Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NOVEL BAG PROTEINS AND
NUCLEIC ACID MOLECULES ENCODING THEM
S'T'ATEMENT AS TO RIGHTS TQ INVENTIONS MADE
UNDER FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with government support
under grant number CA-67329 awarded by the National
Institutes of Health. The United States Government has
certain rights in this invention.
BACKGROUND OF THE INVENTI0~1
FIELD QF TH,E I1~11 ,NTTON
This invention relates generally to the fields of
molecular biology and molecular medicine and more
specifically to a novel family of proteins that can
regulate protein folding. The functions of these proteins
are potentially diverse, including promoting tumor cell
growth and metastasis.
BACKGROUND INFORMATION
The Hsc70/Hsp70-family of molecular chaperones
participate in protein folding reactions, controlling
protein bioactivity, degradation, complex
assembly/disassembly, and translocation across membranes.
These proteins interact with hydrophobic regions within
target proteins via a carboxyl (C)-terminal peptide binding
domain, with substrate binding and release being controlled
by the N-terminal ATP-binding domain of Hsc70/Hsp70.
Hsc70/Hsp70-assisted folding reactions are accomplished by
repeated cycles of peptide binding, refolding, and release,
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which are coupled to ATP hydrolysis by the ATP-binding
domain (ATPase) of Hsc70/Hsp70 and by subsequent nucleotide
exchange. The chaperone activity of mammalian Hsc70/Hsp70
is regulated by partner proteins that either modulate the
peptide binding cycle or that target the actions of these
chaperones to specific proteins and subcellular
compartments. DnaJ-family proteins (Hdj-1/Hsp40; Hdj-2;
Hdj-3) stimulate the ATPase activity of Hsc70/Hsp70,
resulting in the ADP-bound state which binds tightly to
peptide substrates. The Hip protein collaborates with
Hsc70/Hsp70 and DnaJ homologues in stimulating ATP
hydrolysis, and thus also stabilize Hsc70/Hsp70 complexes
with substrate polypeptides, whereas the Hop protein may
provide co-chaperone functions through interactions with
the C-terminal peptide binding domain.
The Bcl-2 associated athanogene-1 (bag-1) is
named from the Greek word athanos, which refers to
anti-cell death. BAG-1 was previously referred to as
Bcl-2-associated protein-1 (BAP-1) in U.S. Patent No.
5,539,094 issued July 23, 1996, which is incorporated
herein by reference. In this earlier patent, BAG-1 is
described as a portion of the human BAG-1 protein, absent
the N-terminal amino acids 1 to 85. In addition, a human
protein essentially identical to human BAG-1 was described
by Zeiner and Gehring, (Pros. Natl. Acad. Sci., USA
92:11465-11469 (1995)). Subsequent to the issuance of U.S.
Patent 5,539,094 the N-terminal amino acid sequence from 1
to 85 of human BAG-1 was reported.
BAG-1 and its longer isoforms BAG-1M (Rap46) and
BAG-1L are recently described Hsc70/Hsp70-regulating
proteins. BAG-1 competes with Hip for binding to the
Hsc70/Hsp70 ATPase domain and promotes substrate release.
BAG-2 also reportedly stimulates Hsc70-mediated ATP
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hydrolysis by accelerating ADP/ATP exchange, analogous to
the prokaryotic GrpE nucleotide exchange protein of the
bacterial Hsc70 homologue, DnaK. Gene transfection studies
indicate that BAG-1 proteins can influence a wide variety
of cellular phenotypes through their interactions with
Hsc70/Hsp70, including increasing resistance to apoptosis,
promoting cell proliferation, enhancing tumor cell
migration and metastasis, and altering transcriptional
activity of steroid hormone receptors.
Despite the notable progress in the art, there
remains an unmet need for the further identification and
isolation of additional homologous BAG protein species, and
the nucleic acid molecules and/or nucleotide sequences
that encode them. Such species would provide additional
means by which the identity and composition of the BAG
domain, that is, the portion of the protein that is
influencing or modulating protein folding, could be
identified. In addition, such species would be useful for
identifying agents that modulate apoptosis as candidates
for therapeutic agents, in particular, anticancer agents.
The present invention satisfies these need, as well as
providing substantial related advantages.
S~J1~ARY OF THE INVENTION
The present invention provides a family of BAG-1
related proteins from humans [BAG-1L (SEQ ID N0:2), BAG-1
(beginning at residue 116 of SEQ ID N0:2) , BAG-2 (SEQ ID
NO: 4), BAG-3 (SEQ ID N0:6) and (SEQ ID N0:20), BAG-4 (SEQ
ID N0:8) and (SEQ ID N0:22) and BAG-5 (SEQ ID N0:10) and
(SEQ TD N0:24)) , the invertebrate C.elegans [BAG-1 (SEQ ID
N0:12), BAG-2 (SEQ ID N0:19)) and the fission yeast S.pombe
[BAG-lA (SEQ ID N0:16), BAG-1B (SEQ ID N0:18)) and the
nucleic acid molecules that encode them.
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Another aspect of the present invention provides
an amino acid sequence present in the family of BAG-1
related proteins, that modulates Hsc70/Hsp70 chaperone
activity, that is, the BAG domain.
Another aspect of the present invention provides
novel polypeptide and nucleic acid compositions and methods
useful in modulating Hsc70/Hsp70 chaperone activity.
Another aspect of the present invention is
directed to methods for detecting agents that modulate the
binding of the BAG family of proteins, such as BAG-1
(beginning at residue 116 of SEQ ID N0:2), and related
proteins with the Hsc70/Hsp70 Family of proteins or with
other proteins that may interact with the BAG-Family
proteins.
Still another aspect of the present invention is
directed to methods for detecting agents that induce the
dissociation of a bound complex formed by the association
of BAG-Family proteins with Hsc70/Hsp70 Family molecule
chaperones or other proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the full length cDNA sequence for
human BAG-1 (SEQ ID N0:1) protein with the corresponding
amino acid sequence (SEQ ID N0:2). Within the full length
sequence are included the overlapping sub-sequences of
BAG-1 (beginning at nucleotide 391), BAG-1M [beginning at
nucleotide 260 of (SEQ ID N0:2)], and BAG-1L [beginning at
nucleotide 46 of (SEQ ID N0:2)].
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Figures 2A and 2B combined shows the full length
cDNA sequence (SEQ ID N0:3) aligned with the corresponding
amino acid residues for human BAG-2 protein (SEQ ID N0:4).
Figure 3 shows a cDNA sequence (SEQ ID N0:5)
5 aligned with the corresponding amino acid residues for
human BAG-3 protein (SEQ ID N0:6).
Figure 4 shows the a cDNA sequence (SEQ ID N0:7)
aligned with the corresponding amino acid residues for
human BAG-4 protein (SEQ ID N0:8).
Figure 5 shows a cDNA sequence (SEQ ID N0:9)
aligned with the corresponding amino acid residues for
human BAG-5 protein (SEQ ID N0:10).
Figure 6A shows the full length cDNA sequence for
C. elegans BAG-1 protein (SEQ ID N0:11).
Figure 6B shows the 210 amino acid sequence for
C. elegans BAG-1 protein (SEQ ID N0:12).
Figure 7A shows the full length cDNA sequence for
C. elegans BAG-2 protein (SEQ ID N0:13).
Figure 7B shows the 458 amino acid sequence for
C. elegans BAG-2 protein (SEQ ID N0:14).
Figure 8A shows the full length cDNA sequence for
S. pombe BAG-lA protein (SEQ ID N0:15).
Figure 88 shows the 195 amino acid sequence for
S. pombe BAG-1A protein (SEQ ID N0:16).
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Figure 9A shows the full length cDNA sequence for
S. pombe BAG-1B protein (SEQ ID N0:17).
Figure 9B shows the 206 amino acid sequence for
S. pombe BAG-1B protein (SEQ ID N0:18).
Figure 10 shows the topologies of the BAG-family
proteins; human BAG proteins, BAG-1 (SEQ ID N0:2), BAG-2
(SEQ ID N0:4), BAG-3 (SEQ ID N0:6), BAG-4 (SEQ ID N0:8),
BAG-5 (SEQ ID NO:10); S.pombe BAG-iA (SEQ ID N0:16)and
BAG-1B (SEQ ID N0:18); and C. elegans BAG-1 (SEQ ID
N0:12)and BAG-2 (SEQ ID N0:19). (A) The relative
positions of the BAG domains are shown in black, ubiquitin-
like regions are represented in gray, WW domain are
represented in strips. Nucleoplasmin-like nuclear
localization sequence are also shown. (B) The amino acid
sequences of the BAG domain for human BAG-1 (SEQ ID N0:2),
BAG-2 (SEQ ID N0:9), BAG-3 (SEQ ID N0:6), BAG-4 (SEQ ID
N0:8), BAG-5 (SEQ ID NO:10), S.pombe BAG-lA (SEQ ID
N0:16)and BAG-1B (SEQ ID N0:18), and C. elegans BAG-1 (SEQ
ID N0:12)and BAG-2 (SEQ ID N0:14) are aligned demonstrating
their homology. Black and gray shading represent identical
and similar amino acids, respectively.
Figure 11 shows assays demonstrating the
interaction of BAG-family proteins with Hsc70/ATPase. (A)
Two-hybrid assays using yeast expressing the indicated
fusion proteins. Blue color indicates a positive
interaction, resulting in activation of the lacZ reporter
gene. (B) In vitro protein assays using GST-fusion
proteins and 35S-labeled in vitro translated proteins. (C)
Co-immunoprecipitation assays using anti-Flag or IgGl
control antibodies and lysates from 293T cells expressing
Flag-tagged BAG-1 (beginning at residue 116 of SEQ ID
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N0:2), BAG-2 (SEQ ID N0:4), BAG-3 (SEQ ID N0:6), Daxx, or
Apaf-1.
Figure 12 shows surface plasmon resonance
analysis of BAG-family protein interactions with
Hsc70/ATPase. (A) SDS-PAGE analysis of purified
recombinant proteins. (B) Representative SPR results of
biosensor chips containing immobilized BAG proteins with
and without maximally bound Hsc70/ATPase.
Figure 13 shows representative SPR results for
biosensor chips containing immobilized BAG-1 (beginning at
residue 116 at SEQ ID N0:2), BAG-1(nC), BAG-2 (SEQ ID N0:4),
or BAG-3 (SEQ ID N0:6) proteins. Hsc70/ATPase was flowed
over the chips (arrow/left) until maximal binding was
reached (response units), then flow was continued without
Hsc70/ATPase (arrow/right). For BAG-2 (SEQ ID N0:4) and
BAG-3 (SEQ ID N0:6), Hsc70 was injected at 0.0175, 0.035,
0.07, 0.19, and 0.28 uM.
Figure 14 shows BAG-family protein modulation of
Hsc70 chaperone activity. (A) Protein refolding assay of
chemically-denatured luciferase by Hsc70 plus DnaJ in the
absence or presence of BAG and BAG-mutant proteins. (B)
Concentration-dependent inhibition of Hsc70-mediated
protein refolding by BAG-family proteins [BAG-1 (beginning
at residue 116 of SEQ ID N0:2), BAG-2 (SEQ ID N0:4), BAG-3
(SEQ ID N0:6)J but not by BAG-mutant (BAG-1 (~C). (C)
Hsc70/Hsp90-mediated refolding of heat-denatured luciferase
was assayed in the presence of (black bars) or absence of
(striped bars) of 1.8 uM Hip, with (lanes 3-10) or without
(lanes 1,2) various BAG-family proteins (l.BUM) as
indicated (mean ~SE; n=3). A control (CNTL) is shown (lane
1) in which Hsc70 was replaced with an equivalent amount of
BSA.
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Figure 15A shows an expanded cDNA sequence for
human BAG-3 protein (SEQ ID N0:19).
Figure 15B shows the corresponding amino acid
residues for the human BAG-3 protein (SEQ ID N0:20) of
Figure 15A.
Figure 15C shows the expanded cDNA sequence (SEQ
ID N0:19) aligned with the corresponding amino acid
residues for human BAG-3 protein of Figure 15A (SEQ ID
N0:20) .
Figure 16A shows an expanded cDNA sequence for
human BAG-4 protein (SEQ ID N0:21).
Figure 16B shows the corresponding amino acid
residues for the human BAG-4 protein of Figure 16A (SEQ ID
N0:22).
Figure 16C shows the expanded cDNA sequence (SEQ
ID N0:21) aligned with the corresponding amino acid
residues for human BAG-4 protein of Figure 16A (SEQ ID
N0:22).
Figure 17A shows an expanded cDNA sequence for
human BAG-5 protein (SEQ ID N0:23).
Figure 17B shows the corresponding amino acid
residues for the human BAG-5 protein of Figure 17A (SEQ ID
N0:29j ,
Figure 17C shows the expanded cDNA sequence (SEQ
ID N0:23) aligned with the corresponding amino acid
residues for human BAG-5 protein of Figure 17A (SEQ ID
N0:29).
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Figure 18 shows the topologies of the BAG-family
proteins; human BAG proteins, BAG-1 (SEQ ID N0:2), BAG-2
(SEQ ID N0:4), expanded BAG-3 (SEQ ID N0:20), expanded
BAG-4 (SEQ ID N0:22), expanded BAG-5 (SEQ ID N0:24);
S.pombe BAG-lA (SEQ ID N0:16)and BAG-1B (SEQ ID NO:1$); and
C. elegans BAG-1 (SEQ ID N0:12)and BAG-2 (SEQ ID N0:14).
The relative positions of the BAG domains are shown in
black, ubiquitin-like regions are represented in gray, WW
domain are represented in strips. Nucleoplasmin-like
nuclear localization sequence are also shown.
Definitions
The term "apoptosis", as used herein, refers to
the process of programmed cell death, although not all
programmed cell deaths occur through apoptosis, as used
herein, "apoptosis" and "programmed cell death" are used
interchangeably.
The term "tumor cell proliferation", as used
herein refers to the ability of tumor cells to grow and
thus expand a tumor mass.
The term "cell migration", as used herein refers
to the role cell motility plays in the invasion and
potentially metastasis by tumor cells.
The term "metastasis", as used herein refers to
the spread of a disease process from one part of the body
to another, as in the appearance of neoplasms in parts of
the body remote from the site of the primary tumor; results
in dissemination of tumor cells by the lymphatics or blood
vessels or by direct extension through serious cavitites or
subarachnoid or other spaces.
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The term "steroid hormone receptor function", as
used herein refers to physiological, cellular and molecular
functioning of receptors sites that bind with steroid
hormones.
5 The term "substantially purified", as used
herein, refers to nucleic acid. or amino acid sequence that
are removed from their natural environment, isolated or
separated, and are at least 60~ free, preferably 75o free,
and most preferably 90a free from other components with
10 which they are naturally associated.
"Nucleic acid molecule" as used herein refers to
an oligonucleotide, nucleotide, or polynucleotide, and
fragments or portions thereof, and to DNA or RNA of genomic
or synthetic origin which may be single or double stranded,
and represent the sense or antisense strand.
"Hybridization", as used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
The terms "complementary" or "complementarity",
as used herein, refer to the natural binding of
polynucleotides under permissive salt and temperature
conditions by base-pairing. For example, the sequence
"A-G-T binds to the complementary sequence "T-C-A".
The term "homology", as used herein, refers to a
degree of complementarity. There may be partial homology
or complete homology (i.e., identity). A partially
complementary sequence is one that at least partially
inhibits an identical sequence from hybridizing to a target
nucleic acid and is referred to using the functional term
"substantially homologous." The inhibition of
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hybridization of the completely complementary sequence to
the target sequence may be examined using a hybridzation
assay (Southern or northern blot, solution hybridization
and the like) under conditions of low stringency. A
substantially homologous sequence or probe will compete for
and inhibit the binding (i.e., the hybridization) of a
completely homologous sequence or probe to the target
sequence under conditions of low stringency.
The term "antisense", as used herein, refers to
nucleotide sequences which are commplementary to a specific
DNA or RNA sequence. The term "antisense strand" is used in
reference to a nucleic acid strand that is complementary to
the "sense" strand. Antisense molecules may be produced by
any method, including synthesis by ligating the genes) of
interest in a reverse orientation to a viral promoter which
permits the synthesis of a complementary strand. Once
introduced into a cell, this transcribed strand combines
with natural sequences produced by the cell to form
duplexes. These duplexes then block either the further
transcription or translation. In this manner, mutant
phenotypes may be generated. The designation "negative" is
sometimes used in reference to the antisense, and
"positive" is sometimes used in reference to the sense
strand.
"Amino acid sequence" as used herein refers to an
oligopeptide, peptide, polypeptide, or protein sequence,
and fragments or portions thereof, and to naturally
occurring or synthetic molecules. Where "amino acid
sequence" is recited herein this term excludes an amino
acid sequence of a naturally occurring protein. "Amino
acid sequence", "polypeptide" or "protein" are not meant to
limit the amino acid sequence to the complete, native amino
acid sequence associated with the recited protein molecule.
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The term "functional fragments" or "fragments",
as used herein, with regard to a protein refers to portions
of that protein that are capable of exhibiting or carrying
out the activity exhibited by the protein as a whole. The
portions may range in size from three amino acid residues
to the entire amino acid sequence minus one amino acid.
For example, a protein "comprising at least a functional
fragment of the amino acid sequence of SEQ ID NO:1",
encompasses the full-length of the protein of SEQ ID N0:1
and portions thereof.
A "derivative" of a BAG protein, as used herein,
refers to an amino acid sequence that is alterd by one or
more amino acids. The derivative may have "conservative"
changes, wherein a substituted amino acid has similar
structural or chemical properties, e.g., substitution of an
apolar amino acid with another apolar amino acid (such as
replacement of leucine with isoleucine). The derivative
may also have "nonconservative" changes, wherein a
substituted amino acid has different but sufficiently
similar structural or chemical properties that permits such
a substitution without adversely effecting the desired
biological activity, e.g., replacement of an amino acid
with an uncharged polar R group with an amino acid with an
apolar R group (such as replacement of glycine with
tryptophan), or alternatively replacement of an amino acid
with a charged R group with an amino acid with an uncharged
Polar R group (such as replacement of lysine with
asparagine).
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Amino Acids - Apolar R Groups
Amino Acid Radical Abbreviations
3-Letter 1-Letter
alanine methyl ala A
valine 2-propyl aal V
leucine 2-methylpropyl leu L
isoleucine 2-butyl ile I
proline propyl* - cyclized pro P
phenylalanine benzyl phe F
trytophan 3-indolylmethl tyr W
methionine ' methylthioethyl I met I M
!
Amino Acids - Uncharged Polar R Groups
Amino Acid Radical 1' Abbreviations
3-Letter 1-Letter
glycine H gly G
serine hydroxymethyl ser S
threonine 1-hydroxyethyl thr T
cysteine thiolmethyl cys C
tyrosine 4-hydroxyphenylmethyl tyr Y
asparagine aminocarbonylmethyl asn N
glutamine aminocarbonylethyl gln Q
Amino Acids - Charged R Groups
Amino Acid Radical ,Abbreviations
3-Letter 1-Letter
aspartic acid carboxymethyl asp D
glutamic acid carboxyethyl glu E
lysine 4-aminobutyl lys K
arginine 3-guanylpropyl arg R
histidine 4-imidazoylmethyl his H
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Similar minor modifications may also include amino acids
deletions or insertions or both. Guidance in determining
which amino acid residues may be modified as indicated
above without abolishing the desired biological
functionality may be determined using computer programs
well known in the art, for example, DNASTAR software. In
addition, the derivative may also result from chemical
modifications to the encoded polypeptide, including but not
limited to the following, replacement of hydrogen by an
alkyl, acyl, or amino group; esterification of a carboxyl
group with a suitable alkyl or aryl moiety; alkylation of
a hydroxyl group to form an ether derivative. Further a
derivative may also result from the substitution of a L
configuration amino acid with its corresponding D
configuration counterpart.
The term "mimetic", as used herein; refers to a
molecule, the structure of which is developed from
knowledge of the structure of a protein/polypeptide or
portions thereof (such as BAG-1) and, as such, is able to
effect some or all of the actions of BAG-1 protein.
"Peptide nucleic acid", as used herein, refers to
a molecule which comprises an oligomer to which an amino
acid residue, such as lysine, and an amino group have been
added. These small molecules, also designated anti-gene
agents, stop transcript elongation by binding to their
complementary strand of nucleic acid (Nielsen, P.E. et al.,
Anticancer Drug Des. 8:53-63 (1993)).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a family of BAG-1
related proteins from humans [BAG-1L (SEQ ID N0:2), BAG-1S
beginning at residue 116 of SEQ ID N0:2, BAG-2 (SEQ ID
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N0:4), BAG-3 (SEQ ID N0:6) and (SEQ ID N0:20), BAG-4 (SEQ
ID N0: 8) and (SEQ ID N0:22) and BAG-5 (SEQ ID N0:10) and
(SEQ ID N0:24)], the invertebrate C.elegans [BAG-1 {SEQ ID
N0:12), BAG-2 (SEQ ID N0:14)] and the fission yeast S.pombe
5 [BAG-lA (SEQ ID N0:16), BAG-1B (SEQ ID N0:18)],
specifically the full length amino acid sequences
comprising human BAG-1L (SEQ ID N0:2), BAG-1 (beginning at
residue 116 of SEQ ID N0:2), and BAG-2 (SEQ ID N0:4) C.
elegans BAG-1 (SEQ ID N0:12), and BAG-2 (SEQ ID N0:14), and
10 S.pombe BAG-lA (SEQ ID N0:16) and BAG-1B (SEQ ID N0:18);
and partial sequences comprising human BAG-3 (SEQ ID NO: 6)
and (SEQ ID N0:20), BAG-4 (SEQ ID N0:8) and (SEQ ID N0:22),
and BAG-5 (SEQ ID NO:10) and (SEQ ID N0:24) and functional
fragments thereof. In particular, the invention provides
15 the amino acid sequences comprising human BAG-2 (SEQ ID
N0:9), BAG-3 (SEQ ID N0:6) and {SEQ ID N0:20), BAG-4 (SEQ
ID N0:8) and (SEQ ID N0:22), and BAG-5 (SEQ ID NO:10) and
(SEQ ID N0:24) proteins.
