U.S. patent application number 09/997701 was filed with the patent office on 2002-08-08 for cell surface glycoproteins.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Baughn, Mariah R., Corley, Neil C., Gorgone, Gina A., Guegler, Karl J., Yue, Henry.
Application Number | 20020107180 09/997701 |
Document ID | / |
Family ID | 22688541 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020107180 |
Kind Code |
A1 |
Yue, Henry ; et al. |
August 8, 2002 |
Cell surface glycoproteins
Abstract
The invention provides human cell surface glycoproteins (CSGP)
and polynucleotides which identify and encode CSGP. The invention
also provides expression vectors, host cells, antibodies, agonists,
and antagonists. The invention also provides methods for
diagnosing, treating, or preventing disorders associated with
expression of CSGP.
Inventors: |
Yue, Henry; (Sunnyvale,
CA) ; Corley, Neil C.; (Mountain View, CA) ;
Guegler, Karl J.; (Menlo Park, CA) ; Gorgone, Gina
A.; (Boulder Creek, CA) ; Baughn, Mariah R.;
(San Leandro, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
22688541 |
Appl. No.: |
09/997701 |
Filed: |
November 30, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09997701 |
Nov 30, 2001 |
|
|
|
09470946 |
Dec 22, 1999 |
|
|
|
09470946 |
Dec 22, 1999 |
|
|
|
09187331 |
Nov 6, 1998 |
|
|
|
Current U.S.
Class: |
424/139.1 ;
435/252.3; 435/320.1; 435/325; 435/69.1; 514/19.4; 514/20.9;
530/395; 536/23.5; 800/8 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 25/14 20180101; A61P 25/24 20180101; A61P 7/02 20180101; A61P
35/00 20180101; A61P 35/02 20180101; A61P 7/00 20180101; A61P 7/06
20180101; A61P 7/04 20180101; A61K 38/00 20130101; A61P 25/16
20180101; C07K 14/47 20130101; A61P 25/28 20180101 |
Class at
Publication: |
514/8 ; 530/395;
536/23.5; 800/8; 435/69.1; 435/325; 435/252.3; 435/320.1 |
International
Class: |
A61K 038/17; A01K
067/00; C07H 021/04; C12P 021/02; C12N 001/21; C07K 014/435; C12N
005/06 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:1-2, b) a polypeptide comprising
a naturally occurring amino acid sequence at least 90% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-2, c) a biologically active fragment of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-2, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-2.
2. An isolated polypeptide of claim 1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-2.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:3-4.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method of producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises an amino
acid sequence selected from the group consisting of SEQ ID
NO:1-2.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:3-4, b) a
polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence
selected from the group consisting of SEQ ID NO:3-4, c) a
polynucleotide complementary to a polynucleotide of a), d) a
polynucleotide complementary to a polynucleotide of b), and e) an
RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-2.
19. A method for treating a disease or condition associated with
decreased expression of functional CSGP, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method of screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional CSGP, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method of screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional CSGP, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, the method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the ab sence of the test compound, wherein a change
in the activity of the polypeptide of claim 1 in the presence of
the test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A diagnostic test for a condition or disease associated with
the expression of CSGP in a biological sample, the method
comprising: a) combining the biological sample with an antibody of
claim 11, under conditions suitable for the antibody to bind the
polypeptide and form an antibody:polypeptide complex, and b)
detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of CSGP in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with
the expression of CSGP in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide consisting of an amino acid
sequence selected from the group consisting of SEQ ID NO:1-2, or an
immunogenic fragment thereof, under conditions to elicit an
antibody response, b) isolating antibodies from said animal, and c)
screening the isolated antibodies with the polypeptide, thereby
identifying a polyclonal antibody which binds specifically to a
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1-2.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal antibody of claim 37
and a suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide consisting of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-2, or an
immunogenic fragment thereof, under conditions to elicit an
antibody response, b) isolating antibody producing cells from the
animal, c) fusing the antibody producing cells with immortalized
cells to form monoclonal antibody-producing hybridoma cells, d)
culturing the hybridoma cells, and e) isolating from the culture
monoclonal antibody which binds specifically to a polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-2.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-2 in a
sample, the method comprising: a) incubating the antibody of claim
11 with a sample under conditions to allow specific binding of the
antibody and the polypeptide, and b) detecting specific binding,
wherein specific binding indicates the presence of a polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-2 in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-2 from a
sample, the method comprising: a) incubating the antibody of claim
11 with a sample under conditions to allow specific binding of the
antibody and the polypeptide, and b) separating the antibody from
the sample and obtaining the purified polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-2.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method of generating an expression profile of a sample which
contains polynucleotides, the method comprising: a) labeling the
polynucleotides of the sample, b) contacting the elements of the
microarray of claim 46 with the labeled polynucleotides of the
sample under conditions suitable for the formation of a
hybridization complex, and c) quantifying the expression of the
polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonucleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, and wherein
said target polynucleotide is a polynucleotide of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have the same sequence, and each distinct physical
location on the substrate contains nucleotide molecules having a
sequence which differs from the sequence of nucleotide molecules at
another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:1.
57. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:2.
58. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:3.
59. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:4.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/470,946, filed Dec. 22, 1999, which is a
divisional application of U.S. application Ser. No. 09/187,331,
filed Nov. 6, 1998, now U.S. Pat. No. 6,043,056, each of which is
entitled CELL SURFACE GLYCOPROTEINS, all of which are hereby
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of cell surface glycoproteins and to the use of these
sequences in the diagnosis, treatment, and prevention of
hematologic, karyotypic, and neuronal disorders.
BACKGROUND OF THE INVENTION
[0003] Proteins expressed on the cell surface function as
stage-specific markers of cell differentiation and as antigenic
determinants for immunological identification of distinct cell
types. In the context of tissue construction and intercellular
communication, cell surface proteins play critical roles in
cell-cell recognition and adhesion, cell motility, and signal
transduction. Cell surface proteins are anchored to the plasma
membrane by one or more membrane-spanning domains or by covalent
attachment to lipophilic membrane-embedded molecules such as
glycosylphosphatidylinositol. Most cell surface proteins are
synthesized as immature, inactive precursors which require
post-translational modifications for activity. Such modifications
include proteolytic processing, glycosylation, oligomerization, and
disulfide bond formation. Proteins destined for the cell surface
generally contain N-terminal signal peptides which are cleaved from
the mature protein.
[0004] Blood group antigens are immunologically defined marker
proteins found on the surface of red blood cells. These markers
provide a means for classification of blood group systems. For
example, the most commonly known blood group system is defined by
the A, B, and 0 antigens. The particular antigen(s) expressed by a
given individual is determined by the allele, or variant, of the
ABO gene that the individual has inherited. The blood type of the
individual is therefore defined by the serologically expressed
antigen(s). The implications of the ABO system on blood transfusion
practice are well known. In addition, the A and B antigens have
potential application in the diagnosis of blood disorders such as
leukemia, thalassemia, and anemia which weaken the expression of
these antigens. (Reviewed in Reid, M. E. and Lomas-Francis, C.
(1997) The Blood Group Antigen Facts Book, Academic Press, San
Diego, Calif., pp. 19-26.)
[0005] Although the ABO system is well known for its clinical
relevance, other blood group systems exist. One such blood group
system is the XG system which is defined by a single antigen,
Xg.sup.a. Xg.sup.a is encoded by one of two allelic forms of the XG
gene. The other XG allele fails to encode a detectable gene
product. (Reviewed in Reid and Lomas-Francis, supra, pp. 251-254.)
The XG gene is situated on the X chromosome at the boundary between
the pseudoautosomal, or X-specific, region and the region which has
homology to Y chromosome sequences. (Ellis, N. A. et al. (1994)
Nat. Genet. 6:394-399.) The latter region is particularly important
for recombination between the X and Y chromosomes during male
meiosis. Lack of recombination results in the failure of X and Y to
segregate and ultimately leads to the generation of male progeny
with an XXY karyotype. XXY individuals suffer from Klinefelter
syndrome, a complex developmental disorder characterized by
infertility, gynecomastia and other manifestations of feminization,
increased height, obesity, mental deficiency, thyroid
abnormalities, diabetes, pulmonary disease, and increased risk of
breast cancer.
[0006] The XG gene has been cloned and sequenced (Ellis, supra, and
Ellis, N. A. et al. (1994) Nat. Genet. 8:285-289). XG RNA is
detectable at low levels in fibroblasts and in some bone marrow
preparations. XG cDNA predicts a proline- and glycine-rich protein
of 180 amino acids with an N-terminal signal peptide. A
transmembrane domain from amino acid 88 through 116 separates the
N-terminal extracellular domain from the C-terminal intracellular
domain. The XG protein may play a role in cell adhesion.
[0007] The surface of white blood cells, like that of red blood
cells, is populated with characteristic glycoproteins. For example,
the plasma cell glycoprotein-1 (PC-1) is expressed on the surface
of plasma cells, which are terminally differentiated,
antibody-secreting B-lymphocytes. PC-1 is also expressed in
nonlymphoid tissue such as kidney, chondrocytes, epididymis, and
hepatocytes. PC-1 was initially isolated from murine plasma cells
as a homodimer with subunits of 115 kilodaltons each (van Driel, I.
R. et al. (1985) Proc. Natl. Acad. Sci. USA 82:8619-8623).
Biochemical and immunological analyses have suggested that murine
PC-1 (mPC-1) is expressed in neuroblastomas. However, molecular
analyses failed to confirm this observation, suggesting that
neuroblastoma tissue contains a glycoprotein having biochemical
similarity to or immunological cross-reactivity with mPC-1. mPC-1
cDNA encodes a predicted protein of 871 amino acids with a short
N-terminal cytoplasmic domain, a single transmembrane domain, and a
large C-terminal extracellular domain of 826 amino acids. Human
PC-1 (hPC-1) is 873 amino acids in length and 80% identical to the
mouse protein (Buckley, M. F. et al. (1990) J. Biol. Chem.
265:17506-17511). hPC-1 possesses the same overall domain structure
as mPC-1. In addition, a soluble form of mPC-1 has been found in
serum and other extracellular fluids (Belli, S. I. et al. (1993)
Eur. J. Biochem. 217:421-428). This soluble form of mPC-1 likely
results from proteolytic cleavage which frees most of the
extracellular domain from the transmembrane domain.
[0008] The extracellular domains of both hPC-1 and mPC-1 have
nucleotide phosphodiesterase (pyrophosphatase) activity (Funakoshi,
I. et al. (1992) Arch. Biochem. Biophys. 295:180-187; Rebbe, N. F.
et al. (1991) Proc. Natl. Acad. Sci. USA 88:5192-5196). In hPC-1,
the enzymatic active site for this activity likely occurs within
the region from amino acids 166 through 225. Phosphodiesterase
activity is associated with the hydrolytic removal of nucleotide
subunits from oligonucleotides. Although the precise physiological
roles of hPC-1 and mPC-1 are not clear, increased hPC-I
phosphodiesterase activity has been correlated with insulin
resistance in patients with noninsulin-dependent diabetes mellitus,
with abnormalities of bone mineralization and calcification, and
with defects in renal tubule function. In addition, it appears that
hPC-1 and mPC-1 are members of a multigene family of transmembrane
phosphodiesterases with extracellular active sites. These enzymes
may play a role in regulating the concentration of
pharmacologically active extracellular compounds such as adenosine
or other nucleotide derivatives in a variety of tissues and cell
types. (Reviewed in Goding, J. W. et al. (1998) Immunol. Rev.
161:11-26.)
[0009] The discovery of new cell surface glycoproteins and the
polynucleotides encoding them satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention, and treatment of hematologic, karyotypic, and neuronal
disorders.
SUMMARY OF THE INVENTION
[0010] The invention features substantially purified polypeptides,
cell surface glycoproteins, referred to collectively as "CSGP" and
individually as "CSGP-1" and "CSGP-2." In one aspect, the invention
provides a substantially purified polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, and fragments thereof.
[0011] The invention further provides a substantially purified
variant having at least 90% amino acid identity to at least one of
the amino acid sequences selected from the group consisting of SEQ
ID NO:1, SEQ ID NO:2, and fragments thereof. The invention also
provides an isolated and purified polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments
thereof. The invention also includes an isolated and purified
polynucleotide variant having at least 90% polynucleotide sequence
identity to the polynucleotide encoding the polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, and fragments thereof.
[0012] Additionally, the invention provides an isolated and
purified polynucleotide which hybridizes under stringent conditions
to the polynucleotide encoding the polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, and fragments thereof. The invention also provides an
isolated and purified polynucleotide having a sequence which is
complementary to the polynucleotide encoding the polypeptide
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof.
[0013] The invention also provides an isolated and purified
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO:3, SEQ ID NO:4, and fragments
thereof. The invention further provides an isolated and purified
polynucleotide variant having at least 90% polynucleotide sequence
identity to the polynucleotide sequence selected from the group
consisting of SEQ ID NO:3, SEQ ID NO:4, and fragments thereof. The
invention also provides an isolated and purified polynucleotide
having a sequence which is complementary to the polynucleotide
comprising a polynucleotide sequence selected from the group
consisting of SEQ ID NO:3, SEQ ID NO:4, and fragments thereof.
