U.S. patent application number 10/837625 was filed with the patent office on 2004-10-14 for isolated human secreted proteins, nucleic acid molecules encoding human secreted proteins, and uses thereof.
This patent application is currently assigned to APPLERA CORPORATION. Invention is credited to Greenberg, Simon, Higgins, Maureen, Ladunga, Steven I., Spier, Eugene, Wang, Yu.
Application Number | 20040203052 10/837625 |
Document ID | / |
Family ID | 26935880 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040203052 |
Kind Code |
A1 |
Ladunga, Steven I. ; et
al. |
October 14, 2004 |
Isolated human secreted proteins, nucleic acid molecules encoding
human secreted proteins, and uses thereof
Abstract
The present invention provides amino acid sequences of peptides
that are encoded by genes within the human genome, the secreted
peptides of the present invention. The present invention
specifically provides isolated peptide and nucleic acid molecules,
methods of identifying orthologs and paralogs of the secreted
peptides, and methods of identifying modulators of the secreted
peptides.
Inventors: |
Ladunga, Steven I.; (Foster
City, CA) ; Spier, Eugene; (Palo Alto, MD) ;
Higgins, Maureen; (Bethesda, MD) ; Greenberg,
Simon; (Foster City, CA) ; Wang, Yu;
(Pittsburg, PA) |
Correspondence
Address: |
CELERA GENOMICS CORP.
ATTN: WAYNE MONTGOMERY, VICE PRES, INTEL PROPERTY
45 WEST GUDE DRIVE
C2-4#20
ROCKVILLE
MD
20850
US
|
Assignee: |
APPLERA CORPORATION
Norwalk
CT
|
Family ID: |
26935880 |
Appl. No.: |
10/837625 |
Filed: |
May 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10837625 |
May 4, 2004 |
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10274266 |
Oct 21, 2002 |
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6753174 |
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10274266 |
Oct 21, 2002 |
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09711681 |
Nov 9, 2000 |
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6503743 |
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60243477 |
Oct 27, 2000 |
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Current U.S.
Class: |
435/6.13 ;
435/320.1; 435/325; 435/6.16; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/04; C07K 014/47 |
Claims
That which is claimed is:
1. An isolated peptide consisting of an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NO:2, wherein
said allelic variant is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid
sequence of an ortholog of an amino acid sequence shown in SEQ ID
NO:2, wherein said ortholog is encoded by a nucleic acid molecule
that hybridizes under stringent conditions to the opposite strand
of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a
fragment of an amino acid sequence shown in SEQ ID NO:2, wherein
said fragment comprises at least 10 contiguous amino acids.
2. An isolated peptide comprising an amino acid sequence selected
from the group consisting of: (a) an amino acid sequence shown in
SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said allelic
variant is encoded by a nucleic acid molecule that hybridizes under
stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of
an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein
said ortholog is encoded by a nucleic acid molecule that hybridizes
under stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino
acid sequence shown in SEQ ID NO:2, wherein said fragment comprises
at least 10 contiguous amino acids.
3. An isolated antibody that selectively binds to a peptide of
claim 2.
4. An isolated nucleic acid molecule consisting of a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
5. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
6. A gene chip comprising a nucleic acid molecule of claim 5.
7. A transgenic non-human animal comprising a nucleic acid molecule
of claim 5.
8. A nucleic acid vector comprising a nucleic acid molecule of
claim 5.
9. A host cell containing the vector of claim 8.
10. A method for producing any of the peptides of claim 1
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide sequence.
11. A method for producing any of the peptides of claim 2
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide sequence.
12. A method for detecting the presence of any of the peptides of
claim 2 in a sample, said method comprising contacting said sample
with a detection agent that specifically allows detection of the
presence of the peptide in the sample and then detecting the
presence of the peptide.
13. A method for detecting the presence of a nucleic acid molecule
of claim 5 in a sample, said method comprising contacting the
sample with an oligonucleotide that hybridizes to said nucleic acid
molecule under stringent conditions and determining whether the
oligonucleotide binds to said nucleic acid molecule in the
sample.
14. A method for identifying a modulator of a peptide of claim 2,
said method comprising contacting said peptide with an agent and
determining if said agent has modulated the function or activity of
said peptide.
15. The method of claim 14, wherein said agent is administered to a
host cell comprising an expression vector that expresses said
peptide.
16. A method for identifying an agent that binds to any of the
peptides of claim 2, said method comprising contacting the peptide
with an agent and assaying the contacted mixture to determine
whether a complex is formed with the agent bound to the
peptide.
17. A pharmaceutical composition comprising an agent identified by
the method of claim 16 and a pharmaceutically acceptable carrier
therefor.
18. A method for treating a disease or condition mediated by a
human secreted protein, said method comprising administering to a
patient a pharmaceutically effective amount of an agent identified
by the method of claim 16.
19. A method for identifying a modulator of the expression of a
peptide of claim 2, said method comprising contacting a cell
expressing said peptide with an agent, and determining if said
agent has modulated the expression of said peptide.
20. An isolated human secreted peptide having an amino acid
sequence that shares at least 70% homology with an amino acid
sequence shown in SEQ ID NO:2.
21. A peptide according to claim 20 that shares at least 90 percent
homology with an amino acid sequence shown in SEQ ID NO:2.
22. An isolated nucleic acid molecule encoding a human secreted
peptide, said nucleic acid molecule sharing at least 80 percent
homology with a nucleic acid molecule shown in SEQ ID NOS:1 or
3.
23. A nucleic acid molecule according to claim 22 that shares at
least 90 percent homology with a nucleic acid molecule shown in SEQ
ID NOS:1 or 3.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of secreted proteins
that are related to the lactate dehydrogenase secreted protein
subfamily, recombinant DNA molecules, and protein production. The
present invention specifically provides novel peptides and proteins
that effect protein phosphorylation and nucleic acid molecules
encoding such peptide and protein molecules, all of which are
useful in the development of human therapeutics and diagnostic
compositions and methods.
BACKGROUND OF THE INVENTION
[0002] Secreted Proteins
[0003] Many human proteins serve as pharmaceutically active
compounds. Several classes of human proteins that serve as such
active compounds include hormones, cytokines, cell growth factors,
and cell differentiation factors. Most proteins that can be used as
a pharmaceutically active compound fall within the family of
secreted proteins. It is, therefore, important in developing new
pharmaceutical compounds to identify secreted proteins that can be
tested for activity in a variety of animal models. The present
invention advances the state of the art by providing many novel
human secreted proteins.
[0004] Secreted proteins are generally produced within cells at
rough endoplasmic reticulum, are then exported to the golgi
complex, and then move to secretory vesicles or granules, where
they are secreted- to the exterior of the cell via exocytosis.
[0005] Secreted proteins are particularly useful as diagnostic
markers. Many secreted proteins are found, and can easily be
measured, in serum. For example, a `signal sequence trap` technique
can often be utilized because many secreted proteins, such as
certain secretory breast cancer proteins, contain a molecular
signal sequence for cellular export. Additionally, antibodies
against particular secreted serum proteins can serve as potential
diagnostic agents, such as for diagnosing cancer.
[0006] Secreted proteins play a critical role in a wide array of
important biological processes in humans and have numerous
utilities; several illustrative examples are discussed herein. For
example, fibroblast secreted proteins participate in extracellular
matrix formation. Extracellular matrix affects growth factor
action, cell adhesion, and cell growth. Structural and quantitative
characteristics of fibroblast secreted proteins are modified during
the course of cellular aging and such aging related modifications
may lead to increased inhibition of cell adhesion, inhibited cell
stimulation by growth factors, and inhibited cell proliferative
ability (Eleftheriou et al., Mutat Res 1991
March-November;256(2-6): 127-38).
[0007] The secreted form of amyloid beta/A4 protein precursor (APP)
functions as a growth and/or differentiation factor. The secreted
form of APP can stimulate neurite extension of cultured
neuroblastoma cells, presumably through binding to a cell surface
receptor and thereby triggering intracellular transduction
mechanisms. (Roch et al., Ann N Y Acad Sci 1993 Sep.
24;695:149-57). Secreted APPs modulate neuronal excitability,
counteract effects of glutamate on growth cone behaviors, and
increase synaptic complexity. The prominent effects of secreted
APPs on synaptogenesis and neuronal survival suggest that secreted
APPs play a major role in the process of natural cell death and,
furthermore, may play a role in the development of a wide variety
of neurological disorders, such as stroke, epilepsy, and
Alzheimer's disease (Mattson et al., Perspect Dev Neurobiol 1998;
5(4):337-52).
[0008] Breast cancer cells secrete a 52K estrogen-regulated protein
(see Rochefort et al., Ann N Y Acad Sci 1986;464:190-201). This
secreted protein is therefore useful in breast cancer
diagnosis.
[0009] Two secreted proteins released by platelets, platelet factor
4 (PF4) and beta-thromboglobulin (betaTG), are accurate indicators
of platelet involvement in hemostasis and thrombosis and assays
that measure these secreted proteins are useful for studying the
pathogenesis and course of thromboembolic disorders (Kaplan, Adv
Exp Med Biol 1978;102:105-19).
[0010] Vascular endothelial growth factor (VEGF) is another example
of a naturally secreted protein. VEGF binds to cell-surface heparan
sulfates, is generated by hypoxic endothelial cells, reduces
apoptosis, and binds to high-affinity receptors that are
up-regulated by hypoxia (Asahara et al., Semin Interv Cardiol 1996
September;1(3):225-32).
[0011] Many critical components of the immune system are secreted
proteins, such as antibodies, and many important functions of the
immune system are dependent upon the action of secreted proteins.
For example, Saxon et al., Biochem Soc Trans 1997 May;25(2):383-7,
discusses secreted IgE proteins.
[0012] For a further review of secreted proteins, see
Nilsen-Hamilton et al., Cell Biol Int Rep 1982
September;6(9):815-36.
[0013] Lactate Dehydrogenase
[0014] Lactate dehydrogenase (LDH), sometimes referred to as lactic
dehydrogenase, is the most clinically important dehydrogenase
occurring in human serum. LDH is clinically important because serum
level of certain isozymes reflect pathological conditions in
particular tissues. Consequently, LDH is most often measured to
evaluate the presence of tissue damage. LDH serves as an indicator
suggestive of disturbances of the cellular integrity induced by
pathological conditions. Since LDH is an enzyme present in
essentially all major organ systems, serum LDH activity is abnormal
in a large number of disorders.
