selective medium consists of 0.67% yeast nitrogen base, 0.5%
casamino acids, 2% glucose, 10 .mu.g/mL adenine and 20 .mu.g/mL
uracil.
[0091] Yeast host cells transformed by vectors containing an ADH2
promoter sequence may be grown for inducing expression in a "rich"
medium. An example of a rich medium is one having 1% yeast extract,
2% peptone, and 1% glucose supplemented with 80 .mu.g/mL uracil.
Derepression of the ADH2 promoter occurs when glucose is exhausted
from the medium.
[0092] Mammalian or insect host cell culture systems may be used to
express recombinant LIR polypeptides. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, Bio/Technology 6:47 (1988). Established cell
lines of mammalian origin also may be employed. Examples of
suitable mammalian host cell lines include the COS-7 line of monkey
kidney cells (ATCC CRL 1651)(Gluzman et al., Cell 23:175, 1981), L
cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary
(CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the
CVI/EBNA cell dine derived from the African green monkey cell line
CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10:2821,
1991). COS-1 (ATCC CRL-1650).
[0093] Transcriptional and translational control sequences for
mammalian host cell expression vectors may be excised from viral
genomes. Commonly used promoter sequences and enhancer sequences
are derived from Polyoma virus, Adenovirus 2, Simian Virus 40
(SV40), and human cytomegalovirus. DNA derived from the SV40 viral
genome, for example, SV40 origin, early and late promoter,
enhancer, splice, and polyadenylation sites may be used to provide
other genetic elements for expression of a structural gene sequence
in a mammalian host cell. Viral early and late promoters are
particularly useful because both are easily obtained from a viral
genome as a fragment which may also contain a viral origin of
replication (Fiers et al., Nature 273:113, 1978). Smaller or larger
SV40 fragments may also be used, provided the approximately 250 bp
sequence extending from the HIND III site toward the Bg/I site
located in the SV40 viral origin of replication site is
included.
[0094] Suitable expression vectors for use in mammalian host cells
can be constructed as disclosed by Okayama and Berg (Mol. Cell.
Biol. 3:280, 1983). One useful system for stable high level
expression of mammalian receptor cDNAs in C127 murine mammary
epithelial cells can be constructed substantially as described by
Cosman et al. (Mol. Immunol. 23:935, 1986). A high expression
vector, PMLSV N1I/N4, described by Cosman et al., Nature 312:768,
1984 has been deposited as ATCC 39890. Additional mammalian
expression vectors are described in EP-A-0367566, and in WO
91/18982. Still additional expression vectors for use in mammalian
host cells include pDC201 (Sims et al., Science 241:585, 1988),
pDC302 (Mosley et al. Cell 59:335, 1989), and pDC406 (McMahan et
al., EMBO J. 10:2821, 1991). Vectors derived from retroviruses also
may be employed. One preferred expression system employs pDC409 as
discussed in Example 5 below.
[0095] For expression of LIR polypeptides the expression vector may
comprise DNA encoding a signal or leader peptide. In place of the
native signal sequence, a heterologous signal sequence may be
added, such as the signal sequence for interleukin-7 (IL-7)
described in U.S. Pat. No. 4,965,195; the signal sequence for
interleukin-2 receptor described in Cosman et al., Nature 312:768,
1984); the interleukin-4 signal peptide described in EP 367,566;
the type I interleukin-1 receptor signal peptide described in U.S.
Pat. No. 4,968,607; and the type II interleukin-1 receptor signal
peptide described in EP 460,846.
[0096] Further contemplated within the present invention are
purified LIR family polypeptides, and processes for their
purification. The purified polypeptides of the present invention
may be purified from the above-described recombinant expression
systems or may be purified from naturally occurring cells. The
desired degree of purity may depend on the intended use of the
protein with a relatively high degree of purity preferred when the
protein is intended for in vivo use. Preferably, LIR polypeptide
purification processes are such that no protein bands corresponding
to proteins other than the desired LIR protein are detectable by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be
recognized by one skilled in the art that multiple bands
corresponding to any LIR polypeptide my be detected by SDS-PAGE,
due to differential glycosylation, variations in post-translational
processing, and the like, as discussed above. Most preferably, any
specific LIR polypeptide is purified to substantial homogeneity, as
indicated by a single protein band upon analysis by SDS-PAGE. The
protein band may be visualized by silver staining, Coomassie blue
staining, or by autoradiography or fluorescence if the protein is
appropriately labeled.
[0097] One process for providing purified LIR polypeptides includes
first culturing a host cell transformed with an expression vector
comprising a DNA sequence that encodes the desired polypeptide
under conditions that promote expressing the desired LIR
polypeptide and then recovering the LIR polypeptide. As the skilled
artisan will recognize, procedures for recovering the polypeptide
will vary according to such factors as the type of host cells
employed and whether the polypeptide is secreted in the culture
medium is extracted from cells.
[0098] When the expression system secretes the polypeptide into the
culture medium, the medium may be first concentrated using a
comrnmercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. Following the
concentration step, the concentrate can be applied to a suitable
purification matrix such as a gel filtration medium. Alternatively,
an anion exchange resin can be employed, such as a resin matrix or
resin substrate having pendant diethylaminoethyl (DEAE) groups. The
matrices can be acrylamide, agarose, dextran, cellulose or other
types commonly employed in protein purification. Similarly, a
purification matrix having cation exchange groups such as
sulfopropyl or carboxymethyl functionalities on an insoluble matrix
can be used. Sulfopropyl groups are preferred. Still other
purification matrices and methods suitable for providing purified
LIR are high performance liquid chromatography using hydrophobic
reversed phase media (RP-HPLC). One skilled in the art will
recognized the any or all of the foregoing purification steps, in
various combinations, can be employed to provide a purified LIR
polypeptide.
[0099] Alternatively, LIR polypeptides can be purified by
immunoaffinity chromatography. An affinity column containing an
antibody that binds a LIR polypeptide may be prepared by
conventional procedures and employed in purifying LIR. Example 5
describes a procedures for generating monoclonal antibodies
directed against P3G2 which may be utilized in immunoaffinity
chromatography.
[0100] Recombinant protein produced in bacterial culture may be
isolated by first disrupting the host cells by any convenient
method, including freeze-thaw cycling, sonication, mechanical
disruption, or use of cell lysing agents and then extracting the
polypeptide from cell pellets if the polypeptide is insoluble, or
from the supernatant fluid if the polypeptide is soluble. After the
initial isolation step, the purification process may include one or
more concentrating, salting out, ion exchange, affinity, or size
exclusion chromatography purification steps. For many application a
final RP-HPLC purification step is beneficial.
[0101] Additional methods for providing LIR polypeptides and
purified LIR polypeptides involves fermenting yeast which express
proteins as a secreted protein. Secreted recombinant protein
resulting from a large-scale fermentation can be purified by
methods analogous to those disclosed by Urdal et al. (J. Chromatog.
296:171, 1984), involving two sequential, reversed-phase HPLC steps
for purification of a recombinant protein on a preparative HPLC
column.
[0102] LIR-P3G2 DNA in pDC406 vector was deposited with the
American Type Culture Collection on Apr. 22, 1997 and assigned
accession No.97995. The deposit was made under the terms of the
Budapest Treaty.
[0103] As described above and shown in Examples 6 and 14, LIR-P3G2
and LIR-pbm8 are MHC class I receptor molecules found on the
surface of certain monocytes, B cells, and NK cells. With respect
to monocytes, the expression of LIRs that are MHC class I binding
proteins suggests that there is some requirement for monocytes to
recognize MHC class I molecules. LIR-P3G2, LIR-pbm8 LIR and certain
additional LIR family members contain cytoplasmic ITIM motifs. By
analogy with the structure and function of known MHC class I
receptor molecules, these LIRs are inhibitory receptors mediating
negative signaling. Indeed, the results demonstrated in Example 11
reveal that LIRS associate with SHP-1 and inhibit FcR-mediated
activation events. Thus, monocytes may express class I receptors in
order to suppress cell-mediated lytic mechanisms. Monocytes rapidly
phagocytes extracellular pathogens via FcR and, monocyte-FcR
engagement induces propagation of immune responses by producing
more systemic mediators, particularly TNF-.alpha., IL-6 and IL-8.
Thus, the LIRs play a role in monocyte and macrophage regulation of
cytolytic and inflammatory responses against self tissues. The
interplay between the FcR activatory signals and LIR inhibitory
signal may allow low levels of self-reactive IgG to exist in
circulation and bind to the monocyte membrane with initiating an
immune response. For example, the expression of these inhibitory
receptors can protect the developing embryo from maternal
antibody-mediated allogeneic recognition.
[0104] With respect to LIRs on cells of the DC lineage, as
described in Example 13
CD33.sup.+CD14.sup.-CD16.sup.-HLA.sup.-DR.sup.+DC co-express
LIR-P3G2 and LIR-pbm8. It is suggested the DC FcR play a role in
binding immune complexes and triggering DC activation signal
following binding. Thus, LIRs expressed on DC may suppress DC
activation through interactions of FcR.
[0105] Many LIR family members lack the ITIM motif and by analogy
with the structure and function of known MHC class I receptors
lacking ITIMs are activatory receptors. Failure of a receptor that
mediates negative signaling could result in autoimmune diseases.
Thus, engaging an LIR family member having ITIM motifs with an
agonistic antibody or ligand can be used to downregulate a cell
function in disease states in which the immune system is overactive
and excessive inflammation or immunopathology is present. On the
other hand, using an antagonistic antibody specific to the ITIM
possessing LIR receptor or a soluble form of the receptor can be
used to block the interaction of the cell surface receptor with the
receptor's ligand to activate the specific immune function in
disease states associated with suppressed immune function. Since
receptors lacking the ITIM motif send activatory signals once
engaged as described above, failure of a receptor that mediates an
activatory signal could result in suppressed immune function.
Engaging the receptor with its agonistic antibody or ligand can be
used to treat diseases associated with the suppressed immune
function. Using an antagonistic antibody specific to the activatory
LIR receptor or a soluble form of the receptor can be used to block
the interaction of the activatory receptor with the receptor's
ligand to downregulate the activatory signaling.
[0106] Since LIR-P3G2 binds to various cells, LIR-P3G2 may be used
to purify or isolate these cells from heterogeneous preparations.
Additionally, P3G2 probes can be used to isolate and identify
related molecules.
[0107] LIR polypeptides of the present invention may be used in
developing treatments for any disorder mediated directly or
indirectly by defective or insufficient amounts of any of the LIR
polypeptides. A therapeutically effective amount of purified LIR
protein is administered by a patient afflicted with such a
disorder. Alternatively, LIR DNA may be employed in developing a
gene therapy approach to treating such disorders. Disclosure herein
of native LIR nucleotide sequence permits the detection of
defective LIR genes, and the replacement thereof with normal
LIR-encoding genes. Defective genes may be detected in vitro
diagnostic assays, and by comparison of the native LIR nucleotide
sequence disclosed herein with that of an LIR gene derived from a
person suspected of harboring a defect in the gene.
[0108] The present invention also provides pharmaceutical
compositions which may include an LIR polypeptide, or fragments or
variants thereof with a physiologically acceptable carrier or
diluent. Such carriers and diluents will be nontoxic to recipients
at the dosages and concentrations employed. Such compositions may
further include buffers, antioxidants such as ascorbic acid, low
molecular weight (less than about ten residues) polypeptides,
proteins, amino acids, carbohydrates including glucose, sucrose or
dextrins, chelating agents such as EDTA, glutathione and other
stabilizers and excipients commonly used in pharmaceutical
compositions. The pharmaceutical compositions of the present
invention may be formulated as a lyophilizate using appropriate
excipient solutions as diluents. The pharmaceutical compositions
may include an LIR polypeptide in any for described herein,
including but not limited to active variants, fragments, and
oligomers. LIR polypeptides may be formulated according to known
methods that are used to prepare pharmaceutically useful
compositions. Components that are commonly employed in
pharmaceutical formulations include those described in Remington's
Pharmaceutical Sciences, 16th ed. (Mack Publishing Company, Easton,
Pa., 1980).
[0109] The pharmaceutical preparations of the present invention may
be administered to a patient, preferably a human, in a manner
appropriate to the indication. Thus, for example, the compositions
can be administered by intravenous injection, local administration,
continuous infusion, sustained release from implants, etc.
Appropriate dosages and the frequency of administration will depend
on such factors as the nature and severity of the indication being
treated, the desired response, the condition of the patient and so
forth.
[0110] In preferred embodiments an LIR polypeptide used in the
pharmaceutical compositions of the present invention is purified
such that the LIR polypeptide is substantially free of other
proteins of natural or endogenous origin, desirably containing less
than about 1% by mass of protein contaminants residual of the
production processes. Such compositions, however, can contain other
proteins added as stabilizers, carriers, excipients or
co-therapeutics.
[0111] LIR encoding DNAs and DNA fragments disclosed herein find
use in the production of LIR polypeptides, as described above. In
one embodiment, such fragments comprise at least about 17
consecutive nucleotides, more preferably at least 30 consecutive
nucleotides, of LIR DNA. DNA and RNA complements of the fragments
have similar utility. Among the uses of LIR nucleic acid fragments
are as probes or primers in polymerase chain reactions. For
example, a probe corresponding to a fragment of DNA encoding the
extracellular domain of LIR may be employed to detect the presence
of LIR nucleic acids in in vitro assays and in other probing assays
such as Northern Blot and Southern blot assays. Cell types
expressing an LIR polypeptide can be identified using LIR family
nucleic acid probes using probing procedures well known in the art.
Those skilled in the art have the knowledge to choose a probe of
suitable length and apply conventional PCR techniques to isolate
and amplify a DNA sequence.
[0112] Nucleic acid fragments may also be used as a probe in cross
species hybridization procedures to isolate LIR DNA from other
mammalian species. As one example, a probe corresponding to the
extracellular domain of an LIR polypeptide may be employed. The
probes may be labeled (e.g., with .sup.32P) by conventional
techniques.
[0113] Other useful fragments of LIR nucleic acids are sense or
antisense oligonucleotides, which may comprise either RNA or DNA,
and which correspond in sequence to an LIR MRNA (sense), to the
complement of an LIR mRNA (antisense), or to the non-coding strand
of a double-stranded LIR DNA, such as P3G2 DNA (antisense). Thus,
an antisense oligonucleotide will form a hybrid duplex with an MRNA
sequence. Such oligonucleotides generally are at least 14
nucleotides, and preferably are from about 14 to about 30
nucleotides. The ability to create an antisense or a sense
oligonucleotide based upon a cDNA sequence for a given protein is
described in, for example, Stein and Cohen, Cancer Res. 48:2659,
1988 and van der Krol et al., BioTechniques 6:958, 1988.
[0114] Binding antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block translation (RNA) or transcription (DNA) by one of several
means, including enhanced degradation of the duplexes, premature
termination of transcription or translation, or by other means.
These oligonucleotides thus may be used to block LIR
expression.
[0115] In one embodiment antisense or sense LIR oligonucleotides
used in binding procedures may encompass oligonucleotides having
modified sugar-phosphodiester backbones (or other sugar linkages,
such as those described in W091/06629) and wherein such sugar
linkages are resistant to endogenous nucleases. Oligonucleotides
having sugar linkages resistant to endogenous nucleases are stable
in vivo (i.e., capable of resisting enzymatic degradation) but
retain sequence specificity to be able to bind to target nucleotide
sequences. Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10448, and other
moieties that increase affinity of the oligonucleotide for a target
nucleic acid sequence, such as poly-(L-lysine). Further still,
intercalating agents, such as ellipticine, and alkylating agents or
metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0116] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. Antisense or sense oligonucleotides are
preferably introduced into a cell containing the target nucleic
acid sequence by inserting the antisense or sense oligonucleotide
into a suitable retroviral vector, then contacting the cell with
the retroviral vector containing the inserted sequence, either in
vivo or ex vivo. Suitable retroviral vectors include, but are not
limited to, those derived from the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see PCT Application US
90/02656).
[0117] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugating the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind its corresponding molecule or receptor, or block
entry of the sense of antisense oligonucleotide or its conjugated
version into the cell.
[0118] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0119] In still a further aspect, the present invention provides
antibodies that specifically bind LIR polypeptides, i.e.,
antibodies bind to LIR polypeptides via an antigen-binding site of
the antibody (as opposed to non-specific binding). Antibodies of
the present invention may be generated using LIR polypeptides or
immunogenic fragments thereof. Polyclonal and monoclonal antibodies
may be prepared by conventional techniques. See, for example,
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Kennet et al. (eds.), Plenum Press, New York 1980; and
Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988. An
exemplary procedure for producing monoclonal antibodies
immunoreactive with P3G2-LIR is further illustrated in Example 5
below.
[0120] Included within the scope of the present invention are
antigen binding fragments of antibodies which specifically bind to
an LIR polypeptide. Such fragments include, but are not limited to,
Fab, F(ab'), and F(ab').sub.2. Antibody variants and derivatives
produced by genetic engineering techniques are contemplated as
within the presented invention.
[0121] The monoclonal antibodies of the present invention include
chimeric antibodies, e.g., humanized versions of murine monoclonal
antibodies. Such antibodies may be prepared by known techniques and
offer the advantage of reduced immunogenicity when the antibodies
are administered to humans. In one embodiment a humanized
monoclonal antibody comprises the variable region of a murine
antibody (or just the antigen binding site thereof) and a constant
region derived from a human antibody. Alternatively, a humanized
antibody fragment may comprise the antigen binding site of a murine
monoclonal antibody and a variable region fragment (lacking the
antigen-binding site) derived from a human antibody. Procedures for
the production of chimeric and further engineered monoclonal
antibodies include those described in Riechmann et al., Nature
332:232, 1988; Lie et al. PNAS 84:3439, 1987; Larrick et al.
Bio/Technology 7:934, 1989; and Winter and Harris TIPS 14:139,
1993.
[0122] As mentioned above, antibodies of the present invention are
useful in in vitro or in vivo assays to detect the presence of LIR
polypeptides and in purifying an LIR polypeptide by affinity
chromatography.
[0123] Additionally, antibodies capable of blocking an LIR from
binding to target cells may be used to inhibit a biological
activity of an LIR polypeptide. More specifically, therapeutic
compositions of an antibody antagonistic to one or more LIR family
members having the ITIM motif may be administered to an individual
in order to block the interaction of a cell surface LIR with its
ligand. The result is an activation of immune function and is
particularly beneficial in disease states in which the immune
system is hyporesponsive or suppressed. Conversely, therapeutic
compositions of an antibody antagonistic to one or more LIR family
members lacking the ITIM motif may be used to obtain the opposite
effect and be beneficial in disease states in which the immune
system is overactive and excessive inflammation or immunopathology
is present.
[0124] Pharmaceutical compositions which include at least one
antibody that is immunoreactive with an LIR polypeptide and a
suitable diluent, excipient, or carrier, are considered with the
present invention. Suitable diluents, excipients, and carriers are
described in the context of pharmaceutical compositions which
include polypeptides of the present invention.
[0125] The following examples are provided to illustrate certain
embodiments of the invention, and are not to be construed as
limiting the scope of the invention.
EXAMPLES
Example 1
Isolating and Expressing Viral Protein
[0126] DNA encoding P3G2 polypeptide of the present invention was
identified by isolating and expressing a viral glycoprotein, UL18,
known to be expressed on cells infected with HCMV, and then
expressing and using a UL18/Fc fusion protein to search for UL18
receptors. DNA encoding UL18 and its amino acid sequence are known
and described in Beck, S., B. G. Barrell, Nature 331:269-272, 1988.
The following describes isolating UL18 and preparing the UL18/Fc
fusion protein.
[0127] Using standard techniques, total RNA was isolated from Human
Foreskin Fibroblasts infected with HCMV (AD169) at three different
transcription stages-immediate early (IE 8 p.i.h.), early (24
p.i.h.) and late (48 p.i.h.). Because UL18 is known to be
transcribed early in the infection, the IE total RNA was
polyA+selected and used to construct an HCMV-IE cDNA library using
a cDNA kit according to the manufacturer's instructions (Pharmacia
TIME SAVER cDNA Kit). In order to isolate the full length UL18
gene, two oligonucleotide primers known to include the terminal
sequences of the UL18 gene were synthesized and used to isolate and
amplify the UL18 gene from the HCMV-IE cDNA library. The primers
had the following sequences and included Not I restriction sites
which incorporate into the PCR product:
1 Not I 5' - TAT GCG GCC GCC ATG ATG ACA ATG TGG T - 3' (SEQ ID
NO:23) 5' - TAT GCG GCC GCC CCT TGC GAT AGC G - 3' (SEQ ID NO:24)
Not I
[0128] The PCR conditions included one 5 minute 95.degree. C. cycle
followed by 30 cycles of 45 seconds at 95.degree., 45 seconds at
58.degree. and 45 seconds at 72.degree., and then one cycle for 5
minutes at 72.degree. C. The PCR product was electrophoresed on a
1% agarose gel and sized using ethidium bromide to visualize the
separated DNA products. The presence of DNA of having the expected
size of approximately 1. lkb was confirmed.
[0129] The pDC409 expression vector, a vector derived from pDC406
(McMahan et al., EMBO J. 10:2821, 1991) but having a single Bgl II
site was selected for the cloning process. The PCR product was
subcloned into a pDC409 expression vector through the Not I sites,
sequenced and the amino acid sequence deduced from the DNA
sequence. The determined nucleotide sequence and amino acid
sequence were identical to the previously published sequences
(ibid.).
[0130] A fusion protein of the extracellular region of UL18 and a
mutein human IgG1 Fc region (UL18:Fc) was prepared by first
isolating cDNA encoding the extracellular region of UL18 using
primers which flank the extracellular region of UL18. The primers
were synthesized with Sal I and Bgl II restriction sites inserted
at the 5' and 3' termini so that the PCR amplified cDNA introduced
Sal I and Bgl II restriction sites at the 5' and 3' ends,
respectively. The primers had the following sequences:
2 5' - ATA GTC GAC AAC GCC ATG ATG ACA ATG TGG TG - 3' (SEQ ID
NO:25) .sub.Sal I 5' - TAA AGA TCT GGG CTC GYP AGC TGT CGG GT - 3'
(SEQ ID NO:26) .sup.Bgl II
[0131] The conditions for the PCR reaction were as described above
except that the template was the full length gene isolated as just
described.
[0132] To prepare a vector construct for expressing fusion protein,
sUL18:Fc, for use in cell binding studies, a DNA fragment encoding
the Fc region of a human IgG1 antibody was isolated from a plasmid
using Bgl II and Not I restriction enzymes. The encoded Fc portion
was the mutein Fc described in U.S. Pat. No. 5,457,035 having
reduced affinity for immunoglobulin receptors. The Bgl II site on
the sUL18 gene was used to ligate the sUL18 gene DNA to the Bgl II
site on the Fc gene to form a sUL18:Fc fusion DNA construction
having an N-terminal Sal I restriction site and a C-terminal Not I
restriction site. This fusion sUL18:Fc DNA construct was then
ligated into pDC409 expression vector at its Sal I and Not I sites
to form a 409/sUL18/Fc DNA construct.
[0133] The monkey cell line COS-1 (ATCC CRL-1650) was used to
confirm expression of the fusion protein. COS-1 cells in 6-well
plates ( 2.times.10.sup.5 cells per well) were transfected with
about 2 .mu.g of the DNA construct 409/sUL18/Fc per well. The cells
were cultured for 2-3 days in 5% FBSDMEM/F12 (available from
GIBCO), then washed twice with PBS, starved for 1 hour in
cysteine/methionine depleted RPMI (available from GIBCO as RPMI
1640) and metabolically labeled with 100 .mu.Ci/mL of
.sup.35S-Met/Cys for 4 hours. The supernatant was spun clear to
remove loose cells and 150 .mu.L of the supernatant was incubated
with 100 .mu.L of RIPA (0.05% Tween 20, 0.1% SDS, 1% Triton X-100,
0.5% deoxycholate in PBS) buffer and 50 .mu.L of 50% Protein
A-Sepharose solid support beads at 4.degree. C. for 1 hour. Protein
A-Sepharose is a Sepharose solid support (available from Pharmacia)
having immobilized Protein A which binds the Fc portion of the
fusion protein. After washing the solid support with RIPA to remove
unbound material, fusion protein bound to the Protein A-Sepharose
solid support was eluted from the Protein A-Sepharose using 35.mu.L
of SDS -PAGE reducing sample buffer and then heated at 100.degree.
C. for 5 minutes. The eluant was then electrophoresed on a 4-20%
SDS polyacrylamide gradient gel with .sup.4C labeled protein
molecular weight markers. After electrophoresis the gel was fixed
with 8% acetic acid and enhanced at room temperature for 20 minutes
with Amplifier available from Amersham. After drying the gel under
vacuum it was exposed to x-ray film. Film analysis confirmed that
the expected protein, a 100-120 kDa protein which includes the
mutein Fc region of IgG and UL18 extracellular domains fused to the
Fc, was expressed.
[0134] Once cells expressing the fusion protein were identified
large scale cultures of transfected cells were grown to accumulate
supernatant from cells expressing the fusion protein. This
procedure involved transfecting COS-1 cells in T175 flasks with 15
.mu.g of the UL18/Fc/409 fusion DNA per flask. After 7 days of
culture in medium containing 0.5% low immunoglobulin bovine serum,
a solution of 0.2% azide was added to the supernatant and the
supernatant was filtered through a 0.22 .mu.m filter. Then
approximately 1 L of culture supernatant was passed through a
BioCad Protein A HPLC protein purification system using a
4.6.times.100 mm Protein A column (POROS 20A from PerSeptive
Biosystems) at 10 mL/min. The Protein A column binds the Fc portion
of the sUL18/Fc fusion protein in the supernatant, immobilizing the
fusion protein and allowing other components of the supernatant to
pass through the column. The column was washed with 30 mL of PBS
solution and bound sUL18/Fc was eluted from the HPLC column with
citric acid adjusted to pH 3.0. Eluted purified sUL18/Fc was
neutralized as it eluted using IM Hepes solution at pH 7.4. The
pooled eluted protein was analyzed using SDS PAGE with silver
staining, confirming expression of the 100-120 kDa UL18/Fc fusion
protein.
Example 2
Screening Cell Lines for Binding to UL18
[0135] The sUL18/Fc protein isolated as described in Example 1 was
used to screen cells lines to which it binds using quantitative
binding studies according to standard flow cytometry methodologies.
For each cell line screened, the procedure involved incubating
approximately 100,000 of the cells blocked with 2% FCS (fetal calf
serum), 5% normal goat serum and 5% rabbit serum in PBS for 1 hour.
Then the blocked cells were incubated with 5 .mu.g/mL of sUL18/Fc
fusion protein in 2% FCS, 5% goat serum and 5% rabbit serum in PBS.
Following the incubation the sample was washed 2 times with FACS
buffer (2% FCS in PBS) and then treated with mouse anti human
Fc/biotin (purchased from Jackson Research) and SAPE
(streptavidin-phycoerythrin purchased from Molecular Probes). This
treatment causes the anti human Fc/biotin to bind to any bound
sUL18/Fc and the SAPE to bind to the anti human Fc/biotin resulting
in a fluorescent identifying label on sUL18/Fc which is bound to
cells. The cells were analyzed for any bound protein using
fluorescent detection flow cytometry. The results indicated that
UL18 binds well to B cell lines CB23, RAJI and MP-1; monocytic cell
lines Thp-1 and U937; and primary B cell and primary monocytes.