[0014] The invention also provides a method for detecting a
polynucleotide in a sample containing nucleic acids, the method
comprising the steps of (a) hybridizing the complement of the
polynucleotide sequence to at least one of the polynucleotides of
the sample, thereby forming a hybridization complex; and (b)
detecting the hybridization complex, wherein the presence of the
hybridization complex correlates with the presence of a
polynucleotide in the sample. In one aspect, the method further
comprises amplifying the polynucleotide prior to hybridization.
[0015] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments
thereof. In another aspect, the expression vector is contained
within a host cell.
[0016] The invention also provides a method for producing a
polypeptide, the method comprising the steps of: (a) culturing the
host cell containing an expression vector containing at least a
fragment of a polynucleotide under conditions suitable for the
expression of the polypeptide; and (b) recovering the polypeptide
from the host cell culture.
[0017] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, and fragments thereof, in conjunction with a suitable
pharmaceutical carrier.
[0018] The invention further includes a purified antibody which
binds to a polypeptide selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, and fragments thereof. The invention also
provides a purified agonist and a purified antagonist to the
polypeptide.
[0019] The invention also provides a method for treating or
preventing a disorder associated with decreased expression or
activity of CSGP, the method comprising administering to a subject
in need of such treatment an effective amount of a pharmaceutical
composition comprising a substantially purified polypeptide having
the amino acid sequence selected from the group consisting of SEQ
ID NO:1, SEQ ID NO:2, and fragments thereof, in conjunction with a
suitable pharmaceutical carrier.
[0020] The invention also provides a method for treating or
preventing a disorder associated with increased expression or
activity of CSGP, the method comprising administering to a subject
in need of such treatment an effective amount of an antagonist of a
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
[0021] FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID
NO:1) and nucleic acid sequence (SEQ ID NO:3) of CSGP-1. The
alignment was produced using MACDNASIS PRO software (Hitachi
Software Engineering, South San Francisco Calif.).
[0022] FIG. 2 shows the amino acid sequence alignment between
CSGP-1 (2297891; SEQ ID NO:1) and human XG (GI 2499136; SEQ ID
NO:5). The alignment was produced using the multisequence alignment
program of LASERGENE software (DNASTAR, Madison Wis.).
[0023] FIGS. 3A, 3B, 3C, and 3D show the amino acid sequence (SEQ
ID NO:2) and nucleic acid sequence (SEQ ID NO:4) of CSGP-2.
[0024] FIGS. 4A and 4B show the amino acid sequence alignment
between amino acid residues 27 through 378 of CSGP-2 (2705267; SEQ
ID NO:2) and amino acid residues 161 through 508 of hPC-1 (GI
189650; SEQ ID NO:6).
[0025] Table 1 shows the programs, their descriptions, references,
and threshold parameters used to analyze CSGP.
DESCRIPTION OF THE INVENTION
[0026] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular machines, materials and methods
described, as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention which will be limited only by the appended
claims.
[0027] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any machines, materials, and methods similar or equivalent to those
described herein can be used to practice or test the present
invention, the preferred machines, materials and methods are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, protocols,
reagents and vectors which are reported in the publications and
which might be used in connection with the invention. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
[0029] "CSGP" refers to the amino acid sequences of substantially
purified CSGP obtained from any species, particularly a mammalian
species, including bovine, ovine, porcine, murine, equine, and
preferably the human species, from any source, whether natural,
synthetic, semi-synthetic, or recombinant.
[0030] The term "agonist" refers to a molecule which, when bound to
CSGP, increases or prolongs the duration of the effect of CSGP.
Agonists may include proteins, nucleic acids, carbohydrates, or any
other molecules which bind to and modulate the effect of CSGP.
[0031] An "allelic variant" is an alternative form of the gene
encoding CS GP. Allelic variants may result from at least one
mutation in the nucleic acid sequence and may result in altered
mRNAs or in polypeptides whose structure or function may or may not
be altered. Any given natural or recombinant gene may have none,
one, or many allelic forms. Common mutational changes which give
rise to allelic variants are generally ascribed to natural
deletions, additions, or substitutions of nucleotides. Each of
these types of changes may occur alone, or in combination with the
others, one or more times in a given sequence.
[0032] "Altered" nucleic acid sequences encoding CSGP include those
sequences with deletions, insertions, or substitutions of different
nucleotides, resulting in a polynucleotide the same as CSGP or a
polypeptide with at least one functional characteristic of CSGP.
Included within this definition are polymorphisms which may or may
not be readily detectable using a particular oligonucleotide probe
of the polynucleotide encoding CSGP, and improper or unexpected
hybridization to allelic variants, with a locus other than the
normal chromosomal locus for the polynucleotide sequence encoding
CSGP. The encoded protein may also be "altered," and may contain
deletions, insertions, or substitutions of amino acid residues
which produce a silent change and result in a functionally
equivalent CSGP. Deliberate amino acid substitutions may be made on
the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the biological or immunological activity
of CSGP is retained. For example, negatively charged amino acids
may include aspartic acid and glutamic acid, positively charged
amino acids may include lysine and arginine, and amino acids with
uncharged polar head groups having similar hydrophilicity values
may include leucine, isoleucine, and valine; glycine and alanine;
asparagine and glutamine; serine and threonine; and phenylalanine
and tyrosine.
[0033] The terms "amino acid" or "amino acid sequence" refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or a
fragment of any of these, and to naturally occurring or synthetic
molecules. In this context, "fragments," "immunogenic fragments,"
or "antigenic fragments" refer to fragments of CSGP which are
preferably at least 5 to about 15 amino acids in length, most
preferably at least 14 amino acids, and which retain some
biological activity or immunological activity of CSGP. Where "amino
acid sequence" is recited to refer to an amino acid sequence of a
naturally occurring protein molecule, "amino acid sequence" and
like terms are not meant to limit the amino acid sequence to the
complete native amino acid sequence associated with the recited
protein molecule.
[0034] "Amplification" relates to the production of additional
copies of a nucleic acid sequence. Amplification is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art.
[0035] The term "antagonist" refers to a molecule which, when bound
to CSGP, decreases the amount or the duration of the effect of the
biological or immunological activity of CSGP. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any
other molecules which decrease the effect of CSGP.
[0036] The term "antibody" refers to intact molecules as well as to
fragments thereof, such as Fab, F(ab').sub.2, and Fv fragments,
which are capable of binding the epitopic determinant. Antibodies
that bind CSGP polypeptides can be prepared using intact
polypeptides or using fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can
be derived from the translation of RNA, or synthesized chemically,
and can be conjugated to a carrier protein if desired. Commonly
used carriers that are chemically coupled to peptides include
bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin
(KLH). The coupled peptide is then used to immunize the animal.
[0037] The term "antigenic determinant" refers to that fragment of
a molecule (i.e., an epitope) that makes contact with a particular
antibody. When a protein or a fragment of a protein is used to
immunize a host animal, numerous regions of the protein may induce
the production of antibodies which bind specifically to antigenic
determinants (given regions or three-dimensional structures on the
protein). An antigenic determinant may compete with the intact
antigen (i.e., the immunogen used to elicit the immune response)
for binding to an antibody.
[0038] The term "antisense" refers to any composition containing a
nucleic acid sequence which is complementary to the "sense" strand
of a specific nucleic acid sequence. Antisense molecules may be
produced by any method including synthesis or transcription. Once
introduced into a cell, the complementary nucleotides combine with
natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0039] The term "biologically active," refers to a protein having
structural, regulatory, or biochemical functions of a naturally
occurring molecule. Likewise, "immunologically active" refers to
the capability of the natural, recombinant, or synthetic CSGP, or
of any oligopeptide thereof, to induce a specific immune response
in appropriate animals or cells and to bind with specific
antibodies.
[0040] The terms "complementary" or "complementarity" refer to the
natural binding of polynucleotides by base pairing. For example,
the sequence "5' A-G-T 3'" bonds to the complementary sequence
"3'T-C-A 5'." Complementarity between two single-stranded molecules
may be "partial," such that only some of the nucleic acids bind, or
it may be "complete," such that total complementarity exists
between the single stranded molecules. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of the hybridization between
the nucleic acid strands. This is of particular importance in
amplification reactions, which depend upon binding between nucleic
acids strands, and in the design and use of peptide nucleic acid
(PNA) molecules.
[0041] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence" refer
broadly to any composition containing the given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation
or an aqueous solution. Compositions comprising polynucleotide
sequences encoding CSGP or fragments of CSGP may be employed as
hybridization probes. The probes may be stored in freeze-dried form
and may be associated with a stabilizing agent such as a
carbohydrate. In hybridizations, the probe may be deployed in an
aqueous solution containing salts (e.g., NaCl), detergents (e.g.,
sodium dodecyl sulfate; SDS), and other components (e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.).
[0042] "Consensus sequence" refers to a nucleic acid sequence which
has been resequenced to resolve uncalled bases, extended using
XL-PCR kit (Perkin-Elmer, Norwalk Conn.) in the 5' andlor the 3'
direction, and resequenced, or which has been assembled from the
overlapping sequences of more than one Incyte Clone using a
computer program for fragment assembly, such as the GELVIEW
fragment assembly system (GCG, Madison Wis.). Some sequences have
been both extended and assembled to produce the consensus
sequence.
[0043] The term "correlates with expression of a polynucleotide"
indicates that the detection of the presence of nucleic acids, the
same or related to a nucleic acid sequence encoding CSGP, by
northern analysis is indicative of the presence of nucleic acids
encoding CSGP in a sample, and thereby correlates with expression
of the transcript from the polynucleotide encoding CSGP.
[0044] A "deletion" refers to a change in the amino acid or
nucleotide sequence that results in the absence of one or more
amino acid residues or nucleotides.
[0045] The term "derivative" refers to the chemical modification of
a polypeptide sequence, or a polynucleotide sequence. Chemical
modifications of a polynucleotide sequence can include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group.
A derivative polynucleotide encodes a polypeptide which retains at
least one biological or immunological function of the natural
molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any similar process that retains at
least one biological or immunological function of the polypeptide
from which it was derived.
[0046] The term "similarity" refers to a degree of complementarity.
There may be partial similarity or complete similarity. The word
"identity" may substitute for the word "similarity." A partially
complementary sequence that at least partially inhibits an
identical sequence from hybridizing to a target nucleic acid is
referred to as "substantially similar." The inhibition of
hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or northern blot, solution hybridization, and the like)
under conditions of reduced stringency. A substantially similar
sequence or hybridization probe will compete for and inhibit the
binding of a completely similar (identical) sequence to the target
sequence under conditions of reduced stringency. This is not to say
that conditions of reduced stringency are such that non-specific
binding is permitted, as reduced stringency conditions require that
the binding of two sequences to one another be a specific (i.e., a
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target sequence which lacks even a
partial degree of complementarity (e.g., less than about 30%
similarity or identity). In the absence of non-specific binding,
the substantially similar sequence or probe will not hybridize to
the second non-complementary target sequence.
[0047] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MEGALIGN program
(DNASTAR, Madison Wis.) which creates alignments between two or
more sequences according to methods selected by the user, e.g., the
clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988)
Gene 73:237-244.) The clustal algorithm groups sequences into
clusters by examining the distances between all pairs. The clusters
are aligned pairwise and then in groups. The percentage similarity
between two amino acid sequences, e.g., sequence A and sequence B,
is calculated by dividing the length of sequence A, minus the
number of gap residues in sequence A, minus the number of gap
residues in sequence B, into the sum of the residue matches between
sequence A and sequence B, times one hundred. Gaps of low or of no
similarity between the two amino acid sequences are not included in
determining percentage similarity. Percent identity between nucleic
acid sequences can also be counted or calculated by other methods
known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J.
(1990) Methods Enzymol. 183:626-645.) Identity between sequences
can also be determined by other methods known in the art, e.g., by
varying hybridization conditions.
[0048] "Human artificial chromosomes" (HACs) are linear
microchromosomes which may contain DNA sequences of about 6 kb to
10 Mb in size, and which contain all of the elements required for
stable mitotic chromosome segregation and maintenance.
[0049] The term "humanized antibody" refers to antibody molecules
in which the amino acid sequence in the non-antigen binding regions
has been altered so that the antibody more closely resembles a
human antibody, and still retains its original binding ability.
[0050] "Hybridization" refers to any process by which a strand of
nucleic acid binds with a complementary strand through base
pairing.
[0051] The term "hybridization complex" refers to a complex formed
between two nucleic acid sequences by virtue of the formation of
hydrogen bonds between complementary bases. A hybridization complex
may be formed in solution (e.g., C.sub.0t or R.sub.0t analysis) or
formed between one nucleic acid sequence present in solution and
another nucleic acid sequence immobilized on a solid support (e.g.,
paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate to which cells or their nucleic acids
have been fixed).
[0052] The words "insertion" or "addition" refer to changes in an
amino acid or nucleotide sequence resulting in the addition of one
or more amino acid residues or nucleotides, respectively, to the
sequence found in the naturally occurring molecule.
[0053] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems.
[0054] The term "microarray" refers to an arrangement of distinct
polynucleotides on a substrate.
[0055] The terms "element" or "array element" in a microarray
context, refer to hybridizable polynucleotides arranged on the
surface of a substrate.
[0056] The term "modulate" refers to a change in the activity of
CSGP. For example, modulation may cause an increase or a decrease
in protein activity, binding characteristics, or any other
biological, functional, or immunological properties of CSGP.
[0057] The phrases "nucleic acid" or "nucleic acid sequence" refer
to a nucleotide, oligonucleotide, polynucleotide, or any fragment
thereof. These phrases also refer to DNA or RNA of genomic or
synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which, when translated, would produce polypeptides retaining some
functional characteristic, e.g., antigenicity, or structural domain
characteristic, e.g., ATP-binding site, of the full-length
polypeptide.
