U.S. patent application number 09/757716 was filed with the patent office on 2001-08-09 for human nucleotide pyrophosphohydrolase-2.
Invention is credited to Hutchinson, Nancy, Lawton, Michael, Magna, Holly, Mitchell, Peter, Murry, Lynn E., Schaffer, Paul, Yocum, Sue.
Application Number | 20010012515 09/757716 |
Document ID | / |
Family ID | 25542484 |
Filed Date | 2001-08-09 |
United States Patent
Application |
20010012515 |
Kind Code |
A1 |
Magna, Holly ; et
al. |
August 9, 2001 |
Human nucleotide pyrophosphohydrolase-2
Abstract
The invention provides a human nucleotide pyrophosphohydrolase-2
(NTPPH-2) and polynucleotides which identify and encode NTPPH-2.
The invention also provides expression vectors, host cells,
agonists, antibodies and antagonists. The invention also provides
methods for treating disorders associated with expression of
NTPPH-2.
Inventors: |
Magna, Holly; (Niantic,
CT) ; Schaffer, Paul; (Groton, CT) ; Lawton,
Michael; (Westbrook, CT) ; Yocum, Sue;
(Baltic, CT) ; Mitchell, Peter; (Mystic, CT)
; Hutchinson, Nancy; (Old Lyme, CT) ; Murry, Lynn
E.; (Portola Valley, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Family ID: |
25542484 |
Appl. No.: |
09/757716 |
Filed: |
January 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09757716 |
Jan 9, 2001 |
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09429516 |
Oct 28, 1999 |
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09429516 |
Oct 28, 1999 |
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08996083 |
Dec 22, 1997 |
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6124095 |
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Current U.S.
Class: |
424/146.1 ;
424/94.6; 435/195; 435/6.16; 435/7.4; 530/387.3; 530/388.26;
536/23.2 |
Current CPC
Class: |
A61P 37/02 20180101;
A61P 19/02 20180101; C12N 9/14 20130101; A61K 38/00 20130101; C12N
9/16 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/146.1 ;
424/94.6; 435/7.4; 435/195; 435/6; 530/388.26; 530/387.3;
536/23.2 |
International
Class: |
A61K 039/395; A61K
038/46; G01N 033/573; C12Q 001/68; C12N 009/14; C07H 021/04; C07K
016/00; C07K 016/40; C12P 021/08 |
Claims
What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: a) an amino acid sequence of
SEQ ID NO:1, b) a naturally occurring amino acid sequence having at
least 90% sequence identity to an amino acid sequence of SEQ ID
NO:1, c) a biologically active fragment of an amino acid sequence
of SEQ ID NO:1, and d) an immunogenic fragment of an amino acid
sequence of SEQ ID NO:1.
2. An isolated polypeptide of claim 1, having a sequence of SEQ ID
NO:1.
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, having a sequence of SEQ
ID NO:2.
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 method for 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.
9. A method of claim 8, wherein the polypeptide has the sequence of
SEQ ID NO:1.
10. An isolated antibody which specifically binds to a polypeptide
of claim 1.
11. An isolated polynucleotide comprising a polynucleotide sequence
selected from the group consisting of: a) a polynucleotide sequence
of SEQ ID NO:2, b) a naturally occurring polynucleotide sequence
having at least 90% sequence identity to a polynucleotide of SEQ ID
NO:2, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence complementary to b), and e) an RNA
equivalent of a)-d).
12. An isolated polynucleotide of claim 11 comprising the
polynucleotide sequence of SEQ ID NO:2.
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 11.
14. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 11, 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 for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 11, 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 an
acceptable excipient.
18. A composition of claim 17, wherein the polypeptide has the
sequence of SEQ ID NO:1.
19. A method for treating a disease or condition associated with
decreased expression of functional NTPPH, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method for 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 an acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional NTPPH, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method for 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 an acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional NTPPH, 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, said method comprising the steps of: 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 for 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.
28. A method for assessing toxicity of a test compound, said 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 11 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 11 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.
29. A diagnostic test for a condition or disease associated with
the expression of NTPPH in a biological sample comprising the steps
of: a) combining the biological sample with an antibody of claim
10, 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.
30. The antibody of claim 10, 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.
31. A composition comprising an antibody of claim 10 and an
acceptable excipient.
32. A method of diagnosing a condition or disease associated with
the expression of NTPPH in a subject, comprising administering to
said subject an effective amount of the composition of claim
31.
33. A composition of claim 31, wherein the antibody is labeled.
34. A method of diagnosing a condition or disease associated with
the expression of NTPPH in a subject, comprising administering to
said subject an effective amount of the composition of claim
33.
35. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 10 comprising: a) immunizing
an animal with a polypeptide of SEQ ID NO:1 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 of
SEQ ID NO:1.
36. An antibody produced by a method of claim 35.
37. A composition comprising the antibody of claim 36 and a
suitable carrier.
38. A method of making a monoclonal antibody with the specificity
of the antibody of claim 10 comprising: a) immunizing an animal
with a polypeptide of SEQ ID NO:1 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 of SEQ ID NO:1.
39. A monoclonal antibody produced by a method of claim 38.
40. A composition comprising the antibody of claim 39 and a
suitable carrier.
41. The antibody of claim 10, wherein the antibody is produced by
screening a Fab expression library.
42. The antibody of claim 10, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
43. A method for detecting a polypeptide of SEQ ID NO:1 in a sample
comprising the steps of: a) incubating the antibody of claim 10
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 of
SEQ ID NO:1 in the sample.
44. A method of purifying a polypeptide of SEQ ID NO:1 from a
sample, the method comprising: a) incubating the antibody of claim
10 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 purified polypeptide of SEQ ID NO:1.
Description
[0001] This is a DIVISIONAL of Ser. No. 09/429,516, filed Oct. 28,
1999, which is a divisional of U.S. Ser. No. 08/996,083, filed Dec.
22, 1997, the contents all of which are hereby incorporated by
reference.
[0002] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure, as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
[0003] This invention relates to nucleic acid and amino acid
sequences of a human nucleotide pyrophosphohydrolase-2 and to the
use of these sequences in the diagnosis, prevention, and treatment
of arthropathies, immunological disorders, and cancers.
BACKGROUND
[0004] Calcium pyrophosphate dihydrate (CPPD) deposition disease is
an arthropathy characterized by the accumulation of CPPD crystals
in articular tissues including synovial fluid. CPPD crystals
contribute significantly to the chronic pain and tissue damage of
joint degeneration, and in vitro they induce neutrophil activation
and fibroblast and chondrocyte mitogenesis as well as the
production of matrix metalloproteinases (MMP) and prostaglandins.
CPPD deposition is associated with acute inflammatory episodes
(pseudogout), chronic arthritis, and degenerative joint disease.
Although only about 10% of the CPPD patient population ever
experience acute inflammatory attacks, the majority of patients
with chronic arthritis of the large joints have CPPD deposition.
CPPD crystals play a significant role in arthritic disease
progression. Synovial fluids containing CPPD crystals sampled from
patients with degenerative joint disease have high concentrations
of cartilage fragments and MMPs, e.g., collagenase and stromelysin.
(Swan, A. B. et al. (1994) Ann. Rheum. Dis. 53:467-470; Lohmander,
L. S. et al. (1993) Arthritis. Rheum. 36:181-189.)
[0005] Deposition of CPPD crystals appears to be related to excess
levels of extracellular calcium, pyrophosphate (PPi), or both.
Whereas elevated calcium levels do not appear to be a major
contributing factor to CPPD deposition in joints, elevated PPi
levels have been observed in the synovial fluids from patients with
CPPD deposition. Synovial fluid PPi may be produced by joint
tissues because PPi levels are higher in synovial fluid than in
plasma and in vitro cartilage explants release PPi into the
extracellular medium. (Ryan, L. M. et al. (1996) J. Rheumatol.
23:214-219.)
[0006] Enzymes that hydrolyze nucleotide triphosphates and release
PPi are called nucleotide pyrophosphohydrolases (NTPPH). NTPPH
activity is found in synovial fluid and correlates with the
production of PPi. Elevated ATP levels have been found in joint
fluids of patients with CPPD deposition, and addition of
extracellular ATP to joint tissues and fluids results in the
production of PPi. (Park, W. I. et al. (1996) J. Rheumatol.
23:665-671.) The levels of molecules with NTPPH activity are higher
in extracts from cartilage containing CPPD crystals than from
cartilage lacking crystals. Matrix vesicles released from articular
cartilage in vitro show high NTPPH activity and produce CPPD in the
presence of calcium and ATP. (Derfus, B. A. et al. (1992)
Arthritis. Rheum. 35:231-240.)
[0007] A protein demonstrating NTPPH activity and having a
molecular weight of 61 kD was recently purified from porcine
articular cartilage explant conditioned medium. (Masuda, I. et al.
(1995) J. Clin. Invest. 95:699-704.) The first 26 residues of the
amino-terminus were sequenced and showed no homology to any protein
in public databases. Antipeptide antibodies were generated against
the 61 kD porcine protein, and the antisera identified the original
61 kD protein and an additional 127 kD vesicle-associated protein
in conditioned medium from cultures of both chondrocytes and
cartilage explants. The 61 kD isoform is believed to be a
catalytically active proteolytic fragment of the 127 kD protein.
Both the 61 kD and the 127 kD isoforms were identified in human
synovial fluids, and a 100 kD protein was identified in human
serum. Using the antipeptide antibody on immunoblots of tissue
extracts, NTPPH expression was found only in articular tissues,
e.g., hyaline cartilage, fibrocartilage, tendon, and ligament, in
which CPPD deposition occurs. (Cardenal, A. et al. (1996) Arthritis
Rheum. 39:252-256; Cardenal, A. et al. (1996) Arthritis Rheum.
39:245-251.) Recently, a partial porcine NTPPH cDNA was isolated.
(Masuda, I. et al. (1997) Gene 197:227-282.)
[0008] A full length human nucleotide pyrophosphohydrolase
(NTPPH-1) has been cloned using a cDNA clone isolated from a
cartilage cDNA library. Northern analysis of human, dog, and rabbit
joint tissue RNA samples indicated elevated levels of NTPPH-1
expression in articular cartilage, and lower, but significant,
levels of expression in synovium, meniscus, tendon and ligament.
Expression studies on additional human tissues demonstrated
significant mRNA levels in skeletal muscle, heart muscle, and bone
marrow; and lower, but detectable, levels in trachea, spinal cord,
thyroid, stomach, testis, uterus, small intestine, colon, thymus,
placenta, lymph, and adrenal tissue.
[0009] The discovery of a new human nucleotide
pyrophosphohydrolase, NTPPH-2, and the polynucleotides encoding it
satisfies a need in the art by providing new compositions which are
useful in the diagnosis, prevention and treatment of anthropathies,
immunological disorders, and cancers.
SUMMARY OF THE INVENTION
[0010] The invention features a substantially purified polypeptide,
human nucleotide pyrophosphohydrolase-2 (NTPPH-2), comprising the
amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID
NO:1.
[0011] The invention further provides a substantially purified
variant of NTPPH-2 having at least 90% amino acid identity to the
amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide
encoding the polypeptide comprising the amino acid of SEQ ID NO:1
or a fragment of SEQ ID NO:1. The invention also includes an
isolated and purified polynucleotide variant having at least 90%
polynucleotide identity to the polynucleotide encoding the
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1.
[0012] Additionally, the invention provides a composition
comprising a polynucleotide encoding the polypeptide comprising the
amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides an isolated and purified
polynucleotide which hybridizes under stringent conditions to the
polynucleotide encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an
isolated and purified polynucleotide which is complementary to the
polynucleotide encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0013] The invention also provides an isolated and purified
polynucleotide comprising SEQ ID NO:2 or a fragment of SEQ ID NO:2,
and an isolated and purified polynucleotide variant having at least
90% polynucleotide identity to the polynucleotide comprising SEQ ID
NO:2 or a fragment of SEQ ID NO:2. The invention provides a useful
fragment of SEQ ID NO:2 selected from the group consisting of
nucleotides 55 through 75, 481 through 507, 646 through 669, 2182
through 4149, 1726 through 4149, 757 through 4149, and 113 through
4149 of SEQ ID NO:2. The invention additionally provides an
isolated and purified polynucleotide which has been deposited as
Accession No. 98615 in the American Type Culture Collection. The
invention also provides an isolated and purified polynucleotide
which is complementary to the polynucleotide comprising SEQ ID NO:2
or a fragment of SEQ ID NO:2.
[0014] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1. In another aspect, the expression vector
is contained within a host cell.
[0015] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1, the method comprising the steps of: (a)
culturing the host cell containing an expression vector containing
at least a fragment of a polynucleotide encoding NTPPH-2 under
conditions suitable for the expression of the polypeptide; and (b)
recovering the polypeptide from the host cell culture.
[0016] The invention also provides a pharmaceutical composition
comprising a substantially purified NTPPH-2 having the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 and a suitable
pharmaceutical carrier.
[0017] The invention further includes a purified antibody which
binds to a polypeptide comprising the amino acid sequence of SEQ ID
NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist
and a purified antagonist of the polypeptide.