Another aspect of the present invention provides
the nucleic molecule and nucleotide sequences that encode
the family of BAG-1 related proteins from humans [BAG-1
(SEQ ID N0:1), BAG-2 (SEQ ID N0:3), BAG-3 (SEQ ID N0:5) and
(SEQ ID N0:19), BAG-4 (SEQ ID N0:7) and (SEQ ID N0:21) and
BAG-5 (SEQ ID N0:9) and (SEQ ID N0:23)], the invertebrate
.C.elegans [BAG-1 (SEQ ID NO:11), BAG-2(SEQ ID N0:13)] and
the fission yeast S.pombe [BAG-lA (SEQ ID N0:15), BAG-1B
(SEQ ID N0:17)].
BAG-1L (SEQ ID N0:2) is a multifunctional protein
that blocks apoptosis, promotes tumor cell metastasis, and
contributes to factor-independent and p53-resistant cell
growth. BAG-1L (SEQ ID N0:2) interacts with several types
of proteins, including Bcl-2, some tyrosine kinase growth
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factor receptors, steroid hormone receptors, and the p53-
induced cell cycle regulator Siah-1A.
BAG-1 is a regulator of Hsc70/Hsp70 family
molecular chaperones. A carboxyl-terminal domain in this
protein binds tightly to the ATPase domains of Hsc70 and
Hsp70 (Kp = 1 nM) (Zeiner; M., Gebauer, M., and Gehring, U.,
EMBO J. 16: 5483-5490, (1997)). BAG-1 modulates the
activity of these molecular chaperones, acting as an
apparent functional antagonist of the Hsp70/Hsc70-
associated protein Hip (3-5)(Hohfeld, J. and Jentsch, S.,
EMBO J. 16: 6209-6216, (1997); Takayama, S., Bimston, D.
N., Matsuzawa, S., Freeman, B. C., Aime-Sempe, C., Xie, Z.,
Morimoto, R. J., and Reed, J. C., EMBO J. 16: 4887-96,
( 1997 ) ; Zeiner, M. , Gebauer, M. , and Gehring, U. , EMBO J.
16: 5483-5490, (1997)). In general, protein refolding is
accomplished by Hsp70/Hsc70 through repeated cycles of
target peptide binding and release, coupled to ATP
hydrolysis (Ellis, R., Curr Biol. 7: 8531-8533, (1997)).
BAG-1 appears to promote substrate release, whereas Hip
stabilizes Hsp70/Hsc70 complex formation with target
peptides (Hohfeld, J., Minami, Y., and Hartl, F.-U., Cell.
83:. 589-598, (1995)). Since each substrate interaction
with Hsc70/Hsp70 is unique in terms of the optimal length
of time the protein target should remain complexed with
Hsc70/Hsp70 for achieving new conformations, the net effect
of BAG-1 can be either enhancement or inhibition of the
refolding reaction.
The 70kd heat shock family proteins (Hsp70/Hsc70)
are essential to a variety of cellular processes and have
been implicated in cancer, yet it is unclear how these
proteins are regulated in vivo. A variety of co-chaperones
have been identified which may target Hsp70/Hsc70 to
different subcellular compartments or promote their
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interactions with specific protein or protein complexes.
BAG-1 appears to represent a novel Hsp70/Hsc70 regulator
which differs functionally from all other mammalian co-
chaperones identified to date, such as members of the
DnaJ-, Hip-, Hop-, and cyclophilin-families of proteins.
Another aspect of the present invention provides
the amino acid sequence of a binding domain of about 90 to
55 amino acids that bind the a Hsc70/Hsp70 ATPase domain.
The BAG domain is situated near the C-terminus, and the
l0 ubiquitin-like domains are situated near the N-terminus.
The BAG family of proteins of the present
invention contain a common conserved C-terminal domain (the
"BAG" domain) that facilitates binding to the ATPase domain
of Hsp70/Hsc70. The carboxyl-terminal domain of BAG-1
binds to the ATPase domain of Hsc70/Hsp70 and regulates its
chaperone function by acting as a ADP-ATP exchange factor.
Other domains of BAG-1 mediate interactions with proteins
such as Bcl-2 and retinoic acid receptors (RARs), allowing
BAG-1 to target Hsc70/Hsp70 to other proteins, presumably
modulating their function by changing their conformations.
Human BAG-1 was previously shown to inhibit
Hsc7o/Hsp70 dependent refolding of denatured protein
substrates in vitro (S. Takayama, et al., EMBO J 16, 4887-
96 (1997); M. Zeiner, M. Gebauer, U. Gehring, EMBD J. 16,
5483-5490 (1997); and J. Hohfeld, S. Jentsch, EMBO J. 16,
6209-6216 (1997)). In Example III, Part A the effects of
recombinant human BAG-1, BAG-2 (SEQ ID N0:4) and BAG-3 (SEQ
ID N0:6) were compared using in vitro protein refolding
assays similar to those employed previously for assessing
BAG-1. The study showed that addition of equimolar amounts
of each of the recombinant proteins to Hsc70 resulted in
significant inhibition of luciferase refolding, with BAG-2
(SEQ ID N0:4) and BAG-3 (SEQ ID N0:6) showing somewhat
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greater inhibitor activity than BAG-1 (Figure 4A). In a
separate luciferase folding study BAG-1, BAG-2 (SEQ ID
N0:4) and BAG-3 (SEQ ID N0:6) once again displayed
inhibition of luciferase refolding, however in this study
varying amounts of BAG-1, BAG-2 (SEQ ID N0:4) and BAG-3
(SEQ ID N0:6) were added relative to Hsc70 which resulting
in concentration-dependent inhibition of Hsc70 chaperone
activity, i.e., luciferase folding (Example III Part A).
Additional follow on studies using the same experimental
protocols as the previous studies, as taught in Example
IIA, have shown that BAG-4 (SEQ ID N0:22) also undergoes
association with Hsc70/ATPase.
Yet another aspect of the present invention
provides a nucleotide sequence having at least about 15
nucleotides and, generally, about 25 nucleotides,
preferably about 35 nucleotides, more preferably about 45
nucleotides, and most preferably about 55 nucleotides that
can hybridize or is complementary under relatively
stringent conditions to a portion of the nucleic acid
sequences shown in Figures 1-9 and Figures 15-17, in
particular the BAG domain as shown in in Figure 1B, e.g.,
nucleotides 552-593 of human BAG-3, or nucleotides 167-221
of human BAG-4.
Yet another aspect of the present invention
provides a compound of the formula,
RN-R1X1R2X2R3X3R4X9R5xsR6X6R~x~XsR9X9R1oX1oRmxy Rc
wherein,
RN is a group of 1 to 552 independently selected
amino acids;
R1 is a group of 3 independently selected amino
acids;
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X1 is an amino acid with a charged or uncharged
R group, such as aspartic acid, glutamic acid, asparagine,
or glutamine;
RZ is a group of 7 independently selected amino
acids;
X2 is an amino acid with a charged R group, such
as glutamic acid;
R3 is a group of 5 independently selected amino
acids;
X3 is an amino acid with an apolar R group, such
as leucine, methionine, or isoleucine;
R4 is a group of 3 independently selected amino
acids;
X4 is an amino acid with charged R group, such as
aspartic acid or glutamine acid;
R5 is a single independently selected amino acid;
XS is an amino acid with apolar or uncharged R
group, such as leucine, valine, methionine, alanine or
threonine;
R5 is a group of 15 independently selected amino
acids;
X6 is an amino acid with a charged or uncharged
R group, such as arginine, lysine, glutamine or aspartic
acid;
R' is a group of 2 independently selected amino
acids;
X' is an amino acid with a charged R group, such
as arginine;
Xe is an amino acid with a charged R group, such
as arginine or lysine;
R9 is a group of 2 independently selected amino
acids;
X9 is an amino acid with an apolar R group, such
as valine;
R1° is a group of 3 independently selected amino
acids;
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X1° is an amino acid with an uncharged R group,
such as glutamine;
R11 is a group of 2 independently selected amino
acids;
5 X11 is an amino acid with an apolar R group, such
as leucine; and
R~ is a group of 1 to 100 independently selected
amino acids.
A nucleotide sequence of at least about 15
10 nucleotides and, generally, about 25 nucleotides,
preferably about 35 nucleotides, more preferably about 45
nucleotides, and most preferably about 55 nucleotides can
be useful, for example, as a primer for the polymerise
chain reaction (PCR) or other similar reaction mediated by
15 a polymerise such as a DNA or RNA polymerise (see PCR
Protocols: A guide to methods and applications, ed. Innis
et al. (Academic Press, Inc., 1990), which is incorporated
herein by reference; see, for example, pages 40-41). In
addition, such a nucleotide sequence of the invention can
20 be useful as a probe in a hybridization reaction such as
Southern or northern blot analysis or in a binding assay
such as a gel shift assay.
A nucleotide sequence of the invention can be
particularly useful as an antisense molecule, which can be
DNA or RNA and can be targeted to all or a portion of the
5'-untranslated region or of the 5'-translated region of a
bag-1 nucleic acid sequence in a cell. For example, an
antisense molecule can be directed to at least a portion of
the sequence shown as the BAG domain in Figure lA, e.g.,
nucleotides 272-319 of human BAG-1L (SEQ ID NO:1), or
nucleotides 79-147 of human BAG-5 (SEQ ID N0:9). Since the
5'-region of a nucleic acid contains elements involved in
the control of expression of an encoded protein, an
antisense molecule directed to the 5'-region of a nucleic
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acid molecule can affect the levels of protein expressed in
a cell.
A nucleotide sequence of the invention also can
be useful as a probe to identify a genetic defect due a
mutation of a gene encoding a BAG protein in a cell. Such
a genetic defect can lead to aberrant expression of a BAG
protein in the cell or to expression of an aberrant BAG
protein, which does not properly associate with a Bcl-2-
related protein or Hsc70/Hsp70 protein in the cell. As a
result, a genetic defect in a gene encoding, for example,
human BAG-1 can result in a pathology characterized by
increased or decreased levels in protein folding.
Further a nucleotide compound or composition as
taught in the present invention can be synthesized using
routine methods or can be purchased from a commercial
source. In addition, a population of such nucleotide
sequences can be prepared by restriction endonuclease or
mild DNAse digestion of a nucleic acid molecule that
contains nucleotides as shown in the nucleotide sequences
shown in Figures 1-9 and Figures 15-17 that encodes the
amino acids sequences also shown in Figures 1-9 and
Figures 15-17. Methods for preparing and using such
nucleotide sequences, for example, as hybridization probes
to screen a library for homologous nucleic acid molecules
are well known in the art (see, for example, Sambrook et
al., Molecular Cloning: A laboratory manual (Cold Spring
Harbor Laboratory Press 1989); Ausubel et al., Current
Protocols in Molecular Biology (Green Publ., NY 1989),
each of which is incorporated herein by reference).
A particular nucleotide sequence can be designed
based, for example, on a comparison of the nucleic acid
molecules encoding any one of the BAG family proteins, as
shown in Figures 1-9 and Figures 15-17, with another in the
family. Such a comparison allows, for example, the
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preparation of a nucleotide sequence that will hybridize to
a conserved region present in both nucleic acid molecules,
thus providing a means to identify homologous nucleic acid
molecules present in other cell types or other organisms.
In addition, such a comparison allows the preparation of a
nucleotide sequence that will hybridize to a unique region
of any of the BAG family nucleotide sequences, such as
those corresponding to the BAG domain, thus allowing
identification of other proteins sharing this motif. In
this regard, it is recognized that, while the human BAG-3
proteins shown as Figures 3 and 20, and human BAG-5
proteins shown as Figures 5 and 24, are only partial
sequences, a variant human BAG-3 or BAG-5 produced, for
example, by alternative splicing can exist and can be
identified using an appropriately designed nucleotide
sequence of the invention as a probe. Such useful probes
readily can be identified by inspection of the sequences
shown in the disclosed Figures by a comparison of the
encoding nucleotide sequences.
If desired, a nucleotide sequence of the
invention can incorporate a detectable moiety such as a
radiolabel, a fluorochrome, a ferromagnetic substance, a
luminescent tag or a detectable binding agent such as
biotin. These and other detectable moieties and methods of
incorporating such moieties into a nucleotide sequence are
well known in the art and are commercially available . A
population of labelled nucleotide sequences can be
prepared, for example, by nick translation of a nucleic
acid molecule of the invention (Sambrook et al., supra,
1989; Ausubel et al., supra, 1989).
One skilled in the art would know that a method
involving hybridization of a nucleotide sequence of the
invention can require that hybridization be performed under
relatively stringent conditions such that nonspecific
background hybridization is minimized. Such hybridization
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conditions can be determined empirically or can be
estimated based, for example, on the relative GC content of
a sequence and the number of mismatches, if known, between
the probe and the target sequence (see, for example,
Sambrook et al., Supra, 1989).
The invention further provides antibodies
specific for human BAG family protein. As used herein, the
term "antibody" includes polyclonal and monoclonal
antibodies, as well as polypeptide fragments of antibodies
that retain a specific binding activity for human BAG-1 of
at least about 1 x 105 M-' . One skilled in the art would
know that anti-BAG-1 antibody fragments such as Fab, F(ab')
and Fv fragments can retain specific binding activity for
human BAG-1 (beginning at residue 116 of SEQ ID N0:2) and,
thus, are included within the definition of an antibody.
In addition, the term "antibody" as used herein includes
naturally occurring antibodies as well as non-naturally
occurring antibodies and fragments that retain binding
activity such as chimeric antibodies or humanized
antibodies. Such non-naturally occurring antibodies can be
constructed using solid phase peptide synthesis, can be
produced recombinantly or can be obtained, for example, by
screening combinatorial libraries consisting of variable
heavy chains and variable light chains as described by Huse
et al., Science 246:1275-1281 (1989), which is incorporated
herein by reference.
One skilled in the art would know that purified
BAG family protein, which can be prepared from natural
sources or synthesized chemically or produced
recombinantly, or portions of a BAG family protein,
including a portion of human BAG family protein such as a
synthetic peptide as described above, can be used as an
immunogen. Such peptides useful for raising an antibody
include, for example, peptide portions of the N-terminal 85
amino acids or the BAG domain cf any of the human BAG
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proteins (see Figure 1B). A particularly advantageous use
of such a protein is for the immunostaining, wherein the
methods provides a process to contrast the immunostaining
of BAG-family proteins in carcinoma cells with adjacent
non-neoplastic prostatic epithelial and basal cells which
are generally present in the same tissue sections. These
results would be correlated with a Gleason grade to
determine whether any of the BAG-family proteins tend to be
expressed at higher or lower levels in histologically
advanced tumors. From this process a determination can be
made as to degree at which the disease is progressing in a
given patient, i.e., a prognosis can be made.
Non-immunogenic fragments or synthetic peptides
of BAG proteins can be made immunogenic by coupling the
hapten to a carrier molecule such bovine serum albumin
(BSA) or keyhole limpet hemocyanin (KLH), as described in
Example IV, below. In addition, various other carrier
molecules and methods for -coupling a hapten to a carrier
molecule axe well known in the art and described, for
example, by Harlow and Lane, Antibodies: A laboratory
manual (Cold Spring Harbor Laboratory Press, 1988), which
is incorporated herein by reference.
EXAMPLES
The following examples are given to enable those
skilled in the art to more clearly understand and to
practice the present invention. They should not be
considered as limiting the scope of the invention, but
merely as being illustrative and representative thereof.
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EXAMPhE I
Isolat i nn anrl C''h~rnnl-cr; .sir; ~n
9f BAG famil~r cDNA Seauences
This example describes methods for isolating and
5 characterizing of BAG-family cDNA sequences from human,
nematode and yeast.
Yeast two-hybrid library screening of a human
Jurkat cell cDNA library was performed as described by
10 Takayama et al., EMBO J.. 16:4887-96 (1997); Matsuzawa et
al., EMBO J.. 17:2736-2747 (1998), which are incorporated
herein by reference) using EGY48 strain yeast transformed
with pGilda-Hsc70/ATPase (67-377 amino acids) and the lacZ
reporter plasmid pSHlB-34. Of the resulting ~5 x 10~
15 transformants, 112 Leu" colonies were obtained after
1 week incubation at 30°C. Assay of ~i-galactosidase ((3-gal)
activity of these colonies resulted in 96 clones. Mating
tests were then performed using RFY206 yeast strain
transformed with pGilda, pGilda mBAG-1 (1-219), or pGilda
20 Hsc70/ATPase. Of these, 66 displayed specific interactions
with Hsc70/ATPase. The pJG4-5 cDNAs were recovered using
KC8 E. coli strain which is auxotrophic for tryptophan
(Trp). DNA sequencing revealed 3 partially overlapping
human BAG-1, 4 identical and one overlapping cDNAs encoding
25 BAG-2, and 2 partially overlapping BAG-3 clones.
Using the above described yeast two-hybrid screen
with the ATPase domain of Hsc70 as ~~bait~~, several human
cDNAs were cloned which encode portions of BAG-1 or of two
other BAG-1-like proteins which are termed BAG-2 (SEQ ID
N0:4) and BAG-3 (SEQ ID N0:6) . The longest of the cDNAs
for BAG-2 (SEQ ID N0:3) and BAG-3 (SEQ ID N0:5) contained
open reading frames (ORFs) of 207 and 162 amino acids,
respectively, followed by stop codons. All BAG-1 (SEQ ID
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NO:1), BAG-2 (SEQ ID N0:3) and BAG-3 (SEQ ID N0:5) cDNAs
obtained by two-hybrid library screening with Hsc70/ATPase
contained a conserved. domain of about 40-50 amino acids
which are termed the ~~BAG" domain and are shown in Figure
10. These results demonstrate that a family of BAG-1-
related proteins all contain a conserved ~45 amino acid
region near their C-terminus that binds Hsc70/Hsp70.
'f'
A search of the translated Genbank database using
the bBLAST and FASTA search programs also identified human
ESTs that provided sequences for further investigation of
BAG-family proteins. The putative BAG-4 (SEQ ID N0:8) and
BAG-5 (SEQ ID NO:10) proteins contain BAG-domains that
share the greatest sequence similarity with the BAG-domain
of BAG-3 (SEQ ID N0:6). These were designated BAG-4
(Accession number AA693697, N74588) and BAG-5 (Accession
number AA456862, N34101). BAG-4 has 62% identity and ~81%
similarity to BAG-3, and BAG-5 has 51% identity and ~75%
similarity to BAG-3.
Additional BAG-family orthologues or homologues
were also identified using computer-based searches and
resulted in BAG-family homologue in the nematode C. elegans
and the fission yeast S. pombe. The C. elegans genome
encodes two apparent BAG-family proteins, which are most
similar in their overall sequences to the human BAG-1
(Afo39713, gi:2773211) (SEQ ID N0:12) and BAG-2 (SEQ ID
N0:14) (~Afo68719, gi;3168927). The S. pombe contains two
BAG-family proteins that share the greatest overall
sequence similarity with human BAG-1 (Alo23S54,gi/3133105
and A1o23634, gi/3150250). The human and C. e2egans BAG-1
proteins as well as S. pombe BAG-lA all have ubiquitin-like
domains near their N-termini (see Figure l0A) of unknown
function.
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The overall predicted amino acid sequences of the
C. elegans BAG-1 (SEQ ID N0:12) and S. pombe BAG-lA (SEQ ID
N0:16) proteins are "'18o identical (~61o similar) and ~17%
identical (~64% similar), respectively, to human BAG-1,
implying origin from a common ancestral gene. The C.
elegans BAG-1 protein (SEQ ID N0:12), however, contains a
5 to 7 amino acid insert in its BAG-domain as compared to
the human, murine, and yeast BAG-1 homologues (see Figure
lOB), and is more similar to BAG-2 (SEQ ID N0:4) in regard
to its BAG-domain. C. elegans and human BAG-2 also may be
derived from a common ancestor as the C-terminal 225 amino
acid region which encompasses both the BAG domain and
upstream region of both C. elegans and human BAG-2 share
"'34% amino acid sequence identity and "'70% similarity: The
human BAG-2 protein (SEQ ID N0:4), however, contains a 9
amino acid insert in its BAG-domain compared to it
C.elegans counterpart (see Figure lOB). Evolutionary-tree
prediction algorithms suggest that human and C. elegans
BAG-2 represent a distinct branch of the BAG-family that is
more evolutionarily distant from the other BAG-family
proteins. None of the predicted BAG-family proteins
contain recognizable regions analogous to those found in
other Hsc70 regulatory proteins, such as the J-domains and
G/F-domains of DnaJ family proteins and the
Tetratricopeptide Repeat (TR) domains of Hip/Hop family
proteins.