[0058] The terms "operably associated" or "operably linked" refer
to functionally related nucleic acid sequences. A promoter is
operably associated or operably linked with a coding sequence if
the promoter controls the translation of the encoded polypeptide.
While operably associated or operably linked nucleic acid sequences
can be contiguous and in the same reading frame, certain genetic
elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the polypeptide but still bind to operator
sequences that control expression of the polypeptide.
[0059] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to 60 nucleotides, preferably about
15 to 30 nucleotides, and most preferably about 20 to 25
nucleotides, which can be used in PCR amplification or in a
hybridization assay or microarray. "Oligonucleotide" is
substantially equivalent to the terms "amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the
art.
[0060] "Peptide nucleic acid" (PNA) refers to an antisense molecule
or anti-gene agent which comprises PF-0631-2 DIV an oligonucleotide
of at least about 5 nucleotides in length linked to a peptide
backbone of amino acid residues ending in lysine. The terminal
lysine confers solubility to the composition. PNAs preferentially
bind complementary single stranded DNA or RNA and stop transcript
elongation, and may be pegylated to extend their lifespan in the
cell.
[0061] The term "sample" is used in its broadest sense. A sample
suspected of containing nucleic acids encoding CSGP, or fragments
thereof, or CSGP itself, may comprise a bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a
cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a
substrate; a tissue; a tissue print; etc.
[0062] The terms "specific binding" or "specifically binding" refer
to that interaction between a protein or peptide and an agonist, an
antibody, or an antagonist. The interaction is dependent upon the
presence of a particular structure of the protein, e.g., the
antigenic determinant or epitope, recognized by the binding
molecule. For example, if an antibody is specific for epitope "A,"
the presence of a polypeptide containing the epitope A, or the
presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will reduce the amount of labeled A that binds
to the antibody.
[0063] The term "stringent conditions" refers to conditions which
permit hybridization between polynucleotides and the claimed
polynucleotides. Stringent conditions can be defined by salt
concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art.
In particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature.
[0064] The term "substantially purified" refers to nucleic acid or
amino acid sequences that are removed from their natural
environment and are isolated or separated, and are at least about
60% free, preferably about 75% free, and most preferably about 90%
free from other components with which they are naturally
associated.
[0065] A "substitution" refers to the replacement of one or more
amino acids or nucleotides by different amino acids or nucleotides,
respectively. "Substrate" refers to any suitable rigid or
semi-rigid support including membranes, filters, chips, slides,
wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers, microparticles and capillaries. The substrate can
have a variety of surface forms, such as wells, trenches, pins,
channels and pores, to which polynucleotides or polypeptides are
bound. "Transformation" describes a process by which exogenous DNA
enters and changes a recipient cell. Transformation may occur under
natural or artificial conditions according to various methods well
known in the art, and may rely on any known method for the
insertion of foreign nucleic acid sequences into a prokaryotic or
eukaryotic host cell. The method for transformation is selected
based on the type of host cell being transformed and may include,
but is not limited to, viral infection, electroporation, heat
shock, lipofection, and particle bombardment. The term
"transformed" cells includes stably transformed cells in which the
inserted DNA is capable of replication either as an autonomously
replicating plasmid or as part of the host chromosome, as well as
transiently transformed cells which express the inserted DNA or RNA
for limited periods of time.
[0066] A "variant" of CSGP polypeptides refers to an amino acid
sequence that is altered by one or more amino acid residues. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties (e.g.,
replacement of leucine with isoleucine). More rarely, a variant may
have "nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
LASERGENE software (DNASTAR).
[0067] The term "variant," when used in the context of a
polynucleotide sequence, may encompass a polynucleotide sequence
related to CSGP. This definition may also include, for example,
"allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a
reference molecule, but will generally have a greater or lesser
number of polynucleotides due to alternate splicing of exons during
mRNA processing. The corresponding polypeptide may possess
additional functional domains or an absence of domains. Species
variants are polynucleotide sequences that vary from one species to
another. The resulting polypeptides generally will have significant
amino acid identity relative to each other. A polymorphic variant
is a variation in the polynucleotide sequence of a particular gene
between individuals of a given species. Polymorphic variants also
may encompass "a single nucleotide polymorphisms" (SNPs) in which
the polynucleotide sequence varies by one base. The presence of
SNPs may be indicative of, for example, a certain population, a
disease state, or a propensity for a disease state.
THE INVENTION
[0068] The invention is based on the discovery of new human cell
surface glycoproteins (CSGP), the polynucleotides encoding CSGP,
and the use of these compositions for the diagnosis, treatment, or
prevention of hematologic, karyotypic, and neuronal disorders.
[0069] Nucleic acids encoding the CSGP-1 of the present invention
were identified in Incyte Clone 2297891H1 from the breast tissue
cDNA library (BRSTNOT05) using a computer search for nucleotide
and/or amino acid sequence alignments. A consensus sequence, SEQ ID
NO:3, was derived from the following overlapping and/or extended
nucleic acid sequences: Incyte Clones 2297891H1 and 2297891R6
(BRSTNOT05), 727622H1 (SYNOOAT01), and shotgun sequences
SAXA00975F1 and SAXA00091F1.
[0070] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1A, 1B, and 1C. CSGP-1 is 195 amino acids in length and has
one potential protein kinase C phosphorylation site at T37. HMM and
SPSCAN analyses predict a signal peptide in CSGP-1 from M1 to G21.
As shown in FIG. 2, CSGP-1 has significant amino acid identity with
human XG (GI 2499136; SEQ ID NO:5). However, CSGP-1 contains a
region of unique amino acid sequence from R126 to G140, suggesting
that CSGP-1 is generated by alternative splicing of XG-encoded RNA.
Fragments of SEQ ID NO:3 from about nucleotide 246 to about
nucleotide 275 and from about nucleotide 651 to about nucleotide
695 are useful in hybridization or amplification technologies to
identify SEQ ID NO:3 and to distinguish between SEQ ID NO:3 and a
related sequence. Northern analysis shows the expression of this
sequence in seven cDNA libraries, three of which are associated
with vascular tissue.
[0071] Nucleic acids encoding the CSGP-2 of the present invention
were identified in Incyte Clone 2705267H1 from the diseased brain
tissue cDNA library (PONSAZT01) using a computer search for
nucleotide and/or amino acid sequence alignments. A consensus
sequence, SEQ ID NO:4, was derived from the following overlapping
and/or extended nucleic acid sequences: incyte Clones 2705267H1 and
2705267T6 (PONSAZT01), 3347532H1 (BRAITUT24), and shotgun sequences
SBDA02672F1 and SBDA04322F1.
[0072] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, as shown in
FIGS. 3A, 3B, 3C, and 3D. CSGP-2 is 438 amino acids in length and
has four potential N-glycosylation sites at N100, N118, N341, and
N431; three potential casein kinase II phosphorylation sites at
S260, S291, and T297; seven potential protein kinase C
phosphorylation sites at S21, T80, T99, S176, S179, T343, and S390;
and two potential tyrosine kinase phosphorylation sites at Y199 and
Y258. HMM and SPSCAN analyses predict a signal peptide in CSGP-2
from M1 to A17 or A22. CSGP-2 has chemical and structural
similarity with hPC-1 (GI 189650; SEQ ID NO:6). As shown in FIGS.
4A and 4B, the region of CSGP-2 from L27 through D378 shares 26%
identity with the extracellular region of hPC-1 from L161 through
E508. In particular, the region of CSGP-2 from D32 through N92
shares 33% identity with the active site region of hPC-1 from D166
through N225. In addition, the potential N-glycosylation sites at
N100 and N341 and the potential phosphorylation sites at S291 and
T343 in CSGP-2 are conserved in hPC-1. Fragments of SEQ ID NO:4
from about nucleotide 150 to about nucleotide 179 and from about
nucleotide 228 to about nucleotide 275 are useful in hybridization
or amplification technologies to identify SEQ ID NO:4 and to
distinguish between SEQ ID NO:4 and a related sequence. Northern
analysis shows the expression of this sequence in eight cDNA
libraries, all of which are derived from brain tissue, including
tumorous and Alzheimer's brain tissue.
[0073] The invention also encompasses CSGP variants. A preferred
CSGP variant is one which has at least about 80%, more preferably
at least about 90%, and most preferably at least about 95% amino
acid sequence identity to the CSGP amino acid sequence, and which
contains at least one functional or structural characteristic of
CSGP.
[0074] The invention also encompasses polynucleotides which encode
CSGP. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising a sequence selected from the
group consisting of SEQ ID NO:3 and SEQ ID NO:4, which encodes
CSGP.
[0075] The invention also encompasses a variant of a polynucleotide
sequence encoding CSGP. In particular, such a variant
polynucleotide sequence will have at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding CSGP. A particular aspect of the invention encompasses a
variant of a polynucleotide sequence comprising a sequence selected
from the group consisting of SEQ ID NO:3 and SEQ ID NO:4 which has
at least about 80%, more preferably at least about 90%, and most
preferably at least about 95% polynucleotide sequence identity to a
nucleic acid sequence selected from the group consisting of SEQ ID
NO:3 and SEQ ID NO:4. Any one of the polynucleotide variants
described above can encode an amino acid sequence which contains at
least one functional or structural characteristic of CSGP.
[0076] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding CSGP, some bearing minimal
similarity to the polynucleotide sequences of any known and
naturally occurring gene, may be produced. Thus, the invention
contemplates each and every possible variation of polynucleotide
sequence that could be made by selecting combinations based on
possible codon choices. These combinations are made in accordance
with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally occurring CSGP, and all such
variations are to be considered as being specifically
disclosed.
[0077] Although nucleotide sequences which encode CSGP and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring CSGP under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding CSGP or its derivatives
possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring codons. Codons may be selected to
increase the rate at which expression of the peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the
frequency with which particular codons are utilized by the host.
Other reasons for substantially altering the nucleotide sequence
encoding CSGP and its derivatives without altering the encoded
amino acid sequences include the production of RNA transcripts
having more desirable properties, such as a greater half-life, than
transcripts produced from the naturally occurring sequence.
[0078] The invention also encompasses production of DNA sequences
which encode CSGP and CSGP derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents well known in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding CSGP or any fragment thereof.
[0079] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:3 and SEQ ID NO:4 and fragments thereof under various
conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger
(1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods
Enzymol. 152:507-511.) For example, stringent salt concentration
will ordinarily be less than about 750 mM NaCl and 75 mM trisodium
citrate, preferably less than about 500 mM NaCl and 50 mM trisodium
citrate, and most preferably less than about 250 mM NaCl and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in
the absence of organic solvent, e.g., formamide, while high
stringency hybridization can be obtained in the presence of at
least about 35% formamide, and most preferably at least about 50%
formamide. Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In a
preferred embodiment, hybridization will occur at 30.degree. C. in
750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more
preferred embodiment, hybridization will occur at 37.degree. C. in
500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment, hybridization will occur at 42.degree. C. in
250 mM NaCl, 25 MM trisodium citrate, 1% SDS, 50% formamide, and
200 .mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0080] The washing steps which follow hybridization can also vary
in stringency. Wash stringency conditions can be defined by salt
concentration and by temperature. As above, wash stringency can be
increased by decreasing salt concentration or by increasing
temperature. For example, stringent salt concentration for the wash
steps will preferably be less than about 30 mM NaCl and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCl
and 1.5 mM trisodium citrate. Stringent temperature conditions for
the wash steps will ordinarily include temperature of at least
about 25.degree. C., more preferably of at least about 42.degree.
C., and most preferably of at least about 68.degree. C. In a
preferred embodiment, wash steps will occur at 25.degree. C. in 30
mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred
embodiment, wash steps will occur at 42.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred
embodiment, wash steps will occur at 68.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on
these conditions will be readily apparent to those skilled in the
art.
[0081] Methods for DNA sequencing are well known in the art and may
be used to practice any of the embodiments of the invention. The
methods may employ such enzymes as the Klenow fragment of DNA
polymerase I, SEQUENASE (US Biochemical, Cleveland Ohio), Taq
polymerase (Perkin-Elmer), thermostable T7 polymerase (Amersham
Pharmacia Biotech, Piscataway N.J.), or combinations of polymerases
and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg Md.).
Preferably, sequence preparation is automated with machines such as
the Hamilton MICROLAB 2200 (Hamilton, Reno Nev.), Peltier thermal
cycler 200 (PTC200; MJ Research, Watertown Mass.) and the ABI
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using
either ABI 373 or 377 DNA sequencing systems (Perkin-Elmer) or the
MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale
Calif.). The resulting sequences are analyzed using a variety of
algorithms which are well known in the art. (See, e.g., Ausubel, F.
M. (1997) Short Protocols in Molecular Biology, John Wiley &
Sons, New York N.Y., unit 7.7; Meyers, R. A. (1995) Molecular
Biology and Biotechnology, Wiley VCH, New York N.Y., pp.
856-853.)