[0018] The invention also provides a method for detecting a
polynucleotide encoding an NTPPH-2 having an amino acid sequence of
SEQ ID NO:1 in a biological sample containing nucleic acids, the
method comprising the steps of: (a) hybridizing the complement of
the polynucleotide encoding the polypeptide comprising SEQ ID NO:1
or a fragment of SEQ ID NO:1 to at least one of the nucleic acids
of the biological 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 encoding NTPPH-2 in the biological sample. In one
aspect, the nucleic acids of the biological sample are amplified by
the polymerase chain reaction prior to the hybridizing step.
[0019] The invention also provides a method for treating or
preventing an arthropathy, comprising administering to a subject in
need of such treatment an effective amount of an antagonist of
NTPPH-2.
[0020] The invention also provides a method for treating or
preventing an immunological disorder comprising administering to a
subject in need of such treatment an effective amount of an
antagonist of NTPPH-2.
[0021] The invention also provides a method for treating or
preventing a cancer comprising administering to a subject in need
of such treatment an effective amount of an antagonist of
NTPPH-2.
[0022] The invention also provides a method for detecting NTPPH-2
in a biological sample comprising the steps of: a) providing a
biological sample; b) combining the biological sample and an
antibody which binds to a polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under suitable
conditions for complex formation to occur between NTPPH-2 and the
antibody; and c) detecting complex formation between NTPPH-2 and
the antibody, thereby establishing the presence of NTPPH-2 in the
biological sample.
[0023] The invention provides a method for screening a library of
small molecules to identify a molecule which binds NTPPH-2, the
method comprising the steps of: a) providing a library of small
molecules; b) combining the library of small molecules with the
polypeptide of SEQ ID NO:2 or a fragment of SEQ ID NO:2 under
suitable conditions for complex formation; and c) detecting complex
formation wherein the presence of the complex identifies a small
molecule which binds NTPPH-2.
[0024] The invention also provides a method for identifying an
agonist, the method comprising the steps of: a) delivering one of
the small molecules identified by screening a library of small
molecules and gamma labeled ATP into cells transformed with a
vector expressing NTPPH-2; b) growing the cells under suitable
conditions; and c) assaying for an increased amount of PPi thereby
establishing that the small molecule is an agonist.
[0025] The invention further provides a method for identifying an
antagonist, the method comprising the steps of: a) delivering one
of the small molecules identified by screening a library of small
molecules and gamma labeled ATP into cells transformed with a
vector expressing NTPPH-2; b) growing the cells under suitable
conditions; and c) assaying for a decreased amount of PPi thereby
establishing that the small molecule is an antagonist.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1J, and 1K show the
amino acid sequence (1388013; SEQ ID NO:1) and nucleic acid
sequence (SEQ ID NO:2) of human nucleotide pyrophosphohydrolase,
NTPPH-2. The alignment was produced using MacDNAsis PRO.TM.
software (Hitachi Software Engineering Co. Ltd. San Bruno,
Calif.).
[0027] FIGS. 2A, 2B, and 2C show sequence alignments between
NTPPH-2 (SEQ ID NO:1) and NTPPH-1 (422069; SEQ ID NO:3).
[0028] FIGS. 3A and 3B show hydrophobicity plots for NTPPH-2 (SEQ
ID NO:1) and NTPPH-1 (SEQ ID NO:3); the positive X axis reflects
amino acid position, and the negative Y axis, hydrophobicity
(MacDNAsis PRO software).
DESCRIPTION OF THE INVENTION
[0029] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents 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.
[0030] 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,
e.g., 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.
[0031] 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 methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies 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
[0032] "NTPPH-2," as used herein, refers to the amino acid
sequences of substantially purified NTPPH-2 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.
[0033] The term "agonist," as used herein, refers to a molecule
which, when bound to NTPPH-2, increases or prolongs the duration of
the effect of NTPPH-2. Agonists may include proteins, nucleic
acids, carbohydrates, or any other molecules which bind to and
modulate the effect of NTPPH-2.
[0034] An "allele" or an "allelic sequence," as these terms are
used herein, is an alternative form of the gene encoding NTPPH-2.
Alleles 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 alleles 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.
[0035] "Altered" nucleic acid sequences encoding NTPPH-2, as
described herein, include those sequences with deletions,
insertions, or substitutions of different nucleotides, resulting in
a polynucleotide the same as NTPPH-2 or a polypeptide with at least
one functional characteristic of NTPPH-2. Included within this
definition are polymorphisms which may or may not be readily
detectable using a particular oligonucleotide probe of the
polynucleotide encoding NTPPH-2, and improper or unexpected
hybridization to alleles, with a locus other than the normal
chromosomal locus for the polynucleotide sequence encoding NTPPH-2.
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 NTPPH-2. 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 NTPPH-2 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.
[0036] The terms "amino acid" or "amino acid sequence," as used
herein, 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" refers to
fragments of NTPPH-2 which are preferably about 5 to about 15 amino
acids in length and which retain some biological activity or
immunological activity of NTPPH-2. Where "amino acid sequence" is
recited herein 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.
[0037] "Amplification," as used herein, 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. (Dieffenbach, C. W. and G. S.
Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y.)
[0038] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to NTPPH-2, decreases the amount or the
duration of the effect of the biological or immunological activity
of NTPPH-2. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of NTPPH-2.
[0039] As used herein, 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 NTPPH-2 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.
[0040] The term "antigenic determinant," as used herein, 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.
[0041] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to a specific DNA or RNA sequence. The term
"antisense strand" is used in reference to a nucleic acid strand
that is complementary to the "sense" strand. Antisense molecules
may be produced by any method including synthesis 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.
[0042] As used herein, 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
NTPPH-2, or of any oligopeptide thereof, to induce a specific
immune response in appropriate animals or cells and to bind with
specific antibodies.
[0043] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base pairing. For
example, the sequence "A-G-T" binds to the complementary sequence
"T-C-A." 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.
[0044] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence," as these
terms are used herein, 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 NTPPH-2 or fragments
of NTPPH-2 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., SDS), and other components (e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.).
[0045] The phrase "consensus sequence," as used herein, refers to a
nucleic acid sequence which has been resequenced to resolve
uncalled bases, extended using XL-PCR.TM. (Perkin Elmer, Norwalk,
Conn.) in the 5' and/or 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.TM. Fragment Assembly system (GCG, Madison, Wis.).
Some sequences have been both extended and assembled to produce the
consensus sequence.
[0046] As used herein, 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 NTPPH-2, by northern analysis is indicative of the
presence of nucleic acids encoding NTPPH-2 in a sample, and thereby
correlates with expression of the transcript from the
polynucleotide encoding NTPPH-2.
[0047] A "deletion," as the term is used herein, 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.
[0048] The term "derivative," as used herein, refers to the
chemical modification of NTPPH-2, of a polynucleotide sequence
encoding NTPPH-2, or of a polynucleotide sequence complementary to
a polynucleotide sequence encoding NTPPH-2. Chemical modifications
of a polynucleotide sequence can include, e.g., 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.
[0049] The term "homology," as used herein, refers to a degree of
complementarity. There may be partial homology or complete
homology. The word "identity" may substitute for the word
"homology." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially homologous."
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 homologous sequence or hybridization
probe will compete for and inhibit the binding of a completely
homologous 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% homology or
identity). In the absence of non-specific binding, the
substantially homologous sequence or probe will not hybridize to
the second non-complementary target sequence.
[0050] "Human artificial chromosomes" (HACs), as described herein,
are linear microchromosomes which may contain DNA sequences of
about 10 kb to 10 mb in size, and which contain all of the elements
required for stable mitotic chromosome segregation and maintenance.
(Harrington, J. J. et al. (1997) Nat Genet. 15:345-355.)
[0051] The term "humanized antibody," as used herein, 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.
[0052] "Hybridization," as the term is used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0053] As used herein, 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).
[0054] "Inflammation" as used herein is interchangeable with
"immune response", with both terms referring to a condition
associated with trauma, immune disorders, and infectious or genetic
diseases and are characterized by production of cytokines,
chemokines, and other signaling molecules which activate cellular
and systemic defense systems.
[0055] The words "insertion" or "addition," as used herein, 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.
[0056] The term "microarray," as used herein, refers to an array of
distinct polynucleotides or oligonucleotides arrayed on a
substrate, such as paper, nylon or any other type of membrane,
filter, chip, glass slide, or any other suitable solid support.
[0057] The term "modulate," as it appears herein, refers to a
change in the activity of NTPPH-2. For example, modulation may
cause an increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of NTPPH-2.
[0058] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to an oligonucleotide, nucleotide,
polynucleotide, or any fragment thereof, 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 are greater than about 60 nucleotides in length, and most
preferably are at least about 100 nucleotides, at least about 1000
nucleotides, or at least about 10,000 nucleotides in length.
[0059] The term "oligonucleotide," as used herein, 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. As used
herein, the term "oligonucleotide" is substantially equivalent to
the terms "amplimers," "primers," "oligomers," and "probes," as
these terms are commonly defined in the art.
[0060] "Peptide nucleic acid" (PNA), as used herein, refers to an
antisense molecule or anti-gene agent which comprises 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 and RNA and
stop transcript elongation, and may be pegylated to extend their
lifespan in the cell. (Nielsen, P. E. et al. (1993) Anticancer Drug
Des. 8:53-63.
[0061] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding NTPPH-2, or fragments thereof, or NTPPH-2 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 solid support); a tissue; a
tissue print; and the like.
[0062] As used herein, 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 recognized by the binding molecule (i.e.,
the antigenic determinant or epitope). 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] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotide
sequences and the claimed polynucleotide sequences. Suitably
stringent conditions can be defined by, e.g., the concentrations of
salt or formamide in the prehybridization and hybridization
solutions, or by the hybridization temperature, and are 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] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In particular, hybridization could occur under high
stringency conditions at 42.degree. C. in 50% formamide, 5X SSPE,
0.3% SDS, and 200 .mu.g/ml sheared and denatured salmon sperm DNA.
Hybridization could occur under reduced stringency conditions as
described above, but in 35% formamide at a reduced temperature of
35.degree. C. The temperature range corresponding to a particular
level of stringency can be further narrowed by calculating the
purine to pyrimidine ratio of the nucleic acid of interest and
adjusting the temperature accordingly. Variations on the above
ranges and conditions are well known in the art.
[0065] The term "substantially purified," as used herein, 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.
[0066] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0067] "Transformation," as defined herein, 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, and refers to cells which transiently
express the inserted DNA or RNA for limited periods of time.
[0068] A "variant" of NTPPH-2, as used herein, refers to an amino
acid sequence that is altered by one or more amino acids. 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, e.g., DNASTAR
software.
THE INVENTION
[0069] The invention is based on the discovery of a new human
nucleotide pyrophosphohydrolase (NTPPH-2), the polynucleotides
encoding NTPPH-2, and the use of these compositions for the
diagnosis, treatment, or prevention of arthropathies, immunological
disorders, and cancers.
[0070] Nucleic acids encoding the NTPPH-2 of the present invention
were identified in Incyte Clones 1388013, 1423393, and 1423402 from
the osteoarthritic chondrocyte cDNA library (SATPF 1008) using a
computer search for nucleotide sequence homology and the partial
porcine cDNA sequence. (Masuda, supra.) A 4.1 kb sequence was
identified in the chondrocyte library using the cDNA insert from
Incyte Clone 1423393 as a hybridization probe. When a 700 bp
restriction fragment from the 5' most coding region of the 4.1 kb
clone was used to rescreen the osteoarthritic chondrocyte library,
the full length gene encoding NTPPH-2 with appropriate Kozak
initiation and signal sequence was obtained. This sequence does not
match any sequence in the public DNA sequence database. The cDNA
for full length NTPPH-2 of this application has been deposited as
Accession Number 98615 at the American Type Culture Collection,
Bethesda, Md. on Dec. 9, 1997.
[0071] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1. NTPPH-2 is 1156
amino acids in length (FIGS. 1A-1K) and has three potential
N-glycosylation sites at N.sub.276, N.sub.308, N.sub.329, 25
potential phosphorylation sites at T.sub.24, S.sub.135, S.sub.229,
T.sub.245, S.sub.267, S.sub.325, T.sub.331, T.sub.372, S.sub.427,
S.sub.434, S.sub.439, T.sub.517, T.sub.523, Y.sub.599, T.sub.608,
S.sub.630, T.sub.750, T.sub.847, S.sub.883, Y.sub.909, S.sub.977,
S.sub.1017, T.sub.1063, S.sub.1068, and T.sub.1149. As shown in
FIGS. 2A, 2B, and 2C, NTPPH-2 has chemical and structural homology
with NTPPH-1 (SEQ ID NO:3). In particular, NTPPH-2 and NTPPH-1
share 50% sequence identity. Fragments of the nucleic acid sequence
of SEQ ID NO:2 useful for designing oligonucleotides or to be used
directly as hybridization probes to distinguish between these
homologous molecules include the fragments from nucleotides 55
through 75, 481 through 507, 646 through 669, 2182 through 4149,
1726 through 4149, 757 through 4149, and 113 through 4149. As
illustrated by FIGS. 3A and 3B, NTPPH-2 and NTPPH-1 have similar
hydrophobicity plots and both show a hydrophobic signal sequence.
