Note: Descriptions are shown in the official language in which they were submitted.
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
G-PROTEIN COUPLED RECEPTOR AND DNA SEQUENCES THEREOF
Field of the Invention
This invention relates to newly identified polypeptides and
polynucleotides encoding such polypeptides, to their use in diagnosis and
in identifying compounds that may be agonists, antagonists that are
potentially useful in therapy, and to production of such polypeptides and
polynucleotides, sometimes hereinafter referred to as high affinity
lysophosphatidic acid receptor (HA-LPA-R). HA-LPA-R belong to the
io class of G-protein coupled receptors.
Background of the Invention
The drug discovery process is currently undergoing a fundamental
revolution as it embraces "functional genomics", that is, high throughput
l; genome- or gene-based biology. This approach as a means to identify
genes and gene products as therapeutic targets is rapidly superceding
earlier approaches based on "positional cloning". A phenotype, that is a
biological function or genetic disease, would be identified and this would
then be tracked back to the responsible gene, based on its genetic map
position.
Functional genomics relies heavily on high-throughput DNA sequencing
technologies and the various tools of bioinformatics to identify gene
sequences of potential interest from the many molecular biology databases
now available. There is a continuing need to identify and characterise
further genes and their related polypeptides/proteins, as targets for drug
discovery.
It is well established that many medically significant biological processes
are mediated by proteins participating in signal transduction pathways that.
involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz,
;o Nature, 1991, 351:353-354). Herein these proteins are referred to as
proteins participating in path4vays with G-proteins or PPG proteins. Some
examples of these proteins include the GPC receptors, such as those for
adrenergic agents and dopamine (Kobilka, B.K., et al., Proc. Natl Acad.
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
-,
- G
Sci., USA, 1987, 84:46-50; Kobilka, B.K., et al., Science, 1987, 238:650
656; Bunzow, J.R., et al., Nature, 1988, 336:783-787), G-proteins
themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and
phosphodiesterase, and actuator proteins, e.g., protein kinase A and
s protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8).
For example, in one form of signal transduction, the effect of hormone
binding is activation of the enzyme, adenylate cyclase, inside the cell.
Enzyme activation by hormones is dependent on the presence of the
nucleotide GTP. GTP also influences hormone binding. A G-protein
connects the hormone receptor to adenylate cyclase. G-protein was shown
to exchange GTP for bound GDP when activated by a hormone receptor.
The GTP-carrying form then binds to activated adenylate cyclase.
Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-
protein to its basal, inactive form. Thus, the G-protein serves a dual role,
as
~s an intermediate that relays the signal from receptor to effector, and as a
clock that controls the duration of the signal.
The membrane protein gene superfamily of G-protein coupled receptors
has been characterized as having seven putative transmembrane domains.
The domains are believed to represent transmembrane a-helices
2o connected by extracellular or cytoplasmic loops. G-protein coupled
receptors (GPC-Rs) include a wide range of biologically active receptors,
such as hormone, viral, growth factor and neuroreceptors.
G-protein coupled receptors (otherwise known as 7TM receptors) have
been characterized as including these seven conserved hydrophobic
~s stretches of about 20 to 30 amino acids, connecting at least eight
divergent
hydrophilic loops. The G-protein family of coupled receptors includes
dopamine receptors which bind to neuroleptic drugs used for treating
psychotic and neurological disorders. Other examples of members of this
family include, but are not limited to, calcitonin, adrenergic, endothelin,
cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine,
thrombin, kinin, follicle stimulating hormone, opsins, endothelial
differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors.
Most G-protein coupled receptors have single conserved cysteine residues
in each of the first two extracellular loops which form disulfide bonds that
;s are believed to stabilize functional protein structure. The 7 transmembrane
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
regions are designated as TM1, TM2, TM3, TM4, TMS, TM6, and TM7.
TM3 has been implicated in signal transduction.
Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine
residues can influence signal transduction of some G-protein coupled
receptors. Most G-protein coupled receptors contain potential
phosphorylation sites within the third cytoplasmic loop and/or the carboxy
terminus. For several G-protein coupled receptors, such as the b-
adrenoreceptor, phosphorylation by protein kinase A and/or specific
receptor kinases mediates receptor desensitization.
io For some receptors, the ligand binding sites of G-protein coupled receptors
are believed to comprise hydrophilic sockets formed by several G-protein
coupled receptor transmembrane domains, said socket being surrounded
by hydrophobic residues of the G-protein coupled receptors. The
hydrophilic side of each G-protein coupled receptor transmembrane helix is
postulated to face inward and form polar ligand binding site. TM3 has been
implicated in several G-protein coupled receptors as having a ligand
binding site, such as the TM3 aspartate residue. TM5 serines, a TM6
asparagine and TM6 or TM7 phenylalanines or tyrosines are also
implicated in ligand binding.
2o G-protein coupled receptors can be intracellularly coupled by
heterotrimeric
G-proteins to various intracellular enzymes, ion channels and transporters
(see, Johnson et al., Endoc. Rev., 1989, 10:317-331) Different G-protein
a-subunits preferentially stimulate particular effectors to modulate various
biological functions in a cell. Phosphorylation of cytoplasmic residues of G-
2s protein coupled receptors have been identified as an important mechanism
for the regulation of G-protein coupling of some G-protein coupled
receptors. G-protein coupled receptors are found in numerous sites within
a mammalian host.
Over the past 15 years, nearly 350 therapeutic agents targeting 7
3o transmembrane (7 TM) receptors have been successfully introduced onto
the market.
Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse
biological activities (Moolenaar, W.H., Curr Opin Cell Biol 7(2):203-10,
1995). The principal effects of LPA are growth related, including induction
of cellular proliferation, alterations in differentiation and survival, and
WO 01/09313 CA 02381437 2002-O1-28 pCT~P00/06854
suppression of apoptosis. LPA also evokes cellular effector functions,
which are dependent on cytoskeletal responses such as contraction,
secretion, adhesion, and chemotaxis (Goetzl, E.J. and An, S., FASEB J.
12(15): 1589-1598, 1998). In N1 E-15 neuronal cell culture assays for
example, LPA induces rapid growth cone collapse and neurite retraction
(Kranenburg, O., Poland, M., van Horck, F.P., Drechsel, D., Hall, A. and
Moolenaar, W.H., Mol. Biol. Cell 10(6): 1851-1857, 1999). In neonatal
Schwann cell cultures, LPA functions as a potent survival factor (Weiner,
J.A., and Chun, J., Proc. Natl. Acad. Sci. USA 96(9): 5233-5238, 1999).
~o Additionally, among other processes LPA has been implicated in the
pathological processes of mesangial proliferative glomerulonephritis
(Inoue, C.N., Epstein, M., Forster, H.G., Hotta, O., Kondo, Y. and linuma,
K., Clin. Sci. 96(4): 431-436, 1999) and atherosclerosis (Siess, W., Zangl,
K.J., Essler, M., Bauer, M., Brandl, R., Corrinth, C., Bittmann, R., Tigyi,
o G. and Aepfelbacher, M., Proc. Natl., Acad. Sci. USA 96(12): 6931-6936,
1999). These LPA activities are attributable to the activation of multiple G
protein-coupled receptors (An, S., Goetzl, E.J. and Lee, H., J. Cell.
Biochem. Suppl. 30-31:147-157, 1998). Therefore, the identification of
further LPA receptors is crucial in the identification of LPA's complex and
2o diverse biological functions and will greatly facilitate the development of
new drugs targeted to interfere with LPA signalling.
Summary of the Invention
The present invention relates to HA-LPA-R, in particular HA-LPA-R
~s polypeptides and HA-LPA-R polynucleotides, recombinant materials and
methods for their production. Such polypeptides and polynucleotides are of
interest in relation to methods of treatment of certain diseases, including,
but not limited to, infections such as bacterial, fungal, protozoan and viral
infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers;
~o diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute
heart failure; hypotension; hypertension; glomerulonephritis, urinary
retention; atherosclerosis, osteoporosis; angina pectoris; myocardial
infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy;
migraine; vomiting; psychotic and neurological disorders, including anxiety,
~s schizophrenia, manic depression, depression, delirium, dementia, and
severe mental retardation; and dyskinesias, such as Huntington's disease
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
or Gilles dela Tourett's syndrome, hereinafter referred to as " diseases of
the invention". In a further aspect, the invention relates to methods for
identifying agonists and antagonists (e.g., inhibitors) using the materials
provided by the invention, and treating conditions associated with HA-
s LPA-R imbalance with the identified compounds. In a still further aspect,
the invention relates to diagnostic assays for detecting diseases associated
with inappropriate HA-LPA-R activity or levels.
Description of the Invention
~o In a first aspect, the present invention relates to HA-LPA-R polypeptides.
Such polypeptides include:
(a) an isolated polypeptide encoded by a polynucleotide comprising the
sequence of SEQ ID N0:1 and/or SEQ ID N0:3 and/or SEQ ID N0:5;
(b) an isolated polypeptide comprising a polypeptide sequence having at
is least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence
of SEQ ID N0:2 and/or SEQ ID N0:4 and/or SEQ ID N0:6;
(c) an isolated polypeptide comprising the polypeptide sequence of SEQ
ID N0:2 and/or SEQ ID N0:4 and/or SEQ ID N0:6;
(d) an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99%
2o identity to the polypeptide sequence of SEQ ID N0:2 and/or SEQ ID
N0:4 and/or SEQ ID N0:6;
(e) the polypeptide sequence of SEQ ID N0:2 and/or SEQ ID N0:4
and/or SEQ ID N0:6; and
(f) an isolated polypeptide having or comprising a polypeptide sequence
2s that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to
the polypeptide sequence of SEQ ID N0:2 and/or SEQ ID N0:4 and/or
SEQ ID N0:6;
(g) fragments and variants of such polypeptides in (a) to (f).
Polypeptides of the present invention are believed to be members of the G
;o protein-coupled receptors family of polypeptides.
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The biological properties of the HA-LPA-R are hereinafter referred to as
"biological activity of HA-LPA-R" or "HA-LPA-R activity". Preferably, a
polypeptide of the present invention exhibits at least one biological
activity of HA-LPA-R.
s Polypeptides of the present invention also includes variants of the
aforementioned polypeptides, including all allelic forms and splice variants.
Such polypeptides vary from the reference polypeptide by insertions,
deletions, and substitutions that may be conservative or non-conservative,
or any combination thereof. Particularly preferred variants are those in
to which several, for instance from 50 to 30, from 30 to 20, from 20 to 10,
from
to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acids are inserted,
substituted, or deleted, in any combination.
Preferred fragments of polypeptides of the present invention include an
isolated polypeptide comprising an amino acid sequence having at least
is 30, 50 or 100 contiguous amino acids from the amino acid sequence of
SEO ID N0:2 and/or SEO ID N0:4 and/or SEO ID N0:6, or an isolated
polypeptide comprising an amino acid sequence having at least 30, 50 or
100 contiguous amino acids truncated or deleted from the amino acid
sequence of SEQ ID N0:2 and/or SEO ID N0:4 and/or SEQ ID N0:6.
2o Preferred fragments are biologically active fragments that mediate the
biological activity of HA-LPA-R, including those with a similar activity or an
improved activity, or with a decreased undesirable activity. Also preferred
are those fragments that are antigenic or immunogenic in an animal,
especially in a human.
