Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1999-10-28
2001-06-26
Saidha, Tekchand (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S195000, C435S252300, C435S320100, C435S021000, C536S023200, C530S350000
Reexamination Certificate
active
06251389
ABSTRACT:
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
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
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.)
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.)
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.)
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.)
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.
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 arthropathies, immunological disorders, and cancers.
SUMMARY OF THE INVENTION
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.
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.
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.
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.
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.
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)
Hutchinson Nancy
Lawton Michael
Magna Holly
Mitchell Peter
Murry Lynn E.
Incyte Genomics Inc.
Incyte Genomics, Inc.
Saidha Tekchand
LandOfFree
Human nucleotide pyrophosphohydrolase-2 does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Human nucleotide pyrophosphohydrolase-2, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Human nucleotide pyrophosphohydrolase-2 will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2484644