The predicted isoelectric points for NTPPH-2 and NTPPH-1 are 8.07
and 8.21, respectively. Membrane-based northern analysis showed the
highest level of NTPPH-2 mRNA expression in cartilage and lower,
but significant, expression in testes, trachea, and bone marrow.
Electronic northern analysis shows the expression of this sequence
in various libraries at least 57% of which involve immunological
response and many of which are cartilage or joint related and at
least 26% of which involve immortalized or cancerous cells and
tissues. Of particular note is the expression of NTPPH-2 in
rheumatoid and osteoarthritic synovial, chondrocyte, and tibial
libraries.
[0072] The invention also encompasses NTPPH-2 variants. A preferred
NTPPH-2 variant is one having at least about 80%, more preferably
at least about 90%, and most preferably at least about 95% amino
acid sequence identity to the NTPPH-2 amino acid sequence and which
retains at least one biological, immunological or other functional
characteristic or activity of NTPPH-2.
[0073] The invention also encompasses polynucleotides which encode
NTPPH-2. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:2,
which encodes an NTPPH-2.
[0074] The invention also encompasses a variant of a polynucleotide
sequence encoding NTPPH-2. 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 NTPPH-2. A particular aspect of the invention encompasses
a variant of SEQ ID NO:2 which has at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to SEQ ID NO:2. In addition,
the amino acid sequences encoded by these variants may have at
least one functional or structural characteristic of NTPPH-2.
[0075] 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 NTPPH-2, some bearing minimal
homology 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 NTPPH-2, and all such variations
are to be considered as being specifically disclosed.
[0076] Although nucleotide sequences which encode NTPPH-2 and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring NTPPH-2 under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding NTPPH-2 or its derivatives
possessing a substantially different codon usage. 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 NTPPH-2 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.
[0077] The invention also encompasses production of DNA sequences
which encode NTPPH-2 and NTPPH-2 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 that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding NTPPH-2 or any fragment
thereof.
[0078] 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:2, or a fragment of SEQ ID NO:2, under various conditions of
stringency as taught in Wahl, G. M. and S. L. Berger (1987; Methods
Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.
152:507-511.)
[0079] Methods for DNA sequencing are well known and generally
available 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.RTM. (US
Biochemical Corp., Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of polymerases and proofreading exonucleases such as
those found in the ELONGASE Amplification System marketed by
GIBCO/BRL (Gaithersburg, Md.). Preferably, the process is automated
with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,
Nev.), Peltier Thermal Cycler (PTC200; M J Research, Watertown,
Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin
Elmer).
[0080] The nucleic acid sequences encoding NTPPH-2 may be extended
utilizing a partial nucleotide sequence and employing various
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 primers to
retrieve unknown sequence adjacent to a known locus. (Sarkar, G.
(1993) PCR Methods Applic. 2:318-322.) In particular, genomic DNA
is first amplified in the presence of a primer to a linker sequence
and a primer specific to the known region. The amplified sequences
are then subjected to a second round of PCR with the same linker
primer and another specific primer internal to the first one.
Products of each round of PCR are transcribed with an appropriate
RNA polymerase and sequenced using reverse transcriptase.
[0081] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region. (Triglia, T. et
al. (1988) Nucleic Acids Res. 16:8186.) The primers may be designed
using commercially available software such as OLIGO 4.06 Primer
Analysis software (National Biosciences Inc., Plymouth, Minn.) 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
72.degree. C. The method uses several restriction enzymes to
generate a suitable fragment in the known region of a gene. The
fragment is then circularized by intramolecular ligation and used
as a PCR template.
[0082] Another method which may be used is capture PCR, which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA. (Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method,
multiple restriction enzyme digestions and ligations may be used to
place an engineered double-stranded sequence into an unknown
fragment of the DNA molecule before performing PCR. Another method
which may be used to retrieve unknown sequences is that of Parker,
J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060). Additionally,
one may use PCR, nested primers, and PromoterFinder.TM. libraries
to walk genomic DNA (Clontech, Palo Alto, Calif.). This process
avoids the need to screen libraries and is useful in finding
intron/exon junctions.
[0083] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable in that they will
include more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially 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 fluorescent dyes (one for each
nucleotide) which are laser activated, 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.TM. and Sequence Navigator.TM., 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 the
sequencing of small pieces of DNA which might be present in limited
amounts in a particular sample.
[0085] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode NTPPH-2 may be used in
recombinant DNA molecules to direct expression of NTPPH-2, 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 these sequences
may be used to clone and express NTPPH-2.
[0086] As will be understood by those of skill in the art, it may
be advantageous to produce NTPPH-2-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0087] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter NTPPH-2 encoding sequences for a variety of reasons
including, but not limited to, alterations which modify 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, site-directed mutagenesis
may be used to insert new restriction sites, alter glycosylation
patterns, change codon preference, produce splice variants,
introduce mutations, and so forth.
[0088] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding NTPPH-2 may be
ligated to a heterologous sequence to encode a fusion protein. For
example, to screen peptide libraries for inhibitors of NTPPH-2
activity, it may be useful to encode a chimeric NTPPH-2 protein
that can be recognized by a commercially available antibody. A
fusion protein may also be engineered to contain a cleavage site
located between the NTPPH-2 encoding sequence and the heterologous
protein sequence, so that NTPPH-2 may be cleaved and purified away
from the heterologous moiety.
[0089] In another embodiment, sequences encoding NTPPH-2 may be
synthesized, in whole or in part, using chemical methods well known
in the art (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, the protein itself may be produced
using chemical methods to synthesize the amino acid sequence of
NTPPH-2, or a fragment thereof. For example, peptide synthesis can
be performed using various solid-phase techniques (Roberge, J. Y.
et al. (1995) Science 269:202-204) and automated synthesis may be
achieved using the ABI 431A Peptide Synthesizer (Perkin Elmer).
[0090] The newly synthesized peptide may be substantially purified
by preparative high performance liquid chromatography. (Creighton,
T. (1983) Proteins, Structures and Molecular Principles, W H
Freeman and Co., New York, N.Y.) The composition of the synthetic
peptides may be confirmed by amino acid analysis or by sequencing.
(the Edman degradation procedure described in Creighton, supra.)
Additionally, the amino acid sequence of NTPPH-2, 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.
[0091] In order to express a biologically active NTPPH-2, the
nucleotide sequences encoding NTPPH-2 or derivatives thereof may be
inserted into appropriate expression vector, i.e., a vector which
contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0092] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding NTPPH-2 and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook, J. et al.
(1989; Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y.) and Ausubel, F. M. et al. (1989; Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y.)
[0093] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding NTPPH-2. 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 virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus 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.
[0094] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector (i.e., enhancers, promoters,
and 5' and 3' untranslated regions) which interact with host
cellular proteins to carry out transcription and translation. Such
elements may vary in their strength and specificity. Depending on
the vector system and host utilized, any number of suitable
transcription and translation elements, including constitutive and
inducible promoters, may be used. For example, when cloning in
bacterial systems, inducible promoters such as the hybrid lacZ
promoter of the Bluescript.RTM. phagemid (Stratagene, La Jolla,
Calif.) or pSport1.TM. plasmid (GIBCO/BRL), and the like, may be
used. The baculovirus polyhedrin promoter may be used in insect
cells. Promoters or enhancers derived from the genomes of plant
cells (e.g., heat shock, RUBISCO, and storage protein genes) or
from plant viruses (e.g., viral promoters or leader sequences) may
be cloned into the vector. In mammalian cell systems, promoters
from mammalian genes or from mammalian viruses are preferable. If
it is necessary to generate a cell line that contains multiple
copies of the sequence encoding NTPPH-2, vectors based on SV40 or
EBV may be used with an appropriate selectable marker.
[0095] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for NTPPH-2. For example,
when large quantities of NTPPH-2 are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, multifunctional E. coli cloning
and expression vectors such as Bluescript.RTM. (Stratagene), in
which the sequence encoding NTPPH-2 may be ligated into the vector
in frame with sequences for the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase so that a hybrid
protein is produced, pIN vectors (Van Heeke, G. and S. M. Schuster
(1989) J. Biol. Chem. 264:5503-5509), and the like. pGEX vectors
(Promega, Madison, Wis.) may also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione.
Proteins made in such systems may be designed to include heparin,
thrombin, or factor XA protease cleavage sites so that the cloned
polypeptide of interest can be released from the GST moiety at
will.
[0096] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used. For reviews, see
Ausubel (supra) and Grant et al. (1987; Methods Enzymol.
153:516-544.)
[0097] In cases where plant expression vectors are used, the
expression of sequences encoding NTPPH-2 may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be 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. (Coruzzi,
G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105.) These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (Hobbs, S. or Murry, L. E. in McGraw
Hill Yearbook of Science and Technology (1992) McGraw Hill, New
York, N.Y., pp. 191-196).
[0098] An insect system may also be used to express NTPPH-2. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding NTPPH-2 may be cloned into a non-essential
region of the virus, such as the polyhedrin gene, and placed under
control of the polyhedrin promoter. Successful insertion of NTPPH-2
will render the polyhedrin gene inactive and produce recombinant
virus lacking coat protein. The recombinant viruses may then be
used to infect, e.g., S. frugiperda cells or Trichoplusia larvae in
which NTPPH-2 may be expressed. (Engelhard, E. K. et al. (1994)
Proc. Nat. Acad. Sci. 91:3224-3227.)
[0099] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding NTPPH-2 may be ligated into
an adenovirus transcription/translation complex consisting of the
late promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing NTPPH-2 in
infected host cells. (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.
[0100] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. 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.
[0101] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding NTPPH-2. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding NTPPH-2 and its initiation codon and
upstream 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 the ATG initiation codon
should be provided. Furthermore, the initiation codon should be in
the correct reading frame to ensure translation of the entire
insert. 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 cell system used, such as those
described in the literature. (Scharf, D. et al. (1994) Results
Probl. Cell Differ. 20:125-162.)
[0102] 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
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and WI38), are available from the American
Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0103] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing NTPPH-2 can be transformed 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
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type.
[0104] 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 genes (Wigler, M. et al.
(1977) Cell 11:223-32) and adenine phosphoribosyltransferase genes
(Lowy, I. et al. (1980) Cell 22:817-23), which can be employed in
tk.sup.- or apf.sup.- cells, respectively. Also, antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for
selection. For example, dhfr confers resistance to methotrexate
(Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14); and als
or pat confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively. (Murry, supra.) Additional
selectable genes have been described, e.g., trpB, which allows
cells to utilize indole in place of tryptophan, or hisD, which
allows cells to utilize histinol in place of histidine. (Hartman,
S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51.)
Recently, the use of visible markers has gained popularity with
such markers as anthocyanins, .beta. glucuronidase and its
substrate GUS, and luciferase and its substrate luciferin. These
markers can be used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributable to a specific vector system. (Rhodes, C. A. et al.
(1995) Methods Mol. Biol. 55:121-131.)
[0105] 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 NTPPH-2 is inserted within a marker gene
sequence, transformed cells containing sequences encoding NTPPH-2
can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding NTPPH-2 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.
[0106] Alternatively, host cells which contain the nucleic acid
sequence encoding NTPPH-2 and express NTPPH-2 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 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.
[0107] The presence of polynucleotide sequences encoding NTPPH-2
can be detected by DNA-DNA or DNA-RNA hybridization or
amplification using probes or fragments or fragments of
polynucleotides encoding NTPPH-2. Nucleic acid amplification based
assays involve the use of oligonucleotides or oligomers based on
the sequences encoding NTPPH-2 to detect transformants containing
DNA or RNA encoding NTPPH-2.
[0108] A variety of protocols for detecting and measuring the
expression of NTPPH-2, using either polyclonal or monoclonal
antibodies specific for the protein, 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
NTPPH-2 is preferred, but a competitive binding assay may be
employed. These and other assays are well described in the art,
e.g., in Hampton, R. et al. (1990; Serological Methods, a
Laboratory Manual, APS Press, St Paul, Minn.) and in Maddox, D. E.
et al. (1983; J. Exp. Med. 158:1211-1216.)
[0109] 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 NTPPH-2 include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding NTPPH-2, 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 Pharmacia & Upjohn (Kalamazoo, Mich.), Promega
(Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio).
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.
[0110] Host cells transformed with nucleotide sequences encoding
NTPPH-2 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 contained
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 NTPPH-2 may be
designed to contain signal sequences which direct secretion of
NTPPH-2 through a prokaryotic or eukaryotic cell membrane. Other
constructions may be used to join sequences encoding NTPPH-2 to
nucleotide sequences encoding a polypeptide domain which will
facilitate purification of soluble proteins. Such purification
facilitating domains include, but are not limited to, metal
chelating peptides such as histidine-tryptophan modules that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAG extension/affinity purification system (Immunex Corp.,
Seattle, Wash.). The inclusion of cleavable linker sequences, such
as those specific for Factor XA or enterokinase (Invitrogen, San
Diego, Calif.), between the purification domain and the NTPPH-2
encoding sequence may be used to facilitate purification. One such
expression vector provides for expression of a fusion protein
containing NTPPH-2 and a nucleic acid encoding 6 histidine residues
preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues facilitate purification by immobilized metal ion
affinity chromatography (IMIAC; described in Porath, J. et al.