[0015] LDH is found in the cytoplasm of cells and catalyzes the
following reversible reaction for the interconversion of pyruvate
and lactate: Pyruvate+NADHLactate+NAD
[0016] This bidirectional reaction can be monitored
spectrophotometrically by measuring either the increase in NADH at
340 nm produced in the lactate-to-pyruvate reaction, or by
measuring the decrease in NADH at 340 nm produced in the
pyruvate-to-lactate reaction.
[0017] Mammalian LDH exists as five tetrameric isozymes composed of
combinations of two different polypeptide subunits with a molecular
weight of 136,700.+-.2,100 daltons per tetramer. These tetrameric
isozymes each differ in catalytic, physical, and immunological
properties. Cahn et al., (1962) designated the two polypeptide
subunits as H (heart) and M (muscle), which combine to form two
pure types of isozymes, H4 and M4, and three hybrids, H3M, H2M2 and
HM3. Type H4 is the most negatively charged at pH 7 and appears
nearest the anode upon zone electrophoresis. Subunit H predominates
in heart muscle and facilitates the aerobic oxidation of pyruvate.
The M subunit predominates in skeletal muscle and liver and is
predominantly involved with anaerobic metabolism and pyruvate
reduction.
[0018] Iodide inhibits LDH; p-mercuribenzoate also inhibits LDH,
but at a slower rate. LDH is activated by a number of organic
compounds that stabilize the enzyme, such as dimethyl sulfoxide,
ethanol, and methanol. Diethystilbestrol and several of its
derivatives also stabilize the enzyme.
[0019] Normal total LDH levels are approximately 105 to 333 IU/L
(international units per liter). Higher than normal LDH levels may
be indicative of such conditions as cerebrovascular accident (e.g.,
CVA, stroke), myocardial infarction, hemolytic anemia (as well as
other forms of anemia), hypotension, infectious mononucleosis,
intestinal ischemia and infarction, liver disease (e.g.,
hepatitis), muscle injury, muscular dystrophy, neoplastic
conditions, pancreatitis, and pulmonary infarction. If the total
LDH level is elevated, specific serum LDH isoenzymes may be
measured to increase the accuracy of diagnosis.
[0020] The most frequent use of LDH isoenzyme analysis is for the
diagnosis of myocardial infarction and other myocardial diseases.
Serum LDH levels are elevated in various myocardial diseases and a
certain pattern of LDH isoenzyme elevation occurs in myocardial
disease. In the absence of hemolysis, an LDH-1/LDH-2 ratio greater
than 1 (referred to as a "flipped" ratio) is usually indicative of
acute myocardial infarction (AMI). This flipped ratio is observed
in about 80% of AMI patients. The normal ratio rarely exceeds 0.80
in the absence of AMI. LDH activity begins to rise 8-12 hrs after
the onset of chest pain, peaking at 24-48 hrs, and elevated levels
may persist for 1 week or more.
[0021] LDH activity and its isoenzyme pattern are also useful
indicators of pathological conditions in the lungs, such as cell
damage or inflammation. Measurement of LDH activity levels and its
isoenzyme pattern in pleural effusion and in bronchoalveolar lavage
fluid may provide substantial information about lung and pulmonary
endothelial cell injury.
[0022] LDH isoenzymes are also useful for evaluating other
physiological conditions and disorders such as muscle trauma, liver
damage, and a variety of malignancies. In these cases, variations
of isoenzyme distribution are useful diagnostic indicators. Serum
LDH levels are also useful for diagnosing ovarian dysgerminoma and
testicular germ cell tumors. Furthermore, LDH is also useful for
establishing the survival duration and rate in Hodgkin's disease
and non-Hodgkin's lymphoma, and in the follow-up of ovarian
dysgerminoma. Additionally, LDH is also useful for a variety of
NAD, NADH, NADP and NADPH related studies.
[0023] For a further review of lactate dehydrogenases, see: Li,
Prog Clin Biol Res 1990;344:75-99; Markert et al., Cell Biochem
Funct 1984 July;2(3):131-4; Huijgen et al., Eur J Clin Chem Clin
Biochem 1997 August;35(8):569-79; Drent et al., Eur Respir J 1996
August;9(8):1736-42; Vanderlinde, Ann Clin Lab Sci 1985
January-February;15(1):13-31; and Wolf, Clin Lab Med 1989
December;9(4):655-65.
[0024] Secreted proteins, particularly members of the lactate
dehydrogenase secreted protein subfamily, are a major target for
drug action and development. Accordingly, it is valuable to the
field of pharmaceutical development to identify and characterize
previously unknown members of this subfamily of secreted proteins.
The present invention advances the state of the art by providing
previously unidentified human secreted proteins that have homology
to members of the lactate dehydrogenase secreted protein
subfamily.
SUMMARY OF THE INVENTION
[0025] The present invention is based in part on the identification
of amino acid sequences of human secreted peptides and proteins
that are related to the lactate dehydrogenase secreted protein
subfamily, as well as allelic variants and other mammalian
orthologs thereof. These unique peptide sequences, and nucleic acid
sequences that encode these peptides, can be used as models for the
development of human therapeutic targets, aid in the identification
of therapeutic proteins, and serve as targets for the development
of human therapeutic agents that modulate secreted protein activity
in cells and tissues that express the secreted protein.
DESCRIPTION OF THE FIGURE SHEETS
[0026] FIG. 1 provides the nucleotide sequence of a cDNA molecule
or transcript sequence that encodes the secreted protein of the
present invention. (SEQ ID NO:1) In addition, structure and
functional information is provided, such as ATG start, stop and
tissue distribution, where available, that allows one to readily
determine specific uses of inventions based on this molecular
sequence.
[0027] FIG. 2 provides the predicted amino acid sequence of the
secreted protein of the present invention. (SEQ ID NO:2) In
addition structure and functional information such as protein
family, function, and modification sites is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence.
[0028] FIG. 3 provides genomic sequences that span the gene
encoding the secreted protein of the present invention. (SEQ ID
NO:3) In addition structure and functional information, such as
intron/exon structure, promoter location, etc., is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence. As indicated by the
data presented in FIG. 3, the map position was determined to be on
chromosome 15.
DETAILED DESCRIPTION OF THE INVENTION
[0029] General Description
[0030] The present invention is based on the sequencing of the
human genome. During the sequencing and assembly of the human
genome, analysis of the sequence information revealed previously
unidentified fragments of the human genome that encode peptides
that share structural and/or sequence homology to
protein/peptide/domains identified and characterized within the art
as being a secreted protein or part of a secreted protein and are
related to the lactate dehydrogenase secreted protein subfamily.
Utilizing these sequences, additional genomic sequences were
assembled and transcript and/or cDNA sequences were isolated and
characterized. Based on this analysis, the present invention
provides amino acid sequences of human secreted peptides and
proteins that are related to the lactate dehydrogenase secreted
protein subfamily, nucleic acid sequences in the form of transcript
sequences, cDNA sequences and/or genomic sequences that encode
these secreted peptides and proteins, nucleic acid variation
(allelic information), tissue distribution of expression, and
information about the closest art known protein/peptide/domain that
has structural or sequence homology to the secreted protein of the
present invention.
[0031] In addition to being previously unknown, the peptides that
are provided in the present invention are selected based on their
ability to be used for the development of commercially important
products and services. Specifically, the present peptides are
selected based on homology and/or structural relatedness to known
secreted proteins of the lactate dehydrogenase secreted protein
subfamily and the expression pattern observed. The art has clearly
established the commercial importance of members of this family of
proteins and proteins that have expression patterns similar to that
of the present gene. Some of the more specific features of the
peptides of the present invention, and the uses thereof, are
described herein, particularly in the Background of the Invention
and in the annotation provided in the Figures, and/or are known
within the art for each of the known lactate dehydrogenase family
or subfamily of secreted proteins.
[0032] Specific Embodiments
[0033] Peptide Molecules The present invention provides nucleic
acid sequences that encode protein molecules that have been
identified as being members of the secreted protein family of
proteins and are related to the lactate dehydrogenase secreted
protein subfamily (protein sequences are provided in FIG. 2,
transcript/cDNA sequences are provided in FIG. 1 and genomic
sequences are provided in FIG. 3). The peptide sequences provided
in FIG. 2, as well as the obvious variants described herein,
particularly allelic variants as identified herein and using the
information in FIG. 3, will be referred herein as the secreted
peptides of the present invention, secreted peptides, or
peptides/proteins of the present invention.
[0034] The present invention provides isolated peptide and protein
molecules that consist of, consist essentially of, or comprise the
amino acid sequences of the secreted peptides disclosed in the FIG.
2, (encoded by the nucleic acid molecule shown in FIG. 1,
transcript/cDNA or FIG. 3, genomic sequence), as well as all
obvious variants of these peptides that are within the art to make
and use. Some of these variants are described in detail below.
[0035] As used herein, a peptide is said to be "isolated" or
"purified" when it is substantially free of cellular material or
free of chemical precursors or other chemicals. The peptides of the
present invention can be purified to homogeneity or other degrees
of purity. The level of purification will be based on the intended
use. The critical feature is that the preparation allows for the
desired function of the peptide, even if in the presence of
considerable amounts of other components (the features of an
isolated nucleic acid molecule is discussed below).
[0036] In some uses, "substantially free of cellular material"
includes preparations of the peptide having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the peptide is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0037] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the peptide in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the secreted peptide having less than
about 30% (by dry weight) chemical precursors or other chemicals,
less than about 20% chemical precursors or other chemicals, less
than about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0038] The isolated secreted peptide can be purified from cells
that naturally express it, purified from cells that have been
altered to express it (recombinant), or synthesized using known
protein synthesis methods. For example, a nucleic acid molecule
encoding the secreted peptide is cloned into an expression vector,
the expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques. Many of these techniques are
described in detail below.
[0039] Accordingly, the present invention provides proteins that
consist of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence
of such a protein is provided in FIG. 2. A protein consists of an
amino acid sequence when the amino acid sequence is the final amino
acid sequence of the protein.