2s Fragments of the polypeptides of the invention may be employed for
producing the corresponding full-length polypeptide by peptide synthesis;
therefore, these variants may be employed as intermediates for
producing the full-length polypeptides of the invention.The polypeptides of
the present invention may be in the form of the "mature" protein or may
~o be a part of a larger protein such as a precursor or a fusion protein. It
is
often advantageous to include an additional amino acid sequence that
contains secretory or leader sequences, pro-sequences, sequences that
aid in purification, for instance multiple histidine residues, or an
additional
sequence for stability during recombinant production.
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
Polypeptides of the present invention can be prepared in any suitable
manner, for instance by isolation form naturally occuring sources, from
genetically engineered host cells comprising expression systems (vide
infra) or by chemical synthesis, using for instance automated peptide
s synthesisers, or a combination of such methods.. Means for preparing
such polypeptides are well understood in the art.
In a further aspect, the present invention relates to HA-LPA-R
polynucleotides. Such polynucleotides include:
io (a) an isolated polynucleotide comprising a polynucleotide sequence
having at least 95%, 96%, 97%, 98%, or 99% identity to the
polynucleotide squence of SEO ID N0:1 and/or SEO ID N0:3 and/or
SEO ID N0:5;
(b) an isolated polynucleotide comprising the polynucleotide of SEQ ID
N0:1 and/or SEO ID N0:3 and/or SEO ID N0:5;
(c) an isolated polynucleotide having at least 95%, 96%, 97%, 98%, or
99% identity to the polynucleotide of SEO ID N0:1 and/or SEQ ID N0:3
and/or SEQ ID N0:5;
(d) the isolated polynucleotide of SEO ID N0:1 and/or SEQ ID N0:3 and/or
2o SEQ ID N0:5;
(e) an isolated polynucleotide comprising a polynucleotide sequence
encoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or
99% identity to the polypeptide sequence of SEQ ID N0:2 and/or SEO ID
N0:4 and/or SEQ ID N0:6;
?s (f) an isolated polynucleotide comprising a polynucleotide sequence
encoding the polypeptide of SEO ID N0:2 and/or SEQ ID N0:4 and/or SEO
ID N0:6;
(g) an isolated polynucleotide having a polynucleotide sequence encoding
a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99%
~o identity to the polypeptide sequence of SEQ ID N0:2 and/or SEQ ID N0:4
and/or SEQ ID N0:6;
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
(h) an isolated polynucleotide encoding the polypeptide of SEO ID N0:2
and/or SEQ ID N0:4 and/or SEQ ID N0:6;
(i) an isolated polynucleotide having or comprising a polynucleotide
sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99
compared to the polynucleotide sequence of SEO ID N0:1 and/or SEQ
ID N0:3 and/or SEO ID N0:5;
(j) an isolated polynucleotide having or comprising a polynucleotide
sequence encoding a polypeptide sequence that has an Identity Index of
0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of
to SEQ ID N0:2 and/or SEQ ID N0:4 and/or SEO ID N0:6; and
polynucleotides that are fragments and variants of the above mentioned
polynucleotides or that are complementary to above mentioned
polynucleotides, over the entire length thereof.
Preferred fragments of polynucleotides of the present invention include an
> j isolated polynucleotide comprising an nucleotide sequence having at least
15, 30, 50 or 100 contiguous nucleotides from the sequence SEQ ID
N0:1 and/or SEQ ID N0:3 and/or SEQ ID N0:5, or an isolated
polynucleotide comprising an sequence having at least 30, 50 or 100
contiguous nucleotides truncated or deleted from the sequence SEQ ID
2o N0:1 and/or SEQ ID N0:3 and/or SEQ ID N0:5.
Preferred variants of polynucleotides of the present invention include
splice variants, allelic variants, and polymorphisms, including
polynucleotides having one or more single nucleotide polymorphisms
(SNPs).
2s Polynucleotides of the present invention also include polynucleotides
encoding polypeptide variants that comprise the amino acid sequence of
SEQ ID N0:2 and/or SEO ID N0:4 and/or SEQ ID N0:6 and in which
several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to
5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acid residues are
3o substituted, deleted or added, in any combination.
In a further aspect, the present invention provides polynucleotides that
are RNA transcripts of the DNA sequences of the present invention.
Accordingly, there is provided an RNA polynucleotide that:
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
(a) comprises an RNA transcript of the DNA sequence encoding the
polypeptide of SEQ ID N0:2 and/or SEQ ID N0:4 and/or SEQ ID N0:6;
(b) is the RNA transcript of the DNA sequence encoding the polypeptide
of SEO ID N0:2 and/or SEQ ID N0:4 and/or SEO ID N0:6;
s (c) comprises an RNA transcript of the DNA sequence of SEO ID N0:1
and/or SEO ID N0:3 and/or SEQ ID N0:5; or
(d) is the RNA transcript of the DNA sequence of SEO ID N0:1 and/or
SEQ ID N0:3 and/or SEQ ID N0:5;
and RNA polynucleotides that are complementary thereto.
~o
The polynucleotide sequence of SEO ID N0:1 and/or SEO ID N0:3 and/or
SEQ ID N0:5 shows homology with U76385 (Guo, Z., Liliom, K., Fischer,
D.J., Bathurst, I.C., Tomei, L.D., Kiefer, M.C. and Tigyi, G., Proc. Natl.
Acad. Sci. USA. 1996 Dec 10;93(25):14367-72). The polynucleotide
is sequence of SEO ID N0:1 and/or SEO ID N0:3 and/or SEQ ID N0:5 is a
cDNA sequence that encodes the polypeptide of SEO ID N0:2 and/or
SEQ ID N0:4 and/or SEO ID N0:6. The polynucleotide sequence
encoding the polypeptide of SEQ ID N0:2 and/or SEQ ID N0:4 and/or
SEQ ID N0:6 may be identical to the polypeptide encoding sequence of
2o SEQ ID N0:1 and/or SEQ ID N0:3 and/or SEQ ID N0:5 or it may be a
sequence other than SEQ ID N0:1 and/or SEQ ID N0:3 and/or SEQ ID
N0:5, which, as a result of the redundancy (degeneracy) of the genetic
code, also encodes the polypeptide of SEO ID N0:2 and/or SEQ ID N0:4
and/or SEQ ID N0:6. The polypeptide of the SEQ ID N0:2 and/or SEQ ID
2s N0:4 and/or SEQ ID N0:6 is related to other proteins of the G protein-
coupled receptors family, having homology and/or structural similarity with
P79945 (Guo, Z., Liliom, K., Fischer, D.J., Bathurst, I.C., Tomei, L.D.,
Kiefer, M.C. and Tigyi, G., Proc Natl Acad Sci U S A. 1996 Dec
10;93(25):14367-72).
Preferred polypeptides and polynucleotides of the present invention are
expected to have, inter alia, similar biological functionslproperties to their
homologous polypeptides and polynucleotides. Furthermore, preferred
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
_ 0 _
polypeptides and polynucleotides of the present invention have at least one
HA-LPA-R activity.
Polynucleotides of the present invention may be obtained using standard
s cloning and screening techniques from a cDNA library derived from mRNA
in cells of human brain, kidney, blood, lung, colon, lymphe nodes, liver or
placenta (see for instance, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1989)). Polynucleotides of the invention can also be
~o obtained from natural sources such as genomic DNA libraries or can be
synthesized using well known and commercially available techniques.
When polynucleotides of the present invention are used for the
recombinant production of polypeptides of the present invention, the
polynucleotide may include the coding sequence for the mature
is polypeptide, by itself, or the coding sequence for the mature polypeptide
in
reading frame with other coding sequences, such as those encoding a
leader or secretory sequence, a pre-, or pro- or prepro- protein sequence,
or other fusion peptide portions. For example, a marker sequence that
facilitates purification of the fused polypeptide can be encoded. In certain
2o preferred embodiments of this aspect of the invention, the marker sequence
is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.)
and described in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824,
or is an HA tag. The polynucleotide may also contain non-coding 5' and 3'
sequences, such as transcribed, non-translated sequences, splicing and
2s polyadenylation signals, ribosome binding sites and sequences that
stabilize mRNA.
Polynucleotides that are identical, or have sufficient identity to a
polynucleotide sequence of SEQ ID N0:1 and/or SEQ ID N0:3 and/or SEQ
ID N0:5, may be used as hybridization probes for cDNA and genomic DNA
30 or as primers for a nucleic acid amplification reaction (for instance,
PCR).
Such probes and primers may be used to isolate full-length cDNAs and
genomic clones encoding polypeptides of the present invention and to
isolate cDNA and genomic clones of other genes (including genes
encoding paralogs from human sources and orthologs and paralogs from
~s species other than human) that have a high sequence similarity to SEQ ID
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_ ,~i _
N0:1 and/or SEO ID N0:3 and/or SEO ID N0:5, typically at least 95%
identity. Preferred probes and primers will generally comprise at least 15
nucleotides, preferably, at least 30 nucleotides and may have at least 50, if
not at least 100 nucleotides. Particularly preferred probes will have
s between 30 and 50 nucleotides. Particularly preferred primers will have
between 20 and 25 nucleotides.
A polynucleotide encoding a polypeptide of the present invention, including
homologs from species other than human, may be obtained by a process
comprising the steps of screening a library under stringent hybridization
to conditions with a labeled probe having the sequence of SEO ID N0:1
and/or SEQ ID N0:3 and/or SEQ ID N0:5 or a fragment thereof, preferably
of at least 15 nucleotides; and isolating full-length cDNA and genomic
clones containing said polynucleotide sequence. Such hybridization
techniques are well known to the skilled artisan. Preferred stringent
1 ~ hybridization conditions include overnight incubation at 42oC in a
solution
comprising: 50% formamide, 5xSSC (150mM NaCI, 15mM trisodium
citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10
dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm
DNA; followed by washing the filters in 0.1 x SSC at about 65oC. Thus the
2o present invention also includes isolated polynucleotides, preferably with a
nucleotide sequence of at least 100, obtained by screening a library under
stringent hybridization conditions with a labeled probe having the sequence
of SEQ ID N0:1 and/or SEQ ID N0:3 and/or SEO ID N0:5 or a fragment
thereof, preferably of at least 15 nucleotides.
2s The skilled artisan will appreciate that, in many cases, an isolated cDNA
sequence will be incomplete, in that the region coding for the polypeptide
does not extend all the way through to the 5' terminus. This is a
consequence of reverse transcriptase, an enzyme with inherently low
"processivity" (a measure of the ability of the enzyme to remain attached
to the template during the polymerisation reaction), failing to complete a
DNA copy of the mRNA template during first strand cDNA synthesis.
There are several methods available and well known to those skilled in
the art to obtain full-length cDNAs, or extend short cDNAs, for example
those based on the method of Rapid Amplification of cDNA ends (RACE)
;; (see, for example, Frohman et al., Proc Nat Acad Sci USA 85, 8998-
9002, 1988). Recent modifications of the technique, exemplified by the
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
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Marathon (trade mark) technology (Clontech Laboratories Inc.) for
example, have significantly simplified the search for longer cDNAs. In the
Marathon (trade mark) technology, cDNAs have been prepared from
mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated
s onto each end. Nucleic acid amplification (PCR) is then carried out to
amplify the "missing" 5' end of the cDNA using a combination of gene
specific and adaptor specific oligonucleotide primers. The PCR reaction
is then repeated using 'nested' primers, that is, primers designed to
anneal within the amplified product (typically an adaptor specific primer
that anneals further 3' in the adaptor sequence and a gene specific
primer that anneals further 5' in the known gene sequence). The
products of this reaction can then be analysed by DNA sequencing and a
full-length cDNA constructed either by joining the product directly to the
existing cDNA to give a complete sequence, or carrying out a separate
~s full-length PCR using the new sequence information for the design of the
5' primer.
