U.S. patent application number 10/282717 was filed with the patent office on 2003-05-01 for cdna clone my1 that encodes a novel human 7-transmembrane receptor.
This patent application is currently assigned to University of Texas System. Invention is credited to Yanagisawa, Masashi.
Application Number | 20030083466 10/282717 |
Document ID | / |
Family ID | 21986561 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030083466 |
Kind Code |
A1 |
Yanagisawa, Masashi |
May 1, 2003 |
cDNA clone MY1 that encodes a novel human 7-transmembrane
receptor
Abstract
MY1 polypeptides and polynucleotides and methods for producing
such polypeptides by recombinant techniques are disclosed. Also
disclosed are methods for utilizing MY1 polypeptides and
polynucleotides in the design of protocols for the treatment of
infections such as bacterial, fungal, protozoan and viral
infections, particularly infections caused by HIV-1 or HIV-2; pain;
cancers; anorexia nervosa; bulimia; cachexia; obesity; diabetes;
asthma; Parkinson's disease; acute heart failure; hypotension;
hypertension; urinary retention; osteoporosis; angina pectoris;
myocardial infarction; ulcers; asthma; allergies; benign prostatic
hypertrophy; and psychotic and neurological disorders, including
anxiety, schizophrenia, manic depression, delirium, dementia,
severe mental retardation and dyskinesias, such as Huntington's
disease or Gilles dela Tourett's syndrome, among others, and
diagnostic assays for such conditions.
Inventors: |
Yanagisawa, Masashi;
(Dallas, TX) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
University of Texas System
|
Family ID: |
21986561 |
Appl. No.: |
10/282717 |
Filed: |
October 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10282717 |
Oct 28, 2002 |
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09676625 |
Oct 2, 2000 |
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09676625 |
Oct 2, 2000 |
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09119788 |
Jul 21, 1998 |
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6166193 |
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60053790 |
Jul 25, 1997 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.1; 536/23.5 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 1/00 20180101; A61P 9/12 20180101; A61P 13/08 20180101; A61P
9/00 20180101; A61P 31/00 20180101; A61P 3/00 20180101; A61P 31/10
20180101; A61P 13/00 20180101; A61P 31/12 20180101; A61P 3/04
20180101; A61P 19/10 20180101; A61P 19/00 20180101; A61P 11/06
20180101; A61P 1/04 20180101; A61P 21/00 20180101; C07K 14/705
20130101; A61P 31/18 20180101; A61P 25/20 20180101; A61K 39/00
20130101; A61P 37/00 20180101; A61P 9/08 20180101; A61K 38/00
20130101; A61P 25/22 20180101; A61P 25/24 20180101; A61P 31/04
20180101 |
Class at
Publication: |
530/350 ;
536/23.5; 435/69.1; 435/320.1; 435/325 |
International
Class: |
A61K 038/17; C07K
014/705; C12N 005/06; C12P 021/02; C07H 021/04 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a nucleotide sequence that
has at least 80% identity over its entire length to a nucleotide
sequence encoding the MY1 polypeptide of SEQ ID NO:2; or a
nucleotide sequence complementary to said isolated
polynucleotide.
2. The polynucleotide of claim 1 wherein said polynucleotide
comprises the nucleotide sequence contained in SEQ ID NO:1 encoding
the MY1 polypeptide of SEQ ID NO2.
3. The polynucleotide of claim 1 wherein said polynucleotide
comprises a nucleotide sequence that is at least 80% identical to
that of SEQ ID NO: 1 over its entire length.
4. The polynucleotide of claim 3 which is polynucleotide of SEQ ID
NO: 1.
5. The polynucleotide of claim 1 which is DNA or RNA.
6. A DNA or RNA molecule comprising an expression system, wherein
said expression system is capable of producing a MY1 polypeptide
comprising an amino acid sequence, which has at least 80% identity
with the polypeptide of SEQ ID NO:2 when said expression system is
present in a compatible host cell.
7. A host cell comprising the expression system of claim 6.
8. A process for producing a MY1 polypeptide comprising culturing a
host of claim 7 under conditions sufficient for the production of
said polypeptide and recovering the polypeptide from the
culture.
9. A process for producing a cell which produces a MY1 polypeptide
thereof comprising transforming or transfecting a host cell with
the expression system of claim 6 such that the host cell, under
appropriate culture conditions, produces a MY1 polypeptide.
10. A MY1 polypeptide comprising an amino acid sequence which is at
least 80% identical to the amino acid sequence of SEQ ID NO:2 over
its entire length.
11. The polypeptide of claim 10 which comprises the amino acid
sequence of SEQ ID NO:2.
12. An antibody immunospecific for the MY1 polypeptide of claim
10.
13. A method for the treatment of a subject in need of enhanced
activity or expression of MY1 polypeptide of claim 10 comprising:
(a) administering to the subject a therapeutically effective amount
of an agonist to said receptor; and/or (b) providing to the subject
an isolated polynucleotide comprising a nucleotide sequence that
has at least 80% identity to a nucleotide sequence encoding the MY1
polypeptide of SEQ ID NO:2 over its entire length; or a nucleotide
sequence complementary to said nucleotide sequence in a form so as
to effect production of said polypeptide activity in vivo.
14. A method for the treatment of a subject having need to inhibit
activity or expression of MY1 polypeptide of claim 10 comprising:
(a) administering to the subject a therapeutically effective amount
of an antagonist to said receptor; and/or (b) administering to the
subject a nucleic acid molecule that inhibits the expression of the
nucleotide sequence encoding said receptor; and/or (c)
administering to the subject a therapeutically effective amount of
a polypeptide that competes with said receptor for its ligand.
15. A process for diagnosing a disease or a susceptibility to a
disease in a subject related to expression or activity of MY1
polypeptide of claim 10 in a subject comprising: (a) determining
the presence or absence of a mutation in the nucleotide sequence
encoding said MY1 polypeptide in the genome of said subject; and/or
(b) analyzing for the presence or amount of the MY1 polypeptide
expression in a sample derived from said subject.
16. A method for identifying agonists to MY1 polypeptide of claim
10 comprising: (a) contacting a cell which produces a MY1
polypeptide with a candidate compound; and (b) determining whether
the candidate compound effects a signal generated by activation of
the MY1 polypeptide.
17. An agonist identified by the method of claim 16.
18. A method for identifying antagonists to MY1 polypeptide of
claim 10 comprising: (a) contacting a cell which produces a MY1
polypeptide with an agonist; and (b) determining whether the signal
generated by said agonist is diminished in the presence of a
candidate compound.
19. An antagonist identified by the method of claim 18.
20. A recombinant host cell produced by a method of claim 9 or a
membrane thereof expressing a MY1 polypeptide.
Description
FIELD OF INVENTION
[0001] This invention relates to newly identified polynucleotides,
polypeptides encoded by them and to the use of such polynucleotides
and polypeptides, and to their production. More particularly, the
polynucleotides and polypeptides of the present invention relate to
G-protein coupled receptor family, hereinafter referred to as MY1.
The invention also relates to inhibiting or activating the action
of such polynucleotides and polypeptides.