(1992) Prot. Exp. Purif. 3: 263-281), while the enterokinase
cleavage site provides a means for purifying NTPPH-2 from the
fusion protein. A discussion of vectors which contain fusion
proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.
12:441-453.)
[0111] Fragments of NTPPH-2 may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154.)
Protein synthesis may be performed by manual techniques or by
automation. Automated synthesis may be achieved, e.g., using the
Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Various
fragments of NTPPH-2 may be synthesized separately and then
combined to produce the full length molecule.
THERAPEUTICS
[0112] Chemical and structural homology exists between NTPPH-2 and
human NTPPH-1 (SEQ ID NO:3). Transcripts hybridizing to the NTPPH-2
cDNA were detected in cartilage, testes, trachea, and bone marrow
tissues. Electronic northern analysis showed expression of NTPPH-2
in tissues with an immunological association (57%; including
synovial and cartilage tissues), and in cancerous tissues (26%).
Therefore, NTPPH-2 appears to play a role in arthropathies,
immunological disorders, and cancers.
[0113] Therefore, in one embodiment, an antagonist of NTPPH-2 may
be administered to a subject to prevent or treat an arthropathy.
Arthropathies include, but are not limited to, Behcet's syndrome,
Charcot osteoarthropathy, CPPD disease, diabetic neuropathic
arthropathy, degenerative joint disease, fibromyalgias,
hemachromatosis, hemophilic arthropathy, Jaccoud's type
arthropathy, lupus erythematosus, mixed connective tissue disease,
Muckle-Wells syndrome, osteoarthritis, progressive systemic
sclerosis, pseudogout, psoriasis, Reiter's syndrome, rheumatoid
arthritis, Sjogren's syndrome, and spondyloarthropathies. In one
aspect, an antibody which specifically binds NTPPH-2 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 NTPPH-2.
[0114] In another embodiment, a vector expressing the complement of
the polynucleotide encoding NTPPH-2 may be administered to a
subject to treat or prevent an arthropathy including, but not
limited to, those described above.
[0115] In another embodiment, an antagonist of NTPPH-2 may be
administered to a subject to prevent or treat an immunological
disorder. Immunological disorders include, but are not limited to,
AIDS, Addison's disease, adult respiratory distress syndrome,
allergies, anemia, asthma, atherosclerosis, bronchitis,
cholecystitis, Crohn's disease, ulcerative colitis, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythema
nodosum, atrophic gastritis, glomerulonephritis, gout, Graves'
disease, hypereosinophilia, irritable bowel syndrome, multiple
sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoporosis, pancreatitis, polymyositis,
scleroderma, autoimmune thyroiditis and ulcerative colitis;
complications of cancer, hemodialysis, and extracorporeal
circulation; viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections; and trauma. Such disorders may be
characterized by the production of cytokines and the multiplication
of leukocytes, macrophages, and other cells which may cause tissue
damage or the inappropriate proliferation of tissues in response to
inflammatory mediators or the generation of granulomatous tissues.
In one aspect, an antibody which specifically binds NTPPH-2 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 NTPPH-2.
[0116] In another embodiment, a vector expressing the complement of
the polynucleotide encoding NTPPH-2 may be administered to a
subject to treat or prevent an immunological disorder including,
but not limited to, those described above.
[0117] In another embodiment, an antagonist of NTPPH-2 may be
administered to a subject to prevent or treat a cancer. Cancers
include, but are not limited to, adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cartilage, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus. In one aspect, an
antibody which specifically binds NTPPH-2 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 NTPPH-2.
[0118] In another embodiment, a vector expressing the complement of
the polynucleotide encoding NTPPH-2 may be administered to a
subject to treat or prevent a cancer including, but not limited to,
those described above.
[0119] 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.
[0120] An antagonist of NTPPH-2 may be produced using methods which
are generally known in the art. In particular, purified NTPPH-2 may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
NTPPH-2. Antibodies to NTPPH-2 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.
[0121] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with NTPPH-2 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 Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
[0122] For the production of antibodies, binding proteins, or
peptides which bind specifically to NTPPH-2, libraries of single
chain antibodies, Fab fragments, other antibody fragments,
non-antibody protein domains, or peptides may be screened. The
libraries could be generated using phage display, other recombinant
DNA methods, or peptide synthesis (Vaughan, T. J. et al.(1996)
Nature Biotechnology 14:309-314). The libraries would be screened
using methods which are well known in the art to identify sequences
which demonstrate specific binding to NTPPH-2.
[0123] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to NTPPH-2 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 NTPPH-2 amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0124] Monoclonal antibodies to NTPPH-2 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. (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.)
[0125] 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.
(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
NTPPH-2-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (Burton D. R. (1991) Proc. Natl. Acad.
Sci. 88:11120-11123.)
[0126] 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. (Orlandi, R. et al. (1989)
Proc. Natl. Acad. Sci. 86: 3833-3837, and Winter, G. et al. (1991)
Nature 349:293-299.)
[0127] Antibody fragments which contain specific binding sites for
NTPPH-2 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. (Huse, W. D. et al. (1989) Science
254:1275-1281.)
[0128] Various immunoassays may be used 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 NTPPH-2 and its specific antibody. A
two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two non-interfering NTPPH-2 epitopes is
preferred, but a competitive binding assay may also be employed.
(Maddox, supra.)
[0129] In another embodiment of the invention, the polynucleotides
encoding NTPPH-2, or any fragment or complement thereof, may be
used for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding NTPPH-2 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 NTPPH-2. Thus, complementary molecules
or fragments may be used to modulate NTPPH-2 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 NTPPH-2.
[0130] 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 which
will express nucleic acid sequence complementary to the
polynucleotides of the gene encoding NTPPH-2. These techniques are
described, e.g., in Sambrook (supra) and in Ausubel (supra).
[0131] Genes encoding NTPPH-2 can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
at polynucleotide or fragment thereof encoding NTPPH-2. 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.
[0132] 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 NTPPH-2. 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. (Gee, J. E. et al. (1994) in
Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,
Futura Publishing Co., Mt. Kisco, N.Y.) A complementary sequence or
antisense molecule may also be designed to block translation of
mRNA by preventing the transcript from binding to ribosomes.
[0133] 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 specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding NTPPH-2.
[0134] 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.
[0135] 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 chemically 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 NTPPH-2. 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.
[0136] 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.
[0137] 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, such as
those described in Goldman, C. K. et al. (1997; Nature
Biotechnology 15:462-466.)
[0138] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, e.g.,
mammals such as dogs, cats, cows, horses, rabbits, monkeys, and
most preferably, humans.
[0139] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of NTPPH-2, antibodies to NTPPH-2, and mimetics, agonists,
antagonists, or inhibitors of NTPPH-2. The compositions may be
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.
[0140] 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.
[0141] 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 Co.,
Easton, Pa.)
[0142] 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.
[0143] 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,
hydroxypropylmethyl-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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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 NTPPH-2, such
labeling would include amount, frequency, and method of
administration.
[0151] 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.
[0152] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays of neoplastic
cells, e.g., or in animal models, usually mice, 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.
[0153] A therapeutically effective dose refers to that amount of
active ingredient, e.g. NTPPH-2 or fragments thereof, antibodies of
NTPPH-2, and agonists, antagonists or inhibitors of NTPPH-2, 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 ED50 (the dose therapeutically effective in 50% of the
population) or LD50 (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 LD50/ED50 ratio.
Pharmaceutical compositions which exhibit large therapeutic indices
are preferred. The data obtained from cell culture assays and
animal studies is used in formulating a range of dosage for human
use. The dosage contained in such compositions is preferably within
a range of circulating concentrations that include the ED50 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.
[0154] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring 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, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on the half-life and clearance rate of the particular
formulation.
[0155] Normal dosage amounts may vary from 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
[0156] In another embodiment, antibodies which specifically bind
NTPPH-2 may be used for the diagnosis of disorders characterized by
expression of NTPPH-2, or in assays to monitor patients being
treated with NTPPH-2 or agonists, antagonists, and inhibitors of
NTPPH-2. Antibodies useful for diagnostic purposes may be prepared
in the same manner as those described above for therapeutics.
Diagnostic assays for NTPPH-2 include methods which utilize the
antibody and a label to detect NTPPH-2 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 joining with a reporter molecule. A wide variety of
reporter molecules, several of which are described above, are known
in the art and may be used.
[0157] A variety of protocols for measuring NTPPH-2, including
ELISAs, RIAs, and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of NTPPH-2 expression.
Normal or standard values for NTPPH-2 expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to NTPPH-2 under
conditions suitable for complex formation. The method for detecting
NTPPH-2 in a biological sample would comprise the steps of: a)
providing a biological sample; b) combining the biological sample
and an anti-NTPPH-2 antibody under conditions which are suitable
for complex formation to occur between NTPPH-2 and the antibody;
and c) detecting complex formation between NTPPH-2 and the
antibody, thereby establishing the presence of NTPPH-2 in the
biological sample. The amount of complex formation then may be
quantified by various methods, preferably by photometric means.
Quantities of NTPPH-2 expressed in subject, control, and disease
samples from biopsied tissues are compared with the standard
values. Deviation between standard and subject values establishes
the parameters for diagnosing disease.
[0158] In another embodiment of the invention, the polynucleotides
encoding NTPPH-2 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 NTPPH-2 may
be correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
NTPPH-2, and to monitor regulation of NTPPH-2 levels during
therapeutic intervention.
[0159] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding NTPPH-2 or closely related molecules may be
used to identify nucleic acid sequences which encode NTPPH-2. 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., the 3' coding region), and the stringency of the
hybridization or amplification (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding NTPPH-2, alleles, or related
sequences.
[0160] Probes may also be used for the detection of related
sequences, and should preferably contain at least 50% of the
nucleotides from any of the NTPPH-2 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:2 or from genomic
sequences including promoter and enhancer elements and introns of
the naturally occurring NTPPH-2.
[0161] Means for producing specific hybridization probes for DNAs
encoding NTPPH-2 include the cloning of polynucleotide sequences
encoding NTPPH-2 or NTPPH-2 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, e.g., 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.
[0162] Polynucleotide sequences encoding NTPPH-2 may be used for
the diagnosis of conditions or disorders which are associated with
expression of NTPPH-2. Examples of such conditions or disorders
include, but are not limited to, arthropathies, e.g., Behcet's
syndrome, Charcot osteoarthropathy, CPPD disease, diabetic
neuropathic arthropathy, degenerative joint disease, fibromyalgias,
hemachromatosis, hemophilic arthropathy, Jaccoud's type
arthropathy, lupus erythematosus, mixed connective tissue disease,
Muckle-Wells syndrome, osteoarthritis, progressive systemic
sclerosis, pseudogout, psoriasis, Reiter's syndrome, rheumatoid
arthritis, Sjogren's syndrome, and spondyloarthropathies;
immunological disorders, e.g., AIDS, Addison's disease, adult
respiratory distress syndrome, allergies, anemia, asthma,
atherosclerosis, bronchitis, cholecystitis, Crohn's disease,
ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, erythema nodosum, atrophic gastritis,
glomerulonephritis, gout, Graves' disease, hypereosinophilia,
irritable bowel syndrome, multiple sclerosis, myasthenia gravis,
myocardial or pericardial inflammation, osteoporosis, pancreatitis,
polymyositis, scleroderma, and autoimmune thyroiditis;
complications of cancer, hemodialysis, and extracorporeal
circulation; viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections; and trauma; and cancers, e.g.,
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and particularly cancers of the adrenal gland,
bladder, bone, bone marrow, brain, breast, cartilage, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver,
lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus.
[0163] The polynucleotide sequences encoding NTPPH-2 may be used in
Southern or northern analysis, dot blot, or other membrane-based
technologies; in PCR technologies; in dipstick, pin, and ELISA
assays; and in microarrays utilizing fluids or tissues from patient
biopsies to detect altered NTPPH-2 expression. Such qualitative or
quantitative methods are well known in the art.
[0164] In a particular aspect, the nucleotide sequences encoding
NTPPH-2 may be useful in assays that detect the presence of
associated disorders, particularly those mentioned above. The
nucleotide sequences encoding NTPPH-2 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
from that of a comparable control sample, the nucleotide sequences
have hybridized with nucleotide sequences in the sample, and the
presence of altered levels of nucleotide sequences encoding NTPPH-2
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 in monitoring the treatment of an individual
patient.
[0165] In order to provide a basis for the diagnosis of a disorder
associated with expression of NTPPH-2, 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 NTPPH-2, 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 from normal
samples 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.
[0166] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to evaluate whether 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.
[0167] With respect to cancer, the presence of a relatively high
amount of transcript 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.
[0168] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding NTPPH-2 may involve the use of PCR.
Such oligomers may be chemically synthesized, generated
enzymatically, or produced in vitro. Oligomers will preferably
consist of two nucleotide sequences, one with sense orientation (5'
to 3') and another with antisense orientation (3' to 5'), employed
under optimized conditions for identification of a specific gene or
condition. The same two oligomers, nested sets of oligomers, or
even a degenerate pool of oligomers may be employed under less
stringent conditions for detection and/or quantitation of closely
related DNA or RNA sequences.
[0169] Methods which may also be used to quantitate the expression
of NTPPH-2 include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (Melby, P. C. et al. (1993) J.