[0040] The present invention further provides proteins that consist
essentially of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists
essentially of an amino acid sequence when such an amino acid
sequence is present with only a few additional amino acid residues,
for example from about 1 to about 100 or so additional residues,
typically from 1 to about 20 additional residues in the final
protein.
[0041] The present invention further provides proteins that
comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2),
for example, proteins encoded by the transcript/cDNA nucleic acid
sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences
provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid
sequence when the amino acid sequence is at least part of the final
amino acid sequence of the protein. In such a fashion, the protein
can be only the peptide or have additional amino acid molecules,
such as amino acid residues (contiguous encoded sequence) that are
naturally associated with it or heterologous amino acid
residues/peptide sequences. Such a protein can have a few
additional amino acid residues or can comprise several hundred or
more additional amino acids. The preferred classes of proteins that
are comprised of the secreted peptides of the present invention are
the naturally occurring mature proteins. A brief description of how
various types of these proteins can be made/isolated is provided
below.
[0042] The secreted peptides of the present invention can be
attached to heterologous sequences to form chimeric or fusion
proteins. Such chimeric and fusion proteins comprise a secreted
peptide operatively linked to a heterologous protein having an
amino acid sequence not substantially homologous to the secreted
peptide. "Operatively linked" indicates that the secreted peptide
and the heterologous protein are fused in-frame. The heterologous
protein can be fused to the N-terminus or C-terminus of the
secreted peptide.
[0043] In some uses, the fusion protein does not affect the
activity of the secreted peptide per se. For example, the fusion
protein can include, but is not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig
fusions. Such fusion proteins, particularly poly-His fusions, can
facilitate the purification of recombinant secreted peptide. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of a protein can be increased by using a heterologous
signal sequence.
[0044] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al., Current
Protocols in Molecular Biology, 1992). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A secreted peptide-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the secreted peptide.
[0045] As mentioned above, the present invention also provides and
enables obvious variants of the amino acid sequence of the proteins
of the present invention, such as naturally occurring mature forms
of the peptide, allelic/sequence variants of the peptides,
non-naturally occurring recombinantly derived variants of the
peptides, and orthologs and paralogs of the peptides. Such variants
can readily be generated using art-known techniques in the fields
of recombinant nucleic acid technology and protein biochemistry. It
is understood, however, that variants exclude any amino acid
sequences disclosed prior to the invention.
[0046] Such variants can readily be identified/made using molecular
techniques and the sequence information disclosed herein. Further,
such variants can readily be distinguished from other peptides
based on sequence and/or structural homology to the secreted
peptides of the present invention. The degree of homology/identity
present will be based primarily on whether the peptide is a
functional variant or non-functional variant, the amount of
divergence present in the paralog family and the evolutionary
distance between the orthologs.
[0047] To determine the percent identity of two amino acid
sequences or two nucleic acid sequences, the sequences are aligned
for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or nucleic acid
sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In a preferred embodiment, at
least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of
a reference sequence is aligned for comparison purposes. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0048] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (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 1, 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). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (Devereux, J., et
al., Nucleic Acids Res. 12(1):387 (1984)) (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. In another embodiment, the percent identity between two amino
acid or nucleotide sequences is determined using the algorithm of
E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0049] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0050] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the secreted peptides of the present invention
as well as being encoded by the same genetic locus as the secreted
peptide provided herein.
[0051] Allelic variants of a secreted peptide can readily be
identified as being a human protein having a high degree
(significant) of sequence homology/identity to at least a portion
of the secreted peptide as well as being encoded by the same
genetic locus as the secreted peptide provided herein. Genetic
locus can readily be determined based on the genomic information
provided in FIG. 3, such as the genomic sequence mapped to the
reference human. As indicated by the data presented in FIG. 3, the
map position was determined to be on chromosome 15. As used herein,
two proteins (or a region of the proteins) have significant
homology when the amino acid sequences are typically at least about
70-80%, 80-90%, and more typically at least about 90-95% or more
homologous. A significantly homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic
acid sequence that will hybridize to a secreted peptide encoding
nucleic acid molecule under stringent conditions as more fully
described below.
[0052] Paralogs of a secreted peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the secreted peptide, as being encoded by a gene
from humans, and as having similar activity or function. Two
proteins will typically be considered paralogs when the amino acid
sequences are typically at least about 60% or greater, and more
typically at least about 70% or greater homology through a given
region or domain. Such paralogs will be encoded by a nucleic acid
sequence that will hybridize to a secreted peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0053] Orthologs of a secreted peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the secreted peptide as well as being encoded by
a gene from another organism. Preferred orthologs will be isolated
from mammals, preferably primates, for the development of human
therapeutic targets and agents. Such orthologs will be encoded by a
nucleic acid sequence that will hybridize to a secreted peptide
encoding nucleic acid molecule under moderate to stringent
conditions, as more fully described below, depending on the degree
of relatedness of the two organisms yielding the proteins.
[0054] Non-naturally occurring variants of the secreted peptides of
the present invention can readily be generated using recombinant
techniques. Such variants include, but are not limited to
deletions, additions and substitutions in the amino acid sequence
of the secreted peptide. For example, one class of substitutions
are conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a secreted peptide by another
amino acid of like characteristics. Typically seen as conservative
substitutions are the replacements, one for another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser and Thr, exchange of the acidic residues Asp
and Glu; substitution between the amide residues Asn and Gln;
exchange of the basic residues Lys and Arg; and replacements among
the aromatic residues Phe and Tyr. Guidance concerning which amino
acid changes are likely to be phenotypically silent are found in
Bowie et al., Science 247:1306-1310 (1990).
[0055] Variant secreted peptides can be fully functional or can
lack function in one or more activities, e.g. ability to bind
substrate, ability to phosphorylate substrate, ability to mediate
signaling, etc. Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. FIG. 2 provides the result of protein
analysis and can be used to identify critical domains/regions.
Functional variants can also contain substitution of similar amino
acids that result in no change or an insignificant change in
function. Alternatively, such substitutions may positively or
negatively affect function to some degree.
[0056] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0057] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)), particularly using the results
provided in FIG. 2. The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant
molecules are then tested for biological activity such as secreted
protein activity or in assays such as an in vitro proliferative
activity. Sites that are critical for binding partner/substrate
binding can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et
al. Science 255:306-312 (1992)).
[0058] The present invention further provides fragments of the
secreted peptides, in addition to proteins and peptides that
comprise and consist of such fragments, particularly those
comprising the residues identified in FIG. 2. The fragments to
which the invention pertains, however, are not to be construed as
encompassing fragments that may be disclosed publicly prior to the
present invention.
[0059] As used herein, a fragment comprises at least 8, 10, 12, 14,
16, or more contiguous amino acid residues from a secreted peptide.
Such fragments can be chosen based on the ability to retain one or
more of the biological activities of the secreted peptide or could
be chosen for the ability to perform a function, e.g. bind a
substrate or act as an immunogen. Particularly important fragments
are biologically active fragments, peptides that are, for example,
about 8 or more amino acids in length. Such fragments will
typically comprise a domain or motif of the secreted peptide, e.g.,
active site or a substrate-binding domain. Further, possible
fragments include, but are not limited to, domain or motif
containing fragments, soluble peptide fragments, and fragments
containing immunogenic structures. Predicted domains and functional
sites are readily identifiable by computer programs well known and
readily available to those of skill in the art (e.g., PROSITE
analysis). The results of one such analysis are provided in FIG.
2.
[0060] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in secreted peptides are
described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the art
(some of these features are identified in FIG. 2).
[0061] Known modifications include, but are not limited to,
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
crosslinks, 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.
[0062] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[0063] Accordingly, the secreted peptides of the present invention
also encompass derivatives or analogs in which a substituted amino
acid residue is not one encoded by the genetic code, in which a
substituent group is included, in which the mature secreted peptide
is fused with another compound, such as a compound to increase the
half-life of the secreted peptide (for example, polyethylene
glycol), or in which the additional amino acids are fused to the
mature secreted peptide, such as a leader or secretory sequence or
a sequence for purification of the mature secreted peptide or a
pro-protein sequence.
[0064] Protein/Peptide Uses
[0065] The proteins of the present invention can be used in
substantial and specific assays related to the functional
information provided in the Figures; to raise antibodies or to
elicit another immune response; as a reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of
the protein (or its binding partner or ligand) in biological
fluids; and as markers for tissues in which the corresponding
protein is preferentially expressed (either constitutively or at a
particular stage of tissue differentiation or development or in a
disease state). Where the protein binds or potentially binds to
another protein or ligand (such as, for example, in a secreted
protein-effector protein interaction or secreted protein-ligand
interaction), the protein can be used to identify the binding
partner/ligand so as to develop a system to identify inhibitors of
the binding interaction. Any or all of these uses are capable of
being developed into reagent grade or kit format for
commercialization as commercial products.
[0066] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
[0067] The potential uses of the peptides of the present invention
are based primarily on the source of the protein as well as the
class/action of the protein. For example, secreted proteins
isolated from humans and their human/mammalian orthologs serve as
targets for identifying agents for use in mammalian therapeutic
applications, e.g. a human drug, particularly in modulating a
biological or pathological response in a cell or tissue that
expresses the secreted protein. A large percentage of
pharmaceutical agents are being developed that modulate the
activity of secreted proteins, particularly members of the lactate
dehydrogenase subfamily (see Background of the Invention). The
structural and functional information provided in the Background
and Figures provide specific and substantial uses for the molecules
of the present invention, particularly in combination with the
expression information provided in FIG. 1. Such uses can readily be
determined using the information provided herein, that which is
known in the art, and routine experimentation.
[0068] The proteins of the present invention (including variants
and fragments that may have been disclosed prior to the present
invention) are useful for biological assays related to secreted
proteins that are related to members of the lactate dehydrogenase
subfamily. Such assays involve any of the known secreted protein
functions or activities or properties useful for diagnosis and
treatment of secreted protein-related conditions that are specific
for the subfamily of secreted proteins that the one of the present
invention belongs to, particularly in cells and tissues that
express the secreted protein.
[0069] The proteins of the present invention are also useful in
drug screening assays, in cell-based or cell-free systems.