The longest of the cDNAs obtained for the BAG-2
and BAG-3 proteins were expressed with N-terminal
transactivation (TA) domains in yeast and tested by yeast
two-hybrid assay for interactions with fusion proteins
consisting of Hsp70/ATPase or a variety of unrelated
proteins (Fas, Siah, Fadd) containing N-terminal LexA DNA-
binding domains. TA-BAG-2 and TA-BAG-3 demonstrated
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positive interactions with LexA-Hsc70/ATPase, resulting in
transactivation of a lacZ reporter gene that was under the
control of LexA operators (Figure 11A). No interactions
with LexA-Fas (cytosolic domain), LexA-Siah, LexA-Fadd, or
LexA were detected (see Figure 11A) demonstrating that the
BAG-2 and BAG-3 proteins interact specifically with
Hsc70/ATPase. Specific two-hybrid interactions between
Hsc70/ATPase and either BAG-2 or BAG-3 were also observed
when BAG-2 and BAG-3 were expressed as LexA DNA-binding
domain fusion proteins and Hsc70/ATPase was fused with a TA
domain (see Figure 11A; right panel). These results
demonstrate that similarly to BAG-1, BAG-2 and BAG-3
specifically interact with Hsc70/ATPase.
In order to determine whether the BAG proteins
are capable of forming heterodimers, coexpression of BAG-2
and BAG-3 in the yeast two-hybrid assay was also performed.
Coexpression of BAG-2 and BAG-3 failed to show interaction
with BAG-1 or a deletion mutant of BAG-1 (OC) which is
missing part of its C-terminal domain required for
Hsp70/Hsc70 binding suggest that these proteins do not form
heterdimers.
In order to deduce the complete ORFs of BAG-2 and
BAG-3, a A-phage cDNA library was screened as follows,
using hybridization probes derived from the two-hybrid
screening. A human jurkat T-cell A-ZapII library cDNA
library (Stratagene) was screened by hybridization using
3'P-labeled purified insert DNA from the longest of the
human BAG-2 (clone #11) and human BAG-3 (clone #28) cDNA
clones. From about one million clones screened, 38 BAG-2
and 23 BAG-3 clones were identified, cloned, and their cDNA
inserts recovered as pSKII plasmids using a helper phage
method (Stratagene). DNA sequencing of ?~-phage derived
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human BAG-2 cDNA clones revealed an ORF encoding a
predicted 211 amino acid protein, preceded by an in-frame
stop codon. The longest human BAG-3 1~-phage cDNA clone
contains a continuous ORF of 682 amino acids followed by a
stop codon, but without an identifiable start codon (see
Figure l0A) .
Although BAG-1L (SEQ ID N0:2), BAG-1 (beginning
at residue 116 of SEQ ID N0:2), BAG-2 (SEQ ID N0:4), and
BAG-3 (SEQ ID N0:6) all contain a homologous BAG domain
near their C-terminus, the N-terminal regions of these
proteins are dissimilar. Using a combination of search
tools (Prosite Search: PP search, using the Prosite pattern
database, BCM Search Launcher, Baylor College of Medicine,
and Blocks Search), it was determined that the BAG-2 N-
terminal region contains potential kinase phosphorylation
sites but otherwise shares no apparent similarity with
other proteins or known functional domains.
In contrast, the predicted N-terminal region
BAG-3 contains a WW domain as shown in Figure 10A. WW
domains have been identified in a wide variety of signaling
proteins, including a Yes kinase adaptor protein (YAP), the
Na'-channel regulator Nedd4, formin-binding proteins,
dystrophin, and the peptidyl prolyl cis-trans-isomerase
Pin-1. These roughly 40 amino acid domains mediate protein
interactions and bind the preferred peptide ligand sequence
xPPxY (Sudol., TIBS, 21: 161-163, 1996, which is
incorporated herein by reference).
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Tn vitro Association of
BA~C_proteins and Hsc70 /A'rpas,~
This example demonstrates that BAG-2 (SEQ IE
5 N0:4) and BAG-3 (SEQ ID N0:6) bind Hsc70/ATPase in various
in vitro assays.
A. Solution binding assax of BgG-2 and 1 A~G-3- to
Hsc70~/ATPase
Association of BAG-2 (SEQ ID N0:4) and BAG-3 (SEQ
10 ID N0:6) with Hsc70/ATPase was determine by an in vitro
protein binding assay where Hsc70/ATPase or BAG-family
proteins were expressed in bacteria as Glutathione S-
Transferase (GST) fusion proteins. Purified cDNA sequences
encoding residues 5 to 211 of human BAG-2 (clone #11) and
15 the C-terminal 135 amino acids of human BAG-3 (clone #28)
(see Figure l0A) were subcloned into the EcoRI/Xho I sites
of pGEX4T-1 prokaryotic expression plasmid (Pharmacia;
Piscataway, NJ). These plasmids as well as pGEX4T-1-BAG-1,
pGEX-4T-1-BAG-1 (DC), and pGEX-4T-1-XL which have been
20 described previously (Takayama et al.,supra (1997); Xie et
al., Bio~hemistrv, 37:6410-6418, (1998), which are
incorporated herein by reference), were expressed in XL-1
blue strain E. Coli (Stratagene, Inc., La Jolla, CA).
Briefly, a single colony was inoculated into 1L of LB media
25 containing 50 ~,g/ml ampicillin and grown at 37°C overnight.
The culture was then diluted by half with fresh
LB/ampicillin and cooled to room temperature for 1 hr,
before inducing with 0.4mM IPTG for 6 h at 25°C.
Cells were recovered and incubated with 0.5 mg/ml
30 lysozyme in 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Tween-20,
0.1% 2-mercaptoethanol, 5 mM EDTA, 1 mM PMSF and a mixture
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of other protease inhibitors obtained from Boehringer
Mannheim (1697498) at room temperature for 0.5 h, followed
by sonication. Cellular debris were pelleted by
centrifugation at 27,500g for 10 min and the resulting
supernatants were incubated with 30 ml of glutathionine-
Sepharose (Pharmacia) at 4°C overnight. The resin was then
washed with 20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% Tween-
20, and 0.1% 2-mercaptoethanol until the OD 280nm reached
<0.01. For removal of GST, the resin with immobilized GST-
fusion protein was incubated with l0U of thrombin
(Boehringer, Inc. ) at 4°C in 20 mM Tris (pH 8. 0) , 150 mM
NaCl, 0.1% Tween-20, 0.1% 2-Mercaptoethanol, and 2.5 mM
CaCl2 overnight. Released proteins were then purified on
Mono Q (HR10/10, Pharmacia) by FPLC using a linear gradient
of 0.5M NaCl at pH 8.0 and dialyzed into chaperone assay
buffer.
The ability of BAG-2 (SEQ ID N0:4) or BAG-3 (SEQ
ID N0:6) to bind Hsc70/ATPase in solution was then
examined. GST control or GST-BAG proteins were immobilized
on glutathione-Sepharose and tested for binding to 35S-
labeled in vitro translated (IVT) proteins.
Immunoprecipitation and in vitro GST-protein binding assays
were performed as described by Takayama et al., supra
(1997), using pCI-Neo flag or pcDNA3-HA into which human
Bag-2 (clone #11) or human BAG-3 (clone #28) had been
subcloned fox in vitro translation of 35S-L-methionine
labeled proteins or expression in 293T cells. As shown in
Figure 11B, 'SS-Hsc70/ATPase bound in vitro to GST-BAG-1,
GST-BAG-2, and GST-BAG-3 but not to GST-BAG-1(~C) or
several other control proteins. BAG-1 (beginning at
residue 116 of SEQ ID N0:2), BAG-2 (SEQ ID N0:4), and BAG-3
(SEQ ID N0:6) also exhibited little or no binding to
themselves or to each other, demonstrating that these
proteins do not strongly homo- or hetero-dimerize or
oligomerize. It should be noted, however, that BAG-2 (SEQ
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ID N0:4) displayed weak interactions with itself in binding
assays and produced a positive result in yeast two-hybrid
experiments, demonstrating that it can have the ability to
self-associate.
B Binc,~~,a of BAG ~r~teins to Hsc70 i~ vivo
The ability of BAG-2 (SEQ ID N0:4) and BAG-3 (SEQ
ID N0:6) proteins to interact in cells with Hsc70 was
tested by expressing these proteins with N-terminal Flag
epitope tags in 293T human epithelial cells using co-
immunoprecipitation assays as described previously
(Takayama et al., supra (1997)). cDNAs encoding the
phage cloned regions of BAG-2 and BAG-3 were subcloned in-
frame into pcDNA3-Flag. Anti-Flag immune complexes
prepared from 293T cells after transfection with plasmids
encoding Flag-BAG-1, Flag-BAG-2, or Flag-BAG-3 were
analyzed by SDS-PAGE/immunoblot assay. As shown in Figure
10C, antiserum specific to Hsc70 detected the presence of
BAG proteins associated with Hsc70, whereas control immune-
complexes prepared with IgGl as well as anti-Flag immune
complexes prepared from cells transfected with Flag-tagged
control proteins, Daxx and Apaf-1, did not contain Hsc70
associated protein. These results further demonstrate that
BAG-family proteins specifically bind to Hsc70.
C BIAcore assay of BAG ~rgtein binding to the ATPasP
domain of Hsc70
BAG-1 (beginning at residue 116 of SEQ ID N0:2)
is known to bind tightly to the ATPase domain of Hsc70
(Stuart et al., J. Biol. Chem., In Press (1998}}. BAG-2
(SEQ ID N0:4) and BAG-3 (SEQ ID N0:6) proteins were
therefore, examined for their ability to bind to
Hsc70/ATPase. The affinity and binding kinetics of BAG-2
(SEQ ID N0:4) and BAG-3 (SEQ ID N0:6) to Hsc70/ATPase was
also compared to that of BAG-1 (beginning at residue 116 of
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SEQ ID N0:2) for Hsc70/ATPase, using a surface plasmon
resonance technique (BIAcore) which has been described
previously (Stuart et al., supra, (1998) which is
incorporated herein by reference).
BAG-family proteins were produced in bacteria and
purified to near homogeneity as shown in Figure 12A and
described above in Example I. The purified BAG-1
(beginning at residue 116 of SEQ ID N0:2), -2 (SEQ ID
N0:4), and -3 (SEQ ID N0:6) proteins were then immobilized
on biosensor chips and tested for their interactions with
Hsc70 in the soluble phase. Kinetic measurements were
performed using a BIAcore-II instrument with CM5 sensor
chip and Amine Coupling Kit (Pharmacia Biosensor AB,
Sweden). Briefly, for immobilization of proteins, the
sensor chip was equilibrated with HK buffer (10 mM Hepes
(pH 7.4), 150 mM KCL) at 5u1/min, then activated by
injecting 17 ~1 of 0.2M N-ethyl-N'-(3-diethylaminopropyl)-
carbodiimide and 0.05M N-hydroxysuccinimide (NHS/EDC)
followed by 35 ~,1 of the protein of interest, in 10 mM
acetate, pH 3.5-4.5. Excess NHS-ester on the surface was
deactivated with 17 ~.1 1M ethanolamine-HCL (pH8.5). After
immobilization, 5~.1 of regeneration buffer (50 mM phosphate
(pH 6.8) and 4M GuHCl) was injected. For binding assays,
Hsp70 (Sigma, H8778) was dissolved in HK buffer, and
injected at 10 ~l/min across the prepared surface at
various concentrations. The surface was regenerated after
each injection with 5 ul of regeneration buffer. The rate
constants xass and Kdiss were generated with BIAevaluation
softward 3.01 (Pharmacia Biosensor AB). Addition of Hsc70
to chips containing BAG-1 (beginning at residue 116 of SEQ
ID N0:2), BAG-2 (SEQ ID N0:4) or BAG-3 (SEQ ID N0:6)
resulted in concentration-dependent binding, as. reflected
by an increase in the Response Units (RU) measured at the
chip surface (shown in Figure 3B). In contrast, Hsc70
failed to display interactions in BIAcore assays with a
variety of control proteins as well as a mutant of BAG-1
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34
lacking a C-terminal portion of the BAG domain which is
required for Hsc70-binding (Figure 3B). Furthermore,
flowing of various control proteins such as GST, BSA and
Bcl-XL over the BAG-1 (beginning at residue 116 of SEQ ID
N0:2), BAG-2 (SEQ ID N0:4), or BAG-3 (SEQ ID N0:6) chips
resulted in negligible interaction. These results further
demonstrate the specificity with which BAG-family proteins
interact with and bind to Hsc70.
The rates of Hsc70 binding to BAG-1 (beginning at
residue 116 of SEQ ID N0:2), BAG-2 (SEQ ID N0:4), and BAG-3
(SEQ ID N0:6) proteins were similar, following pseudo
first-order kinetics with estimated association rate
constants (xa) of 2.1, 2.1 and 2.4 x 105 M-1 sec-1,
respectively. After allowing binding of Hsc70 to
immobilized BAG-1 (beginning at residue 116 of SEQ ID
N0:2), BAG-2 (SEQ ID N0:4), or BAG-3 (SEQ ID N0:6) to reach
plateau levels, the chaperone was removed from the flow
solution and the dissociation rate was monitored. BAG-1
(beginning at residue 116 at SEQ ID N0:2) and BAG-2 (SEQ ID
N0:4) exhibited similar dissociation rates, with relatively
slow loss of Hsc70 from the chip surface, resulting in
estimated dissociation rate constants (xd) of 3.0 and 5.0 x
10-q sec-', respectively (see Figure 3B). In contrast, Hsc70
dissociated more rapidly from biosensor chips containing
BAG-3 (see Figure 3B), yielding an estimated xd of 1.7 x 10-3
sec-1. From the kinetic data, the apparent affinities (x~
- Kd~Ka) were calculated for binding of Hsc70 to BAG-1
(beginning at residue 116 of SEQ ID N0:2), BAG-2 (SEQ ID
N0:4), and BAG-3 (SEQ ID N0:6) and were estimated to equal
about Kp= l.4nM, KD=2.4nM, and Kp=7.4nM, respectively. These
results demonstrate that the interactions of BAG-family
proteins with Hsc70 occur with apparent affinities
sufficient for physiological relevance.
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EXAMPLE III
BAG-famil~r~~oteins i~ghibit
Hsx~70/Hsc70-dependent protein folding
This example demonstrates that BAG-2 (SEQ ID
5 N0:4) and BAG-3 (SEQ ID N0:6) proteins inhibit Hsp70/Hsc70-
dependent refolding of denatured proteins similarly to a
BAG-1 (beginning at residue 116 of SEQ ID N0:2) protein.
The effects of BAG-2 (SEQ ID N0:4) and BAG-3 (SEQ
ID N0:6) protein on Hsp70/Hsc70-dependent protein refolding
10 was determined using in vitro protein refolding assays
similar to those described previously by Takayama et al.,
supra, 1998: Terada et al., J Cell Biol., 139:1089-1095,
1997, which are incorporated herein by reference. Briefly,
luciferase (20~,M) was denatured in 25 mM Hepes-KOH, pH 7.2,
15 50 mM potassium acetate, 5 mM DTT, 6M guanidine
hydrochloride at "25°C for 1 h. Denatured luciferase was
diluted 1:40 into 25 mM Hepes-KOH, pH 7.2, 50 mM potassium
acetate, 5 mM DTT. Hsc70 (1.8 ~M), DnaJ (StressGen, Inc.)
(0.9~M), and various purified recombinant proteins as
20 indicated were added to refolding buffer (30 mM Hepes-KOH,
pH 7.6, 120 mM potassium acetate, 3mM magnesium acetate, 2
mM DTT, 2.5 mM ATP) with 0.2 volume of diluted denatured
luciferase to a final concentration of 0.1 ~,M. Luciferase
activity was measured after 1.5 hr incubation at 35°C.
25 The combination of Hsc70 and DnaJ resulted in
ATP-dependent refolding of chemically denatured firefly
luciferase, with function of over half the denatured enzyme
restored in a 90 minute reaction, as monitored by a
chemiluminescence assay. In contrast, neither Hsc70 nor
30 DnaJ alone were able to induce substantial refolding of
denatured luciferase. Furthermore, little spontaneous
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36
restoration of luciferase activity was observed with
control proteins, BSA, GST or Bcl-XL (see Figure 4A}.
Addition of recombinant purified BAG-1 (beginning
at residue 116 of SEQ ID N0:2), BAG-2 (SEQ ID N0:4), or
BAG-3 (SEQ ID N0:6) to the above assays in amounts
equimolar to Hsc70 (1.8 ~M) resulted in striking inhibition
of luciferase refolding. BAG-2 (SEQ ID N0:4) and BAG-3
(SEQ ID N0:6) displayed somewhat greater inhibitory
activity than BAG-1 (beginning at residue 116 of SEQ ID
N0:2) as shown in Figure 4A. In contrast, the BAG-1 (DC)
protein, which fails to bind Hsc70 as well as several other
control proteins, had no effect on luciferase refolding.
In an additional refolding assay, described
previously by Minami et al . , J Bid . Chem. 271 : 19617-24,
1996), purified Hsc70 and human DnaJ homolog Hdj-1 (Hsp 40)
were used with additional cofactors provided in
reticulocyte lysates (5% v:v) to produce a system capable
of refolding denatured luciferase. Briefly, additional
cofactors included, recombinant Luciferase (Promega:
QuantiLum TM), that had been heat denatured at 42°C for 10
min, 1.8 ~M Hsc70 (Sigma; purified from bovine brain), 0.9
~.M Hsp40, and various recombinant purified proteins.
Luciferase activity was measured (Promega luciferase assay
kit) using a luminometer (EG&G Berthold, MicroLumat
luminometer, Model #LB96P). All results were normalized
relative to non-denatured luciferase that had been
subjected to the same conditions. Control reactions
lacking ATP, Hsc70, or Hsp40 resulted in negligible
luciferase refolding.
Various amounts of purified BAG-1 (beginning at
residue 116 of SEQ ID N0:2), BAG-2 (SEQ ID N0:4), or BAG-3
(SEQ ID N0:6), relative to amounts of Hsc70 were used in
the above-described protein refolding assay. Addition of
BAG-family proteins resulted in a concentration-dependent
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37
inhibition of Hsc70 chaperone activity. Furthermore, the
BAG-2 (SEQ ID N0:4)and BAG-3 (SEQ ID N0:6) inhibition of
Hsc70 chaperone activity was demonstrated to be as potent
as that observed for BAG-1 (beginning at residue 116 of SEQ
ID N0:2) . In contrast, the BAG-1 (QC) mutant as well as
other control proteins did not suppress Hsc70-mediated
refolding of denatured luciferase. These results indicate
that BAG-2 (SEQ ID N0:4} and BAG-3 (SEQ ID N0:6) can
inhibit Hsc70/Hsp70 dependent protein refolding activity to
the same extent as BAG-1 (beginning at residue 116 of SEQ
ID N0:2) .
It is known that BAG-1 competes with Hip for
binding to Hsc70, with these proteins exerting opposite
effects on Hsc70-mediated protein refolding (Hohfeld; J.,
and Jentsch, S., Embo J., 16:6209-6216, 1997, which is
incorporated herein by reference). In order to determine
whether BAG-2 (SEQ ID N0:4) and BAG-3 (SEQ ID N0:6) also
compete with Hip for binding to Hsc70, refolding assays
were performed as described above in the presence of Hip
protein.
Hi.p was purified as His6-protein: The fusion
protein was induced from pET28-Hip (V. Prapapanich et al.,
Mo1 Cell Biol., 18:944-952, 1998, which is incorporated
herein by reference) with 0.1 mM IPTG at 25°C for 6h in BL21
cells. Cells from 1L of culture were resuspended into 50
ml of 50 mM Phosphate buffer (pH 6.8), 150 mM NaCl, and 1%
(v/v) Tween-20 and then incubated with 0.5 mg/ml lysozyme
at 25°C for 0.5h, followed by sonication. After
centrifugation at 27,5008 for 10 min, the resulting
supernatant was mixed with 15 ml nickel resin (Qiagen,
Inc . ) at 4°C for 3 h with 25 mM imidazol . The resin was
then washed with 50 mM phosphate buffer (pH 6.8), 25 mM
imidazol, 150 mM NaCl and O.lo Tween-20 until the OD280nm
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38
reached a value of <0.01. His6-Hip protein was eluted with
250 mM imidazol in washing buffer (Qiagene, Inc.) and
purified on Mono Q (HR10/10 Pharmacia) by FPLC using a
linear gradient of 0.5M NaCl at pH 8.0, followed by
dialysis in chaperone assay buffer.
In the refolding assay reactions, addition of
purified Hip at equimolar concentrations relative to BAG-1
(beginning at residue 116 of SEQ ID N0:2), BAG-2 (SEQ ID
N0:4), or BAG-3 (SEQ ID N0:6) (1.8 ~.M) completely negated
the inhibitory effects of the BAG-family proteins on
refolding of denatured luciferase (see Figure 4C). These
results demonstrate that the suppression of Hsc70 chaperone
activity by BAG-family proteins is reversible, and that Hip
antagonizes the effects of not only BAG-1 (beginning at
residue 116 of SEQ ID N0:2), but also of BAG-2 (SEQ ID
N0:4) and BAG-3 (SEQ ID N0:6).
In summary, these results demonstrate that BAG-
family proteins all contain a conserved BAG domain near
their C-terminus that binds Hsc70/Hsp70, and that human
BAG-family proteins can bind with high affinity to the
ATPase domain of Hsc70 and inhibit its chaperone activity
through a Hip-repressable mechanism.
EXAMPLE IV
~~XPANDED NUCLEIC ACID AND AMINO ACID SEQUENCES
FOR HUMAN BAG-3, BAG-~ AND BAG-5
Following the procedures disclosed herein, the
nucleic acid and amino acids sequences to human BAG-3,
BAG-4 and BAG-5 were further expanded. The expanded
sequences for BAG-3, BAG-4 and BAG-5 are shown in
Figures 15, l6 and 17, respectively, with their respective
sequence identification numbers, "SEQ ID NO"s.