[0082] The nucleic acid sequences encoding CSGP may be extended
utilizing a partial nucleotide sequence and employing various
PCR-based methods known in the art to detect upstream sequences,
such as promoters and regulatory elements. For example, one method
which may be employed, restriction-site PCR, uses universal and
nested primers to amplify unknown sequence from genomic DNA within
a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend
in divergent directions to amplify unknown sequence from a
circularized template. The template is derived from restriction
fragments comprising a known genonic locus and surrounding
sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification
of DNA fragments adjacent to known sequences in human and yeast
artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991)
PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and ligations may be used to insert
an engineered double-stranded sequence into a region of unknown
sequence before performing PCR. Other methods which may be used to
retrieve unknown sequences are known in the art. (See, e.g.,
Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries (Clontech, Palo Alto Calif.) to walk genomic DNA. This
procedure avoids the need to screen libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers
may be designed using commercially available software, such as
OLIGO 4.06 primer analysis software (National Biosciences, Plymouth
MN) or another appropriate program, to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the template at temperatures of about 68.degree.
C. to 72.degree. C.
[0083] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
In addition, random-primed libraries, which often include sequences
containing the 5' regions of genes, are preferable for situations
in which an oligo d(T) library does not yield a full-length cDNA.
Genomic libraries may be useful for extension of sequence into 5'
non-transcribed regulatory regions.
[0084] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different nucleotide-specific, laser-stimulated
fluorescent dyes, and a charge coupled device camera for detection
of the emitted wavelengths. Output/light intensity may be converted
to electrical signal using appropriate software (e.g., GENOTYPER
and SEQUENCE NAVIGATOR, Perkin-Elmer), and the entire process from
loading of samples to computer analysis and electronic data display
may be computer controlled. Capillary electrophoresis is especially
preferable for sequencing small DNA fragments which may be present
in limited amounts in a particular sample.
[0085] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode CSGP may be cloned in
recombinant DNA molecules that direct expression of CSGP, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be produced and used to express
CSGP.
[0086] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter CSGP-encoding sequences for a variety of purposes including,
but not limited to, modification of the cloning, processing, and/or
expression of the gene product. DNA shuffling by random
fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides may be used to engineer the nucleotide sequences.
For example, oligonucleotide-mediated site-directed mutagenesis may
be used to introduce mutations that create new restriction sites,
alter glycosylation patterns, change codon preference, produce
splice variants, and so forth.
[0087] In another embodiment, sequences encoding CSGP may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232.) Alternatively, CSGP itself or a fragment
thereof may be synthesized using chemical methods. For example,
peptide synthesis can be performed using various solid-phase
techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science
269:202-204.) Automated synthesis may be achieved using the ABI 43
1A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of CSGP, or any part thereof, may be altered during direct
synthesis and/or combined with sequences from other proteins, or
any part thereof, to produce a variant polypeptide.
[0088] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g, Chiez, R. M. and
F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition
of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See, e.g., Creighton, T. (1984) Proteins,
Structures and Molecular Properties, W H Freeman, New York
N.Y.)
[0089] In order to express a biologically active CSGP, the
nucleotide sequences encoding CSGP or derivatives thereof may be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for transcriptional and
translational control of the inserted coding sequence in a suitable
host. These elements include regulatory sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3'
untranslated regions in the vector and in polynucleotide sequences
encoding CSGP. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding CSGP. Such
signals include the ATG initiation codon and adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding CSGP and
its initiation codon and upstream regulatory sequences are inserted
into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including an in-frame ATG initiation codon should be provided by
the vector. Exogenous translational elements and initiation codons
may be of various origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
enhancers appropriate for the particular host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.)
[0090] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding CSGP and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995) Current
Protocols in Molecular Biology, John Wiley & Sons, New York
N.Y., ch. 9, 13, and 16.)
[0091] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding CSGP. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic
virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322
plasmids); or animal cell systems. The invention is not limited by
the host cell employed.
[0092] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding CSGP. For example, routine
cloning, subloning, and propagation of polynucleotide sequences
encoding CSGP can be achieved using a multifunctional E. coli
vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding CSGP
into the vector's multiple cloning site disrupts the lacZ gene,
allowing a calorimetric screening procedure for identification of
transformed bacteria containing recombinant molecules. In addition,
these vectors may be useful for in vitro transcription, dideoxy
sequencing, single strand rescue with helper phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.
and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large
quantities of CSGP are needed, e.g. for the production of
antibodies, vectors which direct high level expression of CSGP may
be used. For example, vectors containing the strong, inducible T5
or T7 bacteriophage promoter may be used.
[0093] Yeast expression systems may be used for production of CSGP.
A number of vectors containing constitutive or inducible promoters,
such as alpha factor, alcohol oxidase, and PGH, may be used in the
yeast Saccharomyces cerevisiae or Pichia pastoris. In addition,
such vectors direct either the secretion or intracellular retention
of expressed proteins and enable integration of foreign sequences
into the host genome for stable propagation. (See, e.g., Ausubel,
1995, supra; Grant et al. (1987) Methods Enzymol. 153:516-54; and
Scorer, C. A. et al. (1994) Bio/Technology 12:181-184.)
[0094] Plant systems may also be used for expression of CSGP.
Transcription of sequences encoding CSGP may be driven viral
promoters, e.g., the 35S and 19S promoters of CaMV used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as
the small subunit of RUBISCO or heat shock promoters may be used.
(See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie,
R. et al. (1984) Science 224:83 8-843; and Winter, J. et al. (199
1) Results Probl. Cell Differ. 17:85-105.) These constructs can be
introduced into plant cells by direct DNA transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of Science and Technology (1992) McGraw Hill, New York
N.Y., pp. 191-196.)
[0095] In mammalian cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding CSGP may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential El or E3 region of the viral genome may be used to
obtain infective virus which expresses CSGP in host cells. (See,
e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
81:3655-3659.) In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells. SV40 or EBV-based vectors may
also be used for high-level protein expression.
[0096] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained in and
expressed from a plasrid. HACs of about 6 kb to 10 Mb are
constructed and delivered via conventional delivery methods
(liposomes, polycationic amino polymers, or vesicles) for
therapeutic purposes. (See, e.g., Harrington, J. J. et al. (1997)
Nat. Genet. 15:345-355.)
[0097] For long term production of recombinant proteins in
mammalian systems, stable expression of CSGP in cell lines is
preferred. For example, sequences encoding CSGP can be transformed
into cell lines using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for about 1 to 2 days in enriched media before being switched
to selective media. The purpose of the selectable marker is to
confer resistance to a selective agent, and its presence allows
growth and recovery of cells which successfully express the
introduced sequences.
[0098] Resistant clones of stably transformed cells may be
propagated using tissue culture techniques appropriate to the cell
type.
[0099] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase and adeline
phosphoribosyltransferase genes, for use in tk or apr cells,
respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for
selection. For example, dhfr confers resistance to methotrexate;
neo confers resistance to the aminoglycosides, neomycin and G-418;
and als or pat confer resistance to chlorsulluron and
phosphinotricin acetyltransferase, respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570;
Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.)
Additional selectable genes have been described, e.g., trpB and
hisD, which alter cellular requirements for metabolites. (See,
e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad.
Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins (GFP; Clontech), B glucuronidase and its
substrate B-glucuronide, or luciferase and its substrate luciferin
may be used. These markers can be used not ordy to identify
transformants, but also to quantify the amount of transient or
stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol.
55:121-131.)
[0100] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding CSGP is inserted within a marker gene
sequence, transformed cells containing sequences encoding CSGP can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding CSGP under the control of a single promoter.
Expression of the marker gene in response to induction or selection
usually indicates expression of the tandem gene as well.
[0101] In general, host cells that contain the nucleic acid
sequence encoding CSGP and that express CSGP may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations, PCR amplification, and protein bioassay or
immunoassay techniques which include membrane, solution, or chip
based technologies for the detection and/or quantification of
nucleic acid or protein sequences.
[0102] Immunological methods for detecting and measuring the
expression of CSGP using either specific polyclonal or monoclonal
antibodies are known in the art. Examples of such techniques
include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
CSGP is preferred, but a competitive binding assay may be employed.
35 These and other assays are well known in the art. (See, e.g.,
Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual,
APS Press, St Paul Minn., Sect. IV; Coligan, J. E. et al. (1997)
Current Protocols in Immunology, Greene Pub. Associates and
Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998)
Immunochemical Protocols, Humana Press, Totowa N.J.).
[0103] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding CSGP include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding CSGP, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Amersham Pharmacia Biotech, Promega (Madison Wis.), and
US Biochemical. Suitable reporter molecules or labels which may be
used for ease of detection include radionuclides, enzymes,
fluorescent, chemiluminescent, or chromogenic agents, as well as
substrates, cofactors, inhibitors, magnetic particles, and the
like.
[0104] Host cells transformed with nucleotide sequences encoding
CSGP may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or retained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode CSGP may be designed to
contain signal sequences which direct secretion of CSGP through a
prokaryotic or eukaryotic cell membrane.
[0105] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to specify
protein targeting, folding, and/or activity. Different host cells
which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, HEK293, and W138), are available from the American Type
Culture Collection (ATCC, Manassas, Va.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0106] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding CSGP may be ligated
to a heterologous sequence resulting in translation of a fusion
protein in any of the aforementioned host systems. For example, a
chimeric CSGP protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of CSGP activity.
Heterologous protein and peptide moieties may also facilitate
purification of fusion proteins using commercially available
affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase (GST), maltose binding protein (MBP),
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate fusion proteins on immobilized
glutathione, maltose, phenylarsine oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin
(HA) enable immunoaffniity purification of fusion proteins using
commercially available monoclonal and polyclonal antibodies that
specifically recognize these epitope tags. A fusion protein may
also be engineered to contain a proteolytic cleavage site located
between the CSGP encoding sequence and the heterologous protein
sequence, so that CSGP may be cleaved away from the heterologous
moiety following purification. Methods for fusion protein
expression and purification are discussed in Ausubel (1995, supra,
ch 10). A variety of commercially available kits may also be used
to facilitate expression and purification of fusion proteins.
[0107] In a further embodiment of the invention, synthesis of
radiolabeled CSGP may be achieved in vitro using the TNT rabbit
reticulocyte lysate or wheat germ extract systems (Promega). These
systems couple transcription and translation of protein-coding
sequences operably associated with the T7, T3, or SP6 promoters.
Translation takes place in the presence of a radiolabeled amino
acid precursor, preferably .sup.35S-methionine.
[0108] Fragments of CSGP may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, supra, pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the ABI
431A peptide synthesizer (Perkin-Elmer). Various fragments of CSGP
may be synthesized separately and then combined to produce the full
length molecule.
THERAPEUTICS
[0109] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between regions of CSGP-1 and XG and
between regions of CSGP-2 and hPC-1. In addition, the expression of
CSGP-1 is closely associated with vascular tissue, and the
expression of CSGP-2 is closely associated with brain tissue.
Therefore, CSGP appears to play a role in hematologic, karyotypic,
and neuronal disorders. In the treatment of hematologic,
karyotypic, and neuronal disorders associated with increased CSGP
expression or activity, it is desirable to decrease the expression
or activity of CSGP. In the treatment of the above conditions
associated with decreased CSGP expression or activity, it is
desirable to increase the expression or activity of CSGP.
[0110] Therefore, in one embodiment, CSGP or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of CSGP. Examples of such disorders include, but are not limited
to, a hematologic disorder such as anemia including
.beta.-thalassemia, hemorrhage, thrombosis, embolism,
lymphadenopathy, splenomegaly, phagocytic disorders, hematopoietic
disorders, hemoglobin disorders including sickle cell anemia, bone
marrow disorders, leukemia including chronic myelogenous leukemia
and other myeloproliferative disorders, lymphoma including
non-Hodgkin's lymphoma, Hodgkin's disease, complications related to
blood transfusion, complications related to bone marrow
transplantation, and clotting disorders including von Willebrand's
disease and hemophilia; a karyotypic disorder associated with sex
chromosome imbalance including Klinefelter syndrome and Turner
syndrome; and a neuronal disorder such as akathesia, Alzheimer's
disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder,
catatonia, cerebral neoplasms, dementia, depression, diabetic
neuropathy, Down's syndrome, tardive dyskinesia, dystonias,
epilepsy, Huntington's disease, peripheral neuropathy, multiple
sclerosis, neurofibromatosis, Parkinson's disease, paranoid
psychoses, postherpetic neuralgia, schizophrenia, and Tourette's
disorder.
[0111] In another embodiment, a vector capable of expressing CSGP
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a disorder associated with decreased
expression or activity of CSGP including, but not limited to, those
described above.
[0112] In a further embodiment, a pharmaceutical composition
comprising a substantially purified CSGP in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a disorder associated with decreased expression or
activity of CSGP including, but not limited to, those provided
above.
[0113] In still another embodiment, an agonist which modulates the
activity of CSGP may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of CSGP including, but not limited to, those listed above.
[0114] In a further embodiment, an antagonist of CSGP may be
administered to a subject to treat or prevent a disorder associated
with increased expression or activity of CSGP. Examples of such
disorders include, but are not limited to, those hematologic,
karyotypic, and neuronal disorders described above. In one aspect,
an antibody which specifically binds CSGP may be used directly as
an antagonist or indirectly as a targeting or delivery mechanism
for bringing a pharmaceutical agent to cells or tissue which
express CSGP.
[0115] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding CSGP may be administered
to a subject to treat or prevent a disorder associated with
increased expression or activity of CSGP including, but not limited
to, those described above.