Cell-based systems can be native, i.e., cells that normally express
the secreted protein, as a biopsy or expanded in cell culture. In
an alternate embodiment, cell-based assays involve recombinant host
cells expressing the secreted protein.
[0070] The polypeptides can be used to identify compounds that
modulate secreted protein activity of the protein in its natural
state or an altered form that causes a specific disease or
pathology associated with the secreted protein. Both the secreted
proteins of the present invention and appropriate variants and
fragments can be used in high-throughput screens to assay candidate
compounds for the ability to bind to the secreted protein. These
compounds can be further screened against a functional secreted
protein to determine the effect of the compound on the secreted
protein activity. Further, these compounds can be tested in animal
or invertebrate systems to determine activity/effectiveness.
Compounds can be identified that activate (agonist) or inactivate
(antagonist) the secreted protein to a desired degree.
[0071] Further, the proteins of the present invention can be used
to screen a compound for the ability to stimulate or inhibit
interaction between the secreted protein and a molecule that
normally interacts with the secreted protein, e.g. a substrate or a
component of the signal pathway that the secreted protein normally
interacts (for example, another secreted protein). Such assays
typically include the steps of combining the secreted protein with
a candidate compound under conditions that allow the secreted
protein, or fragment, to interact with the target molecule, and to
detect the formation of a complex between the protein and the
target or to detect the biochemical consequence of the interaction
with the secreted protein and the target.
[0072] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al., Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0073] One candidate compound is a soluble fragment of the receptor
that competes for substrate binding. Other candidate compounds
include mutant secreted proteins or appropriate fragments
containing mutations that affect secreted protein function and thus
compete for substrate. Accordingly, a fragment that competes for
substrate, for example with a higher affinity, or a fragment that
binds substrate but does not allow release, is encompassed by the
invention.
[0074] Any of the biological or biochemical functions mediated by
the secreted protein can be used as an endpoint assay. These
include all of the biochemical or biochemical/biological events
described herein, in the references cited herein, incorporated by
reference for these endpoint assay targets, and other functions
known to those of ordinary skill in the art or that can be readily
identified using the information provided in the Figures,
particularly FIG. 2. Specifically, a biological function of a cell
or tissues that expresses the secreted protein can be assayed.
[0075] Binding and/or activating compounds can also be screened by
using chimeric secreted proteins in which the amino terminal
extracellular domain, or parts thereof, the entire transmembrane
domain or subregions, such as any of the seven transmembrane
segments or any of the intracellular or extracellular loops and the
carboxy terminal intracellular domain, or parts thereof, can be
replaced by heterologous domains or subregions. For example, a
substrate-binding region can be used that interacts with a
different substrate then that which is recognized by the native
secreted protein. Accordingly, a different set of signal
transduction components is available as an end-point assay for
activation. This allows for assays to be performed in other than
the specific host cell from which the secreted protein is
derived.
[0076] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the secreted protein (e.g. binding
partners and/or ligands). Thus, a compound is exposed to a secreted
protein polypeptide under conditions that allow the compound to
bind or to otherwise interact with the polypeptide. Soluble
secreted protein polypeptide is also added to the mixture if the
test compound interacts with the soluble secreted protein
polypeptide, it decreases the amount of complex formed or activity
from the secreted protein target. This type of assay is
particularly useful in cases in which compounds are sought that
interact with specific regions of the secreted protein. Thus, the
soluble polypeptide that competes with the target secreted protein
region is designed to contain peptide sequences corresponding to
the region of interest.
[0077] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the secreted protein, or fragment,
or its target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0078] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtitre
plates, which are then combined with the cell lysates (e.g.,
.sup.35S-labeled) and the candidate compound, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads are washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supernatant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of secreted protein-binding protein found
in the bead fraction quantitated from the gel using standard
electrophoretic techniques. For example, either the polypeptide or
its target molecule can be immobilized utilizing conjugation of
biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies reactive with the protein but which do
not interfere with binding of the protein to its target molecule
can be derivatized to the wells of the plate, and the protein
trapped in the wells by antibody conjugation. Preparations of a
secreted protein-binding protein and a candidate compound are
incubated in the secreted protein-presenting wells and the amount
of complex trapped in the well can be quantitated. Methods for
detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the secreted protein target
molecule, or which are reactive with secreted protein and compete
with the target molecule, as well as enzyme-linked assays which
rely on detecting an enzymatic activity associated with the target
molecule.
[0079] Agents that modulate one of the secreted proteins of the
present invention can be identified using one or more of the above
assays, alone or in combination. It is generally preferable to use
a cell-based or cell free system first and then confirm activity in
an animal or other model system. Such model systems are well known
in the art and can readily be employed in this context.
[0080] Modulators of secreted protein activity identified according
to these drug screening assays can be used to treat a subject with
a disorder mediated by the secreted protein pathway, by treating
cells or tissues that express the secreted protein. These methods
of treatment include the steps of administering a modulator of
secreted protein activity in a pharmaceutical composition to a
subject in need of such treatment, the modulator being identified
as described herein.
[0081] In yet another aspect of the invention, the secreted
proteins can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with the
secreted protein and are involved in secreted protein activity.
[0082] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a secreted
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a secreted protein-dependent complex, the DNA-binding
and activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the secreted protein.
[0083] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a secreted
protein-modulating agent, an antisense secreted protein nucleic
acid molecule, a secreted protein-specific antibody, or a secreted
protein-binding partner) can be used in an animal or other model to
determine the efficacy, toxicity, or side effects of treatment with
such an agent. Alternatively, an agent identified as described
herein can be used in an animal or other model to determine the
mechanism of action of such an agent. Furthermore, this invention
pertains to uses of novel agents identified by the above-described
screening assays for treatments as described herein.
[0084] The secreted proteins of the present invention are also
useful to provide a target for diagnosing a disease or
predisposition to disease mediated by the peptide. Accordingly, the
invention provides methods for detecting the presence, or levels
of, the protein (or encoding mRNA) in a cell, tissue, or organism.
The method involves contacting a biological sample with a compound
capable of interacting with the secreted protein such that the
interaction-can be detected. Such an assay can be provided in a
single detection format or a multi-detection format such as an
antibody chip array.
[0085] One agent for detecting a protein in a sample is an antibody
capable of selectively binding to protein. A biological sample
includes tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject.
[0086] The peptides of the present invention also provide targets
for diagnosing active protein activity, disease, or predisposition
to disease, in a patient having a variant peptide, particularly
activities and conditions that are known for other members of the
family of proteins to which the present one belongs. Thus, the
peptide can be isolated from a biological sample and assayed for
the presence of a genetic mutation that results in aberrant
peptide. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered secreted protein activity in
cell-based or cell-free assay, alteration in substrate or
antibody-binding pattern, altered isoelectric point, direct amino
acid sequencing, and any other of the known assay techniques useful
for detecting mutations in a protein. Such an assay can be provided
in a single detection format or a multi-detection format such as an
antibody chip array.
[0087] In vitro techniques for detection of peptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence using a detection
reagent, such as an antibody or protein binding agent.
Alternatively, the peptide can be detected in vivo in a subject by
introducing into the subject a labeled anti-peptide antibody or
other types of detection agent. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
Particularly useful are methods that detect the allelic variant of
a peptide expressed in a subject and methods which detect fragments
of a peptide in a sample.
[0088] The peptides are also useful in pharmacogenomic analysis.
Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and
Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical
outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes effects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants of the
secreted protein in which one or more of the secreted protein
functions in one population is different from those in another
population. The peptides thus allow a target to ascertain a genetic
predisposition that can affect treatment modality. Thus, in a
ligand-based treatment, polymorphism may give rise to amino
terminal extracellular domains and/or other substrate-binding
regions that are more or less active in substrate binding, and
secreted protein activation. Accordingly, substrate dosage would
necessarily be modified to maximize the therapeutic effect within a
given population containing a polymorphism. As an alternative to
genotyping, specific polymorphic peptides could be identified.
[0089] The peptides are also useful for treating a disorder
characterized by an absence of, inappropriate, or unwanted
expression of the protein. Accordingly, methods for treatment
include the use of the secreted protein or fragments.
[0090] Antibodies
[0091] The invention also provides antibodies that selectively bind
to one of the peptides of the present invention, a protein
comprising such a peptide, as well as variants and fragments
thereof. As used herein, an antibody selectively binds a target
peptide when it binds the target peptide and does not significantly
bind to unrelated proteins. An antibody is still considered to
selectively bind a peptide even if it also binds to other proteins
that are not substantially homologous with the target peptide so
long as such proteins share homology with a fragment or domain of
the peptide target of the antibody. In this case, it would be
understood that antibody binding to the peptide is still selective
despite some degree of cross-reactivity.
[0092] As used herein, an antibody is defined in terms consistent
with that recognized within the art: they are multi-subunit
proteins produced by a mammalian organism in response to an antigen
challenge. The antibodies of the present invention include
polyclonal antibodies and monoclonal antibodies, as well as
fragments of such antibodies, including, but not limited to, Fab or
F(ab').sub.2, and Fv fragments.
[0093] Many methods are known for generating and/or identifying
antibodies to a given target peptide. Several such methods are
described by Harlow, Antibodies, Cold Spring Harbor Press,
(1989).
[0094] In general, to generate antibodies, an isolated peptide is
used as an immunogen and is administered to a mammalian organism,
such as a rat, rabbit or mouse. The full-length protein, an
antigenic peptide fragment or a fusion protein can be used.
Particularly important fragments are those covering functional
domains, such as the domains identified in FIG. 2, and domain of
sequence homology or divergence amongst the family, such as those
that can readily be identified using protein alignment methods and
as presented in the Figures.
[0095] Antibodies are preferably prepared from regions or discrete
fragments of the secreted proteins. Antibodies can be prepared from
any region of the peptide as described herein. However, preferred
regions will include those involved in function/activity and/or
secreted protein/binding partner interaction. FIG. 2 can be used to
identify particularly important regions while sequence alignment
can be used to identify conserved and unique sequence
fragments.