Recombinant polypeptides of the present invention may be prepared by
processes well known in the art from genetically engineered host cells
comprising expression systems. Accordingly, in a further aspect, the
2o present invention relates to expression systems comprising a
polynucleotide or polynucleotides of the present invention, to host cells
which are genetically engineered with such expression sytems and to the
production of polypeptides of the invention by recombinant techniques.
Cell-free translation systems can also be employed to produce such
~s proteins using RNAs derived from the DNA constructs of the present
invention.
For recombinant production, host cells can be genetically engineered to
incorporate expression systems or portions thereof for pofynucleotides of
the present invention. Polynucleotides may be introduced into host cells by
o methods described in many standard laboratory manuals, such as Davis et
al., Basic Methods in Molecular Biology (1986) and Sambrook et al.(ibic~.
Preferred methods of introducing polynucleotides into host cells include, for
instance, calcium phosphate transfection, DEAF-dextran mediated
transfection, transvection, microinjection, cationic lipid-mediated
;s transfection, electroporation, transduction, scrape loading, ballistic
introduction or infection.
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
Representative examples of appropriate hosts include bacterial cells, such
as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis
cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells
such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as
s CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells;
and plant cells.
A great variety of expression systems can be used, for instance,
chromosomal, episomal and virus-derived systems, e.g., vectors derived
from bacterial plasmids, from bacteriophage, from transposons, from yeast
to episomes, from insertion elements, from yeast chromosomal elements,
from viruses such as baculoviruses, papova viruses, such as SV40,
vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and
retroviruses, and vectors derived from combinations thereof, such as those
derived from plasmid and bacteriophage genetic elements, such as
is cosmids and phagemids. The expression systems may contain control
regions that regulate as well as engender expression. Generally, any
system or vector that is able to maintain, propagate or express a
polynucleotide to produce a polypeptide in a host may be used. The
appropriate polynucleotide sequence may be inserted into an expression
2o system by any of a variety of well-known and routine techniques, such as,
for example, those set forth in Sambrook et al., (ibid). Appropriate secretion
signals may be incorporated into the desired polypeptide to allow secretion
of the translated protein into the lumen of the endoplasmic reticulum, the
periplasmic space or the extracellular environment. These signals may be
2s endogenous to the polypeptide or they may be heterologous signals.
If a polypeptide of the present invention is to be expressed for use in
screening assays, it is generally preferred that the polypeptide be
produced at the surface of the cell. In this event, the cells may be
harvested prior to use in the screening assay. If the polypeptide is
~o secreted into the medium, the medium can be recovered in order to
recover and purify the polypeptide. If produced intracellularly. the cells
must first be lysed before the polypeptide is recovered.
Polypeptides of the present invention can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
;s sulfate or ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
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- ~~4 -
interaction chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography is employed for purification. Well
known techniques for refolding proteins may be employed to regenerate
s active conformation when the polypeptide is denatured during intracellular
synthesis, isolation and/or purification.
Polynucleotides of the present invention may be used as diagnostic
reagents, through detecting mutations in the associated gene. Detection of
a mutated form of the gene characterised by the polynucleotide of SEQ ID
to N0:1 and/or SEQ ID N0:3 and/or SEQ ID N0:5 in the cDNA or genomic
sequence and which is associated with a dysfunction will provide a
diagnostic tool that can add to, or define. a diagnosis of a disease, or
susceptibility to a disease, which results from under-expression, over-
expression or altered spatial or temporal expression of the gene.
is Individuals carrying mutations in the gene may be detected at the DNA
level by a variety of techniques well known in the art.
Nucleic acids for diagnosis may be obtained from a subject's cells, such as
from blood, urine, saliva, tissue biopsy or autopsy material. The genomic
DNA may be used directly for detection or it may be amplified enzymatically
2o by using PCR, preferably RT-PCR, or other amplification techniques prior to
analysis. RNA or cDNA may also be used in similar fashion. Deletions and
insertions can be detected by a change in size of the amplified product in
comparison to the normal genotype. Point mutations can be identified by
hybridizing amplified DNA to labeled HA-LPA-R nucleotide sequences.
2s Perfectly matched sequences can be distinguished from mismatched
duplexes by RNase digestion or by differences in melting temperatures.
DNA sequence difference may also be detected by alterations in the
electrophoretic mobility of DNA fragments in gels, with or without
denaturing agents, or by direct DNA sequencing (see, for instance, Myers
3o et al., Science (1985) 230:1242). Sequence changes at specific locations
may also be revealed by nuclease protection assays, such as RNase and
S1 protection or the chemical cleavage method (see Cotton et al., Proc Natl
Acad Sci USA (1985) 85: 4397-4401).
An array of oligonucleotides probes comprising HA-LPA-R polynucleotide
;s sequence or fragments thereof can be constructed to conduct efficient
screening of e.g., genetic mutations. Such arrays are preferably high
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
density arrays or grids. Array technology methods are well known and
have general applicability and can be used to address a variety of
questions in molecular genetics including gene expression, genetic linkage,
and genetic variability, see, for example, M.Chee et al., Science, 274, 610
s 613 (1996) and other references cited therein.
Detection of abnormally decreased or increased levels of polypeptide or
mRNA expression may also be used for diagnosing or determining
susceptibility of a subject to a disease of the invention. Decreased or
increased expression can be measured at the RNA level using any of the
io methods well known in the art for the quantitation of polynucleotides,
such as, for example, nucleic acid amplification, for instance PCR, RT-
PCR, RNase protection, Northern blotting and other hybridization
methods. Assay techniques that can be used to determine levels of a
protein, such as a polypeptide of the present invention, in a sample derived
i s from a host are well-known to those of skill in the art. Such assay
methods
include radioimmunoassays, competitive-binding assays, Western Blot
analysis and ELISA assays.
Thus in another aspect, the present invention relates to a diagonostic kit
comprising:
?o (a) a polynucleotide of the present invention, preferably the nucleotide
sequence of SEO ID N0:1 and/or SEQ ID N0:3 and/or SEO ID N0:5, or a
fragment or an RNA transcript thereof;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide of
?s SEQ ID N0:2 and/or SEQ ID N0:4 and/or SEQ ID N0:6 or a fragment
thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to the
polypeptide of SEQ ID N0:2 andlor SEO ID N0:4 and/or SEQ ID N0:6.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise
~o a substantial component. Such a kit will be of use in diagnosing a
disease or susceptibility to a disease, particularly diseases of the
invention, amongst others.
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
-
The polynucleotide sequences of the present invention are valuable for
chromosome localisation studies. The sequence is specifically targeted to,
and can hybridize with, a particular location on an individual human
chromosome. The mapping of relevant sequences to chromosomes
s according to the present invention is an important first step in correlating
those sequences with gene associated disease. Once a sequence has
been mapped to a precise chromosomal location, the physical position of
the sequence on the chromosome can be correlated with genetic map data.
Such data are found in, for example, V. McKusick, Mendelian Inheritance in
io Man (available on-line through Johns Hopkins University Welch Medical
Library). The relationship between genes and diseases that have been
mapped to the same chromosomal region are then identified through
linkage analysis (co-inheritance of physically adjacent genes). Precise
human chromosomal localisations for a genomic sequence (gene
is fragment etc.) can be determined using Radiation Hybrid (RH) Mapping
(Walter, M. Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P.,
(1994) A method for constructing radiation hybrid maps of whole
genomes, Nature Genetics 7, 22-28). A number of RH panels are
available from Research Genetics (Huntsville, AL, USA) e.g. the
2o GeneBridge4 RH panel (Hum Mol Genet 1996 Mar;S(3):339-46 A
radiation hybrid map of the human genome. Gyapay G, Schmitt K,
Fizames C, Jones H, Vega-Czarny N, Spillett D, Muselet D, Prud'Homme
JF, Dib C, Auffray C, Morissette J, Weissenbach J, Goodfellow PN). To
determine the chromosomal location of a gene using this panel, 93 PCRs
~s are performed using primers designed from the gene of interest on RH
DNAs. Each of these DNAs contains random human genomic fragments
maintained in a hamster background (human / hamster hybrid cell lines).
These PCRs result in 93 scores indicating the presence or absence of
the PCR product of the gene of interest. These scores are compared
with scores created using PCR products from genomic sequences of
known location. This comparison is conducted at
http://www.genome.wi.mit.edu/. The gene of the present invention maps
to human chromosome 6q16.1-16.3.
;s The polynucleotide sequences of the present invention are also valuable
tools for tissue expression studies. Such studies allow the determination of
expression patterns of polynucleotides of the present invention which may
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
give an indication as to the expression patterns of the encoded
polypeptides in tissues, by detecting the mRNAs that encode them. The
techniques used are well known in the art and include in situ hydridisation
techniques to clones arrayed on a grid, such as cDNA microarray
s hybridisation (Schena et al, Science, 270, 467-470, 1995 and Shalon et al,
Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques
such as PCR. A preferred method uses the TAQMAN (Trade mark)
technology available from Perkin Elmer. Results from these studies can
provide an indication of the normal function of the polypeptide in the
~o organism. In addition, comparative studies of the normal expression
pattern of mRNAs with that of mRNAs encoded by an alternative form of
the same gene (for example, one having an alteration in polypeptide coding
potential or a regulatory mutation) can provide valuable insights into the
role
of the polypeptides of the present invention, or that of inappropriate
is expression thereof in disease. Such inappropriate expression may be of a
temporal, spatial or simply quantitative nature.
The polypeptides of the present invention are expressed in human brain,
kidney, blood, lung, colon, lymphe nodes, liver or placenta.
2o A further aspect of the present invention relates to antibodies. The
polypeptides of the invention or their fragments, or cells expressing them,
can be used as immunogens to produce antibodies that are immunospecific
for polypeptides of the present invention. The term "immunospecific"
means that the antibodies have substantially greater affinity for the
~s polypeptides of the invention than their affinity for other related
polypeptides
in the prior art.
Antibodies generated against polypeptides of the present invention may be
obtained by administering the polypeptides or epitope-bearing fragments, or
cells to an animal, preferably a non-human animal, using routine protocols.
3o For preparation of monoclonal antibodies, any technique which provides
antibodies produced by continuous cell line cultures can be used.
Examples include the hybridoma technique (Kohler, G. and Milstein, C.,
Nature (1975) 256:495-497), the trioma technique, the human B-cell
hybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) and
WO 01/09313 CA 02381437 2002-O1-28 p~/Ep00/06854
the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and
Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).
Techniques for the production of single chain antibodies, such as those
described in U.S. Patent No. 4,946,778, can also be adapted to produce
s single chain antibodies to polypeptides of this invention. Also, transgenic
mice, or other organisms, including other mammals, may be used to
express humanized antibodies.
The above-described antibodies may be employed to isolate or to identify
clones expressing the polypeptide or to purify the polypeptides by affinity
to chromatography. Antibodies against polypeptides of the present invention
may also be employed to treat diseases of the invention, amongst others.