[0116] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders described
above. Using this approach, one may be able to achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
[0117] An antagonist of CSGP may be produced using methods which
are generally known in the art. In particular, purified CSGP may be
used to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind CSGP. Antibodies
to CSGP may also be generated using methods that are well known in
the art. Such antibodies may include, but are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies (i.e., those which inhibit dimer formation)
are especially preferred for therapeutic use.
[0118] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with CSGP or with any fragment or oligopeptide thereof
which has immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calnette-Guerin) and Corynebacterium parvum are
especially preferable.
[0119] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to CSGP have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 10 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of CSGP amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0120] Monoclonal antibodies to CSGP may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G.
et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell
Biol. 62:109-120.)
[0121] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci.
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608;
and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively,
techniques described for the production of single chain antibodies
may be adapted, using methods known in the art, to produce
CSGP-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc.
Natl. Acad. Sci. 88:10134-10137.)
[0122] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al.
(1991) Nature 349:293-299.)
[0123] Antibody fragments which contain specific binding sites for
CSGP may also be generated. For example, such fragments include,
but are not limited to, F(ab')2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries may be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity. (See, e.g., Huse, W. D. et al. (1989) Science
246:1275-1281.)
[0124] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between CSGP and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering CSGP epitopes
is preferred, but a competitive binding assay may also be employed
(Pound, supra).
[0125] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for CSGP. Affinity is expressed as an association
constant, K.sub.a, which is defined as the molar concentration of
CSGP-antibody complex divided by the molar concentrations of free
antigen and free antibody under equilibrium conditions. The K.sub.a
determined for a preparation of polyclonal antibodies, which are
heterogeneous in their affinities for multiple CSGP epitopes,
represents the average affinity, or avidity, of the antibodies for
CSGP. The K.sub.a determined for a preparation of monoclonal
antibodies, which are monospecific for a particular CSGP epitope,
represents a true measure of affinity. High-affinity antibody
preparations with K.sub.a ranging from about 10.sup.9 to 10.sup.12
L/mole are preferred for use in immunoassays in which the
CSGP-antibody complex must withstand rigorous manipulations.
Low-affinity antibody preparations with K.sub.a ranging from about
10.sup.6 to 10.sup.7 L/mole are preferred for use in
immunopurification and similar procedures which ultimately require
dissociation of CSGP, preferably in active form, from the antibody
(Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington, DC; Liddell, J. E. and Cryer, A. (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York N.Y.).
[0126] The titer and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody/ml, preferably 5-10 mg specific antibody/ml,
is preferred for use in procedures requiring precipitation of
CSGP-antibody complexes. Procedures for evaluating antibody
specificity, titer, and avidity, and guidelines for antibody
quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
[0127] In another embodiment of the invention, the polynucleotides
encoding CSGP, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding CSGP may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding CSGP. Thus, complementary molecules or
fragments may be used to modulate CSGP activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding CSGP.
[0128] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors to
express nucleic acid sequences complementary to the polynucleotides
encoding CSGP. (See, e.g., Sambrook, supra; Ausubel, 1995,
supra.)
[0129] Genes encoding CSGP can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide, or fragment thereof, encoding CSGP. Such constructs
may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in the absence of integration into the
DNA, such vectors may continue to transcribe RNA molecules until
they are disabled by endogenous nucleases. Transient expression may
last for a month or more with a non-replicating vector, and may
last even longer if appropriate replication elements are part of
the vector system.
[0130] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding CSGP. Oligonucleotides derived from
the transcription initiation site, e.g., between about positions
-10 and +10 from the start site, are preferred. Similarly,
inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules. Recent therapeutic advances using triplex DNA
have been described in the literature. (See, e.g., Gee, J.E. et al.
(1994) in Huber, B. E. and B. I. Carr, Molecular and Immunologic
Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177.) A
complementary sequence or antisense molecule may also be designed
to block translation of niRNA by preventing the transcript from
binding to ribosomes.
[0131] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding CSGP.
[0132] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides, corresponding to the region of the target
gene containing the cleavage site, may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0133] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for cheincally synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding CSGP. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell lines, cells, or tissues.
[0134] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule, or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0135] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C.K. et al. (1997) Nature Biotechnology 15:462-466.)
[0136] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0137] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of CSGP, antibodies to CSGP, and mimetics,
agonists, antagonists, or inhibitors of CSGP. The compositions
maybe administered alone or in combination with at least one other
agent, such as a stabilizing compound, which may be administered in
any sterile, biocompatible pharmaceutical carrier including, but
not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs, or hormones.
[0138] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0139] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing, Easton
Pa.).
[0140] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0141] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropyhnethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0142] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0143] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0144] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0145] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0146] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0147] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0148] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of CSGP, such
labeling would include amount, frequency, and method of
administration.
[0149] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0150] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0151] A therapeutically effective dose refers to that amount of
active ingredient, for example CSGP or fragments thereof,
antibodies of CSGP, and agonists, antagonists or inhibitors of
CSGP, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED.sub.50 (the dose therapeutically effective in
50% of the population) or LD.sub.50 (the dose lethal to 50% of the
population) statistics. The dose ratio of toxic to therapeutic
effects is the therapeutic index, and it can be expressed as the
LD.sub.50/ED.sub.50 ratio. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route
of administration.
[0152] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requing treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0153] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, up to a total dose of about 1 gram, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
[0154] In another embodiment, antibodies which specifically bind
CSGP may be used for the diagnosis of disorders characterized by
expression of CSGP, or in assays to monitor patients being treated
with CSGP or agonists, antagonists, or inhibitors of CSGP.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for CSGP include methods which utilize the antibody and a label to
detect CSGP in human body fluids or in extracts of cells or
tissues. The antibodies may be used with or without modification,
and may be labeled by covalent or non-covalent attachment of a
reporter molecule. A wide variety of reporter molecules, several of
which are described above, are known in the art and may be
used.
[0155] A variety of protocols for measuring CSGP, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of CSGP expression. Normal or
standard values for CSGP expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to CSGP under conditions suitable
for complex formation. The amount of standard complex formation may
be quantitated by various methods, preferably by photometric means.
Quantities of CSGP expressed in subject samples from biopsied
tissues are compared with the standard values. Deviation between
standard and subject values establishes the parameters for
diagnosing disease.
[0156] In another embodiment of the invention, the polynucleotides
encoding CSGP may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of CSGP may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of CSGP, and to
monitor regulation of CSGP levels during therapeutic
intervention.
[0157] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genonic
sequences, encoding CSGP or closely related molecules may be used
to identify nucleic acid sequences which encode CSGP. The
specificity of the probe, whether it is made from a highly specific
region, e.g., the 5' regulatory region, or from a less specific
region, e.g., a conserved motif, and the stringency of the
hybridization or amplification (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding CSGP, allelic variants, or related
sequences.
[0158] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the CSGP encoding sequences. The hybridization
probes of the subject invention may be DNA or RNA and may be
derived from the sequence of SEQ ID NO:3 or SEQ ID NO:4 or from
genomic sequences including promoters, enhancers, and introns of
the CSGP gene.
[0159] Means for producing specific hybridization probes for DNAs
encoding CSGP include the cloning of polynucleotide sequences
encoding CSGP or CSGP derivatives into vectors for the production
of MRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32p or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0160] Polynucleotide sequences encoding CSGP may be used for the
diagnosis of disorders associated with expression of CSGP. Examples
of such disorders include, but are not limited to, a hematologic
disorder such as anemia including .delta.-thalassemia, hemorrhage,
thrombosis, embolism, lymphadenopathy, splenomegaly, phagocytic
disorders, hematopoietic disorders, hemoglobin disorders including
sickle cell anemia, bone marrow disorders, leukemia including
chronic myelogenous leukemia and other myeloproliferative
disorders, lymphoma including non-Hodgkin's lymphoma, Hodgkin's
disease, complications related to blood transfusion, complications
related to bone marrow transplantation, and clotting disorders
including von Willebrand's disease and hemophilia; a karyotypic
disorder associated with sex chromosome imbalance including
Klinefelter syndrome and Turner syndrome; and a neuronal disorder
such as akathesia, Alzheimer's disease, amnesia, amyotrophic
lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms,
dementia, depression, diabetic neuropathy, Down's syndrome, tardive
dyskinesia, dystonias, epilepsy, Huntington's disease, peripheral
neuropathy, multiple sclerosis, neurofibromatosis, Parkinson's
disease, paranoid psychoses, postherpetic neuralgia, schizophrenia,
and Tourette's disorder. The polynucleotide sequences encoding CSGP
may be used in Southern or northern analysis, dot blot, or other
membrane-based technologies; in PCR technologies; in dipstick, pin,
and multiformat ELISA-like assays; and in microarrays utilizing
fluids or tissues from patients to detect altered CSGP expression.
Such qualitative or quantitative methods are well known in the
art.
[0161] In a particular aspect, the nucleotide sequences encoding
CSGP may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding CSGP may be labeled by standard methods and
added to a fluid or tissue sample from a patient under conditions
suitable for the formation of hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is
quantitated and compared with a standard value. If the amount of
signal in the patient sample is significantly altered in comparison
to a control sample then the presence of altered levels of
nucleotide sequences encoding CSGP in the sample indicates the
presence of the associated disorder. Such assays may also be used
to evaluate the efficacy of a particular therapeutic treatment
regimen in animal studies, in clinical trials, or to monitor the
treatment of an individual patient.
[0162] In order to provide a basis for the diagnosis of a disorder
associated with expression of CSGP, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
encoding CSGP, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with values from
an experiment in which a known amount of a substantially purified
polynucleotide is used. Standard values obtained in this manner may
be compared with values obtained from samples from patients who are
symptomatic for a disorder. Deviation from standard values is used
to establish the presence of a disorder.
[0163] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0164] With respect to cancer, the presence of an abnormal amount
of transcript (either under- or overexpressed) in biopsied tissue
from an individual may indicate a predisposition for the
development of the disease, or may provide a means for detecting
the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0165] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding CSGP may involve the use of PCR. These
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment
of a polynucleotide encoding CSGP, or a fragment of a
polynucleotide complementary to the polynucleotide encoding CSGP,
and will be employed under optimized conditions for identification
of a specific gene or condition. Oligomers may also be employed
under less stringent conditions for detection or quantitation of
closely related DNA or RNA sequences.
[0166] Methods which may also be used to quantitate the expression
of CSGP include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P.C. et al. (1993)
J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal.
Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be accelerated by running the assay in an ELISA format
where the oligomer of interest is presented in various dilutions
and a spectrophotometric or colorimetric response gives rapid
quantitation.
[0167] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, and to develop and monitor the activities of
therapeutic agents.
[0168] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalon, D. et al. (1995) PCT application W095/35505;
Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.) In
another embodiment of the invention, nucleic acid sequences
encoding CSGP may be used to generate hybridization probes useful
in mapping the naturally occurring genomic sequence. The sequences
may be mapped to a particular chromosome, to a specific region of a
chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes (HACs), yeast artificial chromosomes (YACs),
bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g.,
Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355; Price, C.
M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991) Trends
Genet. 7:149-154.)
[0169] Fluorescent in situ hybridization (FISH) may be correlated
with other physical chromosome mapping techniques and genetic map
data. (See, e.g., Heinz-Ulhrich, et al. (1995) in Meyers, supra,
pp. 965-968.) Examples of genetic map data can be found in various
scientific journals or at the Online Mendelian Inheritance in Man
(OMIM) site. Correlation between the location of the gene encoding
CSGP on a physical chromosomal map and a specific disorder, or a
predisposition to a specific disorder, may help define the region
of DNA associated with that disorder. The nucleotide sequences of
the invention may be used to detect differences in gene sequences
among normal, carrier, and affected individuals.
[0170] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms by
physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, e.g., ataxia-telangiectasia to 11 q22-23, any
sequences mapping to that area may represent associated or
regulatory genes for further investigation. (See, e.g., Gatti, R.
A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of
the subject invention may also be used to detect differences in the
chromosomal location due to translocation, inversion, etc., among
normal, carrier, or affected individuals.
[0171] In another embodiment of the invention, CSGP, its catalytic
or immunogenic fragments, or oligopeptides thereof can be used for
screening libraries of compounds in any of a variety of drug
screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The formation of binding
complexes between CSGP and the agent being tested may be
measured.
[0172] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate. The test
compounds are reacted with CSGP, or fragments thereof, and washed.
Bound CSGP is then detected by methods well known in the art.
Purified CSGP can also be coated directly onto plates for use in
the aforementioned drug screening techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on a solid support.
[0173] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding CSGP specifically compete with a test compound for binding
CSGP. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
CSGP.
[0174] In additional embodiments, the nucleotide sequences which
encode CSGP may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0175] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0176] I. Construction of cDNA Libraries
[0177] The BRSTNOT05 cDNA library was constructed using RNA
isolated from breast tissue removed from a 58-year-old Caucasian
female during a unilateral extended simple mastectomy. Pathology
for the associated tumor tissue indicated multicentric invasive
grade 4 lobular carcinoma. Patient history included skin cancer,
rheumatic heart disease, osteoarthritis, and tuberculosis. Family
history included cerebrovascular and cardiovascular disease, breast
and prostate cancer, and type I diabetes.
[0178] The PONSAZTO1 cDNA library was constructed using RNA
isolated from diseased pons tissue removed from the brain of a
74-year-old Caucasian male who died from Alzheimer's disease.