UL18 does not bind detectably to T cell lines nor does it bind to
primary T cells.
Example 3
Isolating a P3G2 cDNA and Polypeptide
[0136] The following describes screening cDNA of one of the cell
lines found to bind UL18 and the isolation of a novel polypeptide
expressed by the cell line. A CB23 cDNA library in the mammalian
expression vector pDC406, prepared as described in U.S. Pat. No.
5,350,683 (incorporated herein by reference) was obtained and
plasmid DNA was isolated from pools consisting of approximately
2,000 clones per pool. The isolated DNA was transfected into
CV1-EBNA cells (ATCC CRL 10478) using DEAE-dextran followed by
chloroquine treatment. The CV1-EBNA cells were maintained in
complete medium (Dulbecco's modified Eagles' media containing 10%
(v/v) fetal calf serum, 50 U/mL penicillin, 50 U/mL streptomycin,
and 2 mM L-glutamine) and were plated to a density of approximately
2.times.10.sup.5 cells/well in single-well chambered slides. The
slides had been pre-treated with 1 mL of a solution of 10 .mu.g/mL
human fibronectin in PBS for 30 minutes followed by a single
washing with PBS. Media was removed from adherent cells growing in
a layer and replaced with 1.5 mL complete medium containing 66.6
.mu.M chloroquine sulfate. About 0.2 mL of a DNA solution (2 .mu.g
DNA, 0.5 mg/mL DEAE-dextran in complete medium containing
chloroquine) was added to the cells and the mixture was incubated
at 37 C for about five hours. Following incubation, the media was
removed and the cells were shocked by addition of complete medium
containing 10% DMSO (dimethylsulfoxide) for 2.5 minutes. Shocking
was followed by replacing the solution with fresh complete medium.
The cells were grown in culture for two to three days to permit
transient expression of the inserted DNA sequences. These
conditions led to a 30% to 80% transfection frequency in surviving
CV1-EBNA cells.
[0137] Each slide was incubated with I mL of UL18:Fc at a
concentration of 1 .mu.g/mL in binding buffer (RPMI 1640 containing
25 mg/mL bovine serum albumin, 2 mg/mL sodium azide, 20 mM Hepes at
pH 7.2, and 50 mg/mL nonfat dry milk) at room temperature for 1
hour. The incubated slides were washed with the binding buffer and
then incubated with Fc specific .sup.125I-mouse anti-human IgG (see
Goodwin et al., Cell 73:447-456, 1993). This was followed by a
second wash with buffer after which the slides were fixed with a
2.5% glutaraldehyde/PBS solution, washed with PBS solution and
allowed to air dry. The dried slides were dipped in Kodak GTNB-2
photographic emulsion (6.times.dilution in water). After air
drying, the slides were placed in a dark box and refrigerated.
After three days the slides were developed in Kodak D19 developer,
rinsed in water and fixed in Agfa G433C fixer. The fixed slides
were individually examined under a microscope at 25-40.times.
magnification. Positive cells demonstrating binding of sUL18:Fc
were visualized by the presence of autoradiographic silver grains
against the film background. Two positive pools were identified.
Bacterial clones from each pool were titered and plated to provide
plates containing approximately 200 colonies each. Each plate was
scraped to provide pooled plasmid DNA for transfection into
CV1-EBNA cells and screening as described above. Following
subsequent breakdowns and screenings, two positive individual
colonies were obtained. The cDNA inserts of the two positive clones
were 2922 and 2777 nucleotides in length as determined by automated
DNA sequences. The coding regions of the two inserts, designated
P3G2 and 18A3 were 1953 (nucleotides 310-2262) and 1959
(nucleotides 168-2126) nucleotides, respectively. The two cDNA
clones encode proteins that are substantially similar and probably
represent different alleles of the same gene.
[0138] The cDNA sequence and encoded amino acid of P3G2 are
presented in SEQ ID NO:1 and SEQ ID NO:2, respectively. The cDNA
sequence and encoded amino acid of 18A3 are presented in SEQ ID
NO:3 and SEQ ID NO:4, respectively. The P3G2 amino acid sequence
(SEQ ID NO:2) has a predicted signal peptide of 16 amino acids
(amino acids 1-16); an extracellular domain of 442 amino acids
(amino acids 17458); a transmembrane domain of 25 amino acids
(amino acids 459-483) and, a cytoplasmic domain of 167 amino acids
(amino acids 484-650. The extracellular domain includes four
immunoglobulin-like domains. Ig-like domain I includes
approximately amino acids 17-118; Ig-like domain II includes
approximately amino acids 119-220; Ig-like domain III includes
approximately amino acids 221-318; and Ig-like domain IV includes
approximately amino acids 319-419. Significantly, the cytoplasmic
domain of this polypeptide includes four ITIM motifs, each having
the consensus sequence of YxxL/V. The first ITIM motif pair is
found at amino acids 533-536 and 562-565 and the second pair is
found at amino acids 614-617 and 644-647. The amino acid sequence
of 18A3 is nearly identical having the features describes
above.
[0139] The features of these encoded polypeptides are consistent
with a type I transmembrane glycoprotein.
Example 4
Preparing P3G2 Fusion Protein
[0140] The following describes procedures used to generate a P3G2
fusion protein which was then used to identify cell lines to which
it binds and finally isolate a normal cell-surface P3G2 ligand
which is distinct from UL/18. A fusion protein of the extracellular
region of P3G2 and the mutein human Fc region (sP3G2:Fc) was
prepared by first isolating cDNA encoding the extracellular region
of P3G2 using primers which flank the extracellular region of P3G2.
The primers were synthesized with Sal I and Bgl II restriction
sites inserted at the 5' and 3' termini so that the PCR amplified
cDNA introduced Sal I and Bgl II restriction sites at the 5' and 3'
ends, respectively. The primers had the following sequences:
3 Sal I 5' - TAT GTC GAC CAT GAC CCC CAT CCT CAC GGT - 3' (SEQ ID
NO:5) 10 Bgl II Xa 5' - TAT GGG CTC TGC TCC AGG AGA AGA TCT TCC TTC
TAT AAC CCC CAG GTG CCT T (SEQ ID NO:6)
[0141] The conditions for the PCR reaction were as described above
and the template was the full length gene P3G2 gene isolated as
described in Example 3 above.
[0142] To prepare a vector construct for expressing fusion protein
sP3G2:Fc for use in cell binding studies, the mutein human Fc
region of IgGI was cut from the plasmid described above in Example
1 using Bgl II and Not I restriction enzymes. The Bgl II site on
the sP3G2 gene was used to ligate the sP3G2 gene DNA to the Bgl II
site on the human mutein Fc gene to form a sP3G2/Fc fusion DNA
construction having an N-terminal Sal I restriction site and a
terminal Not I restriction site. This fusion sP3G2:Fc DNA construct
was then ligated into pDC409 expression vector at its Sal I and Not
I sites to form a 409/sP3G2/Fc DNA construct.
[0143] The monkey cell line COS-1 (ATCC CTL-1650) was used to
confirm expression of the fusion protein. COS-1 cells in 6-well
plates (2.times.10.sup.5 cells per well) were transfected with
about 2 .mu.g of the DNA construct 409/sP3G2/Fc per well. The cells
were cultured in 5% FBSIDMEM/F12 (available from GIBCO) and at day
two or three following transfection, the cells were starved for 1
hour in cysteine/methionine depleted RPMI and the transfected cells
were metabolically labeled with 100 .mu.Ci/mL of .sup.35S-Met/Cys
for 4 hours. The supernatant was spun clear to removed loose cells
and debris and 150 .mu.L of the supernatant was incubated with 100
.mu.L of RIPA buffer and 50 82 L of 50% Protein A-Sepharose solid
support beads at 4.degree. C. for 1 hour. After washing the solid
support with RIPA to remove unbound material, fusion protein bound
to the Protein A-Sepharose solid support was eluted from the
Protein A-Sepharose using 30 .mu.L of SDS-PAGE reducing sample
buffer and then heated at 100.degree. C. for 5 minutes. The eluant
was then electrophoresed on a 4-20% SDS polyacrylamide gradient gel
with .sup.14C labeled protein molecular weight markers. After
electrophoresis the gel was fixed with 8% acetic acid and enhanced
at room temperature for 20 minutes with Amplifier available from
Amersham. After drying the gel under vacuum it was exposed to x-ray
film. Film analysis confirmed that the expected protein, having a
molecular weight of 120-130 kDa, was expressed.
[0144] Once fusion protein expression was verified, large scale
cultures of transfected cells were grown to accumulate supernatant
from COS-1 cells expressing the fusion protein as described in
Example 1 above. The P3G2/Fc fusion protein was purified according
to the procedure described in Example 3 above using the BioCad
system and the POROS 20A column from PerSeptive Biosystems. The
pooled eluted protein was analyzed using SDS PAGE with silver
staining, confirming expression.
Example 5
Generating LIR-P3G2 Antibody
[0145] The following example describes generating monoclonal
antibody to P3G2 that was used in flow cytometry analysis to
identify cells on which P3G2 is expressed. Purified P3G2/Fc fusion
protein was prepared by COS-1 cell expression and affinity
purification as described in Example 4. The purified protein or
cells transfected with an expression vector encoding the full
length protein can generate monoclonal antibodies against P3G2
using conventional techniques, for example those techniques
described in U.S. Pat. No. 4,411,993. Briefly BALB-C mice were
immunized at 0, 2 and 6 weeks with 10 .mu.g P3G2/Fc. The primary
immunization was prepared with TITERMAX adjuvant, from Vaxcell,
Inc., and subsequent immunization were prepared with incomplete
Freund's adjuvant (IFA). At 11 weeks, the mice were IV boosted with
34 .mu.g P3G2 in PBS. Three days after the IV boost, splenocytes
were harvested and fused with an Ag8.653 myeloma fusion partner
using 50% aqueous PEG 1500 solution. Hybridoma supernatants were
screened by ELISA using P3G2 transfected COS-1 cells in PBS at
2.times.10.sup.3 cells per well and dried to polystyrene 96-well
microtiter plates as the platecoat antigen. Positive supernatants
were subsequently confirmed by FACS analysis and RIP using P3G2
transfected COS-1 cells. Hybridomas were cloned and followed using
the same assays. Monoclonal cultures were expanded and supernatants
purified by affinity chromatography using BioRad Protein A
agarose.
[0146] The monoclonal antibodies to P3G2/Fc were used to screen
cells and cell lines using standard flow cytometry procedures to
identify cells on which P3G2 is expressed. Cell lines and cells
screened in the flow cytometry analyses were CB23, CB39, RAJI,
AK778, K299, PS-1, U937, THP-1, JURKAT and HSB2. For each cell line
or cell sample screened, the procedure involved incubating
approximately 100,000 of the cells blocked with 2% FCS (fetal calf
serum), 5% normal Goat serum and 5% rabbit serum in PBS with 5
.mu.g of FITC conjugated mouse anti-P3G2 antibody for 1 hour.
Following the incubation the sample was washed 2 times with FACS
buffer (2% FCS in PBS). The cells were analyzed for any bound
protein using fluorescent detection flow cytometry to detect FITC.
The results indicated that LIR-P3G2 antibody binds well to B cell
lines CB23 and RAJI1; monocytic cell lines THP-1 and U937; and
primary B cell and primary monocytes. The highest expression of
LIR-P3G2 was shown on monocytes that stained brightly for CD16 and
less brightly for CD14 and CD64. The antibody does not bind
detectably to T cell lines nor does it bind detectably to primary T
cells.
[0147] In a related experiment, the P3G2 antibody generated as
described above was used in immunoprecipitation experiments. The
immunoprecipitation analyses involved first surface biotinylating
2.5.times.10.sup.6 monocytes by washing the cells with PBS and
suspending the cells in a biotinylation buffer of 10 mM sodium
borate and 150 mM NaCl at pH 8.8, followed by adding 5 .mu.L of a
10 mg/mL solution of biotin-CNHS-ester (D-biotinoyl-e-aminocaproic
acid-N-hydroxysuccinimide ester purchased from Amersham) in DMSO to
the cells. After quenching the reaction with 10 .mu.L of 1 M
ammonium chloride per 1 mL of cells and washing the cells in PBS,
the cells were lysed in 1 mL of 0.5% NP40-PBS and the lysate was
recovered following centrifugation. Then 100 .mu.L of 0.5%NP40-PBS
was added to 150 .mu.L of the lysate and the resulting mixture was
incubated with 2 .mu.g/mL of antibody, at 4.degree. C. for 16
hours. Fifty microliters of 50% Protein A-Sepharose slurry was
added to the antibody mixture and the slurry was shaken at
4.degree. C. for 1 hour. The slurry was centrifuged and the
resulting pellet was washed with 0.75 mL of 0.5% NP40 in PBS six
times. Protein bound to the Protein A-Sepharose was eluted with 30
.mu.L of SDS-PAGE reducing sample buffer and heating at 100.degree.
C. for five minutes.
[0148] The eluted proteins were analyzed using 4-20% gradient
SDS-PAGE with enhanced chemiluminescence (ECL) protein markers.
Then the electrophoreses samples were transferred in a Western Blot
onto nitrocellulose membranes. The membranes were treated with
blocking reagent (0.1% Tween-20 and 3% nonfat dry milk in PBS) for
one hour at room temperature and then they were washed once for 15
minutes followed and twice for 5 minutes with 0.1% Tween-20 in PBS.
The washed membranes were incubated with 10 mL of 1:100
HRP-Streptavidin for 30 minutes and then washed 1 times for 15
minutes followed by 4 times for 5 minutes with 0.1% Tween-20 in
PBS.
[0149] Bound streptavidin HRP was detected with ECL Detection
Reagents purchased from Amersham and used according to
manufacturer's instructions. The developed membranes were exposed
to x-ray film and then visualized. The results showed that LIR-P3G2
was immunoprecipitated from CB23 cells and P3G2 transfected COS-1
cells, indicating that P3G2 is expressed by these cells.
Example 6
Screening Cells and Cell Lines for Binding to P3G2
[0150] The following describes flow cytometry analyses used to
identify cells and cell lines which bind to P3G2. The cells and
cell lines tested were CB23, HSB2, MP-1, Jurkat, primary T cells,
primary B cells, and primary NK cells. For each cell line or cell
line tested the procedure involved washing the cells three times
with FACS buffer (2% FCS in PBS with 0.2% azide) and incubating
each sample (10.sup.5 cells) in 100 pL blocking buffer (2% FCS, 5%
NGS, 5% rabbit serum in PBS) for one hour. For each cell line 4
test samples were prepared, one each having 0, 2, 5, or 10 .mu.g of
W6/32 (ATCC HB-95) in 100 .mu.L blocking buffer added to the
samples, respectively. W6/32 is an antibody against MHC Class I
heavy chains (an anti HLA-A, B, and C molecule). Following the
addition of the W6/32 solution, the samples were incubated on ice
for 1 hour and then washed three times with 200 .mu.L of FACS
buffer. Then 5 .mu.g of P3G2/Fc in blocking buffer was added to
each sample and they were incubated on ice for one hour. The
P3G2/Fc competes with W6/32 for binding sites on the cells.
[0151] Following the incubation, the cells were washed three times
with 200 .mu.L of FACS buffer and treated with mouse anti human
Fcfbiotin and SAPE for 45 minutes. This treatment causes the anti
human Fc/biotin to bind to any cell bound sP3G2/Fc and the SAPE to
bind to the anti human Fc/Biotin. Since the SAPE is a fluorescing
compound its detection using appropriate excitation and emission
conditions positively identifies cell bound P3G2/Fc. Finally the
treated cells were washed three times with FACS buffer and
subjected to flow cytometry to identify cells bound to protein.
[0152] The results demonstrated that W6/32 competed with P3G2 for
binding to all cells and cell lines tested. The P3G2 binding was
totally blocked at 5 .mu.g W6/32 indicating that W6/32 and P3G2 are
binding to the same or overlapping sites on the MHC Class I heavy
chains.
Example 7
Screening HSB2 cDNA Library to Isolate a P3G2 Binding Ligand
[0153] The following describes screening a cDNA library from of one
of the cell lines, HSB-2, a T lymphoblastic leukemia cell line,
found to bind P3G2, and identifying a P3G2 binding ligand. An HSB2
cDNA library in the mammalian expression vector pDC302, was
prepared as generally described in U.S. Pat. No. 5,516,658 and
specifically in Kozlosky et al. Oncogene 10.299-306, 1995. Briefly,
mRNA was isolated from sorted HSB-2 cells and a first cDNA strand
was synthesized using 5 .mu.g polyA.sup.+and the reverse
transcriptase AMV RTase from Life Science. The second cDNA strand
was synthesized using DNA polymerase I from BRL at concentration of
1.5 U/.mu.L. Using standard techniques as described in Haymerle et
al., Nucl. Acids Res. 14:8615, 1986, the cDNA was ligated into the
appropriate site of the pDC302 vector.
[0154] E.coli. strain DH5.alpha. cells were transformed with the
cDNA library in pDC302. After amplifying the library a titer check
indicated that there was a total of 157,200 clones. The transformed
cells were plated into 15 different plates. Plasmid DNA was
isolated from pools consisting of approximately 2,000 clones per
pool. The isolated DNA was transfected into CV1-EBNA cells (ATCC
CRL 10478) using DEAE-dextran followed by chloroquine treatment.
The CV1-EBNA cells were maintained in complete medium (Dulbecco's
modified Eagles' media containing 10% (v/v) fetal calf serum, 50
U/mL penicillin, 50 U/mL streptomycin, and 2 mM L-glutamine) and
were plated to a density of approximately 2.times.10.sup.5
cells/well in single-well chambered slides. The slides had been
pre-treated with 1 mL of a solution of 10 .mu.g/mL human
fibronectin in PBS for 30 minutes followed by a single washing with
PBS. Media was removed from adherent cells growing in a layer and
replaced with 1.5 mL complete medium containing 66.6 .mu.M
chloroquine sulfate. About 0.2 mL of a DNA solution (2 .mu.g DNA,
0.5 mg/mL DEAE-dextran in complete medium containing chloroquine)
was added to the cells and mixture was incubated at 37 C for about
five hours. Following incubation media was removed and the cells
were shocked by adding complete medium containing 10% DMSO for 2.5
minutes. After shocking the cells the complete medium was replaced
with fresh complete medium and the cells were grown in culture for
three days to permit transient expression of the inserted DNA
sequences. These conditions led to a 30% to 80% transfection
frequency in surviving CV1-EBNA cells.
[0155] Each slide was incubated with 1 mL of P3G2:Fc at a
concentration of 0.45 .mu.g/mL in binding buffer (RPMI 1640
containing 25 mg/mL bovine serum albumin, 2 mg/iL sodium azide, 20
mM Hepes at pH 7.2, and 50 mg/mL nonfat dry milk) at room
temperature for 1 hour. After incubating the slides, they were
washed with binding buffer and then incubated with Fc specific
.sup.125-mouse anti-human IgG (see Goodwin et al. Cell 73:447456,
1993). This was followed by a second wash with buffer after which
the slides were fixed with a 2.5% glutaraldehyde/PBS solution,
washed in PBS and allowed to air dry. The slides were dipped in
Kodak GTNB-2 photographic emulsion (6x dilution in water). After
air drying the slides were placed in a dark box and refrigerated.
After three days the slides were developed in Kodak D19 developer,
rinsed in water and fixed in Agfa G433C fixer. The fixed slides
were individually examined under a microscope at 25-40.times.
magnification. Positive pools demonstrating binding of sP3G2:Fc
were visualized by the presence of autoradiographic silver grains
against the film background. Two positive pools were titered and
plated to provide plates containing approximately 200 colonies
each. Each plate was scraped to provide pooled plasmid DNA for
transfection into CV1-EBNA cells and screening as described above.
Following subsequent breakdowns and screenings, one positive
individual colony was obtained for each pool. The cDNA insert of
the positive clones were identified as HLA-B44 and HLA-A2, class I
MHC antigens.
Example 8
Northern Blot Analysis
[0156] Since the experiments described in Example 4 resulted in the
detection of LIR-P3G2 surface expression on a number of cell lines,
conventional Northern Blot analysis procedures were used to study
the expression of LIR-P3G2 and any LIR-P3G2 related mRNAs in
different tissue types. The cell lines selected for Northern Blot
analysis were RAJI, PBT, PBM, YT, HEP3B, HELA, KB, KG-1, IMTLH,
HPT, HFF, THP-1, and U937. The following describes the Northern
Blot analysis and the analysis results.
[0157] The cDNA encoding the extracellular region of P3G2 was
isolated using primers which flank the extracellular region of P3G2
and having the following sequences:
4 Sal I 5' -TAT GTC GAG CAT GAG CCC CAT CCT GAG GGT - 3' (SEQ ID
NO:5) Bgl II 5' - TAT AGA TCT ACC CCC AGG TGG CTT CCC AGA CCA (SEQ
ID NO:27)
[0158] The PCR template was the full length P3G2 gene isolated as
described in Example 3 above. The conditions for the PCR reaction
were as follows: One cycle at 95.degree. C. for 5 minutes; 30
cycles which included 95.degree. C. for 45 seconds, 64 .degree. C.
for 45 seconds and 72.degree. C. for 45 seconds; and, one cycle at
72.degree. C. for 5 minutes. The PCR product was cloned into PCR II
vector, purchased from Invitrogen, in accordance with the
supplier's instructions. The isolated DNA encoding the
extracellular region of P3G2 was used to make a riboprobe with the
Ambion MAXISCRIPT Kit according to the manufacturer's
instructions.
[0159] Northern blots containing poly A+selected RNA or total RNA
from a variety of human cell lines were prepared by resolving RNA
samples on a 1.1% agarose-formaldehyde gel, blotting onto Hybond-N
as recommended by the manufacturer (Amersham Corporation) and
staining with methylene blue to monitor RNA concentrations. The
blots were prepared using 1 .mu.g of the PolyA+RNA or 10 .mu.g of
total RNA and each blot was probed with 10.sup.6 cpm/mL RNA
extracellular P3G2 riboprobe, prepared as just described, at
63.degree. C. for 16 hours. The probed blots were washed with
2.times.SSC at 63.degree. C. for 30 minutes 2 times; 1.times.SSC at
63.degree. C. for 30 minutes 2 times; and, 0.1.times.SSC at
63.degree. C. for 5 minutes 2 times.
[0160] The probed blots were autoradiographically developed. The
developed blots showed that the P3G2 RNA hybridized to a 3.5 kb RNA
expressed by RAJI, CB23 and U937; an approximately 1.5kb RNA
expressed by THP-1; and multiple RNAs ranging from 1.5 kb to 3.5 kb
expressed by PBM. These results suggest that different genes having
extracellular domains similar in structure to that of P3G2 may be
expressed by peripheral blood monocytes.
Example 9
Probing PBM cDNA Library to Isolate LIR Polypeptides
[0161] The following describes steps taken to screen a peripheral
blood monocyte cDNA library to isolate polypeptides relating to the
P3G2 polypeptide using conventional Southern Blot methodologies. A
peripheral blood monocyte cDNA library was prepared using
substantially the same procedures described in Example 7.
[0162] DNA from an initial 15 pools of cDNA having 10,000 clones
per pool was digested with Bgl II restriction enzyme and
electrophoresed on a 1% agarose gel at 100 V for 2 hours. Southern
Blots were prepared by electroblotting the electrophoresed DNA in
0.55% TBE buffer onto Hybond membranes. The blotted DNA was
denatured in 0.5 M NaOH in 0.6M NaCl solution for 5 minutes and
then neutralized in 0.5 M TRIS in 1.5 M NaCl at pH 7.8 for 5
minutes. The membranes were placed in a STRATALINKER UV crosslinker
for 20 seconds to crosslink the blotted DNA to the membrane. The
membrane and bound DNA were placed in pre-hybridization solution of
10.times. Denhart's Solution, 0.05M TRIS at pH 7.5, 0.9M NaCl, 0.1%
sodium pyrophosphate, 1% SDS and 200 .mu.g/mL salmon sperm DNA at
63.degree. C. for 2 hours and then the bound DNA was probed with
.sup.32P labeled probe of DNA encoding the extracellular region of
LIR-P3G2, including the signal peptide and Sal I and Bgl II
restriction sites. The concentration of the DNA probe in
hybridization solution was 10.sup.6 CPM per mL of hybridization
solution. The probed blots were incubated for 16 hours at
63.degree. C. and then washed with 2.times.SSC at 63.degree. C. for
1 hour with one solution change; 1.times. with SSC at 63.degree. C.
for one hour with one solution change; and, with 0.1.times.SSC at
68.degree. C. for 45 minutes with one solution change. After drying
the blots they were autoradiographically developed and visualized
for DNA bands which hybridized to the P3G2 extracellular DNA
probe.
[0163] The results of the autoradiography visualization indicated
that all pools contained DNA which hybridized to the probe. One
pool showing 7 positive DNA bands was selected and subsequently
subdivided to 10 pools having 3,000 clones per pool. Applying
subsequent Southern Blotting methodologies to the 10 pools resulted
in one pool showing 9 positively hybridizing DNA sequences. Single
hybridizing clones were isolated by standard colony hybridization
techniques.
[0164] Duplicate bacterial colonies on filters were probed with the
P3G2 extracellular probe described above at a concentration of
500,000 cpm/mL at 63.degree. C. for 16 hours. The hybridized
filters were washed with 2.times.SSC at 63.degree. C. for 30
minutes; with 1.times. SSC at 63.degree. C. for 30 minutes; and
finally with 0.1 .times.SSC at 68.degree. C. for 15 minutes.
[0165] Forty-eight clones were visualized as hybridizing on
duplicate filters by autoradiography and DNA obtained from these
clones using standard DNA preparation methodologies was digested
with Bgl H. Then Southern Blots of the digests were obtained and
probed with the P3G2 extracellular probe described above. Seven
different sized cloned inserts were identified as positively
hybridizing to the P3G2 probe. The nucleotide sequence of each of
the inserts was obtained using automated sequencing technology. Of
the 8 different cloned inserts, one was identical in sequence to
LIR-P3G2. The others were identified as DNA encoding polypeptides
of the new LIR family of polypeptides. The nucleotide sequences
(cDNA) of the isolated LIR family members are presented in SEQ ID
NO:7 (designated pbm25), SEQ ID NO:9 (designated pbm8), SEQ ID
NO:11 (designated pbm36-2), SEQ ID NO:13 (designated pbm364); SEQ
ID NO:15 (designated pbmhh); SEQ ID NO:17 (designated pbm2) and SEQ
ID NO:19 (designated pbm17). The amino acid sequences encoded
thereby are presented in SEQ ID NO:8 (designated pbm25), SEQ ID
NO:10 (designated pbm8), SEQ ID NO:12 (designated pbm36-2), SEQ ID
NO:14 (designated pbm364), SEQ ID NO:16 (designated pbmhh); SEQ ID
NO:18 (designated pbm2); and SEQ ID NO:20 (designated pbm17).
Example 10
Screening a Human Dendritic Cell cDNA Library for LIR cDNA
Sequences
[0166] The following describes the isolation and identification of
an LIR family member by screening a human bone marrow-derived
dendritic cell cDNA library in the .lambda. Zap vector with a
radiolabeled Hh0779 cDNA fragment. The Hh0779 cDNA fragment is a
0.7kb insert of the Hh0779 clone previously isolated from a human
dendritic cell cDNA library and obtained by restriction digestion
with the enzymes PstI and Spel. The Hh0779 cDNA fragment was
labeled with [a-.sup.32P]dCTP using the DECAprime II DNA labeling
kit purchased from Ambion.