[0096] An antigenic fragment will typically comprise at least 8
contiguous amino acid residues. The antigenic peptide can comprise,
however, at least 10, 12, 14, 16 or more amino acid residues. Such
fragments can be selected on a physical property, such as fragments
correspond to regions that are located on the surface of the
protein, e.g., hydrophilic regions or can be selected based on
sequence uniqueness (see FIG. 2).
[0097] Detection on an antibody of the present invention can be
facilitated by coupling (i.e., physically linking) the antibody to
a detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Exanples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0098] Antibody Uses
[0099] The antibodies can be used to isolate one of the proteins of
the present invention by standard techniques, such as affinity
chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the natural protein from cells and
recombinantly produced protein expressed in host cells. In
addition, such antibodies are useful to detect the presence of one
of the proteins of the present invention in cells or tissues to
determine the pattern of expression of the protein among various
tissues in an organism and over the course of normal development.
Further, such antibodies can be used to detect protein in situ, in
vitro, or in a cell lysate or supernatant in order to evaluate the
abundance and pattern of expression. Also, such antibodies can be
used to assess abnormal tissue distribution or abnormal expression
during development or progression of a biological condition.
Antibody detection of circulating fragments of the full length
protein can be used to identify turnover.
[0100] Further, the antibodies can be used to assess expression in
disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by an inappropriate
tissue distribution, developmental expression, level of expression
of the protein, or expressed/processed form, the antibody can be
prepared against the normal protein. If a disorder is characterized
by a specific mutation in the protein, antibodies specific for this
mutant protein can be used to assay for the presence of the
specific mutant protein.
[0101] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. The diagnostic uses can be applied, not only in
genetic testing, but also in monitoring a treatment modality.
Accordingly, where treatment is ultimately aimed at correcting
expression level or the presence of aberrant sequence and aberrant
tissue distribution or developmental expression, antibodies
directed against the protein or relevant fragments can be used to
monitor therapeutic efficacy.
[0102] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic proteins
can be used to identify individuals that require modified treatment
modalities. The antibodies are also useful as diagnostic tools as
an immunological marker for aberrant protein analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0103] The antibodies are also useful for tissue typing. Thus,
where a specific protein has been correlated with expression in a
specific tissue, antibodies that are specific for this protein can
be used to identify a tissue type.
[0104] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the secreted peptide
to a binding partner such as a substrate. These uses can also be
applied in a therapeutic context in which treatment involves
inhibiting the protein's function. An antibody can be used, for
example, to block binding, thus modulating (agonizing or
antagonizing) the peptides activity. Antibodies can be prepared
against specific fragments containing sites required for function
or against intact protein that is associated with a cell or cell
membrane. See FIG. 2 for structural information relating to the
proteins of the present invention.
[0105] The invention also encompasses kits for using antibodies to
detect the presence of a protein in a biological sample. The kit
can comprise antibodies such as a labeled or labelable antibody and
a compound or agent for detecting protein in a biological sample;
means for determining the amount of protein in the sample; means
for comparing the amount of protein in the sample with a standard;
and instructions for use. Such a kit can be supplied to detect a
single protein or epitope or can be configured to detect one of a
multitude of epitopes, such as in an antibody detection array.
Arrays are described in detail below for nuleic acid arrays and
similar methods have been developed for antibody arrays.
[0106] Nucleic Acid Molecules
[0107] The present invention further provides isolated nucleic acid
molecules that encode a secreted peptide or protein of the present
invention (cDNA, transcript and genomic sequence). Such nucleic
acid molecules will consist of, consist essentially of, or comprise
a nucleotide sequence that encodes one of the secreted peptides of
the present invention, an allelic variant thereof, or an ortholog
or paralog thereof.
[0108] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
However, there can be some flanking nucleotide sequences, for
example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less,
particularly contiguous peptide encoding sequences and peptide
encoding sequences within the same gene but separated by introns in
the genomic sequence. The important point is that the nucleic acid
is isolated from remote and unimportant flanking sequences such
that it can be subjected to the specific manipulations described
herein such as recombinant expression, preparation of probes and
primers, and other uses specific to the nucleic acid sequences.
[0109] Moreover, an "isolated" nucleic acid molecule, such as a
transcript/cDNA molecule, can be substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0110] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0111] Accordingly, the present invention provides nucleic acid
molecules that consist of the nucleotide sequence shown in FIG. 1
or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists
of a nucleotide sequence when the nucleotide sequence is the
complete nucleotide sequence of the nucleic acid molecule.
[0112] The present invention further provides nucleic acid
molecules that consist essentially of the nucleotide sequence shown
in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3,
genomic sequence), or any nucleic acid molecule that encodes the
protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule
consists essentially of a nucleotide sequence when such a
nucleotide sequence is present with only a few additional nucleic
acid residues in the final nucleic acid molecule.
[0113] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in FIG. 1 or
3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that ericodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises
a nucleotide sequence when the nucleotide sequence is at least part
of the final nucleotide sequence of the nucleic acid molecule. In
such a fashion, the nucleic acid molecule can be only the
nucleotide sequence or have additional nucleic acid residues, such
as nucleic acid residues that are naturally associated with it or
heterologous nucleotide sequences. Such a nucleic acid molecule can
have a few additional nucleotides or can comprises several hundred
or more additional nucleotides. A brief description of how various
types of these nucleic acid molecules can be readily made/isolated
is provided below.
[0114] In FIGS. 1 and 3, both coding and non-coding sequences are
provided. Because of the source of the present invention, humans
genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1),
the nucleic acid molecules in the Figures will contain genomic
intronic sequences, 5' and 3' non-coding sequences, gene regulatory
regions and non-coding intergenic sequences. In general such
sequence features are either noted in FIGS. 1 and 3 or can readily
be identified using computational tools known in the art. As
discussed below, some of the non-coding regions, particularly gene
regulatory elements such as promoters, are useful for a variety of
purposes, e.g. control of heterologous gene expression, target for
identifying gene activity modulating compounds, and are
particularly claimed as fragments of the genomic sequence provided
herein.
[0115] The isolated nucleic acid molecules can encode the mature
protein plus additional amino or carboxyl-terminal amino acids, or
amino acids interior to the mature peptide (when the mature form
has more than one peptide chain, for instance). Such sequences may
play a role in processing of a protein from precursor to a mature
form, facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0116] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the secreted
peptide alone, the sequence encoding the mature peptide and
additional coding sequences, such as a leader or secretory sequence
(e.g., a pre-pro or pro-protein sequence), the sequence encoding
the mature peptide, with or without the additional coding
sequences, plus additional non-coding sequences, for example
introns and non-coding 5' and 3' sequences such as transcribed but
non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals),
ribosome binding and stability of mRNA. In addition, the nucleic
acid molecule may be fused to a marker sequence encoding, for
example, a peptide that facilitates purification.
[0117] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0118] The invention further provides nucleic acid molecules that
encode fragments of the peptides of the present invention as well
as nucleic acid molecules that encode obvious variants of the
secreted proteins of the present invention that are described
above. Such nucleic acid molecules may be naturally occurring, such
as allelic variants (same locus), paralogs (different locus), and
orthologs (different organism), or may be constructed by
recombinant DNA methods or by chemical synthesis. Such
non-naturally occurring variants may be made by mutagenesis
techniques, including those applied to nucleic acid molecules,
cells, or organisms. Accordingly, as discussed above, the variants
can contain nucleotide substitutions, deletions, inversions and
insertions. Variation can occur in either or both the coding and
non-coding regions. The variations can produce both conservative
and non-conservative amino acid substitutions.
[0119] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred
non-coding fragments include, but are not limited to, promoter
sequences, enhancer sequences, gene modulating sequences and gene
termination sequences. Such fragments are useful in controlling
heterologous gene expression and in developing screens to identify
gene-modulating agents. A promoter can readily be identified as
being 5' to the ATG start site in the genomic sequence provided in
FIG. 3.
[0120] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could at
least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length
of the fragment will be based on its intended use. For example, the
fragment can encode epitope bearing regions of the peptide, or can
be useful as DNA probes and primers. Such fragments can be isolated
using the known nucleotide sequence to synthesize an
oligonucleotide probe. A labeled probe can then be used to screen a
cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in
PCR reactions to clone specific regions of gene.
[0121] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 20, 25, 40, 50 or
more consecutive nucleotides.
[0122] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. As described in the Peptide
Section, these variants comprise a nucleotide sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous to the
nucleotide sequence shown in the Figure sheets or a fragment of
this sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to the nucleotide sequence shown in the Figure sheets
or a fragment of the sequence. Allelic variants can readily be
determined by genetic locus of the encoding gene.
[0123] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45 C, followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 50-65 C. Examples of
moderate to low stringency hybridization conditions are well known
in the art.
[0124] Nucleic Acid Molecule Uses
[0125] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in FIG. 2 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in FIG. 2.
[0126] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in the Figures.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed,
fragments are not to be construed as encompassing fragments
disclosed prior to the present invention.
[0127] The nucleic acid molecules are also useful as primers for
PCR to amplify any given region of a nucleic acid molecule and are
useful to synthesize antisense molecules of desired length and
sequence.
[0128] The nucleic acid molecules are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the peptide sequences. Vectors
also include insertion vectors, used to integrate into another
nucleic acid molecule sequence, such as into the cellular genome,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding sequence can be replaced via
homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0129] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0130] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods.
[0131] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0132] The nucleic acid molecules are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from the nucleic acid molecules described herein.
[0133] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0134] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0135] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0136] The nucleic acid molecules are also useful as hybridization
probes for determining the presence, level, form and distribution
of nucleic acid expression. Accordingly, the probes can be used to
detect the presence of, or to determine levels of, a specific
nucleic acid molecule in cells, tissues, and in organisms. The
nucleic acid whose level is determined can be DNA or RNA.
Accordingly, probes corresponding to the peptides described herein
can be used to assess expression and/or gene copy number in a given
cell, tissue, or organism. These uses are relevant for diagnosis of
disorders involving an increase or decrease in secreted protein
expression relative to normal results.
[0137] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA include Southern hybridizations and in situ
hybridization.
[0138] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a secreted protein, such
as by measuring a level of a secreted protein-encoding nucleic acid
in a sample of cells from a subject e.g., mRNA or genomic DNA, or
determining if a secreted protein gene has been mutated.