[0179] Frozen tissue from each of the above sources was homogenized
and lysed in guanidinium isothiocyanate solution using a Polytron
PT-3 000 homogenizer (Brinkmann Instruments, Westbury N.Y.). The
lysate was centrifuged over a CsCl cushion to isolate RNA. The RNA
was extracted with acid phenol, precipitated with sodium acetate
and ethanol, resuspended in RNase-free water, and treated with
DNase. The RNA was re-extracted with acid phenol and reprecipitated
as described above. Poly(A+) RNA was isolated using the OLIGOTEX
mRNA purification kit (QIAGEN, Chatsworth Calif.).
[0180] Poly(A+) RNA was used for cDNA synthesis and construction of
the cDNA library according to the recommended protocols in the
SUPERSCRIPT plasmid system (Life Technologies). The cDNAs were
fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia
Biotech), and those cDNAs exceeding 400 bp were used for library
construction. cDNAs used to construct BRSTNOT05 were ligated into
PSPORT1 (Life Technologies), and cDNAs used to construct PONSAZT01
were ligated into pINCY (Incyte Pharmaceuticals, Palo Alto Calif.).
Recombinant plasmids were transformed into DH5.alpha. competent
cells (Life Technologies).
[0181] II. Isolation of CDNA Clones
[0182] For each of the above cDNA libraries, plasmid DNA was
released from host cells and purified using the R.E.A.L. PREP 96
plasmid kit (QIAGEN). The recommended protocol was employed except
for the following changes: 1) the bacteria were cultured in 1 ml of
sterile Terrific Broth (Life Technologies) with carbenicillin at 25
mg/l and glycerol at 0.4%; 2) after the cultures were incubated for
19 hours, the cells were lysed with 0.3 ml of lysis buffer; and 3)
following isopropanol precipitation, the plasmid DNA pellets were
each resuspended in 0.1 ml of distilled water. The DNA samples were
stored at4.degree. C.
[0183] III. Sequencing and Analysis
[0184] The cDNAs were prepared for sequencing using the ABI
CATALYST 800 (Perkin-Elmer) or the HYDRA microdispenser (Robbins
Scientific) or MICROLAB 2200 (Hamilton) systems in combination with
the PTC-200 thermal cyclers (MJ Research). The cDNAs were sequenced
using the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer)
and standard ABI protocols, base calling software, and kits. In one
alternative, cDNAs were sequenced using the MEGABACE 1000 DNA
sequencing system (Molecular Dynamics). In another alternative, the
cDNAs were amplified and sequenced using the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Perkin-Elmer). In
yet another alternative, cDNAs were sequenced using solutions and
dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs
were determined using standard methods (reviewed in Ausubel, 1997,
supra, unit 7.7). Some of the cDNA sequences were selected for
extension using the techniques disclosed in Example V.
[0185] The polynucleotide sequences derived from cDNA, extension,
and shotgun sequencing were assembled and analyzed using a
combination of software programs which utilize algorithms well
known to those skilled in the art. Table 1 summarizes the software
programs, descriptions, references, and threshold parameters used.
The first column of Table 1 shows the tools, programs, and
algorithms used, the second column provides a brief description
thereof, the third column presents the references which are
incorporated by reference herein, and the fourth column presents,
where applicable, the scores, probability values, and other
parameters used to evaluate the strength of a match between two
sequences (the higher the probability the greater the homology).
Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software Engineering, South San Francisco Calif.) and LASERGENE
software (DNASTAR).
[0186] The polynucleotide sequences were validated by removing
vector, linker, and polyA sequences and by masking ambiguous bases,
using algorithms and programs based on BLAST, dynamic programing,
and dinucleotide nearest neighbor analysis. The sequences were then
queried against a selection of public databases such as GenBank
primate, rodent, mammalian, vertebrate, and eukaryote databases,
and BLOCKS to acquire annotation, using programs based on BLAST,
FASTA, and BLIMPS. The sequences were assembled into full length
polynucleotide sequences using programs based on Phred, Phrap, and
Consed, and were screened for open reading frames using programs
based on GeneMark, BLAST, and FASTA. The full length polynucleotide
sequences were translated to derive the corresponding full length
amino acid sequences, and these full length sequences were
subsequently analyzed by querying against databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS,
Prosite, and Hidden Markov Model (HMM)-based protein family
databases such as PFAM. HMM is a probalistic approach which
analyzes consensus primary structures of gene families. (See, e.g.,
Eddy, S.R. (1996) Cur. Opin. Str. Biol. 6:361-365.)
[0187] The programs described above for the assembly and analysis
of full length polynucleotide and amino acid sequences were also
used to identify polynucleotide sequence fragments from SEQ ID NO:3
and SEQ ID NO:4. Fragments from about 20 to about 4000 nucleotides
which are useful in hybridization and amplification technologies
were described in The Invention section above.
[0188] IV. Northern Analysis
[0189] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and
16.)
[0190] Analogous computer techniques applying BLAST were used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ database (Incyte Pharmaceuticals). This
analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or similar. The basis of the search is the
product score, which is defined as: 1 % sequence identity .times. %
maximum BLAST score 100
[0191] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0192] The results of northern analyses are reported as a
percentage distribution of libraries in which the transcript
encoding CSGP occurred. Analysis involved the categorization of
cDNA libraries by organ/tissue and disease. The organ/tissue
categories included cardiovascular, dermatologic, developmental,
endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal,
nervous, reproductive, and urologic. The disease/condition
categories included cancer, inflammation/trauma, cell
proliferation, neurological, and pooled. For each category, the
number of libraries expressing the sequence of interest was counted
and divided by the total number of libraries across all categories.
Percentage values of tissue-specific and disease- or
condition-specific expression are reported in The Invention.
[0193] V. Extension of CSGP Encoding Polynucleotides
[0194] The full length nucleic acid sequences of SEQ ID NO:3 and
SEQ ID NO:4 were produced by extension of an appropriate fragment
of the full length molecule using oligonucleotide primers designed
from this fragment. One primer was synthesized to initiate 5'
extension of the known fragment, and the other primer, to initiate
3' extension of the known fragment. The initial primers were
designed using OLIGO 4.06 software (National Biosciences), or
another appropriate program, to be about 22 to 30 nucleotides in
length, to have a GC content of about 50% or more, and to anneal to
the target sequence at temperatures of about 68.degree. C. to about
72.degree. C. Any stretch of nucleotides which would result in
hairpin structures and primer-primer dimerizations was avoided.
[0195] Selected human cDNA libraries were used to extend the
sequence. If more than one extension was necessary or desired,
additional or nested sets of primers were designed.
[0196] High fidelity amplification was obtained by PCR using
methods well known in the art. PCR was performed in 96-well plates
using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction
buffer containing Mg.sup.2+, (NH.sub.4).sub.2SO.sub.4, and
.beta.-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA
polymerase (Stratagene), with the following parameters for primer
pair PCI A and PCI B: Step 1: 94.degree. C., 3 min; Step 2:
94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min; Step 4:
68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68.degree. C., 5 min; Step 7: storage at 4.degree. C. In
the alternative, the parameters for primer pair T7 and SK+ were as
follows: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C., 15
sec; Step 3: 57.degree. C., 1 min; Step 4: 68.degree. C., 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68.degree. C.,
5 min; Step 7: storage at 4.degree. C.
[0197] The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% (v/v)
PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1X TE and
0.5 .mu.l of undiluted PCR product into each well of an opaque
fluorimeter plate (Corning Costar, Acton Mass.), allowing the DNA
to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of
the sample and to quantify the concentration of DNA. A 5 .mu.l to
10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a 1% agarose mini-gel to determine which
reactions were successful in extending the sequence.
[0198] The extended nucleotides were desalted and concentrated,
transferred to 384-well plates, digested with CviJI cholera virus
endonuclease (Molecular Biology Research, Madison Wis.), and
sonicated or sheared prior to religation into pUC 18 vector
(Amersham Pharmacia Biotech). For shotgun sequencing, the digested
nucleotides were separated on low concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar
ACE (Promega). Extended clones were religated using T4 ligase (New
England Biolabs, Beverly Mass.) into pUC 18 vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction site overhangs, and transfected into competent
E. coli cells. Transformed cells were selected on
antibiotic-containing media, individual colonies were picked and
cultured overnight at 37.degree. C. in 384-well plates in LB/2x
carb liquid media.
[0199] The cells were lysed, and DNA was amplified by PCR using Taq
DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase
(Stratagene) with the following parameters: Step 1: 94.degree. C.,
3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min;
Step 4: 72.degree. C., 2 min; Step 5: steps 2, 3, and 4 repeated 29
times; Step 6: 72.degree. C., 5 min; Step 7: storage at 4.degree.
C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as
described above. Samples with low DNA recoveries were reamplified
using the same conditions as described above. Samples were diluted
with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit
(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle sequencing ready reaction kit (Perkin-Elmer).
[0200] In like manner, the nucleotide sequences of SEQ ID NO:3 and
SEQ ID NO:4 are used to obtain 5' regulatory sequences using the
procedure above, oligonucleotides designed for such extension, and
an appropriate genomic library.
[0201] VI. Labeling and Use of Individual Hybridization Probes
[0202] Hybridization probes derived from SEQ ID NO:3 and SEQ ID
NO:4 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although
the labeling of oligonucleotides, consisting of about 20 base
pairs, is specifically described, essentially the same procedure is
used with larger nucleotide fragments. Oligonucleotides are
designed using state-of-the-art software such as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of
each oligomer, 250 .mu.Ci of [.gamma.-.sup.32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide
kinase (DuPont NEN, Boston Mass.). The labeled oligonucleotides are
substantially purified using a SEPHADEX G-25 superfine size
exclusion dextran bead column (Amersham Pharmacia Biotech). An
aliquot containing 10.sup.7 counts per minute of the labeled probe
is used in a typical membrane-based hybridization analysis of human
genomic DNA digested with one of the following endonucleases: Ase
I, Bgl II, Eco RI, Pst I, Xbal, or Pvu II (DuPont NEN).
[0203] The DNA from each digest is fractionated on a 0.7% agarose
gel and transferred to nylon membranes (Nytran Plus, Schleicher
& Schuell, Durham N.H.). Hybridization is carried out for 16
hours at 40.degree. C. To remove nonspecific signals, blots are
sequentially washed at room temperature under increasingly
stringent conditions up to 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate. After XOMAT-AR film (Eastman Kodak,
Rochester N.Y.) is exposed to the blots to film for several hours,
hybridization patterns are compared visually.
[0204] VII. Microarrays
[0205] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced by hand or
using available methods and machines and contain any appropriate
number of elements. After hybridization, nonhybridized probes are
removed and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0206] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments suitable for hybridization can be selected using software
well known in the art such as LASERGENE software (DNASTAR).
Full-length cDNAs, ESTs, or fragments thereof corresponding to one
of the nucleotide sequences of the present invention, or selected
at random from a cDNA library relevant to the present invention,
are arranged on an appropriate substrate, e.g., a glass slide. The
cDNA is fixed to the slide using, e.g., UV cross-lining followed by
thermal and chemical treatments and subsequent drying. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al.
(1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and
used for hybridization to the elements on the substrate. The
substrate is analyzed by procedures described above.
[0207] VIII. Complementary Polynucleotides
[0208] Sequences complementary to the CSGP-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring CSGP. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the
coding sequence of CSGP. To inhibit transcription, a complementary
oligonucleotide is designed from the most unique 5' sequence and
used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary oligonucleotide is designed to prevent
ribosomal binding to the CSGP-encoding transcript.
[0209] IX. Expression of CSGP
[0210] Expression and purification of CSGP is achieved using
bacterial or virus-based expression systems. For expression of CSGP
in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express CSGP upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of CSGP
in eukaryotic cells is achieved by infecting insect or manunalian
cell lines with recombinant Autographica californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding CSGP by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda
(S19) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter requires additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.)
[0211] In most expression systems, CSGP is synthesized as a fusion
protein with, e.g., glutathione S-transferase (GST) or a peptide
epitope tag, such as FLAG or 6-His, permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from
crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma
japonicum, enables the purification of fusion proteins on
immobilized glutathione under conditions that maintain protein
activity and antigenicity (Amersham Pharmacia Biotech). Following
purification, the GST moiety can be proteolytically cleaved from
CSGP at specifically engineered sites. FLAG, an 8-amino acid
peptide, enables immunoaffinity purification using commercially
available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues,
enables purification on metal-chelate resins (QIAGEN). Methods for
protein expression and purification are discussed in Ausubel (1995,
supra, ch 10 and 16). Purified CSGP obtained by these methods can
be used directly in the following activity assay.
[0212] X. Demonstration of CSGP Activity
[0213] An assay for CSGP activity measures the expression of CSGP
on the cell surface. cDNA encoding CSGP is transfected into a cell
line that does not express endogenous CSGP. Cell surface proteins
of transfectants are labeled with biotin (de la Fuente, M. A. et
al. (1997) Blood 90:2398-2405). Immunoprecipitations are performed
using CSGP-specific antibodies, and immunoprecipitated samples are
analyzed using SDS-PAGE and immunoblotting techniques. The ratio of
labeled immunoprecipitant to unlabeled immunoprecipitant is
proportional to the amount of CSGP expressed on the cell
surface.