Immunol. Methods 159:235-244, and 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.
[0170] 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 (to produce a transcript image) and to identify
genetic variants, mutations, and polymorphisms. This information
may be used in determining gene function, in understanding the
genetic basis of a disorder, in diagnosing a disorder, and in
developing and monitoring the activities of therapeutic agents.
[0171] In one embodiment, the microarray is prepared and used
according to methods known in the art, such as those described in
published PCT application WO95/11995 (Chee et al.), Lockhart, D. J.
et al. (1996; Nat. Biotech. 14:1675-1680), and Schena, M. et al.
(1996; Proc. Natl. Acad. Sci. 93:10614-10619.)
[0172] The microarray is preferably composed of a large number of
unique single-stranded nucleic acid sequences, usually either
synthetic antisense oligonucleotides or fragments of cDNAs, fixed
to a solid support. The oligonucleotides are preferably about 6 to
60 nucleotides in length, more preferably about 15 to 30
nucleotides in length, and most preferably about 20 to 25
nucleotides in length. For a certain type of microarray, it may be
preferable to use oligonucleotides which are about 7 to 10
nucleotides in length. The microarray may contain oligonucleotides
which cover the known 5' or 3' sequence, or may contain sequential
oligonucleotides which cover the full length sequence or unique
oligonucleotides selected from particular areas along the length of
the sequence. Polynucleotides used in the microarray may be
oligonucleotides specific to a gene or genes of interest in which
at least a fragment of the sequence is known or oligonucleotides
specific to one or more unidentified cDNAs common to a particular
cell or tissue type or to a normal, developmental, or disease
state. In certain situations, it may be appropriate to use pairs of
oligonucleotides on a microarray. The pairs will be identical,
except for one nucleotide preferably located in the center of the
sequence. The second oligonucleotide in the pair (mismatched by
one) serves as a control. The number of oligonucleotide pairs may
range from about 2 to 1,000,000.
[0173] In order to produce oligonucleotides to a known sequence for
a microarray, the gene of interest is examined using a computer
algorithm which starts at the 5' end, or, more preferably, at the
3' end of the nucleotide sequence. The algorithm identifies
oligomers of defined length that are unique to the gene, have a GC
content within a range suitable for hybridization, and lack
predicted secondary structure that may interfere with
hybridization. In one aspect, the oligomers are synthesized at
designated areas on a substrate using a light-directed chemical
process. The substrate may be paper, nylon, any other type of
membrane, filter, chip, glass slide, or any other suitable solid
support.
[0174] In one aspect, the oligonucleotides may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, such as that described in
published PCT application WO95/251116 (Baldeschweiler et al.). In
another aspect, a grid array analogous to a dot or slot blot
(HYBRIDOT.RTM. apparatus, GIBCO/BRL) may be used to arrange and
link cDNA fragments or oligonucleotides to the surface of a
substrate using a vacuum system or thermal, UV, mechanical or
chemical bonding procedures. In yet another aspect, an array may be
produced by hand or by using available devices, materials, and
machines (including Brinkmann.RTM. multichannel pipettors or
robotic instruments), and may contain 8, 24, 96, 384, 1536, or 6144
oligonucleotides, or any other multiple from 2 to 1,000,000 which
lends itself to the efficient use of commercially available
instrumentation.
[0175] In order to conduct sample analysis using the microarrays,
polynucleotides are extracted from a biological sample. The
biological samples may be obtained from any bodily fluid (blood,
urine, saliva, phlegm, gastric juices, etc.), cultured cells,
biopsies, or other tissue preparations. To produce probes, the
polynucleotides extracted from the sample are used to produce
nucleic acid sequences which are complementary to the nucleic acids
on the microarray. If the microarray consists of cDNAs, antisense
RNAs (aRNA) are appropriate probes. Therefore, in one aspect, mRNA
is used to produce cDNA which, in turn and in the presence of
fluorescent nucleotides, is used to produce fragment or
oligonucleotide aRNA probes. These fluorescently labeled probes are
incubated with the microarray so that the probe sequences hybridize
to the cDNA oligonucleotides of the microarray. In another aspect,
nucleic acid sequences used as probes can include polynucleotides,
fragments, and complementary or antisense sequences produced using
restriction enzymes, PCR technologies, and Oligolabeling or
TransProbe kits (Pharmacia & Upjohn) well known in the area of
hybridization technology.
[0176] Incubation conditions are adjusted so that hybridization
occurs with precise complementarity or with various degrees of
complementarity between hybridizing sequences. After removal of
nonhybridized probes, a scanner is used to determine the levels and
patterns of fluorescence. The scanned images are examined to
determine the degree of complementarity and the relative abundance
of each oligonucleotide sequence on the microarray. A detection
system may be used to measure the absence, presence, and amount of
hybridization for all of the distinct sequences simultaneously.
This data may be used for large scale correlation studies or for
functional analysis of the sequences, mutations, variants, or
polymorphisms among samples. (Heller, R. A. et al. (1997) Proc.
Natl. Acad. Sci. 94:2150-2155.)
[0177] In another embodiment of the invention, nucleic acid
sequences encoding NTPPH-2 may be used to generate hybridization
probes useful for 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, such as human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA
libraries, such as those reviewed in Price, C. M. (1993; Blood Rev.
7:127-134) and Trask, B. J. (1991; Trends Genet. 7:149-154.)
[0178] Fluorescent in situ hybridization (FISH, as described in
Verma et al. (1988) Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York, N.Y.) may be correlated with
other physical chromosome mapping techniques and genetic map data.
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 NTPPH-2 on a
physical chromosomal map and a specific disorder, or predisposition
to a specific disorder, may help define the region of DNA
associated with that disorder. The nucleotide sequences of the
subject invention may be used to detect differences in gene
sequences between normal, carrier, and affected individuals.
[0179] 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, or parts
thereof, 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., AT to 11q22-23 (Gatti, R. A. et al. (1988)
Nature 336:577-580), any sequences mapping to that area may
represent associated or regulatory genes for further investigation.
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.
[0180] In another embodiment of the invention, NTPPH-2, its
catalytic or immunogenic fragments, or oligopeptides thereof can be
used for screening libraries of compounds such as agonists or
antagonists 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 NTPPH-2
and the agent being tested may be measured.
[0181] The method provided for screening a library of small
molecules to identify a molecule which binds NTPPH-2 comprises: a)
providing a library of small molecules; b) combining the library of
small molecules with the polypeptide of SEQ ID NO:2 or a fragment
of SEQ ID NO:2 under conditions which are suitable for complex
formation; and c) detecting complex formation wherein the presence
of the complex identifies a small molecule which binds NTPPH-2. The
method for identifying one of these small molecules which binds
NTPPH-2 as an agonist comprises delivering a small molecule which
binds NTPPH-2 and gamma labeled ATP into cells transformed with a
vector expressing NTPPH-2, growing the cells under suitable
conditions, and assaying for PPi. An increased amount of PPi
establishes that the small molecule is an agonist which increases
NTPPH-2 activity. The method for identifying an antagonist
comprises delivering a small molecule which binds NTPPH-2 and gamma
labeled ATP into cells transformed with a vector expressing
NTPPH-2, growing the cells under suitable conditions, and assaying
the media for PPi. A reduced amount of PPi establishes that the
small molecule is an antagonist which reduces NTPPH-2 activity.
[0182] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO84/03564. In this method, large numbers
of different small test compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. The test
compounds are reacted with NTPPH-2, or fragments thereof, and
washed. Bound NTPPH-2 is then detected by methods well known in the
art. Purified NTPPH-2 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.
[0183] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding NTPPH-2 specifically compete with a test compound for
binding NTPPH-2. In this manner, antibodies can be used to detect
the presence of any peptide which shares one or more antigenic
determinants with NTPPH-2.
[0184] In additional embodiments, the nucleotide sequences which
encode NTPPH-2 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.
[0185] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0186] I SATPF 1008 cDNA Library Construction
[0187] The SATPF 1008 cDNA library was made from cartilage tissue
obtained from four donors with end stage osteoarthritis. The
osteoarthritic patients had received steroids such as prednisone
and a variety of non-steroidal anti-inflammatory drugs. There was
no stimulation with IL-1 (Pfizer Inc., Groton, Conn.).
[0188] The osteoarthritic cartilage tissue was harvested at joint
replacement surgery and placed in Dulbecco's Modified Eagle Medium
(D-MEM; Gibco/BRL) supplemented with antibiotics (penicillin,
streptomycin, and gentamicin) and transported to Pfizer
laboratories. The cartilage was removed aseptically from the
underlying bone, rinsed in D-MEM and diced into small pieces
(.about.4 mm.sup.2), and placed in 100 mm petri dishes containing
20 ml of Neuman and Tytell's serum free medium (Gibco/BRL). Using
the protocol of Mitchell et al. (1996) J. Clin. Invest. 97:761-768,
the cartilage from each patient was digested with 4 mg/ml pronase
(Sigma, St Louis, Mo.) for 1.5 hours, then subsequently digested
with 3 mg/ml bacterial collagenase (Sigma) for 1.5 hours. The
digested cartilage was filtered through a cell strainer to remove
undigested material, and the cells were pelleted by centrifugation.
The cell pellet was washed once with phosphate buffered saline
(PBS) and then dissolved in 5 ml of buffer consisting of 5 M
guanidine isothiocyanate, 10 mM EDTA, 50 mM Tris (pH 7.5) and 8%
.beta. mercaptoethanol. A five-fold volume of 4M LiCl was added to
the buffer, and the mixture was stored in the refrigerator
overnight. After centrifugation, the precipitate was washed once
with 3 M LiCl and recentrifuged. The second precipitate was
dissolved in a solution consisting of 0.1% sodium dodecyl sulfate,
1 mM EDTA and 10 mM Tris (pH 7.5). The suspension was frozen at
-70.degree. C. and then vortexed during thawing (Cathala et al
(1983; DNA 2:329-335).
[0189] Total RNA was extracted twice with phenol chloroform, once
with chloroform, and then, precipitated with ethanol. Equal amounts
of RNA from the four donors were combined and ethanol precipitated,
resulting in 112 .mu.g pooled RNA. Following centrifugation, the
RNA pellet was redissolved in DEPC-treated, distilled, deionized
water (DEPC-ddH.sub.2O) and run over a CsCl gradient. The RNA was
extracted with acid phenol (1X at pH 4.0, catalog #972Z, Ambion,
Austin, Tex.), precipitated with ethanol and resuspended in
DEPC-ddH.sub.2O. The RNA was treated with RNase-free Dnase
(Epicentre Technologies, Madison, Wis.) for 15 minutes, extracted
with chloroform, precipitated and washed with ethanol, and
dissolved in DEPC-ddH.sub.2O.
[0190] The mRNA was handled according to the recommended protocols
in the SuperScript Plasmid System for cDNA synthesis and plasmid
cloning (Cat. #18248-013, Gibco/BRL). The cDNAs were fractionated
on a Sepharose CL4B column (Cat. #275105-01; Pharmacia), and those
cDNAs exceeding 400 bp were ligated into pINCY 1 (Incyte). The
plasmid pINCY 1 was subsequently transformed into DH5.varies..TM.
competent cells (Cat. #18258-012; Gibco/BRL).
[0191] II Isolation and Sequencing of cDNA Clones
[0192] Plasmid DNA was released from the cells and purified using
the REAL Prep 96 plasmid kit (Catalog #26173, QIAGEN, Inc.,
Chatsworth, Calif.). The recommended protocol was employed except
for the following changes 1) the bacteria were cultured in 1 ml of
sterile Terrific Broth (Catalog #22711, GIBCO/BRL) with
carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after
inoculation, the cultures were incubated for 19 hours and at the
end of incubation, the cells were lysed with 0.3 ml of lysis
buffer; and 3) following isopropanol precipitation, the plasmid DNA
pellet was resuspended in 0.1 ml of distilled water. After the last
step in the protocol, samples were transferred to a 96-well block
for storage at 4.degree. C.
[0193] The cDNAs were sequenced by the method of Sanger, et al.
(1975, J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200
(Hamilton, Reno, Nev.) in combination with Peltier Thermal Cyclers
(PTC200 from M J Research, Watertown, Mass.) and Applied Biosystems
377 DNA Sequencing Systems; and the reading frame was
determined.
[0194] III Homology Searching of cDNA Clones and Their Deduced
Proteins
[0195] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, BLOCKS, and Pima II databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of homology using BLAST (Basic Local Alignment
Search Tool). (Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and
Altschul et al. (1990) J. Mol. Biol. 215:403-410.)
[0196] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms such as the one described in Smith, T. et
al. (1992; Protein Engineering 5:35-51), could have been used when
dealing with primary sequence patterns and secondary structure gap
penalties. The sequences disclosed in this application have lengths
of at least 49 nucleotides and have no more than 12% uncalled bases
(where N is recorded rather than A, C, G, or T).
[0197] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-10 for peptides.
[0198] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam), and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp), for homology.
[0199] IV Cloning of Full Length NTPPH-2
[0200] A 2.3 kb cDNA sequence which encodes a partial porcine NTPPH
(L. M. Ryan, Medical College of Wisconsin, Milwaukee, Wis.) was
searched against NCBI public EST and in-house databases. Homologous
sequences were found in the osteoarthritic cartilage libraries.