BACKGROUND OF THE INVENTION
[0002] 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, Nature, 1991, 351:353-354).
Herein, these proteins are referred to as proteins participating in
pathways 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. 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 protein kinase C (Simon, M. I., et al.,
Science, 1991, 252:802-8).
[0003] 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 an
intermediate that relays the signal from receptor to effector, and
as a clock that controls the duration of the signal.
[0004] 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 connected by extracellular or cytoplasmic
loops. G-protein coupled receptors include a wide range of
biologically active receptors, such as hormone, viral, growth
factor and neuroreceptors.
[0005] G-protein coupled receptors (otherwise known as 7TM
receptors) have been characterized as including these seven
conserved hydrophobic 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.
[0006] Most G-protein coupled receptors have single conserved
cysteine residues in each of the first two extracellular loops
which form disulfide bonds that are believed to stabilize
functional protein structure. The 7 transmembrane regions are
designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been
implicated in signal transduction.
[0007] 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.
[0008] 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.
[0009] 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-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.
[0010] Over the past 15 years, nearly 350 therapeutic agents
targeting 7 transmembrane (7 TM) receptors have been successfully
introduced onto the market. This indicates that these receptors
have an established, proven history as therapeutic targets.
Clearly, there is a need for identification and characterization of
further receptors which can play a role in preventing, ameliorating
or correcting dysfunctions or 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; anorexia nervosa; bulimia; cachexia; obesity; diabetes;
asthma; Parkinson's disease; acute heart failure; hypotension;
hypertension; urinary retention; osteoporosis; angina pectoris;
myocardial infarction; ulcers; asthma; allergies; benign prostatic
hypertrophy; and psychotic and neurological disorders, including
anxiety, schizophrenia, manic depression, delirium, dementia,
severe mental retardation and dyskinesias, such as Huntington's
disease or Gilles dela Tourett's syndrome, among others.
SUMMARY OF THE INVENTION
[0011] In one aspect, the invention relates to MY1 polypeptides and
recombinant materials and methods for their production. Another
aspect of the invention relates to methods for using such MY1
polypeptides and polynucleotides. Such uses include the treatment
of infections such as bacterial, fungal, protozoan and viral
infections, particularly infections caused by HIV-1 or HIV-2; pain;
cancers; anorexia nervosa; bulimia; cachexia; obesity; diabetes;
asthma; Parkinson's disease; acute heart failure; hypotension;
hypertension; urinary retention; osteoporosis; angina pectoris;
myocardial infarction; ulcers; asthma; allergies; benign prostatic
hypertrophy; and psychotic and neurological disorders, including
anxiety, schizophrenia, manic depression, delirium, dementia,
severe mental retardation and dyskinesias, such as Huntington's
disease or Gilles dela Tourett's syndrome, among others.
[0012] In still another aspect, the invention relates to methods to
identify agonists and antagonists using the materials provided by
the invention, and treating conditions associated with MY1
imbalance with the identified compounds. Yet another aspect of the
invention relates to diagnostic assays for detecting diseases
associated with inappropriate MY1 activity or levels.
DESCRIPTION OF THE INVENTION
[0013] Definitions
[0014] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0015] "MY1" refers, among others, to a polypeptide comprising the
amino acid sequence set forth in SEQ ID NO:2, or an allelic variant
thereof.
[0016] "Receptor Activity" or "Biological Activity of the Receptor"
refers to the metabolic or physiologic function of said MY1
including similar activities or improved activities or these
activities with decreased undesirable side-effects. Also included
are antigenic and immunogenic activities of said MY1.
[0017] "MY1 gene" refers to a polynucleotide comprising the
nucleotide sequence set forth in SEQ ID NO:1 or allelic variants
thereof and/or their complements.
[0018] "Antibodies" as used herein includes polyclonal and
monoclonal antibodies, chimeric, single chain, and humanized
antibodies, as well as Fab fragments, including the products of an
Fab or other immunoglobulin expression library.
[0019] "Isolated" means altered "by the hand of man" from the
natural state. If an "isolated" composition or substance 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 animal 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.
[0020] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxribonucleotide, which may be unmodified RNA or DNA or
modified 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, 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. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications has been 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 viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0021] "Polypeptide" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds, 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 processes, such as
posttranslational processing, or by chemical modification
techniques which 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 can
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 in 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 be cyclic, with or without branching.
Cyclic, branched and branched cyclic polypeptides may result from
posttranslation natural processes or may be made by synthetic
methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
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,
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, W. H. Freeman and Company, New York, 1993 and
Wold, F., Posttranslational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983;
Seifter et al., "Analysis for protein modifications and nonprotein
cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al.,
"Protein Synthesis: Posttranslational Modifications and Aging", Ann
NY Acad Sci (1992) 663:48-62.
[0022] "Variant" as the term is used herein, is a polynucleotide or
polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the 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 polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences 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, additions, deletions in any
combination. A substituted or inserted amino acid residue may or
may not be one encoded by the genetic code. A variant of a
polynucleotide or polypeptide may be a naturally occurring such as
an allelic variant, or it may be a 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.
[0023] "Identity" is a measure of the identity of nucleotide
sequences or amino acid sequences. In general, the sequences are
aligned so that the highest order match is obtained. "Identity" per
se has an art-recognized meaning and can be calculated using
published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed.,
Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA,
PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje,
G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). While
there exist a number of methods to measure identity between two
polynucleotide or polypeptide sequences, the term "identity" is
well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J
Applied Math (1988) 48:1073). Methods commonly employed to
determine identity or similarity between two sequences include, but
are not limited to, those disclosed in Guide to Huge Computers,
Martin J. Bishop, ed., Academic Press, San Diego, 1994, and
Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073.
Methods to determine identity and similarity are codified in
computer programs. Preferred computer program methods to determine
identity and similarity between two sequences include, but are not
limited to, GCS program package (Devereux, J., et al., Nucleic
Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul,
S. F. et al., J Molec Biol (1990) 215:403).
[0024] As an illustration, by a polynucleotide having a nucleotide
sequence having at least, for example, 95% "identity" to a
reference nucleotide sequence of SEQ ID NO: 1 is intended that the
nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence of SEQ ID NO: 1. In other words, to
obtain a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5 or 3 terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0025] Similarly, by a polypeptide having an amino acid sequence
having at least, for example, 95% identity to a reference amino
acid sequence of SEQ ID NO:2 is intended that the amino acid
sequence of the polypeptide is identical to the reference sequence
except that the polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the reference amino
acid of SEQ ID NO: 2. In other words, to obtain a polypeptide
having an amino acid sequence at least 95% identical to a reference
amino acid sequence, up to 5% of the amino acid residues in the
reference sequence may be deleted or substituted with another amino
acid, or a number of amino acids up to 5% of the total amino acid
residues in the reference sequence may be inserted into the
reference sequence. These alterations of the reference sequence may
occur at the amino or carboxy terminal positions of the reference
amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0026] Polypeptides of the Invention
[0027] In one aspect, the present invention relates to MY1
polypeptides (or MY1 proteins). The MY1 polypeptides include the
polypeptide of SEQ ID NO:2; as well as polypeptides comprising the
amino acid sequence of SEQ ID NO:2; and polypeptides comprising the
amino acid sequence which have at least 80% identity to that of SEQ
ID NO:2 over its entire length, and still more preferably at least
90% identity, and even still more preferably at least 95% identity
to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly
preferred. Also included within MY1 polypeptides are polypeptides
having the amino acid sequence which have at least 80% identity to
the polypeptide having the amino acid sequence of SEQ ID NO: 2 over
its entire length, and still more preferably at least 90% identity,
and even still more preferably at least 95% identity to SEQ ID NO:
2. Furthermore, those with at least 97-99% are highly preferred.
Preferably, MY1 polypeptides exhibit at least one biological
activity of the receptor.