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SEQUENCE LISTING
<110> Reed, John C.
Takayama, Shinichi
The Burnham Institute
<120> Novel BAG Proteins and Nucleic Acid Molecules Encoding
Them
<130> FP-LJ 3696
<190>
<191>
<i50> 09/150,489
<151> 1998-09-09
<160> 29
<170> PatentIn Ver. 2.0
<210> 1
<211> 1291
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (96)..(1080)
<400> 1
acgccgcgct cagcttccat cgctgggcgg tcaacaagtg cgggc ctg get cag cgc 57
Leu Ala Gln Arg
1
ggg ggg gcg cgg aga ccg cga ggc gac cgg gag cgg ctg ggt tcc cgg 105
Gly Gly Ala Arg Arg Pro Arg Gly Asp Arg Glu Arg Leu Gly Ser Arg
10 15 20
ctg cgc gcc ctt cgg cca ggc cgg gag ccg cgc cag tcg gag ccc ccg 153
Leu Arg Ala Leu Arg Pro Gly Arg Glu Pro Arg Gln Ser Glu Pro Pro
25 30 35
gcc cag cgt ggt ccg cct ccc tct cgg cgt cca cct gcc cgg agt act 201
Ala Gln Arg Gly Pro Pro Pro Ser Arg Arg Pro Pro Ala Arg Ser Thr
90 95 50
gcc agc ggg cat gac cga ccc acc agg ggc gcc gcc gcc ggc get cgc 299
1
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Ala Ser Gly His Asp Arg Pro Thr Arg Gly Ala Ala Ala Gly Ala Arg
55 60 65
agg ccg cgg atg aag aag aaa acc cgg cgc cgc tcg acc cgg agc gag 297
Arg Pro Arg Met Lys Lys Lys Thr Arg Arg Arg Sex Thr Arg Ser Glu
0 75 g0
gag ttg acc cgg agc gag gag ttg acc ctg agt gag gaa gcg acc tgg 395
Glu Leu Thr Arg Ser Glu Glu Leu Thr Leu Ser Glu Glu Ala Thr Trp
85 90 95 100
agt gaa gag gcg acc cag agt gag gag gcg acc cag ggc gaa gag atg 393
Ser Glu Glu Ala Thr Gln Ser Glu Glu Ala Thr Gln Gly Glu Glu Met
105 I10 115
aat cgg agc cag gag gtg acc cgg gac gag gag tcg acc cgg agc gag 491
Asn Arg Ser Gln Glu Val Thr Arg Asp Glu Glu Ser Thr Arg Ser Glu
I20 125 130
gag gtg acc agg gag gaa atg gcg gca get ggg ctc acc gtg act gtc 989
Glu Val Thr Arg Glu Glu Met Ala Ala Ala Gly Leu Thr Val Thr Val
135 190 245
acc cac agc aat gag aag cac gac ctt cat gtt acc tcc cag cag ggc 537
Thr His Ser Asn Glu Lys His Asp Leu His Val Thr Ser Gln Gln Gly
150 155 160
agc agt gaa cca gtt gtc caa gac ctg gcc cag gtt gtt gaa gag gtc 585
Ser Ser Glu Pro Val Val Gln Asp Leu Ala Gln Val Val Glu Glu Val
165 170 175 180
ata ggg gtt cca cag tct ttt cag aaa ctc ata ttt aag gga aaa tct 633
Ile Gly Val Pro Gln Ser Phe Gln Lys Leu Ile Phe Lys Gly Lys Ser
185 190 195
ctg aag gaa atg gaa aca ccg ttg tca gca ctt gga ata caa gat ggt 681
Leu Lys Glu Met Glu Thr Pro Leu Ser Ala Leu Gly Ile Gln Asp Gly
200 205 210
tgc cgg gtc atg tta att ggg aaa aag aac agt cca cag gaa gag gtt 729
Cys Arg Val Met Leu Ile Gly Lys Lys Asn Ser Pro Gln Glu Glu Val
215 220 225
gaa cta aag aag ttg aaa cat ttg gag aag tct gtg gag aag ata get 777
Glu Leu Lys Lys Leu Lys His Leu Glu Lys Ser Val Glu Lys Ile Ala
230 235 290
gac cag ctg gaa gag ttg aat aaa gag ctt act gga atc cag cag ggt 825
2
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Asp Gln Leu Glu Glu Leu Asn Lys Glu Leu Thr Gly Ile Gln Gln Gly
295 250 255 260
ttt ctg ccc aag gat ttg caa get gaa get ctc tgc aaa ctt gat agg 873
Phe Leu Pro Lys Asp Leu Gln Ala Glu Ala Leu Cys Lys Leu Asp Arg
265 270 275
aga gta aaa gcc aca ata gag cag ttt atg aag atc ttg gag gag att 921
Arg Val Lys Ala Thr Ile Glu Gln Phe Met Lys Ile Leu Glu Glu Ile
280 285 290
gac aca ctg atc ctg cca gaa aat ttc aaa gac agt aga ttg aaa agg 969
Asp Thr Leu Ile Leu Pro Glu Asn Phe Lys Asp Ser Arg Leu Lys Arg
295 300 305
aaa ggc ttg gta aaa aag gtt cag gca ttc cta gcc gag tgt gac aca 1017
Lys Gly Leu Val Lys Lys Val Gln Ala Phe Leu Ala Glu Cys Asp Thr
310 315 320
gtg gag cag aac atc tgc cag gag act gag cgg ctg cag tct aca aac 1065
Val Glu Gln Asn Ile Cys Gln Glu Thr Glu Arg Leu Gln Ser Thr Asn
325 330 335 390
ttt gcc ctg gcc gag tgaggtgtag cagaaaaagg ctgtgctgcc ctgaagaatg 1120
Phe Ala Leu Ala Glu
395
gcgccaccag ctctgccgtc tctggatcgg aatttacctg atttcttcag ggctgctggg 1180
ggcaactggc catttgccaa ttttcctact ctcacactgg ttctcaatga aaaatagtgt 1240
ctttgtgatt tgagtaaagc tcctattctg tttttcacaa aaaaaaaaaa a 1291
<210> 2
<211> 345
<212> PRT
<213> Homo sapiens
<400> 2
Leu Ala Gln Arg Gly Gly Ala Arg Arg Pro Arg Gly Asp Arg Glu Arg
1 5 10 15
Leu Gly Ser Arg Leu Arg Ala Leu Arg Pro Gly Arg Glu Pro Arg Gln
20 25 30
Ser Glu Pro Pro Ala Gln Arg Gly Pro Pro Pro Ser Arg Arg Pro Pro
35 90 95
3
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Ala Arg Ser Thr Ala Ser Gly His Asp Arg Pro Thr Arg Gly Ala Ala
50 55 60
Ala Gly Ala Arg Arg Pro Arg Met Lys Lys Lys Thr Arg Arg Arg Ser
65 70 75 80
Thr Arg Ser Glu Glu Leu Thr Arg Ser Glu Glu Leu Thr Leu Ser Glu
85 90 95
Glu Ala Thr Trp Ser Glu Glu Ala Thr Gln Ser Glu Glu Ala Thr Gln
100 105 110
Gly Glu Glu Met Asn Arg Ser Gln Glu Val Thr Arg Asp Glu Glu Ser
115 120 125
Thr Arg Ser Glu Glu Val Thr Arg Glu Glu Met Ala Ala Ala Gly Leu
130 135 lqp
Thr Val Thr Val Thr His Ser Asn Glu Lys His Asp Leu His Val Thr
195 150 155 160
Ser Gln Gln Gly Ser Ser Glu Pro Val Val Gln Asp Leu Ala Gln Val
165 170 175
Val Glu Glu Val Ile Gly Val Pro Gln Ser Phe Gln Lys Leu Ile Phe
1-80 185 190
Lys Gly Lys Ser Leu Lys Glu Met Glu Thr Pro Leu Ser Ala Leu Gly
195 200 205
Ile Gln Asp Gly Cys Arg Val Met Leu Ile Gly Lys Lys Asn Ser Pro
210 215 220
Gln Glu Glu Val Glu Leu Lys Lys Leu Lys His Leu Glu Lys Ser Val
225 230 235 290
Glu Lys Ile Ala Asp Gln Leu Glu Glu Leu Asn Lys Glu Leu Thr Gly
295 250 255
Ile Gln Gln Gly Phe Leu Pro Lys Asp Leu Gln Ala Glu Ala Leu Cys
260 265 270
Lys Leu Asp Arg Arg Val Lys Ala Thr Ile Glu Gln Phe Met Lys Ile
275 280 285
Leu Glu G1u Ile Asp Thr Leu Ile Leu Pro Glu Asn Phe Lys Asp Ser
290 295 300
9
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Arg Leu Lys Arg Lys Gly Leu Val Lys Lys Val Gln Ala Phe Leu Ala
305 310 315
320
Glu Cys Asp Thr Vas Glu Gln Asn Ile Cys Gln Glu Thr Glu Arg Leu
325 330 335
Gln Ser Thr Asn Phe Ala Leu Ala Glu
340 345
<210> 3
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<220>
<221> CDS
<222> (160)..(792)
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gcagccgcgg tgtcgcgaag tcctcccggg ttgcccccgc ggcgtcagag ggagggcggg 60
cgccgcgttg gtgacggcga ccctgcagcc caaggagcgc tccactcgct gccgccggag 120
ggccggtgac ctcttggcta ccccgcgtcg gaggcttag atg get cag gcg aag 174
Met Ala Gln Ala Lys
1 5
atc aac get aaa gcc aac gag ggg cgc ttc tgc cgc tcc tcc tcc atg 222
Ile Asn Ala Lys Ala Asn Glu Gly Arg Phe Cys Arg Ser Ser Ser Met
10 15 20
get gac cgc tcc agc cgc ctg ctg gag agc ctg gac cag ctg gag ctc 270
Ala Asp Arg Ser Ser Arg Leu Leu Glu Ser Leu Asp Gln Leu Glu Leu
25 30 35
agg gtt gaa get ttg aga gaa gca gca act get gtt gag caa gag aaa 318
Arg Val Glu Ala Leu Arg Glu Ala Ala Thr Ala Val Glu Gln Glu Lys
90 45 50
gaa atc ctt ctg gaa atg atc cac agt atc caa aat agc cag gac atg 366
Glu Ile Leu Leu Glu Met Ile His Ser Ile Gln Asn Ser Gln Asp Met
55 60 65
agg cag atc agt gac gga gaa aga gaa gaa tta aat ctg act gca aac 419
Arg Gln Ile Ser Asp Gly Glu Arg Glu Glu Leu Asn Leu Thr Ala Asn
70 75 80 85
5
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cgt ttg atg gga aga act ctc acc gtt gaa gtg tca gta gaa aca att 462
Arg Leu Met Gly Arg Thr Leu Thr Val Glu Val Ser Val Glu Thr Ile
90 95 100
aga aac ccc cag cag caa gaa tcc cta aag cat gcc aca agg att att 510
Arg Asn Pro Gln Gln Gln Glu Ser Leu Lys His Ala Thr Arg Ile Ile
105 110 115
gat gag gtg gtc aat aag ttt ctg gat gat ttg gga aat gcc aag agt 558
Asp Glu Val Val Asn Lys Phe Leu Asp Asp Leu G.ly Asn Ala Lys Ser
120 125 130
cat tta atg tcg ctc tac agt gca tgt tca tct gag gtg cca cat ggg 606
His Leu Met Ser Leu Tyr Ser Ala Cys Ser Ser Glu Val Pro His Gly
135 190 145
cca gtt gat cag aag ttt caa tcc ata gta att ggc tgt get ctt gaa 654
Pro Val Asp Gln Lys Phe Gln Ser Ile Val Ile Gly Cys Ala Leu Glu
150 155 160 165
gat cag aag aaa att aag aga aga tta gag act ctg ctt aga aat att 702
Asp Gln Lys Lys Ile Lys Arg Arg Leu Glu Thr Leu Leu Arg Asn Ile
170 175 180
gaa aac tct gac aag gcc atc aag cta tta gag cat tct aaa gga get 750
Glu Asn Ser Asp Lys Ala Ile Lys Leu Leu Glu His Ser Lys Gly Ala
185 190 195
ggt tcc aaa act ctg caa caa aat get gaa agc aga ttc aat 792
Gly Ser Lys Thr Leu Gln Gln Asn Ala Glu Ser Arg Phe Asn
200 205 210
tagtcttcaa acctaagagc atttacacaa tacacaaggt gtaaaaatga taaaatacta 852
ttttaattga taactagttc tttgttaggt ataaccactt agttgacact gatagttgtt 912
tcagatgagg aaaatattcc atcaagtatc ttcagttttg tgaataacaa aactagcaat 972
attttaatta tctatctaga gattttttag attgaattct tgtcttgtac taggatctag 1032
catatttcac tattctgtgg atgaatacat agtttgtggg gaaaacaaac gttcagctag 1092
gggcaaaaag catgactgct ttttcctgtc tggcatggaa tcacgcagtc accttgggca 1152
tttagtttac tagaaattct ttactgg 1179
6
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<210> 4
<211> 211
<212> PRT
<213> Homo sapiens
<400> 9
Met Ala Gln Ala Lys Ile Asn Ala Lys Ala Asn Glu Gly Arg Phe Cys
1 5 10 15
Arg Ser Ser Ser Met Ala Asp Arg Ser Ser Arg Leu Leu Glu Ser Leu
20 25 30
Asp Gln Leu Glu Leu Arg Val Glu Ala Leu Arg Glu Ala Ala Thr Ala
35 40 45
Val Glu Gln Glu Lys Glu Ile Leu Leu Glu Met Ile His Ser Ile Gln
50 , 55 60
Asn Ser Gln Asp Met Arg Gln Ile Ser Asp Gly Glu Arg Glu Glu Leu
65 70 75 80
Asn Leu Thr Ala Asn Arg Leu Met Gly Arg Thr Leu Thr Val Glu Val
85 90 95
Ser Val Glu Thr Ile Arg Asn Pro Gln Gln Gln Glu Ser Leu Lys His
100 105 110
Ala Thr Arg Ile Ile Asp Glu Val Val Asn Lys Phe Leu Asp Asp Leu
115 120 125
Gly Asn Ala Lys Ser His Leu Met Ser Leu Tyr Ser Ala Cys Ser Ser
130 135 190
Glu Val Pro His Gly Pro Val Asp Gln Lys Phe Gln Ser Iie Val Ile
195 150 155 160
Gly Cys Ala Leu Glu Asp Gln Lys Lys Ile Lys Arg Arg Leu Glu Thr
165 170 175
Leu Leu Arg Asn Ile Glu Asn Ser Asp Lys Ala Ile Lys Leu Leu Glu
180 1B5 190
His Ser Lys Gly Ala Gly Ser Lys Thr Leu Gln Gln Asn Ala Glu Ser
195 200 205
Arg Phe Asn
210
7
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<210> 5
<211> 2528
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (1)..(2031)
<400> 5
gcg gag ctc cgc atc caa ccc cgg gcc gcg gcc aac ttc tct gga ctg 48
Ala Glu Leu Arg Ile Gln Pro Arg Ala Ala Ala Asn Phe Ser Gly Leu
1 5 10 15
gac cag aag ttt cta gcc ggc cag ttg cta cct ccc ttt atc tcc tcc 96
Asp Gln Lys Phe Leu Ala Gly Gln Leu Leu Pro Pro Phe Ile Ser Ser
20 25 30
ttc ccc tct ggc agc gag gag get att tcc aga cac ttc cac ccc tct 149
Phe Pro Ser Gly Ser Glu Glu Ala Ile Ser Arg His Phe His Pro Ser
35 40 45
ctg gcc acg tca ccc ccg cct tta att cat aaa ggt gcc cgg cgc cgg 192
Leu Ala Thr Ser Pro Pro Pro Leu Ile His Lys Gly Ala Arg Arg Arg
50 55 60
ctt ccc gga cac gtc ggc ggc gga gag ggg ccc acg gcg gcg gcc cgg 240
Leu Pro Gly His Val Gly Gly Gly Glu Gly Pro Thr Ala Ala Ala Arg
65 70 75 80
cca gag act cgg cgc ccg gag cca gcg ccc cgc acc cgc gcc cca gcg 288
Pro Glu Thr Arg Arg Pro Glu Pro Ala Pro Arg Thr Arg Ala Pro Ala
85 90 95
ggc aga ccc caa ccc agc atg agc gcc gcc acc cac tcg ccc atg atg 336
Gly Arg Pro Gln Pro Ser Met Ser Ala Ala Thr His Ser Pro Met Met
100 105 110
cag gtg gcg tcc ggc aac ggt gac cgc gac cct ttg ccc ccc gga tgg 384
Gln Val Ala Ser Gly Asn Gly Asp Arg Asp Pro Leu Pro Pro Gly Trp
115 120 125
gag atc aag atc gac ccg cag acc ggc tgg ccc ttc ttc gtg gac cac 432
Glu Ile Lys Ile Asp Pro Gln Thr Gly Trp Pro Phe Phe Val Asp His
130 135 190
aac agc cgc acc act acg tgg aac gac ccg cgc gtg ccc tct gag ggc 980
8
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
Asn Ser Arg Thr Thr Thr Trp Asn Asp Pro Arg Val Pro Ser Glu Gly
145 150 155 160
ccc aag gag act cca tcc tct gcc aat ggc cct tcc cgg gag ggc tct 528
Pro Lys Glu Thr~Pro Ser Ser Ala Asn Gly Pro Ser Arg Glu Gly Ser
165 170 i75
agg ctg ccg cct get agg gaa ggc cac cct gtg tac ccc cag ctc cga 576
Arg Leu Pro Pro Ala Arg Glu Gly His Pro Val Tyr Pro Gln Leu Arg
180 185 190
cca ggc tac att ccc att cct gtg ctc cat gaa ggc get gag aac cgg 629
Pro Gly Tyr Ile Pro Ile Pro Val Leu His Glu Gly Ala Glu Asn Arg
195 200 205
cag gtg cac cct ttc cat gtc tat ccc cag cct ggg atg cag cga ttc 672
Gln Val His Pro Phe His Val Tyr Pro Gln Pro Gly Met Gln Arg Phe
210 215 220
cga act gag gcg gca gca gcg get cct cag agg tcc cag tca cct ctg 720
Arg Thr Glu Ala Ala Ala Ala Ala Pro Gln Arg Ser Gln Ser Pro Leu
225 230 235 290
cgg ggc atg cca gaa acc act cag cca gat aaa cag tgt gga cag gtg 76g
Arg Gly Met Pro Glu Thr Thr Gln Pro Asp Lys Gln Cys Gly Gln Val
245 250 255
gca gcg gcg gcg gca gcc cag ccc cca gcc tcc cac gga cct gag cgg 816
Ala Ala Ala Ala Ala Ala Gln Pro Pro Ala Ser His Gly Pro Glu Arg
260 265 27p
tcc cag tct cca get gcc tct gac tgc tca tcc tca tcc tcc tcg gcc 869
Ser Gln Ser Pro Ala Ala Ser Asp Cys Ser Ser Ser Ser Ser Ser Ala
275 280 285
agc ctg cct tcc tcc ggc agg agc agc ctg ggc agt cac cag ctc ccg 912
Ser Leu Pro Ser Ser Gly Arg Ser Ser Leu Gly Ser His Gln Leu Pro
290 295 300
cgg ggg tac atc tcc att ccg gtg ata cac gag cag aac gtt acc cgg 960
Arg Gly Tyr Ile Ser Ile Pro Val Ile His Glu Gln Asn Val Thr Arg
305 310 315 320
cca gca gcc cag ccc tcc ttc cac aaa gcc cag aag acg cac tac cca 1008
Pro Ala Ala Gln Pro Ser Phe His Lys Ala Gln Lys Thr His Tyr Pro
325 330 335
gcg cag agg ggt gag tac cag acc cac cag cct gtg tac cac aag atc 1056
9
CA 02342027 2001-03-08
WO 00/14106 PCTNS99/21053
Ala Gln Arg Gly Glu Tyr Gln Thr His Gln Pro Val Tyr His Lys Ile
390 345 350
cag ggg gat gac tgg gag ccc cgg ccc ctg cgg gcg gca tcc ccg ttc 1104
Gln Gly Asp Asp Trp Glu Pro Arg Pro Leu Arg Ala Ala Ser Pro Phe
355 360 365
agg tca tct gtc cag ggt gca tcg agc cgg gag ggc tca cca gcc agg 1152
Arg Ser Ser Val Gln Gly Ala Ser Ser Arg Glu Gly Ser Pro Ala Arg
370 375 380
agc agc acg cca ctc cac tcc ccc tcg ccc atc cgt gtg cac acc gtg 1200
Ser Ser Thr Pro Leu His Ser Pro Ser Pro Ile Arg Val His Thr Val
385 390 395 900
gtc gac agg cct cag cag ccc atg acc cat cga gaa act gca cct gtt 1248
Val Asp Arg Pro Gln Gln Pro Met Thr His Arg Glu Thr Ala Pro Val
905 410 915