Polypeptides and polynucleotides of the present invention may also be
used as vaccines. Accordingly, in a further aspect, the present invention
is relates to a method for inducing an immunological response in a mammal
that comprises inoculating the mammal with a polypeptide of the present
invention, adequate to produce antibody and/or T cell immune response,
including, for example, cytokine-producing T cells or cytotoxic T cells, to
protect said animal from disease, whether that disease is already
zo established within the individual or not. An immunological response in a
mammal may also be induced by a method comprises delivering a
polypeptide of the present invention via a vector directing expression of
the polynucleotide and coding for the polypeptide in vivo in order to
induce such an immunological response to produce antibody to protect
said animal from diseases of the invention. One way of administering the
vector is by accelerating it into the desired cells as a coating on particles
or otherwise. Such nucleic acid vector may comprise DNA, RNA, a
modified nucleic acid, or a DNA/RNA hybrid. For use a vaccine, a
polypeptide or a nucleic acid vector will be normally provided as a
~o vaccine formulation (composition). The formulation may further comprise
a suitable carrier. Since a polypeptide may be broken down in the
stomach, it is preferably administered parenterally (for instance,
subcutaneous, intramuscular, intravenous, or intradermal injection).
Formulations suitable for parenteral administration include aqueous and
;, non-aqueous sterile injection solutions that may contain anti-oxidants,
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
buffers, bacteriostats and solutes that render the formulation instonic with
the blood of the recipient; and aqueous and non-aqueous sterile
suspensions that may include suspending agents or thickening agents.
The formulations may be presented in unit-dose or multi-dose containers,
for example, sealed ampoules and vials and may be stored in a freeze-
dried condition requiring only the addition of the sterile liquid carrier
immediately prior to use. The vaccine formulation may also include
adjuvant systems for enhancing the immunogenicity of the formulation,
such as oil-in water systems and other systems known in the art. The
to dosage will depend on the specific activity of the vaccine and can be
readily determined by routine experimentation.
Polypeptides of the present invention have one or more biological functions
that are of relevance in one or more disease states, in particular the
diseases of the invention hereinbefore mentioned. It is therefore useful to
is to identify compounds that stimulate or inhibit the function or level of
the
polypeptide. Accordingly, in a further aspect, the present invention
provides for a method of screening compounds to identify those that
stimulate or inhibit the function or level of the polypeptide. Such methods
identify agonists or antagonists that may be employed for therapeutic and
2o prophylactic purposes for such diseases of the invention as hereinbefore
mentioned. Compounds may be identified from a variety of sources, for
example, cells, cell-free preparations, chemical libraries, collections of
chemical compounds, and natural product mixtures. Such agonists or
antagonists so-identified may be natural or modified substrates, ligands,
2s receptors, enzymes, etc., as the case may be, of the polypeptide; a
structural or functional mimetic thereof (see Coligan et al., Current
Protocols in Immunology 1 (2):Chapter 5 (1991 )) or a small molecule.
The screening method may simply measure the binding of a candidate
compound to the polypeptide, or to cells or membranes bearing the
3o polypeptide, or a fusion protein thereof, by means of a label directly or
indirectly associated with the candidate compound. Alternatively, the
screening method may involve measuring or detecting (qualitatively or
quantitatively) the competitive binding of a candidate compound to the
polypeptide against a labeled competitor (e.g. agonist or antagonist).
;5 Further, these screening methods may test whether the candidate
compound results in a signal generated by activation or inhibition of the
polypeptide, using detection systems appropriate to the cells bearing the
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
polypeptide. Inhibitors of activation are generally assayed in the
presence of a known agonist and the effect on activation by the agonist
by the presence of the candidate compound is observed. Further, the
screening methods may simply comprise the steps of mixing a candidate
s compound with a solution containing a polypeptide of the present
invention, to form a mixture, measuring a HA-LPA-R activity in the
mixture, and comparing the HA-LPA-R activity of the mixture to a control
mixture which contains no candidate compound.
Polypeptides of the present invention may be employed in conventional
to low capacity screening methods and also in high-throughput screening
(HTS) formats. Such HTS formats include not only the well-established
use of 96- and, more recently, 384-well micotiter plates but also emerging
methods such as the nanowell method described by Schullek et al, Anal
Biochem., 246, 20-29, (1997).
~s Fusion proteins, such as those made from Fc portion and HA-LPA-R
polypeptide, as hereinbefore described, can also be used for
high-throughput screening assays to identify antagonists for the
polypeptide of the present invention (see D. Bennett et al., J Mol
Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,
20 270(16):9459-9471 (1995)).
One screening technique includes the use of cells which express receptor
of this invention (for example. transfected CHO cells) in a system which
measures extracellular pH or intracellular calcium changes caused by
receptor activation. In this technique. compounds may be contacted with
2s cells expressing the receptor polypeptide of the present invention. A
second messenger response. e.g., signal transduction, pH changes, or
changes in calcium level, is then measured to determine whether the
potential compound activates or inhibits the receptor.
Another method involves screening for receptor inhibitors by determining
3o inhibition or stimulation of receptor-mediated cAMP and/or adenylate
cyclase accumulation. Such a method involves transfecting a eukaryotic
cell with the receptor of this invention to express the receptor on the cell
surface. The cell is then exposed to potential antagonists in the presence
of the receptor of this invention. The amount of cAMP accumulation is then.
;; measured. If the potential antagonist binds the receptor, and thus inhibits
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
-, ,
receptor binding, the levels of receptor-mediated cAMP, or adenylate
cyciase, activity will be reduced or increased.
Another methods for detecting agonists or antagonists for the receptor of
the present invention is the yeast based technology as described in U.S.
s Patent 5,482,835.
Screening techniques
The polynucleotides, polypeptides and antibodies to the polypeptide of the
present invention may also be used to configure screening methods for
~o detecting the effect of added compounds on the production of mRNA and
polypeptide in cells. For example, an ELISA assay may be constructed
for measuring secreted or cell associated levels of polypeptide using
monoclonal and polyclonal antibodies by standard methods known in the
art. This can be used to discover agents that may inhibit or enhance the
is production of polypeptide (also called antagonist or agonist, respectively)
from suitably manipulated cells or tissues.
A polypeptide of the present invention may be used to identify membrane
bound or soluble receptors, if any, through standard receptor binding
techniques known in the art. These include, but are not limited to, ligand
2o binding and crosslinking assays in which the polypeptide is labeled with a
radioactive isotope (for instance, X251), chemically modified (for instance,
biotinylated), or fused to a peptide sequence suitable for detection or
purification, and incubated with a source of the putative receptor (cells,
cell membranes, cell supernatants, tissue extracts, bodily fluids). Other
2s methods include biophysical techniques such as surface plasmon
resonance and spectroscopy. These screening methods may also be
used to identify agonists and antagonists of the polypeptide that compete
with the binding of the polypeptide to its receptors, if any. Standard
methods for conducting such assays are well understood in the art.
;o Examples of antagonists of polypeptides of the present invention include
antibodies or, in some cases, oligonucleotides or proteins that are closely
related to the ligands, substrates, receptors, enzymes, etc., as the case
may be, of the polypeptide, e.g., a fragment of the ligands, substrates,
receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
the present invention but do not elicit a response, so that the activity of
the
polypeptide is prevented.
Screening methods may also involve the use of transgenic technology
and HA-LPA-R gene. The art of constructing transgenic animals is well
s established. For example, the HA-LPA-R gene may be introduced
through microinjection into the male pronucleus of fertilized oocytes,
retroviral transfer into pre- or post-implantation embryos, or injection of
genetically modified, such as by electroporation, embryonic stem cells
into host blastocysts. Particularly useful transgenic animals are so-called
to "knock-in" animals in which an animal gene is replaced by the human
equivalent within the genome of that animal. Knock-in transgenic animals
are useful in the drug discovery process, for target validation, where the
compound is specific for the human target. Other useful transgenic
animals are so-called "knock-out" animals in which the expression of the
is animal ortholog of a polypeptide of the present invention and encoded by
an endogenous DNA sequence in a cell is partially or completely
annulled. The gene knock-out may be targeted to specific cells or
tissues, may occur only in certain cells or tissues as a consequence of
the limitations of the technology, or may occur in all, or substantially all,
2o cells in the animal. Transgenic animal technology also offers a whole
animal expression-cloning system in which introduced genes are
expressed to give large amounts of polypeptides of the present invention
Screening kits for use in the above described methods form a further
aspect of the present invention. Such screening kits comprise:
~s (a) a polypeptide of the present invention;
(b) a recombinant cell expressing a polypeptide of the present invention;
(c) a cell membrane expressing a polypeptide of the present invention; or
(d) an antibody to a polypeptide of the present invention:
which polypeptide is preferably that of SEQ ID N0:2 and/or SEO ID N0:4
~o and/or SEQ ID N0:6.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise
a substantial component.
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
Glossary
The following definitions are provided to facilitate understanding of certain
terms used frequently hereinbefore.
"Antibodies" as used herein includes polyclonal and monoclonal
s antibodies, chimeric, single chain, and humanized antibodies, as well as
Fab fragments, including the products of an
Fab or other immunoglobulin expression library.
"Isolated" means altered "by the hand of man" from its natural state, i.e.,
if it occurs in nature, it has been changed or removed from its original
environment, or both. For example, a polynucleotide or a polypeptide
naturally present in a living organism is not "isolated," but the same
polynucleotide or polypeptide separated from the coexisting materials of
its natural state is "isolated", as the term is employed herein. Moreover,
a polynucleotide or polypeptide that is introduced into an organism by
is transformation, genetic manipulation or by any other recombinant method
is "isolated" even if it is still present in said organism, which organism
may be living or non-living.
"Polynucleotide" generally refers to any polyribonucleotide (RNA) or
polydeoxribonucleotide (DNA), which may be unmodified or modified
~o RNA or DNA. "Polynucleotides" include, without limitation, single- and
double-stranded DNA, DNA that is a mixture of single- and double-
stranded regions, single- and double-stranded RNA, and RNA that is
mixture of single- and double-stranded regions, hybrid molecules
comprising DNA and RNA that may be single-stranded or, more typically,
2s double-stranded or a mixture of single- and double-stranded regions. In
addition, "polynucleotide" refers to triple-stranded regions comprising
RNA or DNA or both RNA and DNA. The term "polynucleotide" also
includes DNAs or RNAs containing one or more modified bases and
DNAs or RNAs with backbones modified for stability or for other reasons.
30 "Modified" bases include, for example, tritylated bases and unusual bases
such as inosine. A variety of modifications may be made to DNA and
RNA; thus, "polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found in
nature, as well as the chemical forms of DNA and RNA characteristic of
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
_ -~ ,~
viruses and cells. "Polynucleotide" also embraces relatively short
polynucleotides, often referred to as oligonucleotides.
"Polypeptide" refers to any polypeptide comprising two or more amino.
acids joined to each other by peptide bonds or modified peptide bonds,
s i.e., peptide isosteres. "Polypeptide" refers to both short chains,
commonly referred to as peptides, oligopeptides or oligomers, and to
longer chains, generally referred to as proteins. Polypeptides may
contain amino acids other than the 20 gene-encoded amino acids.
"Polypeptides" include amino acid sequences modified either by natural
to processes, such as post-translational processing, or by chemical
modification techniques that are well known in the art. Such
modifications are well described in basic texts and in more detailed
monographs, as well as in a voluminous research literature.