[0028] The MY1 polypeptides may be in the form of the "mature"
protein or may be a part of a larger protein such as a fusion
protein. It is often advantageous to include an additional amino
acid sequence which contains secretory or leader sequences,
pro-sequences, sequences which aid in purification such as multiple
histidine residues, or an additional sequence for stability during
recombinant production.
[0029] Fragments of the MY1 polypeptides are also included in the
invention. A fragment is a polypeptide having an amino acid
sequence that entirely is the same as part, but not all, of the
amino acid sequence of the aforementioned MY1 polypeptides. As with
MY1 polypeptides, fragments may be "free-standing," or comprised
within a larger polypeptide of which they form a part or region,
most preferably as a single continuous region. Representative
examples of polypeptide fragments of the invention, include, for
example, fragments from about amino acid number 1-20, 21-40, 41-60,
61-80, 81-100, and 101 to the end of MY1 polypeptide. In this
context "about" includes the particularly recited ranges larger or
smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or
at both extremes.
[0030] Preferred fragments include, for example, truncation
polypeptides having the amino acid sequence of MY1 polypeptides,
except for deletion of a continuous series of residues that
includes the amino terminus, or a continuous series of residues
that includes the carboxyl terminus or deletion of two continuous
series of residues, one including the amino terminus and one
including the carboxyl terminus. Also preferred are fragments
characterized by structural or functional attributes such as
fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions. Other
preferred fragments are biologically active fragments. Biologically
active fragments are those that mediate receptor activity,
including those with a similar activity or an improved activity, or
with a decreased undesirable activity. Also included are those that
are antigenic or immunogenic in an animal, especially in a
human.
[0031] Preferably, all of these polypeptide fragments retain the
biological activity of the receptor, including antigenic activity.
Variants of the defined sequence and fragments also form part of
the present invention. Preferred variants are those that vary from
the referents by conservative amino acid substitutions--i.e., those
that substitute a residue with another of like characteristics.
Typical such substitutions are among Ala, Val, Leu and Ile; among
Ser and Thr; among the acidic residues Asp and Glu; among Asn and
Gln; and among the basic residues Lys and Arg; or aromatic residues
Phe and Tyr. Particularly preferred are variants in which several,
5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in
any combination.
[0032] The MY1 polypeptides of the invention can be prepared in any
suitable manner. Such polypeptides include isolated naturally
occurring polypeptides, recombinantly produced polypeptides,
synthetically produced polypeptides, or polypeptides produced by a
combination of these methods. Means for preparing such polypeptides
are well understood in the art.
[0033] Polynucleotides of the Invention
[0034] Another aspect of the invention relates to MY1
polynucleotides. MY1 polynucleotides include isolated
polynucleotides which encode the MY1 polypeptides and fragments,
and polynucleotides closely related thereto. More specifically, MY1
polynucleotide of the invention include a polynucleotide comprising
the nucleotide sequence contained in SEQ ID NO:1 encoding a MY1
polypeptide of SEQ ID NO: 2, and polynucleotide having the
particular sequence of SEQ ID NO:1. MY1 polynucleotides further
include a polynucleotide comprising a nucleotide sequence that has
at least 80% identity over its entire length to a nucleotide
sequence encoding the MY1 polypeptide of SEQ ID NO:2, and a
polynucleotide comprising a nucleotide sequence that is at least
80% identical to that of SEQ ID NO:1 over its entire length. In
this regard, polynucleotides at least 90% identical are
particularly preferred, and those with at least 95% are especially
preferred. Furthermore, those with at least 97% are highly
preferred and those with at least 98-99% are most highly preferred,
with at least 99% being the most preferred. Also included under MY1
polynucleotides are a nucleotide sequence which has sufficient
identity to a nucleotide sequence contained in SEQ ID NO:1 to
hybridize under conditions useable for amplification or for use as
a probe or marker. The invention also provides polynucleotides
which are complementary to such MY1 polynucleotides.
[0035] MY1 of the invention is structurally related to other
proteins of the G-protein coupled receptor family, as shown by the
results of sequencing the cDNA of Table 1 (SEQ ID NO:1) encoding
human MY1. The cDNA sequence of SEQ ID NO:1 contains an open
reading frame (nucleotide number 114 to 1445) encoding a
polypeptide of 445 amino acids SEQ ID NO:2. Amino acid sequence of
Table 2 (SEQ ID NO:2) has about 30.6% identity (using FASTA) in 219
amino acid residues with substance-P receptor, NK1 (Accession #
P30547, Gorbuley, V. et al, Biochim. Biophys. ACTA, 1131: 99-102,
1992). Furthermore, MY1 (SEQ ID NO:2) is 29.6% identical to human
neuromedin K receptor, NK3 over 206 amino acid residues (Accession
# P29371, Takahashi, K et al, Eur. J. biochem., 204: 1025-1033,
1992). Furthermore, MY1 (SEQ ID NO:2) is 29.1% identical to human
putative tachykinin receptor over 206 amino acid residues
(Accession # P30098, Xie G. X et al, Proc. Natl. Acad. Sci. U.S.A.
89:4124-4128, 1992). Nucleotide sequence of Table 1 (SEQ ID NO:1)
has about 97.16% identity (using BLAST) in 388 nucleotide residues
with Soares fetal liver spleen 1NFLS DNA (Accession # W86471,
Wilson, R. K. et al., WashU-Merck ESt project, Unpublished, 1995).
Furthermore, MY1 (SEQ ID NO:1) is 99.39% identical to human fetal
brain cDNA over 330 nucleotide residues (Accession # D81887, T.
Fugiwara et al., Otsuka GEN research Institute, Unpublished, 1995).
Furthermore, MY1 (SEQ ID NO: 1) is 80.82% identical to Soares fetal
liver spleen 1NFLS DNA over 146 nucleotide residues (Accession #
W86548, Wilson, R. K. et al, WashU-Merck EST project, Unpublished,
1997). Thus, MY1 polypeptides and polynucleotides of the present
invention are expected to have, inter alia, similar biological
functions/properties to their homologous polypeptides and
polynucleotides, and their utility is obvious to anyone skilled in
the art.