tcc cag cct gaa aac aaa cca gaa agt aag cca ggc cca gtt gga cca 1296
Ser Gln Pro Glu Asn Lys Pro Glu Ser Lys Pro Gly Pro Val Gly Pro
420 425 430
gaa ctc cct cct gga cac atc cca att caa gtg atc cgc aaa gag gtg 1349
Glu Leu Pro Pro Gly His Ile Pro Ile Gln Val Ile Arg Lys Glu Val
435 490 495
gat tct aaa cct gtt tcc cag aag ccc cca cct ccc tct gag aag gta 1392
Asp Ser Lys Pro Val Ser Gln Lys Pro Pro Pro Pro Ser Glu Lys Val
450 455 960
gag gtg aaa gtt ccc cct get cca gtt cct tgt cct cct ccc agc cct 1940
Glu Val Lys Val Pro Pro Ala Pro Val Pro Cys Pro Pro Pro Ser Pro
465 970 475 480
ggc cct tct get gtc ccc tct tcc ccc aag agt gtg get aca gaa gag 1988
Gly Pro Ser Ala Val Pro Ser Ser Pro Lys Ser Val Ala Thr Glu Glu
485 990 495
agg gca gcc ccc agc act gcc cct gca gaa get aca cct cca aaa cca 1536
Arg Ala Ala Pro Ser Thr Ala Pro Ala Glu Ala Thr Pro Pro Lys Pro
500 505 510
gga gaa gcc gag get ccc cca aaa cat cca gga gtg ctg aaa gtg gaa 1589
Gly Glu Ala Glu Ala Pro Pro Lys His Pro Gly Val Leu Lys Val Glu
515 520 525
gcc atc ctg gag aag gtg cag ggg ctg gag cag get gta gac aac ttt 1632
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
Ala Ile Leu Glu Lys Val Gln Gly Leu Glu Gln Ala Val Asp Asn Phe
530 535 540
gaa ggc aag aag act gac aaa aag tac ctg atg atc gaa gag tat ttg 1680
Glu Gly Lys Lys Thr Asp Lys Lys Tyr Leu Met Ile Glu Glu Tyr Leu
595 550 555 560
acc aaa gag ctg ctg gcc ctg gat tca gtg gac ccc gag gga cga gcc 1728
Thr Lys Glu Leu Leu Ala Leu Asp Ser Val Asp Pro Glu Gly Arg Ala
565 570 575
gat gtg cgt cag gcc agg aga gac ggt gtc agg aag gtt cag acc atc 1776
Asp Val Arg Gln Ala Arg Arg Asp Gly Val Arg Lys Val Gln Thr Ile
580 585 590
ttg gaa aaa ctt gaa cag aaa gcc att gat gtc cca ggt caa gtc cag 1824
Leu Glu Lys Leu Glu Gln Lys Ala Ile Asp Val Pro Gly Gln Val Gln
595 600 605
gtc tat gaa ctc cag ccc agc aac ctt gaa gca gat cag cca ctg cag 1872
Val Tyr Glu Leu Gln Pro Ser Asn Leu Glu Ala Asp Gln Pro Leu Gln
610 615 620
gca atc atg gag atg ggt gcc gtg gca gca gac aag ggc aag aaa aat 1920
Ala Ile Met Glu Met Gly Ala Val Ala Ala Asp Lys Gly Lys Lys Asn
625 630 635 640
get gga aat gca gaa gat ccc cac aca gaa acc cag cag cca gaa gcc 1968
Ala Gly Asn Ala Glu Asp Pro His Thr Glu Thr Gln Gln Pro Glu Ala
695 650 655
aca gca gca gcg act tca aac ccc agc agc atg aca gac acc cct ggt 2016
Thr Ala Ala Ala Thr Ser Asn Pro Ser Ser Met Thr Asp Thr Pro Gly
660 665 670
aac cca gca gca ccg tagcctctgc cctgtaaaag tcagactcgg aaccgatgtg 2071
Asn Pro Ala Ala Pro
675
tgctttaggg attttagttg catgcatttc agagacttta ggtcagttgg ttttgattag 2131
ctgcttggta tgcagtactt gggtgaggca aacactataa agggctaaaa gggaaaatga 2191
tgcttttctt caatattctt actcttgtac aattaangaa gttgcttgtt gtttgagaag 2251
tttaaccccg ttgcttgttc tgcagccctg tcnacttggg cacccccacc acctgttagc 2311
tgtggttgtg cactgtcttt tgtagctctg gactggaggg gtagatgggg agtcaattac 2371
11
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
ccatcacata aatatgaaac atttatcaga aatgttgcca ttttaatgag atgattttct 2431
tcatctcata attaaaatac ctgactttag agagagtaaa atgtgccagg agccatagga 2491
atatctgtat gttggatgac tttaatgcta catttth 2528
<210> 6
<211> 677
<212> PRT
<213> Homo sapiens
<400> 6
Ala Glu Leu Arg Ile Gln Pro Arg Ala Ala Ala Asn Phe Ser Gly Leu
1 5 10 15
Asp Gln Lys Phe Leu Ala Gly Gln Leu Leu Pro Pro Phe Ile Ser Ser
20 25 30
Phe Pro Ser Gly Ser Glu Glu Ala Ile Ser Arg His Phe His Pro Ser
35 90 45
Leu Ala Thr Ser Pro Pro Pro Leu Ile His Lys Gly Ala Arg Arg Arg
50 55 60
Leu Pro Gly His Val Gly Gly Gly Glu Gly Pro Thr Ala Ala Ala Arg
65 70 75 80
Pro Glu Thr Arg Arg Pro Glu Pro Ala Pro Arg Thr Arg Ala Pro Ala
85 90 95
Gly Arg Pro Gln Pro Ser Met Ser Ala Ala Thr His Ser Pro Met Met
100 105 110
Gln Val Ala Ser Gly Asn Gly Asp Arg Asp Pro Leu Pro Pro Gly,Trp
115 120 125
Glu Ile Lys Ile Asp Pro G1n Thr Gly Trp Pro Phe Phe Val Asp His
130 135 140
Asn Ser Arg Thr Thr Thr Trp Asn Asp Pro Arg Val Pro Ser Glu Gly
195 150 155 160
Pro Lys Glu Thr Pro Ser Ser Ala Asn Gly Pro Ser Arg Glu Gly Ser
165 170 175
Arg Leu Pro Pro Ala Arg Glu Gly His Pro Val Tyr Pro Gln Leu Arg
12
CA 02342027 2001-03-08
WO 00/14106 PCTNS99/21053
180 185 190
Pro Gly Tyr Ile Pro Ile Pro Val Leu His Glu Gly Ala Glu Asn Arg
195 200 205
Gln Val His Pro Phe His Val Tyr Pro Gln Pro Gly Met Gln Arg Phe
210 215 220
Arg Thr Glu Ala Ala Ala Ala Ala Pro Gln Arg Ser Gln Ser Pro Leu
225 230 235 2q0
Arg Gly Met Pro Glu Thr Thr Gln Pro Asp Lys Gln Cys Gly Gln Val
245 250 255
Ala Ala Ala Ala Ala Ala Gln Pro Pro Ala Ser His Gly Pro Glu Arg
260 265 270
Ser Gln Ser Pro Ala Ala Ser Asp Cys Ser Ser Ser Sex Ser Ser Ala
275 280 285
Ser Leu Pro Ser Ser Gly Arg Ser Ser Leu Gly Ser His Gln Leu Pro
290 295 300
Arg Gly Tyr Ile Ser Ile Pro Val Ile His Glu Gln Asn Val Thr Arg
305 310 315 320
Pro Ala Ala Gln Pro Ser Phe His Lys Ala Gln Lys Thr His Tyr Pro
325 330 335
Ala Gln Arg Gly Glu Tyr Gln Thr His Gln Pro Val Tyr His Lys Ile
340 395 350
Gln Gly Asp Asp Trp Glu Pro Arg Pro Leu Arg Ala Ala Ser Pro Phe
355 360 365
Arg Ser Ser Val Gln Gly Ala Ser Ser Arg Glu Gly Ser Pro Ala Arg
370 375 380
Ser Ser Thr Pro Leu His Ser Pro Ser Pro Ile Arg Val His Thr Val
385 390 395 900
Val Asp Arg Pro Gln Gln Pro Met Thr His Arg Glu Thr Ala Pro Val
905 910 915
Ser Gln Pro Glu Asn Lys Pro Glu Ser Lys Pro Gly Pro Val Gly Pro
420 925 930
Glu Leu Pro Pro Gly His Ile Pro Ile Gln Val Ile Arg Lys Glu Val
13
CA 02342027 2001-03-08
WO 00/14106 PCTNS99/2I053
935 990 495
Asp Ser Lys Pro Val Ser Gln Lys Pro Pro Pro Pro Ser Glu Lys Val
950 955 460
Glu Val Lys Val Pro Pro~Ala Pro Val Pro Cys Pro Pro Pro Ser Pro
465 970 475 980
Gly Pro Ser Ala Val Pro Ser Ser Pro Lys Ser Val Ala Thr Glu Glu
985 490 995
Arg Ala Ala Pro Ser Thr Ala Pro Ala Glu Ala Thr Pro Pro Lys Pro
500 505 510
Gly Glu Ala Glu Ala Pro Pro Lys His Pro Gly Val Leu Lys Val Glu
515 520 525
Ala Ile Leu Glu Lys Val Gln Gly Leu Glu Gln Ala Val Asp Asn Phe
530 535 590
Glu Gly Lys Lys Thr Asp Lys Lys Tyr Leu Met Ile Glu Glu Tyr Leu
595 550 555 560
Thr Lys Glu Leu Leu Ala Leu Asp Ser Val Asp Pro Glu Gly Arg Ala
565 570 575
Asp Val Arg Gln Ala Arg Arg Asp Gly Val Arg Lys Val Gln Thr Ile
580 585 590
Leu Glu Lys Leu Glu Gln Lys Ala Ile Asp Val Pro Gly Gln Val Gln
595 600 605
Val Tyr Glu Leu Gln Pro Ser Asn Leu Glu Ala Asp Gln Pro Leu Gln
610 615 620
Ala Ile Met Glu Met Gly Ala Val Ala Ala Asp Lys Gly Lys Lys Asn
625 630 635 640
Ala Gly Asn Ala Glu Asp Pro His Thr Glu Thr Gln Gln Pro Glu Ala
695 650 655
Thr Ala Ala Ala Thr Ser Asn Pro Ser Ser Met Thr Asp Thr Pro Gly
660 665 670
Asn Pro Ala Ala Pro
675
19
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/Z1053
<210> 7
<211> 1010
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (323)..(1009)
<400> 7
acgatatcct gtaagaccaa gaattgcaag gccagagttt gaattcttat acaaatggag 60
cgtatggtcc aacatacccc ccaggccctg gggcaaatac tgcctcatac tcaggggctt 120
attatgcacc tggttatact cagaccagtt actccacaga agttccaagt acttaccgtt 180
catctggcaa cagcccaact ccagtctctc gttggatcta tccccagcag gactgtcaag 240
actgaagcac cccctcttaa ggggcaggtt ccaggatatc cgccttcaca gaaccctgga 300
atgaccctgc cccattatcc tt atg gag atg gta atc gta gtg ttc cac aat 352
Met Glu Met Val Ile Val Val Phe His Asn
1 5 10
cac ggc cga ctg tac gac cac aag aaa gat gcg tgg get tct cct ggt 900
His Gly Arg Leu Tyr Asp His' Lys Lys Asp Ala Trp Ala Ser Pro Gly
15 20 25
get tat gga atg ggt gge cgt tat ecc tgg cct tca tca geg ecc tca 448
Ala Tyr Gly Met Gly Gly Arg Tyr Pro Trp Pro Ser Ser Ala Pro Ser
30 35 90
gca cca ccc ggc aat ctc tac atg act gaa agt act tca cca tgg cct 496
Ala Pro Pro Gly Asn Leu Tyr Met Thr Glu Ser Thr Ser Pro Trp Pro
45 SO 55
agc agt ggc tct ccc cag tca ccc cct tca ccc cca gtc cag cag ccc 599
Ser Ser Gly Ser Pro Gln Ser Pro Pro Ser Pro Pro Val Gln Gln Pro
60 65 70
aag gat tct tca tac ccc tat agc caa tca gat caa agc atg aac cgg 592
Lys Asp Ser Ser Tyr Pro Tyr Ser Gln Ser Asp Gln Ser Met Asn Arg
75 80 85 90
cac aac ttt cct tgc agt gtc cat cag tac gaa tcc tcg ggg aca gtg 690
His Asn Phe Pro Cys Ser Val His Gln Tyr Glu Ser Ser Gly Thr Val
95 100 105
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
aac aat gat gat tca gat ctt ttg gat tcc caa gtc cag tat agt get 688
Asn Asn Asp Asp Sex Asp Leu Leu Asp Ser Gln Val Gln Tyr Ser Ala
110 115 120
gag cct cag ctg tat ggt aat gcc acc agt gac cat ccc aac aat caa 736
Glu Pro Gln Leu Tyr Gly Asn Ala Thr Ser Asp His Pro Asn Asn Gln
125 130 135
gat caa agt agc agt ctt cct gaa gaa tgt gta cct tca gat gaa agt 789
Asp Gln Ser Ser Ser Leu Pro Glu Glu Cys Val Pro Ser Asp Glu Ser
140 195 150
act cct ccg agt att aaa aaa atc ata cat gtg ctg gag aag gtc cag 832
Thr Pro Pro Ser Ile Lys Lys Ile Ile His Val Leu Glu Lys Val Gln
155 160 165 170
tat ctt gaa caa gaa gta gaa gaa ttt gta gga aaa aag aca gac aaa 880
Tyr Leu Glu Gln Glu Val Glu Glu Phe Val Gly Lys Lys Thr Asp Lys
175 180 185
gca tac tgg ctt ctg gaa gaa atg cta acc aag gaa ctt ttg gaa ctg 928
Ala Tyr Trp Leu Leu Glu Glu Met Leu Thr Lys Glu Leu Leu Glu Leu
190 195 200
gat tca gtt gaa act ggg ggc cag gac tct gta cgg cag gcc aga aaa 976
Asp Ser Val Glu Thr Gly Gly Gln Asp Ser Val Arg Gln Ala Arg Lys
205 210 215
gag get gtt tgt aag att cag gcc ata ttg gaa a 1010
Glu Ala Val Cys Lys Ile Gln Ala Ile Leu Glu
220 225
<210> 8
<211> 229
<212> PRT
<213> Homo sapiens
<400> 8
Met Glu Met Val Ile Val Val Phe His Asn His Gly Arg Leu Tyr Asp
1 5 10 15
His Lys Lys Asp Ala Trp Ala Ser Pro Gly Ala Tyr Gly Met Gly Gly
20 25 30
Arg Tyr Pro Trp Pro Ser Ser Ala Pro Ser Ala Pro Pro Gly Asn Leu
35 90 45
16
CA 02342027 2001-03-08
WO 00/14106 PCTNS99/21053
Tyr Met Thr Glu Ser Thr Ser Pro Trp Pro Ser Ser Gly Ser Pro Gln
50 55 60
Ser Pro Pro Ser Pro Pro Val Gln Gln Pro Lys Asp Ser Ser Tyr Pro
65 70 75 80
Tyr Ser Gln Sex Asp Gln Ser Met Asn Arg His Asn Phe Pro Cys Ser
85 90 95
Val His Gln Tyr Glu Ser Ser Gly Thr Val Asn Asn Asp Asp Ser Asp
100 105 110
Leu Leu Asp Ser Gln Val Gln Tyr Ser Ala Glu Pro Gln Leu Tyr Gly
115 120 125
Asn Ala Thr Ser Asp His Pro Asn Asn Gln Asp Gln Ser Ser Ser Leu
130 135 140
Pro Glu Glu Cys Val Pro Ser Asp Glu Ser Thr Pro Pro Ser Ile Lys
145 150 155 160
Lys Ile Ile His Val Leu Glu Lys Val Gln Tyr Leu Glu Gln Glu Val
165 170 175
Glu Glu Phe Val Gly Lys Lys Thr Asp Lys Ala Tyr Trp Leu Leu Glu
180 185 190
Glu Met Leu Thr Lys Glu Leu Leu Glu Leu Asp Ser Val Glu Thr Gly
195 200 205
Gly Gln Asp Ser Val Arg Gln Ala Arg Lys Glu Ala Val Cys Lys Ile
210 215 220
Gln Ala Ile Leu Glu
225
<210> 9
<211> 689
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (3)..(482)
<220>
<221> unsure
17
CA 02342027 2001-03-08
WO 00/14106 PCT/US991Z1053
<222> (105)
<223> any amino acid
<900> 9
ga gaa.ata a~a aat gaa ctt ctc caa.gca caa aac cct tct gaa ttg 47
Glu Ile Lys Asn Glu Leu Leu Gln Ala Gln Asn Pro Ser Glu Leu
1 5 10 15
tac ctg agc tcc aaa aca gaa ttg cag ggt tta att gga cag ttg gat 95
Tyr Leu Ser Ser Lys Thr Glu Leu Gln Gly Leu Ile Gly Gln Leu Asp
20 25 30
gag gta agt ntt gaa aaa aac ccc tgc atc cgg gaa gcc agg aga aga 193
Glu Val Ser Xaa Glu Lys Asn Pro Cys Ile Arg Glu Ala Arg Arg Arg
35 40 45
gca gtg atc gag gtg caa act ctg atc aca tat att gac ttg aag gag 191
Ala Val Ile Glu Val Gln Thr Leu Ile Thr Tyr Ile Asp Leu Lys Glu
50 55 60
gcc ctt gag aaa aga aag ctg ttt get tgt gag gag cac cca tcc cat 239
Ala Leu Glu Lys Arg Lys Leu Phe Ala Cys Glu Glu His Pro Ser His
65 70 75
aaa gcc gtc tgg aac gtc ctt gga aac ttg tct gag atc cag gga gaa 287
Lys Ala Val Trp Asn Val Leu Gly Asn Leu Ser Glu Ile Gln Gly Glu
80 85 90 95
gtt ctt tca ttt gat gga aat cga acc gat aag aac tac atc cgg ctg 335
Val Leu Ser Phe Asp Gly Asn Arg Thr Asp Lys Asn Tyr Ile Arg Leu
100 105 110
gaa gag ctg ctc acc aag cag ctg cta gcc ctg gat get gtt gat ccg 383
Glu Glu Leu Leu Thr Lys Gln Leu Leu Ala Leu Asp Ala Val Asp Pro
115 120 125
cag gga gaa gag aag tgt aag get gcc agg aaa caa get gtg agg ctt 431
Gln Gly Glu Glu Lys Cys Lys Ala Ala Arg Lys Gln Ala Val Arg Leu
130 135 190
gcg cag aat att ctc agc tat ctc gac ctg aaa tct gat gaa tgg gag 979
Ala Gln Asn Ile Leu Ser Tyr Leu Asp Leu Lys Ser Asp Glu Trp Glu
145 150 155
tac tgaaatacca gagatctcac ttttgatact gttttgcact tcatatgtgc 532
Tyr
160
18
CA 02342027 2001-03-08
WO 00/14106 PCTNS99/21053
ttctatgtat agagagcttt cagttcattg atttatacgt gcatatttca gtctcagtat 592
ttatgattga agcaaattct attcagtatc tgctgctttt gatgttgcaa gacaaatatc 652
attacagcac gttaactttt ccattcggat caaaaa~a 689
<210> 10
<211> 160
<212> PRT
<213> Homo sapiens
<400> 10
Glu Ile Lys Asn Glu Leu Leu Gln Ala Gln Asn Pro Ser Glu Leu Tyr
1 5 10 15
Leu Ser Ser Lys Thr Glu Leu Gln Gly Leu Ile Gly Gln Leu Asp Glu
20 25 30
Val Ser Xaa Glu Lys Asn Pro Cys Ile Arg Glu Ala Arg Arg Arg Ala
35 90 95
Val Ile Glu. Val Gln Thr Leu Ile Thr Tyr Ile Asp Leu Lys Glu Ala
50 55 60
Leu Glu Lys Arg Lys Leu Phe Ala Cys Glu Glu His Pro Ser His Lys
65 70 75 80
Ala Val Trp Asn Val Leu Gly Asn Leu Ser Glu Ile Gln Gly Flu Val
85 90 95
Leu Ser Phe Asp Gly Asn Arg Thr Asp Lys Asn Tyr Ile Arg Leu Glu
100 105 110
Glu Leu Leu Thr Lys Gln Leu Leu Ala Leu Asp Ala Val Asp Pro Gln
115 120 125
Gly Glu Glu Lys Cys Lys Ala Ala Arg Lys Gln Ala Val Arg Leu Ala
130 135 190
Gln Asn Ile Leu Ser Tyr Leu Asp Leu Lys Ser Asp Glu Trp Glu Tyr
195 150 155 160
<210> 11
<211> 296
<212> DNA
<213> Caenorhabditis elegans
19
CA 02342027 2001-03-08
WO 00/14106 PCTNS99/21053
<900> 11
atgtctttcc gcctcttcgt tgaaatattt cactttcttt tccagctttt tccccatctc 60
gacctgcttt ggtttttcga gaaaaccacg ttccaaatca gcgacatctc tcaaattgag 120
atcataggct ttttgaagat tgctcaaatt atgcttctca tattgcatga gcattttgaa 180
gcccgcgtca tcaaccaaag cattttttcc acccatcaca atgattttat cattttcttt 290
aaaatt 246
<210> 12
<211> 210
<212> PRT
<213> Caenorhabditis elegans
<900> 12
Met Lys Val Asn Val Ser Cys Ser Ser Val Gln Thr Thr Ile Asp Ile
1 5 10 15
