REGULATION OF AGGRECAN CATABOLISM

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DESCRIPTION (provided by applicant): Aggrecan is a large hybrid proteoglycan of the cartilage extracellular matrix, which is critical to cartilage functional properties. The interglobular domain (IGD) in mammalian aggrecan contains aggrecanase and matrix metalloproteinase cleavage sites, and additional sites for aggrecanase-mediated cleavage have been described in the CS-2 domain. We have constructed the first full-sized aggrecan expression vector to surmount the limitations of smaller aggrecan subdomain constructs used previously as mutagenizable aggrecanase substrates. We propose the hypothesis that highly conserved proteinase cleavage sites in aggrecan are important in regulation of aggrecan turnover. We further hypothesize that the chondrocyte can regulate aggrecan catabolism by altering glycosylation patterns on the aggrecan substrate. To address these hypotheses, we propose the following Specific Aims: Specific Aim 1: To determine amino acid residues required for recognition and cleavage by aggrecanase. Comparison of known aggrecanase cleavage sites in aggrecan and brevican have revealed conservation of clusters of flanking residues. We will establish whether these flanking residues are required for cleavage by aggrecanase by replacing conserved residues and assaying for cleavage at each site. Specific Aim 2: To determine the functional relationships between cleavage sites in aggrecan. Cleavage at the MMP site in the IGD has been shown to inhibit aggrecanase cleavage within the IGD, suggesting the involvement of motifs in the G1 domain in aggrecanase substrate recognition. We will mutagenize these G1 motifs and assay for aggrecanase cleavage at the IGD site. There is also evidence that cleavage of aggrecan C-terminal sites precedes, and may be a prerequisite for cleavage within the IGD. We will mutagenize aggrecanase sites within the CS-2 domain and determine directly whether cleavage at the IGD is inhibited. Specific Aim 3: To determine whether susceptibility to aggrecanase cleavage may be regulated by known and potential sites for KS substitution of aggrecan. We will determine the optimal cell type for expression of KS-containing aggrecan. We will mutagenize threonine residues in the IGD and "nodal" region of the CS-2 domain to prevent KS substitution and assay for aggrecanase susceptibility. Wild-type and mutant aggrecan constructs will be expressed in chondrocytes isolated from cartilage of different aged animals, to determine if there is an age-specific pattern of glycosylation that influences aggrecanase susceptibility.