[0167] The .lambda. Zap cDNA library was plated at a density of
20,000 pfu per plate to provide a total of 480,000 plagues for the
initial screening. The .lambda. Zap cDNA was blotted in duplicate
onto Hybond membranes, purchased from Amersham, and then denatured
in a solution of 0.5N NaOH and 0.5M NaCl for 5 minutes. The
membranes were neutralized in a solution of 0.5M Tris (pH 7.8) and
1.5M NaCl for 5 minutes, and then washed in 2.times. SSC for 3
minutes. The cDNA was crosslinked to the Hybond membranes using a
STRATALINKER UV crosslinker in the auto setting.
[0168] The membranes were pre-hybridized at 65.degree. C. for 2.25
hours in hybridization buffer containing 10.times.Denhardt's, 0.05M
Tris (pH 7.5), 0.9M NaCl, 0.1% sodium pyrophosphate, 1% SDS and 4
mg/mL heat denatured salmon sperm DNA. After the pre-hybridization,
the radiolabeled Hh0779 cDNA was added to the hybridization buffer
to a final concentration of 0.54.times.10.sup.6 cpm/mL. After 24
hours of hybridization, the membranes were washed in
0.25.times.SSC, 0.25% SDS at 65.degree. C. for 1.5 hours. The blots
were then exposed to autoradiographic film to visual positive
clones.
[0169] A total of 146 positive clones showing hybridization signals
in both membranes of a duplicate set were identified, isolated, and
saved for future use. Of the 146 clones, 35 were selected for
secondary screening. The selected clones were plated at low density
and single clones were isolated after hybridization to the HH0779
probe using the hybridization conditions described above. The
plasmids were then isolated from the .lambda. Zap clones using the
VCSM13 helper phage purchased from Stratagene. The plasmid DNA was
analyzed by restriction digestion and PCR, and the clones
containing the 24 largest inserts were selected and sequenced. Of
the 24 sequenced clones, 6 encoded LIR-P3G2, 3 encoded LIR-pbm2, 8
encoded LIR-pbm364 and LIR-pbm36-2 , 1 encoded LIR-pbm8, 2 encoded
LIR-pmbhh, and 1 encoded a novel sequence designated LIR-pbmnew.
Three clones were identified as encoding amino acid sequences that
are not relevant to the LIR polypeptide family.
Example 11
Association of LIR-P3G2 and LIR-pbm8 with Tyrosine Phosphatase,
SHP-1
[0170] The following describes the tests performed to demonstrate
that LIR-P3G2 and LIR-pbm8 associate with SHP-1. Human monocytes
were cultured in RPMI medium supplemented with 10% FBS,
concentrated by centrifugation and finally subdivided into two
aliquots. One aliquot was stimulated with a solution of 50 Mm/mL
sodium pervanadate for 5 minutes. The second aliquot was not
stimulated. After stimulation, the cells in each aliquot were
immediately lysed in RIPA buffer containing 1% NP-40, 0.5% sodium
deoxycholate, 50 mM Tris pH8, 2 mM EDTA, 0.5 mM sodium
orthovanadate, 5mM sodium fluoride, 25mM .beta.-glycerol phosphate,
and protease inhibitors. Samples of 24.times.10.sup.6 cell
equivalents were incubated for 2 hours at 4.degree. C. with either
5 .mu.g/mL of anti-SHP-1 antibody purchased from Transduction
Laboratories, or 5 .mu.g/mL of an isotype-matched antibody control
(anti-Flag-M5 IgGI). The resulting immunocomplexes were
precipitated by incubation with protein G-agarose (Boehringer
Mannheim), washed, and resuspended in 40 mL of 2.times. SDS-PAGE
sample buffer. Twenty microliters of each immunoprecipitate were
loaded onto electrophoresis gels, electrophoresed under reducing
conditions, and transferred to nitrocellulose membranes purchased
from Amersham. Western blots were probed with anti-LIR-P3G2
monoclonal antibody sera and anti-LIR-pbm8 monoclonal antibody
antisera and the immunocomplexes were detected by enhanced
chemiluminescence (NEN).
[0171] A protein having a molecular weight of approximately 120
kDa, corresponding to LIR-P3G2 was readily detected in SHP-1
immunoprecipitates, but not the immunoprecipitates generated with
the anti-Flag-M5 antibody control. Similarly, a protein of
90-100kDa, corresponding to LIR-pbm8, was detected in SHP-1
immunoprecipitates, but not in the control immunoprecipitates.
Neither the LIR-P3G2 band nor the LIR-pbm8 band was seen in the
absence of sodium pervanadate treatment. This confirms that
tyrosine phosphorylation of LIR-P3G2 is essential for the
association of LIR-P3G2 and SHP-1 and phosphorylation of LIR-pbm8
is essential for the association of LIR-pbm8 and SHP-1.
[0172] To study the inhibition of Fc.gamma.RI-mediated tyrosine
phosphorylation events upon LIR coligation, peripheral blood
monocytes were incubated with or without 10 .mu.g/mL of F(ab).sub.2
version of a number of antibodies (.alpha.-LIR-1+.alpha.-LIR-2,
.alpha.-CD11c, .alpha.CD14, .alpha.CD64,
.alpha.-CD64+.alpha.-LIR-1, .alpha.-CD64+.alpha.-LIR-2,
.alpha.-CD64+.alpha.-LIR-1+.alpha.-LIR-2,
.alpha.CD64+.alpha.-CD11c, .alpha.-CD64+.alpha.-CD 14). This was
followed with crosslinking with 30 .mu./mL of polyclonal
F(ab).sub.2 goat anti-mouse. Cell lysates were immunoprecipitated
overnight with anti-phosphotyrosine conjugated agarose,
electrophoresed, and transferred onto nitrocellulose Western
blotting was performed using a combination of PY-20 and 4G10
HRP-conjugated anti-phosphotyrosine mAbs. This data demonstrates
the specific inhibition of Fc.gamma.RI-mediated tyrosine
phosphorylation events upon LIR-P3G2 and LIR-pbm8 coligation.
Example 12
Generating Antibodies Immunoreactive with LIR Polypeptides
[0173] The following describes generating monoclonal antibody
immunoreactive with LIR family members. A purified LIR polypeptide
is prepared by COS-1 cell expression and affinity purification as
described in Example 4. The purified protein or cells transfected
with an expression vector encoding the full length protein can
generate monoclonal antibodies against the LIR polypeptide using
conventional techniques, for example those techniques described in
U.S. Pat. No. 4,411,993. Briefly BALB-C mice are immunized at 0, 2
and 6 weeks with 10 .mu.g of the LIR polypeptide. The primary
immunization is prepared with TITERMAX adjuvant and subsequent
immunizations are prepared with incomplete Freund's adjuvant (IFA).
At 11 weeks, the mice are IV boosted with 3-4 .mu.g the LIR
polypeptide in PBS. Three days after the IV boost, splenocytes are
harvested and fused with an Ag8.653 myeloma fusion partner using
50% aqueous PEG 1500 solution. Hybridoma supernatants are screened
by ELISA using the LIR transfected cells in PBS at 7.times.10.sup.3
cells per well and dried to polystyrene 96-well microtiter plates
as the platecoat antigen. Positive supernatants are subsequently
confirmed by FACS analysis and RIP using LIR transfected cells.
Hybridomas are cloned and followed in the same manner of screening.
Monoclonal cultures are expanded and supernatants purified by
affinity chromatography.
Example 13
Flow Cytometric Analysis For Expression of LIR-P3G2 and LIR-pbm8 on
Lymphoid and Myeloid Cells
[0174] In order to compare the differential expression and
distribution of LIR-P3G2 and LIR-pbm8 on lymphocyte populations,
freshly isolated peripheral blood mononuclear cells (PBMC) were
stained with PE-labeled anti-CD3, anti-CD19, or anti-CD56 mAb in
the presence of either biotin labeled anti-LIR-P3G2 or anti
LIR-pbm8 mAb. Then the stained cells were treated with APC-labeled
streptavidin. Density plots representing 5.times.10.sup.4 events
were collected on a FACScaliber (from Beckton Dickinson). The
results demonstrated that LIR-P3G2 is expressed on 80%-95% of
CD19+B cells, on 5%-15% CD3.sup.+ T cells, and on 10%-30%
CD56.sup.+ NK cells. On the cells examiner from the same 12 donors,
LIR-pbm8 expression was not detected on CD19.sup.+ B cells,
CD3.sup.+ T cells, and CD56.sup.+NK cells.
[0175] Countercurrent elutriated fractions containing a high
percentage of circulating monocytes and dendritic cells (DC) were
obtained. The monocytes were characterized according to the
phenotypes subsets CD14.sup.+CD16.sup.- and CD14.sup.+CD16.sup.+.
The peripheral blood DC were characterized with the phenotype
CD33.sup.+CD14.sup.-CD16.sup.-HLA-D- R.sup.+ The monocytes subsets
and DC's were stained with FITC-labeled antiCD14, PE-labeled anti
CD3, perCp-labeled antiHLA-DR, and either biotin-labeled anti-CD16,
anti-LIR-P3G2, or anti LIR-pbm8. Then the stained cells were
treated with APC-labeled streptavidin. Both monocyte subsets
co-express similar levels of LIR-P3G2 and LIR-pbm8, with the
highest LIR-P3G2 and LIR-pbm8 expression detected on the
CD14.sup.+CD16.sup.+subset. Blood DC express lower levels of
LIR-P3G2 and LIR-pbm8 compared to monocytes. The results of these
experiments demonstrate the LIR-P3G2 is expressed on lymphocytes,
monocytes and DC, and LIR-pbm8 is expressed on monocytes and
DC.
Example 14
Screening LIR-P3G2 and LIR-pbm8 Binding to HLA Class I Alleles
[0176] The following describes flow cytometry analyses used to
screen LIR-P3G2 and LIR-pbm8 for binding to HLA Class I alleles.
The B lymphoblastoid class I-deficient 721.221 cell line,
untransfected or transfected with a panel of HLA class I alleles
was used for staining. LIR-P3G2/Fc and LIR-pbm8/Fc fusion proteins
were used in the binding studies and both bound detectably to seven
of the eleven HLA-A, HLA-B and HLA-C alleles that were tested. In
general, LIR-P3G2/Fc and LIR-pbm8/Fc bind with higher affinity to
HLA-B alleles than to HLA-A or HLA-C alleles. W6/32 (ATCC HB-95),
an antibody against MHC Class I heavy chains (an anti HLA-A, B, and
C molecule) inhibits LIR-P3G2/Fc and LIR-pbm8/Fc binding to all
class I transfectants. Finally, LIR-P3G2 and LIR-pbm8 binding does
not correlate with the MHC class I expression levels. Thus,
LIR-P3G2 and LIR-pbm8 bind to several HLA-A, -B, and -C alleles,
and recognize a similar broad spectrum of MHC class I
specificities.
Example 15
Isolation of LIR-9m1, LIR-9m, LIR-9s1, LIR-9s2 and LIR-10
[0177] In the course of high throughput sequencing of a human
dendritic cell cDNA library, it was noted that the sequence of an
incomplete cDNA (clone ss4894) was strikingly similar to the
nucleotide sequences of LIRs 6a, 6b and 7, thus suggesting that
ss4894 was a member of the LIR gene family. To obtain the remainder
of this cDNA clone, the Rapid Amplification cDNA Extension system
(RACE) was used to amplify a human leukocyte cDNA library (Chenchik
et al., A new method for full-length cDNA cloning by PCR, In A
Laboratory Guide to RNA: Isolation, Analysis, and Synthesis, Ed.
Kreig, P. A. (Wiley-Liss, Inc.), pages 273-321). The first round of
amplification employed one primer corresponding to the RACE adapter
at the 5' end of the cDNAs, and a second primer corresponding to
sequences near the 3' end of ss4894. This effort yielded several
clones that contained sequence that was highly homologous though
not identical to that of ss4894 and that extended upstream beyond
an initiating methionine codon. These clones, however, lacked some
of the sequence at the 3' end of the coding region. In an effort to
obtain an entire coding region, another round of RACE sequencing
was performed, this time using a first primer from near the 5' end
of the first RACE products, and a second primer corresponding to
the 3' adapter. This effort yielded five clones containing LIR
inserts, four of which are closely related and appear to encode
variants of the same gene. These four closely related cDNA
sequences were designated LIR-9m1, LIR-9m2, LIR-9s1 and LIR-9s2
(SEQ ID NOS:29, 31, 33 and 35). The fifth of the clones obtained
using this last set of primers represented a different gene, which
has been designated LIR-10 (SEQ ID NO:37).
[0178] All four of the LIR-9 clones encode variants of the same
protein, and are presumed to be the products of alternative
splicing. The proteins encoded by LIR-9m1 (SEQ ID NO:30) and
LIR-9s1 (SEQ ID NO:34) contain a 12 amino acid insert that is
absent from LIR-9m2 (SEQ ID NO:32) and LIR-9s2 (SEQ ID NO:36). The
soluble forms of the LIR-9 protein, i.e., LIR-9s1 and LIR-9s2,
diverge near their carboxy termini from the membrane forms, i.e.,
LIRs-9m1 and -9m2. This divergence presumably is due to different
exons being used by the soluble and membrane forms to encode that
region of the protein.
Sequence CWU 1
1
39 1 2922 DNA human CDS (310)..(2262) 1 agggccacgc gtgcatgcgt
cgactggaac gagacgacct gctgtgaccc ccttgtgggc 60 actccattgg
ttttatggcg cctctacttt ctggagtttg tgtaaaacaa aaatattatg 120
gtctttgtgc acatttacat caagctcagc ctgggcggca cagccagatg cgagatgcgt
180 ctctgctgat ctgagtctgc ctgcagcatg gacctgggtc ttccctgaag
catctccagg 240 gctggaggga cgactgccat gcaccgaggg ctcatccatc
cacagagcag ggcagtggga 300 ggagacgcc atg acc ccc atc ctc acg gtc ctg
atc tgt ctc ggg ctg 348 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu
Gly Leu 1 5 10 agt ctg ggc ccc cgg acc cac gtg cag gca ggg cac ctc
ccc aag ccc 396 Ser Leu Gly Pro Arg Thr His Val Gln Ala Gly His Leu
Pro Lys Pro 15 20 25 acc ctc tgg gct gaa cca ggc tct gtg atc acc
cag ggg agt cct gtg 444 Thr Leu Trp Ala Glu Pro Gly Ser Val Ile Thr
Gln Gly Ser Pro Val 30 35 40 45 acc ctc agg tgt cag ggg ggc cag gag
acc cag gag tac cgt cta tat 492 Thr Leu Arg Cys Gln Gly Gly Gln Glu
Thr Gln Glu Tyr Arg Leu Tyr 50 55 60 aga gaa aag aaa aca gca ccc
tgg att aca cgg atc cca cag gag ctt 540 Arg Glu Lys Lys Thr Ala Pro
Trp Ile Thr Arg Ile Pro Gln Glu Leu 65 70 75 gtg aag aag ggc cag
ttc ccc atc cca tcc atc acc tgg gaa cat gca 588 Val Lys Lys Gly Gln
Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala 80 85 90 ggg cgg tat
cgc tgt tac tat ggt agc gac act gca ggc cgc tca gag 636 Gly Arg Tyr
Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu 95 100 105 agc
agt gac ccc ctg gag ctg gtg gtg aca gga gcc tac atc aaa ccc 684 Ser
Ser Asp Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro 110 115
120 125 acc ctc tca gcc cag ccc agc ccc gtg gtg aac tca gga ggg aat
gta 732 Thr Leu Ser Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn
Val 130 135 140 acc ctc cag tgt gac tca cag gtg gca ttt gat ggc ttc
att ctg tgt 780 Thr Leu Gln Cys Asp Ser Gln Val Ala Phe Asp Gly Phe
Ile Leu Cys 145 150 155 aag gaa gga gaa gat gaa cac cca caa tgc ctg
aac tcc cag ccc cat 828 Lys Glu Gly Glu Asp Glu His Pro Gln Cys Leu
Asn Ser Gln Pro His 160 165 170 gcc cgt ggg tcg tcc cgc gcc atc ttc
tcc gtg ggc ccc gtg agc ccg 876 Ala Arg Gly Ser Ser Arg Ala Ile Phe
Ser Val Gly Pro Val Ser Pro 175 180 185 agt cgc agg tgg tgg tac agg
tgc tat gct tat gac tcg aac tct ccc 924 Ser Arg Arg Trp Trp Tyr Arg
Cys Tyr Ala Tyr Asp Ser Asn Ser Pro 190 195 200 205 tat gag tgg tct
cta ccc agt gat ctc ctg gag ctc ctg gtc cta ggt 972 Tyr Glu Trp Ser
Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly 210 215 220 gtt tct
aag aag cca tca ctc tca gtg cag cca ggt cct atc gtg gcc 1020 Val
Ser Lys Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala 225 230
235 cct gag gag acc ctg act ctg cag tgt ggc tct gat gct ggc tac aac
1068 Pro Glu Glu Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr
Asn 240 245 250 aga ttt gtt ctg tat aag gac ggg gaa cgt gac ttc ctt
cag ctc gct 1116 Arg Phe Val Leu Tyr Lys Asp Gly Glu Arg Asp Phe
Leu Gln Leu Ala 255 260 265 ggc gca cag ccc cag gct ggg ctc tcc cag
gcc aac ttc acc ctg ggc 1164 Gly Ala Gln Pro Gln Ala Gly Leu Ser
Gln Ala Asn Phe Thr Leu Gly 270 275 280 285 cct gtg agc cgc tcc tac
ggg ggc cag tac aga tgc tac ggt gca cac 1212 Pro Val Ser Arg Ser
Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His 290 295 300 aac ctc tcc
tcc gag tgg tcg gcc ccc agc gac ccc ctg gac atc ctg 1260 Asn Leu
Ser Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu 305 310 315
atc gca gga cag ttc tat gac aga gtc tcc ctc tcg gtg cag ccg ggc
1308 Ile Ala Gly Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro
Gly 320 325 330 ccc acg gtg gcc tca gga gag aac gtg acc ctg ctg tgt
cag tca cag 1356 Pro Thr Val Ala Ser Gly Glu Asn Val Thr Leu Leu
Cys Gln Ser Gln 335 340 345 gga tgg atg caa act ttc ctt ctg acc aag
gag ggg gca gct gat gac 1404 Gly Trp Met Gln Thr Phe Leu Leu Thr
Lys Glu Gly Ala Ala Asp Asp 350 355 360 365 cca tgg cgt cta aga tca
acg tac caa tct caa aaa tac cag gct gaa 1452 Pro Trp Arg Leu Arg
Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu 370 375 380 ttc ccc atg
ggt cct gtg acc tca gcc cat gcg ggg acc tac agg tgc 1500 Phe Pro
Met Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys 385 390 395
tac ggc tca cag agc tcc aaa ccc tac ctg ctg act cac ccc agt gac
1548 Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser
Asp 400 405 410 ccc ctg gag ctc gtg gtc tca gga ccg tct ggg ggc ccc
agc tcc ccg 1596 Pro Leu Glu Leu Val Val Ser Gly Pro Ser Gly Gly
Pro Ser Ser Pro 415 420 425 aca aca ggc ccc acc tcc aca tct ggc cct
gag gac cag ccc ctc acc 1644 Thr Thr Gly Pro Thr Ser Thr Ser Gly
Pro Glu Asp Gln Pro Leu Thr 430 435 440 445 ccc acc ggg tcg gat ccc
cag agt ggt ctg gga agg cac ctg ggg gtt 1692 Pro Thr Gly Ser Asp
Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val 450 455 460 gtg atc ggc
atc ttg gtg gcc gtc atc cta ctg ctc ctc ctc ctc ctc 1740 Val Ile
Gly Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu 465 470 475
ctc ctc ttc ctc atc ctc cga cat cga cgt cag ggc aaa cac tgg aca
1788 Leu Leu Phe Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp
Thr 480 485 490 tcg acc cag aga aag gct gat ttc caa cat cct gca ggg
gct gtg ggg 1836 Ser Thr Gln Arg Lys Ala Asp Phe Gln His Pro Ala
Gly Ala Val Gly 495 500 505 cca gag ccc aca gac aga ggc ctg cag tgg
agg tcc agc cca gct gcc 1884 Pro Glu Pro Thr Asp Arg Gly Leu Gln
Trp Arg Ser Ser Pro Ala Ala 510 515 520 525 gat gcc cag gaa gaa aac
ctc tat gct gcc gtg aag cac aca cag cct 1932 Asp Ala Gln Glu Glu
Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro 530 535 540 gag gat ggg
gtg gag atg gac act cgg agc cca cac gat gaa gac ccc 1980 Glu Asp
Gly Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro 545 550 555
cag gca gtg acg tat gcc gag gtg aaa cac tcc aga cct agg aga gaa
2028 Gln Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg
Glu 560 565 570 atg gcc tct cct cct tcc cca ctg tct ggg gaa ttc ctg
gac aca aag 2076 Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe
Leu Asp Thr Lys 575 580 585 gac aga cag gcg gaa gag gac agg cag atg
gac act gag gct gct gca 2124 Asp Arg Gln Ala Glu Glu Asp Arg Gln
Met Asp Thr Glu Ala Ala Ala 590 595 600 605 tct gaa gcc ccc cag gat
gtg acc tac gcc cag ctg cac agc ttg acc 2172 Ser Glu Ala Pro Gln
Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr 610 615 620 ctt aga cgg
aag gca act gag cct cct cca tcc cag gaa ggg ccc tct 2220 Leu Arg
Arg Lys Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser 625 630 635
cca gct gtg ccc agc atc tac gcc act ctg gcc atc cac tag 2262 Pro
Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 640 645 650
cccagggggg gacgcagacc ccacactcca tggagtctgg aatgcatggg agctgccccc
2322 ccagtggaca ccattggacc ccacccagcc tggatctacc ccaggagact
ctgggaactt 2382 ttaggggtca ctcaattctg cagtataaat aactaatgtc
tctacaattt tgaaataaag 2442 caacagactt ctcaataatc aatgaagtag
ctgagaaaac taagtcagaa agtgcattaa 2502 actgaatcac aatgtaaata
ttacacatca agcgatgaaa ctggaaaact acaagccacg 2562 aatgaatgaa
ttaggaaaga aaaaaagtag gaaatgaatg atcttggctt tcctataaga 2622
aatttagggc agggcacggt ggctcacgcc tgtaattcca gcactttggg aggccgaggc
2682 gggcagatca cgagttcagg agatcgagac catcttggcc aacatggtga
aaccctgtct 2742 ctcctaaaaa tacaaaaatt agctggatgt ggtggcagtg
cctgtaatcc cagctatttg 2802 ggaggctgag gcaggagaat cgcttgaacc
agggagtcag aggtttcagt gagccaagat 2862 cgcaccactg ctctccagcc
tggcgacaag caggtcgtct cgttccagtc gacggcccat 2922 2 650 PRT human 2
Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5