Leu Glu Glu Asn Gln Gly Glu Asp Glu Ser Ile Leu Thr Leu Gly Gln
20 25 30
Leu Arg Asp Arg Ile Ala Thr Asp Asn Asp Val Asp Val Glu Thr Met
35 90 95
Lys Leu Leu His Arg Gly Lys Phe Leu Gln Gly Ala Asp Asp Val Ser
50 55 60
Leu Ser Thr Leu Asn Phe Lys Glu Asn Asp Lys Ile Ile Val Met Gly
65 70 75 80
Gly Lys Asn Ala Leu Val Asp Asp Ala Gly Phe Lys Met Leu Met Gln
85 90 95
Tyr Glu Lys His Asn Leu Ser Asn Leu Gln Lys Ala Tyr Asp Leu Asn
100 105 110
Leu Arg Asp Val Ala Asp Leu Glu Arg Gly Phe Leu Glu Lys Pro Lys
115 120 125
Gln Val Glu Met Gly Lys Lys Leu Glu Lys Lys Val Lys Tyr Phe Asn
130 135 140
Glu Glu Ala Glu Arg His Leu Glu Thr Leu Asp Gly Met Asn Ile Ile
195 150 155 160
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
Thr Glu Thr Thr Pro Glu Asn Gln Ala Lys Arg Asn Arg Glu Lys Arg
165 170 175
Lys Thr Leu Val Asn Gly Ile Gln Thr Leu Leu Asn Gln Asn Asp Ala
180 185. 190
Leu Leu Arg Arg Leu Gln Glu Tyr Gln Ser Val Leu Asn Gly Asp Ile
195 200 205
Pro Glu
210
<210> 13
<211> 1377
<212> DNA
<213> Caenorhabditis elegans
<220>
<221> CDS
<222> (1)..(1377)
<900> 13
atg cca gtc gtg aac ata cca atc aaa ata ctt ggt cag aat caa tca 98
Met Pro Val Val Asn Ile Pro Ile Lys Ile Leu Gly Gln Asn Gln Ser
1 5 10 15
cat agt cga agt aac tcc tcg tct tct gtt gac aac gat cga aat caa 96
His Ser Arg Ser Asn Ser Ser Ser Ser Val Asp Asn Asp Arg Asn Gln
20 25 30
cca cca cag cag cca cct caa ccg caa cca caa cag caa tct cag caa 199
Pro Pro Gln Gln Pro Pro Gln Pro Gln Pro Gln Gln Gln Ser Gln Gln
35 90 45
caa tac cag cag get cca aac gtg aat acc aat atg cat cat tcc aac 192
Gln Tyr Gln Gln Ala Pro Asn Val Asn Thr Asn Met His His Ser Asn
50 55 60
gga ttc tca cct aac ttc cca tct cgt agt cct att ccg gac ttt ccc 290
Gly Phe Ser Pro Asn Phe Pro Ser Arg Ser Pro Ile Pro Asp Phe Pro
65 70 75 80
agt ttt tca tct ggg ttc cca aac gat tct gaa tgg tct tcg aat ttc 288
Ser Phe Ser Ser Gly Phe Pro Asn Asp Ser Glu Trp Ser Ser Asn Phe
85 90 95
21
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
ccg tcg ttt cca aat ttc cca agt gga ttc tca aat gga agt tct aat 336
Pro Ser Phe Pro Asn Phe Pro Ser Gly Phe Ser Asn Gly Ser Ser Asn
100 105 110
ttc cct gat ttt cca aga ttc gga aga. gat gga gga cta tcg cca aac 389
Phe Pro Asp Phe Pro Arg Phe Gly Arg Asp Gly Gly Leu Ser Pro Asn
115 120 125
cca ccg atg caa gga tac agg aga agt cca aca cca aca tca act caa 432
Pro Pro Met Gln Gly Tyr Arg Arg Ser Pro Thr Pro Thr Ser Thr Gln
130 135 190
tct cca act tct aca tta aga cgc aac tct cag cag aat caa get cct 980
Ser Pro Thr Ser Thr Leu Arg Arg Asn Ser Gln Gln Asn Gln Ala Pro
145 150 155 160
cca caa tat tct cag caa caa cca caa caa get caa caa cgt cag aca 52B
Pro Gln Tyr Ser Gln Gln Gln Pro Gln Gln Ala Gln Gln Arg Gln Thr
165 170 175
act cct ccg tca aca aaa get tca tct cga cca cca tct cgt act cgt 576
Thr Pro Pro Ser Thr Lys Ala Ser Ser Arg Pro Pro Ser Arg Thr Arg
180 185 190
gaa cca aag gaa cct gag gta ccc gag aga cca gca gtt att cca ttg 629
Glu Pro Lys Glu Pro Glu Val Pro Glu Arg Pro Ala Val Ile Pro Leu
195 200 205
cca tat gag aag aag gag aaa cca ctg gag aag aaa ggt agt cgt gat 672
Pro Tyr Glu Lys Lys Glu Lys Pro Leu Glu Lys Lys Gly Ser Arg Asp
210 215 220
tct gga aag ggt gat gag aac ctt gaa gag aac att gcc aag atc acg 720
Ser Gly Lys Gly Asp Glu Asn Leu Glu Glu Asn Ile Ala Lys Ile Thr
225 230 235 290
atc gga aag aat aat tgc gag tta tgt ccg gaa caa gaa acg gac ggc 768
Ile Gly Lys Asn Asn Cys Glu Leu Cys Pro Glu Gln Glu Thr Asp Gly
245 250 255
gac cca tct cca cta acc tcc cca atc acc gaa gga aag cca aag aga 816
Asp Pro Ser Pro Leu Thr Ser Pro Ile Thr Glu Gly Lys Pro Lys Arg
260 265 270
gga aag aaa ctt caa cgt aat caa agt gtt gtt gat ttc aat gcc aag 869
Gly Lys Lys Leu Gln Arg Asn Gln Ser Val Val Asp Phe Asn Ala Lys
275 280 285
22
CA 02342027 2001-03-08
WO 00114106 PCT/US99/21053
aca att gtt act ttg gat aaa att gaa tta caa gtt gag cag ttg aga 912
Thr Ile Val Thr Leu Asp Lys Ile Glu Leu Gln Val Glu Gln Leu Arg
290 295 300
aaa aaa get get gaa ctc gaa atg gaa aaa gag caa att ctt cgt tct 960
Lys Lys Ala Ala Glu Leu Glu Met Glu Lys Glu Gln Ile Leu Arg Ser
305 310 315 320
cta gga gaa atc agt gtt cat aac tgc atg ttc aaa ctg gaa gaa tgt 1008
Leu Gly Glu Ile Ser Val His Asn Cys Met Phe Lys Leu Glu Glu Cys
325 330 335
gat cgt gaa gag att gaa gca atc act gac cga ttg aca aaa aga aca 1056
Asp Arg Glu Glu Ile Glu Ala Ile Thr Asp Arg Leu Thr Lys Arg Thr
340 395 350
aag aca gtt caa gtt gtt gtc gaa act cca cga aat gaa gaa cag aaa 1104
Lys Thr Val Gln Val Val Val Glu Thr Pro Arg Asn Glu Glu Gln Lys
355 360 365
aaa gca ctg gaa gat gca act ttg atg atc gat gaa gtc gga gaa atg 1152
Lys Ala Leu Glu Asp Ala Thr Leu Met Ile Asp Glu Val Gly Glu Met
370 375 380
atg cat tcg aat att gaa aag get aag ctg tgc cta caa acc tac atg 1200
Met His Ser Asn Ile Glu Lys Ala Lys Leu Cys Leu Gln Thr Tyr Met
385 390 395 900
aac gcc tgt tcg tac gaa gaa act get gga gcc acc tgc caa aac ttc 1248
Asn Ala Cys Ser Tyr Glu Glu Thr Ala Gly Ala Thr Cys Gln Asn Phe
405 410 415
ttg aag atc ata att cag tgc get get gat gat cag aaa cgc atc aag 1296
Leu Lys Ile Ile Ile Gln Cys Ala Ala Asp Asp Gln Lys Arg Ile Lys
420 425 430
cgt cgt ctg gaa aat ctg atg tct caa att gag aat get gag aga acg 1349
Arg Arg Leu Glu Asn Leu Met Ser Gln Ile Glu Asn Ala Glu Arg Thr
435 990 445
aaa gca gat ttg atg gat gat caa agc gaa tag 1377
Lys Ala Asp Leu Met Asp Asp Gln Ser Glu
450 955
<210> 19
<211> 958
<212> PRT
23
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
<213> Caenorhabditis elegans
<400> 19
Met Pro Val Val Asn Ile Pro Ile Lys Ile Leu G1y Gln Asn Gln Ser
1 5 10 15
His Ser Arg Ser Asn Ser Ser Ser Ser Val Asp Asn Asp Arg Asn Gln
20 25 30
Pro Pro Gln Gln Pro Pro Gln Pro Gln Pro Gln Gln Gln Ser Gln Gln
35 90 95
Gln Tyr Gln Gln Ala Pro Asn Val Asn Thr Asn Met His His Ser Asn
50 55 60
Gly Phe Ser Pro Asn Phe Pro Ser Arg Ser Pro Ile Pro Asp Phe Pro
65 70 75 80
Ser Phe Ser Ser Gly Phe Pro Asn Asp Ser Glu Trp Ser Ser Asn Phe
85 90 95
Pro Ser Phe Pro Asn Phe Pro Ser Gly Phe Ser Asn Gly Ser Ser Asn
100 105 110
Phe Pro Asp Phe Pro Arg Phe Gly Arg Asp Gly Gly Leu Ser Pro Asn
115 220 125
Pro Pro Met Gln Gly Tyr Arg Arg Ser Pro Thr Pro Thr Ser Thr Gln
130 135 140
Ser Pro Thr Ser Thr Leu Arg Arg Asn Ser Gln Gln Asn Gln Ala Pro
195 150 155 160
Pro Gln Tyr Ser Gln Gln Gln Pro Gln Gln Ala Gln Gln Arg Gln Thr
165 170 175
Thr Pro Pro Ser Thr Lys Ala Ser Ser Arg Pro Pro Ser Arg Thr Arg
180 185 190
Glu Pro Lys Glu Pro Glu Val Pro Glu Arg Pro Ala Val Ile Pro Leu
195 200 205
Pro Tyr Glu Lys Lys Glu Lys Pro Leu Glu Lys Lys Gly Ser Arg Asp
210 2i5 220
Ser Gly Lys Gly Asp Glu Asn Leu Glu Glu Asn Ile Ala Lys Ile Thr
225 230 235 290
29
CA 02342027 2001-03-08
WO 00/14106 PCTNS99/21053
Ile Gly Lys Asn Asn Cys Glu Leu Cys Pro Glu Gln Glu Thr Asp Gly
295 250 255
Asp Pro Ser Pro Leu Thr Ser Pro Ile Thr Glu Gly Lys Pro Lys Arg
260 265 270
Gly Lys Lys Leu Gln Arg Asn Gln Ser Val Val Asp Phe Asn Ala Lys
275 280 285
Thr Ile Val Thr Leu Asp Lys Ile Glu Leu Gln Val Glu Gln Leu Arg
290 295 300
Lys Lys Ala Ala Glu Leu Glu Met Glu Lys Glu Gln Ile Leu Arg Ser
305 310 315 320
Leu Gly Glu Ile Ser Val His Asn Cys Met Phe Lys Leu Glu Glu Cys
325 330 335
Asp Arg Glu Glu Ile Glu Ala Ile Thr Asp Arg Leu Thr Lys Arg Thr
390 345 350
Lys Thr Val Gln Val Val Val Glu Thr Pro Arg Asn Glu Glu Gln Lys
35S 360 . 365
Lys Ala Leu Glu Asp Ala Thr Leu Met Ile Asp Glu Val Gly Glu Met
370 375 380
Met His Ser Asn Ile Glu Lys Ala Lys Leu Cys Leu Gln Thr Tyr Met
385 390 39S 400
Asn Ala Cys Ser Tyr Glu Glu Thr Ala Gly Ala Thr Cys Gln Asn Phe
405 910 915
Leu Lys Ile Ile Ile Gln Cys Ala Ala Asp Asp Gln Lys Arg Ile Lys
420 925 430
Arg Arg Leu Glu Asn Leu Met Ser Gln Ile Glu Asn Ala Glu Arg Thr
935 940 495
Lys Ala Asp Leu Met Asp Asp Gln Ser Glu
450 455
<210> 15
<211> 588
<212> DNA
<213> Schizosaccharamyces pombe
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
<220>
<221> CDS
<222> (1)..(588)
<400> 15
atg tca gaa aag act agc aca gtt aca ata cac tat gga aat cag cga 98
Met Ser Glu Lys Thr Ser Thr Val Thr Ile His Tyr Gly Asn Gln Arg
1 S 10 15
ttt ccg gta gca gtc aat cta aat gag acg tta agt gaa ctg att gat 96
Phe Pro Val Ala Val Asn Leu Asn Glu Thr Leu Ser Glu Leu Ile Asp
20 25 30
gat tta ctt gaa acg act gag att tct gag aag aaa gtc aag ctt ttt 199
Asp Leu Leu Glu Thr Thr Glu Ile Ser Glu Lys Lys Val Lys Leu Phe
35 40 95
tac get ggc aag cgt tta aaa gac aaa aaa gcc tcg tta tca aaa ttg 192
Tyr Ala Gly Lys Arg Leu Lys Asp Lys Lys Ala Ser Leu Ser Lys Leu
50 55 60
ggt tta aaa aat cat agt aaa att cta tgt ata aga cca cat aag caa 290
Gly Leu Lys Asn His Ser Lys Ile Leu Cys Ile Arg Pro His Lys Gln
65 70 75 80
caa cga ggt tcc aag gaa aaa gac acg gtt gag ccc get ccg aaa gcg 288
Gln Arg Gly Ser Lys Glu Lys Asp Thr Val Glu Pro Ala Pro Lys Ala
85 90 95
gaa gcg gag aat cct gta ttt tcg cgt att tct gga gaa ata aaa gcc 336
Glu Ala Glu Asn Pro Val Phe Ser Arg Ile Ser Gly Glu Ile Lys Ala
100 105 110
atc gat cag tat gtt gac aaa gaa ctt tcc ccc atg tac gac aat tac 389
Ile Asp Gln Tyr Val Asp Lys Glu Leu Ser Pro Met Tyr Asp Asn Tyr
115 120 125
gta aat aaa ccg tcg aac gat cca aag cag aaa aac aaa cag aaa cta 932
Val Asn Lys Pro Ser Asn Asp Pro Lys Gln Lys Asn Lys Gln Lys Leu
130 135 140
atg ata agt gaa cta ctt tta caa cag ctt tta aaa ttg gat gga gtt 980
Met Ile Ser Glu Leu Leu Leu Gln Gln Leu Leu Lys Leu Asp Gly Val
195 150 155 160
gac gta ctg ggc agc gag aaa ttg cgt ttt gaa cgg aag caa ctt gtt 528
Asp Val Leu Gly Ser Glu Lys Leu Arg Phe Glu Arg Lys Gln Leu Val
165 170 175
26
CA 02342027 2001-03-08
WO 00/14106 PCTNS99/21053
tct aag atc caa aaa atg ttg gat cac gtt gac caa aca agc caa gaa 576
Ser Lys Ile Gln Lys Met Leu Asp His Val Asp Gln Thr Ser Gln Glu
180 185 190
gtg gcc gca tag
588
Val Ala Ala
195
<210> 16
<211> 195
<212> PRT
<213> Schizosaccharomyces pombe
<900> 16
Met Ser Glu Lys Thr Ser Thr Val Thr Ile His Tyr Gly Asn Gln Arg
1 5 10 15
Phe Pro Val Ala Val Asn Leu Asn Glu Thr Leu Ser Glu Leu Ile Asp
20 25 30
Asp Leu Leu Glu Thr Thr Glu Ile Ser Glu Lys Lys Val Lys Leu Phe
35 90 45
Tyr Ala Gly Lys Arg Leu Lys Asp Lys Lys Ala Ser Leu Ser Lys Leu
50 55 60
Gly Leu Lys Asn His Ser Lys Ile Leu Cys Ile Arg Pro His Lys Gln
65 70 75 80
Gln Arg Gly Ser Lys Glu Lys Asp Thr Val Glu Pro Ala Pro Lys Ala
85 90 95
Glu Ala Glu Asn Pro Val Phe Ser Arg Ile Ser Gly Glu Ile Lys Ala
100 105 110
Ile Asp Gln Tyr Val Asp Lys Glu Leu Ser Pro Met Tyr Asp Asn Tyr
115 120 125
Val Asn Lys Pro Ser Asn Asp Pro Lys Gln Lys Asn Lys Gln Lys Leu
130 135 190
Met Ile Ser Glu Leu Leu Leu Gln Gln Leu Leu Lys Leu Asp Gly Val
145 150 155 160
Asp Val Leu Gly Ser Glu Lys Leu Arg Phe Glu Arg Lys Gln Leu Val
165 170 175
27
CA 02342027 2001-03-08
WO 00/14106 PCT/US99121053
Ser Lys Ile Gln Lys Met Leu Asp His Val Asp Gln Thr Ser Gln Glu
180 185 190
Val Ala Ala
195
<210> 17
<211> 621
<212> DNA
<213> Schizosaccharomyces pombe
<220>
<221> CDS
<222> (1)..(621)
<900> 17
atg tct ttt ttt acc cag ttg tgt tct atg gat aaa aaa tat tgg atc 98
Met Ser Phe Phe Thr Gln Leu Cys Ser Met Asp Lys Lys Tyr Trp Ile
1 5 10 15
tct cta get gta ttg tca gtt act gtt ttg att agc gca tta ttg aaa 96
Ser Leu Ala Val Leu Ser Val Thr Val Leu Ile Ser Ala Leu Leu Lys
20 25 30
aag aga get act gaa acc gaa gat att gtc gtt gtt cat tac gat ggc 149
Lys Arg Ala Thr Glu Thr Glu Asp Ile Val Val Val His Tyr Asp Gly
35 90 95
gaa aag ttg aat ttt gtg ttg cga caa cca agg ctg aat atg gtt tct 192
Glu Lys Leu Asn Phe Val Leu Arg Gln Pro Arg Leu Asn Met Val Ser
50 55 60
tac act agt ttt ctt cgt cgc gtg tgc aac gca ttt tca gta atg ccc 240
Tyr Thr Ser Phe Leu Arg Arg Val Cys Asn Ala Phe Ser Val Met Pro
65 70 75 80
gac aaa gcg tct ctc aag tta aac ggg gtg acc ctc aag gat ggt tca 288
Asp Lys Ala Ser Leu Lys Leu Asn Gly Val Thr Leu Lys Asp Gly Ser
85 90 95
ctt tcc gac caa aat gtg caa aat gga agt gaa tta gag ctc gaa tta 336
Leu Ser Asp Gln Asn Val Gln Asn Gly Ser Glu Leu Glu Leu Glu Leu
100 105 110
ccc aaa ctg agc ccg gca atg caa caa att gaa gca tat ata gat gag 384
Pro Lys Leu Ser Pro Ala Met Gln Gln Ile Glu Ala Tyr Ile Asp Glu
ze
CA 02342027 2001-03-08
wo oona~o6 rc~rnrs99mos3
115 120 125
ctt caa cag gat ctc gtc cct aaa att gaa gcc ttc tgc caa tcg tct 932
Leu Gln Gln Asp Leu Val Pro Lys Ile Glu Ala Phe Cys Gln Ser Ser
130 135 140
ccc get tcg gca caa gat gtt caa gat ttg cat aca cgc ctt agt gaa 980
Pro Ala Ser Ala Gln Asp Val Gln Asp Leu His Thr Arg Leu Ser Glu
145 150 155 160
aca ttg ttg get agg atg ata aaa tta gat get gtt aat gtt gaa gac 528
Thr Leu Leu Ala Arg Met Ile Lys Leu Asp Ala Val Asn Val Glu Asp
165 170 175
gac cca gaa get cgt ett aaa aga aaa gaa get att cgt tta tct eaa 576
Asp Pro Glu Ala Arg Leu Lys Arg Lys Glu Ala Ile Arg Leu Ser Gln
180 185 190
caa tat ttg agt aaa cta gat tcc acc aag aat caa aac aaa tga 621
Gln Tyr Leu Ser Lys Leu Asp Ser Thr Lys Asn Gln Asn Lys
195 200 205
<210> 18
<211> 206
<212> PRT
<213> Schizosaccharomyces pombe
<400> 18
Met Ser Phe Phe Thr Gln Leu Cys Ser Met Asp Lys Lys Tyr Trp Ile
1 5 10 15
Ser Leu Ala Val Leu Ser Val Thr Val Leu Ile Ser Ala Leu Leu Lys
20 25 30
Lys Arg Ala Thr Glu Thr Glu Asp Ile Val Val Val His Tyr Asp Gly
35 90 95
Glu Lys Leu Asn Phe Val Leu Arg Gln Pro Arg Leu Asn Met Val Ser
50 55 60
Tyr Thr Ser Phe Leu Arg Arg Val Cys Asn Ala Phe Ser Val Met Pro
65 70 75 80
Asp Lys Ala Ser Leu Lys Leu Asn Gly Val Thr Leu Lys Asp Gly Ser
85 90 95
Leu Ser Asp Gln Asn Val Gln Asn Gly Ser Glu Leu Glu Leu Glu Leu
29
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
100 105 110
Pro Lys Leu Ser Pro Ala Met Gln Gln Ile Glu Ala Tyr Ile Asp Glu
115 120 125
Leu Gln Gln Asp Leu Val Pro Lys Ile Glu Ala Phe Cys Gln Ser Ser
130 135 190
Pro Ala Ser Ala Gln Asp Val Gln Asp Leu His Thr Arg Leu Ser Glu
195 150 155 160