[0214] XI. Functional Assays
[0215] CSGP function is assessed by expressing the sequences
encoding CSGP at physiologically elevated levels in mammalian cell
culture systems. cDNA is subcloned into a mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT (Life
Technologies) and pCR3.1 (Invitrogen, Carlsbad CA), both of which
contain the cytomegalovirus promoter. 5-10 .mu.g of recombinant
vector are transiently transfected into a human cell line,
preferably of endothelial or hematopoietic origin, using either
liposome formulations or electroporation. 1-2 .mu.g of an
additional plasmid containing sequences encoding a marker protein
are co-transfected. Expression of a marker protein provides a means
to distinguish transfected cells from nontransfected cells and is a
reliable predictor of cDNA expression from the recombinant vector.
Marker proteins of choice include, e.g., Green Fluorescent Protein
(GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an automated, laser optics-based technique, is used to
identify transfected cells expressing GFP or CD64-GFP, and to
evaluate properties, for example, their apoptotic state. FCM
detects and quantifies the uptake of fluorescent molecules that
diagnose events preceding or coincident with cell death. These
events include changes in nuclear DNA content as measured by
staining of DNA with propidium iodide; changes in cell size and
granularity as measured by forward light scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by
decrease in bromodeoxyuridine uptake; alterations in expression of
cell surface and intracellular proteins as measured by reactivity
with specific antibodies; and alterations in plasma membrane
composition as measured by the binding of fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry
are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New
York N.Y.
[0216] The influence of CSGP on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding CSGP and either CD64 or CD64-GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions of human immunoglobulin G (IgG). Transfected
cells are efficiently separated from nontransfected cells using
magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the
cells using methods well known by those of skill in the art.
Expression of mRNA encoding CSGP and other genes of interest can be
analyzed by northern analysis or microarray techniques.
[0217] XII. Production of CSGP Specific Antibodies
[0218] CSGP substantially purified using polyacrylamide gel
electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods
Enzymol. 182:488-495), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols.
[0219] Alternatively, the CSGP amino acid sequence is analyzed
using LASERGENE software (DNASTAR) to determine regions of high
immunogenicity, and a corresponding oligopeptide is synthesized and
used to raise antibodies by means known to those of skill in the
art. Methods for selection of appropriate epitopes, such as those
near the C-terminus or in hydrophilic regions are well described in
the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
[0220] Typically, oligopeptides 15 residues in length are
synthesized using an ABI 431A peptide synthesizer (Perkin-Elmer)
using fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis
Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995,
supra.) Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. Resulting antisera are tested for
antipeptide activity by, for example, binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radio-iodinated goat anti-rabbit
IgG.
[0221] XIII. Purification of Naturally Occurring CSGP Using
Specific Antibodies
[0222] Naturally occurring or recombinant CSGP is substantially
purified by immunoaffinity chromatography using antibodies specific
for CSGP. An immunoaffinity column is constructed by covalently
coupling anti-CSGP antibody to an activated chromatographic resin,
such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech).
After the coupling, the resin is blocked and washed according to
the manufacturer's instructions.
[0223] Media containing CSGP are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of CSGP (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/CSGP binding (e.g., a buffer of pH
2 to pH 3, or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and CSGP is collected.
[0224] XIV. Identification of Molecules Which Interact with
CSGP
[0225] CSGP, or biologically active fragments thereof, are labeled
with .sup.125I Bolton-Hunter reagent. (See, e.g., Bolton et al.
(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed
in the wells of a multi-well plate are incubated with the labeled
CSGP, washed, and any wells with labeled CSGP complex are assayed.
Data obtained using different concentrations of CSGP are used to
calculate values for the number, affinity, and association of CSGP
with the candidate molecules.
[0226] Various modifications and variations of the described
methods and systems 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 described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
1TABLE 1 PF-0631-2 DIV Program Description Reference Parameter
Threshold ABI A program that removes vector sequences and masks
Perkin-Elmer Applied Biosystems, FACTURA ambiguous bases in nucleic
acid sequences. Foster City, CA. ABI/ A Fast Data Finder useful in
comparing and annotating Perkin-Elmer Applied Biosystems, Mismatch
<50% PARACEL amino acid or nucleic acid sequences. Foster City,
CA; Paracel Inc., Pasadena, CA. FDF ABI A program that assembles
nucleic acid sequences. Perkin-Elmer Applied Biosystems, Auto
Foster City, CA. Assembler BLAST A Basic Local Alignment Search
Tool useful in sequence Altschul, S.F. et al. (1990) J. Mol. Biol.
ESTs: Probability similarity search for amino acid and nucleic acid
sequences. 215:403-410; Altschul, S.F. et al. (1997) value = 1.0E-8
or less BLAST includes five functions: blastp, blastn, blastx,
Nucleic Acids Res. 25: 3389-3402. Full Length sequences: tblastn,
and tblastx. Probability value = 1.0E-10 or less FASTA A Pearson
and Lipman algorithm that searches for Pearson, W.R. and D.J.
Lipman (1988) Proc. ESTs: fasta E value = similarity between a
query sequence and a group of Natl. Acad Sci. 85:2444-2448;
Pearson, W.R. 1.06E-6 Assembled ESTs: sequences of the same type.
FASTA comprises as least (1990) Methods Enzymol. 183: 63-98; and
fasta Identity = 95% or five functions: fasta, tfasta, fastx,
tfastx, and ssearch. Smith, T.F. and M.S. Waterman (1981) Adv.
greater and Match length = Appl. Math. 2:482-489. 200 bases or
greater; fastx E value = 1.0E-8 or less Full Length sequences:
fastx score = 100 or greater BLIMPS A BLocks IMProved Searcher that
matches a sequence Henikoff, S and J.G. Henikoff, Nucl. Acid Score
= 1000 or greater; against those in BLOCKS and PRINTS databases to
search Res., 19:6565-72, 1991. J.G. Henikoff and S. Ratio of
Score/Strength = for gene families, sequence homology, and
structural Henikoff (1996) Methods Enzymol. 0.75 or larger;
fingerprint regions. 266:88-105; and Attwood, T.K. et al. (1997)
and Probability value = J. Chem. Inf. Comput. Sci. 37: 417-424.
1.0E-3 or less PFAM A Hidden Markov Models-based application useful
for Krogh, A. et al. (1994) J. Mol. Biol., Score = 10-50 bits,
protein family search. 235:1501-1531; Sonnhammer, E.L.L. et al.
depending on individual (1988) Nucleic Acids Res. 26:320-322.
protein families Profile An algorithm that searches for structural
and sequence Gribskov, M. et al. (1988) CABIOS 4:61-66; Score = 4.0
or greater Scan motifs in protein sequences that match sequence
patterns Gribskov, et al. (1989) Methods Enzymol. defined in
Prosite. 183:146-159; Bairoch, A. et al. (1997) Nucleic Acids Res.
25:217-221. Phred A base-calling algorithm that examines automated
Ewing, B. et al. (1998) Genome sequencer traces with high
sensitivity and probability. Res. 8:175-185; Ewing, B. and P. Green
(1998) Genome Res. 8:186-194. Phrap A Phils Revised Assembly
Program including SWAT and Smith, T.F. and M.S. Waterman (1981)
Adv. Score = 120 or greater; CrossMatch, programs based on
efficient implementation of Appl. Math. 2:482-489; Smith, T.F. and
M.S. Match length = the Smith-Waterman algorithm, useful in
searching Waterman (1981) J. Mol. Biol. 147:195-197; 56 or greater
sequence homology and assembling DNA sequences. and Green, P.,
University of Washington, Seattle, WA. Consed A graphical tool for
viewing and editing Phrap assemblies Gordon, D. et al. (1998)
Genome Res. 8:195-202. SPScan A weight matrix analysis program that
scans protein Nielson, H. et al. (1997) Protein Engineering Score =
5 or greater sequences for the presence of secretory signal
peptides. 10:1-6; Claverie, J.M. and S. Audic (1997) CABIOS 12:
431-439. Motifs A program that searches amino acid sequences for
patterns Bairoch et al. supra; Wisconsin that matched those defined
in Prosite. Package Program Manual, version 9, page M51-59,
Genetics Computer Group, Madison, WI.
[0227]
Sequence CWU 1
1
6 1 195 PRT Homo sapiens 2297891 1 Met Glu Ser Trp Trp Gly Leu Pro
Cys Leu Ala Phe Leu Cys Phe 1 5 10 15 Leu Met His Ala Arg Gly Gln
Arg Asp Phe Asp Leu Ala Asp Ala 20 25 30 Leu Asp Asp Pro Glu Pro
Thr Lys Lys Pro Asn Ser Asp Ile Tyr 35 40 45 Pro Lys Pro Lys Pro
Pro Tyr Tyr Pro Gln Pro Glu Asn Pro Asp 50 55 60 Ser Gly Gly Asn
Ile Tyr Pro Arg Pro Lys Pro Arg Pro Gln Pro 65 70 75 Gln Pro Gly
Asn Ser Gly Asn Ser Gly Gly Tyr Phe Asn Asp Val 80 85 90 Asp Arg
Asp Asp Gly Arg Tyr Pro Pro Arg Pro Arg Pro Arg Pro 95 100 105 Pro
Ala Gly Gly Gly Gly Gly Gly Tyr Ser Ser Tyr Gly Asn Ser 110 115 120
Asp Asn Thr His Gly Arg Gly Gly Tyr Arg Pro Asn Ser Arg Tyr 125 130
135 Gly Asn Thr Tyr Gly Gly Asp His His Ser Thr Tyr Gly Asn Pro 140
145 150 Glu Gly Asn Met Val Ala Lys Ile Val Ser Pro Ile Val Ser Val
155 160 165 Val Val Val Thr Leu Leu Gly Ala Ala Ala Ser Tyr Phe Lys
Leu 170 175 180 Asn Asn Arg Arg Asn Cys Phe Arg Thr His Glu Pro Glu
Asn Val 185 190 195 2 438 PRT Homo sapiens 2705267 2 Met Ala Val