Modifications may occur anywhere in a polypeptide, including the peptide
backbone, the amino acid side-chains and the amino or carboxyl termini.
It will be appreciated that the same type of modification may be present
to the same or varying degrees at several sites in a given polypeptide.
Also, a given polypeptide may contain many types of modifications.
Polypeptides may be branched as a result of ubiquitination, and they may
2o be cyclic, with or without branching. Cyclic, branched and branched
cyclic polypeptides may result from post-translation natural processes or
may be made by synthetic methods. Modifications include acetylation,
acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment
of flavin, covalent attachment of a heme moiety, covalent attachment of a
~s nucleotide or nucleotide derivative, covalent attachment of a lipid or
lipid
derivative, covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation, formation of covalent.
cross-links, formation of cystine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
o hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to
proteins such as arginylation, and ubiquitination (see, for instance,
Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton,
;s W. H. Freeman and Company, New York, 1993; Wold, F., Post-
translational Protein Modifications: Perspectives and Prospects, 1-12, in
Post-translational Covalent Modification of Proteins, B. C. Johnson, Ed.,
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
-~s _
Academic Press, New York, 1983; Seifter et al., "Analysis for protein
modifications and nonprotein cofactors", Meth Enzymol, 182, 626-646,
1990, and Rattan et al., "Protein Synthesis: Post-translational
Modifications and Aging", Ann NY Acad Sci, 663, 48-62, 1992).
s "Fragment" of a polypeptide sequence refers to a polypeptide sequence
that is shorter than the reference sequence but that retains essentially the
same biological function or activity as the reference polypeptide.
"Fragment" of a polynucleotide sequence refers to a polynucloetide
sequence that is shorter than the reference sequence of SEQ ID N0:1
~o and/or SEQ ID N0:3 and/or SEQ ID N0:5..
"Variant" refers to a polynucleotide or polypeptide that differs from a
reference polynucleotide or polypeptide, but retains the essential
properties thereof. A typical variant of a polynucleotide differs in
nucleotide sequence from the reference polynucleotide. Changes in the
is nucleotide sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions, additions,
deletions, fusions and truncations in the polypeptide encoded by the
reference sequence, as discussed below. A typical variant of a
~o polypeptide differs in amino acid sequence from the reference
polypeptide. Generally, alterations are limited so that the sequences of
the reference polypeptide and the variant are closely similar overall and,
in many regions, identical. A variant and reference polypeptide may differ
in amino acid sequence by one or more substitutions, insertions,
2s deletions in any combination. A substituted or inserted amino acid
residue may or may not be one encoded by the genetic code. Typical
conservative substitutions include Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn,
Gln;
Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of a polynucleotide or
polypeptide may be naturally occurring such as an allele, or it may be a
3o variant that is not known to occur naturally. Non-naturally occurring
variants of polynucleotides and polypeptides may be made by
mutagenesis techniques or by direct synthesis. Also included as variants
are polypeptides having one or more post-translational modifications, for
instance glycosylation, phosphorylation, methylation, ADP ribosylation
;s and the like. Embodiments include methylation of the N-terminal amino
acid, phosphorylations of serines and threonines and modification of C-
terminal glycines.
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
- ?6 -
"Allele" refers to one of two or more alternative forms of a gene occuring
at a given locus in the genome.
"Polymorphism" refers to a variation in nucleotide sequence (and
encoded polypeptide sequence, if relevant) at a given position in the
s genome within a population.
"Single Nucleotide Polymorphism" (SNP) refers to the occurence of
nucleotide variability at a single nucleotide position in the genome, within
a population. An SNP may occur within a gene or within intergenic
regions of the genome. SNPs can be assayed using Allele Specific
io Amplification (ASA). For the process at least 3 primers are required. A
common primer is used in reverse complement to the polymorphism
being assayed. This common primer can be between 50 and 1500 bps
from the polymorphic base. The other two (or more) primers are identical
to each other except that the final 3' base wobbles to match one of the
two (or more) alleles that make up the polymorphism. Two (or more)
PCR reactions are then conducted on sample DNA, each using the
common primer and one of the Allele Specific Primers.
"Splice Variant" as used herein refers to cDNA molecules produced from
RNA molecules initially transcribed from the same genomic DNA
2o sequence but which have undergone alternative RNA splicing.
Alternative RNA splicing occurs when a primary RNA transcript
undergoes splicing, generally for the removal of introns, which results in
the production of more than one mRNA molecule each of that may
encode different amino acid sequences. The term splice variant also
2s refers to the proteins encoded by the above cDNA molecules.
"Identity" reflects a relationship between two or more polypeptide
sequences or two or more polynucleotide sequences, determined by
comparing the sequences. In general, identity refers to an exact
nucleotide to nucleotide or amino acid to amino acid correspondence of
~o the two polynucleotide or two polypeptide sequences, respectively, over
the length of the sequences being compared.
"% Identity" - For sequences where there is not an exact
correspondence, a "% identity" may be determined. in general, the two
sequences to be compared are aligned to give a maximum correlation
;s between the sequences. This may include inserting -"gaps" in either one
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
or both sequences, to enhance the degree of alignment. A % identity
may be determined over the whole length of each of the sequences being
compared (so-called global alignment), that is particularly suitable for
sequences of the same or very similar length, or over shorter, defined
s lengths (so-called local alignment), that is more suitable for sequences of
unequal length.
"Similarity" is a further, more sophisticated measure of the relationship
between two polypeptide sequences. In general, "similarity" means a
comparison between the amino acids of two polypeptide chains, on a
io residue by residue basis, taking into account not only exact
correspondences between a between pairs of residues, one from each of
the sequences being compared (as for identity) but also, where there is
not an exact correspondence, whether, on an evolutionary basis. one
residue is a likely substitute for the other. This likelihood has an
~s associated "score" from which the "% similarity" of the two sequences
can then be determined.
Methods for comparing the identity and similarity of two or more
sequences are well known in the art. Thus for instance, programs
available in the Wisconsin Sequence Analysis Package, version 9.1
20 (Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984, available from
Genetics Computer Group, Madison, Wisconsin, USA), for example the
programs BESTFIT and GAP, may be used to determine the % identity
between two polynucleotides and the % identity and the % similarity
between two polypeptide sequences. BESTFIT uses the "local
25 homology" algorithm of Smith and Waterman (J Mol Biol, 147,195-197,
1981, Advances in Applied Mathematics, 2, 482-489, 1981 ) and finds the
best single region of similarity between two sequences. BESTFIT is
more suited to comparing two polynucleotide or two polypeptide
sequences that are dissimilar in length, the program assuming that the
~o shorter sequence represents a portion of the longer. In comparison. GAP
aligns two sequences, finding a "maximum similarity", according to the
algorithm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970).
GAP is more suited to comparing sequences that are approximately the
same length and an alignment is expected over the entire length.
Preferably, the parameters "Gap Weight" and "Length Weight" used in
each program are 50 and 3, for polynucleotide sequences and 12 and 4
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
_ .8 _
for polypeptide sequences, respectively. es being compared are optimally
aligned.
Other programs for determining identity and/or similarity between
sequences are also known in the art, for instance the BLAST family of
programs (Altschul S F et al, J Mol Biol, 215, 403-410, 1990, Altschul S F
et al, Nucleic Acids Res., 25:389-3402, 1997, available from the National
Center for Biotechnology Information (NCB/), Bethesda, Maryland, USA
and accessible through the home page of the NCB/ at
www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, Methods in
~o Enzymology, 183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat
Acad Sci USA, 85, 2444-2448,1988, available as part of the Wisconsin
Sequence Analysis Package).
Preferably, the BLOSUM62 amino acid substitution matrix (Henikoff S
and Henikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is
is used in polypeptide sequence comparisons including where nucleotide
sequences are first translated into amino acid sequences before
comparison.
Preferably, the program BESTFIT is used to determine the % identity of a
query polynucleotide or a polypeptide sequence with respect to a
2o reference polynucleotide or a polypeptide sequence, the query and the
reference sequence being optimally aligned and the parameters of the
program set at the default value, as hereinbefore described.
"Identity Index" is a measure of sequence relatedness which may be
used to compare a candidate sequence (polynucleotide or polypeptide)
2s and a reference sequence. Thus, for instance, a candidate
polynucleotide sequence having, for example, an Identity Index of 0.95
compared to a reference polynucleotide sequence is identical to the
reference sequence except that the candidate polynucleotide sequence
may include on average up to five differences per each 100 nucleotides
~o of the reference sequence. Such differences are selected from the group
consisting of at least one nucleotide deletion, substitution, including
transition and transversion, or insertion. These differences may occur at
the 5' or 3' terminal positions of the reference polynucleotide sequence or
anywhere between these terminal positions, interspersed either
;5 individually among the nucleotides in the reference sequence or in one or
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
_ ?a _
J
more contiguous groups within the reference sequence. In other words,
to obtain a polynucleotide sequence having an Identity Index of 0.95
compared to a reference polynucleotide sequence, an average of up to 5
in every 100 of the nucleotides of the in the reference sequence may be
s deleted, substituted or inserted, or any combination thereof, as
hereinbefore described. The same applies mutatis mutandis for other
values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.
Similarly, for a polypeptide, a candidate polypeptide sequence having, for
example, an Identity Index of 0.95 compared to a reference polypeptide
to sequence is identical to the reference sequence except that the
polypeptide sequence may include an average of up to five differences
per each 100 amino acids of the reference sequence. Such differences
are selected from the group consisting of at least one amino acid
deletion, substitution, including conservative and non-conservative
is substitution, or insertion. These differences may occur at the amino- or
carboxy-terminal positions of the reference polypeptide sequence or
anywhere between these terminal positions, interspersed either
individually among the amino acids in the reference sequence or in one
or more contiguous groups within the reference sequence. In other
?o words, to obtain a polypeptide sequence having an Identity Index of 0.95
compared to a reference polypeptide sequence, an average of up to 5 in
every 100 of the amino acids in the reference sequence may be deleted,
substituted or inserted, or any combination thereof, as hereinbefore
described. The same applies mutatis mutandis for other values of the
Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.
The relationship between the number of nucleotide or amino acid
differences and the Identity Index may be expressed in the following
equation:
na s xa - (xa ~ I),
;o in which:
na is the number of nucleotide or amino acid differences,
xa is the total number of nucleotides or amino acids in SEQ ID N0:1
and/or SEQ ID N0:3 andlor SEQ ID N0:5 or SE0 ID N0:2 andlor SEQ
ID N0:4 andlor SEQ ID N0:6, respectively,
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
- 30 -
I is the Identity Index ,
~ is the symbol for the multiplication operator, and
in which any non-integer product of xa and I is rounded down to the
nearest integer prior to subtracting it from xa.
s "Homolog" is a generic term used in the art to indicate a polynucleotide or
polypeptide sequence possessing a high degree of sequence relatedness
to a reference sequence. Such relatedness may be quantified by
determining the degree of identity and/or similarity between the two
sequences as hereinbefore defined. Falling within this generic term are
to the terms "ortholog", and "paralog". "Ortholog" refers to a polynucleotide
or polypeptide that is the functional equivalent of the polynucleotide or
polypeptide in another species. "Paralog" refers to a polynucleotideor
polypeptide that within the same species which is functionally similar.
is "Fusion protein" refers to a protein encoded by two, unrelated, fused
genes or fragments thereof. Examples have been disclosed in US
5541087, 5726044. In the case of Fc-HA-LPA-R, employing an
immunoglobulin Fc region as a part of a fusion protein is advantageous
for performing the functional expression of Fc-HA-LPA-R or fragments of
2o HA-LPA-R, to improve pharmacokinetic properties of such a fusion
protein when used for therapy and to generate a dimeric Fc-HA-LPA-R
The Fc- HA-LPA-R DNA construct comprises in 5' to 3' direction, a
secretion cassette, i.e. a signal sequence that triggers export from a
mammalian cell, DNA encoding an immunoglobulin Fc region fragment,
2s as a fusion partner, and a DNA encoding Fc-HA-LPA-R or fragments
thereof. In some uses it would be desirable to be able to alter the intrinsic
functional properties (complement binding, Fc-Receptor binding) by
mutating the functional Fc sides while leaving the rest of the fusion
protein untouched or delete the Fc part completely after expression.