Thr Leu Leu Ala Arg Met Ile Lys Leu Asp Ala Val Asn Val Glu Asp
165 170 175
Asp Pro Glu Ala Arg Leu Lys Arg Lys Glu Ala Il.e Arg Leu Ser Gln
180 185 190
Gln Tyr Leu Ser Lys Leu Asp Ser Thr Lys Asn Gln Asn Lys
195 200 205
<210> 19
<211> 2539
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (307)..(2039)
<900> 19
gcggagctcc gcatccaacc ccgggccgcg gccaacttct ctggactgga ccagaagttt 60
ctagccggcc agttgctacc tccctttatc tcctccttcc cctctggcag cgaggaggct 120
atttccagac acttccaccc ctctctggcc acgtcacccc cgcctttaat tcataaaggt 180
gcccggcgcc ggcttcccgg acacgtcggc ggcggagagg ggcccacggc ggcggcccgg 240
ccagagactc ggcgcccgga gccagcgccc cgcacccgcg ccccagcggg cagaccccaa 300
cccagc atg agc gcc gcc acc cac tcg ccc atg atg cag gtg gcg tcc 398
Met Ser Ala Ala Thr His Ser Pro Met Met Gln Val Ala Ser
1 5 10
ggc aac ggt gac cgc gac cct ttg ccc ccc gga tgg gag atc aag atc 396
Gly Asn Gly Asp Arg Asp Pro Leu Pro Pro Gly Trp Glu Ile Lys Ile
15 20 25 30
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
gac ccg cag acc ggc tgg ccc ttc ttc gtg gac cac aac agc cgc acc 999
Asp Pro Gln Thr Gly Trp Pro Phe Phe Val Asp His Asn Ser Arg Thr
35 90 95
act acg tgg aac gac ccg cgc gtg ccc tct gag ggc ccc aag gag act 492
Thr Thr Trp Asn Asp Pro Arg Val Pro Ser Glu Gly Pro Lys Glu Thr
50 55 60
cca tcc tct gcc aat ggc cct tcc cgg gag ggc tct agg ctg ccg cct 590
Pro Ser Ser Ala Asn Gly Pro Ser Arg Glu Gly Ser Arg Leu Pro Pro
65 70 75
get agg gaa ggc cac cct gtg tac ccc cag ctc cga cca ggc tac att 588
Ala Arg Glu Gly His Pro Val Tyr Pro Gln Leu Arg Pro Gly Tyr Ile
80 85 90
ccc att cct gtg ctc cat gaa ggc get gag aac cgg cag gtg cac cct 636
Pro Ile Pro Val Leu His Glu Gly Ala Glu Asn Arg Gln Val His Pro
95 100 105 110
ttc cat gtc tat ccc cag cct ggg atg cag cga ttc cga act gag gcg 689
Phe His Val Tyr Pro Gln Pro Gly Met Gln Arg Phe Arg Thr Glu Ala
115 120 125
gca gca gcg get cct cag agg tcc cag tca cct ctg cgg ggc atg cca 732
Ala Ala Ala Ala Pro Gln Arg Ser Gln Ser Pro Leu Arg Gly Met Pro
130 135 190
gaa acc act cag cca gat aaa cag tgt gga cag gtg gca gcg gcg gcg 780
Glu Thr Thr Gln Pro Asp Lys Gln Cys Gly Gln Val Ala Ala Ala Ala
145 150 155
gca gcc cag ccc cca gcc tcc cac gga cct gag cgg tcc cag tct cca 828
Ala Ala Gln Pro Pro Ala Ser His Gly Pro Glu Arg Ser Gln Ser Pro
160 165 170
get gcc tct gac tgc tca tcc tca tcc tcc tcg gcc agc ctg cct tcc 896
Ala Ala Ser Asp Cys Ser Ser Ser Ser Ser Ser Ala Ser Leu Pro Ser
175 180 185 190
tcc ggc agg agc agc ctg ggc agt cac cag ctc ccg cgg ggg tac atc 929
Ser Gly Arg Ser Ser Leu Gly Ser His Gln Leu Pro Arg Gly Tyr Ile
195 200 205
tcc att ccg gtg ata cac gag cag aac gtt acc cgg cca gca gcc cag 972
Ser Ile Pro Val Ile His Glu Gln Asn Val Thr Arg Pro Ala Ala Gln
210 215 220
31
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
ccc tcc ttc cac aaa gcc cag aag acg cac tac cca gcg cag agg ggt 1020
Pro Ser Phe His Lys Ala Gln Lys Thr His Tyr Pro Ala Gln Arg Gly
225 230 235
gag tac cag acc cac cag cct gtg tac cac aag atc cag ggg gat gac 1068
Glu Tyr Gln Thr His Gln Pro Val Tyr His Lys Ile Gln Gly Asp Asp
240 245 250
tgg gag ccc cgg ccc ctg cgg gcg gca tcc ccg ttc agg tca tct gtc 1116
Trp Glu Pro Arg Pro Leu Arg Ala Ala Ser Pro Phe Arg Ser Ser Val
255 260 265 270
cag ggt gca tcg agc cgg gag ggc tca cca gcc agg agc agc acg cca 1169
Gln Gly Ala Ser Ser Arg Glu Gly Ser Pro Ala Arg Ser Ser Thr Pro
275 280 285
ctc cac tcc ccc tcg ccc atc cgt gtg cac acc gtg gtc gac agg cct 1212
Leu His Ser Pro Ser Pro Ile Arg Val His Thr Val Val Asp Arg Pro
290 295 300
cag cag ccc atg acc cat cga gaa act gca cct gtt tcc cag cct gaa 1260
Gln Gln Pro Met Thr His Arg Glu Thr Ala Pro Val Ser Gln Pro Glu
305 310 315
aac aaa cca gaa agt aag cca ggc cca gtt gga cca gaa ctc cct cct 1308
Asn Lys Pro Glu Ser Lys Pro Gly Pro Val Gly Pro Glu Leu Pro Pro
320 325 330
gga cac atc cca att caa gtg atc cgc aaa gag gtg gat tct aaa cct 1356
Gly His Ile Pro Ile Gln Val Ile Arg Lys Glu Val Asp Ser Lys Pro
335 390 345 350
gtt tcc cag aag ccc cca cct ccc tct gag aag gta gag gtg aaa gtt 1909
Val Ser Gln Lys Pro Pro Pro Pro Ser Glu Lys Val Glu Val Lys Val
355 360 365
ccc cct get cca gtt cct tgt cct cct ccc agc cct ggc cct tct get 1452
Pro Pro Ala Pro Val Pro Cys Pro Pro Pro Ser Pro Gly Pro Ser Ala
370 375 380
gtc ccc tct tcc ccc aag agt gtg get aca gaa gag agg gca gcc ccc 1500
Val Pro Ser Ser Pro Lys Ser Val Ala Thr Glu Glu Arg Ala Ala Pro
385 390 395
agc act gcc cct gca gaa get aca cct cca aaa cca gga gaa gcc gag 1598
Ser Thr Ala Pro Ala Glu Ala Thr Pro Pro Lys Pro Gly Glu Ala Glu
900 905 910
32
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
get ccc cca aaa cat cca gga gtg ctg aaa gtg gaa gcc atc ctg gag 1596
Ala Pro Pro Lys His Pro Gly Val Leu Lys Val Glu Ala Ile Leu Glu
415 920 925 930
aag gtg cag ggg ctg gag cag get gta gac aac ttt gaa- ggc aag aag 1649
Lys Val Gln Gly Leu Glu Gln Ala Val Asp Asn Phe Glu Gly Lys Lys
935 940 495
act gac aaa aag tac ctg atg atc gaa gag tat ttg acc aaa gag ctg 1692
Thr Asp Lys Lys Tyr Leu Met Ile Glu Glu Tyr Leu Thr Lys Glu Leu
950 455 460
ctg gcc ctg gat tca gtg gac ccc gag gga cga gcc gat gtg cgt cag 1790
Leu Ala Leu Asp Ser Val Asp Pro Glu Gly Arg Ala Asp Val Arg Gln
465 970 975
gcc agg aga gac ggt gtc agg aag gtt cag acc atc ttg gaa aaa ctt 1788
Ala Arg Arg Asp Gly Val Arg Lys Val Gln Thr Ile Leu Glu Lys Leu
980 485 490
gaa cag aaa gcc att gat gtc cca ggt caa gtc cag gtc tat gaa ctc 1836
Glu Gln Lys Ala Ile Asp Val Pro Gly Gln Val Gln Val Tyr Glu Leu
495 500 505 510
cag ccc agc aac ctt gaa gca gat cag cca ctg cag gca atc atg gag 1884
Gln Pro Ser Asn Leu Glu Ala Asp Gln Pro Leu Gln Ala Ile Met Glu
515 520 525
atg ggt gcc gtg gca gca gac aag ggc aag aaa aat get gga aat gca 1932
Met Gly Ala Val Ala Ala Asp Lys Gly Lys Lys Asn Ala Gly Asn Ala
530 535 590
gaa gat ccc cac aca gaa acc cag cag cca gaa gcc aca gca gca gcg 1980
Glu Asp Pro His Thr Glu Thr Gln Gln Pro Glu Ala Thr Ala Ala Ala
545 550 555
act tca aac ccc agc agc atg aca gac acc cct ggt aac cca gca gca 2028
Thr Ser Asn Pro Ser Ser Met Thr Asp Thr Pro Gly Asn Pro Ala Ala
560 565 570
ccg tag cctctgccct gtaaaaatca gactcggaac cgatgtgtgc tttagggaat 2084
Pro
575
tttaagttgc atgcatttca gagactttaa gtcagttggt ttttattagc tgcttggtat 2149
gcagtaactt gggtggaggc aaaacactaa taaaagggct aaaaaggaaa atgatgcttt 2209
33
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
tcttctatat tcttactctg tacaaataaa gaagttgctt gttgtttgag aagtttaacc 2269
ccgttgcttg ttctgcagcc ctgtctactt gggcaccccc accacctgtt agctgtggtt 2329
gtgcactgtc ttttgtagct ctggactgga ggggtagatg gggagtcaat tacccatcac 2389
ataaatatga aacatttatc agaaatgttg ccattttaat gagatgattt tcttcatctc 2999
ataattaaaa tacctgactt tagagagagt aaaatgtgcc aggagccata ggaatatctg 2504
tatgttggat gactttaatg ctacattttc 2539
<210> 20
<211> 575
<212> PRT
<213> Homo sapiens
<400> 20
Met Ser Ala Ala Thr His Ser Pro Met Met Gln Val Ala Ser Gly Asn
1 5 10 15
Gly Asp Arg Asp Pro Leu Pro Pro Gly Trp Glu Ile Lys Ile Asp Pro
20 25 30
Gln Thr Gly Trp Pro Phe Phe Val Asp His Asn Ser Arg Thr Thr Thr
35 40 95
Trp Asn Asp Pro Arg Val Pro Ser Glu Gly Pro Lys Glu Thr Pro Ser
50 55 60
Ser Ala Asn Gly Pro Ser Arg Glu Gly Ser Arg Leu Pro Pro Ala Arg
65 70 75 80
Glu Gly His Pro Val Tyr Pro Gln Leu Arg Pro Gly Tyr Ile Pro Ile
85 90 95
Pro Val Leu His Glu Gly Ala Glu Asn Arg Gln Val His Pro Phe His
100 105 110
Val Tyr Pro Gln Pro Gly Met Gln Arg Phe Arg Thr Glu Ala Ala Ala
115 120 125
Ala Ala Pro Gln Arg Ser Gln Ser Pro Leu Arg Gly Met Pro Glu Thr
130 135 190
Thr Gln Pro Asp Lys Gln Cys Gly Gln Val Ala Ala Ala Ala Ala Ala
39
CA 02342027 2001-03-08
WO 00/14106 PCTNS99/21053
. 195 150 155 160
Gln Pro Pro Ala Ser His Gly Pro Glu Arg Ser Gln Ser Pro Ala Ala
165 170 175
Ser Asp Cys Ser Ser~Ser Ser Ser Ser Ala Ser Leu Pro Ser Ser Gly
180 185 190
Arg Ser Ser Leu Gly Ser His Gln Leu Pro Arg Gly Tyr Ile Ser Ile
195 200 205
Pro Val Ile His Glu Gln Asn Val Thr Arg Pro Ala Ala Gln Pro Ser
210 215 220
Phe His Lys Ala Gln Lys Thr His Tyr Pro Ala Gln Arg Gly Glu Tyr
225 230 235 240
Gln Thr His Gln Pro Val Tyr His Lys Ile Gln Gly Asp Asp Trp Glu
295 250 255
Pro Arg Pro Leu Arg Ala Ala Ser Pro Phe Arg Ser Ser Val Gln Gly
260 265 270
Ala Ser Ser Arg Glu Gly Ser Pro Ala Arg Ser Ser Thr Pro Leu His
275 280 285
Ser Pro Ser Pro Ile Arg Val His Thr Val Val Asp Arg Pro Gln Gln
290 295 300
Pro Met Thr His Arg Glu Thr Ala Pro Val Ser Gln Pro Glu Asn Lys
305 310 315 320
Pro Glu Ser Lys Pro Gly Pro Val Gly Pro Glu Leu Pro Pro Gly His
325 330 335
Ile Pro Ile Gln Val Ile Arg Lys Glu Val Asp Ser Lys Pro Val Ser
390 345 350
Gln Lys Pro Pro Pro Pro Ser Glu Lys Val Glu Val Lys Val Pro Pro
355 360 365
Ala Pro Val Pro Cys Pro Pro Pro Ser Pro Gly Pro Ser Ala Val Pro
370 375 380
Ser Ser Pro Lys Ser Val Ala Thr Glu Glu Arg Ala Ala Pro Ser Thr
385 390 395 900
Ala Pro Ala Glu Ala Thr Pro Pro Lys Pro Gly Glu Ala Glu Ala Pro
CA 02342027 2001-03-08
WO PCT/US99/21053
00/14106
905 410 915
Pro LysHis ProGly ValLeuLys ValGluAla IleLeuGlu LysVal
420 925 430
Gln GlyLeu GluGln AlaValAsp AsnPheGlu GlyLysLys ThrAsp
935 940 445
Lys LysTyr LeuMet IleGluGlu TyrLeuThr LysGluLeu LeuAla
450 955 460
Leu AspSer ValAsp ProGluGly ArgAlaAsp ValArgGln AlaArg
465 970 975 480
Arg AspGly ValArg LysValGln ThrIleLeu GluLysLeu GluGln
485 490 495
Lys AlaIle AspVal ProGlyGln ValGlnVal TyrGluLeu GlnPro
500 505 510
Ser AsnLeu GluAla AspGlnPro LeuGlnAla IleMetGlu MetGly
515 520 525
Ala ValAla AlaAsp LysGlyLys LysAsnAla GlyAsnAla GluAsp
530 535 540
Pro HisThr GluThr GlnGlnPro GluAlaThr AlaAlaAla ThrSer
595 550 555 560
Asn ProSer SerMet ThrAspThr ProGlyAsn ProAlaAia Pro
565 570 575
<210> 21
<211> 1966
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (93)..(1916)
<900> 21
cggtgggagc ggggcgggaa gcgcttcagg gcagcggatc cc atg tcg gcc ctg 59
Met Ser Ala Leu
1
agg cgc tcg ggc tac ggc ccc agt gac ggt ccg tcc tac ggc cgc tac 102
36
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
Arg Arg Ser Gly Tyr Gly Pro Ser Asp Gly Pro Ser Tyr Gly Arg Tyr
10 15 20
tac ggg cct ggg ggt gga gat gtg ccg gta cac cca cct cca ccc tta 150
Tyr Gly Pro Gly Gly Gly Asp Val Pro Val His Pro Pro Pro Pro Leu
25 30 35
tat cct ctt cgc cct gaa cct ccc cag cct ccc att tcc tgg cgg gtg 198
Tyr Pro Leu Arg Pro Glu Pro Pro Gln Pro Pro Ile Ser Trp Arg Val
40 95 50
cgc ggg ggc ggc ccg gcg gag acc acc tgg ctg gga gaa ggc gga gga 246
Arg Gly Gly Gly Pro Ala Glu Thr Thr Trp Leu Gly Glu Gly Gly Gly
55 60 65
ggc gat ggc tac tat ccc tcg gga ggc gcc tgg cca gag cct ggt cga 299
Gly Asp Gly Tyr Tyr Pro Ser Gly Gly Ala Trp Pro Glu Pro Gly Arg
70 75 80
gcc gga gga agc cac cag gag cag cca cca tat cct agc tac aat tct 392
Ala Gly Gly Ser His Gln Glu Gln Pro Pro Tyr Pro Ser Tyr Asn Ser
85 90 95 100
aac tat tgg aat tct act gcg aga tct agg get cct tac cca agt aca 390
Asn Tyr Trp Asn Ser Thr Ala Arg Ser Arg Ala Pro Tyr Pro Ser Thr
105 110 115
tat cct gta aga cca gaa ttg caa ggc cag agt ttg aat tct tat aca 938
Tyr Pro Val Arg Pro Glu Leu Gln Gly Gln Ser Leu Asn Ser Tyr Thr
120 125 130
aat gga gcg tat ggt cca aca tac ccc cca ggc cct ggg gca aat act 986
Asn Gly Ala Tyr Gly Pro Thr Tyr Pro Pro Gly Pro Gly Ala Asn Thr
135 140 195
gcc tca tac tca ggg get tat tat gca cct ggt tat act cag acc agt 534
Ala Ser Tyr Ser Gly Ala Tyr Tyr Ala Pro Gly Tyr Thr Gln Thr Ser
150 155 160
tac tcc aca gaa gtt cca agt act tac cgt tca tct ggc aac agc cca 582
Tyr Ser Thr Glu Val Pro Ser Thr Tyr Arg Ser Ser Gly Asn Ser Pro
165 170 175 180
act cca gtc tct cgt tgg atc tat ccc cag cag gac tgt cag act gaa 630
Thr Pro Val Ser Arg Trp Ile Tyr Pro Gln Gln Asp Cys Gln Thr Glu
185 190 195
gca ccc cct ctt agg ggg cag gtt cca gga tat ccg cct tca cag aac 678
37
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
Ala Pro Pro Leu Arg Gly Gln Val Pro Gly Tyr Pro Pro Ser Gln Asn
200 205 210
cct gga atg acc ctg ccc cat tat cct tat gga gat ggt aat cgt agt 726
Pro Gly Met Thr Leu Pro His Tyr Pro Tyr Gly Asp Gly Asn Arg Ser
215 220 225
gtt cca caa tca gga ccg act gta cga cca caa gaa gat gcg tgg get 774
Val Pro Gln Ser Gly Pro Thr Val Arg Pro Gln Glu Asp Ala Trp Ala
230 235 240
tct cct ggt get tat gga atg ggt ggc cgt tat ccc tgg cct tca tca 822
Ser Pro Gly Ala Tyr Gly Met Gly Gly Arg Tyr Pro Trp Pro Ser Ser
245 250 255 260
gcg ccc tca gca cca ccc ggc aat ctc tac atg act gaa agt act tca 870
Ala Pro Ser Ala Pro Pro Gly Asn Leu Tyr Met Thr Glu Ser Thr Ser
265 270 275
cca tgg cct agc agt ggc tct ccc cag tca ccc cct tca ccc cca gtc 918
Pro Trp Pro Ser Ser Gly Ser Pro Gln Ser Pro Pro Ser Pro Pro Val
280 285 290
cag cag ccc aag gat tct tca tac ccc tat agc caa tca gat caa agc 966
Gln Gln Pro Lys Asp Ser Ser Tyr Pro Tyr Ser Gln Ser Asp Gln Ser
295 300 305
atg aac cgg cac aac ttt cct tgc agt gtc cat cag tac gaa tcc tcg 1019
Met Asn Arg His Asn Phe Pro Cys Ser Val His Gln Tyr Glu Sex Ser
310 315 320
ggg aca gtg atc aat gaa gat tca gat ctt ttg gat tcc caa gtc cag 1062
Gly Thr Val Ile Asn Glu Asp Ser Asp Leu Leu Asp Ser Gln Val Gln
325 330 335 340
tat agt get gag cct cag ctg tat ggt aat gcc acc agt gac cat ccc 1110
Tyr Ser Ala Glu Pro Gln Leu Tyr Gly Asn Ala Thr Ser Asp His Pro
395 350 355
aac aat caa gat caa agt agc agt ctt cct gaa gaa tgt gta cct tca 1158
Asn Asn Gln Asp Gln Ser Ser Ser Leu Pro Glu Glu Cys Val Pro Ser
360 365 370
gat gaa agt act cct ccg agt att aaa aaa atc ata cat gtg ctg gag 1206
Asp Glu Ser Thr Pro Pro Ser Ile Lys Lys Ile Ile His Val Leu Glu
375 380 385
aag gtc cag tat ctt gaa caa gaa gta gaa gaa ttt gta gga aaa aag 1259
38
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
Lys Val Gln Tyr Leu Glu Gln Glu Val Glu Glu Phe Val Gly Lys Lys
390 395 900
aca gac aaa gca tac tgg ctt ctg gaa gaa atg cta acc aag gaa ctt 1302