Lys Leu Gly Thr Leu Leu Leu Ala Leu Ala Leu Gly 1 5 10 15 Leu Ala
Gln Pro Ala Ser Ala Arg Arg Lys Leu Leu Val Phe Leu 20 25 30 Leu
Asp Gly Phe Arg Ser Asp Tyr Ile Ser Asp Glu Ala Leu Glu 35 40 45
Ser Leu Pro Gly Phe Lys Glu Ile Val Ser Arg Gly Val Lys Val 50 55
60 Asp Tyr Leu Thr Pro Asp Phe Pro Ser Leu Ser Tyr Pro Asn Tyr 65
70 75 Tyr Thr Leu Met Thr Gly Arg His Cys Glu Val His Gln Met Ile
80 85 90 Gly Asn Tyr Met Trp Asp Pro Thr Thr Asn Lys Ser Phe Asp
Ile 95 100 105 Gly Val Asn Lys Asp Ser Leu Met Pro Leu Trp Trp Asn
Gly Ser 110 115 120 Glu Pro Leu Trp Val Thr Leu Thr Lys Ala Lys Arg
Lys Val Tyr 125 130 135 Met Tyr Tyr Trp Pro Gly Cys Glu Val Glu Ile
Leu Gly Val Arg 140 145 150 Pro Thr Tyr Cys Leu Glu Tyr Lys Asn Val
Pro Thr Asp Ile Asn 155 160 165 Phe Ala Asn Ala Val Ser Asp Ala Leu
Asp Ser Phe Lys Ser Gly 170 175 180 Arg Ala Asp Leu Ala Ala Ile Tyr
His Glu Arg Ile Asp Val Glu 185 190 195 Gly His His Tyr Gly Pro Ala
Ser Pro Gln Arg Lys Asp Ala Leu 200 205 210 Lys Ala Val Asp Thr Val
Leu Lys Tyr Met Thr Lys Trp Ile Gln 215 220 225 Glu Arg Gly Leu Gln
Asp Arg Leu Asn Val Ile Ile Phe Ser Asp 230 235 240 His Gly Met Thr
Asp Ile Phe Trp Met Asp Lys Val Ile Glu Leu 245 250 255 Asn Lys Tyr
Ile Ser Leu Asn Asp Leu Gln Gln Val Lys Asp Arg 260 265 270 Gly Pro
Val Val Ser Leu Trp Pro Ala Pro Gly Lys His Ser Glu 275 280 285 Ile
Tyr Asn Lys Leu Ser Thr Val Glu His Met Thr Val Tyr Glu 290 295 300
Lys Glu Ala Ile Pro Ser Arg Phe Tyr Tyr Lys Lys Gly Lys Phe 305 310
315 Val Ser Pro Leu Thr Leu Val Ala Asp Glu Gly Trp Phe Ile Thr 320
325 330 Glu Asn Arg Glu Met Leu Pro Phe Trp Met Asn Ser Thr Gly Arg
335 340 345 Arg Glu Gly Trp Gln Arg Gly Trp His Gly Tyr Asp Asn Glu
Leu 350 355 360 Met Asp Met Arg Gly Ile Phe Leu Thr Leu Gly Pro Gly
Arg Arg 365 370 375 Gly Asn Asp Gln Met Leu Ser Asp Pro Ile Pro Lys
Glu Val Ser 380 385 390 Leu Arg Gly Pro Thr Gly Ala Arg Arg Gly Cys
Arg Asp Phe Leu 395 400 405 Thr Asp Pro Leu Tyr Glu Pro Ser Arg Ala
Asn Pro Ala Gly Leu 410 415 420 His Glu Thr Ser Phe Ala Gly Phe Leu
Ser Asn Ala Ser Trp Val 425 430 435 Trp Gln Met 3 935 DNA Homo
sapiens 2297891 3 tgcagattgg ttggggcagc ccggggaggc tggctccgac
acacgactga gtgtgcctac 60 actggtccca caggttttca gctgtggagt
ttgggatctg agcttggagc ccatttgttt 120 ctggcagttc cgctcatatt
ttccacttga agacatcgcc tcccttcctt ccaagctggg 180 agaccagaag
tcaacaacag gagggtggag aggccgggtc tcacaatccg cttggctggg 240
gagtccactg aggttcttgc atcctgaagc aaaccatgga gagctggtgg ggacttccct
300 gtcttgcgtt cctgtgtttt ctaatgcacg cccgaggtca aagagacttt
gatttggcag 360 atgcccttga tgaccctgaa cccaccaaga agccaaactc
agatatctac ccaaagccaa 420 aaccacctta ctacccacag cccgagaatc
ccgacagcgg tggaaatatc tacccaaggc 480 caaagccacg ccctcaaccc
cagcctggca attccggcaa cagtggaggt tacttcaatg 540 atgtggaccg
tgatgacgga cgctacccgc ccaggcccag gccacggccg cctgcaggag 600
gtggcggcgg tggctactcc agttatggca actccgacaa cacgcacgga agagggggct
660 atagacccaa ctctcgttat ggaaatactt atggtggaga tcaccattca
acgtatggca 720 atccagaagg caatatggta gcaaaaatcg tgtctcccat
cgtatccgtg gtggtggtga 780 cactgctggg agcagcagcc agttatttca
aactaaacaa taggagaaat tgtttcagga 840 cccatgaacc agaaaatgtc
tgaagatgtt aagatcccct gattactttg ggaaaaacaa 900 ctaaaacaag
aaccgtgttt atcaaaaaaa aaaaa 935 4 1438 DNA Homo sapiens 2705267 4
gagagaatag ctacagattc tccatcctca gtctttgcaa ggcgacagct gtgccagccg
60 ggctctggca ggctcctggc agcatggcag tgaagcttgg gaccctcctg
ctggcccttg 120 ccctgggcct ggcccagcca gcctctgccc gccggaagct
gctggtgttt ctgctggatg 180 gttttcgctc agactacatc agtgatgagg
cgctggagtc attgcctggt ttcaaagaga 240 ttgtgagcag gggagtaaaa
gtggattact tgactccaga cttccctagt ctctcgtatc 300 ccaattatta
taccctaatg actggccgcc attgtgaagt ccatcagatg atcgggaact 360
acatgtggga ccccaccacc aacaagtcct ttgacattgg cgtcaacaaa gacagcctaa
420 tgcctctctg gtggaatgga tcagaacctc tgtgggtcac tctgaccaag
gccaaaagga 480 aggtctacat gtactactgg ccaggctgtg aggttgagat
tctgggtgtc agacccacct 540 actgcctaga atataaaaat gtcccaacgg
atatcaattt tgccaatgca gtcagcgatg 600 ctcttgactc cttcaagagt
ggccgggccg acctggcagc catataccat gagcgcattg 660 acgtggaagg
ccaccactac gggcctgcat ctccgcagag gaaagatgcc ctcaaggctg 720
tagacactgt cctgaagtac atgaccaagt ggatccagga gcggggcctg caggaccgcc
780 tgaacgtcat tattttctcg gatcacggaa tgaccgacat tttctggatg
gacaaagtga 840 ttgagctgaa taagtacatc agcctgaatg acctgcagca
agtgaaggac cgcgggcctg 900 ttgtgagcct ttggccggcc cctgggaaac
actctgagat atataacaaa ctgagcacag 960 tggaacacat gactgtctac
gagaaagaag ccatcccaag caggttctat tacaagaaag 1020 gaaagtttgt
ctctcctttg actttagtgg ctgatgaagg ctggttcata actgagaatc 1080
gagagatgct tccgttttgg atgaacagca ccggcaggcg ggaaggttgg cagcgtggat
1140 ggcacggcta cgacaacgag ctcatggaca tgcggggcat cttcctgact
ctcggacctg 1200 gtaggcgagg aaatgaccag atgctctcag accccattcc
caaggaagtg tctctaaggg 1260 gccctacggg tgccaggaga ggctgcaggg
atttcctcac agaccctctt tatgagccaa 1320 gcagagcaaa cccagccggt
ctccatgaaa catcttttgc tggcttcctt tcaaatgctt 1380 cttgggtttg
gcaaatgtag ccaaatactg tgccgtgtaa attttaaatc ctgcagca 1438 5 180 PRT
Homo sapiens g2499136 5 Met Glu Ser Trp Trp Gly Leu Pro Cys Leu Ala
Phe Leu Cys Phe 1 5 10 15 Leu Met His Ala Arg Gly Gln Arg Asp Phe
Asp Leu Ala Asp Ala 20 25 30 Leu Asp Asp Pro Glu Pro Thr Lys Lys
Pro Asn Ser Asp Ile Tyr 35 40 45 Pro Lys Pro Lys Pro Pro Tyr Tyr
Pro Gln Pro Glu Asn Pro Asp 50 55 60 Ser Gly Gly Asn Ile Tyr Pro
Arg Pro Lys Pro Arg Pro Gln Pro 65 70 75 Gln Pro Gly Asn Ser Gly
Asn Ser Gly Gly Tyr Phe Asn Asp Val 80 85 90 Asp Arg Asp Asp Gly
Arg Tyr Pro Pro Arg Pro Arg Pro Arg Pro 95 100 105 Pro Ala Gly Gly
Gly Gly Gly Gly Tyr Ser Ser Tyr Gly Asn Ser 110 115 120 Asp Asn Thr
His Gly Gly Asp His His Ser Thr Tyr Gly Asn Pro 125 130 135 Glu Gly
Asn Met Val Ala Lys Ile Val Ser Pro Ile Val Ser Val 140 145 150 Val
Val Val Thr Leu Leu Gly Ala Ala Ala Ser Tyr Phe Lys Leu 155 160 165
Asn Asn Arg Arg Asn Cys Phe Arg Thr His Glu Pro Glu Asn Val 170 175
180 6 873 PRT Homo sapiens g189650 6 Met Asp Val Gly Glu Glu Pro
Leu Glu Lys Ala Ala Arg Ala Arg 1 5 10 15 Thr Ala Lys Asp Pro Asn
Thr Tyr Lys Val Leu Ser Leu Val Leu 20 25 30 Ser Val Cys Val Leu
Thr Thr Ile Leu Gly Cys Ile Phe Gly Leu 35 40 45 Lys Pro Ser Cys
Ala Lys Glu Val Lys Ser Cys Lys Gly Arg Cys 50 55 60 Phe Glu Arg
Thr Phe Gly Asn Cys Arg Cys Asp Ala Ala Cys Val 65 70 75 Glu Leu
Gly Asn Cys Cys Leu Asp Tyr Gln Glu Thr Cys Ile Glu 80 85 90 Pro
Glu His Ile Trp Thr Cys Asn Lys Phe Arg Cys Gly Glu Lys 95 100 105
Arg Leu Thr Arg Ser Leu Cys Ala Cys Ser Asp Asp Cys Lys Asp 110 115
120 Lys Gly Asp Cys Cys Ile Asn Tyr Ser Ser Val Cys Gln Gly Glu 125
130 135 Lys Ser Trp Val Glu Glu Pro Cys Glu Ser Ile Asn Glu Pro Gln
140 145 150 Cys Pro Ala Gly Phe Glu Thr Pro Pro Thr Leu Leu Phe Ser
Leu 155 160 165 Asp Gly Phe Arg Ala Glu Tyr Leu His Thr Trp Gly Gly
Leu Leu 170 175 180 Pro Val Ile Ser Lys Leu Lys Lys Cys Gly Thr Tyr
Thr Lys Asn 185 190 195 Met Arg Pro Val Tyr Pro Thr Lys Thr Phe Pro
Asn His Tyr Ser 200 205 210 Ile Val Thr Gly Leu Tyr Pro Glu Ser His
Gly Ile Ile Asp Asn 215 220 225 Lys Met Tyr Asp Pro Lys Met Asn Ala
Ser Phe Ser Leu Lys Ser 230 235 240 Lys Glu Lys Phe Asn Pro Glu Trp
Tyr Lys Gly Glu Pro Ile Trp 245 250 255 Val Thr Ala Lys Tyr Gln Gly
Leu Lys Ser Gly Thr Phe Phe Trp 260 265 270 Pro Gly Ser Asp Val Glu
Ile Asn Gly Ile Phe Pro Asp Ile Tyr 275 280 285 Lys Met Tyr Asn Gly
Ser Val Pro Phe Glu Glu Arg Ile Leu Ala 290 295 300 Val Leu Gln Trp
Leu Gln Leu Pro Lys Asp Glu Arg Pro His Phe 305 310 315 Tyr Thr Leu
Tyr Leu Glu Glu Pro Asp Ser Ser Gly His Ser Tyr 320 325 330 Gly Pro
Val Ser Ser Glu Val Ile Lys Ala Leu Gln Arg Val Asp 335 340 345 Gly
Met Val Gly Met Leu Met Asp Gly Leu Lys Glu Leu Asn Leu 350 355 360
His Arg Cys Leu Asn Leu Ile Leu Ile Ser Asp His Gly Met Glu 365 370
375 Gln Gly Ser Cys Lys Lys Tyr Ile Tyr Leu Asn Lys Tyr Leu Gly 380
385 390 Asp Val Lys Asn Ile Lys Val Ile Tyr Gly Pro Ala Ala Arg Leu
395 400 405 Arg Pro Ser Asp Val Pro Asp Lys Tyr Tyr Ser Phe Asn Tyr
Glu 410 415 420 Gly Ile Ala Arg Asn Leu Ser Cys Arg Glu Pro Asn Gln
His Phe 425 430 435 Lys Pro Tyr Leu Lys His Phe Leu Pro Lys Arg Leu
His Phe Ala 440 445 450 Lys Ser Asp Arg Ile Glu Pro Leu Thr Phe Tyr
Leu Asp Pro Gln 455 460 465 Trp Gln Leu Ala Leu Asn Pro Ser Glu Arg
Lys Tyr Cys Gly Ser 470 475 480 Gly Phe His Gly Ser Asp Asn Val Phe
Ser Asn Met Gln Ala Leu 485 490 495 Phe Val Gly Tyr Gly Pro Gly Phe
Lys His Gly Ile Glu Ala Asp 500 505 510 Thr Phe Glu Asn Ile Glu Val
Tyr Asn Leu Met Cys Asp Leu Leu 515 520 525 Asn Leu Thr Pro Ala Pro
Asn Asn Gly Thr His Gly Ser Leu Asn 530 535 540 His Leu Leu Lys Asn
Pro Val Tyr Thr Pro Lys His Pro Lys Glu 545 550 555 Val His Pro Leu
Val Gln Cys Pro Phe Thr Arg Asn Pro Arg Asp 560 565 570 Asn Leu Gly
Cys Ser Cys Asn Pro Ser Ile Leu Pro Ile Glu Asp 575 580 585 Phe Gln
Thr Gln Phe Asn Leu Thr Val Ala Glu Glu Lys Ile Ile 590 595 600 Lys
His Glu Thr Leu Pro Tyr Gly Arg Pro Arg Val Leu Gln Lys 605 610 615
Glu Asn Thr Ile Cys Leu Leu Ser Gln His Gln Phe Met Ser Gly 620 625
630 Tyr Ser Gln Asp Ile Leu Met Pro Leu Trp Thr Ser Tyr Thr Val 635
640 645 Asp Arg Asn Asp Ser Phe Ser Thr Glu Asp Phe Ser Asn Cys Leu
650 655 660 Tyr Gln Asp Phe Arg Ile Pro Leu Ser Pro Val His Lys Cys
Ser 665 670 675 Phe Tyr Lys Asn Asn Thr Lys Val Ser Tyr Gly Phe Leu
Ser Pro 680 685 690 Pro Gln Leu Asn Lys Asn Ser Ser Gly Ile Tyr Ser
Glu Ala Leu 695 700 705 Leu Thr Thr Asn Ile Val Pro Met Tyr Gln Ser
Phe Gln Val Ile 710 715 720 Trp Arg Tyr Phe His Asp Thr Leu Leu Arg
Lys Tyr Ala Glu Glu 725 730 735 Arg Asn Gly Val Asn Val Val Ser Gly
Pro Val Phe Asp Phe Asp 740 745 750 Tyr Asp Gly Arg Cys Asp Ser Leu
Glu Asn Leu Arg Gln Lys Arg 755 760 765 Arg Val Ile Arg Asn Gln Glu
Ile Leu Ile Pro Thr His Phe Phe 770 775 780 Ile Val Leu Thr Ser Cys
Lys Asp Thr Ser Gln Thr Pro Leu His 785 790 795 Cys Glu Asn Leu Asp
Thr Leu Ala Phe Ile Leu Pro His Arg Thr 800 805 810 Asp Asn Ser Glu
Ser Cys Val His Gly Lys His Asp Ser Ser Trp 815 820 825 Val Glu Glu
Leu Leu Met Leu His Arg Ala Arg Ile Thr Asp Val 830 835 840 Glu His
Ile Thr Gly Leu Ser Phe Tyr Gln Gln Arg Lys Glu Pro 845 850 855 Val
Ser Asp Ile Leu Lys Leu Lys Thr His Leu Pro Thr Phe Ser 860 865 870
Gln Glu Asp
* * * * *