All publications and references, including but not limited to patents and
patent applications, cited in this specification are herein incorporated by
reference in their entirety as if each individual publication or reference
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
- 31 -
were specifically and individually indicated to be incorporated by
reference herein as being fully set forth. Any patent application to which
this application claims priority is also incorporated by reference herein in
its entirety in the manner described above for publications and
s references.
Examples
Example 1
Mammalian Cell Expression
to The receptors of the present invention are expressed in either human
embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. To
maximize receptor expression, typically all 5' and 3' untranslated regions
(UTRs) are removed from the receptor cDNA prior to insertion into a pCDN
or pCDNA3 vector. The cells are transfected with individual receptor
is cDNAs by lipofectin and selected in the presence of 400 mg/ml 6418. After
3 weeks of selection, individual clones are picked and expanded for further
analysis. HEK293 or CHO cells transfected with the vector alone serve as
negative controls. To isolate cell lines stably expressing the individual
receptors, about 24 clones are typically selected and analyzed by Northern
2o blot analysis. Receptor mRNAs are generally detectable in about 50% of
the 6418-resistant clones analyzed.
Example 2
Ligand bank for binding and functional assays.
A bank of over 600 putative receptor ligands has been assembled for
2s screening. The bank comprises: transmitters, hormones and chemokines
known to act via a human seven transmembrane (7TM) receptor; naturally
occurring compounds which may be putative agonists for a human 7TM
receptor, non-mammalian, biologically active peptides for which a
mammalian counterpart has not yet been identified; and compounds not
3o found in nature, but which activate 7TM receptors with unknown natural
ligands. This bank is used to initially screen the receptor for known ligands,
using both functional (i.e . calcium, cAMP, microphysiometer, oocyte
electrophysiology, etc, see below) as well as binding assays.
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
- JG -
Example 3
Ligand Binding Assays
Ligand binding assays provide a direct method for ascertaining receptor
pharmacology and are adaptable to a high throughput format. The purified
ligand for a receptor is radiolabeled to high specific activity (50-2000
Ci/mmol) for binding studies. A determination is then made that the
process of radiolabeling does not diminish the activity of the ligand towards
its receptor. Assay conditions for buffers, ions, pH and other modulators
~o such as nucleotides are optimized to establish a workable signal to noise
ratio for both membrane and whole cell receptor sources. For these
assays, specific receptor binding is defined as total associated radioactivity
minus the radioactivity measured in the presence of an excess of unlabeled
competing ligand. Where possible, more than one competing ligand is
i> used to define residual nonspecific binding.
Example 4
Functional Assay in Xenopus Oocytes
Capped RNA transcripts from linearized plasmid templates encoding the
receptor cDNAs of the invention are synthesized in vitro with RNA
polymerases in accordance with standard procedures. In vitro transcripts
are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes
are removed from adult female toads. Stage V defolliculated oocytes are
obtained, and RNA transcripts (10 ng/oocyte) are injected in a 50 n1 bolus
?5 using a microinjection apparatus. Two electrode voltage clamps are used
to measure the currents from individual Xenopus oocytes in response to
agonist exposure. Recordings are made in Ca2+ free Barth's medium at
room temperature. The Xenopus system can be used to screen known
ligands and tissuelcell extracts for activating ligands.
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
Example 5
Microphysiometric Assays
Activation of a wide variety of secondary messenger systems results in
extrusion of small amounts of acid from a cell. The acid formed is largely
s as a result of the increased metabolic activity required to fuel the
intracellular signaling process. The pH changes in the media surrounding
the cell are very small but are detectable by the CYTOSENSOR
microphysiometer (Molecular Devices Ltd., Menlo Park, CA). The
CYTOSENSOR is thus capable of detecting the activation of a receptor
io which is coupled to an energy utilizing intracellular signaling pathway
such
as the G-protein coupled receptor of the present invention.
Example 6
Extract/Cell Supernatant Screening
is A large number of mammalian receptors exist for which there remains, as
yet, no cognate activating ligand (agonist). Thus, active ligands for these
receptors may not be included within the ligands banks as identified to date.
Accordingly, the 7TM receptor of the invention is also functionally screened
(using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc.,
2o functional screens) against tissue extracts to identify natural ligands.
Extracts that produce positive functional responses can be sequencially
subfractionated until an activating ligand is isolated identified.
Example 7
~s Calcium and cAMP Functional Assays
7TM receptors which are expressed in HEK 293 cells have been shown to
be coupled functionally to activation of PLC and calcium mobilization and/or
cAMP stimuation or inhibition. Basal calcium levels in the HEK 293 cells in.
receptor-transfected or vector control cells were observed to be in the
normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant
receptors are loaded with fura 2 and in a single day > 150 selected ligands
WO 01/09313 CA 02381437 2002-O1-28 PCT/EP00/06854
- 34 -
or tissue/cell extracts are evaluated for agonist induced calcium
mobilization. Similarly, HEK 293 cells expressing recombinant receptors
are evaluated for the stimulation or inhibition of cAMP production using
standard cAMP quantitation assays. Agonists presenting a calcium
s transient or cAMP flucuation are tested in vector control cells to determine
if
the response is unique to the transfected cells expressing receptor.
WO 01/09313 CA 02381437 2002-O1-28 p~/EppO/06854
SEQUENCE LISTING
<110> Merck Patent GmbH
j
<120> New G-protein coupled recectcr
<130> HALPAARKDWS
<190>
< 1 .? 1 >
<1nC> c
Ij <170> PatentIn Ver. 2.1
<21C>
<211> 111C
<2';2> DNA
?p <<13> Homo sapiens
tLLJ>
<221> :~.isc_teature
<222> !'3) .i'_110)
2j <223> v9ainly translated ORF
<220>
<221> GDS
<222> (,3)..(1110)
<400>
ataaatatta cactccctcc accattccag catcctgacc tcagtccatt gcttagatat 60
agttttgaaa cc atg get ccc act ggt ttg agt tcc ttg acc gtg aat agt 111
3j Met Ala Pro Thr Gly Leu Se. Ser Leu Thr Va'_ Asn Ser
.C
aCa - rgCt gtg CCC aCa aCa CCa gCa gCa tt~ aag agC C,... aaC . ___ _JG
mhr _..a Val Pr0 Thr Thr Pr0 i-:la A-a Phe ~',iS SEr _..eu =.Sn _,2~,: ~rG
-.. -J 20 25
ctt cap atc acc c't tct get ata ate ata ttc a~~ ctg t~_ gtg ..._ 207
Leu Gln .1E T::r LeU Ser Ala .'°_ M~. Ile ?.~2 .1e Lel: Phe ~~'al
Je=
~:0 :3
4j
ttt C... ggg aaC ttg gtt gt~ tgC ~_~ atg . - taC Caa aaa ~C= ~CC 2~J
Dhc :,a;~ Gi'Y _ASn Leu Val '~'a1 G'JS :J=l:: Mt''~ ~.'a~- ~'!" G_.': LyS Ala
.-.__
a .. _ . .
.. _
j0 atg agC tCt gCa at~ aaC atC CtC ...-t gCC % , ..ta gC~ '-.._ gCa ''.7,aC
.5~3
"e. .. J Ser ~ I1 c ,.~.,Sn _ ~ a Lel: L2',: Ala Je=' _.°U i:'_a P~:e
.la .'-.S:
w:~a -' --
_J 7J
atC ttg Ctt gCa gtg Ctg aaC atg CCC ttt ~r"C Ctg gta aCt att Ct~ .~J~1
jj Met T e'~ LeU Ala '~jdl Leu ASn rev PrG Phe Al' a L°u '~aiJ i'hr Ile
L2;_
.r c~ fy. Gv
a~. .u_... ~vC tgg att ttt .rug CGG tW tt~ , Gv'.. tr'.~ ur G , ..m
'Jr; ~ T ~_P ~rl,c ~ I -y~ . iS '_ n~ : ).C ~~~ t~r~.~'. 'JG_ Ser~ .'T'~S'_~
~y==
WU 01109313 CA 02381437 2002-0l-28 PC'lyEpQO/06854
95 1CC 105
ttL ttc tgg tta LLt gtg ata gaa gga gta gcC atc ctg ctc atc att 447
Phe Phe Trp Leu Phe Val 112 Glu Gly Val Aia ile Leu Leu Ile Ile
~ 110 115 i20 125
agc ata gaL agg Ltc ctt att ata gtc cag acg Cag gat aag cta aac 495
Ser Ile Asp Arg Phe Leu Ile Ile Vai Gln Arc Gln Asp Lys Leu Asn
.30 135 i4C
cca _tat aga get aag gtt cLg atL gCa gtL _~_ tag gca act tcc LtL 543
Dre Tyr Arg Aia l.ys Vai Leu Ile Aia Val Ser Trp Aia Thr Ser Phe
145 15C 155
1~ tgt gta get ttL cct tta gec gta gga aae ccc gac ctg cag ata cct 591
Cys Val Ala Phe Pro Leu Ala Val G'_.y Asn Prc Asp Leu Gln Ile Prc
1 6C 165 :7C
LCC Cga gCL CCC Cag tgt gtg ttL ggg ta~ aCa aCC dat CCa ggC LaC 639
~0 Ser Arg Aia Pro Gln Cys Va'~ Phe G~-y Tyr Thr Thr Asn Pro Gly Tyr
175 18C 185
cag get tar gtg att ttg att tcL cLc att tct ttc LLc ata ccc ttc 687
Gln Ala TVr Val Ile Leu Iie Ser Leu Ile Ser Phe Phe I12 Pro Phe
~5 lap 195 20C 2C5
CtQ -gLa aLa Ctg =aC tCd tLt atg gCC dta CtC aaC aCC Ctt Cgg CaC 735
Leu Va'_ Ile Leu Tyr Se_- D:ne Me. G'y Ile Leu ~.sn Thr Leu Arg His
210 215 220
aaL gcc tLg agg atc cat agc tac cct gaa get ata tgc ctc agc cag 783
Asn Ala Leu Arg Ile His Se. Tyr Pro Giu Gly Ile Cys Leu Ser Gin
225 23C 235
3~gcc agcaaa ctgggtcLcaLg agLcLgcagaga cctLtccagatg agc 831
Ala SerLys LeuGlyLeuMeL SerLe:.Gln.=. ProPheGlnMeL Ser
,
245 250
att gacatg ggcLttaaaaca cgtgccttcacc actattttgatt ctc 879
40Ile AsoMet GlysheLysThr ArgA'~aPheThr ThrI'_eLeuIle Leu
255 2o'C 265
ttt getgtc ttcattgtctgc tgggccccaLLc accacttacagc ctt 927
P ' '~ PheI 'la'''wsTrpA'_aPrc'.~ Tt-:r1'~rTyrSer Lev
he A1a' 1e _
~.a~
4527C 275
C)t~gCadCa ttCagtaagCaC _~_taCtaL.. CaCaaCtLLttt gag 975
,
Val Alamhr PheSerLys::isPheT;'rTyrG_n HisAsnPhePhe Glu
29C 295 3OC
?o
aLL agCaCC tggCtdCtgLgg CtCt. LaCC__ dagLCtgCattg daL iO23
v
I' SerThr Trol,euLev_ Lev.s TVr~__ LysSerAlaLeu As.~.