[0139] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate secreted protein nucleic acid
expression.
[0140] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the secreted protein gene, particularly
biological and pathological processes that are mediated by the
secreted protein in cells and tissues that express it. The method
typically includes assaying the ability of the compound to modulate
the expression of the secreted protein nucleic acid and thus
identifying a compound that can be used to treat a disorder
characterized by undesired secreted protein nucleic acid
expression. The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing the
secreted protein nucleic acid or recombinant cells genetically
engineered to express specific nucleic acid sequences.
[0141] Thus, modulators of secreted protein gene expression can be
identified in a method wherein a cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of secreted protein mRNA in the presence of the
candidate compound is compared to the level of expression of
secreted protein mRNA in the absence of the candidate compound. The
candidate compound can then be identified as a modulator of nucleic
acid expression based on this comparison and be used, for example
to treat a disorder characterized by aberrant nucleic acid
expression. When expression of mRNA is statistically significantly
greater in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
nucleic acid expression. When nucleic acid expression is
statistically significantly less in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of nucleic acid expression.
[0142] The invention further provides methods of treatment, with
the nucleic acid as a target, using a compound identified through
drug screening as a gene modulator to modulate secreted protein
nucleic acid expression in cells and tissues that express the
secreted protein. Modulation includes both up-regulation (i.e.
activation or agonization) or down-regulation (suppression or
antagonization) or nucleic acid expression.
[0143] Alternatively, a modulator for secreted protein nucleic acid
expression can be a small molecule or drug identified using the
screening assays described herein as long as the drug or small
molecule inhibits the secreted protein nucleic acid expression in
the cells and tissues that express the protein.
[0144] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the secreted protein gene in clinical trials or in a
treatment regimen. Thus, the gene expression pattern can serve as a
barometer for the continuing effectiveness of treatment with the
compound, particularly with compounds to which a patient can
develop resistance. The gene expression pattern can also serve as a
marker indicative of a physiological response of the affected cells
to the compound. Accordingly, such monitoring would allow either
increased administration of the compound or the administration of
alternative compounds to which the patient has not become
resistant. Similarly, if the level of nucleic acid expression falls
below a desirable level, administration of the compound could be
commensurately decreased.
[0145] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in secreted protein nucleic acid
expression, and particularly in qualitative changes that lead to
pathology. The nucleic acid molecules can be used to detect
mutations in secreted protein genes and gene expression products
such as mRNA. The nucleic acid molecules can be used as
hybridization probes to detect naturally occurring genetic
mutations in the secreted protein gene and thereby to determine
whether a subject with the mutation is at risk for a disorder
caused by the mutation. Mutations include deletion, addition, or
substitution of one or more nucleotides in the gene, chromosomal
rearrangement, such as inversion or transposition, modification of
genomic DNA, such as aberrant methylation patterns or changes in
gene copy number, such as amplification. Detection of a mutated
form of the secreted protein gene associated with a dysfunction
provides a diagnostic tool for an active disease or susceptibility
to disease when the disease results from overexpression,
underexpression, or altered expression of a secreted protein.
[0146] Individuals carrying mutations in the secreted protein
gene-can be detected at the nucleic acid level by a variety of
techniques. Genomic DNA can be analyzed directly or can be
amplified by using PCR prior to analysis. RNA or cDNA can be used
in the same way. In some uses, detection of the mutation involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988);
and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which
can be particularly useful for detecting point mutations in the
gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).
This method can include the steps of collecting a sample of cells
from a patient, isolating nucleic acid (e.g., genomic, mRNA or
both) from the cells of the sample, contacting the nucleic acid
sample with one or more primers which specifically hybridize to a
gene under conditions such that hybridization and amplification of
the gene (if present) occurs, and detecting the presence or absence
of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[0147] Alternatively, mutations in a secreted protein gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0148] Further, sequence-specific ribozymes (U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
Perfectly matched sequences can be distinguished from mismatched
sequences by nuclease cleavage digestion assays or by differences
in melting temperature.
[0149] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method. Furthermore, sequence differences
between a mutant secreted protein gene and a wild-type gene can be
determined by direct DNA sequencing. A variety of automated
sequencing procedures can be utilized when performing the
diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448),
including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen et al., Adv.
Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.
Biotechnol. 38:147-159 (1993)).
[0150] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.,
Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al., Nature
313:495 (1985)). Examples of other techniques for detecting point
mutations include selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0151] The nucleic acid molecules are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules described herein can be
used to assess the mutation content of the secreted protein gene in
an individual in order to select an appropriate compound or dosage
regimen for treatment.
[0152] Thus nucleic acid molecules displaying genetic variations
that affect treatment provide a diagnostic target that can be used
to tailor treatment in an individual. Accordingly, the production
of recombinant cells and animals containing these polymorphisms
allow effective clinical design of treatment compounds and dosage
regimens.
[0153] The nucleic acid molecules are thus useful as antisense
constructs to control secreted protein gene expression in cells,
tissues, and organisms. A DNA antisense nucleic acid molecule is
designed to be complementary to a region of the gene involved in
transcription, preventing transcription and hence production of
secreted protein. An antisense RNA or DNA nucleic acid molecule
would hybridize to the mRNA and thus block translation of mRNA into
secreted protein.
[0154] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of secreted protein
nucleic acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired secreted protein nucleic
acid expression. This technique involves cleavage by means of
ribozymes containing nucleotide sequences complementary to one or
more regions in the mRNA that attenuate the ability of the mRNA to
be translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the secreted protein, such as
substrate binding.
[0155] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in secreted
protein gene expression. Thus, recombinant cells, which include the
patient's cells that have been engineered ex vivo and returned to
the patient, are introduced into an individual where the cells
produce the desired secreted protein to treat the individual.
[0156] The invention also encompasses kits for detecting the
presence of a secreted protein nucleic acid in a biological sample.
For example, the kit can comprise reagents such as a labeled or
labelable nucleic acid or agent capable of detecting secreted
protein nucleic acid in a biological sample; means for determining
the amount of secreted protein nucleic acid in the sample; and
means for comparing the amount of secreted protein nucleic acid in
the sample with a standard. The compound or agent can be packaged
in a suitable container. The kit can further comprise instructions
for using the kit to detect secreted protein mRNA or DNA.
[0157] Nucleic Acid Arrays
[0158] The present invention further provides nucleic acid
detection kits, such as arrays or microarrays of nucleic acid
molecules that are based on the sequence information provided in
FIGS. 1 and 3 (SEQ ID NOS:1 and 3).
[0159] As used herein "Arrays" or "Microarrays" refers to an array
of distinct polynucleotides or oligonucleotides synthesized on a
substrate, such as paper, nylon or other type of membrane, filter,
chip, glass slide, or any other suitable solid support. In one
embodiment, the microarray is prepared and used according to the
methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT
application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996;
Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.
Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated
herein in their entirety by reference. In other embodiments, such
arrays are produced by the methods described by Brown et al., U.S.
Pat. No. 5,807,522.
[0160] The microarray or detection kit is preferably composed of a
large number of unique, single-stranded nucleic acid sequences,
usually either synthetic antisense oligonucleotides or fragments of
cDNAs, fixed to a solid support. The oligonucleotides are
preferably about 6-60 nucleotides in length, more preferably 15-30
nucleotides in length, and most preferably about 20-25 nucleotides
in length. For a certain type of microarray or detection kit, it
may be preferable to use oligonucleotides that are only 7-20
nucleotides in length. The microarray or detection kit may contain
oligonucleotides that cover the known 5', or 3', sequence,
sequential oligonucleotides which cover the full length sequence;
or unique oligonucleotides selected from particular areas along the
length of the sequence. Polynucleotides used in the microarray or
detection kit may be oligonucleotides that are specific to a gene
or genes of interest.
[0161] In order to produce oligonucleotides to a known sequence for
a microarray or detection kit, the gene(s) of interest (or an ORF
identified from the contigs of the present invention) is typically
examined using a computer algorithm which starts at the 5' or at
the 3' end of the nucleotide sequence. Typical algorithms will then
identify oligomers of defined length that are unique to the gene,
have a GC content within a range suitable for hybridization, and
lack predicted secondary structure that may interfere with
hybridization. In certain situations it may be appropriate to use
pairs of oligonucleotides on a microarray or detection kit. The
"pairs" will be identical, except for one nucleotide that
preferably is located in the center of the sequence. The second
oligonucleotide in the pair (mismatched by one) serves as a
control. The number of oligonucleotide pairs may range from two to
one million. The oligomers are synthesized at designated areas on a
substrate using a light-directed chemical process. The substrate
may be paper, nylon or other type of membrane, filter, chip, glass
slide or any other suitable solid support.
[0162] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application WO95/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or
any other number between two and one million which lends itself to
the efficient use of commercially available instrumentation.
[0163] In order to conduct sample analysis using a microarray or
detection kit, the RNA or DNA from a biological sample is made into
hybridization probes. The mRNA is isolated, and cDNA is produced
and used as a template to make antisense RNA (aRNA). The aRNA is
amplified in the presence of fluorescent nucleotides, and labeled
probes are incubated with the microarray or detection kit so that
the probe sequences hybridize to complementary oligonucleotides of
the microarray or detection kit. Incubation conditions are adjusted
so that hybridization occurs with precise complementary matches or
with various degrees of less complementarity. After removal of
nonhybridized probes, a scanner is used to determine the levels and
patterns of fluorescence. The scanned images are examined to
determine degree of complementarity and the relative abundance of
each oligonucleotide sequence on the microarray or detection kit.
The biological samples may be obtained from any bodily fluids (such
as blood, urine, saliva, phlegm, gastric juices, etc.), cultured
cells, biopsies, or other tissue preparations. A detection system
may be used to measure the absence, presence, and amount of
hybridization for all of the distinct sequences simultaneously.
This data may be used for large-scale correlation studies on the
sequences, expression patterns, mutations, variants, or
polymorphisms among samples.
[0164] Using such arrays, the present invention provides methods to
identify the expression of the secreted proteins/peptides of the
present invention. In detail, such methods comprise incubating a
test sample with one or more nucleic acid molecules and assaying
for binding of the nucleic acid molecule with components within the
test sample. Such assays will typically involve arrays comprising
many genes, at least one of which is a gene of the present
invention and or alleles of the secreted protein gene of the
present invention.