Thr Asp Lys Ala Tyr Trp Leu Leu Glu Glu Met Leu Thr Lys Glu Leu
905- 410 415 420
ttg gaa ctg gat tca gtt gaa act ggg ggc cag gac tct gta cgg cag 1350
Leu Glu Leu Asp Ser Val Glu Thr Gly Gly Gln Asp Ser Val Arg Gln
425 930 935
gcc aga aaa gag get gtt tgt aag att cag gcc ata ctg gaa aaa tta 1398
Ala Arg Lys Glu Ala Val Cys Lys Ile Gln Ala Ile Leu Glu Lys Leu
490 445 450
gaa aaa aaa gga tta tga aaggatttag aacaaagtgg aagcctgtta 1996
Glu Lys Lys Gly Leu
955
ctaacttgac caaagaacac ttgattaggt taattaccct ctttttgaaa tgcctgttga 1506
tgacaagaag caatacattc cagcttttcc tttgatttta tacttgaaaa actggcaaag 1566
gaatggaaga atattttagt catgaagttg ttttcagttt tcagacgaat gaatgtaata 1626
ggaaactatg gagttaccaa tattgccaag tagactcact ccttaaaaaa tttatggata 1686
tctacaagct gcttattacc agcaggaggg aaacacactt cacacaacag gcttatcaga 1796
aacctaccag atgaaactgg atataatttg agacaaacag gatgtgtttt tttaaacatc 1806
tggatatctt gtcacatttt tgtacattgt gactgctttc aacatatact tcatgtgtaa 1866
ttatagctta gactttagcc ttcttggact tctgttttgt tttgttattt gcagtttaca 1926
aatatagtat tattctctaa aaaaaaaaaa aaaaaaaaaa 1966
<210> 22
<211> 457
<212> PRT
<213> Homo Sapiens
<400> 22
Met Ser Ala Leu Arg Arg Ser Gly Tyr Gly Pro Ser Asp Gly Pro Sex
1 5 10 15
Tyr Gly Arg Tyr Tyr Gly Pro Gly Gly Gly Asp Val Pro Val His Pro
39
CA 02342027 2001-03-08
WO 00/14106 PCT/US99I21053
20 25 30
Pro Pro Pro Leu Tyr Pro Leu Arg Pro Glu Pro Pro Gln Pro Pro Ile
35 q0 95
Ser Trp Arg Val Arg Gly Gly Gly Pro Ala Glu Thr Thr Trp Leu Gly
50 55 60
Glu Gly Gly Gly Gly Asp Gly Tyr Tyr Pro Ser Gly Gly Ala Trp Pro
65 70 75 80
Glu Pro Gly Arg Ala Gly Gly Ser His Gln Glu Gln Pro Pro Tyr Pro
85 90 95
Ser Tyr Asn Ser Asn Tyr Trp Asn Ser Thr Ala Arg Ser Arg Ala Pro
100 105 110
Tyr Pro Ser Thr Tyr Pro Val Arg Pro Glu Leu Gln Gly Gln Ser Leu
115 120 125
Asn Ser Tyr Thr Asn Gly Ala Tyr Gly Pro Thr Tyr Pro Pro Gly Pro
130 135 140
Gly Ala Asn Thr Ala Ser Tyr Ser Gly Ala Tyr Tyr Ala Pro Gly Tyr
195 150 155 160
Thr Gln Thr Ser Tyr Ser Thr Glu Val Pro Ser Thr Tyr Arg Ser Ser
165 170 175
Gly Asn Ser Pro Thr Pro Val Ser Arg Trp Ile Tyr Pro Gln Gln Asp
180 185 190
Cys Gln Thr Glu Ala Pro Pro Leu Arg Gly Gln Val Pro Gly Tyr Pro
195 200 205
Pro Ser Gln Asn Pro Gly Met Thr Leu Pro His Tyr Pro Tyr Gly Asp
210 215 220
Gly Asn Arg Ser Val Pro Gln Ser Gly Pro Thr Val Arg Pro Gln Glu
225 230 235 2qp
Asp Ala Trp Ala Ser Pro Gly Ala Tyr Gly Met Gly Gly Arg Tyr Pro
295 250 255
Trp Pro Ser Ser Ala Pro Ser Ala Pro Pro Gly Asn Leu Tyr Met Thr
260 265 270
Glu Ser Thr Ser Pro Trp Pro Ser Ser Gly Ser Pro Gln Ser Pro Pro
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
275 280 285
Ser Pro Pro Val Gln Gln Pro Lys Asp Ser Ser Tyr Pro Tyr Ser Gln
290 295 300
Ser Asp Gln Ser Met 'Asn Arg His Asn Phe Pro Cys Ser Val His Gln
305 310 315 320
Tyr Glu Ser Ser Gly Thr Val Ile Asn Glu Asp Ser Asp Leu Leu Asp
325 330 335
Ser Gln Val Gln Tyr Ser Ala Glu Pro Gln Leu Tyr Gly Asn Ala Thr
340 345 350
Ser Asp His Pro Asn Asn Gln Asp Gln Ser Ser Ser Leu Pro Glu Glu
355 360 365
Cys Val Pro Ser Asp Glu Ser Thr Pro Pro Ser Ile Lys Lys Ile Ile
370 375 380
His Val Leu Glu Lys Val Gln Tyr Leu Glu Gln Glu Val Glu Glu Phe
385 390 395 400
Val Gly Lys Lys Thr Asp Lys Ala Tyr Trp Leu Leu Glu Glu Met Leu
405 410 415
Thr Lys Glu Leu Leu Glu Leu Asp Ser Val Glu Thr Gly Gly Gln Asp
420 425 930
Ser Val Arg Gln Ala Arg Lys Glu Ala Val Cys Lys Ile Gln Ala Ile
935 990 495
Leu Glu Lys Leu Glu Lys Lys Gly Leu
950 455
<210>23
<211>4308
<212>DNA
<213>Homo sapiens
<220>
<221> CDS
<222> (247)..(1590)
<900> 23
cccccccccc cccccccccc ccngaagacg cccggagcgg ctgctgcagc cagtagcggc 60
91
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
cccttcaccg gctgccccgc tcagacctag tcgggagggg tgcgaggcat gcagctgggg 120
gcccagctcc ggtgccgcac cccgtaaagg gctgatcttc cacctcgcca cctcagccac 180
gggacgccaa gaccgcatcc aattcagact tcttttggtg cttgtgaaac tgaacacaac 290
aaaagt atg gat atg gga aac caa cat cct tct att agt agg ctt cag 288
Met Asp Met Gly Asn Gln His Pro Ser Ile Ser Arg Leu Gln
1 5 10
gaa atc caa aag gaa gta aaa agt gta gaa cag caa gtt atc ggc ttc 336
Glu Ile Gln Lys Glu Val Lys Ser Val Glu Gln Gln Val Ile Gly Phe
15 20 25 30
agt ggt ctg tca gat gac aag aat tac aag aaa ctg gag agg att cta 389
Ser Gly Leu Ser Asp Asp Lys Asn Tyr Lys Lys Leu Glu Arg Ile Leu
35 40 95
aca aaa cag ctt ttt gaa ata gac tct gta gat act gaa gga aaa gga 432
Thr Lys Gln Leu Phe Glu Ile Asp Ser Val Asp Thr Glu Gly Lys Gl.y
50 55 60
gat att cag caa get agg aag cgg gca gca cag gag aca gaa cgt ctt 480
Asp Ile Gln Gln Ala Arg Lys Arg Ala Ala Gln Glu Thr Glu Arg Leu
65 70 75
ctc aaa gag ttg gag cag aat gca aac cac cca cac cgg att gaa ata 528
Leu Lys Glu Leu Glu Gln Asn Ala Asn His Pro His Arg Ile Glu Ile
80 85 90
cag aac att ttt gag gaa gcc cag tcc ctc gtg aga gag aaa att gtg 576
Gln Asn Ile Phe Glu Glu Ala Gln Ser Leu Val Arg Glu Lys Ile Val
95 100 105 110
cca ttt tat aat gga ggc aac tgc gta act gat gag ttt gaa gaa ggc 629
Pro Phe Tyr Asn Gly Gly Asn Cys Val Thr Asp Glu Phe Glu Glu Gly
115 120 125
atc caa gat atc att ctg agg ctg aca cat gtt aaa act gga gga aaa 672
Ile Gln Asp Ile Ile Leu Arg Leu Thr His Val Lys Thr Gly Gly Lys
130 135 140
atc tcc ttg cgg aaa gca agg tat cac act tta acc aaa atc tgt gcg 720
Ile Ser Leu Arg Lys Ala Arg Tyr His Thr Leu Thr Lys Ile Cys Ala
195 150 155
gtg caa gag ata atc gaa gac tgc atg aaa aag cag cct tcc ctg ccg 768
Val Gln Glu Ile Ile Glu Asp Cys Met Lys Lys Gln Pro Ser Leu Pro
92
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
160 165 170
ctt tcc gag gat gca cat cct tcc gtt gcc aaa atc aac ttc gtg atg 816
Leu Ser Glu Asp Ala His Pro Ser Val Ala Lys Ile Asn Phe Val Met
175 1$0 185 190
tgt gag gtg aac aag gcc cga ggg gtc ctg att gca ctt ctg atg ggt 864
Cys Glu Val Asn Lys Ala Arg Gly Val Leu Ile Ala Leu Leu Met Gly
195 200 205
gtg aac aac aat gag acc tgc agg cac tta tcc tgt gtg ctc tcg ggg 912
Val Asn Asn Asn Glu Thr Cys Arg His Leu Ser Cys Val Leu Ser Gly
210 215 220
ctg atc get gac etg gat get cta gat gtg tgc ggc egg aca gaa atc 960
Leu Ile Ala Asp Leu Asp Ala Leu Asp Val Cys Gly Arg Thr Glu Ile
225 230 235
aga aat tat cgg agg gag gta gta gaa gat atc aac aaa tta ttg aaa 1008
Arg Asn Tyr Arg Arg Glu Val Val Glu Asp Ile Asn Lys Leu Leu Lys
240 245 250
tat ctg gat ttg gaa gag gaa gca gac aca act aaa gca ttt gac ctg 1056
Tyr Leu Asp Leu Glu Glu Glu Ala Asp Thr Thr Lys Ala Phe Asp Leu
255 260 265 270
aga cag aat cat tcc att tta aaa ata gaa aag gtc ctc aag aga atg 1109
Arg Gln Asn His Ser Ile Leu Lys Ile Glu Lys Val Leu Lys Arg Met
275 280 285
aga gaa ata aaa aat gaa ctt ctc caa gca caa aac cct tct gaa ttg 1152
Arg Glu Ile Lys Asn Glu Leu Leu Gln Ala Gln Asn Pro Ser Glu Leu
290 295 300
tac ctg agc tcc aaa aca gaa ttg cag ggt tta att gga cag ttg gat 1200
Tyr Leu Ser Ser Lys Thr Glu Leu Gln Gly Leu Ile Gly Gln Leu Asp
305 310 315
gag gta agt ctt gaa aaa aac ccc tgc atc cgg gaa gcc agg aga aga 1298
Glu Val Ser Leu Glu Lys Asn Pro Cys Ile Arg Glu Ala Arg Arg Arg
320 325 330
gca gtg atc gag gtg caa act ctg atc aca tat att gac ttg aag gag 1296
Ala Val Ile Glu Val Gln Thr Leu Ile Thr Tyr Ile Asp Leu Lys Glu
335 390 345 350
gcc ctt gag aaa aga aag ctg ttt get tgt gag gag cac cca tcc cat 1349
Ala Leu Glu Lys Arg Lys Leu Phe Ala Cys Glu Glu His Pro Ser His
93
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
355 360 365
aaa gcc gtc tgg aac gtc ctt gga aac ttg tct gag atc cag gga gaa 1392
Lys Ala Val Trp Asn Val Leu Gly Asn Leu Ser Glu Ile Gln Gly Giu
370 375 380
gtt ctt tca ttt gat gga aat cga acc gat aag aac tac atc cgg ctg 1490
Val Leu Ser Phe Asp Gly Asn Arg Thr Asp Lys Asn Tyr Ile Arg Leu
385 390 395
gaa gag ctg ctc acc aag cag ctg cta gcc ctg gat get gtt gat ccg 1983
Glu Glu Leu Leu Thr Lys Gln Leu Leu Ala Leu Asp Ala Val Asp Pro
900 405 410
cag gga gaa gag aag tgt aag get gcc agg aaa caa get gtg agg ctt 1536
Gln Gly Glu Glu Lys Cys Lys Ala Ala Arg Lys Gln Ala Val Arg Leu
915 920 925 430
gcg cag aat att ctc agc tat ctc gac ctg aaa tct gat gaa tgg gag 1584
Ala Gln Asn Ile Leu Ser Tyr L.eu Asp Leu Lys Ser Asp Glu Trp Glu
935 440 445
tac tga aataccagag atctcacttt tgatactgtt ttgcacttca tatgtgcttc 1690
Tyr
tatgtataga gagctttcag ttcattgatt tatacgtgca tatttcagtc tcagtattta 1700
tgattgaagc aaattctatt cagtatctgc tgcttttgat gttgcaagac aaatatcatt 1760
acagcacgtt aacttttcca ttcggatcat tatctgtatg atgtggtgtg gtttgtttgg 1820
tttgtccttt tttttgcgtt tt.taatcaga aaacaaaata gaggcagctt ttgtagattt 1880
taaatgggtt gtgcaagcat taaaatgcag gtctttcaga atctagaact aggcataacc 1940
ttacataata ctaggaaaat tatgagaaag gggaaatttt tggttaaata agagtaaggt 2000
tcaaacacaa gcagtacatg ttctgtttca ttatgctcga tagaaggctt ttttttcact 2060
tataaggcct gattggtcct acccagctta acggggtggg gtttttttgt ttgttcagac 2120
agtctgttct tttgtaaaca tttttagttg gaaaaacagc atctgcattt tccccatcct 2180
ctacgtttta gagaggaatc ttgtttttgt gtgcaacata agaaaattat gaaaactaat 2240
agccaaaaaa cctttgagat tgcattaaag agaagggata aaggaccagc aataatacct 2300
tgtaagttgc ttttgtttgt aaaatctgag cttatagttt tccttagtga gtaaattcat 2360
99
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
aaggatggga acatttaaat taagttaatg ggcctttaaa aaaaaaaaag gaaacactca 2920
tacctgtagt tggaggatga atactggaga cgggttacca atgtcaggtt atactaaaac 2980
taaatcagaa agtctgaatg tagcacataa tggttctctt ctgttgtcca aggctgtaaa 2540
atggacagcc ttgtcacacc tccccggtgc tgttttacaa cgtgagggta gacgctgtca 2600
gtaacccaga gggaccaggc cttcctaggt tttctaggca gtcagctgtt aaccactcac 2660
ttagtaaatg tcataactac acctgctcca ggaccaatca gtgaaacctg ctcggaatta 2720
aaggcttcct ctgggtgcct gctgaacaac tgagctcatg tcatgggcat gtggtggttt 2780
ctctgttgcc tgaaagagcc attaaagtca gtcgtgcgtg aagcatctct cttctaaagg 2840
atgtgtattt ccataaatgc tttctgagga tccggtacaa aatgatttcc caaagttctg 2900
aagtgccttg agaacatgtg ggtccgagtg ttataacaga ctcctccccc gggtcacctt 2960
ttgcctggtc atcctgttag agtacatctt tggaaatcca gggtaatatt ctctttcaga 3020
gatgctcatt gtgtaactct gtgtagggag atagtcactt taaacagctc aaagtagcta 3080
gctaaaggag tagccttaaa tacctaaaag atgacagaag catagccctt aacaaatctt 3190
cagcttgtct ctcagtattt cccaatcatg aaaatccctt gctatgtctt tcctactaga 3200
aatgttctag aatcgctgga cggtggggtc agagggcagt cggtatttag gccgtgagct 3260
tcccatacta ctgcaggtcc aactcctggc aaccgcgggc tcaaggcagg tcattggaat 3320
ccacgttttg gccacagtag ttgtaggatt g.cttttctgt atcataattt tagaatgctc 3380
ttaaaatctt gaggaagagt ttttattttt tatttatttt tgagatggag tctctgttgc 3440
ccaggctgca gtgcagtggt gccatctcag ctcactgcaa cctccacctc ccaggttcaa 3500
gcgattctcc tgcctcagcc acctgagtag ctgggagtac aggcatgtgg caccatgcct 3560
ggctaatttt tgtattttta atagagttga gatttcacca tgatggtcag gctggtctcg 3620
aactcctgac ctcgtgatcc gcccgcctcg gccccccaaa gtgctgggat taacgggtgt 3680
gagccacggc gcccagccca ggaagagttt ttaaattaga gctctgttta attataccac 3790
tgggaaatca tggttacgct tcaggcatat tcttccccag agtactactt acattttaaa 3800
45
CA 02342027 2001-03-08
WO 00/14106 PCT/US99121053
tttcattttg taaagttaaa tgtcagcatt ccctttaaaa gtgtccattg ttctttgaaa 3860
gtagacgttt cagtcattct tttcaaacaa gtgtttgtgt accttttgcc aagctgtggg 3920
catcgtgtgt gagtacaggg tgctcagctc ttccaccgtc attttgaatt gttcacatgg 3980
gtaattggtc atggaaatga tcagattgac cttgattgac tgtcaggcat ggctttgttt 4090
ctagtttcaa tctgttctcg ttccttgtac cggattattc tactcctgca atgaaccctg 4100
ttgacaccgg atttagctct tgtcggcctt cgtggggagc tgtttgtgtt aatatgagct 9160
actgcatgta attcttaaac tgggcttgtc acattgtatt gtatttttgt gatctgtaat 9220
gaaaagaatc tgtactgcaa gtaaaaccta ctccccaaaa atgtgtggct ttgggtctgc 4280
attaaacgct gtagtccatg ttcatgcc q3pg
<210> 29
<211> 447
<212> PRT
<213> Homo sapiens
<900> 24
Met Asp Met Gly Asn Gln His Pro Ser Ile Ser Arg Leu Gin Glu Ile
1 5 10 15
Gln Lys Glu Val Lys Ser Val Glu Gln Gln Val Ile Gly Phe Ser Gly
20 25 30
Leu Ser Asp Asp Lys Asn Tyr Lys Lys Leu Glu Arg Ile Leu Thr Lys
35 40 95
Gln Leu Phe Glu Ile Asp Ser Val Asp Thr Glu Gly Lys Gly Asp Ile
50 55 60
Gln Gln Ala Arg Lys Arg Ala Ala Gln Glu Thr Glu Arg Leu Leu Lys
65 70 75 80
Glu Leu Glu Gln Asn Ala Asn His Pro His Arg Ile Glu Ile Gln Asn
85 90 95
Ile Phe Glu Glu Ala Gln Ser Leu Val Arg Glu Lys Ile Val Pro Phe
100 105 110
Tyr Asn Gly Gly Asn Cys Val Thr Asp Glu Phe Glu Glu Gly Ile Gln
96
CA 02342027 2001-03-08
WO 00/14106 PCT/US99/21053
115 lzo 125
Asp Ile Ile Leu Arg Leu Thr His Val Lys Thr Gly Gly Lys Ile Ser
130 135 140
Leu Arg Lys Ala Arg Tyr His Thr Leu Thr Lys Ile Cys Ala Val Gln
195 150 155 160
Glu Ile Ile Glu Asp Cys Met Lys Lys Gln Pro Ser Leu Pro Leu Ser
165 170 175
Glu Asp Ala His Pro Ser Val Ala Lys Ile Asn Phe Val Met Cys Glu
180 185 190
Val Asn Lys Ala Arg Gly Val Leu Ile Ala Leu Leu Met Gly Val Asn
195 200 205
Asn Asn Glu Thr Cys Arg His Leu Ser Cys Val Leu Ser Gly Leu Ile
210 215 220
Ala Asp Leu Asp Ala Leu Asp Val Cys Gly Arg Thr Glu Ile Arg Asn
225 230 235 290
Tyr Arg Arg Glu Val Val Glu Asp Ile Asn Lys Leu Leu Lys Tyr Leu
245 250 255
Asp Leu Glu Glu Glu Ala Asp Thr Thr Lys Ala Phe Asp Leu Arg Gln
260 265 270
Asn His Ser Ile Leu Lys Ile Glu Lys Val Leu Lys Arg Met Arg Glu
275 280 285
Ile Lys Asn Glu Leu Leu Gln Ala Gln Asn Pro Ser Glu Leu Tyr Leu
290 295 300
Ser Ser Lys Thr Glu Leu Gln Gly Leu Ile Gly Gln Leu Asp Glu Val
305 310 315 320
Ser Leu Glu Lys Asn Pro Cys Ile Arg Glu Ala Arg Arg Arg Ala Val
325 330 335
Ile Glu Val Gln Thr Leu Ile Thr Tyr Ile Asp Leu Lys Glu Ala Leu
390 395 350
Glu Lys Arg Lys Leu Phe Ala Cys Glu Glu His Pro Ser His Lys Ala
355 360 365
Val Trp Asn Val Leu Gly Asn Leu Ser Glu Ile Gln Gly Glu Val Leu
97
CA 02342027 2001-03-08
WO 00/1410b PCT/US99/21053
370 375 380
Ser PheAspGly AsnArgThr AspLysAsn TyrIle ArgLeuGlu Glu
385 390 395 400
Leu LeuThrLys GlnLeuLeu AlaLeuAsp AlaVal AspProGln Gly
905 410 415
Glu GluLysCys LysAlaAla ArgLysGln AlaVal ArgLeuAla Gln
920 425 430
Asn IleLeuSer TyrLeuAsp LeuLysSer AspGlu TrpGluTyr
435 490 445
98