1TABLE 1.sup.a 1 TTCCATCCTA ATACGACTCA CTATAGGGCT SEQ ID NO: 1
CAAGCGGGCC CGCGCAGGTC 51 AGTGCTCATG GGGCAGGCGG AGAGGAGCTT
GCAGCATTGA GCGGAACCGG 101 ACTTGACCCC GTGATGTCCG GCACCAAATT
GGAGGACTCC CCCCCTTGTC 151 GCAACTGGTC ATCTGCTTCG GAGCTGAATG
AAACTCAAGA GCCCTTTTTA 201 AACCCCACCG ACTATGACGA CGAGGAATTC
CTGCGGTACC TGTGGAGGGA 251 ATACCTGCAC CCGAAAGAAT ATGAGTGGGT
CCTGATCGCC GGGTACATCA 301 TCGTGTTCGT CGTGGCTCTC ATTGGGAACG
TCCTGGTTTG TGTGGCAGTG 351 TGGAAGAACC ACCACATGAG GACGGTAACC
AACTACTTCA TAGTCAATCT 401 TTCTCTGGCT GATGTGCTCG TGACCATCAC
CTGCCTTCCA GCCACACTGG 451 TCGTGGATAT CACTGAGACC TGGTTTTTTG
GACAGTCCCT TTGCAAAGTG 501 ATTCCTTATC TACAGACCGT GTCGGTGTCT
GTGTCTGTCC TCACACTGAG 551 CTGTATCGCC TTGGATCCGT GGTATGCAAT
CTGTCACCCT TTGATGTTTA 601 AGAGCACAGC AAAGCGGGCC CGTAACAGCA
TTGTCATCAT CTGGATTGTC 651 TCCTGCATTA TAATGATTCC TCAGGCCATC
GTCATGGAGT GCAGCACCGT 701 GTTCCCAGGC TTAGCCAATA AAACCACCCT
CTTTACGGTG TGTGATGAGC 751 GCTCGGGTGG TGAAATTTAT CCCAAGATGT
ACCACATCTG TTTCTTTCTC 801 GTGACATACA TGGCACCACT GTGTCTCATG
GTGTTGCCTT ATCTGCAAAT 851 ATTTCGCAAA CTCTGGTGTC GACAGATCCC
TGGAACATCA TCTGTAGTTC 901 AGAGAAAATG GAAGCCCCTG CAGCCTGTTT
CACAGCCTCG AGGGCCAGGA 951 CAGCCAACGA AGTCCCGGAT GGGCGCTGTG
GCGGCTGAAA TAAAGCAGAT 1001 CCGAGCCAGA AGGAAAACAG CCCGGATGTT
ATGGTTGTG CTTTTGGTAT 1051 TTGCAATTTG CTATCTACCA ATTAG4ATCC
TCAATGTGCT AAAGAGAGTA 1101 TTTGGGATGT TTGCCCATAC TGAAGACAGA
GAGACTGTGT ATGCCTGGTT 1151 TACCTTTTCA CACTGGCTTG TATATGCCAA
TAGTGCTGCG AATCCAATTA. .sup.aA nucicotide sequence of a human MY
1.
[0036]
2TABLE 2.sup.b 1 MSGTKLEDSP PCRNWSSASE LNETQEPFLN SEQ ID NO: 2
PTDYDDEEFL RYLWREYLHP 51 KEYEWVLIAG YIIVFVVALI GNVLVCVAVW
KNHHMRTVTN YFIVNLSLAD 101 VLVTITCLPA TLVVDITETW FFGQSLCKVI
PYLQTVSVSV SVLTLSCIAL 151 DRWYAICHPL MFKSTAKRAR NSIVIIWIVS
CIIMIPQAIV MECSTVFPGL 201 ANKTTLFTVC DERWGGEIYP KMYHICFFLV
TYMAPLCLMV LAYLQIFRKL 251 WCRQIPGTSS VVQRKWKPLQ PVSQPRGPGQ
PTKSRMGAVA AEIKQIRARR 301 KTARMLMVVL LVFAICYLPI SILNVLKRVF
GMFAHTEDRE TVYAWFTFSH 351 WLVYANSAAN PIIYNFLSGK FREEFKAAFS
CCCLGVHHRQ EDRLTRGRTS 401 TESRKSLTTQ ISNFDNISKL SEQVVLTSIS T
LPAANGAOP LQNW*. .sup.b An amino acid sequence of a human MYI.
[0037] One polynucleotide of the present invention encoding MY1 may
be obtained using standard cloning and screening, from a cDNA
library derived from mRNA in cells of human fetal brain using the
expressed sequence tag (EST) analysis (Adams, M. D., et al.,
Science (1991) 252:1651-1656; Adams, M. D. et al., Nature, (1992)
355:632-634; Adams, M. D., et al., Nature (1995) 377 Supp:3-174).
Polynucleotides of the invention can also be obtained from natural
sources such as genomic DNA libraries or can be synthesized using
well known and commercially available techniques.
[0038] The nucleotide sequence encoding MY1 polypeptide of SEQ ID
NO:2 may be identical to the polypeptide encoding sequence
contained in Table 1 (nucleotide number 114 to 1445 of SEQ ID
NO:1), or it may be a sequence, which as a result of the redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of
SEQ ID NO:2.
[0039] When the polynucleotides of the invention are used for the
recombinant production of MY1 polypeptide, the polynucleotide may
include the coding sequence for the mature polypeptide or a
fragment thereof, by itself; the coding sequence for the mature
polypeptide or fragment 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 which facilitates
purification of the fused polypeptide can be encoded. In certain
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 polyadenylation signals,
ribosome binding sites and sequences that stabilize mRNA.
[0040] Further preferred embodiments are polynucleotides encoding
MY1 variants comprising the amino acid sequence of MY1 polypeptide
of Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1
amino acid residues are substituted, deleted or added, in any
combination.
[0041] The present invention further relates to polynucleotides
that hybridize to the herein above-described sequences. In this
regard, the present invention especially relates to polynucleotides
which hybridize under stringent conditions to the herein
above-described polynucleotides. As herein used, the term
"stringent conditions" means hybridization will occur only if there
is at least 80%, and preferably at least 90%, and more preferably
at least 95%, yet even more preferably 97-99% identity between the
sequences.
[0042] Polynucleotides of the invention, which are identical or
sufficiently identical to a nucleotide sequence contained in SEQ ID
NO:1 or a fragment thereof, may be used as hybridization probes for
cDNA and genomic DNA, to isolate full-length cDNAs and genomic
clones encoding MY1 and to isolate cDNA and genomic clones of other
genes (including genes encoding homologs and orthologs from species
other than human) that have a high sequence similarity to the MY1
gene. Such hybridization techniques are known to those of skill in
the art. Typically these nucleotide sequences are 80% identical,
preferably 90% identical, more preferably 95% identical to that of
the referent. The probes generally will comprise at least 15
nucleotides. Preferably, such probes will have at least 30
nucleotides and may have at least 50 nucleotides. Particularly
preferred probes will range between 30 and 50 nucleotides.