[0165] Conditions for incubating a nucleic acid molecule with a
test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid molecule used in the assay. One
skilled in the art will recognize that any one of the commonly
available hybridization, amplification or array assay formats can
readily be adapted to employ the novel fragments of the Human
genome disclosed herein. Examples of such assays can be found in
Chard, T, An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
[0166] The test samples of the present invention include cells,
protein or membrane extracts of cells. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing nucleic acid
extracts or of cells are well known in the art and can be readily
be adapted in order to obtain a sample that is compatible with the
system utilized.
[0167] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0168] Specifically, the invention provides a compartmentalized kit
to receive, in close confinement, one or more containers which
comprises: (a) a first container comprising one of the nucleic acid
molecules that can bind to a fragment of the Human genome disclosed
herein; and (b) one or more other containers comprising one or more
of the following: wash reagents, reagents capable of detecting
presence of a bound nucleic acid.
[0169] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers, strips of
plastic, glass or paper, or arraying material such as silica. Such
containers allows one to efficiently transfer reagents from one
compartment to another compartment such that the samples and
reagents are not cross-contaminated, and the agents or solutions of
each container can be added in a quantitative fashion from one
compartment to another. Such containers will include a container
which will accept the test sample, a container which contains the
nucleic acid probe, containers which contain wash reagents (such as
phosphate buffered saline, Tris-buffers, etc.), and containers
which contain the reagents used to detect the bound probe. One
skilled in the art will readily recognize that the previously
unidentified secreted protein gene of the present invention can be
routinely identified using the sequence information disclosed
herein can be readily incorporated into one of the established kit
formats which are well known in the art, particularly expression
arrays.
[0170] Vectors/Host Cells
[0171] The invention also provides vectors containing the nucleic
acid molecules described herein. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule, which can transport
the nucleic acid molecules. When the vector is a nucleic acid
molecule, the nucleic acid molecules are covalently linked to the
vector nucleic acid. With this aspect of the invention, the vector
includes a plasmid, single or double stranded phage, a single or
double stranded RNA or DNA viral vector, or artificial chromosome,
such as a BAC, PAC, YAC, OR MAC.
[0172] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the nucleic acid molecules. Alternatively, the
vector may integrate into the host cell genome and produce
additional copies of the nucleic acid molecules when the host cell
replicates.
[0173] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
nucleic acid molecules. The vectors can function in prokaryotic or
eukaryotic cells or in both (shuttle vectors).
[0174] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. Thus, the second nucleic acid
molecule may provide a trans-acting factor interacting with the
cis-regulatory control region to allow transcription of the nucleic
acid molecules from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself. It is understood,
however, that in some embodiments, transcription and/or translation
of the nucleic acid molecules can occur in a cell-free system.
[0175] The regulatory sequence to which the nucleic acid molecules
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. Coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0176] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0177] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al, Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989).
[0178] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1989).
[0179] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0180] The nucleic acid molecules can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0181] The vector containing the appropriate nucleic acid molecule
can be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0182] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein, increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enterokinase. Typical fusion expression vectors
include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New
England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,
N.J.) which fuse glutathione S-transferase (GST), maltose E binding
protein, or protein A, respectively, to the target recombinant
protein. Examples of suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in Enzymology 185:60-89 (1990)).
[0183] Recombinant protein expression can be maximized in host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128).
Alternatively, the sequence of the nucleic acid molecule of
interest can be altered to provide preferential codon usage for a
specific host cell, for example E. coli. (Wada et al., Nucleic
Acids Res. 20:2111-2118 (1992)).
[0184] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kujan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0185] The nucleic acid molecules can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
[0186] In certain embodiments of the invention, the nucleic acid
molecules described herein are expressed in mammalian cells using
mammalian expression vectors. Examples of mammalian expression
vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC
(Kaufman et al., EMBO J. 6:187-195 (1987)).
[0187] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
nucleic acid molecules. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance
propagation or expression of the nucleic acid molecules described
herein. These are found for example in Sambrook, J., Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
[0188] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the nucleic
acid molecule sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0189] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0190] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook,
et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989).
[0191] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the nucleic acid molecules can be introduced
either alone or with other nucleic acid molecules that are not
related to the nucleic acid molecules such as those providing
trans-acting factors for expression vectors. When more than one
vector is introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the nucleic acid molecule
vector.
[0192] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0193] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the nucleic acid molecules described
herein or may be on a separate vector. Markers include tetracycline
or ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0194] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0195] Where secretion of the peptide is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as kinases, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0196] Where the peptide is not secreted into the medium, which is
typically the case with kinases, the protein can be isolated from
the host cell by standard disruption procedures, including freeze
thaw, sonication, mechanical disruption, use of lysing agents and
the like. The peptide can then be recovered and purified by
well-known purification methods including ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0197] It is also understood that depending upon the host cell in
recombinant production of the peptides described herein, the
peptides can have various glycosylation patterns, depending upon
the cell, or maybe non-glycosylated as when produced in bacteria.
In addition, the peptides may include an initial modified
methionine in some cases as a result of a host-mediated
process.
[0198] Uses of Vectors and Host Cells
[0199] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a secreted protein or peptide that can be further
purified to produce desired amounts of secreted protein or
fragments. Thus, host cells containing expression vectors are
useful for peptide production.
[0200] Host cells are also useful for conducting cell-based assays
involving the secreted protein or secreted protein fragments, such
as those described above as well as other formats known in the art.
Thus, a recombinant host cell expressing a native secreted protein
is useful for assaying compounds that stimulate or inhibit secreted
protein function.
[0201] Host cells are also useful for identifying secreted protein
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations are useful to assay compounds that have a desired
effect on the mutant secreted protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native secreted protein.
[0202] Genetically engineered host cells can be further used to
produce non-human transgenic animals. A transgenic animal is
preferably a mammal, for example a rodent, such as a rat or mouse,
in which one or more of the cells of the animal include a
transgene. A transgene is exogenous DNA which is integrated into
the genome of a cell from which a transgenic animal develops and
which remains in the genome of the mature animal in one or more
cell types or tissues of the transgenic animal. These animals are
useful for studying the function of a secreted protein and
identifying and evaluating modulators of secreted protein activity.
Other examples of transgenic animals include non-human primates,
sheep, dogs, cows, goats, chickens, and amphibians.
[0203] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the
secreted protein nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0204] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
secreted protein to particular cells.
[0205] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0206] In another embodiment, transgenic non-human animals can be
produced which contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS
89:6232-6236 (1992). Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. Science
251:1351-1355 (1991). If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0207] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. Nature 385:810-813 (1997) and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring born of this female foster animal will
be a clone of the animal from which the cell, e.g., the somatic
cell, is isolated.
[0208] Transgenic animals containing recombinant cells that express
the peptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
effect substrate binding, secreted protein activation, and signal
transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo secreted protein function,
including substrate interaction, the effect of specific mutant
secreted proteins on secreted protein function and substrate
interaction, and the effect of chimeric secreted proteins. It is
also possible to assess the effect of null mutations, that is,
mutations that substantially or completely eliminate one or more
secreted protein functions.
[0209] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the above-described modes for carrying out
the invention which are obvious to those skilled in the field of
molecular biology or related fields are intended to be within the
scope of the following claims.