[0201] A 4.1 kb sequence was identified in using the chondrocyte
library and the cDNA insert from Incyte Clone 1423393 as a
hybridization probe. When a 700 bp restriction fragment from the 5'
most coding region of the 4.1 kb clone was used to rescreen the
osteoarthritic cartilage library, the full length cDNA encoding
NTPPH-2 was identified. The full length cDNA was sequenced and
found to contain an appropriate Kozak initiation and signal
sequence. The cloned polynucleotide sequence was deposited with The
American Type Culture Collection as Accession No. 98615 on Dec. 9,
1997.
[0202] V Northern Analysis
[0203] Human multiple tissue northern blots were obtained from
Clontech (Palo Alto, Calif.). Human cartilage was treated with +/-
human recombinant IL-1.varies.. For RNA preparation, chondrocytes
were isolated via sequential digestion for 1.5 hours with pronase
(4 mg/ml) followed by bacterial collagenase (3 mg/ml) for 3-5
hours. The chondrocytes were pelleted, and the cells lysed in
guanidinium isothiocyanate. RNA was precipitated with LiCl as
described in Mitchell et al. (supra.) Northern blot analysis was
carried out using DNA probes labeled with a random primer kit
(Pharmacia Biotech Inc. Piscataway, N.J.). The blots were
hybridized overnight at 42.degree. C. essentially as described in
Sambrook et al. (supra); then washed three times in
3.times.SSC/0.1% SDS at room temperature and once in 0.3.times.
SSC/0.1% SDS at 60.degree. C. for 15 minutes.
[0204] Membrane-based northern analyses of human, dog and rabbit
joint tissue RNA samples demonstrated the highest levels of NTPPH-2
mRNA expression in cartilage and lower, but significant, expression
levels in testes, trachea, and bone marrow.
[0205] Computer techniques analogous to northern analysis were also
performed using BLAST. (Altschul (1993) supra, Altschul (1990)
supra.) The basis of the search is the product score which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0206] 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 1-2% error; and at 70, the match will be exact.
Homologous molecules are usually identified by selecting those
which show product scores between 15 and 40, although lower scores
may identify related molecules.
[0207] Electronic northern analysis shows the expression of this
sequence in various libraries at least 57% of which involve
immunological response and at least 26% of which are immortalized
or cancerous. Of particular note is the expression of NTPPH-2 in
rheumatoid and osteoarthritic synovial, chondrocyte, and tibial
libraries.
[0208] VI Extension of NTPPH-2 Encoding Polynucleotides
[0209] The nucleic acid sequence encoding NTPPH-2 was used to
design oligonucleotide primers for obtaining 5' regulatory
sequences using an appropriate genomic library. One primer was
synthesized to initiate extension in the antisense direction, and
the other was synthesized to extend sequence in the sense
direction. Primers were used to facilitate the extension of the
known sequence "outward" generating amplicons containing new,
unknown nucleotide sequence for the region of interest. The initial
primers were designed from the cDNA using OLIGO 4.06 (National
Biosciences), or another appropriate program, to be about 22 to
about 30 nucleotides in length, to have a GC content of 50% or
more, and to anneal to the target sequence at temperatures of about
68.degree. about 72.degree. C. Any stretch of nucleotides which
would result in hairpin structures and primer-primer dimerizations
was avoided.
[0210] Selected human cDNA libraries (GIBCO/BRL) were used to
extend the sequence If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0211] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. Beginning with 40 pmol of each
primer and the recommended concentrations of all other components
of the kit, PCR was performed using the Peltier Thermal Cycler
(PTC200; M. J. Research, Watertown, Mass.) and the following
parameters:
[0212] Step 1 94.degree. C. for 1 min (initial denaturation)
[0213] Step 2 65.degree. C. for 1 min
[0214] Step3 68.degree. C. for 6 min
[0215] Step 4 94.degree. C. for 15 sec
[0216] Step 5 65.degree. C. for 1 min
[0217] Step 6 68.degree. C. for 7 min
[0218] Step 7 Repeat step 4-6 for 15 additional cycles
[0219] Step 8 94.degree. C. for 15 sec
[0220] Step 9 65.degree. C. for 1 min
[0221] Step 10 68.degree. C. for 7:15 min
[0222] Step 11 Repeat step 8-10 for 12 cycles
[0223] Step 12 72.degree. C. for 8 min
[0224] Step 13 4.degree. C. (and holding)
[0225] A 5-10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a low concentration (about 0.6-0.8%) agarose
mini-gel to determine which reactions were successful in extending
the sequence. Bands thought to contain the largest products were
excised from the gel, purified using QIAQuick.TM. (QIAGEN), and
trimmed of overhangs using Klenow enzyme to facilitate religation
and cloning.
[0226] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2-3 hours or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium (Sambrook et al., supra.) After
incubation for one hour at 37.degree. C., the E. coli mixture was
plated on Luria Bertani (LB)-agar (Sambrook et al., supra)
containing 2.times. Carb. The following day, several colonies were
randomly picked from each plate and cultured in 150 .mu.l of liquid
LB/2.times. Carb medium placed in an individual well of an
appropriate, commercially-available, sterile 96-well microtiter
plate. The following day, 5 .mu.l of each overnight culture was
transferred into a non-sterile 96-well plate and after dilution
1:10 with water, 5 .mu.l of each sample was transferred into a PCR
array.
[0227] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions:
[0228] Step 1 94.degree. C. for 60 sec
[0229] Step 2 94.degree. C. for 20 sec
[0230] Step 3 55.degree. C. for 30 sec
[0231] Step 4 72.degree. C. for 90 sec
[0232] Step 5 Repeat steps 2-4 for an additional 29 cycles
[0233] Step 6 72.degree. C. for 180 sec
[0234] Step 7 4.degree. C. (and holding)
[0235] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0236] VII Labeling and Use of Individual Hybridization Probes
[0237] Hybridization probes derived from SEQ ID NO:2 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 (National
Biosciences), labeled by combining 50 pmol of each oligomer and 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham) and
T4 polynucleotide kinase (DuPont NEN.RTM., Boston, Mass.). The
labeled oligonucleotides are substantially purified with Sephadex
G-25 superfine resin column (Pharmacia & Upjohn). A 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, Xba 1, or Pvu II; DuPont NEN.RTM.).
[0238] The DNA from each digest is fractionated on a 0.7 percent
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.times. saline sodium
citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR.TM. film
(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimager
cassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,
hybridization patterns are compared visually.
[0239] VIII Microarrays
[0240] To produce oligonucleotides for a microarray, the nucleotide
sequence described herein is examined using a computer algorithm
which starts at the 3' end of the nucleotide sequence. The
algorithm identifies oligomers of defined length that are unique to
the gene, have a GC content within a range suitable for
hybridization, and lack predicted secondary structure that would
interfere with hybridization. The algorithm identifies 20
sequence-specific oligonucleotides of 20 nucleotides in length
(20-mers). A matched set of oligonucleotides is created in which
one nucleotide in the center of each sequence is altered. This
process is repeated for each gene in the microarray, and double
sets of twenty 20 mers are synthesized and arranged on the surface
of the silicon chip using a light-directed chemical process (Chee,
M. et al., PCT/WO095/11995).
[0241] In the alternative, a chemical coupling procedure and an ink
jet device are used to synthesize oligomers on the surface of a
substrate (Baldeschweiler, J. D. et al., PCT/WO95/25116). In
another alternative, a "gridded" array analogous to a dot (or slot)
blot is used to arrange and link cDNA fragments or oligonucleotides
to the surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array may be produced
by hand or using available materials and machines and contain grids
of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.
After hybridization, the microarray is washed to remove
nonhybridized probes, and a scanner is used to determine the levels
and patterns of fluorescence. The scanned images are examined to
determine degree of complementarity and the relative abundance of
each oligonucleotide sequence on the microarray.
[0242] IX Complementary Polynucleotides
[0243] Sequence complementary to the NTPPH-2-encoding sequence, or
any part thereof, is used to decrease or inhibit expression of
naturally occurring NTPPH-2. Although use of oligonucleotides
comprising from about 15 to about 30 base-pairs is described,
essentially the same procedure is used with smaller or larger
sequence fragments. Appropriate oligonucleotides are designed using
Oligo 4.06 software and the sequence encoding NTPPH-2. 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
NTPPH-2-encoding transcript.
[0244] X Expression of NTPPH-2
[0245] The cDNA encoding NTPPH-2 is used to express both
full-length and truncated forms of recombinant NTPPH-2. Expression
of NTPPH-2 is accomplished by subcloning the cDNAs into appropriate
vectors and transforming the vectors into host cells. In this case,
the cloning vector was used to express NTPPH-2 in the baculovirus
Fast-BAC system (GIBCO/BRL). Upstream of the cloning site, this
vector contains a promoter for polyhedron coat protein. Infection
of an insect cell line such as SF9 with the recombinant baculovirus
results in the expression of NTPPH-2. Signal residues direct the
secretion of NTPPH-2 into the culture media which can be used
directly in the following assay for activity.
[0246] XI Demonstration of NTPPH-2 Activity
[0247] Human nucleotide pyrophosphohydrolase-2 activity is analyzed
using thymidine monophosphate paranitrophenyl ester or [.sup.32P]
gamma labeled ATP as substrate. Media are chromatographed and peak
fractions are analyzed kinetically as described in Cardenal, A. et
al. (1996; Arthritis Rheum. 39:252-256.)
[0248] XII Production of NTPPH-2 Specific Antibodies
[0249] The amino acid sequence deduced from the cDNA encoding
NTPPH-2 is analyzed using DNASTAR software (DNASTAR, Inc.) to
determine regions of high immunogenicity and a corresponding
oligopeptide is synthesized and used to raise anti-NTPPH-2
antibodies. The selection of appropriate peptide sequences and the
techniques for antibody production can occur by means known to
those of skill in the art. Selection of appropriate epitopes, such
as those near the C-terminus or in hydrophilic regions, is
described by Ausubel et al. (supra), and others.
[0250] Typically, the oligopeptides are 15 residues in length,
synthesized using an Applied Biosystems Peptide Synthesizer Model
431A using fmoc-chemistry, and coupled to keyhole limpet hemocyanin
(KLH, Sigma, St. Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester. (MBS; Ausubel et
al., supra.) Rabbits are immunized with the oligopeptide-KLH
complex in complete Freund's adjuvant. The resulting antisera are
tested for antipeptide activity, e.g., by binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radio iodinated, goat anti-rabbit
IgG.
[0251] XIII Purification of Naturally Occurring NTPPH-2 Using
Specific Antibodies
[0252] Naturally occurring or recombinant NTPPH-2 is substantially
purified by immunoaffinity chromatography using antibodies specific
for NTPPH-2. An immunoaffinity column is constructed by covalently
coupling NTPPH-2 antibody to an activated chromatographic resin,
such as CNBr-activated Sepharose (Pharmacia & Upjohn). After
the coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0253] Media containing NTPPH-2 is passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of NTPPH-2 (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/NTPPH-2 binding (eg, a buffer of
pH 2-3 or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and NTPPH-2 is collected.
[0254] XIV Identification of Molecules which Interact with
NTPPH-2
[0255] NTPPH-2 or biologically active fragments thereof are labeled
with .sup.125I Bolton-Hunter reagent. (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
NTPPH-2, washed and any wells with labeled NTPPH-2 complex are
assayed. Data obtained using different concentrations of NTPPH-2
are used to calculate values for the number, affinity, and
association of NTPPH-2 with the candidate molecules.
[0256] Various modifications and variations of the described method
and system of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
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.