[0043] In one embodiment, to obtain a polynucleotide encoding MY1
polypeptide, including homologs and orthologs from species other
than human, comprises the steps of screening an appropriate library
under stingent hybridization conditions with a labeled probe having
the SEQ ID NO: 1 or a fragment thereof; and isolating full-length
cDNA and genomic clones containing said polynucleotide sequence.
Thus in another aspect, MY1 polynucleotides of the present
invention further include a nucleotide sequence comprising a
nucleotide sequence that hybridize under stringent condition to a
nucleotide sequence having SEQ ID NO: 1 or a fragment thereof. Also
included with MY1 polypeptides are polypeptide comprising amino
acid sequence encoded by nucleotide sequence obtained by the above
hybridization condition. Such hybridization techniques are well
known to those of skill in the art. Stringent hybridization
conditions are as defined above or, alternatively, conditions under
overnight incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 microgram/ml denatured, sheared salmon
sperm DNA, followed by washing the filters in 0.1.times.SSC at
about 65.degree. C.
[0044] The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials for
discovery of treatments and diagnostics to animal and human
disease.
[0045] Vectors, Host Cells, Expression
[0046] The present invention also relates to vectors which comprise
a polynucleotide or polynucleotides of the present invention, and
host cells which are genetically engineered with vectors of the
invention and to the production of polypeptides of the invention by
recombinant techniques. Cell-free translation systems can also be
employed to produce such proteins using RNAs derived from the DNA
constructs of the present invention.
[0047] For recombinant production, host cells can be genetically
engineered to incorporate expression systems or portions thereof
for polynucleotides of the present invention. Introduction of
polynucleotides into host cells can be effected by methods
described in many standard laboratory manuals, such as Davis et
al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium
phosphate transfection, DEAE-dextran mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction or infection.
[0048] 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 CHO, COS, HeLa,
C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant
cells.
[0049] A great variety of expression systems can be used. Such
systems include, among others, chromosomal, episomal and
virus-derived systems, e.g., vectors derived from bacterial
plasmids, from bacteriophage, from transposons, from yeast
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 cosmids and phagemids. The expression
systems may contain control regions that regulate as well as
engender expression. Generally, any system or vector suitable to
maintain, propagate or express polynucleotides to produce a
polypeptide in a host may be used. The appropriate nucleotide
sequence may be inserted into an expression system by any of a
variety of well-known and routine techniques, such as, for example,
those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (supra).
[0050] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the desired polypeptide. These signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0051] If the MY1 polypeptide is to be expressed for use in
screening assays, generally, it is 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 MY1
polypeptide is 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.
[0052] MY1 polypeptides can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic 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 active conformation when the polypeptide is
denatured during isolation and or purification.
[0053] Diagnostic Assays
[0054] This invention also relates to the use of MY1
polynucleotides for use as diagnostic reagents. Detection of a
mutated form of MY1 gene 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 expression of MY1.
Individuals carrying mutations in the MY1 gene may be detected at
the DNA level by a variety of techniques.
[0055] 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 may
be amplified enzymatically by using 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 MY1 nucleotide sequences. Perfectly
matched sequences can be distinguished from mismatched duplexes by
RNase digestion or by differences in melting temperatures. DNA
sequence differences may also be detected by alterations in
electrophoretic mobility of DNA fragments in gels, with or without
denaturing agents, or by direct DNA sequencing. See, e.g., Myers 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. In
another embodiment, an array of oligonucleotides probes comprising
MY1 nucleotide sequence or fragments thereof can be constructed to
conduct efficient screening of e.g., genetic mutations. 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, Vol 274, pp
610-613 (1996)).
[0056] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to infections such as bacterial,
fungal, protozoan and viral infections, particularly infections
caused by HIV-1 or HIV-2; pain; cancers; anorexia nervosa; bulimia;
cachexia; obesity; diabetes; asthma; Parkinson's disease; acute
heart failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; ulcers;
asthma; allergies; benign prostatic hypertrophy; and psychotic and
neurological disorders, including anxiety, schizophrenia, manic
depression, delirium, dementia, severe mental retardation and
dyskinesias, such as Huntington's disease or Gilles dela Tourett's
syndrome, among others, through detection of mutation in the MY1
gene by the methods described.
[0057] In addition, infections such as bacterial, fungal, protozoan
and viral infections, particularly infections caused by HIV-1 or
HIV-2; pain; cancers; anorexia nervosa; bulimia; cachexia; obesity;
diabetes; asthma; Parkinson's disease; acute heart failure;
hypotension; hypertension; urinary retention; osteoporosis; angina
pectoris; myocardial infarction; ulcers; asthma; allergies; benign
prostatic hypertrophy; and psychotic and neurological disorders,
including anxiety, schizophrenia, manic depression, delirium,
dementia, severe mental retardation and dyskinesias, such as
Huntington's disease or Gilles dela Tourett's syndrome, among
others, can be diagnosed by methods comprising determining from a
sample derived from a subject an abnormally decreased or increased
level of MY1 polypeptide or MY1 mRNA. Decreased or increased
expression can be measured at the RNA level using any of the
methods well known in the art for the quantitation of
polynucleotides, such as, for example, 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 an MY1, in a sample derived 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.
[0058] Thus in another aspect, the present invention relates to a
diagonostic kit for a disease or suspectability to a disease,
particularly infections such as bacterial, fungal, protozoan and
viral infections, particularly infections caused by HIV-1 or HIV-2;
pain; cancers; anorexia nervosa; bulimia; cachexia; obesity;
diabetes; asthma; Parkinson's disease; acute heart failure;
hypotension; hypertension; urinary retention; osteoporosis; angina
pectoris; myocardial infarction; ulcers; asthma; allergies; benign
prostatic hypertrophy; and psychotic and neurological disorders,
including anxiety, schizophrenia, manic depression, delirium,
dementia, severe mental retardation and dyskinesias, such as
Huntington's disease or Gilles dela Tourett's syndrome, among
others, which comprises:
[0059] (a) a MY1 polynucleotide, preferably the nucleotide sequence
of SEQ ID NO: 1, or a fragment thereof;
[0060] (b) a nucleotide sequence complementary to that of (a);
[0061] (c) a MY1 polypeptide, preferably the polypeptide of SEQ ID
NO: 2, or a fragment thereof; or
[0062] (d) an antibody to a MY1 polypeptide, preferably to the
polypeptide of SEQ ID NO: 2.
[0063] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0064] Chromosome Assays
[0065] The nucleotide sequences of the present invention are also
valuable for chromosome identification. 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 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, for example, in V. McKusick, Mendelian Inheritance in 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 (coinheritance of physically adjacent
genes). The differences in the cDNA or genomic sequence between
affected and unaffected individuals can also be determined. If a
mutation is observed in some or all of the affected individuals but
not in any normal individuals, then the mutation is likely to be
the causative agent of the disease.
[0066] Antibodies
[0067] The polypeptides of the invention or their fragments or
analogs thereof, or cells expressing them can also be used as
immunogens to produce antibodies immunospecific for the MY1
polypeptides. The term "immunospecific" means that the antibodies
have substantiall greater affinity for the polypeptides of the
invention than their affinity for other related polypeptides in the
prior art.
