The major clinical features of osteoarthritis are focal loss of articular cartilage and osteophytic outgrowth of subchondral bone. Loss of articular cartilage is preceded by changes in the content and composition of cartilage matrix proteoglycan aggregates. Mature chondrocytes, although biosynthetically active, appear unable to maintain or repair matrix aggregates so that cartilage degeneration is progressive. Proteoglycan aggregates are composed of monomers, link protein and hyaluronate in ternary complex. Monomers bind to hyaluronate (and link protein) via a specific binding region and this process immobilizes the molecules and possibly protects them from proteolytic attack. An understanding of how proteoglycans acquire hyaluronate binding properties and what occurs during loss of binding is therefore central to the question of matrix maintenance in normal and diseased cartilages. Three closely related systems will be studied (1) rabbit chondrocytes in monolayer culture which secrete a """"""""precursor"""""""" form of proteoglycan with a low affinity for hyaluronate, which can be markedly enhanced by treatment in vitro with mild alkali. We plan to study conditions of culture which modify precursor affinity and the molecular basis of the alkali induced change in binding activity. (2) a cartilage explant system in which the hyaluronate binding activity of proteoglycans is lost. This appears to be due to limited proteoysis of the binding region domain and we plan to investigate the change in binding region structure. (3) Purified stromelysin, a neutral metalloendopeptidase, will be incubated with binding region to determine its effect on the binding properties and structure of this protein. Essential methodologies are established in our laboratory (culture systems, proteoglycan isolation, immunoassay, binding affinity assays, electrophoresis and blotting) and we are developing reversed-phase HPLC for mapping of peptides in proteoglycan core protein. The long-term objective of these studies is to increase our understanding of the extracellular processing of cartilage proteoglycans, particularly in relation to modification of hyaluronate binding activity. Such knowledge might suggest methods for the useful intervention in joint disease at the level of improving matrix repair by mature chondrocytes.
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