10 15 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu
Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val
Thr Leu Arg 35 40 45 Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg
Leu Tyr Arg Glu Lys 50 55 60 Lys Thr Ala Pro Trp Ile Thr Arg Ile
Pro Gln Glu Leu Val Lys Lys 65 70 75 80 Gly Gln Phe Pro Ile Pro Ser
Ile Thr Trp Glu His Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Gly
Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp 100 105 110 Pro Leu Glu
Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120 125 Ala
Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Thr Leu Gln 130 135
140 Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly
145 150 155 160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His
Ala Arg Gly 165 170 175 Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val
Ser Pro Ser Arg Arg 180 185 190 Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp
Ser Asn Ser Pro Tyr Glu Trp 195 200 205 Ser Leu Pro Ser Asp Leu Leu
Glu Leu Leu Val Leu Gly Val Ser Lys 210 215 220 Lys Pro Ser Leu Ser
Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu 225 230 235 240 Thr Leu
Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255
Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln 260
265 270 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val
Ser 275 280 285 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His
Asn Leu Ser 290 295 300 Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp
Ile Leu Ile Ala Gly 305 310 315 320 Gln Phe Tyr Asp Arg Val Ser Leu
Ser Val Gln Pro Gly Pro Thr Val 325 330 335 Ala Ser Gly Glu Asn Val
Thr Leu Leu Cys Gln Ser Gln Gly Trp Met 340 345 350 Gln Thr Phe Leu
Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg 355 360 365 Leu Arg
Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380
Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser 385
390 395 400 Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro
Leu Glu 405 410 415 Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser
Pro Thr Thr Gly 420 425 430 Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln
Pro Leu Thr Pro Thr Gly 435 440 445 Ser Asp Pro Gln Ser Gly Leu Gly
Arg His Leu Gly Val Val Ile Gly 450 455 460 Ile Leu Val Ala Val Ile
Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 Leu Ile Leu
Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 Arg
Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505
510 Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln
515 520 525 Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu
Asp Gly 530 535 540 Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp
Pro Gln Ala Val 545 550 555 560 Thr Tyr Ala Glu Val Lys His Ser Arg
Pro Arg Arg Glu Met Ala Ser 565 570 575 Pro Pro Ser Pro Leu Ser Gly
Glu Phe Leu Asp Thr Lys Asp Arg Gln 580 585 590 Ala Glu Glu Asp Arg
Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala 595 600 605 Pro Gln Asp
Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg 610 615 620 Lys
Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val 625 630
635 640 Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 645 650 3 2777 DNA
human CDS (168)..(2126) 3 agctcagcct gggcggcaca gccagatgcg
agatgcgtct ctgctgatct gagtctgcct 60 gcagcatgga cctgggtctt
ccctgaagca tctccagggc tggagggacg actgccatgc 120 accgagggct
catccatcca cagagcaggg cagtgggagg agacgcc atg acc ccc 176 Met Thr
Pro 1 atc ctc acg gtc ctg atc tgt ctc ggg ctg agt ctg ggc ccc agg
acc 224 Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly Pro Arg
Thr 5 10 15 cac gtg cag gca ggg cac ctc ccc aag ccc acc ctc tgg gct
gaa cca 272 His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp Ala
Glu Pro 20 25 30 35 ggc tct gtg atc acc cag ggg agt cct gtg acc ctc
agg tgt cag ggg 320 Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu
Arg Cys Gln Gly 40 45 50 ggc cag gag acc cag gag tac cgt cta tat
aga gaa aag aaa aca gca 368 Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr
Arg Glu Lys Lys Thr Ala 55 60 65 ctc tgg att aca cgg atc cca cag
gag ctt gtg aag aag ggc cag ttc 416 Leu Trp Ile Thr Arg Ile Pro Gln
Glu Leu Val Lys Lys Gly Gln Phe 70 75 80 ccc atc cca tcc atc acc
tgg gaa cat gca ggg cgg tat cgc tgt tac 464 Pro Ile Pro Ser Ile Thr
Trp Glu His Ala Gly Arg Tyr Arg Cys Tyr 85 90 95 tat ggt agc gac
act gca ggc cgc tca gag agc agt gac ccc ctg gag 512 Tyr Gly Ser Asp
Thr Ala Gly Arg Ser Glu Ser Ser Asp Pro Leu Glu 100 105 110 115 ctg
gtg gtg aca gga gcc tac atc aaa ccc acc ctc tca gcc cag ccc 560 Leu
Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser Ala Gln Pro 120 125
130 agc ccc gtg gtg aac tca gga ggg aat gta atc ctc cag tgt gac tca
608 Ser Pro Val Val Asn Ser Gly Gly Asn Val Ile Leu Gln Cys Asp Ser
135 140 145 cag gtg gca ttt gat ggc ttc agt ctg tgt aag gaa gga gaa
gat gaa 656 Gln Val Ala Phe Asp Gly Phe Ser Leu Cys Lys Glu Gly Glu
Asp Glu 150 155 160 cac cca caa tgc ctg aac tcc cag ccc cat gcc cgt
ggg tcg tcc cgc 704 His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg
Gly Ser Ser Arg 165 170 175 gcc atc ttc tcc gtg ggc ccc gtg agc ccg
agt cgc agg tgg tgg tac 752 Ala Ile Phe Ser Val Gly Pro Val Ser Pro
Ser Arg Arg Trp Trp Tyr 180 185 190 195 agg tgc tat gct tat gac tcg
aac tct ccc tat gag tgg tct cta ccc 800 Arg Cys Tyr Ala Tyr Asp Ser
Asn Ser Pro Tyr Glu Trp Ser Leu Pro 200 205 210 agt gat ctc ctg gag
ctc ctg gtc cta ggt gtt tct aag aag cca tca 848 Ser Asp Leu Leu Glu
Leu Leu Val Leu Gly Val Ser Lys Lys Pro Ser 215 220 225 ctc tca gtg
cag cca ggt cct atc gtg gcc cct gag gag acc ctg act 896 Leu Ser Val
Gln Pro Gly Pro Ile Val Ala Pro Glu Glu Thr Leu Thr 230 235 240 ctg
cag tgt ggc tct gat gct ggc tac aac aga ttt gtt ctg tat aag 944 Leu
Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val Leu Tyr Lys 245 250
255 gac ggg gaa cgt gac ttc ctt cag ctc gct ggc gca cag ccc cag gct
992 Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln Pro Gln Ala
260 265 270 275 ggg ctc tcc cag gcc aac ttc acc ctg ggc cct gtg agc
cgc tcc tac 1040 Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val
Ser Arg Ser Tyr 280 285 290 ggg ggc cag tac aga tgc tac ggt gca cac
aac ctc tcc tcc gag tgg 1088 Gly Gly Gln Tyr Arg Cys Tyr Gly Ala
His Asn Leu Ser Ser Glu Trp 295 300 305 tcg gcc ccc agt gac ccc ctg
gac atc ctg atc gca gga cag ttc tat 1136 Ser Ala Pro Ser Asp Pro
Leu Asp Ile Leu Ile Ala Gly Gln Phe Tyr 310 315 320 gac aga gtc tcc
ctc tcg gtg cag ccg ggc ccc acg gtg gcc tca gga 1184 Asp Arg Val
Ser Leu Ser Val Gln Pro Gly Pro Thr Val Ala Ser
Gly 325 330 335 gag aac gtg acc ctg ctg tgt cag tca cag gga tgg atg
caa act ttc 1232 Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp
Met Gln Thr Phe 340 345 350 355 ctt ctg acc aag gag ggg gca gct gat
gac cca tgg cgt cta aga tca 1280 Leu Leu Thr Lys Glu Gly Ala Ala
Asp Asp Pro Trp Arg Leu Arg Ser 360 365 370 acg tac caa tct caa aaa
tac cag gct gaa ttc ccc atg ggt cct gtg 1328 Thr Tyr Gln Ser Gln
Lys Tyr Gln Ala Glu Phe Pro Met Gly Pro Val 375 380 385 acc tca gcc
cat gcg ggg acc tac agg tgc tac ggc tca cag agc tcc 1376 Thr Ser
Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Gln Ser Ser 390 395 400
aaa ccc tac ctg ctg act cac ccc agt gac ccc ctg gag ctc gtg gtc
1424 Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu Leu Val
Val 405 410 415 tca gga ccg tct ggg ggc ccc agc tcc ccg aca aca ggc
ccc acc tcc 1472 Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr
Gly Pro Thr Ser 420 425 430 435 aca tct gca ggc cct gag gac cag ccc
ctc acc ccc acc ggg tcg gat 1520 Thr Ser Ala Gly Pro Glu Asp Gln
Pro Leu Thr Pro Thr Gly Ser Asp 440 445 450 ccc cag agt ggt ctg gga
agg cac ctg ggg gtt gtg atc ggc atc ttg 1568 Pro Gln Ser Gly Leu
Gly Arg His Leu Gly Val Val Ile Gly Ile Leu 455 460 465 gtg gcc gtc
atc cta ctg ctc ctc ctc ctc ctc ctc ctc ttc ctc atc 1616 Val Ala
Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe Leu Ile 470 475 480
ctc cga cat cga cgt cag ggc aaa cac tgg aca tcg acc cag aga aag
1664 Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg
Lys 485 490 495 gct gat ttc caa cat cct gca ggg gct gtg ggg cca gag
ccc aca gac 1712 Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro
Glu Pro Thr Asp 500 505 510 515 aga ggc ctg cag tgg agg tcc agc cca
gct gcc gat gcc cag gaa gaa 1760 Arg Gly Leu Gln Trp Arg Ser Ser
Pro Ala Ala Asp Ala Gln Glu Glu 520 525 530 aac ctc tat gct gcc gtg
aag cac aca cag cct gag gat ggg gtg gag 1808 Asn Leu Tyr Ala Ala
Val Lys His Thr Gln Pro Glu Asp Gly Val Glu 535 540 545 atg gac act
cgg cag agc cca cac gat gaa gac ccc cag gca gtg acg 1856 Met Asp
Thr Arg Gln Ser Pro His Asp Glu Asp Pro Gln Ala Val Thr 550 555 560
tat gcc gag gtg aaa cac tcc aga cct agg aga gaa atg gcc tct cct
1904 Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser
Pro 565 570 575 cct tcc cca ctg tct ggg gaa ttc ctg gac aca aag gac
aga cag gcg 1952 Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys
Asp Arg Gln Ala 580 585 590 595 gaa gag gac agg cag atg gac act gag
gct gct gca tct gaa gcc ccc 2000 Glu Glu Asp Arg Gln Met Asp Thr
Glu Ala Ala Ala Ser Glu Ala Pro 600 605 610 cag gat gtg acc tac gcc
cag ctg cac agc ttg acc ctc aga cgg gag 2048 Gln Asp Val Thr Tyr
Ala Gln Leu His Ser Leu Thr Leu Arg Arg Glu 615 620 625 gca act gag
cct cct cca tcc cag gaa ggg ccc tct cca gct gtg ccc 2096 Ala Thr
Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro 630 635 640
agc atc tac gcc act ctg gcc atc cac tag cccagggggg gacgcagacc 2146
Ser Ile Tyr Ala Thr Leu Ala Ile His 645 650 ccacactcca tggagtctgg
aatgcatggg agctgccccc ccagtggaca ccattggacc 2206 ccacccagcc
tggatctacc ccaggagact ctgggaactt ttaggggtca ctcaattctg 2266
cagtataaat aactaatgtc tctacaattt tgaaataaag caatagactt ctcaataatc
2326 aatgaagtag ctgagaaaac taagtcagaa agtgcattaa actgaatcac
aatgtaaata 2386 ttacacatca agcgatgaaa ctggaaaact acaagccacg
aatgaatgaa ttaggaaaga 2446 aaaaaagtag gaaatgaatg atcttggctt
tcctataaga aatttagggc agggcacggt 2506 ggctcacgcc tgtaattcca
gcactttggg aggccgaggc gggcagatca cgagttcagg 2566 agatcgagac
catcttggcc aacatggtga aaccctgtct ctcctaaaaa tacaaaaatt 2626
agctggatgt ggtggcagtg cctgtaatcc cagctatttg ggaggctgag gcaggagaat
2686 cgcttgaacc agggagtcag aggtttcagt gagccaagat cgcaccactg
ctctccagcc 2746 tggcgacaga gggagactcc atctcaaatt a 2777 4 652 PRT
human 4 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu
Gly 1 5 10 15 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro
Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser
Pro Val Thr Leu Arg 35 40 45 Cys Gln Gly Gly Gln Glu Thr Gln Glu
Tyr Arg Leu Tyr Arg Glu Lys 50 55 60 Lys Thr Ala Leu Trp Ile Thr
Arg Ile Pro Gln Glu Leu Val Lys Lys 65 70 75 80 Gly Gln Phe Pro Ile
Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr
Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp 100 105 110 Pro
Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120
125 Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Ile Leu Gln
130 135 140 Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ser Leu Cys Lys
Glu Gly 145 150 155 160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln
Pro His Ala Arg Gly 165 170 175 Ser Ser Arg Ala Ile Phe Ser Val Gly
Pro Val Ser Pro Ser Arg Arg 180 185 190 Trp Trp Tyr Arg Cys Tyr Ala
Tyr Asp Ser Asn Ser Pro Tyr Glu Trp 195 200 205 Ser Leu Pro Ser Asp
Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys 210 215 220 Lys Pro Ser
Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu 225 230 235 240
Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245
250 255 Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala
Gln 260 265 270 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly
Pro Val Ser 275 280 285 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly
Ala His Asn Leu Ser 290 295 300 Ser Glu Trp Ser Ala Pro Ser Asp Pro
Leu Asp Ile Leu Ile Ala Gly 305 310 315 320 Gln Phe Tyr Asp Arg Val
Ser Leu Ser Val Gln Pro Gly Pro Thr Val 325 330 335 Ala Ser Gly Glu
Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met 340 345 350 Gln Thr
Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg 355 360 365
Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met 370
375 380 Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly
Ser 385 390 395 400 Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser
Asp Pro Leu Glu 405 410 415 Leu Val Val Ser Gly Pro Ser Gly Gly Pro
Ser Ser Pro Thr Thr Gly 420 425 430 Pro Thr Ser Thr Ser Ala Gly Pro
Glu Asp Gln Pro Leu Thr Pro Thr 435 440 445 Gly Ser Asp Pro Gln Ser
Gly Leu Gly Arg His Leu Gly Val Val Ile 450 455 460 Gly Ile Leu Val
Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu 465 470 475 480 Phe
Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr 485 490
495 Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu
500 505 510 Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala
Asp Ala 515 520 525 Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr
Gln Pro Glu Asp 530 535 540 Gly Val Glu Met Asp Thr Arg Gln Ser Pro
His Asp Glu Asp Pro Gln 545 550 555 560 Ala Val Thr Tyr Ala Glu Val
Lys His Ser Arg Pro Arg Arg Glu Met 565 570 575 Ala Ser Pro Pro Ser
Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp 580 585 590 Arg Gln Ala
Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser 595 600 605 Glu
Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu 610 615
620 Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro
625 630 635 640 Ala Val Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 645
650 5 30 DNA human 5 tatgtcgacc atgaccccca tcctcacggt 30 6 52 DNA
human 6 tatgggctct gctccaggag aagatcttcc ttctataacc cccaggtgcc tt
52 7 1605 DNA human CDS (93)..(1412) 7 gagcctccaa gtgtccacac
cctgtgtgtc ctctgtcctg ccagcaccga gggctcatcc 60 atccacagag
cagtgcagtg ggaggagacg cc atg acc ccc atc ctc acg gtc 113 Met Thr
Pro Ile Leu Thr Val 1 5 ctg atc tgt ctc ggg ctg agc ctg gac ccc agg
acc cac gtg cag gca 161 Leu Ile Cys Leu Gly Leu Ser Leu Asp Pro Arg
Thr His Val Gln Ala 10 15 20 ggg ccc ctc ccc aag ccc acc ctc tgg
gct gag cca ggc tct gtg atc 209 Gly Pro Leu Pro Lys Pro Thr Leu Trp
Ala Glu Pro Gly Ser Val Ile 25 30 35 acc caa ggg agt cct gtg acc
ctc agg tgt cag ggg agc ctg gag acg 257 Thr Gln Gly Ser Pro Val Thr
Leu Arg Cys Gln Gly Ser Leu Glu Thr 40 45 50 55 cag gag tac cat cta
tat aga gaa aag aaa aca gca ctc tgg att aca 305 Gln Glu Tyr His Leu
Tyr Arg Glu Lys Lys Thr Ala Leu Trp Ile Thr 60 65 70 cgg atc cca
cag gag ctt gtg aag aag ggc cag ttc ccc atc cta tcc 353 Arg Ile Pro
Gln Glu Leu Val Lys Lys Gly Gln Phe Pro Ile Leu Ser 75 80 85 atc
acc tgg gaa cat gca ggg cgg tat tgc tgt atc tat ggc agc cac 401 Ile
Thr Trp Glu His Ala Gly Arg Tyr Cys Cys Ile Tyr Gly Ser His 90 95
100 act gca ggc ctc tca gag agc agt gac ccc ctg gag ctg gtg gtg aca
449 Thr Ala Gly Leu Ser Glu Ser Ser Asp Pro Leu Glu Leu Val Val Thr
105 110 115 gga gcc tac agc aaa ccc acc ctc tca gct ctg ccc agc cct
gtg gtg 497 Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro
Val Val 120 125 130 135 acc tca gga agg aat gtg acc atc cag tgt gac
tca cag gtg gca ttt 545 Thr Ser Gly Arg Asn Val Thr Ile Gln Cys Asp
Ser Gln Val Ala Phe 140 145 150 gat ggc ttc att ctg tgt aag gaa gga
gaa gat gaa cac cca caa tgc 593 Asp Gly Phe Ile Leu Cys Lys Glu Gly
Glu Asp Glu His Pro Gln Cys 155 160 165 ctg aac tcc cat tcc cat gcc
cgt ggg tca tcc cgg gcc atc ttc tcc 641 Leu Asn Ser His Ser His Ala
Arg Gly Ser Ser Arg Ala Ile Phe Ser 170 175 180 gtg ggc ccc gtg agc
cca agt cgc agg tgg tcg tac agg tgc tat ggt 689 Val Gly Pro Val Ser
Pro Ser Arg Arg Trp Ser Tyr Arg Cys Tyr Gly 185 190 195 tat gac tcg
cgc gct ccc tat gtg tgg tct cta ccc agt gat ctc ctg 737 Tyr Asp Ser
Arg Ala Pro Tyr Val Trp Ser Leu Pro Ser Asp Leu Leu 200 205 210 215
ggg ctc ctg gtc cca ggt gtt tct aag aag cca tca ctc tca gtg cag 785
Gly Leu Leu Val Pro Gly Val Ser Lys Lys Pro Ser Leu Ser Val Gln 220
225 230 ccg ggt cct gtc gtg gcc cct ggg gag aag ctg acc ttc cag tgt
ggc 833 Pro Gly Pro Val Val Ala Pro Gly Glu Lys Leu Thr Phe Gln Cys
Gly 235 240 245 tct gat gcc ggc tac gac aga ttt gtt ctg tac aag gag
tgg gga cgt 881 Ser Asp Ala Gly Tyr Asp Arg Phe Val Leu Tyr Lys Glu
Trp Gly Arg 250 255 260 gac ttc ctc cag cgc cct ggc cgg cag ccc cag
gct ggg ctc tcc cag 929 Asp Phe Leu Gln Arg Pro Gly Arg Gln Pro Gln
Ala Gly Leu Ser Gln 265 270 275 gcc aac ttc acc ctg ggc cct gtg agc
cgc tcc tac ggg ggc cag tac 977 Ala Asn Phe Thr Leu Gly Pro Val Ser
Arg Ser Tyr Gly Gly Gln Tyr 280 285 290 295 aca tgc tcc ggt gca tac
aac ctc tcc tcc gag tgg tcg gcc ccc agc 1025 Thr Cys Ser Gly Ala
Tyr Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser 300 305 310 gac ccc ctg
gac atc ctg atc aca gga cag atc cgt gcc aga ccc ttc 1073 Asp Pro
Leu Asp Ile Leu Ile Thr Gly Gln Ile Arg Ala Arg Pro Phe 315 320 325
ctc tcc gtg cgg ccg ggc ccc aca gtg gcc tca gga gag aac gtg acc
1121 Leu Ser Val Arg Pro Gly Pro Thr Val Ala Ser Gly Glu Asn Val
Thr 330 335 340 ctg ctg tgt cag tca cag gga ggg atg cac act ttc ctt
ttg acc aag 1169 Leu Leu Cys Gln Ser Gln Gly Gly Met His Thr Phe
Leu Leu Thr Lys 345 350 355 gag ggg gca gct gat tcc ccg ctg cgt cta
aaa tca aag cgc caa tct 1217 Glu Gly Ala Ala Asp Ser Pro Leu Arg
Leu Lys Ser Lys Arg Gln Ser 360 365 370 375 cat aag tac cag gct gaa
ttc ccc atg agt cct gtg acc tcg gcc cac 1265 His Lys Tyr Gln Ala
Glu Phe Pro Met Ser Pro Val Thr Ser Ala His 380 385 390 gcg ggg acc
tac agg tgc tac ggc tca ctc agc tcc aac ccc tac ctg 1313 Ala Gly
Thr Tyr Arg Cys Tyr Gly Ser Leu Ser Ser Asn Pro Tyr Leu 395 400 405
ctg act cac ccc agt gac ccc ctg gag ctc gtg gtc tca gga gca gct
1361 Leu Thr His Pro Ser Asp Pro Leu Glu Leu Val Val Ser Gly Ala
Ala 410 415 420 gag acc ctc agc cca cca caa aac aag tcc gac tcc aag
gct ggt gag 1409 Glu Thr Leu Ser Pro Pro Gln Asn Lys Ser Asp Ser
Lys Ala Gly Glu 425 430 435 tga ggagatgctt gccgtgatga cgctgggcac
agagggtcag gtcctgtcaa 1462 gaggagctgg gtgtcctggg tggacatttg
aagaattata ttcattccaa cttgaagaat 1522 tattcaacac ctttaacaat
gtatatgtga agtactttat tctttcatat tttaaaaata 1582 aaagataatt
atccatgaga aaa 1605 8 439 PRT human 8 Met Thr Pro Ile Leu Thr Val
Leu Ile Cys Leu Gly Leu Ser Leu Asp 1 5 10 15 Pro Arg Thr His Val
Gln Ala Gly Pro Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro
Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 40 45 Cys
Gln Gly Ser Leu Glu Thr Gln Glu Tyr His Leu Tyr Arg Glu Lys 50 55
60 Lys Thr Ala Leu Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys
65 70 75 80 Gly Gln Phe Pro Ile Leu Ser Ile Thr Trp Glu His Ala Gly
Arg Tyr 85 90 95 Cys Cys Ile Tyr Gly Ser His Thr Ala Gly Leu Ser
Glu Ser Ser Asp 100 105 110 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr
Ser Lys Pro Thr Leu Ser 115 120 125 Ala Leu Pro Ser Pro Val Val Thr
Ser Gly Arg Asn Val Thr Ile Gln 130 135 140 Cys Asp Ser Gln Val Ala
Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly 145 150 155 160 Glu Asp Glu
His Pro Gln Cys Leu Asn Ser His Ser His Ala Arg Gly 165 170 175 Ser
Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180 185
190 Trp Ser Tyr Arg Cys Tyr Gly Tyr Asp Ser Arg Ala Pro Tyr Val Trp
195 200 205 Ser Leu Pro Ser Asp Leu Leu Gly Leu Leu Val Pro Gly Val
Ser Lys 210 215 220 Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Val Val
Ala Pro Gly Glu 225 230 235 240 Lys Leu Thr Phe Gln Cys Gly Ser Asp
Ala Gly Tyr Asp Arg Phe Val 245 250 255 Leu Tyr Lys Glu Trp Gly Arg
Asp Phe Leu Gln Arg Pro Gly Arg Gln 260 265 270 Pro Gln Ala Gly Leu
Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 Arg Ser Tyr
Gly Gly Gln Tyr Thr Cys Ser Gly Ala Tyr Asn Leu Ser 290 295 300 Ser
Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly 305 310
315 320 Gln Ile Arg Ala Arg Pro Phe Leu Ser Val Arg Pro Gly Pro Thr