[0068] Antibodies generated against the MY1 polypeptides can be
obtained by administering the polypeptides or epitope-bearing
fragments, analogs or cells to an animal, preferably a nonhuman,
using routine protocols. 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 the EBV-hybridoma technique
(Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96,
Alan R. Liss, Inc., 1985).
[0069] Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can also be adapted to produce single
chain antibodies to polypeptides of this invention. Also,
transgenic mice, or other organisms including other mammals, may be
used to express humanized antibodies.
[0070] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptide or to purify the
polypeptides by affinity chromatography. Antibodies against MY1
polypeptides may also be employed to treat infections such as
bacterial, fungal, protozoan and viral infections, particularly
infections caused by HIV-1 or HIV-2; pain; cancers; anorexia
nervosa; bulimia; cachexia; obesity; diabetes; asthma; Parkinson's
disease; acute heart failure; hypotension; hypertension; urinary
retention; osteoporosis; angina pectoris; myocardial infarction;
ulcers; asthma; allergies; benign prostatic hypertrophy; and
psychotic and neurological disorders, including anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental
retardation and dyskinesias, such as Huntington's disease or Gilles
dela Tourett's syndrome, among others.
[0071] Vaccines
[0072] Another aspect of the invention relates to a method for
inducing an immunological response in a mammal which comprises
inoculating the mammal with MY1 polypeptide, or a fragment thereof,
adequate to produce antibody and/or T cell immune response to
protect said animal from infections such as bacterial, fungal,
protozoan and viral infections, particularly infections caused by
HIV-1 or HIV-2; pain; cancers; anorexia nervosa; bulimia; cachexia;
obesity; diabetes; asthma; Parkinson's disease; acute heart
failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; ulcers;
asthma; allergies; benign prostatic hypertrophy; and psychotic and
neurological disorders, including anxiety, schizophrenia, manic
depression, delirium, dementia, severe mental retardation and
dyskinesias, such as Huntington's disease or Gilles dela Tourett's
syndrome, among others.
[0073] Yet another aspect of the invention relates to a method of
inducing immunological response in a mammal which comprises,
delivering MY1 polypeptide via a vector directing expression of MY1
polynucleotide in vivo in order to induce such an immunological
response to produce antibody to protect said animal from
diseases.
[0074] Further aspect of the invention relates to an
immunological/vaccine formulation (composition) which, when
introduced into a mammalian host, induces an immunological response
in that mammal to a MY1 polypeptide wherein the composition
comprises a MY1 polypeptide or MY1 gene. The vaccine formulation
may further comprise a suitable carrier. Since MY1 polypeptide may
be broken down in the stomach, it is preferably administered
parenterally (including subcutaneous, intramuscular, intravenous,
intradermal etc. injection). Formulations suitable for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the formulation instonic with the blood of
the recipient; and aqueous and non-aqueous sterile suspensions
which 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 dosage will depend on the
specific activity of the vaccine and can be readily determined by
routine experimentation.
[0075] Screening Assays
[0076] The MY1 polypeptide of the present invention may be employed
in a screening process for compounds which bind the receptor and
which activate (agonists) or inhibit activation of (antagonists)
the receptor polypeptide of the present invention. Thus,
polypeptides of the invention may also be used to assess the
binding of small molecule substrates and ligands in, for example,
cells, cell-free preparations, chemical libraries, and natural
product mixtures. These substrates and ligands may be natural
substrates and ligands or may be structural or functional mimetics.
See Coligan et al., Current Protocols in Immunology 1(2):Chapter 5
(1991).
[0077] MY1 polypeptides are responsible for many biological
functions, including many pathologies. Accordingly, it is desirous
to find compounds and drugs which stimulate MY1 on the one hand and
which can inhibit the function of MY1 on the other hand. In
general, agonists are employed for therapeutic and prophylactic
purposes for such conditions as infections such as bacterial,
fungal, protozoan and viral infections, particularly infections
caused by HIV-1 or HIV-2; pain; cancers; anorexia nervosa; bulimia;
cachexia; obesity; diabetes; asthma; Parkinson's disease; acute
heart failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; ulcers;
asthma; allergies; benign prostatic hypertrophy; and psychotic and
neurological disorders, including anxiety, schizophrenia, manic
depression, delirium, dementia, severe mental retardation and
dyskinesias, such as Huntington's disease or Gilles dela Tourett's
syndrome, among others. Antagonists may be employed for a variety
of therapeutic and prophylactic purposes for such conditions as
infections such as bacterial, fungal, protozoan and viral
infections, particularly infections caused by HIV-1 or HIV-2; pain;
cancers; anorexia nervosa; bulimia; cachexia; obesity; diabetes;
asthma; Parkinson's disease; acute heart failure; hypotension;
hypertension; urinary retention; osteoporosis; angina pectoris;
myocardial infarction; ulcers; asthma; allergies; benign prostatic
hypertrophy; and psychotic and neurological disorders, including
anxiety, schizophrenia, manic depression, delirium, dementia,
severe mental retardation and dyskinesias, such as Huntington's
disease or Gilles dela Tourett's syndrome, among others.
[0078] In general, such screening procedures involve producing
appropriate cells which express the receptor polypeptide of the
present invention on the surface thereof. Such cells include cells
from mammals, yeast, Drosophila or E. coli. Cells expressing the
receptor (or cell membrane containing the expressed receptor) are
then contacted with a test compound to observe binding, or
stimulation or inhibition of a functional response.
[0079] 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 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.
[0080] Another method involves screening for receptor inhibitors by
determining 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
receptor binding, the levels of receptor-mediated cAMP, or
adenylate cyclase, activity will be reduced or increased.
[0081] Another method for detecting agonists or antagonists for the
receptor of the present invention is the yeast based technology as
described in U.S. Pat. No. 5,482,835.
[0082] The assays may simply test binding of a candidate compound
wherein adherence to the cells bearing the receptor is detected by
means of a label directly or indirectly associated with the
candidate compound or in an assay involving competition with a
labeled competitor. Further, these assays may test whether the
candidate compound results in a signal generated by activation of
the receptor, using detection systems appropriate to the cells
bearing the receptor at their surfaces. 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.
[0083] Further, the assays may simply comprise the steps of mixing
a candidate compound with a solution containing a MY1 polypeptide
to form a mixture, measuring MY1 activity in the mixture, and
comparing the MY1 activity of the mixture to a standard.
[0084] The MY1 cDNA, protein and antibodies to the protein may also
be used to configure assays for detecting the effect of added
compounds on the production of MY1 mRNA and protein in cells. For
example, an ELISA may be constructed for measuring secreted or cell
associated levels of MY1 protein using monoclonal and polyclonal
antibodies by standard methods known in the art, and this can be
used to discover agents which may inhibit or enhance the production
of MY1 (also called antagonist or agonist, respectively) from
suitably manipulated cells or tissues. Standard methods for
conducting screening assays are well understood in the art.
