Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-08-18
2001-05-22
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S069100, C435S072000, C435S097000, C435S100000, C435S101000, C435S105000, C435S193000, C435S254200, C435S320100, C435S440000, C536S023200, C530S350000
Reexamination Certificate
active
06235510
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION
The drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics”, that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superceding earlier approaches based on “positional cloning”. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
Functional genomics relies heavily on high-throughput DNA sequencing technologies and the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterize further genes and their related polypeptides/proteins, as targets for drug discovery.
Mammalian connective tissues contain proteins, glycoproteins and large complex glycoconjugates, such as proteoglycans. Control of the synthesis, post-translational modification, organization and degradation of these components appears very important in maintaining tissue homeostasis. Uncontrolled matrix deposition or degradation results in loss of tissue function in virtually all known pathology.
Articular cartilage is a matrix-rich, avascular, aneural tissue that allows the fluid articulation of synovial joints. Chondrocytes are the only cells within this tissue and are responsible for cartilage matrix biosynthesis and assembly and also play a role in matrix catabolism in diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA). Cartilage matrix is rich in carbohydrate-containing proteoglycans as well as glycoproteins. In both OA and RA, there appears to be changes in the synthesis and degradation of the protein cores as well as changes in the glycosylation patterns of these molecules.
One of the major articular cartilage matrix components is the large proteoglycan, aggrecan. Aggrecan is a glycosoaminoglycan (GAG)-rich molecule that contains chondroitin sulfate and keratan sulfate chains as well as N-(asparagine) and O-(serine and threonine) linked oligosaccahrides. The tissue also contains several glycoproteins, some of which are also O-glycosylated.
Chondroitin sulfate is the predominant GAG on aggrecan. It is a repeating polymer of N-acetylgalactosamine and glucuronic acid. These chondroitin sulfate chains are variably sulfated on N-acetylgalactosamine residues in either the 4, 6 or disulfated in both the 4 and 6 positions. In OA, the length of the chondroitin sulfate chains are shorter and the sulfation pattern changes with an increase in 4-sulfation and a reduction in the terminal N-acetylgalactosamine 4,6-disulfation (Plass et al. J. Biol. Chem. 273:12642-12649, 1998). These structural changes in the molecule could result in a matrix with lower water binding capacity and may influence interactions with other matrix components, ultimately resulting in a destabilized matrix.
Keratan sulfate is the other major GAG on aggrecan and is covalently attached to the core protein through either an N- or O-linkage (Hascall and Midura in Keratan sulfate chemistry, biology, chemical pathology 1989 eds. H. Greiling and J. Scott, London: The Biochemical Society, pps 66-75). Serine or threonine post-translational glycosylation to form keratan sulfate is initiated by transfer of N-acetylgalactosamine by specific enzymes. The O-linked keratan sulfate pattern changes in the intraglobular domain of aggrecan with aging (Barry et al., J. Biol. Chem. 270:20516-20524, 1995). One of these variable glycosylation sites is prone to cleavage by the chondrocyte-derived proteinase, aggrecanase (Arner et al, J. Biol. Chem. 274:6594-6601, 1999). Aggrecanase-cleaved aggrecan fragments accumulate at sites of articular cartilage damage in OA and RA (Lark et al., J. Clin. Invest. 100:93-96, 1997) and cleavage activity could be influenced by the glycosylation state of the aggrecan substrate in this intraglobular domain region. Furthermore, keratan sulfate levels appear to be modulated in patients with OA and has been proposed as a potential marker to monitor cartilage matrix turnover (Lohmander, Baillere's Clin. Rhem. 11:711-726, 1997). Changes in the circulating andjoint fluid levels of keratan sulfate could reflect changes in the pattern or levels of enzymes that are responsible for transferring sugar moieties onto protein core. In addition to keratan sulfate, aggrecan contains O-linked oligosaccharides which may influence its ability to interact with other components to maintain a stable extracellular matrix. Careful analysis of 0-linked oligosaccharide changes in aggrecan as cartilage destruction progresses has not been done; however, it is possible that these oligosaccharide patterns could also have profound effects on cartilage matrix structure.
Several UDP-GalNac:polypeptide N-acetylgalactosaminyltransferases (ppGaNTase, EC 2.4.1.41) regulate the initiation of mucin-type O-linked glycosylation where N-acetylgalactosamine is transferred to the hydroxyl group of serine and threonine residues (Clausen and Bennet, Glycobiology 6:635-546, 1996). To date, five members of this family (ppGaNTase-T1, -T2, -T3, -T4 and -T5) have been reported in mammalian cells (Clausen and Bennet, Glycobiology 6:635-546, 1996, Bennet et al. J. Biol. Chem. 273:30472-30481, 1998, Ten Hagen et al. J. Biol. Chem. 273:27749-27754, 1998). Five additional members have been reported in Caenorabditis elegans (Hagen and Nehrke, J. Biol. Chem. 273: 8268-8277, 1998). Present data based on some of the characterized enzymes suggest that there are differences in substrate specificity as well as the expression pattern of these enzymes (Clausen and Bennet, Glycobiology 6:635-546, 1996). Conservation from worms to humans underscores the functional importance and diversity of this family of enzymes. Families of enzymes responsible for carbohydrate addition in glycosaminoglycan synthesis and glycoprotein post-translational modification have been described, and new members of these families continue to emerge. A new member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family of enzymes termed ppGaNTase-T6 is described herein.
SUMMARY OF THE INVENTION
The present invention relates to ppGaNTase-T6, in particular ppGaNTase-T6 polypeptides and ppGaNTase-T6 polynucleotides, recombinant materials and methods for their production. Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to, chronic and acute inflammation, rheumatoid arthritis, osteoarthritis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, allergy, asthma, renal disorders, restenosis, fibrosis, brain injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer (e.g., lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, hereinafter referred to as “diseases of the invention.” In a further aspect, the invention relates to methods for identifying agonists and antagonists (e.g., inhibitors) using the materials provided by the invention, and treating conditions associated with ppGaNTase-T6 imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate ppGaNTase-T6 activity or levels.
DESCRIPTION OF THE INVENTION
In a first aspect, the present invention relates to ppGaNTase-T6 polypeptides. Such polypeptides include:
(a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO:1;
Kumar Sanjay
Lark Michael W
Van Horn Marion M
Han William T.
King William T.
Prouty Rebecca E.
Rao Manjunath N.
Ratner & Prestia
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