Val 325 330 335 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln
Gly Gly Met 340 345 350 His Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala
Asp Ser Pro Leu Arg 355 360 365 Leu Lys Ser
Lys Arg Gln Ser His Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380 Ser
Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390
395 400 Leu Ser Ser Asn Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu
Glu 405 410 415 Leu Val Val Ser Gly Ala Ala Glu Thr Leu Ser Pro Pro
Gln Asn Lys 420 425 430 Ser Asp Ser Lys Ala Gly Glu 435 9 2221 DNA
human CDS (184)..(1980) 9 gctcactgcc acacgcagct cagcctgggc
ggcacagcca gatgcgagat gcgtctctgc 60 tgatctgagt ctgcctgcag
catggacctg ggtcttccct gaagcatctc cagggctgga 120 gggacgactg
ccatgcaccg agggctcatc catccgcaga gcagggcagt gggaggagac 180 gcc atg
acc ccc atc gtc aca gtc ctg atc tgt ctc ggg ctg agt ctg 228 Met Thr
Pro Ile Val Thr Val Leu Ile Cys Leu Gly Leu Ser Leu 1 5 10 15 ggc
ccc agg acc cac gtg cag aca ggg acc atc ccc aag ccc acc ctg 276 Gly
Pro Arg Thr His Val Gln Thr Gly Thr Ile Pro Lys Pro Thr Leu 20 25
30 tgg gct gag cca gac tct gtg atc acc cag ggg agt ccc gtc acc ctc
324 Trp Ala Glu Pro Asp Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu
35 40 45 agt tgt cag ggg agc ctt gaa gcc cag gag tac cgt cta tat
agg gag 372 Ser Cys Gln Gly Ser Leu Glu Ala Gln Glu Tyr Arg Leu Tyr
Arg Glu 50 55 60 aaa aaa tca gca tct tgg att aca cgg ata cga cca
gag ctt gtg aag 420 Lys Lys Ser Ala Ser Trp Ile Thr Arg Ile Arg Pro
Glu Leu Val Lys 65 70 75 aac ggc cag ttc cac atc cca tcc atc acc
tgg gaa cac aca ggg cga 468 Asn Gly Gln Phe His Ile Pro Ser Ile Thr
Trp Glu His Thr Gly Arg 80 85 90 95 tat ggc tgt cag tat tac agc cgc
gct cgg tgg tct gag ctc agt gac 516 Tyr Gly Cys Gln Tyr Tyr Ser Arg
Ala Arg Trp Ser Glu Leu Ser Asp 100 105 110 ccc ctg gtg ctg gtg atg
aca gga gcc tac cca aaa ccc acc ctc tca 564 Pro Leu Val Leu Val Met
Thr Gly Ala Tyr Pro Lys Pro Thr Leu Ser 115 120 125 gcc cag ccc agc
cct gtg gtg acc tca gga gga agg gtg acc ctc cag 612 Ala Gln Pro Ser
Pro Val Val Thr Ser Gly Gly Arg Val Thr Leu Gln 130 135 140 tgt gag
tca cag gtg gca ttt ggc ggc ttc att ctg tgt aag gaa gga 660 Cys Glu
Ser Gln Val Ala Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly 145 150 155
gaa gat gaa cac cca caa tgc ctg aac tcc cag ccc cat gcc cgt ggg 708
Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly 160
165 170 175 tcg tcc cgc gcc atc ttc tcc gtg ggc ccc gtg agc ccg aat
cgc agg 756 Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Asn
Arg Arg 180 185 190 tgg tcg cac agg tgc tat ggt tat gac ttg aac tct
ccc tat gtg tgg 804 Trp Ser His Arg Cys Tyr Gly Tyr Asp Leu Asn Ser
Pro Tyr Val Trp 195 200 205 tct tca ccc agt gat ctc ctg gag ctc ctg
gtc cca ggt gtt tct aag 852 Ser Ser Pro Ser Asp Leu Leu Glu Leu Leu
Val Pro Gly Val Ser Lys 210 215 220 aag cca tca ctc tca gtg cag ccg
ggt cct gtc gtg gcc cct ggg gaa 900 Lys Pro Ser Leu Ser Val Gln Pro
Gly Pro Val Val Ala Pro Gly Glu 225 230 235 agc ctg acc ctc cag tgt
gtc tct gat gtc ggc tat gac aga ttt gtt 948 Ser Leu Thr Leu Gln Cys
Val Ser Asp Val Gly Tyr Asp Arg Phe Val 240 245 250 255 ctg tac aag
gag ggg gaa cgt gac ctt cgc cag ctc cct ggc cgg cag 996 Leu Tyr Lys
Glu Gly Glu Arg Asp Leu Arg Gln Leu Pro Gly Arg Gln 260 265 270 ccc
cag gct ggg ctc tcc cag gcc aac ttc acc ctg ggc cct gtg agc 1044
Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275
280 285 cgc tcc tac ggg ggc cag tac aga tgc tac ggt gca tac aac ctc
tcc 1092 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala Tyr Asn
Leu Ser 290 295 300 tcc gag tgg tcg gcc ccc agc gac ccc ctg gac atc
ctg atc aca gga 1140 Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp
Ile Leu Ile Thr Gly 305 310 315 cag atc cat ggc aca ccc ttc atc tca
gtg cag cca ggc ccc aca gtg 1188 Gln Ile His Gly Thr Pro Phe Ile
Ser Val Gln Pro Gly Pro Thr Val 320 325 330 335 gcc tca gga gag aac
gtg acc ctg ctg tgt cag tca tgg cgg cag ttc 1236 Ala Ser Gly Glu
Asn Val Thr Leu Leu Cys Gln Ser Trp Arg Gln Phe 340 345 350 cac act
ttc ctt ctg acc aag gcg gga gca gct gat gcc cca ctc cgt 1284 His
Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg 355 360
365 cta aga tca ata cac gaa tat cct aag tac cag gct gaa ttc ccc atg
1332 Leu Arg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro
Met 370 375 380 agt cct gtg acc tca gcc cac gcg ggg acc tac agg tgc
tac ggc tca 1380 Ser Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg
Cys Tyr Gly Ser 385 390 395 ctc aac tcc gac ccc tac ctg ctg tct cac
ccc agt gag ccc ctg gag 1428 Leu Asn Ser Asp Pro Tyr Leu Leu Ser
His Pro Ser Glu Pro Leu Glu 400 405 410 415 ctc gtg gtc tca gga ccc
tcc atg ggt tcc agc ccc cca ccc acc ggt 1476 Leu Val Val Ser Gly
Pro Ser Met Gly Ser Ser Pro Pro Pro Thr Gly 420 425 430 ccc atc tcc
aca cct gca ggc cct gag gac cag ccc ctc acc ccc act 1524 Pro Ile
Ser Thr Pro Ala Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr 435 440 445
ggg tcg gat ccc caa agt ggt ctg gga agg cac ctg ggg gtt gtg atc
1572 Gly Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val
Ile 450 455 460 ggc atc ttg gtg gcc gtc gtc cta ctg ctc ctc ctc ctc
ctc ctc ctc 1620 Gly Ile Leu Val Ala Val Val Leu Leu Leu Leu Leu
Leu Leu Leu Leu 465 470 475 ttc ctc atc ctc cga cat cga cgt cag ggc
aaa cac tgg aca tcg acc 1668 Phe Leu Ile Leu Arg His Arg Arg Gln
Gly Lys His Trp Thr Ser Thr 480 485 490 495 cag aga aag gct gat ttc
caa cat cct gca ggg gct gtg ggg cca gag 1716 Gln Arg Lys Ala Asp
Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu 500 505 510 ccc aca gac
aga ggc ctg cag tgg agg tcc agc cca gct gcc gac gcc 1764 Pro Thr
Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala 515 520 525
cag gaa gaa aac ctc tat gct gcc gtg aag gac aca cag cct gaa gat
1812 Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys Asp Thr Gln Pro Glu
Asp 530 535 540 ggg gtg gag atg gac act cgg gct gct gca tct gaa gcc
ccc cag gat 1860 Gly Val Glu Met Asp Thr Arg Ala Ala Ala Ser Glu
Ala Pro Gln Asp 545 550 555 gtg acc tac gcc cag ctg cac agc ttg acc
ctc aga cgg aag gca act 1908 Val Thr Tyr Ala Gln Leu His Ser Leu
Thr Leu Arg Arg Lys Ala Thr 560 565 570 575 gag cct cct cca tcc cag
gaa agg gaa cct cca gct gag ccc agc atc 1956 Glu Pro Pro Pro Ser
Gln Glu Arg Glu Pro Pro Ala Glu Pro Ser Ile 580 585 590 tac gcc acc
ctg gcc atc cac tag cccggagggt acgcagactc cacactcagt 2010 Tyr Ala
Thr Leu Ala Ile His 595 agaaggagac tcaggactgc tgaaggcacg ggagctgccc
ccagtggaca ccaatgaacc 2070 ccagtcagcc tggaccccta acaaagacca
tgaggagatg ctgggaactt tgggactcac 2130 ttgattctgc agtcgaaata
actaatatcc ctacattttt taattaaagc aacagacttc 2190 tcaataaaag
caggtcgtct cgttccaatc t 2221 10 598 PRT human 10 Met Thr Pro Ile
Val Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg
Thr His Val Gln Thr Gly Thr Ile Pro Lys Pro Thr Leu Trp 20 25 30
Ala Glu Pro Asp Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Ser 35
40 45 Cys Gln Gly Ser Leu Glu Ala Gln Glu Tyr Arg Leu Tyr Arg Glu
Lys 50 55 60 Lys Ser Ala Ser Trp Ile Thr Arg Ile Arg Pro Glu Leu
Val Lys Asn 65 70 75 80 Gly Gln Phe His Ile Pro Ser Ile Thr Trp Glu
His Thr Gly Arg Tyr 85 90 95 Gly Cys Gln Tyr Tyr Ser Arg Ala Arg
Trp Ser Glu Leu Ser Asp Pro 100 105 110 Leu Val Leu Val Met Thr Gly
Ala Tyr Pro Lys Pro Thr Leu Ser Ala 115 120 125 Gln Pro Ser Pro Val
Val Thr Ser Gly Gly Arg Val Thr Leu Gln Cys 130 135 140 Glu Ser Gln
Val Ala Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly Glu 145 150 155 160
Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly Ser 165
170 175 Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Asn Arg Arg
Trp 180 185 190 Ser His Arg Cys Tyr Gly Tyr Asp Leu Asn Ser Pro Tyr
Val Trp Ser 195 200 205 Ser Pro Ser Asp Leu Leu Glu Leu Leu Val Pro
Gly Val Ser Lys Lys 210 215 220 Pro Ser Leu Ser Val Gln Pro Gly Pro
Val Val Ala Pro Gly Glu Ser 225 230 235 240 Leu Thr Leu Gln Cys Val
Ser Asp Val Gly Tyr Asp Arg Phe Val Leu 245 250 255 Tyr Lys Glu Gly
Glu Arg Asp Leu Arg Gln Leu Pro Gly Arg Gln Pro 260 265 270 Gln Ala
Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Arg 275 280 285
Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala Tyr Asn Leu Ser Ser 290
295 300 Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly
Gln 305 310 315 320 Ile His Gly Thr Pro Phe Ile Ser Val Gln Pro Gly
Pro Thr Val Ala 325 330 335 Ser Gly Glu Asn Val Thr Leu Leu Cys Gln
Ser Trp Arg Gln Phe His 340 345 350 Thr Phe Leu Leu Thr Lys Ala Gly
Ala Ala Asp Ala Pro Leu Arg Leu 355 360 365 Arg Ser Ile His Glu Tyr
Pro Lys Tyr Gln Ala Glu Phe Pro Met Ser 370 375 380 Pro Val Thr Ser
Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Leu 385 390 395 400 Asn
Ser Asp Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu 405 410
415 Val Val Ser Gly Pro Ser Met Gly Ser Ser Pro Pro Pro Thr Gly Pro
420 425 430 Ile Ser Thr Pro Ala Gly Pro Glu Asp Gln Pro Leu Thr Pro
Thr Gly 435 440 445 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly
Val Val Ile Gly 450 455 460 Ile Leu Val Ala Val Val Leu Leu Leu Leu
Leu Leu Leu Leu Leu Phe 465 470 475 480 Leu Ile Leu Arg His Arg Arg
Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 Arg Lys Ala Asp Phe
Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510 Thr Asp Arg
Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln 515 520 525 Glu
Glu Asn Leu Tyr Ala Ala Val Lys Asp Thr Gln Pro Glu Asp Gly 530 535
540 Val Glu Met Asp Thr Arg Ala Ala Ala Ser Glu Ala Pro Gln Asp Val
545 550 555 560 Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys
Ala Thr Glu 565 570 575 Pro Pro Pro Ser Gln Glu Arg Glu Pro Pro Ala
Glu Pro Ser Ile Tyr 580 585 590 Ala Thr Leu Ala Ile His 595 11 2446
DNA human CDS (171)..(1040) 11 cgcagctcaa cctgagctac acagccagat
gcgagatgct tctctgctga tctgagtctg 60 cctgcagcat ggaccttggt
cttccctgaa gcatctccag ggctggaggg acgactgcca 120 tgcacctagg
gcttatccat ccgcagagca gggcagtggg aggagacgct atg acc 176 Met Thr 1
ccc atc ctc acg gtc ctg atc tgt ctc ggg ctg agt ctg ggc ccc cgg 224
Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly Pro Arg 5
10 15 acc cac gtg cag gca ggg acc ctc ccc aag ccc aca ctc tgg gct
gag 272 Thr His Val Gln Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp Ala
Glu 20 25 30 cca ggc tct gtg atc acc cag ggg agt ccc gtg acc ctc
tgg tgt cag 320 Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu
Trp Cys Gln 35 40 45 50 ggg atc ctg gag acc cag gag tac cgt ctg tat
aga gaa aag aaa aca 368 Gly Ile Leu Glu Thr Gln Glu Tyr Arg Leu Tyr
Arg Glu Lys Lys Thr 55 60 65 gca ccc tgg att aca cgg atc cca cag
gag att gtg aag aag ggc cag 416 Ala Pro Trp Ile Thr Arg Ile Pro Gln
Glu Ile Val Lys Lys Gly Gln 70 75 80 ttc ccc atc ccg tcc atc acc
tgg gaa cac acc ggg cgg tat cgc tgt 464 Phe Pro Ile Pro Ser Ile Thr
Trp Glu His Thr Gly Arg Tyr Arg Cys 85 90 95 ttc tac ggt agc cac
act gca ggc tgg tca gag ccc agt gac ccc ctg 512 Phe Tyr Gly Ser His
Thr Ala Gly Trp Ser Glu Pro Ser Asp Pro Leu 100 105 110 gag ctg gtg
gtg aca gga gcc tac atc aaa ccc acc ctc tcg gct cta 560 Glu Leu Val
Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser Ala Leu 115 120 125 130
ccc agc cct gtg gtg acc tca gga ggg aac gtg acc ctc cat tgt gtc 608
Pro Ser Pro Val Val Thr Ser Gly Gly Asn Val Thr Leu His Cys Val 135
140 145 tca cag gtg gca ttt ggc agc ttc att ctg tgt aag gaa gga gaa
gat 656 Ser Gln Val Ala Phe Gly Ser Phe Ile Leu Cys Lys Glu Gly Glu
Asp 150 155 160 gaa cac cca caa tgc ctg aac tca cag ccc cgt acc cat
ggg tgg tcc 704 Glu His Pro Gln Cys Leu Asn Ser Gln Pro Arg Thr His
Gly Trp Ser 165 170 175 cgg gcc atc ttc tct gtg ggc ccc gtg agc ccg
agt cgc agg tgg tcg 752 Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro
Ser Arg Arg Trp Ser 180 185 190 tac agg tgc tat gct tat gac tcg aac
tct ccc cat gtg tgg tct cta 800 Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn
Ser Pro His Val Trp Ser Leu 195 200 205 210 ccc agt gat ctc ctg gag
ctc ctg gtc cca gga gca gct gag acc ctc 848 Pro Ser Asp Leu Leu Glu
Leu Leu Val Pro Gly Ala Ala Glu Thr Leu 215 220 225 agc cca cca caa
aac aag tcc gat tcc aag gct gga gca gct aac acc 896 Ser Pro Pro Gln
Asn Lys Ser Asp Ser Lys Ala Gly Ala Ala Asn Thr 230 235 240 ctc agc
cca tca caa aac aag act gcc tca cac ccc cag gat tac aca 944 Leu Ser
Pro Ser Gln Asn Lys Thr Ala Ser His Pro Gln Asp Tyr Thr 245 250 255
gtg gag aat ctc atc cgc atg ggc ata gct ggc ttg gtc ctg gtg gtc 992
Val Glu Asn Leu Ile Arg Met Gly Ile Ala Gly Leu Val Leu Val Val 260
265 270 ctc ggg att ctg cta ttt gag gct cag cac agc cag aga agc ctc
tga 1040 Leu Gly Ile Leu Leu Phe Glu Ala Gln His Ser Gln Arg Ser
Leu 275 280 285 gatgcagccg ggaggtgaac agcagagaga agaatgtacc
cttcagagtg gtggagcctt 1100 gggaacagat ctgatgatgc caggaggttc
cgggagacaa tttagggctg atgctatctg 1160 gactgtctgc caatcatttt
tagagggagg aatcagtgtt ggattgcaga gacattttct 1220 ggagtgatcc
atgaaggacc attaacatgt gatacctttc ctctctatta atgttgactt 1280
cccttggttg gatcctcttc tttccccacc cccagacaga catgaggcta catcccacat
1340 ggcagcgttg ggtccacacc tctgcacatc tgtgtgctct ggtccatggt
gtgtaacaca 1400 gtcttcttta ttactcattg ccatactccc tggtgtgctt
tactgagcct ccatctcttc 1460 aattcagagt tccaaacgtg cttcagtaac
taaatcaatg ggagagtatc ggatttcaac 1520 caggaaaaga taaatccacc
ctgatgccct gacaccctct ctgaacccta cgagcccttc 1580 cctccttctc
acatgctacc tgtgcagctt ctccttagat cattgtgtaa ccatcactgc 1640
catcctgttc cacacatggt catcacccta cacccattca gcagccactc cccattccct
1700 cttccctcca gcacctgcta accacaaatg tgctttctgt ctctacggat
ttgcctattc 1760 tgtctgaaaa catttcaatc tcctttgacc tgtgagctcc
tcacttcgag acttcctgcc 1820 tttccaggca gaaccaaagt acaccacgtc
aaaagcaatg ataggcattt gcagtgtgtt 1880 ggtgatccac gaaaggaaaa
tcacggaagc aggatagaaa tccagctgca gacaagacct 1940 caggtcgatg
aatcttgaca agcagttgag ctgttttttt ctactcacct aggacagtca 2000
ggcagaagta tgcaaaatga ctggggctga ttcttttctg aattgtcgca aacagcaaga
2060 ggacttgagt cctagcatta aagagttcaa catgtctagg tccaagacca
ctgttgtgtt 2120 tgaaggatgt aaaaccctgc tgcataggat ggaatatttg
gagggaggat cctgaaaaac 2180 atgagggatc aaatagtcct caactttcta
ggacaaaggg agcagctatt tgccatctac 2240 cctccagaat aaagaaatct
tatcattcac catctaccct ctagaataaa gaaatcttat 2300 cattcgccat
ctaccctgta gaataaagaa atcttatcat tcaccgtcta ccctctagag 2360
taaacaaatc ttatcattca ccatctaccc tctagaataa agaaatctta tcattcgcca
2420
tctaccctct agaataaaga aatctt 2446 12 289 PRT human 12 Met Thr Pro
Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro
Arg Thr His Val Gln Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp 20 25
30 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Trp
35 40 45 Cys Gln Gly Ile Leu Glu Thr Gln Glu Tyr Arg Leu Tyr Arg
Glu Lys 50 55 60 Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu
Ile Val Lys Lys 65 70 75 80 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp
Glu His Thr Gly Arg Tyr 85 90 95 Arg Cys Phe Tyr Gly Ser His Thr
Ala Gly Trp Ser Glu Pro Ser Asp 100 105 110 Pro Leu Glu Leu Val Val
Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120 125 Ala Leu Pro Ser
Pro Val Val Thr Ser Gly Gly Asn Val Thr Leu His 130 135 140 Cys Val
Ser Gln Val Ala Phe Gly Ser Phe Ile Leu Cys Lys Glu Gly 145 150 155
160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro Arg Thr His Gly
165 170 175 Trp Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser
Arg Arg 180 185 190 Trp Ser Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser
Pro His Val Trp 195 200 205 Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu
Val Pro Gly Ala Ala Glu 210 215 220 Thr Leu Ser Pro Pro Gln Asn Lys
Ser Asp Ser Lys Ala Gly Ala Ala 225 230 235 240 Asn Thr Leu Ser Pro
Ser Gln Asn Lys Thr Ala Ser His Pro Gln Asp 245 250 255 Tyr Thr Val
Glu Asn Leu Ile Arg Met Gly Ile Ala Gly Leu Val Leu 260 265 270 Val
Val Leu Gly Ile Leu Leu Phe Glu Ala Gln His Ser Gln Arg Ser 275 280
285 Leu 13 1910 DNA human CDS (183)..(1652) 13 ctcactgcca
cacgcagctc aacctgagct acacagccag atgcgagatg cttctctgct 60
gatctgagtc tgcctgcagc atggaccttg gtcttccctg aagcatctcc agggctggag
120 ggacgactgc catgcaccga gggctcatcc atccgcagag cagggcagtg
ggaggagacg 180 ct atg acc ccc atc gtc aca gtc ctg atc tgt ctc agg
ctg agt ctg 227 Met Thr Pro Ile Val Thr Val Leu Ile Cys Leu Arg Leu
Ser Leu 1 5 10 15 ggc ccc cgg acc cac gtg cag gca ggg acc ctc ccc
aag ccc aca ctc 275 Gly Pro Arg Thr His Val Gln Ala Gly Thr Leu Pro
Lys Pro Thr Leu 20 25 30 tgg gct gag cca ggc tct gtg atc acc cag
ggg agt ccc gtg acc ctc 323 Trp Ala Glu Pro Gly Ser Val Ile Thr Gln
Gly Ser Pro Val Thr Leu 35 40 45 tgg tgt cag ggg atc ctg gag acc
cag gag tac cgt ctg tat aga gaa 371 Trp Cys Gln Gly Ile Leu Glu Thr
Gln Glu Tyr Arg Leu Tyr Arg Glu 50 55 60 aag aaa aca gca ccc tgg
att aca cgg atc cca cag gag att gtg aag 419 Lys Lys Thr Ala Pro Trp
Ile Thr Arg Ile Pro Gln Glu Ile Val Lys 65 70 75 aag ggc cag ttc
ccc atc cca tcc atc acc tgg gaa cac aca ggg cgg 467 Lys Gly Gln Phe
Pro Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg 80 85 90 95 tat cgc
tgt ttc tac ggt agc cac act gca ggc tgg tca gag ccc agt 515 Tyr Arg
Cys Phe Tyr Gly Ser His Thr Ala Gly Trp Ser Glu Pro Ser 100 105 110
gac ccc ctg gag ctg gtg gtg aca gga gcc tac atc aaa ccc acc ctc 563
Asp Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu 115
120 125 tca gct cta ccc agc cct gtg gtg acc tca gga ggg aac gtg acc
ctc 611 Ser Ala Leu Pro Ser Pro Val Val Thr Ser Gly Gly Asn Val Thr
Leu 130 135 140 cat tgt gtc tca cag gtg gca ttt ggc agc ttc att ctg
tgt aag gaa 659 His Cys Val Ser Gln Val Ala Phe Gly Ser Phe Ile Leu
Cys Lys Glu 145 150 155 gga gaa gat gaa cac cca caa tgc ctg aac tca
cag ccc cgt acc cat 707 Gly Glu Asp Glu His Pro Gln Cys Leu Asn Ser
Gln Pro Arg Thr His 160 165 170 175 ggg tgg tcc cgg gcc atc ttc tct
gtg ggc ccc gtg agc ccg agt cgc 755 Gly Trp Ser Arg Ala Ile Phe Ser
Val Gly Pro Val Ser Pro Ser Arg 180 185 190 agg tgg tcg tac agg tgc
tat gct tat gac tcg aac tct ccc cat gtg 803 Arg Trp Ser Tyr Arg Cys
Tyr Ala Tyr Asp Ser Asn Ser Pro His Val 195 200 205 tgg tct cta ccc
agt gat ctc ctg gag ctc ctg gtc cta ggt gtt tct 851 Trp Ser Leu Pro
Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser 210 215 220 aag aag
cca tca ctc tca gtg cag cca ggt cct ata gtg gcc cct ggg 899 Lys Lys
Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Gly 225 230 235
gag agc ctg acc ctc cag tgt gtt tct gat gtc agc tac gac aga ttt 947
Glu Ser Leu Thr Leu Gln Cys Val Ser Asp Val Ser Tyr Asp Arg Phe 240
245 250 255 gtt ctg tat aag gag gga gaa cgt gac ttc ctc cag ctc cct
ggc cca 995 Val Leu Tyr Lys Glu Gly Glu Arg Asp Phe Leu Gln Leu Pro
Gly Pro 260 265 270 cag ccc cag gct ggg ctc tcc cag gcc aac ttc acc
ctg ggc cct gtg 1043 Gln Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe
Thr Leu Gly Pro Val 275 280 285 agc cgc tcc tac ggg ggc cag tac aga
tgc tcc ggt gca tac aac ctc 1091 Ser Arg Ser Tyr Gly Gly Gln Tyr
Arg Cys Ser Gly Ala Tyr Asn Leu 290 295 300 tcc tcc gag tgg tcg gcc
ccc agc gac ccc ctg gac atc ctg atc gca 1139 Ser Ser Glu Trp Ser
Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala 305 310 315 gga cag ttc
cgt ggc aga ccc ttc atc tcg gtg cat ccg ggc ccc acg 1187 Gly Gln
Phe Arg Gly Arg Pro Phe Ile Ser Val His Pro Gly Pro Thr 320 325 330
335 gtg gcc tca gga gag aac gtg acc ctg ctg tgt cag tca tgg ggg ccg
1235 Val Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp Gly
Pro 340 345 350 ttc cac act ttc ctt ctg acc aag gcg gga gca gct gat
gcc ccc ctc 1283 Phe His Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala
Asp Ala Pro Leu 355 360 365 cgt ctc aga tca ata cac gaa tat cct aag
tac cag gct gaa ttc cct 1331 Arg Leu Arg Ser Ile His Glu Tyr Pro
Lys Tyr Gln Ala Glu Phe Pro 370 375 380 atg agt cct gtg acc tca gcc
cac tcg ggg acc tac agg tgc tac ggc 1379 Met Ser Pro Val Thr Ser
Ala His Ser Gly Thr Tyr Arg Cys Tyr Gly 385 390 395 tca ctc agc tcc
aac ccc tac ctg ctg tct cac ccc agt gac tcc ctg 1427 Ser Leu Ser