[0085] Examples of potential MY 1 antagonists include antibodies
or, in some cases, oligonucleotides or proteins which are closely
related to the ligand of the MY1, e.g., a fragment of the ligand,
or small molecules which bind to the receptor but do not elicit a
response, so that the activity of the receptor is prevented.
[0086] Thus in another aspect, the present invention relates to a
screening kit for identifying agonists, antagonists, ligands,
receptors, substrates, enzymes, etc. for MY1 polypeptides; or
compounds which decrease or enhance the production of MY1
polypeptides, which comprises:
[0087] (a) a MY1 polypeptide, preferably that of SEQ ID NO:2;
[0088] (b) a recombinant cell expressing a MY1 polypeptide,
preferably that of SEQ ID NO:2;
[0089] (c) a cell membrane expressing a MY1 polypeptide; preferably
that of SEQ ID NO: 2; or
[0090] (d) antibody to a MY1polypeptide, preferably that of SEQ ID
NO: 2. It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0091] Prophylactic and Therapeutic Methods
[0092] This invention provides methods of treating abnormal
conditions such as, infections such as bacterial, fungal, protozoan
and viral infections, particularly infections caused by HIV-1 or
HIV-2; pain; cancers; anorexia nervosa; bulimia; cachexia; obesity;
diabetes; asthma; Parkinson's disease; acute heart failure;
hypotension; hypertension; urinary retention; osteoporosis; angina
pectoris; myocardial infarction; ulcers; asthma; allergies; benign
prostatic hypertrophy; and psychotic and neurological disorders,
including anxiety, schizophrenia, manic depression, delirium,
dementia, severe mental retardation and dyskinesias, such as
Huntington's disease or Gilles dela Tourett's syndrome, among
others, related to both an excess of and insufficient amounts of
MY1 activity.
[0093] If the activity of MY1 is in excess, several approaches are
available. One approach comprises administering to a subject an
inhibitor compound (antagonist) as hereinabove described along with
a pharmaceutically acceptable carrier in an amount effective to
inhibit activation by blocking binding of ligands to the MY1, or by
inhibiting a second signal, and thereby alleviating the abnormal
condition. In another approach, soluble forms of MY1 polypeptides
still capable of binding the ligand in competition with endogenous
MY1 may be administered. Typical embodiments of such competitors
comprise fragments of the MY1 polypeptide.
[0094] In still another approach, expression of the gene encoding
endogenous MY1 can be inhibited using expression blocking
techniques. Known such techniques involve the use of antisense
sequences, either internally generated or separately administered.
See, for example, O'Connor, J Neurochem (1991) 56:560 in
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988). Alternatively, oligonucleotides
which form triple helices with the gene can be supplied. See, for
example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney et
al., Science (1988) 241:456; Dervan et al., Science (1991)
251:1360. These oligomers can be administered per se or the
relevant oligomers can be expressed in vivo.
[0095] For treating abnormal conditions related to an
under-expression of MY 1 and its activity, several approaches are
also available. One approach comprises administering to a subject a
therapeutically effective amount of a compound which activates MY1,
i.e., an agonist as described above, in combination with a
pharmaceutically acceptable carrier, to thereby alleviate the
abnormal condition. Alternatively, gene therapy may be employed to
effect the endogenous production of MY1 by the relevant cells in
the subject. For example, a polynucleotide of the invention may be
engineered for expression in a replication defective retroviral
vector, as discussed above. The retroviral expression construct may
then be isolated and introduced into a packaging cell transduced
with a retroviral plasmid vector containing RNA encoding a
polypeptide of the present invention such that the packaging cell
now produces infectious viral particles containing the gene of
interest. These producer cells may be administered to a subject for
engineering cells in vivo and expression of the polypeptide in
vivo. For overview of gene therapy, see Chapter 20, Gene Therapy
and other Molecular Genetic-based Therapeutic Approaches, (and
references cited therein) in Human Molecular Genetics, T Strachan
and A P Read, BIOS Scientific Publishers Ltd (1996). Another
approach is to administer a therapeutic amount of MY1 polypeptides
in combination with a suitable pharmaceutical carrier.
[0096] Formulation and Administration
[0097] Peptides, such as the soluble form of MY1 polypeptides, and
agonists and antagonist peptides or small molecules, may be
formulated in combination with a suitable pharmaceutical carrier.
Such formulations comprise a therapeutically effective amount of
the polypeptide or compound, and a pharmaceutically acceptable
carrier or excipient. Such carriers include but are not limited to,
saline, buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. Formulation should suit the mode of
administration, and is well within the skill of the art. The
invention further relates to pharmaceutical packs and kits
comprising one or more containers filled with one or more of the
ingredients of the aforementioned compositions of the
invention.
[0098] Polypeptides and other compounds of the present invention
may be employed alone or in conjunction with other compounds, such
as therapeutic compounds.
[0099] Preferred forms of systemic administration of the
pharmaceutical compositions include injection, typically by
intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal
and transdermal administration using penetrants such as bile salts
or fusidic acids or other detergents. In addition, if properly
formulated in enteric or encapsulated formulations, oral
administration may also be possible. Administration of these
compounds may also be topical and/or localized, in the form of
salves, pastes, gels and the like.
[0100] The dosage range required depends on the choice of peptide,
the route of administration, the nature of the formulation, the
nature of the subject's condition, and the judgment of the
attending practitioner. Suitable dosages, however, are in the range
of 0.1-100 .mu.g/kg of subject. Wide variations in the needed
dosage, however, are to be expected in view of the variety of
compounds available and the differing efficiencies of various
routes of administration. For example, oral administration would be
expected to require higher dosages than administration by
intravenous injection. Variations in these dosage levels can be
adjusted using standard empirical routines for optimization, as is
well understood in the art.
[0101] Polypeptides used in treatment can also be generated
endogenously in the subject, in treatment modalities often referred
to as "gene therapy" as described above. Thus, for example, cells
from a subject may be engineered with a polynucleotide, such as a
DNA or RNA, to encode a polypeptide ex vivo, and for example, by
the use of a retroviral plasmid vector. The cells are then
introduced into the subject.
EXAMPLES
[0102] The examples below are carried out using standard
techniques, which are well known and routine to those of skill in
the art, except where otherwise described in detail. The examples
illustrate, but do not limit the invention.
Example 1
Mammalian Cell Expression
[0103] 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 cDNAs by lipofectin and
selected in the presence of 400 mg/ml G418. 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 blot analysis. Receptor mRNAs are
generally detectable in about 50% of the G418-resistant clones
analyzed.
Example 2
Ligand Bank for Binding and Functional Assays
[0104] A bank of over 200 putative receptor ligands has been
assembled for 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 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.
Example 3
Ligand Binding Assays
[0105] 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 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 used to
define residual nonspecific binding.
Example 4
Functional Assay in Xenopus Oocytes
[0106] 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 nl bolus 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 tissue/cell extracts for activating ligands.
Example 5
Microphysiometric Assays
[0107] 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 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, Calif.). The CYTOSENSOR is thus capable of
detecting the activation of a receptor 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
[0108] 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., 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
Calcium and cAMP Functional Assays
[0109] 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 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 transient or cAMP flucuation are tested in
vector control cells to determine if the response is unique to the
transfected cells expressing receptor.