e ''
v 3C5 _ .,=7 315
CCg CtgatC taCLdCLggagg at.albdaa.__ Ca_gatgCttgC C 1C7':
,
Pr0 LeUile '.T'V=='yrm_,-C~,.r71 ;,1'cL,/Sa:~._:11SAspA~dCyS ~2U
~a
32C ~~~ 330
QcC atCctg ~.._dagtCCtC= d<7CuCttt.~.CCy C~3~CLCCC:CIgLCdCa J.~L
60A=T .'~~et:~e=PrcLysSer_.._LSw:~r~eLevPrc C_,~Leu
..~~ =~1~' ..',
5
2
WO 01/09313 CA 02381437 2002-O1-28 p~~pp0/06854
caaagcgacg gatacgtcct agtgctgtct atgtgtgtgg ggaacatcgg acggtggtgt 1180
ga "g2
j
<210> 2
<211> 396
<212> PRT
<213> Homo Sapiens
<900> 2
Met Ala Pro Thr Gly Leu Ser Ser Leu Thr Vai Asn Ser Thr Ala Val
1 5 10 i5
Ij
Prc Thr Thr Pro Ala Ala Phe Lys Ser Leu Asn Leu Pro Leu Gln Ile
25 3C
Thr Leu Ser Ala Ile Met Ile the Ile Leu Phe Va1 Ser Phe Leu Gly
?0 35 90 45
Asn Leu Val Val Cys Leu Met Val T_~~r Gin Lys Ala Aia Met Arg Ser
5C 55 6C
Ala Ile Asn Ile Leu Leu Ala Ser Leu Ala Phe Ala Asp Met Leu Leu
65 70 75 80
Ala Val Leu Asn Met Pro Phe Ala Leu Vai Thr Ile Leu Thr Thr Arg
85 90 95
Trp Ile Phe G1y Lys Phe Phe Cys Arg Va'_ Ser Ala Met Phe ?he Trp
1O0 1.,5 110
Leu Phe Val Ile Glu Gly Val Ala Ile Leu Leu Ile Ile Ser ile Asp
3j 115 120 125
Arg Phe Leu Ile Ile Val G1n Arg Gin Asp Lys Leu Asn Pro Tyr Arg
13C 135 1.~0
Ala Lys Val Leu Iie Ala Vai Ser Trp Ala Thr Ser Phe Cys 'Jai Ala
195 150 _.~ 160
Phe Pro Leu Ala '!al Gly Asn Pro Asp Leu ~ln Ile Pro Ser Arg Ala
6 5 1 % 0 ~ -.
Pro Gin Cys Val Phe Gly Tyr Thr T::r Asn ?ro Giy Tyr G1W-:la Tyr
180 185 190
Val Ile Leu Ile Ser Leu Ile Ser Phe Phe Iie Pro Phe Lee 'Jal Ile
SO 195 20O 20'5
Leu Tyr Ser Phe Met Gly Ile Leu Asn Thr Leu A=a His Asn A'~ L=~.:
L10 21~ G20
5j Arg Ile His Ser Tyr Pro Glu Gly Iie Cys Leu Ser Gln Ala Ser Lys
225 230 235 290
Leu Gly Leu Met Ser Leu Gln Arg Pro Phe Gln Lfet Ser Ile Asp Met
2~5 250 255
Gly; :hc Lys Tnr arA1a Pn2 Thr Thr Il~ Leu iie Lcu F::e Ri3 Val
3
WO 01/09313 CA 02381437 2002-O1-28 p~~ppp/p6854
260 ~~G - .
Phe I1e Vai Cys Trp Ala Pro Phe Thr Thr Tyr Ser Leu Vai A~_a Thr
275 280 285
Phe Ser Lys His Phe Tyr Tyr Gln His Asn Phe Phe Glu I1e Ser Thr
2g0 295 300
Try Leu Leu Trp Leu Cys Tyr Leu Lys Ser Ala Leu Asn Pro Leu Ile
305 310 315 320
Tyr Tyr Trp Arg Ile Lys Lys Phe His Asp Ala Cys Leu Asp Met Met
325 330 335
1~ Pro Lys Ser Phe Lys Phe Leu Prc G.~. Leu
390 3~'-
<210> 3
<211> 1182
<212> DNA
<213> Homo sapiens
?5 <220>
<221> CDS
<222> (1)..(1182)
<220>
feature
<221> misc
_
<222> (1). (1182)
<223> Extended
ORF-1
<900> 3
atg aat att aca cctccaccattccag catcctgacctc agtcca 98
ctc
Met Asn Iie Thr ProProProPheGln HisProAspLeu SerPro
Leu
5 1 _
0 ..
ett aga tat agt tttgaaacea=gget cccactggtttg agttcc 96
ttg - -
Leu Leu Arg Tyr PheGluThrMatAla ProThrG1yLee SerSer
Ser
20 =5
ace gtg aat agt acagetgtac~caca acaccagca?ca ._.aag _..
tt g
Leu Thr 'Jal Asn ThrAlaVa_PrcTi:rThr:roAla=-- -::~
Ser
35 ~~ 45
c cta aac ttg cttcagatcaccctt tctgetata~=g atattC 1gL
cct
a
g LeuGlnIleThrLeu SerAlaiieMet _'_ePhe
Ser Leu Asn Leu
Pro
50 55 50
att ctg ttt gtg tttcttgggaacttg gttgttr_gcctc atggtt 2_-'-:
tct
Ile Leu Phe Val PheLeuGlyAsnLeu ~Ja'~ValCysLeu Met'Jal
Ser
65
70 ~5 80
tac caa aaa get atgaggtctgcaatt aacatcctc~~~ gccagc 28
gcc
Gln Lys Ala Ala MetArgSerA'_aIle AsnIleLeuL2~.:AlaSer
T
yr
85 90 y5
Cta gCt rtt gCa atgttgCtt~Cagtg CtgdaC3tg~__ _t=gCC ~?=
gaC
~ : LeuLeuAlaVai Lei.:AsnMetFre rh Ala
Le~.: Ala Phe et
Ala Asp
100 105 110
4
WO 01/09313 CA 02381437 2002-O1-28 p~/Ep00/p6854
ctg gtaactatt cttact acccgatggatt~_~ .ggaaattc'~~ tgt 38i
_ __
Leu ValThrIle LeuThr ThrArgTrpIie?he GlyLysPhePhe Cys
115 i20 125
agg gtatctget atgttt ttctggttatttgtg atagaaggagta gcc 432
r
Arc ValSerAla MetPhe PheTrpLeuPheVal IleGluGlyVal Aia
130 135 1~0
10atc ctgctcatc attagc atagata~gttcctt attatagtccag agg 980
Ile LeuLeuIle IieSer IleAspArgPheLeu IleIleValGln Arg
145 150 .55 160
cag gataagcta aaccca tatagagetaaggtt ctgattgcagtt tct 528
1~Gln AspLysLeu AsnPrc TyrArgAlaLysVa'1LeuI1eAlaVal Ser
165 17C '~75
tgg gcaacttcc ttttgt gtagett_~ccttta gccgtaggaaac c~.~576
Trp AlaThrSer ?heCys ValAlaPieProLeu AlaValGlyAsr.?rc
?0 180 1S5 19C
gac ctgcagata ccttcc cgagetceccagtgt gtgtttgggtac aca 624
Asp LeuGlnIle ProSer ArgAlaPrcGlnC,;sValPheGlyTyr '_"hr
195 20C 205
ac aatccaggc taccag gettatg att=tg ~__tctctcatt .,._
c
Thr AsnProG1y TyrG'_~._1aTyrVa'_IleLe~~I-~eSerLeu=~~eSer
210 215 220
30ttc ttcataccc ttcctg gtaatactgtactca tttatgggcata ctc 72G
Phe PheIlePro PheLeu ValIleLeuTyrSer PheMetGlyIle Leu
225 23C 235 290
aac acccttcgg cacaat gccttgagaatccat acctaccctgaa ggt "66
3~Asn ThrLeuArg HisAsn AlaLeuArcIle::1sSerTyrProGlu ;'~y
295 250 255
at tgcctcagc caggcc agcaaactgggtctc atgagtctgcag aga 816
a
Ile CysLeuSer GlnAla SerLysLeuGlyLeu MetSerLeuGl_~.Arg
40 260 205 27C
cct ttccagatg agcatt gacatgggctttaaa acacgtgcct,...acc 96
Pro PheGlnMet SerIle AspMetG~wPheLys T'-:rArgAlaP::e~:;r
275 280 285
45
act at ttgatt ctcttt getgtcttcattarc ', tgggeccca tt..912
t
Thr IleLeuIle LeuPhe .':1aValP:-:eIle'dalCysTrpAlaPro ?he
290 295
50acc acttacagc cttgtg gcaacat..~ag-_aag cacttttactat cag ._.,
_
Thr ThrT Ser LeuVa'_AlaThrPheSerLys HisPheTyrTyr ,__..
vr
305 _ 310 315 320
cac aactttttt gagatt agcacctggctactg tggctctgctac ctc
-.
5~His AsnPhePhe G1uIle SerThrT-cLeuLeu TrpLeuCysTyr Leu
325 330 335
aa tctgcattg aatccg ctgatc___tac-gg aggattaagaaa =tc '_~~.,_
c
Lv= SerrlaLeu AsnPrc LeuI1-Tyr"',T=~ A=g=_
rG C P
Ly I.y Ph_
60~ 39C J ?5U
...