Ser Asn Pro Tyr Leu Leu Ser His Pro Ser Asp Ser Leu 400 405 410 415
gag ctc atg gtc tca gga gca gct gag acc ctc agc cca cca caa aac
1475 Glu Leu Met Val Ser Gly Ala Ala Glu Thr Leu Ser Pro Pro Gln
Asn 420 425 430 aag tcc gat tcc aag gct gga gca gct aac acc ctc agc
cca tca caa 1523 Lys Ser Asp Ser Lys Ala Gly Ala Ala Asn Thr Leu
Ser Pro Ser Gln 435 440 445 aac aag act gcc tca cac ccc cag gat tac
aca gtg gag aat ctc atc 1571 Asn Lys Thr Ala Ser His Pro Gln Asp
Tyr Thr Val Glu Asn Leu Ile 450 455 460 cgc atg ggc ata gct ggc ttg
gtc ctg gtg gtc ctc ggg att ctg cta 1619 Arg Met Gly Ile Ala Gly
Leu Val Leu Val Val Leu Gly Ile Leu Leu 465 470 475 ttt gag gct cag
cac agc cag aga agc ctc tga gatgcagccg ggaggtgaac 1672 Phe Glu Ala
Gln His Ser Gln Arg Ser Leu 480 485 agcagagaga agaatgtacc
cttcagagtg gtggagcctt gggaacagat ctgatgatgc 1732 caggaggttc
cgggagacaa tttagggctg atgttatctg gactgtctgc caatcatttt 1792
tagagggagg aatcagtgtt ggattgcaga gacattttct ggagtgatcc atgaaggacc
1852 attaacatgt gatacctttc ctctctatta atgttgactt cccttggttg
gatcctct 1910 14 489 PRT human 14 Met Thr Pro Ile Val Thr Val Leu
Ile Cys Leu Arg Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln
Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly
Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Trp 35 40 45 Cys Gln
Gly Ile Leu Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60
Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu Ile Val Lys Lys 65
70 75 80 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Thr Gly
Arg Tyr 85 90 95 Arg Cys Phe Tyr Gly Ser His Thr Ala Gly Trp Ser
Glu Pro Ser Asp 100 105 110 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr
Ile Lys Pro Thr Leu Ser 115 120 125 Ala Leu Pro Ser Pro Val Val Thr
Ser Gly Gly Asn Val Thr Leu His 130 135 140 Cys Val Ser Gln Val Ala
Phe Gly Ser Phe Ile Leu Cys Lys Glu Gly 145 150 155 160 Glu Asp Glu
His Pro Gln Cys Leu Asn Ser Gln Pro Arg Thr His Gly 165 170 175 Trp
Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180 185
190 Trp Ser Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro His Val Trp
195 200 205 Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val
Ser Lys 210 215 220 Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val
Ala Pro Gly Glu 225 230 235 240 Ser Leu Thr Leu Gln Cys Val Ser Asp
Val Ser Tyr Asp Arg Phe Val 245 250 255 Leu Tyr Lys Glu Gly Glu Arg
Asp Phe Leu Gln Leu Pro Gly Pro Gln 260 265 270 Pro Gln Ala Gly Leu
Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 Arg Ser Tyr
Gly Gly Gln Tyr Arg Cys Ser Gly Ala Tyr Asn Leu Ser 290 295 300 Ser
Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310
315 320 Gln Phe Arg Gly Arg Pro Phe Ile Ser Val His Pro Gly Pro Thr
Val 325 330 335 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp
Gly Pro Phe 340 345 350 His Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala
Asp Ala Pro Leu Arg 355 360 365 Leu Arg Ser Ile His Glu Tyr Pro Lys
Tyr Gln Ala Glu Phe Pro Met 370 375 380 Ser Pro Val Thr Ser Ala His
Ser Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390 395 400 Leu Ser Ser Asn
Pro Tyr Leu Leu Ser His Pro Ser Asp Ser Leu Glu 405 410 415 Leu Met
Val Ser Gly Ala Ala Glu Thr Leu Ser Pro Pro Gln Asn Lys 420 425 430
Ser Asp Ser Lys Ala Gly Ala Ala Asn Thr Leu Ser Pro Ser Gln Asn 435
440 445 Lys Thr Ala Ser His Pro Gln Asp Tyr Thr Val Glu Asn Leu Ile
Arg 450 455 460 Met Gly Ile Ala Gly Leu Val Leu Val Val Leu Gly Ile
Leu Leu Phe 465 470 475 480 Glu Ala Gln His Ser Gln Arg Ser Leu 485
15 1725 DNA human CDS (40)..(1491) 15 ctcatccatc cgcagagcag
ggcagtggga ggagacgcc atg acc ccc atc ctc 54 Met Thr Pro Ile Leu 1 5
acg gtc ctg atc tgt ctc ggg ctg agt ctg ggc ccc agg acc cac gtg 102
Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly Pro Arg Thr His Val 10
15 20 cag gca ggg cac ctc ccc aag ccc acc ctc tgg gct gag cca ggc
tct 150 Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly
Ser 25 30 35 gtg atc atc cag gga agt cct gtg acc ctc agg tgt cag
ggg agc ctt 198 Val Ile Ile Gln Gly Ser Pro Val Thr Leu Arg Cys Gln
Gly Ser Leu 40 45 50 cag gct gag gag tac cat cta tat agg gaa aac
aaa tca gca tcc tgg 246 Gln Ala Glu Glu Tyr His Leu Tyr Arg Glu Asn
Lys Ser Ala Ser Trp 55 60 65 gtt aga cgg ata caa gag cct ggg aag
aat ggc cag ttc ccc atc cca 294 Val Arg Arg Ile Gln Glu Pro Gly Lys
Asn Gly Gln Phe Pro Ile Pro 70 75 80 85 tcc atc acc tgg gaa cac gca
ggg cgg tat cac tgt cag tac tac agc 342 Ser Ile Thr Trp Glu His Ala
Gly Arg Tyr His Cys Gln Tyr Tyr Ser 90 95 100 cac aat cac tca tca
gag tac agt gac ccc ctg gag ctg gtg gtg aca 390 His Asn His Ser Ser
Glu Tyr Ser Asp Pro Leu Glu Leu Val Val Thr 105 110 115 gga gcc tac
agc aaa ccc acc ctc tca gct ctg ccc agc cct gtg gtg 438 Gly Ala Tyr
Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val 120 125 130 acc
tta gga ggg aac gtg acc ctc cag tgt gtc tca cag gtg gca ttt 486 Thr
Leu Gly Gly Asn Val Thr Leu Gln Cys Val Ser Gln Val Ala Phe 135 140
145 gac ggc ttc att ctg tgt aag gaa gga gaa gat gaa cac cca caa cgc
534 Asp Gly Phe Ile Leu Cys Lys Glu Gly Glu Asp Glu His Pro Gln Arg
150 155 160 165 ctg aac tcc cat tcc cat gcc cgt ggg tgg tcc tgg gcc
atc ttc tcc 582 Leu Asn Ser His Ser His Ala Arg Gly Trp Ser Trp Ala
Ile Phe Ser 170 175 180 gtg ggc ccc gtg agc ccg agt cgc agg tgg tcg
tac agg tgc tat gct 630 Val Gly Pro Val Ser Pro Ser Arg Arg Trp Ser
Tyr Arg Cys Tyr Ala 185 190 195 tat gac tcg aac tct ccc tat gtg tgg
tct cta ccc agt gat ctc ctg 678 Tyr Asp Ser Asn Ser Pro Tyr Val Trp
Ser Leu Pro Ser Asp Leu Leu 200 205 210 gag ctc ctg gtc cca ggt gtt
tct aag aag cca tca ctc tca gtg cag 726 Glu Leu Leu Val Pro Gly Val
Ser Lys Lys Pro Ser Leu Ser Val Gln 215 220 225 cca ggt cct atg gtg
gcc ccc ggg gag agc ctg acc ctc cag tgt gtc 774 Pro Gly Pro Met Val
Ala Pro Gly Glu Ser Leu Thr Leu Gln Cys Val 230 235 240 245 tct gat
gtc ggc tac gac aga ttt gtt ctg tat aag gag gga gaa cgt 822 Ser Asp
Val Gly Tyr Asp Arg Phe Val Leu Tyr Lys Glu Gly Glu Arg 250 255 260
gac ttc ctc cag cgc cct ggt tgg cag ccc cag gct ggg ctc tcc cag 870
Asp Phe Leu Gln Arg Pro Gly Trp Gln Pro Gln Ala Gly Leu Ser Gln 265
270 275 gcc aac ttc acc ctg ggc cct gtg agc ccc tcc cac ggg ggc cag
tac 918 Ala Asn Phe Thr Leu Gly Pro Val Ser Pro Ser His Gly Gly Gln
Tyr 280 285 290 aga tgc tac agt gca cac aac ctc tcc tcc gag tgg tcg
gcc ccc agt 966 Arg Cys Tyr Ser Ala His Asn Leu Ser Ser Glu Trp Ser
Ala Pro Ser 295 300 305 gac ccc ctg gac atc ctg atc aca gga cag ttc
tat gac aga ccc tct 1014 Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln
Phe Tyr Asp Arg Pro Ser 310 315 320 325 ctc tcg gtg cag ccg gtc ccc
aca gta gcc cca gga aag aac gtg acc 1062 Leu Ser Val Gln Pro Val
Pro Thr Val Ala Pro Gly Lys Asn Val Thr 330 335 340 ctg ctg tgt cag
tca cgg ggg cag ttc cac act ttc ctt ctg acc aag 1110 Leu Leu Cys
Gln Ser Arg Gly Gln Phe His Thr Phe Leu Leu Thr Lys 345 350 355 gag
ggg gca ggc cat ccc cca ctg cat ctg aga tca gag cac caa gct 1158
Glu Gly Ala Gly His Pro Pro Leu His Leu Arg Ser Glu His Gln Ala 360
365 370 cag cag aac cag gct gaa ttc cgc atg ggt cct gtg acc tca gcc
cac 1206 Gln Gln Asn Gln Ala Glu Phe Arg Met Gly Pro Val Thr Ser
Ala His 375 380 385 gtg ggg acc tac aga tgc tac agc tca ctc agc tcc
aac ccc tac ctg 1254 Val Gly Thr Tyr Arg Cys Tyr Ser Ser Leu Ser
Ser Asn Pro Tyr Leu 390 395 400 405 ctg tct ctc ccc agt gac ccc ctg
gag ctc gtg gtc tca gaa gca gct 1302 Leu Ser Leu Pro Ser Asp Pro
Leu Glu Leu Val Val Ser Glu Ala Ala 410 415 420 gag acc ctc agc cca
tca caa aac aag aca gac tcc acg act aca tcc 1350 Glu Thr Leu Ser
Pro Ser Gln Asn Lys Thr Asp Ser Thr Thr Thr Ser 425 430 435 cta ggc
caa cac ccc cag gat tac aca gtg gag aat ctc atc cgc atg 1398 Leu
Gly Gln His Pro Gln Asp Tyr Thr Val Glu Asn Leu Ile Arg Met 440 445
450 ggt gtg gct ggc ttg gtc ctg gtg gtc ctc ggg att ctg cta ttt gag
1446 Gly Val Ala Gly Leu Val Leu Val Val Leu Gly Ile Leu Leu Phe
Glu 455 460 465 gct cag cac agc cag aga agc cta caa gat gca gcc ggg
agg tga 1491 Ala Gln His Ser Gln Arg Ser Leu Gln Asp Ala Ala
Gly Arg 470 475 480 acagcagaga ggacaatgca tccttcagcg tggtggagcc
tcagggacag atctgatgat 1551 cccaggaggc tctggaggac aatctaggac
ctacattatc tggactgtat gctggtcatt 1611 tctagagaca gcaatcaata
tttgagtgta aggaaactgt ctggggtgat tcctagaaga 1671 tcattaaact
gtggtacatt tttttgtcta aaaagcaggt cgtctcgttc caag 1725 16 483 PRT
human 16 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser
Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys
Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Ile Gln Gly
Ser Pro Val Thr Leu Arg 35 40 45 Cys Gln Gly Ser Leu Gln Ala Glu
Glu Tyr His Leu Tyr Arg Glu Asn 50 55 60 Lys Ser Ala Ser Trp Val
Arg Arg Ile Gln Glu Pro Gly Lys Asn Gly 65 70 75 80 Gln Phe Pro Ile
Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr His 85 90 95 Cys Gln
Tyr Tyr Ser His Asn His Ser Ser Glu Tyr Ser Asp Pro Leu 100 105 110
Glu Leu Val Val Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu 115
120 125 Pro Ser Pro Val Val Thr Leu Gly Gly Asn Val Thr Leu Gln Cys
Val 130 135 140 Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu
Gly Glu Asp 145 150 155 160 Glu His Pro Gln Arg Leu Asn Ser His Ser
His Ala Arg Gly Trp Ser 165 170 175 Trp Ala Ile Phe Ser Val Gly Pro
Val Ser Pro Ser Arg Arg Trp Ser 180 185 190 Tyr Arg Cys Tyr Ala Tyr
Asp Ser Asn Ser Pro Tyr Val Trp Ser Leu 195 200 205 Pro Ser Asp Leu
Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys Pro 210 215 220 Ser Leu
Ser Val Gln Pro Gly Pro Met Val Ala Pro Gly Glu Ser Leu 225 230 235
240 Thr Leu Gln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val Leu Tyr
245 250 255 Lys Glu Gly Glu Arg Asp Phe Leu Gln Arg Pro Gly Trp Gln
Pro Gln 260 265 270 Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro
Val Ser Pro Ser 275 280 285 His Gly Gly Gln Tyr Arg Cys Tyr Ser Ala
His Asn Leu Ser Ser Glu 290 295 300 Trp Ser Ala Pro Ser Asp Pro Leu
Asp Ile Leu Ile Thr Gly Gln Phe 305 310 315 320 Tyr Asp Arg Pro Ser
Leu Ser Val Gln Pro Val Pro Thr Val Ala Pro 325 330 335 Gly Lys Asn
Val Thr Leu Leu Cys Gln Ser Arg Gly Gln Phe His Thr 340 345 350 Phe
Leu Leu Thr Lys Glu Gly Ala Gly His Pro Pro Leu His Leu Arg 355 360
365 Ser Glu His Gln Ala Gln Gln Asn Gln Ala Glu Phe Arg Met Gly Pro
370 375 380 Val Thr Ser Ala His Val Gly Thr Tyr Arg Cys Tyr Ser Ser
Leu Ser 385 390 395 400 Ser Asn Pro Tyr Leu Leu Ser Leu Pro Ser Asp
Pro Leu Glu Leu Val 405 410 415 Val Ser Glu Ala Ala Glu Thr Leu Ser
Pro Ser Gln Asn Lys Thr Asp 420 425 430 Ser Thr Thr Thr Ser Leu Gly
Gln His Pro Gln Asp Tyr Thr Val Glu 435 440 445 Asn Leu Ile Arg Met
Gly Val Ala Gly Leu Val Leu Val Val Leu Gly 450 455 460 Ile Leu Leu
Phe Glu Ala Gln His Ser Gln Arg Ser Leu Gln Asp Ala 465 470 475 480
Ala Gly Arg 17 1625 DNA human CDS (30)..(1376) 17 cacagctggg
gcccctggga ggagacgcc atg atc ccc acc ttc acg gct ctg 53 Met Ile Pro
Thr Phe Thr Ala Leu 1 5 ctc tgc ctc ggg ctg agt ctg ggc ccc agg acc
cac atg cag gca ggg 101 Leu Cys Leu Gly Leu Ser Leu Gly Pro Arg Thr
His Met Gln Ala Gly 10 15 20 ccc ctc ccc aaa ccc acc ctc tgg gct
gag cca ggc tct gtg atc agc 149 Pro Leu Pro Lys Pro Thr Leu Trp Ala
Glu Pro Gly Ser Val Ile Ser 25 30 35 40 tgg ggg aac tct gtg acc atc
tgg tgt cag ggg acc ctg gag gct cgg 197 Trp Gly Asn Ser Val Thr Ile
Trp Cys Gln Gly Thr Leu Glu Ala Arg 45 50 55 gag tac cgt ctg gat
aaa gag gaa agc cca gca ccc tgg gac aga cag 245 Glu Tyr Arg Leu Asp
Lys Glu Glu Ser Pro Ala Pro Trp Asp Arg Gln 60 65 70 aac cca ctg
gag ccc aag aac aag gcc aga ttc tcc atc cca tcc atg 293 Asn Pro Leu
Glu Pro Lys Asn Lys Ala Arg Phe Ser Ile Pro Ser Met 75 80 85 aca
gag gac tat gca ggg aga tac cgc tgt tac tat cgc agc cct gta 341 Thr
Glu Asp Tyr Ala Gly Arg Tyr Arg Cys Tyr Tyr Arg Ser Pro Val 90 95
100 ggc tgg tca cag ccc agt gac ccc ctg gag ctg gtg atg aca gga gcc
389 Gly Trp Ser Gln Pro Ser Asp Pro Leu Glu Leu Val Met Thr Gly Ala
105 110 115 120 tac agt aaa ccc acc ctt tca gcc ctg ccg agt cct ctt
gtg acc tca 437 Tyr Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Leu
Val Thr Ser 125 130 135 gga aag agc gtg acc ctg ctg tgt cag tca cgg
agc cca atg gac act 485 Gly Lys Ser Val Thr Leu Leu Cys Gln Ser Arg
Ser Pro Met Asp Thr 140 145 150 ttt ctt ctg atc aag gag cgg gca gcc
cat ccc cta ctg cat ctg aga 533 Phe Leu Leu Ile Lys Glu Arg Ala Ala
His Pro Leu Leu His Leu Arg 155 160 165 tca gag cac gga gct cag cag
cac cag gct gaa ttc ccc atg agt cct 581 Ser Glu His Gly Ala Gln Gln
His Gln Ala Glu Phe Pro Met Ser Pro 170 175 180 gtg acc tca gtg cac
ggg ggg acc tac agg tgc ttc agc tca cac ggc 629 Val Thr Ser Val His
Gly Gly Thr Tyr Arg Cys Phe Ser Ser His Gly 185 190 195 200 ttc tcc
cac tac ctg ctg tca cac ccc agt gac ccc ctg gag ctc ata 677 Phe Ser
His Tyr Leu Leu Ser His Pro Ser Asp Pro Leu Glu Leu Ile 205 210 215
gtc tca gga tcc ttg gag ggt ccc agg ccc tca ccc aca agg tcc gtc 725
Val Ser Gly Ser Leu Glu Gly Pro Arg Pro Ser Pro Thr Arg Ser Val 220
225 230 tca aca gct gca ggc cct gag gac cag ccc ctc atg cct aca ggg
tca 773 Ser Thr Ala Ala Gly Pro Glu Asp Gln Pro Leu Met Pro Thr Gly
Ser 235 240 245 gtc ccc cac agt ggt ctg aga agg cac tgg gag gta ctg
atc ggg gtc 821 Val Pro His Ser Gly Leu Arg Arg His Trp Glu Val Leu
Ile Gly Val 250 255 260 ttg gtg gtc tcc atc ctg ctt ctc tcc ctc ctc
ctc ttc ctc ctc ctc 869 Leu Val Val Ser Ile Leu Leu Leu Ser Leu Leu
Leu Phe Leu Leu Leu 265 270 275 280 caa cac tgg cgt cag gga aaa cac
agg aca ttg gcc cag aga cag gct 917 Gln His Trp Arg Gln Gly Lys His
Arg Thr Leu Ala Gln Arg Gln Ala 285 290 295 gat ttc caa cgt cct cca
ggg gct gcc gag cca gag ccc aag gac ggg 965 Asp Phe Gln Arg Pro Pro
Gly Ala Ala Glu Pro Glu Pro Lys Asp Gly 300 305 310 ggc cta cag agg
agg tcc agc cca gct gct gac gtc cag gga gaa aac 1013 Gly Leu Gln
Arg Arg Ser Ser Pro Ala Ala Asp Val Gln Gly Glu Asn 315 320 325 ttc
tgt gct gcc gtg aag aac aca cag cct gag gac ggg gtg gaa atg 1061
Phe Cys Ala Ala Val Lys Asn Thr Gln Pro Glu Asp Gly Val Glu Met 330
335 340 gac act cgg cag agc cca cac gat gaa gac ccc cag gca gtg acg
tat 1109 Asp Thr Arg Gln Ser Pro His Asp Glu Asp Pro Gln Ala Val
Thr Tyr 345 350 355 360 gcc aag gtg aaa cac tcc aga cct agg aga gaa
atg gcc tct cct ccc 1157 Ala Lys Val Lys His Ser Arg Pro Arg Arg
Glu Met Ala Ser Pro Pro 365 370 375 tcc cca ctg tct ggg gaa ttc ctg
gac aca aag gac aga cag gca gaa 1205 Ser Pro Leu Ser Gly Glu Phe
Leu Asp Thr Lys Asp Arg Gln Ala Glu 380 385 390 gag gac aga cag atg
gac act gag gct gct gca tct gaa gcc ccc cag 1253 Glu Asp Arg Gln
Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Pro Gln 395 400 405 gat gtg
acc tac gcc cgg ctg cac agc ttt acc ctc aga cag aag gca 1301 Asp
Val Thr Tyr Ala Arg Leu His Ser Phe Thr Leu Arg Gln Lys Ala 410 415
420 act gag cct cct cca tcc cag gaa ggg gcc tct cca gct gag ccc agt
1349 Thr Glu Pro Pro Pro Ser Gln Glu Gly Ala Ser Pro Ala Glu Pro
Ser 425 430 435 440 gtc tat gcc act ctg gcc atc cac taa tccagggggg
acccagaccc 1396 Val Tyr Ala Thr Leu Ala Ile His 445 cacaagccat
ggagactcag gaccccagaa ggcatggaag ctgcctccag tagacatcac 1456
tgaaccccag ccagcccaga cccctgacac agaccactag aagattccgg gaacgttggg
1516 agtcacctga ttctgcaaag ataaataata tccctgcatt atcaaaataa
agtagcagac 1576 ctctcaattc acaatgagtt aactgataaa acaaaacaga
agtcaaaaa 1625 18 448 PRT human 18 Met Ile Pro Thr Phe Thr Ala Leu
Leu Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Met Gln
Ala Gly Pro Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly
Ser Val Ile Ser Trp Gly Asn Ser Val Thr Ile Trp 35 40 45 Cys Gln
Gly Thr Leu Glu Ala Arg Glu Tyr Arg Leu Asp Lys Glu Glu 50 55 60
Ser Pro Ala Pro Trp Asp Arg Gln Asn Pro Leu Glu Pro Lys Asn Lys 65
70 75 80 Ala Arg Phe Ser Ile Pro Ser Met Thr Glu Asp Tyr Ala Gly
Arg Tyr 85 90 95 Arg Cys Tyr Tyr Arg Ser Pro Val Gly Trp Ser Gln
Pro Ser Asp Pro 100 105 110 Leu Glu Leu Val Met Thr Gly Ala Tyr Ser
Lys Pro Thr Leu Ser Ala 115 120 125 Leu Pro Ser Pro Leu Val Thr Ser
Gly Lys Ser Val Thr Leu Leu Cys 130 135 140 Gln Ser Arg Ser Pro Met
Asp Thr Phe Leu Leu Ile Lys Glu Arg Ala 145 150 155 160 Ala His Pro
Leu Leu His Leu Arg Ser Glu His Gly Ala Gln Gln His 165 170 175 Gln
Ala Glu Phe Pro Met Ser Pro Val Thr Ser Val His Gly Gly Thr 180 185
190 Tyr Arg Cys Phe Ser Ser His Gly Phe Ser His Tyr Leu Leu Ser His
195 200 205 Pro Ser Asp Pro Leu Glu Leu Ile Val Ser Gly Ser Leu Glu
Gly Pro 210 215 220 Arg Pro Ser Pro Thr Arg Ser Val Ser Thr Ala Ala
Gly Pro Glu Asp 225 230 235 240 Gln Pro Leu Met Pro Thr Gly Ser Val
Pro His Ser Gly Leu Arg Arg 245 250 255 His Trp Glu Val Leu Ile Gly
Val Leu Val Val Ser Ile Leu Leu Leu 260 265 270 Ser Leu Leu Leu Phe
Leu Leu Leu Gln His Trp Arg Gln Gly Lys His 275 280 285 Arg Thr Leu
Ala Gln Arg Gln Ala Asp Phe Gln Arg Pro Pro Gly Ala 290 295 300 Ala
Glu Pro Glu Pro Lys Asp Gly Gly Leu Gln Arg Arg Ser Ser Pro 305 310
315 320 Ala Ala Asp Val Gln Gly Glu Asn Phe Cys Ala Ala Val Lys Asn
Thr 325 330 335 Gln Pro Glu Asp Gly Val Glu Met Asp Thr Arg Gln Ser
Pro His Asp 340 345 350 Glu Asp Pro Gln Ala Val Thr Tyr Ala Lys Val
Lys His Ser Arg Pro 355 360 365 Arg Arg Glu Met Ala Ser Pro Pro Ser
Pro Leu Ser Gly Glu Phe Leu 370 375 380 Asp Thr Lys Asp Arg Gln Ala
Glu Glu Asp Arg Gln Met Asp Thr Glu 385 390 395 400 Ala Ala Ala Ser
Glu Ala Pro Gln Asp Val Thr Tyr Ala Arg Leu His 405 410 415 Ser Phe
Thr Leu Arg Gln Lys Ala Thr Glu Pro Pro Pro Ser Gln Glu 420 425 430
Gly Ala Ser Pro Ala Glu Pro Ser Val Tyr Ala Thr Leu Ala Ile His 435
440 445 19 2194 DNA human CDS (67)..(1962) 19 tctctgtcct gccagcactg
agggctcatc cctctgcaga gcgcggggtc accggaagga 60 gacgcc atg acg ccc
gcc ctc aca gcc ctg ctc tgc ctt ggg ctg agt 108 Met Thr Pro Ala Leu
Thr Ala Leu Leu Cys Leu Gly Leu Ser 1 5 10 ctg ggc ccc agg acc cgc
gtg cag gca ggg ccc ttc ccc aaa ccc acc 156 Leu Gly Pro Arg Thr Arg
Val Gln Ala Gly Pro Phe Pro Lys Pro Thr 15 20 25 30 ctc tgg gct gag
cca ggc tct gtg atc agc tgg ggg agc ccc gtg acc 204 Leu Trp Ala Glu
Pro Gly Ser Val Ile Ser Trp Gly Ser Pro Val Thr 35 40 45 atc tgg
tgt cag ggg agc ctg gag gcc cag gag tac caa ctg gat aaa 252 Ile Trp
Cys Gln Gly Ser Leu Glu Ala Gln Glu Tyr Gln Leu Asp Lys 50 55 60
gag gga agc cca gag ccc ttg gac aga aat aac cca ctg gaa ccc aag 300
Glu Gly Ser Pro Glu Pro Leu Asp Arg Asn Asn Pro Leu Glu Pro Lys 65
70 75 aac aag gcc aga ttc tcc atc cca tcc atg aca cag cac cat gca
ggg 348 Asn Lys Ala Arg Phe Ser Ile Pro Ser Met Thr Gln His His Ala
Gly 80 85 90 aga tac cgc tgc cac tat tac agc tct gca ggc tgg tca
gag ccc agc 396 Arg Tyr Arg Cys His Tyr Tyr Ser Ser Ala Gly Trp Ser
Glu Pro Ser 95 100 105 110 gac ccc ctg gag ctg gtg atg aca gga gcc
tat agc aaa ccc acc ctc 444 Asp Pro Leu Glu Leu Val Met Thr Gly Ala
Tyr Ser Lys Pro Thr Leu 115 120 125 tca gcc ctg ccc agc cct gtg gtg
gcc tca ggg ggg aat atg acc ctc 492 Ser Ala Leu Pro Ser Pro Val Val
Ala Ser Gly Gly Asn Met Thr Leu 130 135 140 cga tgt ggc tca cag aag
aga tat cac cat ttt gtt ctg atg aag gaa 540 Arg Cys Gly Ser Gln Lys
Arg Tyr His His Phe Val Leu Met Lys Glu 145 150 155 gga gaa cac cag
ctc ccc cgg acc ctg gac tca cag cag ctc cac agt 588 Gly Glu His Gln
Leu Pro Arg Thr Leu Asp Ser Gln Gln Leu His Ser 160 165 170 ggg ggg
ttc cag gcc ctg ttc cct gtg ggc ccc gtg aac ccc agc cac 636 Gly Gly
Phe Gln Ala Leu Phe Pro Val Gly Pro Val Asn Pro Ser His 175 180 185
190 agg tgg agg ttc aca tgc tat tac tat tat atg aac acc ccc cgg gtg
684 Arg Trp Arg Phe Thr Cys Tyr Tyr Tyr Tyr Met Asn Thr Pro Arg Val
195 200 205 tgg tcc cac ccc agt gac ccc ctg gag att ctg ccc tca ggc
gtg tct 732 Trp Ser His Pro Ser Asp Pro Leu Glu Ile Leu Pro Ser Gly
Val Ser 210 215 220 agg aag ccc tcc ctc ctg acc ctg cag ggc cct gtc
ctg gcc cct ggg 780 Arg Lys Pro Ser Leu Leu Thr Leu Gln Gly Pro Val
Leu Ala Pro Gly 225 230 235 cag agt ctg acc ctc cag tgt ggc tct gat