Sequence CWU 1
1
3 1 1156 PRT Homo sapiens misc_feature Incyte Clone No. 1388013 1
Met Ala Ser Leu Leu Pro Leu Leu Cys Leu Cys Val Val Ala Ala His 1 5
10 15 Leu Ala Gly Ala Arg Asp Ala Thr Pro Thr Glu Glu Pro Met Ala
Thr 20 25 30 Ala Leu Gly Leu Glu Arg Arg Ser Val Tyr Thr Gly Gln
Pro Ser Pro 35 40 45 Ala Leu Glu Asp Trp Glu Glu Ala Ser Glu Trp
Thr Ser Trp Phe Asn 50 55 60 Val Asp His Pro Gly Gly Asp Gly Asp
Phe Glu Ser Leu Ala Ala Ile 65 70 75 80 Arg Phe Tyr Tyr Gly Pro Ala
Arg Val Cys Pro Arg Pro Leu Ala Leu 85 90 95 Glu Ala Arg Thr Thr
Asp Trp Ala Leu Pro Ser Ala Val Gly Glu Arg 100 105 110 Val His Leu
Asn Pro Thr Arg Gly Phe Trp Cys Leu Asn Arg Glu Gln 115 120 125 Pro
Arg Gly Arg Arg Cys Ser Asn Tyr His Val Arg Phe Arg Cys Pro 130 135
140 Leu Glu Ala Ser Trp Gly Ala Trp Gly Pro Trp Gly Pro Cys Ser Gly
145 150 155 160 Ser Cys Gly Pro Gly Arg Arg Leu Arg Arg Arg His Cys
Pro Ser Pro 165 170 175 Ala Gly Asp Ala Cys Pro Gly Arg Pro Leu Glu
Ala Gln Lys Cys Val 180 185 190 Arg Pro Arg Cys Pro Gly Cys Ser Leu
Asp Thr Cys Glu Cys Pro Asp 195 200 205 His Ile Leu Leu Gly Ser Val
Val Thr Pro Ser Gly Gln Pro Leu Leu 210 215 220 Gly Ala Arg Val Ser
Leu Arg Asp Gln Pro Gly Thr Val Ala Thr Ser 225 230 235 240 Asp Ala
His Gly Thr Phe Arg Val Pro Gly Val Cys Ala Asp Ser Arg 245 250 255
Ala Asn Ile Arg Ala Gln Met Asp Gly Phe Ser Ala Gly Glu Ala Gln 260
265 270 Ala Gln Ala Asn Gly Ser Ile Ser Val Val Thr Ile Ile Leu Asp
Lys 275 280 285 Leu Glu Lys Pro Tyr Leu Val Lys His Pro Glu Ser Arg
Val Arg Glu 290 295 300 Ala Gly Gln Asn Val Thr Phe Cys Cys Lys Ala
Ser Gly Thr Pro Met 305 310 315 320 Pro Lys Lys Tyr Ser Trp Phe His
Asn Gly Thr Leu Leu Asp Arg Arg 325 330 335 Ala His Gly Tyr Gly Ala
His Leu Glu Leu Arg Gly Leu Arg Pro Asp 340 345 350 Gln Ala Gly Ile
Tyr His Cys Lys Ala Trp Asn Glu Ala Gly Ala Val 355 360 365 Arg Ser
Gly Thr Ala Arg Leu Thr Val Leu Ala Pro Gly Gln Pro Ala 370 375 380
Cys Asp Pro Arg Pro Arg Glu Tyr Leu Ile Lys Leu Pro Glu Asp Cys 385
390 395 400 Gly Gln Pro Gly Ser Gly Pro Ala Tyr Leu Asp Val Gly Leu
Cys Pro 405 410 415 Asp Thr Arg Cys Pro Ser Leu Ala Gly Ser Ser Pro
Arg Cys Gly Asp 420 425 430 Ala Ser Ser Arg Cys Cys Ser Val Arg Arg
Leu Glu Arg Arg Glu Ile 435 440 445 His Cys Pro Gly Tyr Val Leu Pro
Val Lys Val Val Ala Glu Cys Gly 450 455 460 Cys Gln Lys Cys Leu Pro
Pro Arg Gly Leu Val Arg Gly Arg Val Val 465 470 475 480 Ala Ala Asp
Ser Gly Glu Pro Leu Arg Phe Ala Arg Ile Leu Leu Gly 485 490 495 Gln
Glu Pro Ile Gly Phe Thr Ala Tyr Gln Gly Asp Phe Thr Ile Glu 500 505
510 Val Pro Pro Ser Thr Gln Arg Leu Val Val Thr Phe Val Asp Pro Ser
515 520 525 Gly Glu Phe Met Asp Ala Val Arg Val Leu Pro Phe Asp Pro
Arg Gly 530 535 540 Ala Gly Val Tyr His Glu Val Lys Ala Met Arg Lys
Lys Ala Pro Val 545 550 555 560 Ile Leu His Thr Ser Gln Ser Asn Thr
Ile Pro Leu Gly Glu Leu Glu 565 570 575 Asp Glu Ala Pro Leu Gly Glu
Leu Val Leu Pro Ser Gly Ala Phe Arg 580 585 590 Arg Ala Asp Gly Lys
Pro Tyr Ser Gly Pro Val Glu Ala Arg Val Thr 595 600 605 Phe Val Asp
Pro Arg Asp Leu Thr Ser Ala Ala Ser Ala Pro Ser Asp 610 615 620 Leu
Arg Phe Val Asp Ser Asp Gly Glu Leu Ala Pro Leu Arg Thr Tyr 625 630
635 640 Gly Met Phe Ser Val Asp Leu Arg Ala Pro Gly Ser Ala Glu Gln
Leu 645 650 655 Gln Val Gly Pro Val Ala Val Arg Val Ala Ala Ser Gln
Ile His Met 660 665 670 Pro Gly His Val Glu Ala Leu Lys Leu Trp Ser
Leu Asn Pro Glu Thr 675 680 685 Gly Leu Trp Glu Glu Glu Ser Gly Phe
Arg Arg Glu Gly Ser Ser Gly 690 695 700 Pro Arg Val Arg Arg Glu Glu
Arg Val Phe Leu Val Gly Asn Val Glu 705 710 715 720 Ile Arg Glu Arg
Arg Leu Phe Asn Leu Asp Val Pro Glu Arg Arg Arg 725 730 735 Cys Phe
Val Lys Val Arg Ala Tyr Ala Asn Asp Lys Phe Thr Pro Ser 740 745 750
Glu Gln Val Glu Gly Val Val Val Thr Leu Val Asn Leu Glu Pro Ala 755
760 765 Pro Gly Phe Ser Ala Asn Pro Arg Ala Trp Gly Arg Phe Asp Ser
Ala 770 775 780 Val Thr Gly Pro Asn Gly Ala Cys Leu Pro Ala Phe Cys
Asp Ala Asp 785 790 795 800 Arg Pro Asp Ala Tyr Thr Ala Leu Val Thr
Ala Thr Leu Gly Gly Glu 805 810 815 Glu Leu Glu Pro Ala Pro Ser Leu
Pro Arg Pro Leu Pro Ala Thr Val 820 825 830 Gly Val Thr Gln Pro Tyr
Leu Asp Arg Leu Gly Tyr Arg Arg Thr Asp 835 840 845 His Asp Asp Pro
Ala Phe Lys Arg Asn Gly Phe Arg Ile Asn Leu Ala 850 855 860 Lys Pro
Arg Pro Gly Asp Pro Ala Glu Ala Asn Gly Pro Val Tyr Pro 865 870 875
880 Trp Arg Ser Leu Arg Glu Cys Gln Gly Ala Pro Val Thr Ala Ser His
885 890 895 Phe Arg Phe Ala Arg Val Glu Ala Asp Lys Tyr Glu Tyr Asn
Val Val 900 905 910 Pro Phe Arg Glu Gly Thr Pro Ala Ser Trp Thr Gly
Asp Leu Leu Ala 915 920 925 Trp Trp Pro Asn Pro Gln Glu Phe Arg Ala
Cys Phe Leu Lys Val Lys 930 935 940 Ile Gln Gly Pro Gln Glu Tyr Met
Val Arg Ser His Asn Ala Gly Gly 945 950 955 960 Ser His Pro Arg Thr
Arg Gly Gln Leu Tyr Gly Leu Arg Asp Ala Arg 965 970 975 Ser Val Arg
Asp Pro Glu Arg Pro Gly Thr Ser Ala Ala Cys Val Glu 980 985 990 Phe
Lys Cys Ser Gly Met Leu Phe Asp Gln Arg Gln Val Asp Arg Thr 995
1000 1005 Leu Val Thr Ile Met Pro Gln Gly Ser Cys Arg Arg Val Ala
Val Asn 1010 1015 1020 Gly Leu Leu Arg Asp Tyr Leu Thr Arg His Pro
Pro Pro Val Pro Ala 1025 1030 1035 1040 Glu Asp Pro Ala Ala Phe Ser
Met Leu Ala Pro Leu Asp Pro Leu Gly 1045 1050 1055 His Asn Tyr Gly
Val Tyr Thr Val Thr Asp Gln Ser Pro Arg Leu Ala 1060 1065 1070 Lys
Glu Ile Ala Ile Gly Arg Cys Phe Asp Gly Ser Ser Asp Gly Phe 1075
1080 1085 Ser Arg Glu Met Lys Ala Asp Ala Gly Thr Ala Val Thr Phe
Gln Cys 1090 1095 1100 Arg Glu Pro Pro Ala Gly Arg Pro Ser Leu Phe
Gln Arg Leu Leu Glu 1105 1110 1115 1120 Ser Pro Ala Thr Ala Leu Gly
Asp Ile Arg Arg Glu Met Ser Glu Ala 1125 1130 1135 Ala Gln Ala Gln
Ala Arg Ala Ser Gly Pro Leu Arg Thr Arg Arg Gly 1140 1145 1150 Arg
Val Arg Gln 1155 2 4183 DNA Homo sapiens misc_feature Incyte Clone
No. 1388013 2 gcccgagcac gccgcggagc ccggacctcc ctcggacgct
ctgccccggc catggcgtcg 60 ctgctgccac tgctctgtct ctgtgtcgtc
gctgcgcacc tggcgggggc ccgagacgcc 120 acccccaccg aggagccaat
ggcgactgca ctgggcctgg aaagacggtc cgtgtacacc 180 ggccagccct
caccagccct ggaggactgg gaagaggcca gcgagtggac gtcctggttc 240
aacgtggacc accccggagg cgacggcgac ttcgagagcc tggctgccat ccgcttctac
300 tacgggccag cgcgcgtgtg cccgcgaccg ctggcgctgg aggcgcgcac
cacggactgg 360 gccctgccgt ccgccgtcgg cgagcgcgtg cacttgaacc
ccacgcgcgg cttctggtgc 420 ctcaaccgcg agcaaccgcg tggccgccgc
tgctccaact accacgtgcg cttccgctgc 480 ccactagaag cctcgtgggg
cgcgtggggc ccgtggggtc cctgctcggg gagctgtggg 540 ccaggccgtc
gcttgcgccg ccgccactgc ccaagccccg ctggggatgc gtgtcccggg 600
cgtcctctgg aggcgcagaa gtgcgtgcgg cctcggtgtc cagggtgcag ccttgacacc
660 tgtgaatgcc cggaccacat cctcctgggc tcggtggtca ccccatctgg
gcaaccactg 720 ctaggagcca gggtctccct gcgagaccag cctggcactg
tggccaccag cgatgctcac 780 ggaaccttcc gggtgcctgg tgtctgtgct
gacagccgcg ccaacatcag ggcccagatg 840 gatggcttct ctgcagggga
ggcccaggcc caggccaacg gatccatctc tgtggtcacc 900 atcatccttg
ataagttgga gaagccgtac ctggtgaaac accctgagtc ccgagtgcga 960
gaggctggcc agaatgtgac tttctgctgc aaagcctccg ggacccccat gcccaagaaa
1020 tactcctggt tccacaatgg gaccctgctg gacaggcgag ctcatgggta
cggggcccac 1080 ctggagctsc ggggactgcg cccagaccag gctggcatct
accactgcaa ggcatggaat 1140 gaggcgggtg ccgtgcgctc gggcactgcc
cggctcactg tacttgcccc aggccagcca 1200 gcctgcgacc cccggccccg
agagtacctg atcaagctcc ctgaggactg tggtcagcca 1260 ggtagtggcc
ctgcctacct ggatgtgggc ctctgtcccg acacccgctg ccccagcctg 1320
gcaggctcca gcccccgctg cggggacgcc agctcccgct gctgctctgt gcgccgtctg
1380 gagagaaggg agattcactg ccctggctac gtcctcccag tgaaggtggt
ggcagagtgt 1440 ggctgccaga agtgtctgcc ccctcggggg ctggtccggg
gccgtgttgt ggctgctgac 1500 tccggggagc cgctacgctt cgccaggatt
ctgctgggcc aggagcccat cggcttcacc 1560 gcctaccagg gcgactttac
cattgaggtg ccgccctcca cccagcggct ggtggtgact 1620 tttgtggacc
ccagcggtga gttcatggac gctgtccggg tcttgccttt tgatcctcga 1680
ggtgccggcg tgtaccacga ggtcaaggcc atgcggaaga aagccccggt cattttacat
1740 accagccaga gcaacacgat ccccctgggc gagctggaag atgaggcgcc
cctgggcgag 1800 ctggtcctgc cttctggcgc tttccgcaga gccgacggca
aaccctactc ggggcctgtg 1860 gaggcccggg tgacgttcgt ggacccccga
gacctcacct cggcggcgtc tgcccccagt 1920 gacctgcgct tcgtggacag
cgacggcgag ctggctccac tgcgcaccta cggcatgttc 1980 tccgtggacc
tccgtgcgcc cggctccgcg gagcagctgc aggtggggcc ggtggccgtg 2040
cgggtggccg ccagccagat ccacatgcca ggccacgtgg aggccctcaa gctgtggtcg
2100 ctgaaccccg agaccggctt gtgggaggag gagagcggct tccggcgcga