Sequence CWU 1
1
4 1 1146 DNA Homo sapiens 1 atgagttgga ctgtgcctgt tgtgcgggcc
agccagagaa tgagctcggt gggagcgaat 60 ttcctatgcc tggggatggc
cctgtgtctg cgtcaagcaa cgcgcatccc gctcaacggc 120 acctggctct
tcacacccgt gagcaagatg gcgactgtga agagtgagct tattgagcgt 180
ttcacttccg agaagcccgt tcatcacagt aaggtctcca tcataggaac tggatcggtg
240 ggcatggcct gcgctatcag catcttatta aaaggcttga gtgatgaact
tgcccttgtg 300 gatcttgatg aagacaaact gaagggtgag acgatggatc
ttcaacatgg cagccctttc 360 acgaaaatgc caaatattgt ttgtagcaaa
gattactttg tcacagcaaa ctccaaccta 420 gtgattatca cagcaggtgc
acgccaagaa aagggagaaa cgcgccttaa tttagtccag 480 cgaaatgtgg
ccatcttcaa gttaatgatt tccagtattg tccagtacag cccccactgc 540
aaactgatta ttgtttccaa tccagtggat atcttaactt atgtagcttg gaagttgagt
600 gcatttccca aaaaccgtat tattggaagc ggctgtaatc tggatactgc
tcgttttcgt 660 ttcttgattg gacaaaagct tggtatccat tctgaaagct
gccatggatg gatcctcgga 720 gagcatggag actcaagtgt tcctgtgtgg
agtggagtga acatagctgg tgtccctttg 780 aaggatctga actctgatat
aggaactgat aaagatcctg agcaatggaa aaatgtccac 840 aaagaagtga
ctgcaactgc ctatgagatt attaaaatga aaggttatac ttcttgggcc 900
attggcctat ctgtggccga tttaacagaa agtattttga agaatcttag gagaatacat
960 ccagtttcca ccataactaa gggcctctat ggaatagatg aagaagtatt
cctcagtatt 1020 ccttgtatcc tgggagagaa cggtattacc aaccttataa
agataaagct gacccctgaa 1080 gaagaggccc atctgaaaaa aagtgcaaaa
acactctggg aaattcagaa taagcttaag 1140 ctttaa 1146 2 381 PRT Homo
sapiens 2 Met Ser Trp Thr Val Pro Val Val Arg Ala Ser Gln Arg Met
Ser Ser 1 5 10 15 Val Gly Ala Asn Phe Leu Cys Leu Gly Met Ala Leu
Cys Leu Arg Gln 20 25 30 Ala Thr Arg Ile Pro Leu Asn Gly Thr Trp
Leu Phe Thr Pro Val Ser 35 40 45 Lys Met Ala Thr Val Lys Ser Glu
Leu Ile Glu Arg Phe Thr Ser Glu 50 55 60 Lys Pro Val His His Ser
Lys Val Ser Ile Ile Gly Thr Gly Ser Val 65 70 75 80 Gly Met Ala Cys
Ala Ile Ser Ile Leu Leu Lys Gly Leu Ser Asp Glu 85 90 95 Leu Ala
Leu Val Asp Leu Asp Glu Asp Lys Leu Lys Gly Glu Thr Met 100 105 110
Asp Leu Gln His Gly Ser Pro Phe Thr Lys Met Pro Asn Ile Val Cys 115
120 125 Ser Lys Asp Tyr Phe Val Thr Ala Asn Ser Asn Leu Val Ile Ile
Thr 130 135 140 Ala Gly Ala Arg Gln Glu Lys Gly Glu Thr Arg Leu Asn
Leu Val Gln 145 150 155 160 Arg Asn Val Ala Ile Phe Lys Leu Met Ile
Ser Ser Ile Val Gln Tyr 165 170 175 Ser Pro His Cys Lys Leu Ile Ile
Val Ser Asn Pro Val Asp Ile Leu 180 185 190 Thr Tyr Val Ala Trp Lys
Leu Ser Ala Phe Pro Lys Asn Arg Ile Ile 195 200 205 Gly Ser Gly Cys
Asn Leu Asp Thr Ala Arg Phe Arg Phe Leu Ile Gly 210 215 220 Gln Lys
Leu Gly Ile His Ser Glu Ser Cys His Gly Trp Ile Leu Gly 225 230 235
240 Glu His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Val Asn Ile Ala
245 250 255 Gly Val Pro Leu Lys Asp Leu Asn Ser Asp Ile Gly Thr Asp
Lys Asp 260 265 270 Pro Glu Gln Trp Lys Asn Val His Lys Glu Val Thr
Ala Thr Ala Tyr 275 280 285 Glu Ile Ile Lys Met Lys Gly Tyr Thr Ser
Trp Ala Ile Gly Leu Ser 290 295 300 Val Ala Asp Leu Thr Glu Ser Ile
Leu Lys Asn Leu Arg Arg Ile His 305 310 315 320 Pro Val Ser Thr Ile
Thr Lys Gly Leu Tyr Gly Ile Asp Glu Glu Val 325 330 335 Phe Leu Ser
Ile Pro Cys Ile Leu Gly Glu Asn Gly Ile Thr Asn Leu 340 345 350 Ile
Lys Ile Lys Leu Thr Pro Glu Glu Glu Ala His Leu Lys Lys Ser 355 360
365 Ala Lys Thr Leu Trp Glu Ile Gln Asn Lys Leu Lys Leu 370 375 380
3 3144 DNA Homo sapiens 3 cagaagtggt atcaaagttg caccaatctg
gccagaaact catgctcaga tactctgttt 60 aacaagagat cagctgggat
gatggatgag accaggggat ttccatagca ctaggaagtc 120 cccaagacca
cgacctcaac ttaacttttt tatcatcagc agccccacac agcctggcat 180
ccagtaggca cccaatactg atgggcttta taagaacacc aaaagtgcac tctgggcctt
240 gatttatact accagtaggc atggggagac aaggaatgcc agtctgtgat
tccagcaaga 300 ggcaagacga aaatccattt attcagcagc taatacatgt
agcagggtga ttcattttcc 360 aaaaatatgc agtagtatgc tgagatttag
tgctggagtg actcccagcc ggcccctacc 420 ctgctgaagc cccagccgac
tgtcccaggg tgggtcctgg ccaggactgg caccaaggca 480 gtggaatcct
ttcaaaatag gcctccagcg ccatagacca gaaagcaggc gcccaattct 540
gggacactgg gagatggtgc aggcagccac cagtatgcaa agcatagagc cctatccttc
600 ctggcagggt cacggttcag aaaacgtaac cttattttca tctgtagtta
acagaagcat 660 gtttcctcag cattcatatt aaaaataaaa aactagtaat
tccagggatc tcggttagcc 720 tcttaagcat gtcaactatt gataatactc
ttagcaaaac taactccaga aatcacttgc 780 tagagaagca gcccttcact
gccttagtct gagcacccac ggaaagcacg tgttggagac 840 tctggcaagg
cccgcgcagc cgcagtagag ggccaggggg cggggcacgc gcacctgccg 900
tagagtgctg aaggtcctgc caacggctct cttggcgtct caacgttcgg atcagcagct
960 tttttccatt ctctctctcc acttcttcag tgagcagcca tgagttggac
tgtgcctgtt 1020 gtgcgggcca gccagagaat gagctcggtg ggagcgaatt
tcctatgcct ggggatggcc 1080 ctgtgtctgc gtcaagcaac gcgcatcccg
ctcaacggca cctggctctt cacacccgtg 1140 agcaagatgg cgactgtgaa
gagtgagctt attgagcgtt tcacttccga gaagcccgtt 1200 catcacagta
aggtctccat cataggaact ggatcggtgg gcatggcctg cgctatcagc 1260
atcttattaa aaggcttgag tgatgaactt gcccttgtgg atcttgatga agacaaactg
1320 aagggtgaga cgatggatct tcaacatggc agccctttca cgaaaatgcc
aaatattgtt 1380 tgtagcaaag attactttgt cacagcaaac tccaacctag
tgattatcac agcaggtgca 1440 cgccaagaaa agggagaaac gcgccttaat
ttagtccagc gaaatgtggc catcttcaag 1500 ttaatgattt ccagtattgt
ccagtacagc ccccactgca aactgattat tgtttccaat 1560 ccagtggata
tcttaactta tgtagcttgg aagttgagtg catttcccaa aaaccgtatt 1620
attggaagcg gctgtaatct ggatactgct cgttttcgtt tcttgattgg acaaaagctt
1680 ggtatccatt ctgaaagctg ccatggatgg atcctcggag agcatggaga
ctcaagtgtt 1740 cctgtgtgga gtggagtgaa catagctggt gtccctttga
aggatctgaa ctctgatata 1800 ggaactgata aagatcctga gcaatggaaa
aatgtccaca aagaagtgac tgcaactgcc 1860 tatgagatta ttaaaatgaa
aggttatact tcttgggcca ttggcctatc tgtggccgat 1920 ttaacagaaa
gtattttgaa gaatcttagg agaatacatc cagtttccac cataactaag 1980
ggcctctatg gaatagatga agaagtattc ctcagtattc cttgtatcct gggagagaac
2040 ggtattacca accttataaa gataaagctg acccctgaag aagaggccca
tctgaaaaaa 2100 agtgcaaaaa cactctggga aattcagaat aagcttaagc
tttaaagttg cctaaaacta 2160 ccattccgaa attattgaag agatcataga
tacaggatta tataacgaaa ttttgaataa 2220 acttgaattc ctaaaagatg
gaaacaggaa agtaggtaga gtgattttcc tatttattta 2280 gtcctccagc
tcttttattg agcatccacg tgctggacga tacttattta caattcctaa 2340
gtatttttgg tacctctgat gtagcagcac ttgccatgtt atatatatgt agttggcatt
2400 tggttcccaa aaagtaggat gtaggtattt attgtgttct agaaattccg
actcttttca 2460 ttagatatat gctatttctt tcattcttgc tggtttatac
ctatgttcat ttatatgctg 2520 taaaaaagta gtagcttctt ctacaatgta
aaaataaatg tacatacaaa aaaatgcagt 2580 agtatataca atcttttgtt
ttgcttcctt tgatagttaa taaattccgt ttgttgaatc 2640 aataaaatac
ggcgagccat atgatttgct tcatgagaaa ccagatcaca aacccaaagg 2700
cttaaaacag atagacatta aaatggatat tggccatacc ttcccagcat aatgatgaat
2760 gatgaagcct tggttccaac tgttgaagtg ctcatgactc ccaatctgca
tctgaagttt 2820 ctggagcagc gtctgatctg ccccctcacc caccgcatgc
atcgtggcgc acacgtcatc 2880 caggatgctc atgatgccag gagggttctg
atgggagcaa gaaggcaagg ccctagtcac 2940 tgacctctcc aaaacagaca
atgcttatca ggtcctttct taggcaagga aacagctccc 3000 cagacttaag
ataccatctt gaaagtcagt attccccttc aggcaagaat gcaattaata 3060
gtcccaaata gggagtcacc acttagatct aaagagggag gaaactccac tttccaaaaa
3120 gctgtttaac tccctgaagt ttta 3144 4 331 PRT Mus musculus 4 Met
Ala Thr Leu Lys Asp Gln Leu Ile Val Asn Leu Leu Lys Glu Glu 1 5 10
15 Gln Ala Pro Gln Asn Lys Ile Thr Val Val Gly Val Gly Ala Val Gly
20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp
Glu Leu 35 40 45 Ala Leu Val Asp Val Met Glu Asp Lys Leu Lys Gly
Glu Met Met Asp 50 55 60 Leu Gln His Gly Ser Leu Phe Leu Lys Thr
Pro Lys Ile Val Ser Ser 65 70 75 80 Lys Asp Tyr Cys Val Thr Ala Asn
Ser Lys Leu Val Ile Ile Thr Ala 85 90 95 Gly Ala Arg Gln Gln Glu
Gly Glu Ser Arg Leu Asn Leu Val Gln Arg 100 105 110 Asn Val Asn Ile
Phe Lys Phe Ile Ile Pro Asn Ile Val Lys Tyr Ser 115 120 125 Pro His
Cys Lys Leu Leu Ile Val Ser Asn Pro Val Asp Ile Leu Thr 130 135 140
Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145
150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met
Gly Glu 165 170 175 Arg Leu Gly Val His Ala Leu Ser Cys His Gly Trp
Val Leu Gly Glu 180 185 190 His Gly Asp Ser Ser Val Pro Val Trp Ser
Gly Val Asn Val Ala Gly 195 200 205 Val Ser Leu Lys Ser Leu Asn Pro
Glu Leu Gly Thr Asp Ala Asp Lys 210 215 220 Glu Gln Trp Lys Glu Val
His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys
Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val 245 250 255 Ala
Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro 260 265
270 Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Asn Glu Asp Val Phe
275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp
Val Val 290 295 300 Lys Val Thr Leu Thr Pro Glu Glu Glu Ala Arg Leu
Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu
Leu Gln 325 330
* * * * *
References