gtc ggc tac gac aga ttt 828 Gln Ser Leu Thr Leu Gln Cys Gly Ser Asp
Val Gly Tyr Asp Arg Phe 240 245 250 gtt ctg tat aag gag ggg gaa cgt
gac ttc ctc cag cgc cct ggc cag 876 Val Leu Tyr Lys Glu Gly Glu Arg
Asp Phe Leu Gln Arg Pro Gly Gln 255 260 265 270 cag ccc cag gct ggg
ctc tcc cag gcc aac ttc acc ctg ggc cct gtg 924 Gln Pro Gln Ala Gly
Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val 275 280 285 agc ccc tcc
aat ggg ggc cag tac agg tgc tac ggt gca cac aac ctc 972 Ser Pro Ser
Asn Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu 290 295 300 tcc
tcc gag tgg tcg gcc ccc agc gac ccc ctg aac atc ctg atg gca 1020
Ser Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asn Ile Leu Met Ala 305
310 315 gga cag atc tat gac acc gtc tcc ctg tca gca cag ccg ggc ccc
aca 1068 Gly Gln Ile Tyr Asp Thr Val Ser Leu Ser Ala Gln Pro Gly
Pro Thr 320 325 330 gtg gcc tca gga gag aac gtg acc ctg ctg tgt cag
tca tgg tgg cag 1116 Val Ala Ser Gly Glu Asn Val Thr Leu Leu Cys
Gln Ser Trp Trp Gln 335 340 345 350 ttt gac act ttc ctt ctg acc aaa
gaa ggg gca gcc cat ccc cca ctg 1164 Phe Asp Thr Phe Leu Leu Thr
Lys Glu Gly Ala Ala His Pro Pro Leu 355 360 365 cgt ctg aga tca atg
tac gga gct cat aag tac cag gct gaa ttc ccc 1212 Arg Leu Arg Ser
Met Tyr Gly Ala His Lys Tyr Gln Ala Glu Phe Pro 370 375 380 atg agt
cct gtg acc tca gcc cac gcg ggg acc tac agg tgc tac ggc 1260 Met
Ser Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly 385 390
395 tca cgc agc tcc aac ccc tac ctg ctg tct cac ccc agt gag ccc ctg
1308 Ser Arg Ser Ser Asn Pro Tyr Leu Leu Ser His Pro Ser Glu Pro
Leu 400 405 410 gag ctc gtg gtc tca
gga cac tct gga ggc tcc agc ctc cca ccc aca 1356 Glu Leu Val Val
Ser Gly His Ser Gly Gly Ser Ser Leu Pro Pro Thr 415 420 425 430 ggg
ccg ccc tcc aca cct ggt ctg gga aga tac ctg gag gtt ttg att 1404
Gly Pro Pro Ser Thr Pro Gly Leu Gly Arg Tyr Leu Glu Val Leu Ile 435
440 445 ggg gtc tcg gtg gcc ttc gtc ctg ctg ctc ttc ctc ctc ctc ttc
ctc 1452 Gly Val Ser Val Ala Phe Val Leu Leu Leu Phe Leu Leu Leu
Phe Leu 450 455 460 ctc ctc cga cgt cag cgt cac agc aaa cac agg aca
tct gac cag aga 1500 Leu Leu Arg Arg Gln Arg His Ser Lys His Arg
Thr Ser Asp Gln Arg 465 470 475 aag act gat ttc cag cgt cct gca ggg
gct gcg gag aca gag ccc aag 1548 Lys Thr Asp Phe Gln Arg Pro Ala
Gly Ala Ala Glu Thr Glu Pro Lys 480 485 490 gac agg ggc ctg ctg agg
agg tcc agc cca gct gct gac gtc cag gaa 1596 Asp Arg Gly Leu Leu
Arg Arg Ser Ser Pro Ala Ala Asp Val Gln Glu 495 500 505 510 gaa aac
ctc tat gct gcc gtg aag gac aca cag tct gag gac ggg gtg 1644 Glu
Asn Leu Tyr Ala Ala Val Lys Asp Thr Gln Ser Glu Asp Gly Val 515 520
525 gag ctg gac agt cag agc cca cac gat gaa gac ccc cac gca gtg acg
1692 Glu Leu Asp Ser Gln Ser Pro His Asp Glu Asp Pro His Ala Val
Thr 530 535 540 tat gcc ccg gtg aaa cac tcc agt cct agg aga gaa atg
gcc tct cct 1740 Tyr Ala Pro Val Lys His Ser Ser Pro Arg Arg Glu
Met Ala Ser Pro 545 550 555 cct tcc cca ctg tct ggg gaa ttc ctg gac
aca aag gac aga cag gca 1788 Pro Ser Pro Leu Ser Gly Glu Phe Leu
Asp Thr Lys Asp Arg Gln Ala 560 565 570 gaa gag gac aga cag atg gac
act gag gct gct gca tct gaa gcc tcc 1836 Glu Glu Asp Arg Gln Met
Asp Thr Glu Ala Ala Ala Ser Glu Ala Ser 575 580 585 590 cag gat gtg
acc tac gcc cag ctg cac agc ttg acc ctt aga cgg aag 1884 Gln Asp
Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys 595 600 605
gca act gag cct cct cca tcc cag gaa ggg gaa cct cca gct gag ccc
1932 Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Glu Pro Pro Ala Glu
Pro 610 615 620 agc atc tac gcc act ctg gcc atc cac tag cccggggggt
acgcagaccc 1982 Ser Ile Tyr Ala Thr Leu Ala Ile His 625 630
cacactcagc agaaggagac tcaggactgc tgaaggacgg gagctgcccc cagtggacac
2042 cagtgaaccc cagtcagcct ggacccctaa cacagaccat gaggagacgc
tgggaacttg 2102 tgggactcac ctgactcaaa gatgactaat atcgtcccat
tttggaaata aagcaacaga 2162 cttctcaagc aggtcgtctc gttccaagat ct 2194
20 631 PRT human 20 Met Thr Pro Ala Leu Thr Ala Leu Leu Cys Leu Gly
Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr Arg Val Gln Ala Gly Pro Phe
Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Ser
Trp Gly Ser Pro Val Thr Ile Trp 35 40 45 Cys Gln Gly Ser Leu Glu
Ala Gln Glu Tyr Gln Leu Asp Lys Glu Gly 50 55 60 Ser Pro Glu Pro
Leu Asp Arg Asn Asn Pro Leu Glu Pro Lys Asn Lys 65 70 75 80 Ala Arg
Phe Ser Ile Pro Ser Met Thr Gln His His Ala Gly Arg Tyr 85 90 95
Arg Cys His Tyr Tyr Ser Ser Ala Gly Trp Ser Glu Pro Ser Asp Pro 100
105 110 Leu Glu Leu Val Met Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser
Ala 115 120 125 Leu Pro Ser Pro Val Val Ala Ser Gly Gly Asn Met Thr
Leu Arg Cys 130 135 140 Gly Ser Gln Lys Arg Tyr His His Phe Val Leu
Met Lys Glu Gly Glu 145 150 155 160 His Gln Leu Pro Arg Thr Leu Asp
Ser Gln Gln Leu His Ser Gly Gly 165 170 175 Phe Gln Ala Leu Phe Pro
Val Gly Pro Val Asn Pro Ser His Arg Trp 180 185 190 Arg Phe Thr Cys
Tyr Tyr Tyr Tyr Met Asn Thr Pro Arg Val Trp Ser 195 200 205 His Pro
Ser Asp Pro Leu Glu Ile Leu Pro Ser Gly Val Ser Arg Lys 210 215 220
Pro Ser Leu Leu Thr Leu Gln Gly Pro Val Leu Ala Pro Gly Gln Ser 225
230 235 240 Leu Thr Leu Gln Cys Gly Ser Asp Val Gly Tyr Asp Arg Phe
Val Leu 245 250 255 Tyr Lys Glu Gly Glu Arg Asp Phe Leu Gln Arg Pro
Gly Gln Gln Pro 260 265 270 Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr
Leu Gly Pro Val Ser Pro 275 280 285 Ser Asn Gly Gly Gln Tyr Arg Cys
Tyr Gly Ala His Asn Leu Ser Ser 290 295 300 Glu Trp Ser Ala Pro Ser
Asp Pro Leu Asn Ile Leu Met Ala Gly Gln 305 310 315 320 Ile Tyr Asp
Thr Val Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Ala 325 330 335 Ser
Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp Trp Gln Phe Asp 340 345
350 Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu Arg Leu
355 360 365 Arg Ser Met Tyr Gly Ala His Lys Tyr Gln Ala Glu Phe Pro
Met Ser 370 375 380 Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys
Tyr Gly Ser Arg 385 390 395 400 Ser Ser Asn Pro Tyr Leu Leu Ser His
Pro Ser Glu Pro Leu Glu Leu 405 410 415 Val Val Ser Gly His Ser Gly
Gly Ser Ser Leu Pro Pro Thr Gly Pro 420 425 430 Pro Ser Thr Pro Gly
Leu Gly Arg Tyr Leu Glu Val Leu Ile Gly Val 435 440 445 Ser Val Ala
Phe Val Leu Leu Leu Phe Leu Leu Leu Phe Leu Leu Leu 450 455 460 Arg
Arg Gln Arg His Ser Lys His Arg Thr Ser Asp Gln Arg Lys Thr 465 470
475 480 Asp Phe Gln Arg Pro Ala Gly Ala Ala Glu Thr Glu Pro Lys Asp
Arg 485 490 495 Gly Leu Leu Arg Arg Ser Ser Pro Ala Ala Asp Val Gln
Glu Glu Asn 500 505 510 Leu Tyr Ala Ala Val Lys Asp Thr Gln Ser Glu
Asp Gly Val Glu Leu 515 520 525 Asp Ser Gln Ser Pro His Asp Glu Asp
Pro His Ala Val Thr Tyr Ala 530 535 540 Pro Val Lys His Ser Ser Pro
Arg Arg Glu Met Ala Ser Pro Pro Ser 545 550 555 560 Pro Leu Ser Gly
Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu 565 570 575 Asp Arg
Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Ser Gln Asp 580 585 590
Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys Ala Thr 595
600 605 Glu Pro Pro Pro Ser Gln Glu Gly Glu Pro Pro Ala Glu Pro Ser
Ile 610 615 620 Tyr Ala Thr Leu Ala Ile His 625 630 21 2061 DNA
human CDS (67)..(1839) 21 tttgtgtcct gccaggcacc gtggtctcat
ccgcctgcac agctgagtcc agtgggagct 60 gacgcc atg acc ctc acc ctc tca
gtc ctg att tgc ctc ggg ctg agt 108 Met Thr Leu Thr Leu Ser Val Leu
Ile Cys Leu Gly Leu Ser 1 5 10 gtg ggc ccc agg acc tgc gtg cag gca
ggc acc ctc ccc aaa ccc acc 156 Val Gly Pro Arg Thr Cys Val Gln Ala
Gly Thr Leu Pro Lys Pro Thr 15 20 25 30 ctc tgg gct gag cca gcc tct
gtg ata gct cgg ggg aag ccc gtg acc 204 Leu Trp Ala Glu Pro Ala Ser
Val Ile Ala Arg Gly Lys Pro Val Thr 35 40 45 ctc tgg tgt cag ggg
ccc ctg gag act gag gag tac cgt ctg gat aag 252 Leu Trp Cys Gln Gly
Pro Leu Glu Thr Glu Glu Tyr Arg Leu Asp Lys 50 55 60 gag gga ctc
cca tgg gcc cgg aag aga cag aac cca ctg gag cct gga 300 Glu Gly Leu
Pro Trp Ala Arg Lys Arg Gln Asn Pro Leu Glu Pro Gly 65 70 75 gcc
aag gcc aag ttc cac att cca tcc acg gtg tat gac agt gca ggg 348 Ala
Lys Ala Lys Phe His Ile Pro Ser Thr Val Tyr Asp Ser Ala Gly 80 85
90 cga tac cgc tgc tac tat gag acc cct gca ggc tgg tca gag ccc agt
396 Arg Tyr Arg Cys Tyr Tyr Glu Thr Pro Ala Gly Trp Ser Glu Pro Ser
95 100 105 110 gac ccc ctg gag ctg gtg gcg aca gga ttc tat gca gaa
ccc act ctt 444 Asp Pro Leu Glu Leu Val Ala Thr Gly Phe Tyr Ala Glu
Pro Thr Leu 115 120 125 tta gcc ctg ccg agt cct gtg gtg gcc tca gga
gga aat gtg acc ctc 492 Leu Ala Leu Pro Ser Pro Val Val Ala Ser Gly
Gly Asn Val Thr Leu 130 135 140 cag tgt gat aca ctg gac gga ctt ctc
acg ttt gtt ctt gtt gag gaa 540 Gln Cys Asp Thr Leu Asp Gly Leu Leu
Thr Phe Val Leu Val Glu Glu 145 150 155 gaa cag aag ctc ccc agg acc
ctg tac tca cag aag ctc ccc aaa ggg 588 Glu Gln Lys Leu Pro Arg Thr
Leu Tyr Ser Gln Lys Leu Pro Lys Gly 160 165 170 cca tcc cag gcc ctg
ttc cct gtg ggt ccc gtg acc ccc agc tgc agg 636 Pro Ser Gln Ala Leu
Phe Pro Val Gly Pro Val Thr Pro Ser Cys Arg 175 180 185 190 tgg agg
ttc aga tgc tat tac tat tac agg aaa aac cct cag gtg tgg 684 Trp Arg
Phe Arg Cys Tyr Tyr Tyr Tyr Arg Lys Asn Pro Gln Val Trp 195 200 205
tcg aac ccc agt gac ctc ctg gag att ctg gtc cca ggc gtg tct agg 732
Ser Asn Pro Ser Asp Leu Leu Glu Ile Leu Val Pro Gly Val Ser Arg 210
215 220 aag ccc tcc ctc ctg atc ccg cag ggc tct gtc gtg gcc cgc gga
ggc 780 Lys Pro Ser Leu Leu Ile Pro Gln Gly Ser Val Val Ala Arg Gly
Gly 225 230 235 agc ctg acc ctg cag tgt cgc tct gat gtc ggc tat gac
ata ttc gtt 828 Ser Leu Thr Leu Gln Cys Arg Ser Asp Val Gly Tyr Asp
Ile Phe Val 240 245 250 ctg tac aag gag ggg gaa cat gac ctc gtc cag
ggc tct ggc cag cag 876 Leu Tyr Lys Glu Gly Glu His Asp Leu Val Gln
Gly Ser Gly Gln Gln 255 260 265 270 ccc cag gct ggg ctc tcc cag gcc
aac ttc acc ctg ggc cct gtg agc 924 Pro Gln Ala Gly Leu Ser Gln Ala
Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 cgc tcc cac ggg ggc cag
tac aga tgc tac ggt gca cac aac ctc tcc 972 Arg Ser His Gly Gly Gln
Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser 290 295 300 cct agg tgg tcg
gcc ccc agc gac ccc ctg gac atc ctg atc gca gga 1020 Pro Arg Trp
Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310 315 ctg
atc cct gac ata ccc gcc ctc tcg gtg cag ccg ggc ccc aag gtg 1068
Leu Ile Pro Asp Ile Pro Ala Leu Ser Val Gln Pro Gly Pro Lys Val 320
325 330 gcc tca gga gag aac gtg acc ctg ctg tgt cag tca tgg cat cag
ata 1116 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp His
Gln Ile 335 340 345 350 gac act ttc ttt ttg acc aag gag ggg gca gcc
cat ccc ccg ctg tgt 1164 Asp Thr Phe Phe Leu Thr Lys Glu Gly Ala
Ala His Pro Pro Leu Cys 355 360 365 cta aag tca aag tac cag tct tat
aga cac cag gct gaa ttc tcc atg 1212 Leu Lys Ser Lys Tyr Gln Ser
Tyr Arg His Gln Ala Glu Phe Ser Met 370 375 380 agt cct gtg acc tca
gcc cag ggt gga acc tac cga tgc tac agc gca 1260 Ser Pro Val Thr
Ser Ala Gln Gly Gly Thr Tyr Arg Cys Tyr Ser Ala 385 390 395 atc agg
tcc tac ccc tac ctg ctg tcc agc cct agt tac ccc cag gag 1308 Ile
Arg Ser Tyr Pro Tyr Leu Leu Ser Ser Pro Ser Tyr Pro Gln Glu 400 405
410 ctc gtg gtc tca gga ccc tct ggg gat ccc agc ctc tca cct aca ggc
1356 Leu Val Val Ser Gly Pro Ser Gly Asp Pro Ser Leu Ser Pro Thr
Gly 415 420 425 430 tcc acc ccc aca cct ggc cct gag gac cag ccc ctc
acc ccc acg ggg 1404 Ser Thr Pro Thr Pro Gly Pro Glu Asp Gln Pro
Leu Thr Pro Thr Gly 435 440 445 ttg gat ccc cag agt ggt ctg gga agg
cac ctg ggg gtt gtg act ggg 1452 Leu Asp Pro Gln Ser Gly Leu Gly
Arg His Leu Gly Val Val Thr Gly 450 455 460 gtc tca gtg gcc ttc gtc
ctg ctg ctg ttc ctc ctc ctc ttc ctc ctc 1500 Val Ser Val Ala Phe
Val Leu Leu Leu Phe Leu Leu Leu Phe Leu Leu 465 470 475 ctc cga cat
cgg cat cag agc aaa cac agg aca tcg gcc cat ttc tac 1548 Leu Arg
His Arg His Gln Ser Lys His Arg Thr Ser Ala His Phe Tyr 480 485 490
cgt cct gca ggg gct gcg ggg cca gag ccc aag gac cag ggc ctg cag
1596 Arg Pro Ala Gly Ala Ala Gly Pro Glu Pro Lys Asp Gln Gly Leu
Gln 495 500 505 510 aag agg gcc agc cca gtt gct gac atc cag gag gaa
att ctc aat gct 1644 Lys Arg Ala Ser Pro Val Ala Asp Ile Gln Glu
Glu Ile Leu Asn Ala 515 520 525 gcc gtg aag gac aca cag ccc aag gac
ggg gtg gag atg gat gct cgg 1692 Ala Val Lys Asp Thr Gln Pro Lys
Asp Gly Val Glu Met Asp Ala Arg 530 535 540 gct gct gca tct gaa gcc
ccc cag gat gtg acc tac gcc cag ctg cac 1740 Ala Ala Ala Ser Glu
Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His 545 550 555 agc ttg acc
ctc aga cgg gag gca act gag cct cct cca tcc cag gaa 1788 Ser Leu
Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu 560 565 570
agg gaa cct cca gct gaa ccc agc atc tac gcc ccc ctg gcc atc cac
1836 Arg Glu Pro Pro Ala Glu Pro Ser Ile Tyr Ala Pro Leu Ala Ile
His 575 580 585 590 tag cccacggggg acccagatct catactcaac agaaggagac
tcagagactc 1889 cagaaggcac aggagctgcc cccagtggac accaatgaac
cccagccagc ctggacccct 1949 aacaaagacc accaggacat cctgggaact
ctgggactca ctagattctg cagtcaaaga 2009 tgactaatat ccttgcattt
ttgaaatgaa gccacagact tctcaataaa tc 2061 22 590 PRT human 22 Met
Thr Leu Thr Leu Ser Val Leu Ile Cys Leu Gly Leu Ser Val Gly 1 5 10
15 Pro Arg Thr Cys Val Gln Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp
20 25 30 Ala Glu Pro Ala Ser Val Ile Ala Arg Gly Lys Pro Val Thr
Leu Trp 35 40 45 Cys Gln Gly Pro Leu Glu Thr Glu Glu Tyr Arg Leu
Asp Lys Glu Gly 50 55 60 Leu Pro Trp Ala Arg Lys Arg Gln Asn Pro
Leu Glu Pro Gly Ala Lys 65 70 75 80 Ala Lys Phe His Ile Pro Ser Thr
Val Tyr Asp Ser Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Glu Thr
Pro Ala Gly Trp Ser Glu Pro Ser Asp Pro 100 105 110 Leu Glu Leu Val
Ala Thr Gly Phe Tyr Ala Glu Pro Thr Leu Leu Ala 115 120 125 Leu Pro
Ser Pro Val Val Ala Ser Gly Gly Asn Val Thr Leu Gln Cys 130 135 140
Asp Thr Leu Asp Gly Leu Leu Thr Phe Val Leu Val Glu Glu Glu Gln 145
150 155 160 Lys Leu Pro Arg Thr Leu Tyr Ser Gln Lys Leu Pro Lys Gly
Pro Ser 165 170 175 Gln Ala Leu Phe Pro Val Gly Pro Val Thr Pro Ser
Cys Arg Trp Arg 180 185 190 Phe Arg Cys Tyr Tyr Tyr Tyr Arg Lys Asn
Pro Gln Val Trp Ser Asn 195 200 205 Pro Ser Asp Leu Leu Glu Ile Leu
Val Pro Gly Val Ser Arg Lys Pro 210 215 220 Ser Leu Leu Ile Pro Gln
Gly Ser Val Val Ala Arg Gly Gly Ser Leu 225 230 235 240 Thr Leu Gln
Cys Arg Ser Asp Val Gly Tyr Asp Ile Phe Val Leu Tyr 245 250 255 Lys
Glu Gly Glu His Asp Leu Val Gln Gly Ser Gly Gln Gln Pro Gln 260 265
270 Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Arg Ser
275 280 285 His Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser
Pro Arg 290 295 300 Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile
Ala Gly Leu Ile 305 310 315 320 Pro Asp Ile Pro Ala Leu Ser Val Gln
Pro Gly Pro Lys Val Ala Ser 325 330 335 Gly Glu Asn Val Thr Leu Leu
Cys Gln Ser Trp His Gln Ile Asp Thr 340 345 350 Phe Phe Leu Thr Lys
Glu Gly Ala Ala His Pro Pro Leu Cys Leu Lys 355 360 365 Ser Lys Tyr
Gln Ser Tyr Arg His Gln Ala Glu Phe Ser Met Ser Pro 370 375 380 Val
Thr Ser Ala Gln Gly Gly Thr Tyr Arg Cys Tyr Ser Ala Ile Arg 385 390
395 400 Ser Tyr Pro Tyr Leu Leu Ser Ser Pro Ser Tyr Pro Gln Glu Leu
Val 405
410 415 Val Ser Gly Pro Ser Gly Asp Pro Ser Leu Ser Pro Thr Gly Ser
Thr 420 425 430 Pro Thr Pro Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr
Gly Leu Asp 435 440 445 Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val
Val Thr Gly Val Ser 450 455 460 Val Ala Phe Val Leu Leu Leu Phe Leu
Leu Leu Phe Leu Leu Leu Arg 465 470 475 480 His Arg His Gln Ser Lys
His Arg Thr Ser Ala His Phe Tyr Arg Pro 485 490 495 Ala Gly Ala Ala
Gly Pro Glu Pro Lys Asp Gln Gly Leu Gln Lys Arg 500 505 510 Ala Ser
Pro Val Ala Asp Ile Gln Glu Glu Ile Leu Asn Ala Ala Val 515 520 525
Lys Asp Thr Gln Pro Lys Asp Gly Val Glu Met Asp Ala Arg Ala Ala 530
535 540 Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser
Leu 545 550 555 560 Thr Leu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser
Gln Glu Arg Glu 565 570 575 Pro Pro Ala Glu Pro Ser Ile Tyr Ala Pro
Leu Ala Ile His 580 585 590 23 28 DNA human 23 tatgcggccg
ccatgatgac aatgtggt 28 24 25 DNA human 24 tatgcggccg ccccttgcga
tagcg 25 25 31 DNA human 25 atagtcgaca acgccatcat gagatgtggt g 31
26 29 DNA human 26 taaagatctg ggctcgttag ctgtcgggt 29 27 33 DNA
human 27 tatagatcta cccccaggtg ccttcccaga cca 33 28 42 PRT human
VARIANT (2) Xaa is Gly or Arg 28 Leu Xaa Leu Ser Xaa Xaa Pro Arg
Thr Xaa Xaa Gln Xaa Gly Xaa Xaa 1 5 10 15 Pro Xaa Pro Thr Leu Trp
Ala Glu Pro Xaa Ser Phe Ile Xaa Xaa Ser 20 25 30 Asp Pro Lys Leu
Xaa Leu Val Xaa Thr Gly 35 40 29 1016 DNA human CDS (69)..(968) 29
ctgagtctgc ctgtggcatg gacctgcatc ttccctgaag catctccagg gctgaaaaat
60 cactgacc atg gca cca tgg tct cat cca tct gca cag ctg cag cca gtg
110 Met Ala Pro Trp Ser His Pro Ser Ala Gln Leu Gln Pro Val 1 5 10
gga gga gac gcc gtg agc cct gcc ctc atg gtt ctg ctc tgc ctc ggg 158
Gly Gly Asp Ala Val Ser Pro Ala Leu Met Val Leu Leu Cys Leu Gly 15
20 25 30 ctg agt ctg ggc ccc agg acc cac gtg cag gca ggg aac ctc
tcc aaa 206 Leu Ser Leu Gly Pro Arg Thr His Val Gln Ala Gly Asn Leu
Ser Lys 35 40 45 gcc acc ctc tgg gct gag cca ggc tct gtg atc agc
cgg ggg aac tct 254 Ala Thr Leu Trp Ala Glu Pro Gly Ser Val Ile Ser
Arg Gly Asn Ser 50 55 60 gtg acc atc cgg tgt cag ggg acc ctg gag
gcc cag gaa tac cgt ctg 302 Val Thr Ile Arg Cys Gln Gly Thr Leu Glu
Ala Gln Glu Tyr Arg Leu 65 70 75 gtt aaa gag gga agc cca gaa ccc
tgg gac aca cag aac cca ctg gag 350 Val Lys Glu Gly Ser Pro Glu Pro
Trp Asp Thr Gln Asn Pro Leu Glu 80 85 90 ccc aag aac aag gcc aga
ttc tcc atc cca tcc atg aca gag cac cat 398 Pro Lys Asn Lys Ala Arg
Phe Ser Ile Pro Ser Met Thr Glu His His 95 100 105 110 gca ggg aga
tac cgc tgt tac tac tac agc cct gca ggc tgg tca gag 446 Ala Gly Arg
Tyr Arg Cys Tyr Tyr Tyr Ser Pro Ala Gly Trp Ser Glu 115 120 125 ccc
agc gac ccc ctg gag ctg gtg gtg aca gga ttc tac aac aaa ccc 494 Pro
Ser Asp Pro Leu Glu Leu Val Val Thr Gly Phe Tyr Asn Lys Pro 130 135
140 acc ctc tca gcc ctg ccc agt cct gtg gtg acc tca gga gag aac gtg
542 Thr Leu Ser Ala Leu Pro Ser Pro Val Val Thr Ser Gly Glu Asn Val
145 150 155 acc ctc cag tgt ggc tca cgg ctg aga ttc gac agg ttc att
ctg act 590 Thr Leu Gln Cys Gly Ser Arg Leu Arg Phe Asp Arg Phe Ile
Leu Thr 160 165 170 gag gaa gga gac cac aag ctc tcc tgg acc ttg gac
tca cag ctg acc 638 Glu Glu Gly Asp His Lys Leu Ser Trp Thr Leu Asp
Ser Gln Leu Thr 175 180 185 190 ccc agt ggg cag ttc cag gcc ctg ttc
cct gtg ggc cct gtg acc ccc 686 Pro Ser Gly Gln Phe Gln Ala Leu Phe
Pro Val Gly Pro Val Thr Pro 195 200 205 agc cac agg tgg atg ctc aga
tgc tat ggc tct cgc agg cat atc ctg 734 Ser His Arg Trp Met Leu Arg
Cys Tyr Gly Ser Arg Arg His Ile Leu 210 215 220 cag gta tgg tca gaa
ccc agt gac ctc ctg gag att ccg gtc tca gga 782 Gln Val Trp Ser Glu
Pro Ser Asp Leu Leu Glu Ile Pro Val Ser Gly 225 230 235 gca gct gat
aac ctc agt ccg tca caa aac aag tct gac tct ggg act 830 Ala Ala Asp
Asn Leu Ser Pro Ser Gln Asn Lys Ser Asp Ser Gly Thr 240 245 250 gcc
tca cac ctt cag gat tac gca gta gag aat ctc atc cgc atg ggc 878 Ala
Ser His Leu Gln Asp Tyr Ala Val Glu Asn Leu Ile Arg Met Gly 255 260
265 270 atg gcc ggc ttg atc ctg gtg gtc ctt ggg att ctg ata ttt cag
gat 926 Met Ala Gly Leu Ile Leu Val Val Leu Gly Ile Leu Ile Phe Gln
Asp 275 280 285 tgg cac agc cag aga agc ccc caa gct gca gct gga agg
tga 968 Trp His Ser Gln Arg Ser Pro Gln Ala Ala Ala Gly Arg 290 295
300 acagaagaga gaacaatgca ccattgaatg ctggagcctt ggaagcga 1016 30
299 PRT human 30 Met Ala Pro Trp Ser His Pro Ser Ala Gln Leu Gln
Pro Val Gly Gly 1 5 10 15 Asp Ala Val Ser Pro Ala Leu Met Val Leu
Leu Cys Leu Gly Leu Ser 20 25 30 Leu Gly Pro Arg Thr His Val Gln
Ala Gly Asn Leu Ser Lys Ala Thr 35 40 45 Leu Trp Ala Glu Pro Gly
Ser Val Ile Ser Arg Gly Asn Ser Val Thr 50 55 60 Ile Arg Cys Gln
Gly Thr Leu Glu Ala Gln Glu Tyr Arg Leu Val Lys 65 70 75 80 Glu Gly