[0110] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
Sequence CWU 1
1
2 1 1633 DNA HOMO SAPIENS 1 ttccatccta atacgactca ctatagggct
caagcgggcc cgggcaggtc agtgctcatg 60 gggcaggcgg agaggagctt
gcagcattga gcggaaccgg acttgagccc gtgatgtccg 120 gcaccaaatt
ggaggactcc cccccttgtc gcaactggtc atctgcttcg gagctgaatg 180
aaactcaaga gcccttttta aaccccaccg actatgacga cgaggaattc ctgcggtacc
240 tgtggaggga atacctgcac ccgaaagaat atgagtgggt cctgatcgcc
gggtacatca 300 tcgtgttcgt cgtggctctc attgggaacg tcctggtttg
tgtggcagtg tggaagaacc 360 accacatgag gacggtaacc aactacttca
tagtcaatct ttctctggct gatgtgctcg 420 tgaccatcac ctgccttcca
gccacactgg tcgtggatat cactgagacc tggttttttg 480 gacagtccct
ttgcaaagtg attccttatc tacagaccgt gtcggtgtct gtgtctgtcc 540
tcacactgag ctgtatcgcc ttggatcggt ggtatgcaat ctgtcaccct ttgatgttta
600 agagcacagc aaagcgggcc cgtaacagca ttgtcatcat ctggattgtc
tcctgcatta 660 taatgattcc tcaggccatc gtcatggagt gcagcaccgt
gttcccaggc ttagccaata 720 aaaccaccct ctttacggtg tgtgatgagc
gctggggtgg tgaaatttat cccaagatgt 780 accacatctg tttctttctg
gtgacataca tggcaccact gtgtctcatg gtgttggctt 840 atctgcaaat
atttcgcaaa ctctggtgtc gacagatccc tggaacatca tctgtagttc 900
agagaaaatg gaagcccctg cagcctgttt cacagcctcg agggccagga cagccaacga
960 agtcccggat gggcgctgtg gcggctgaaa taaagcagat ccgagccaga
aggaaaacag 1020 cccggatgtt gatggttgtg cttttggtat ttgcaatttg
ctatctacca attagcatcc 1080 tcaatgtgct aaagagagta tttgggatgt
ttgcccatac tgaagacaga gagactgtgt 1140 atgcctggtt taccttttca
cactggcttg tatatgccaa tagtgctgcg aatccaatta 1200 tttataattt
tctcagtgga aaatttcgag aggaatttaa agctgcgttt tcttgctgtt 1260
gccttggagt tcaccatcgc caggaggatc ggctcaccag gggacgaact agcacagaga
1320 gccggaagtc cttgaccact caaatcagca actttgataa catatcaaaa
ctttctgagc 1380 aagttgtgct cactagcata agcacactcc cagcagccaa
tggagcagga ccacttcaaa 1440 actggtagaa tatttattca tatgacaagg
atacctgagt aaaactatcc tttttaaaat 1500 cactgggagc agaaatttta
ttatcctatg atgtgaagct aaaattactt gtggatcttt 1560 ttttttttta
atctattgct ctttggaaat aaaaaaaaag tcagtaaaaa aaaaaaaaaa 1620
aaaaaaaaaa aaa 1633 2 444 PRT Homo sapiens 2 Met Ser Gly Thr Lys
Leu Glu Asp Ser Pro Pro Cys Arg Asn Trp Ser 1 5 10 15 Ser Ala Ser
Glu Leu Asn Glu Thr Gln Glu Pro Phe Leu Asn Pro Thr 20 25 30 Asp
Tyr Asp Asp Glu Glu Phe Leu Arg Tyr Leu Trp Arg Glu Tyr Leu 35 40
45 His Pro Lys Glu Tyr Glu Trp Val Leu Ile Ala Gly Tyr Ile Ile Val
50 55 60 Phe Val Val Ala Leu Ile Gly Asn Val Leu Val Cys Val Ala
Val Trp 65 70 75 80 Lys Asn His His Met Arg Thr Val Thr Asn Tyr Phe
Ile Val Asn Leu 85 90 95 Ser Leu Ala Asp Val Leu Val Thr Ile Thr
Cys Leu Pro Ala Thr Leu 100 105 110 Val Val Asp Ile Thr Glu Thr Trp
Phe Phe Gly Gln Ser Leu Cys Lys 115 120 125 Val Ile Pro Tyr Leu Gln
Thr Val Ser Val Ser Val Ser Val Leu Thr 130 135 140 Leu Ser Cys Ile
Ala Leu Asp Arg Trp Tyr Ala Ile Cys His Pro Leu 145 150 155 160 Met
Phe Lys Ser Thr Ala Lys Arg Ala Arg Asn Ser Ile Val Ile Ile 165 170
175 Trp Ile Val Ser Cys Ile Ile Met Ile Pro Gln Ala Ile Val Met Glu
180 185 190 Cys Ser Thr Val Phe Pro Gly Leu Ala Asn Lys Thr Thr Leu
Phe Thr 195 200 205 Val Cys Asp Glu Arg Trp Gly Gly Glu Ile Tyr Pro
Lys Met Tyr His 210 215 220 Ile Cys Phe Phe Leu Val Thr Tyr Met Ala
Pro Leu Cys Leu Met Val 225 230 235 240 Leu Ala Tyr Leu Gln Ile Phe
Arg Lys Leu Trp Cys Arg Gln Ile Pro 245 250 255 Gly Thr Ser Ser Val
Val Gln Arg Lys Trp Lys Pro Leu Gln Pro Val 260 265 270 Ser Gln Pro
Arg Gly Pro Gly Gln Pro Thr Lys Ser Arg Met Gly Ala 275 280 285 Val
Ala Ala Glu Ile Lys Gln Ile Arg Ala Arg Arg Lys Thr Ala Arg 290 295
300 Met Leu Met Val Val Leu Leu Val Phe Ala Ile Cys Tyr Leu Pro Ile
305 310 315 320 Ser Ile Leu Asn Val Leu Lys Arg Val Phe Gly Met Phe
Ala His Thr 325 330 335 Glu Asp Arg Glu Thr Val Tyr Ala Trp Phe Thr
Phe Ser His Trp Leu 340 345 350 Val Tyr Ala Asn Ser Ala Ala Asn Pro
Ile Ile Tyr Asn Phe Leu Ser 355 360 365 Gly Lys Phe Arg Glu Glu Phe
Lys Ala Ala Phe Ser Cys Cys Cys Leu 370 375 380 Gly Val His His Arg
Gln Glu Asp Arg Leu Thr Arg Gly Arg Thr Ser 385 390 395 400 Thr Glu
Ser Arg Lys Ser Leu Thr Thr Gln Ile Ser Asn Phe Asp Asn 405 410 415
Ile Ser Lys Leu Ser Glu Gln Val Val Leu Thr Ser Ile Ser Thr Leu 420
425 430 Pro Ala Ala Asn Gly Ala Gly Pro Leu Gln Asn Trp 435 440
* * * * *