WO 01/09313 CA 02381437 2002-O1-28 p~/EppO/06854
cat gat get tgc ctg gac atg atg cct aag tec t~.. aag ttt ttg ccg 1109
His Asp Ala Cys Leu Asp Met Met Pro Lys Ser Phe Lys Phe Lev ?ro
355 360 365
cag ctc cct ggt cac aca aag cga cgg ata cgt cct agt get gtc tat 1152
Gln Leu Pro Gly His Thr Lys Arg Arg Ile Arg Pro Ser Aia Va'_ Tyr
370 375 380
gtg tgt ggg gaa cat cgg acg gtg gtg tga 1182
Val Cys G1y Glu His Arg Thr Val Vai
385 390
<210> 4
l5 <211> 393
<212> PRT
<213> Homo Sapiens
<900> 4
Met Asn Tie Thr Leu Pro Pro Pro Phe Gln His ?ro Asp L2u Ser ?ro
5 10 i5
Leu Leu Arg Tyr Ser Phe Glu Thr Met Ala Pro TI-:r Gly Leu Ser Ser
20 25 3C
Leu Thr Val Asn Ser Thr Ala Val Pro Thr Thr Pro Ala Ala Phe Lys
35 40 45
Ser Leu Asn Leu Pro Leu G'_r. Ile Thr Leu Ser A'_a Ile Met .1e ?he
5p .,., 00
Ile Leu Phe Val Ser Phe Leu Gly Asn Lev 'Jal '~:al Cys Leu Me. ~.'al
65 70 75 80
Tyr Gin Lys Aia Aia Met nrg Ser Ala Iie Asn lie Leu Leu Ala Ser
85 90 95
Leu Ala Phe Ala Asp Met Leu Leu Aia Val Leu Asn Met Pro Phe Ala
100 105 11C
Leu Val Thr Ile Leu Thr Thr Arg Trp Ile Phe Giy Lys Phe Phe Cys
115 12C 125
Arg Val Ser Ala Met Phe Phe Trp L2u Phe Vai Ile G'_u G1,,- ~Ia~: =.lo
13C -~3~ 19C
Ile Leu Leu Ile Iie Ser Ile Asp Arg Phe Leu T_le Ile Val Gln Arg
145 150 155 '.60
Gln Asp Lys Leu Asn Pro Tyr Arg Ala Lys Val Leu Ile Ala Val Ser
165 17C 1~5
Trp Ala Thr Ser Phe Cys Val Ala Phe Fro Leu Ala Val Gly Asn ?ro
180 185 19C
Aso Leu G1 r Ile Pro Ser Fr g Ala P:c G1.~. Cys '% a' Phe G_'; T:~- "'~r
' 195 200 2C5
Thr ~.sr. Prc Gly Tyr Gln Ala Tyr 'Jal I12 Leu ~'_e Ser Leu Ile Ser
210 215 220
Phe Phe Ile Pro Phe Leu Vai Ile Leu Tyr Ser Phe Met Giy I12 Leu
225 230 235 290
Asn Thr Leu Arg His Asn Ala Leu Arg Ile His Ser Tyr Pro Glu Giy
295 250 255
Ile Cys Le'.: Ser G'_n Ala Ser Lys Leu Gly Leu Met Ser Leu G1~: Arg
260 255 270
Pro °he Gln Met Ser Ile Asp Met G~~;~ Phe Lys Thr Arg Aia Phe Thr
275 280 285
Thr Ile Leu Ile Leu Phe Ala Val Fhe Iie V al Cys Trp Ala Pro ?he
290 295 300
Thr Thr Tvr Ser Leu Val A1a Thr Phe Ser Lys His Phe TVr Tyr Gln
305 J 31C 315 32C
H'_~ ~Sr'. ?.rW ~:'?° G1U T!e Ser T.nr li: _,.~'~ L°U _'rp
LEL1 W~ TVr Leli
BLS ._"
6
WD 01/09313 CA 02381437 2002-O1-28 p~/EppO/06854
acc cga tgg att ttt ggg aaa t~.. ttc tg~ agg gta tet get atg ttt 480
Thr Arg Trp Ile Phe Gly Lys Phe P!-:e Cys Arg Val Ser Ala Met Phe
145 150 155 160
ttc tgg tta ttt gtg ata gaa gga gta gcc atc ctg ctc atc att agc 528
Phe Trp Leu Phe Val Ile Giu Gly Val A'_a Iie Leu Leu Ile Ile Ser
16 5 _ ", 17 5
l0 ata gat agg ttc ctt att ata gtc cag agg cag gat aag cta aac cca 5~6
Iie Asp Arg Phe Leu Ile Ile Val Gln Arg Gin =.sp Lys Leu Asn ?ro
180 185 19C
tat aga get aag gtt ctg att gca gtt tct tgg gca act tcc ttt tgt 629
Tyr Arg Ala Lys Val Leu Ile Aia Val Ser Trp Aia Thr Ser Phe Cys
195 200 205
gta get ttt cct tta gcc gta gga aac ccc gac ctg cag ata cct tcc 672
Val Ala Phe Pro Leu Ala Val Gly Asn Prc Asp Leu Gln Ile Pro Ser
210 215 220
cga __get ccc cag tgt gtg ttt gag tac aca acc aat cca ggc tac cag 720
Arg Ala Pro Gln Cys Val Phe Giyr Ty~- T:~.. Trr Asn Pro Gi f Tyr Gln
225 230 _35 24G
get tat gtg att ttg att tct ctc att tc= ttc ttc ata ecc ttc ctg 768
Ala Tyr Val Ile Leu Ile Ser Leu I--a Ser she Pi:e Iie Pro Phe Leu
245 25~~ 255
gtd atd Ctg taC ti:d ttt atg gg': a=a C''..'~ dCC o::C Ctt : gg CaC aat ~JIt7
Val Ile Leu Tyr Ser Phe Met Gly I~_e Lev As.~. '_'!-:r Leu Arg His Asn
260 20~ 270
gcc ttg agg atc cat agc tac cct gaa ggt ata tgc ctc agc cag gcc 864
Ala Leu Arg Ile His Ser Tyr Pro Giu G'!_; Ile Cys Leu Ser Gln Ala
275 280 285
ag~ -aaa ctg ggt ctc atg agt ctg cac aga cct =,.~ cag atg agc att ~'_G
Ser Lys Leu Gly Leu Met Ser Leu G=r. Ark °rc =~a Gln Met Ser =le
290 295
gac atg ggc ttt aaa aca cgt gcc ttc acc act att ttg att ctc ttt 960
Asp Met Gly Phe Lys Thr Arg Ala ?'~:e T':r Thr Tle Leu Ile Leu Phe
305 310 . 3i5 - 320
get gtc ttc att gtc tgc tgg ge~ cca t__ acc act tac agc ctt gtg 1008
Ala Val Phe Iie Val Cys Trp Aia °~c =.._ '_"!:r ...- T;r Ser ~eu
~Jal
3 2 5 .. _ _ ..
SO gca aca ttc agt aag cac ttt tac tat cacac oac ttt ttt gag att 1056
Ala Thr Phe Ser Lys His Phe Ty- T;rr G1.~. His :.sn Phe Phe Glu I1e
340 3=5 35C
agc -acc tgg cta ctg tgg ctc tgc tac ctc aag tct gca ttg aat ccg 1109
Ser Thr Trp Leu Leu Trp Leu Cys Tyr Leu Lys Ser Ala Leu Asn Pro
355 360 365
ctg ' r 'atc tac tac tgg agg att aag aaa ttc cat gat get tgc ctg oac _.52
Leu Ile Tyr Tyr Trp Arg Ile Lys Lys °~°- His Asp Aia Cys
Leu Asp
370 375 ~~0
7
WO 01/09313 CA 02381437 2002-0l-28 PCT/Ep00/06854
atg dtg CCt dag tCC ttC aag ttt ttg CCg C3g :.~.. CCt Cgt CaC aCa 12~~
Met Met Pre Lys Ser Phe Lys Phe Leu Pro Gi~: Leu Pro Gly His Thr
385 39C 395 90C
aag cga cgg ata cgt cct agt get gtc tat gtg tgt ggg gaa cat egg 1298
Lys Arg Arg Ile Arg Pro Ser Ala Val Tyr Val Cys Gly Glu His Arg
9C5 410 915
acg gtg gtg tga '-260
IO Thr Val Val
920
<210> 6
l5 <211> 919
<212> PRT
<213> Homo sapiens
<400> n
ZO Met Val Phe Ser Ala Val Leu Thr Ala Phe His Thr Gly Thr Ser Asn
1 5 10 15
Thr Thr Phe Val Val Tyr Glu Asn Thr Tyr Met Asn Ile Thr Leu Prc
20 =5 30
Pro Pro Phe Gln His Pro Asp Leu Ser Pro Leu Leu Arg Tyr Ser Phe
~5 ~ 35 90 95
Glu Thr Met Aia Pro Thr Gl y Leu Ser Ser Leu T':r Val As.~. Ser Thr
50 ... 00
Ala Val Pro Thr Thr Pro Ala Ala Phe Lys Ser Leu Asn Leu Pro Leu
65 70 ~5 80
30 Gin Iie Thr Leu Ser Ala Iie :~iet I:e P::e ~ie Leu Pipe Val Ser Phe
85 9C 95
Leu Gly Asn Leu Val Val Cys Leu Met Val Tyr Gln Lys Ala Ala Met
100 105 110
Arg Ser Ala Ile Asn I1e Leu Leu Aia Ser Leu Ala Phe Ala Asp Met
35 115 ".20 i25
Leu Leu Ala Val Leu Asn Met Pro Phe Ala Leu Val Thr Ile Leu Thr
13G '_35 190
Thr Arg Trp Ile Phe Gly Lys Phe Phe Cys Arg 'Jal Ser Ala Met Phe
195 _.,., lcv
15G
40 Phe Trp Leu Phe Val I1e Glee Gly V~'_ Aia ~'_e Leu Leu Ii= .1e Ser
165 170 175
_1e Asp Arg Phe Leu Iie .1 a 'Jal G'_n Arg .:1:~ :=_sn_ Lys Leu Asn Pro
180 lay i9C
Tyr Arg Ala Lys Val Leu Ile Ala Va'~ Ser Trp Ala Thr Ser Phe Cys
45 195 200 205
Val Ala Phe Pro Leu Ala 'Jal Giy Asn Pro Asc Leu G1n I1e Pro Se-
210 G15 22C
Arg %:~ a Pro G1 n Cys Val . he !=ly T:,w Th: .'~. _-._~: ?r c Gi;~ ~yJr .._..
225 ~ ~3C ~.._
50 Al Tyr Val Iie Leu =le 3a- ~.e.: __.. Se. ?he c.._ _-~e °ro Phe Leu
a
295 250 255
Val I 1 a Leu Tyr Ser ?he ~~?et Gl y I';e Leu Asr. Tt-:r ~eu Arg Hi s Asr.
260 265 270
Ala Leu Arg Ile His Ser Tyr Pro Glu Gly Ile Cys Leu Ser Gln Ala
55 275 280 285
Ser Lys Leu Gly Leu P9et Se= Leu Gln Arg ?ro Phe ~ln Met Ser Ile
290 295 300
Asc Met Gly Phe Lyrs ':'hr =.rg Ala Fi-:e T~:= T:~:_- Iie ~e~,: _ie Leu Phe
305 310 ._.. 32'J
60 r.lc 'Jai Pale 11°_ Val Cy5 Trp F1 d PrG P;l~ '"~'7r T:'lr Tyr Ccr
Lel: Val
~L~ 3
WO 01109313 CA 02381437 2002-O1-28 p~~pp0/06854
Ala Thr Phe Ser Lys His Phe Tyr Tyr G1 ~ Hls As~: Phe Phe G~;. _.e
340 3;5 350
Ser Thr Trp Leu Leu Trp Leu Cys T~_~r Leu Lys Ser Ala Leu As.~. Prc
355 360 365
~ Leu _Ile Tyr Tyr Trp Arg Ile Lys Lys Phe His Asp Ala Cys Leu Asp
370 375 380
Met Met Fro Lys Ser Phe Lys Phe Leu Pro Gl~ Leu Pro Gly His Thr
385 390 395 40C
Lys Arg Arg Ile Arg Pro Ser Ala Val Tyr Val Cys Gly Glu His Arg
405 4I0 415
Thr Val 'Jal
1J
9