Ser Pro Glu Pro Trp Asp Thr Gln Asn Pro Leu Glu Pro Lys 85 90 95
Asn Lys Ala Arg Phe Ser Ile Pro Ser Met Thr Glu His His Ala Gly 100
105 110 Arg Tyr Arg Cys Tyr Tyr Tyr Ser Pro Ala Gly Trp Ser Glu Pro
Ser 115 120 125 Asp Pro Leu Glu Leu Val Val Thr Gly Phe Tyr Asn Lys
Pro Thr Leu 130 135 140 Ser Ala Leu Pro Ser Pro Val Val Thr Ser Gly
Glu Asn Val Thr Leu 145 150 155 160 Gln Cys Gly Ser Arg Leu Arg Phe
Asp Arg Phe Ile Leu Thr Glu Glu 165 170 175 Gly Asp His Lys Leu Ser
Trp Thr Leu Asp Ser Gln Leu Thr Pro Ser 180 185 190 Gly Gln Phe Gln
Ala Leu Phe Pro Val Gly Pro Val Thr Pro Ser His 195 200 205 Arg Trp
Met Leu Arg Cys Tyr Gly Ser Arg Arg His Ile Leu Gln Val 210 215 220
Trp Ser Glu Pro Ser Asp Leu Leu Glu Ile Pro Val Ser Gly Ala Ala 225
230 235 240 Asp Asn Leu Ser Pro Ser Gln Asn Lys Ser Asp Ser Gly Thr
Ala Ser 245 250 255 His Leu Gln Asp Tyr Ala Val Glu Asn Leu Ile Arg
Met Gly Met Ala 260 265 270 Gly Leu Ile Leu Val Val Leu Gly Ile Leu
Ile Phe Gln Asp Trp His 275 280 285 Ser Gln Arg Ser Pro Gln Ala Ala
Ala Gly Arg 290 295 31 1007 DNA human CDS (95)..(958) 31 caggtgtcag
atgtgtctct gctgatctga gtctgcctgt ggcatggacc tgcatcttcc 60
ctgaagcatc tccagggctg aaaaatcact gacc atg gca cca tgg tct cat cca
115 Met Ala Pro Trp Ser His Pro 1 5 tct gca cag ctg cag cca gtg gga
gga gac gcc gtg agc cct gcc ctc 163 Ser Ala Gln Leu Gln Pro Val Gly
Gly Asp Ala Val Ser Pro Ala Leu 10 15 20 atg gtt ctg ctc tgc ctc
ggg aac ctc tcc aaa gcc acc ctc tgg gct 211 Met Val Leu Leu Cys Leu
Gly Asn Leu Ser Lys Ala Thr Leu Trp Ala 25 30 35 gag cca ggc tct
gtg atc agc cgg ggg aac tct gtg acc atc cgg tgt 259 Glu Pro Gly Ser
Val Ile Ser Arg Gly Asn Ser Val Thr Ile Arg Cys 40 45 50 55 cag ggg
acc ctg gag gcc cag gaa tac cgt ctg gtt aaa gag gga agc 307 Gln Gly
Thr Leu Glu Ala Gln Glu Tyr Arg Leu Val Lys Glu Gly Ser 60 65 70
cca gaa ccc tgg gac aca cag aac cca ctg gag ccc aag aac aag gcc 355
Pro Glu Pro Trp Asp Thr Gln Asn Pro Leu Glu Pro Lys Asn Lys Ala 75
80 85 aga ttc tcc atc cca tcc atg aca gag cac cat gca ggg aga tac
cgc 403 Arg Phe Ser Ile Pro Ser Met Thr Glu His His Ala Gly Arg Tyr
Arg 90 95 100 tgt tac tac tac agc cct gca ggc tgg tca gag ccc agc
gac ccc ctg 451 Cys Tyr Tyr Tyr Ser Pro Ala Gly Trp Ser Glu Pro Ser
Asp Pro Leu 105 110 115 gag ctg gtg gtg aca gga ttc tac aac aaa ccc
acc ctc tca gcc ctg 499 Glu Leu Val Val Thr Gly Phe Tyr Asn Lys Pro
Thr Leu Ser Ala Leu 120 125 130 135 ccc agt cct gtg gtg acc tca gga
gag aac gtg acc ctc cag tgt ggc 547 Pro Ser Pro Val Val Thr Ser Gly
Glu Asn Val Thr Leu Gln Cys Gly 140 145 150 tca cgg ctg aga ttc gac
agg ttc att ctg act gag gaa gga gac cac 595 Ser Arg Leu Arg Phe Asp
Arg Phe Ile Leu Thr Glu Glu Gly Asp His 155 160 165 aag ctc tcc tgg
acc ttg gac tca cag ctg acc ccc agt ggg cag ttc 643 Lys Leu Ser Trp
Thr Leu Asp Ser Gln Leu Thr Pro Ser Gly Gln Phe 170 175 180 cag gcc
ctg ttc cct gtg ggc cct gtg acc ccc agc cac agg tgg atg 691 Gln Ala
Leu Phe Pro Val Gly Pro Val Thr Pro Ser His Arg Trp Met 185 190 195
ctc aga tgc tat ggc tct cgc agg cat atc ctg cag gta tgg tca gaa 739
Leu Arg Cys Tyr Gly Ser Arg Arg His Ile Leu Gln Val Trp Ser Glu 200
205 210 215 ccc agt gac ctc ctg gag att ccg gtc tca gga gca gct gat
aac ctc 787 Pro Ser Asp Leu Leu Glu Ile Pro Val Ser Gly Ala Ala Asp
Asn Leu 220 225 230 agt ccg tca caa aac aag tct gac tct ggg act gcc
tca cac ctt cag 835 Ser Pro Ser Gln Asn Lys Ser Asp Ser Gly Thr Ala
Ser His Leu Gln 235 240 245 gat tac gca gta gag aat ctc atc cgc atg
ggc atg gcc ggc ttg atc 883 Asp Tyr Ala Val Glu Asn Leu Ile Arg Met
Gly Met Ala Gly Leu Ile 250 255 260 ctg gtg gtc ctt ggg att ctg ata
ttt cag gat tgg cac agc cag aga 931 Leu Val Val Leu Gly Ile Leu Ile
Phe Gln Asp Trp His Ser Gln Arg 265 270 275 agc ccc caa gct gca gct
gga agg tga acagaagaga gaacaatgca 978 Ser Pro Gln Ala Ala Ala Gly
Arg 280 285 ccattgaatg ctggagcctt ggaagcgaa 1007 32 287 PRT human
32 Met Ala Pro Trp Ser His Pro Ser Ala Gln Leu Gln Pro Val Gly Gly
1 5 10 15 Asp Ala Val Ser Pro Ala Leu Met Val Leu Leu Cys Leu Gly
Asn Leu 20 25 30 Ser Lys Ala Thr Leu Trp Ala Glu Pro Gly Ser Val
Ile Ser Arg Gly 35 40 45 Asn Ser Val Thr Ile Arg Cys Gln Gly Thr
Leu Glu Ala Gln Glu Tyr 50 55 60 Arg Leu Val Lys Glu Gly Ser Pro
Glu Pro Trp Asp Thr Gln Asn Pro 65 70 75 80 Leu Glu Pro Lys Asn Lys
Ala Arg Phe Ser Ile Pro Ser Met Thr Glu 85 90 95 His His Ala Gly
Arg Tyr Arg Cys Tyr Tyr Tyr Ser Pro Ala Gly Trp 100 105 110 Ser Glu
Pro Ser Asp Pro Leu Glu Leu Val Val Thr Gly Phe Tyr Asn 115 120 125
Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val Thr Ser Gly Glu 130
135 140 Asn Val Thr Leu Gln Cys Gly Ser Arg Leu Arg Phe Asp Arg Phe
Ile 145 150 155 160 Leu Thr Glu Glu Gly Asp His Lys Leu Ser Trp Thr
Leu Asp Ser Gln 165 170 175 Leu Thr Pro Ser Gly Gln Phe Gln Ala Leu
Phe Pro Val Gly Pro Val 180 185 190 Thr Pro Ser His Arg Trp Met Leu
Arg Cys Tyr Gly Ser Arg Arg His 195 200 205 Ile Leu Gln Val Trp Ser
Glu Pro Ser Asp Leu Leu Glu Ile Pro Val 210 215 220 Ser Gly Ala Ala
Asp Asn Leu Ser Pro Ser Gln Asn Lys Ser Asp Ser 225 230 235 240 Gly
Thr Ala Ser His Leu Gln Asp Tyr Ala Val Glu Asn Leu Ile Arg 245 250
255 Met Gly Met Ala Gly Leu Ile Leu Val Val Leu Gly Ile Leu Ile Phe
260 265 270 Gln Asp Trp His Ser Gln Arg Ser Pro Gln Ala Ala Ala Gly
Arg 275 280 285 33 956 DNA human CDS (115)..(912) 33 ctcagcctgg
gctacacagc caggtgtcag atgtgtctct gctgatctga gtctgcctgt 60
ggcatggacc tgcatcttcc ctgaagcatc tccagggctg aaaaatcact gacc atg 117
Met 1 gca cca tgg tct cat cca tct gca cag ctg cag cca gtg gga gga
gac 165 Ala Pro Trp Ser His Pro Ser Ala Gln Leu Gln Pro Val Gly Gly
Asp 5 10 15 gcc gtg agc cct gcc ctc atg gtt ctg ctc tgc ctc ggg ctg
agt ctg 213 Ala Val Ser Pro Ala Leu Met Val Leu Leu Cys Leu Gly Leu
Ser Leu 20 25 30 ggc ccc agg acc cac gtg cag gca ggg aac ctc tcc
aaa gcc acc ctc 261 Gly Pro Arg Thr His Val Gln Ala Gly Asn Leu Ser
Lys Ala Thr Leu 35 40 45 tgg gct gag cca ggc tct gtg atc agc cgg
ggg aac tct gtg acc atc 309 Trp Ala Glu Pro Gly Ser Val Ile Ser Arg
Gly Asn Ser Val Thr Ile 50 55 60 65 cgg tgt cag ggg acc ctg gag gcc
cag gaa tac cgt ctg gtt aaa gag 357 Arg Cys Gln Gly Thr Leu Glu Ala
Gln Glu Tyr Arg Leu Val Lys Glu 70 75 80 gga agc cca gaa ccc tgg
gac aca cag aac cca ctg gag ccc aag aac 405 Gly Ser Pro Glu Pro Trp
Asp Thr Gln Asn Pro Leu Glu Pro Lys Asn 85 90 95 aag gcc aga ttc
tcc atc cca tcc atg aca gag cac cat gca ggg aga 453 Lys Ala Arg Phe
Ser Ile Pro Ser Met Thr Glu His His Ala Gly Arg 100 105 110 tac cgc
tgt tac tac tac agc cct gca ggc tgg tca gag ccc agc gac 501 Tyr Arg
Cys Tyr Tyr Tyr Ser Pro Ala Gly Trp Ser Glu Pro Ser Asp 115 120 125
ccc ctg gag ctg gtg gtg aca gga ttc tac aac aaa ccc acc ctc tca 549
Pro Leu Glu Leu Val Val Thr Gly Phe Tyr Asn Lys Pro Thr Leu Ser 130
135 140 145 gcc ctg ccc agt cct gtg gtg acc tca gga gag aac gtg acc
ctc cag 597 Ala Leu Pro Ser Pro Val Val Thr Ser Gly Glu Asn Val Thr
Leu Gln 150 155 160 tgt ggc tca cgg ctg aga ttc gac agg ttc att ctg
act gag gaa gga 645 Cys Gly Ser Arg Leu Arg Phe Asp Arg Phe Ile Leu
Thr Glu Glu Gly 165 170 175 gac cac aag ctc tcc tgg acc ttg gac tca
cag ctg acc ccc agt ggg 693 Asp His Lys Leu Ser Trp Thr Leu Asp Ser
Gln Leu Thr Pro Ser Gly 180 185 190 cag ttc cag gcc ctg ttc cct gtg
ggc cct gtg acc ccc agc cac agg 741 Gln Phe Gln Ala Leu Phe Pro Val
Gly Pro Val Thr Pro Ser His Arg 195 200 205 tgg atg ctc aga tgc tat
ggc tct cgc agg cat atc ctg cag gta tgg 789 Trp Met Leu Arg Cys Tyr
Gly Ser Arg Arg His Ile Leu Gln Val Trp 210 215 220 225 tca gaa ccc
agt gac ctc ctg gag att ccg gtc tca ggt gag gaa gcc 837 Ser Glu Pro
Ser Asp Leu Leu Glu Ile Pro Val Ser Gly Glu Glu Ala 230 235 240 aca
gtc ttc tct agt aca att cag gga agc cag aca ggt tgt gga gag 885 Thr
Val Phe Ser Ser Thr Ile Gln Gly Ser Gln Thr Gly Cys Gly Glu 245 250
255 ctt tac agg cag ggc agc ccc tgc taa gaaagacaaa aaggggaagg 932
Leu Tyr Arg Gln Gly Ser Pro Cys 260 265 agaacacaga aatcctaggg acac
956 34 265 PRT human 34 Met Ala Pro Trp Ser His Pro Ser Ala Gln Leu
Gln Pro Val Gly Gly 1 5 10 15 Asp Ala Val Ser Pro Ala Leu Met Val
Leu Leu Cys Leu Gly Leu Ser 20 25 30 Leu Gly Pro Arg Thr His Val
Gln Ala Gly Asn Leu Ser Lys Ala Thr 35 40 45 Leu Trp Ala Glu Pro
Gly Ser Val Ile Ser Arg Gly Asn Ser Val Thr 50 55
60 Ile Arg Cys Gln Gly Thr Leu Glu Ala Gln Glu Tyr Arg Leu Val Lys
65 70 75 80 Glu Gly Ser Pro Glu Pro Trp Asp Thr Gln Asn Pro Leu Glu
Pro Lys 85 90 95 Asn Lys Ala Arg Phe Ser Ile Pro Ser Met Thr Glu
His His Ala Gly 100 105 110 Arg Tyr Arg Cys Tyr Tyr Tyr Ser Pro Ala
Gly Trp Ser Glu Pro Ser 115 120 125 Asp Pro Leu Glu Leu Val Val Thr
Gly Phe Tyr Asn Lys Pro Thr Leu 130 135 140 Ser Ala Leu Pro Ser Pro
Val Val Thr Ser Gly Glu Asn Val Thr Leu 145 150 155 160 Gln Cys Gly
Ser Arg Leu Arg Phe Asp Arg Phe Ile Leu Thr Glu Glu 165 170 175 Gly
Asp His Lys Leu Ser Trp Thr Leu Asp Ser Gln Leu Thr Pro Ser 180 185
190 Gly Gln Phe Gln Ala Leu Phe Pro Val Gly Pro Val Thr Pro Ser His
195 200 205 Arg Trp Met Leu Arg Cys Tyr Gly Ser Arg Arg His Ile Leu
Gln Val 210 215 220 Trp Ser Glu Pro Ser Asp Leu Leu Glu Ile Pro Val
Ser Gly Glu Glu 225 230 235 240 Ala Thr Val Phe Ser Ser Thr Ile Gln
Gly Ser Gln Thr Gly Cys Gly 245 250 255 Glu Leu Tyr Arg Gln Gly Ser
Pro Cys 260 265 35 997 DNA human CDS (73)..(834) 35 tgatctgagt
ctgcctgtgg catggacctg catcttccct gaagcatctc cagggctgaa 60
aaatcactga cc atg gca cca tgg tct cat cca tct gca cag ctg cag cca
111 Met Ala Pro Trp Ser His Pro Ser Ala Gln Leu Gln Pro 1 5 10 gtg
gga gga gac gcc gtg agc cct gcc ctc atg gtt ctg ctc tgc ctc 159 Val
Gly Gly Asp Ala Val Ser Pro Ala Leu Met Val Leu Leu Cys Leu 15 20
25 ggg aac ctc tcc aaa gcc acc ctc tgg gct gag cca ggc tct gtg atc
207 Gly Asn Leu Ser Lys Ala Thr Leu Trp Ala Glu Pro Gly Ser Val Ile
30 35 40 45 agc cgg ggg aac tct gtg acc atc cgg tgt cag ggg acc ctg
gag gcc 255 Ser Arg Gly Asn Ser Val Thr Ile Arg Cys Gln Gly Thr Leu
Glu Ala 50 55 60 cag gaa tac cgt ctg gtt aaa gag gga agc cca gaa
ccc tgg gac aca 303 Gln Glu Tyr Arg Leu Val Lys Glu Gly Ser Pro Glu
Pro Trp Asp Thr 65 70 75 cag aac cca ctg gag ccc aag aac aag gcc
aga ttc tcc atc cca tcc 351 Gln Asn Pro Leu Glu Pro Lys Asn Lys Ala
Arg Phe Ser Ile Pro Ser 80 85 90 atg aca gag cac cat gca ggg aga
tac cgc tgt tac tac tac agc cct 399 Met Thr Glu His His Ala Gly Arg
Tyr Arg Cys Tyr Tyr Tyr Ser Pro 95 100 105 gca ggc tgg tca gag ccc
agc gac ccc ctg gag ctg gtg gtg aca gga 447 Ala Gly Trp Ser Glu Pro
Ser Asp Pro Leu Glu Leu Val Val Thr Gly 110 115 120 125 ttc tac aac
aaa ccc acc ctc tca gcc ctg ccc agt cct gtg gtg acc 495 Phe Tyr Asn
Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val Thr 130 135 140 tca
gga gag aac gtg acc ctc cag tgt ggc tca cgg ctg aga ttc gac 543 Ser
Gly Glu Asn Val Thr Leu Gln Cys Gly Ser Arg Leu Arg Phe Asp 145 150
155 agg ttc att ctg act gag gaa gga gac cac aag ctc tcc tgg acc ttg
591 Arg Phe Ile Leu Thr Glu Glu Gly Asp His Lys Leu Ser Trp Thr Leu
160 165 170 gac tca cag ctg acc ccc agt ggg cag ttc cag gcc ctg ttc
cct gtg 639 Asp Ser Gln Leu Thr Pro Ser Gly Gln Phe Gln Ala Leu Phe
Pro Val 175 180 185 ggc cct gtg acc ccc agc cac agg tgg atg ctc aga
tgc tat ggc tct 687 Gly Pro Val Thr Pro Ser His Arg Trp Met Leu Arg
Cys Tyr Gly Ser 190 195 200 205 cgc agg cat atc ctg cag gta tgg tca
gaa ccc agt gac ctc ctg gag 735 Arg Arg His Ile Leu Gln Val Trp Ser
Glu Pro Ser Asp Leu Leu Glu 210 215 220 att ccg gtc tca ggt gag gaa
gcc aca gtc ttc tct agt aca att cag 783 Ile Pro Val Ser Gly Glu Glu
Ala Thr Val Phe Ser Ser Thr Ile Gln 225 230 235 gga agc cag aca ggt
tgt gga gag ctt tac agg cag ggc agc ccc tgc 831 Gly Ser Gln Thr Gly
Cys Gly Glu Leu Tyr Arg Gln Gly Ser Pro Cys 240 245 250 taa
gaaagacaaa aaggggaagg agaacacaga aatcctaggg acacaaattc 884
agggtgagga aaacaaagca agggctgggc acagtggctc acacgtgtaa tctcagcact
944 ttgggaggcc gaggcaggtg gatcacctga tgtcaggagt tcaagaccag cct 997
36 253 PRT human 36 Met Ala Pro Trp Ser His Pro Ser Ala Gln Leu Gln
Pro Val Gly Gly 1 5 10 15 Asp Ala Val Ser Pro Ala Leu Met Val Leu
Leu Cys Leu Gly Asn Leu 20 25 30 Ser Lys Ala Thr Leu Trp Ala Glu
Pro Gly Ser Val Ile Ser Arg Gly 35 40 45 Asn Ser Val Thr Ile Arg
Cys Gln Gly Thr Leu Glu Ala Gln Glu Tyr 50 55 60 Arg Leu Val Lys
Glu Gly Ser Pro Glu Pro Trp Asp Thr Gln Asn Pro 65 70 75 80 Leu Glu
Pro Lys Asn Lys Ala Arg Phe Ser Ile Pro Ser Met Thr Glu 85 90 95
His His Ala Gly Arg Tyr Arg Cys Tyr Tyr Tyr Ser Pro Ala Gly Trp 100
105 110 Ser Glu Pro Ser Asp Pro Leu Glu Leu Val Val Thr Gly Phe Tyr
Asn 115 120 125 Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val Thr
Ser Gly Glu 130 135 140 Asn Val Thr Leu Gln Cys Gly Ser Arg Leu Arg
Phe Asp Arg Phe Ile 145 150 155 160 Leu Thr Glu Glu Gly Asp His Lys
Leu Ser Trp Thr Leu Asp Ser Gln 165 170 175 Leu Thr Pro Ser Gly Gln
Phe Gln Ala Leu Phe Pro Val Gly Pro Val 180 185 190 Thr Pro Ser His
Arg Trp Met Leu Arg Cys Tyr Gly Ser Arg Arg His 195 200 205 Ile Leu
Gln Val Trp Ser Glu Pro Ser Asp Leu Leu Glu Ile Pro Val 210 215 220
Ser Gly Glu Glu Ala Thr Val Phe Ser Ser Thr Ile Gln Gly Ser Gln 225
230 235 240 Thr Gly Cys Gly Glu Leu Tyr Arg Gln Gly Ser Pro Cys 245
250 37 1451 DNA human CDS (1)..(1347) 37 ccc aag ccc acc ctc tgg
gct aag cca ggc tct gtg atc agc tgg aga 48 Pro Lys Pro Thr Leu Trp
Ala Lys Pro Gly Ser Val Ile Ser Trp Arg 1 5 10 15 agc ccc atg acc
atg tgg tgt cag ggg acc ctg gaa gcc cag gag tac 96 Ser Pro Met Thr
Met Trp Cys Gln Gly Thr Leu Glu Ala Gln Glu Tyr 20 25 30 cat ctg
tat aaa gag gga agc aca gag ccc tgg gac aga acg aat cca 144 His Leu
Tyr Lys Glu Gly Ser Thr Glu Pro Trp Asp Arg Thr Asn Pro 35 40 45
ctg gag acc agg aac aag gcc aga tac tcc atc cca tcc atg aca cag 192
Leu Glu Thr Arg Asn Lys Ala Arg Tyr Ser Ile Pro Ser Met Thr Gln 50
55 60 cac cat gca gtg aga tat cag tgt tac tat ctc agc cct gca ggc
tgg 240 His His Ala Val Arg Tyr Gln Cys Tyr Tyr Leu Ser Pro Ala Gly
Trp 65 70 75 80 tca gag ccc agt gac ccc ctg gag ctg gtg atg aca gga
ttc tac agc 288 Ser Glu Pro Ser Asp Pro Leu Glu Leu Val Met Thr Gly
Phe Tyr Ser 85 90 95 aaa ccc acc ctc tca gcc ctg ccc agc cct gtg
gtg gcc tca ggg ggg 336 Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Val
Val Ala Ser Gly Gly 100 105 110 aaa gtg acc ctc cga tgt ggc tca cag
aag gga tat cac cat ttt gtt 384 Lys Val Thr Leu Arg Cys Gly Ser Gln
Lys Gly Tyr His His Phe Val 115 120 125 ctg atg aag gaa gga gaa cac
cag ctc ccc cgg acc ctg gac tca cag 432 Leu Met Lys Glu Gly Glu His
Gln Leu Pro Arg Thr Leu Asp Ser Gln 130 135 140 cag ctc cac agt ggg
ggg ttc cag gcc ctg ttc cct gtg ggc ccc gtg 480 Gln Leu His Ser Gly
Gly Phe Gln Ala Leu Phe Pro Val Gly Pro Val 145 150 155 160 acc ccc
agc cac agg tgg agg ttc aca tgc tat tac tat tat atg aac 528 Thr Pro
Ser His Arg Trp Arg Phe Thr Cys Tyr Tyr Tyr Tyr Met Asn 165 170 175
acc ccc cag gtg tgg tcc cac ccc agt gac ccc ctg gag att ctg ccc 576
Thr Pro Gln Val Trp Ser His Pro Ser Asp Pro Leu Glu Ile Leu Pro 180
185 190 tca gga cag agc tct ccc cct gtc ctg gcc cct gga gag acc ctg
acc 624 Ser Gly Gln Ser Ser Pro Pro Val Leu Ala Pro Gly Glu Thr Leu
Thr 195 200 205 ctc cag tgt ggc tct gat gtc ggc tac gac aga ttc act
ctg tac aag 672 Leu Gln Cys Gly Ser Asp Val Gly Tyr Asp Arg Phe Thr
Leu Tyr Lys 210 215 220 gag ggg gaa tgt gac ttc ctc cag cgc cct ggc
cag cag ccc cag gct 720 Glu Gly Glu Cys Asp Phe Leu Gln Arg Pro Gly
Gln Gln Pro Gln Ala 225 230 235 240 ggg ctc tcc cag gcc aac ttc acc
ctg ggc cct gtg agg ggc tcc cac 768 Gly Leu Ser Gln Ala Asn Phe Thr
Leu Gly Pro Val Arg Gly Ser His 245 250 255 ggg ggc cag tac aga tgc
tcc ggt gca cac aac ctc tcc tcc gag tgg 816 Gly Gly Gln Tyr Arg Cys
Ser Gly Ala His Asn Leu Ser Ser Glu Trp 260 265 270 tcg gcc ccc agt
gac ccc ctg gac atc ctg atc gca gga cag atc cct 864 Ser Ala Pro Ser
Asp Pro Leu Asp Ile Leu Ile Ala Gly Gln Ile Pro 275 280 285 ggc aga
ccc tcc ctc tcg gtg cag ttg tgg ccc aca gtg gcc tca gga 912 Gly Arg
Pro Ser Leu Ser Val Gln Leu Trp Pro Thr Val Ala Ser Gly 290 295 300
gag aac gtg acc ctg ctg tgt caa tca caa gag tgg atg cac act ttc 960
Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Glu Trp Met His Thr Phe 305
310 315 320 ctt ctg acc aag gag ggg gca gcc cat ccc ctg ctg tgt ctg
aga tca 1008 Leu Leu Thr Lys Glu Gly Ala Ala His Pro Leu Leu Cys
Leu Arg Ser 325 330 335 aag tac gga gct cat aag tac cag gct gaa ttc
ccc atg agt cct gtg 1056 Lys Tyr Gly Ala His Lys Tyr Gln Ala Glu
Phe Pro Met Ser Pro Val 340 345 350 acc tca gcc cac acg ggg acc tac
agg tgc tac ggc tca ctc agc tcc 1104 Thr Ser Ala His Thr Gly Thr
Tyr Arg Cys Tyr Gly Ser Leu Ser Ser 355 360 365 gac ccc tac ctg ctg
tct cac ccc agt ggc ccc gtg gag ctc gtg gtc 1152 Asp Pro Tyr Leu
Leu Ser His Pro Ser Gly Pro Val Glu Leu Val Val 370 375 380 tca gcc
tca cac ctt cag gat tac gca gtg gag aat ctc atc cac atg 1200 Ser
Ala Ser His Leu Gln Asp Tyr Ala Val Glu Asn Leu Ile His Met 385 390
395 400 ggc gtg gct ggc ttg atc ctg gtg gtc ctc ggg att ctg tca ttt
gag 1248 Gly Val Ala Gly Leu Ile Leu Val Val Leu Gly Ile Leu Ser
Phe Glu 405 410 415 gct tgg cac agc cag aga agc ttc cca aga tgc agc
cgg gag gtg aac 1296 Ala Trp His Ser Gln Arg Ser Phe Pro Arg Cys
Ser Arg Glu Val Asn 420 425 430 agc aga gag gat aat gta ctt tat aga
gtc gtg aag cct cag gaa cag 1344 Ser Arg Glu Asp Asn Val Leu Tyr
Arg Val Val Lys Pro Gln Glu Gln 435 440 445 atc tgatgatccc
aggaggtgct ggaagaaaat ctagggccga tgctatctgg 1397 Ile actgtctgct
ggtcatttcc agaggaagga atcaatgtcc gagtgcaggg acat 1451 38 449 PRT
human 38 Pro Lys Pro Thr Leu Trp Ala Lys Pro Gly Ser Val Ile Ser
Trp Arg 1 5 10 15 Ser Pro Met Thr Met Trp Cys Gln Gly Thr Leu Glu
Ala Gln Glu Tyr 20 25 30 His Leu Tyr Lys Glu Gly Ser Thr Glu Pro
Trp Asp Arg Thr Asn Pro 35 40 45 Leu Glu Thr Arg Asn Lys Ala Arg
Tyr Ser Ile Pro Ser Met Thr Gln 50 55 60 His His Ala Val Arg Tyr
Gln Cys Tyr Tyr Leu Ser Pro Ala Gly Trp 65 70 75 80 Ser Glu Pro Ser
Asp Pro Leu Glu Leu Val Met Thr Gly Phe Tyr Ser 85 90 95 Lys Pro
Thr Leu Ser Ala Leu Pro Ser Pro Val Val Ala Ser Gly Gly 100 105 110
Lys Val Thr Leu Arg Cys Gly Ser Gln Lys Gly Tyr His His Phe Val 115
120 125 Leu Met Lys Glu Gly Glu His Gln Leu Pro Arg Thr Leu Asp Ser
Gln 130 135 140 Gln Leu His Ser Gly Gly Phe Gln Ala Leu Phe Pro Val
Gly Pro Val 145 150 155 160 Thr Pro Ser His Arg Trp Arg Phe Thr Cys
Tyr Tyr Tyr Tyr Met Asn 165 170 175 Thr Pro Gln Val Trp Ser His Pro
Ser Asp Pro Leu Glu Ile Leu Pro 180 185 190 Ser Gly Gln Ser Ser Pro
Pro Val Leu Ala Pro Gly Glu Thr Leu Thr 195 200 205 Leu Gln Cys Gly
Ser Asp Val Gly Tyr Asp Arg Phe Thr Leu Tyr Lys 210 215 220 Glu Gly
Glu Cys Asp Phe Leu Gln Arg Pro Gly Gln Gln Pro Gln Ala 225 230 235
240 Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Arg Gly Ser His
245 250 255 Gly Gly Gln Tyr Arg Cys Ser Gly Ala His Asn Leu Ser Ser
Glu Trp 260 265 270 Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala
Gly Gln Ile Pro 275 280 285 Gly Arg Pro Ser Leu Ser Val Gln Leu Trp
Pro Thr Val Ala Ser Gly 290 295 300 Glu Asn Val Thr Leu Leu Cys Gln
Ser Gln Glu Trp Met His Thr Phe 305 310 315 320 Leu Leu Thr Lys Glu
Gly Ala Ala His Pro Leu Leu Cys Leu Arg Ser 325 330 335 Lys Tyr Gly
Ala His Lys Tyr Gln Ala Glu Phe Pro Met Ser Pro Val 340 345 350 Thr
Ser Ala His Thr Gly Thr Tyr Arg Cys Tyr Gly Ser Leu Ser Ser 355 360
365 Asp Pro Tyr Leu Leu Ser His Pro Ser Gly Pro Val Glu Leu Val Val
370 375 380 Ser Ala Ser His Leu Gln Asp Tyr Ala Val Glu Asn Leu Ile
His Met 385 390 395 400 Gly Val Ala Gly Leu Ile Leu Val Val Leu Gly
Ile Leu Ser Phe Glu 405 410 415 Ala Trp His Ser Gln Arg Ser Phe Pro
Arg Cys Ser Arg Glu Val Asn 420 425 430 Ser Arg Glu Asp Asn Val Leu
Tyr Arg Val Val Lys Pro Gln Glu Gln 435 440 445 Ile 39 8 PRT human
39 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5
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References