AGGRECAN SUBSTRATE CONSTRUCTS FOR AGGRECANASE CATABOLISM

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Aggrecan is a large hybrid proteoglycan of the cartilage extracellular matrix, which is critical to cartilage functional properties. We have constructed an expression vector coding for full-length bovine aggrecan and have demonstrated expression of aggrecan in transiently transfected COS-7 cells. We propose to construct a series of mutagenized aggrecan expression vectors that can be used to analyze aggrecanase-mediated aggrecan catabolism such as normally occurs during development, and maturation, and which is accelerated during aging and osteoarthritis. The interglobular domain (IGD) in mammalian aggrecan contains a site susceptible to cleavage by matrix metalloproteinases as well as at an additional site by an enzyme activity referred to as aggrecanase. Additional sites for aggrecanase-mediated cleavage have been described in the CS-2 domain of aggrecan. There is evidence that N-linked or O-linked glycosylation may influence the susceptibility of aggrecanase sites to cleavage, and that glycosylation patterns may vary with age. Aggrecan turnover, therefore, may be regulated by the glycosylation pattern near the aggrecanase cleavage site. Using the aggrecan expression construct described above, we propose to these the HYPOTHESIS that these highly conserved proteinase cleavage sites, as well as flanking sites for age- specific glycosylation, are important in regulation of turnover of aggrecan in normal cartilage. An understanding of sites which are most susceptible to proteolytic cleavage will enhance efforts to antagonize aggrecan degradation in osteoarthritic cartilage. To address this hypothesis we propose the following aims: Specific Aim 1: Construction of mutagenized full-length aggrecan expression vectors. We will mutagenize aggrecanase cleavage sites within the IGD and aggrecanase sites within the CS-2 domain for experiments to ascertain their respective contributions to aggrecan turnover. We will generate cleavage site mutants, and mutations in conserved glycosylation sites, and transiently express recombinant aggrecan in rat chondrosarcoma cells (RCS) or in primary bovine chondrocytes. Specific Aim 2: Development of assays to analyze aggrecan catabolism using specifically mutagenized substrates. Recombinant aggrecan produced in these cells will be tested as a substrate using RA-stimulated RCS cells cultured in agarose as an aggrecanase source. Cleavage products will be characterized by Western blot analysis using cleavage neoepitope-specific antisera.