ggggtcctcg 2160 ggcccccggg tgcgccggga ggagcgcgtc ttcctggtgg
gcaacgtgga gatccgggag 2220 cggcgcctgt tcaatctgga cgtgcctgag
cgccgccgct gcttcgtgaa ggtgcgcgcc 2280 tacgccaacg acaagttcac
ccccagcgag caggtggagg gcgtggtggt cacgctggtc 2340 aatctggagc
ccgcccccgg cttctccgcc aacccccgtg cctggggccg ctttgacagc 2400
gcggtcaccg gccccaatgg cgcctgcctc cccgccttct gcgacgccga caggccagac
2460 gcctacaccg ccctggtcac cgccaccctg ggcggcgagg agctggagcc
ggccccttcc 2520 ttgccccgcc cactcccggc caccgtgggc gtcacccagc
cctacctgga caggctgggg 2580 taccgtcgga cggaccacga cgatcccgcc
ttcaagcgta acggcttccg catcaacctc 2640 gccaagccca ggccaggtga
ccccgccgag gccaatgggc ctgtgtaccc gtggcgcagc 2700 ctgcgggaat
gccagggggc cccggtgact gccagccact tccgcttcgc cagggtggag 2760
gcggacaagt acgagtacaa cgtggtcccc ttccgagagg gcacacctgc ctcctggact
2820 ggcgatctcc tggcctggtg gcccaacccg caggagttcc gggcctgctt
cctcaaggtg 2880 aagatccagg gtccccagga gtatatggtc cgctcccaca
acgcaggggg cagccaccca 2940 cgcacccgcg gccagctcta cggacttcgg
gatgcccgga gtgtgcgaga ccccgagcgt 3000 ccgggcacct cggcagcctg
cgtggagttc aagtgcagcg ggatgctgtt cgaccagcgg 3060 caggtggaca
ggacgctggt gaccattatg ccccagggca gctgccggcg cgtggccgtc 3120
aacggactcc ttcgggatta cctgacccgg caccccccac cggtgcccgc ggaggaccca
3180 gctgccttct ccatgctggc ccccctagac cctctgggcc acaactatgg
cgtctacact 3240 gtcactgacc agagcccacg cttggccaag gagatcgcca
ttggccgctg ctttgatggt 3300 tcctctgacg gyttctccag agagatgaag
gctgatgccg gcacagccgt caccttccag 3360 tgccgggagc caccggccgg
acgacccagc ctcttccaga ggctgctgga gtccccggcg 3420 acagcacttg
gtgacatccg cagggagatg agcgaggcgg cgcaggcaca ggcccgggcc 3480
tcaggtcccc tccgcacccg ccggggtagg gtccggcagt gacctgggca ggggcctcgc
3540 tttcccacct ccctccagac tcctttgacc ccaggaagtt ttgcccctcc
ttcttctcca 3600 gacagccccc tccccaggtg tctgggtccc ctttcccgcc
cctttccaga actcagagtc 3660 agacaagaac ccagagcatc cgatggtaga
aacaccagga agacaattgt tgctgtgtgg 3720 tatggaatgg agtttgcggt
gactctgggg ccagcaccca ggggacgacg ttcaacccta 3780 gcctgaaggg
acccgctccc agctcagaag ccgtctctga cttctcgtgc gtattttgac 3840
cctgatttca atcttctacc cttgggagtt ctggcgtttg gcacaaagtc ccctctgcct
3900 gtttggagct cagtgctaga ccaggtcccc tgccccgagc tttgtttttg
gggttattta 3960 ttgaaacaaa gtgtggggag ctggttgtgg gtgtgagtgg
gggtgtgggg tccaggctgg 4020 gcccagtgaa aaggaggaag gggttcccat
gcgggggagg ctctggggct gaggggaaca 4080 attctcacgt gtttggtgct
tagagacctg cccggggcgt tgggcaggcc ctccgggggc 4140 tgaattaaaa
atgctttatt tccaaaaaaa aaaaaaaaan aaa 4183 3 1184 PRT Homo sapiens
misc_feature Incyte Clone No. 422069 3 Met Val Gly Thr Lys Ala Trp
Val Phe Ser Phe Leu Val Leu Glu Val 1 5 10 15 Thr Ser Val Leu Gly
Arg Gln Thr Met Leu Thr Gln Ser Val Arg Arg 20 25 30 Val Gln Pro
Gly Lys Lys Asn Pro Ser Ile Phe Ala Lys Pro Ala Asp 35 40 45 Thr
Leu Glu Ser Pro Gly Glu Trp Thr Thr Trp Phe Asn Ile Asp Tyr 50 55
60 Pro Gly Gly Lys Gly Asp Tyr Glu Arg Leu Asp Ala Ile Arg Phe Tyr
65 70 75 80 Tyr Gly Asp Arg Val Cys Ala Arg Pro Leu Arg Leu Glu Ala
Arg Thr 85 90 95 Thr Asp Trp Thr Pro Ala Gly Ser Thr Gly Gln Val
Val His Gly Ser 100 105 110 Pro Arg Glu Gly Phe Trp Cys Leu Asn Arg
Glu Gln Arg Pro Gly Gln 115 120 125 Asn Cys Ser Asn Tyr Thr Val Arg
Phe Leu Cys Pro Pro Gly Ser Leu 130 135 140 Arg Arg Asp Thr Glu Arg
Ile Trp Ser Pro Trp Ser Pro Trp Ser Lys 145 150 155 160 Cys Ser Ala
Ala Cys Gly Gln Thr Gly Val Gln Thr Arg Thr Arg Ile 165 170 175 Cys
Leu Ala Glu Met Val Ser Leu Cys Ser Glu Ala Ser Glu Glu Gly 180 185
190 Gln His Cys Met Gly Gln Asp Cys Thr Ala Cys Asp Leu Thr Cys Pro
195 200 205 Met Gly Gln Val Asn Ala Asp Cys Asp Ala Cys Met Cys Gln
Asp Phe 210 215 220 Met Leu His Gly Ala Val Ser Leu Pro Gly Gly Ala
Pro Ala Ser Gly 225 230 235 240 Ala Ala Ile Tyr Leu Leu Thr Lys Thr
Pro Lys Leu Leu Thr Gln Thr 245 250 255 Asp Ser Asp Gly Arg Phe Arg
Ile Pro Gly Leu Cys Pro Asp Gly Lys 260 265 270 Ser Ile Leu Lys Ile
Thr Lys Val Lys Phe Ala Pro Ile Val Leu Thr 275 280 285 Met Pro Lys
Thr Ser Leu Lys Ala Ala Thr Ile Lys Ala Glu Phe Val 290 295 300 Arg
Ala Glu Thr Pro Tyr Met Val Met Asn Pro Glu Thr Lys Ala Arg 305 310
315 320 Arg Ala Gly Gln Ser Val Ser Leu Cys Cys Lys Ala Thr Gly Lys
Pro 325 330 335 Arg Pro Asp Lys Tyr Phe Trp Tyr His Asn Asp Thr Leu
Leu Asp Pro 340 345 350 Ser Leu Tyr Lys His Glu Ser Lys Leu Val Leu
Arg Lys Leu Gln Gln 355 360 365 His Gln Ala Gly Glu Tyr Phe Cys Lys
Ala Gln Ser Asp Ala Gly Ala 370 375 380 Val Lys Ser Lys Val Ala Gln
Leu Ile Val Ile Ala Ser Asp Glu Thr 385 390 395 400 Pro Cys Asn Pro
Val Pro Glu Ser Tyr Leu Ile Arg Leu Pro His Asp 405 410 415 Cys Phe
Gln Asn Ala Thr Asn Ser Phe Tyr Tyr Asp Val Gly Arg Cys 420 425 430
Pro Val Lys Thr Cys Ala Gly Gln Gln Asp Asn Gly Ile Arg Cys Arg 435
440 445 Asp Ala Val Gln Asn Cys Cys Gly Ile Ser Lys Thr Glu Glu Arg
Glu 450 455 460 Ile Gln Cys Ser Gly Tyr Thr Leu Pro Thr Lys Val Ala
Lys Glu Cys 465 470 475 480 Ser Cys Gln Arg Cys Thr Glu Thr Arg Ser
Ile Val Arg Gly Arg Val 485 490 495 Ser Ala Ala Asp Asn Gly Glu Pro
Met Arg Phe Gly His Val Tyr Met 500 505 510 Gly Asn Ser Arg Val Ser
Met Thr Gly Tyr Lys Gly Thr Phe Thr Leu 515 520 525 His Val Pro Gln
Asp Thr Glu Arg Leu Val Leu Thr Phe Val Asp Arg 530 535 540 Leu Gln
Lys Phe Val Asn Thr Thr Lys Val Leu Pro Phe Asn Lys Lys 545 550 555
560 Gly Ser Ala Val Phe His Glu Ile Lys Met Leu Cys Arg Lys Glu Pro
565 570 575 Ile Thr Leu Glu Ala Met Glu Thr Asn Ile Ile Pro
Leu Gly Glu Val 580 585 590 Val Gly Glu Asp Pro Met Ala Glu Leu Glu
Ile Pro Ser Arg Ser Phe 595 600 605 Tyr Arg Gln Asn Gly Glu Pro Tyr
Ile Gly Lys Val Lys Ala Ser Val 610 615 620 Thr Phe Leu Asp Pro Arg
Asn Ile Ser Thr Ala Thr Ala Ala Gln Thr 625 630 635 640 Asp Leu Asn
Phe Ile Asn Asp Glu Gly Asp Thr Phe Pro Leu Arg Thr 645 650 655 Tyr
Gly Met Phe Ser Val Asp Phe Arg Asp Glu Val Thr Ser Glu Pro 660 665
670 Leu Asn Ala Gly Lys Val Lys Val His Leu Asp Ser Thr Gln Val Lys
675 680 685 Met Pro Glu His Ile Ser Thr Val Lys Leu Trp Ser Leu Asn
Pro Asp 690 695 700 Thr Gly Leu Trp Glu Glu Glu Gly Asp Phe Lys Phe
Glu Asn Gln Arg 705 710 715 720 Arg Asn Lys Arg Glu Asp Arg Thr Phe
Leu Val Gly Asn Leu Glu Ile 725 730 735 Arg Glu Arg Arg Leu Phe Asn
Leu Asp Val Pro Glu Ser Arg Arg Cys 740 745 750 Phe Val Lys Val Arg
Ala Tyr Arg Ser Glu Arg Phe Leu Pro Ser Glu 755 760 765 Gln Ile Gln
Gly Val Val Ile Ser Val Ile Asn Leu Glu Pro Arg Thr 770 775 780 Gly
Phe Leu Ser Asn Pro Arg Ala Trp Gly Arg Phe Asp Ser Val Ile 785 790
795 800 Thr Gly Pro Asn Gly Ala Cys Val Pro Ala Phe Cys Asp Asp Gln
Ser 805 810 815 Pro Asp Ala Tyr Ser Ala Tyr Val Leu Ala Ser Leu Ala
Gly Glu Glu 820 825 830 Leu Gln Ala Val Glu Ser Ser Pro Lys Phe Asn
Pro Asn Ala Ile Gly 835 840 845 Val Pro Gln Pro Tyr Leu Asn Lys Leu
Asn Tyr Arg Arg Thr Asp His 850 855 860 Glu Asp Pro Arg Val Lys Lys
Thr Ala Phe Gln Ile Ser Met Ala Lys 865 870 875 880 Pro Arg Pro Asn
Ser Ala Glu Glu Ser Asn Gly Pro Ile Tyr Ala Phe 885 890 895 Glu Asn
Leu Arg Ala Cys Glu Glu Ala Pro Pro Ser Ala Ala His Phe 900 905 910
Arg Phe Tyr Gln Ile Glu Gly Asp Arg Tyr Asp Tyr Asn Thr Val Pro 915
920 925 Phe Asn Glu Asp Asp Pro Met Ser Trp Thr Glu Asp Tyr Leu Ala
Trp 930 935 940 Trp Pro Lys Pro Met Glu Phe Arg Ala Cys Tyr Ile Lys
Val Lys Ile 945 950 955 960 Val Gly Pro Leu Glu Val Asn Val Arg Ser
Arg Asn Met Gly Gly Thr 965 970 975 His Arg Arg Thr Val Gly Lys Leu
Tyr Gly Ile Arg Asp Val Arg Ser 980 985 990 Thr Arg Asp Arg Asp Gln
Pro Asn Val Ser Ala Ala Cys Leu Glu Phe 995 1000 1005 Lys Cys Ser
Gly Met Leu Tyr Asp Gln Asp Arg Val Asp Arg Thr Leu 1010 1015 1020
Val Lys Val Ile Pro Gln Gly Ser Cys Arg Arg Ala Ser Val Asn Pro
1025 1030 1035 1040 Met Leu His Glu Tyr Leu Val Asn His Leu Pro Leu
Ala Val Asn Asn 1045 1050 1055 Asp Thr Ser Glu Tyr Thr Met Leu Ala
Pro Leu Asp Pro Leu Gly His 1060 1065 1070 Asn Tyr Gly Ile Tyr Thr
Val Thr Asp Gln Asp Pro Arg Thr Ala Lys 1075 1080 1085 Glu Ile Ala
Leu Gly Arg Cys Phe Asp Gly Thr Ser Asp Gly Ser Ser 1090 1095 1100
Arg Ile Met Lys Ser Asn Val Gly Val Ala Leu Thr Phe Asn Cys Val
1105 1110 1115 1120 Glu Arg Gln Val Gly Arg Gln Ser Ala Phe Gln Tyr
Leu Gln Ser Thr 1125 1130 1135 Pro Ala Gln Ser Pro Ala Ala Gly Thr
Val Gln Gly Arg Val Pro Ser 1140 1145 1150 Arg Arg Gln Gln Arg Ala
Ser Arg Gly Gly Gln Arg Gln Ser Gly Val 1155 1160 1165 Val Ala Ser
Leu Arg Phe Pro Arg Val Ala Gln Gln Pro Leu Ile Asn 1170 1175
1180
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