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Synovial fluid was collected from patients with recent knee injury and from patients with early or late stage osteoarthritis. Chondroitin sulfate-substituted aggrecan fragments present in these fluids, and in normal bovine synovial fluid, were purified by cesium chloride gradient centrifugation, enzymically deglycosylated and fractionated by gel filtration on Superose-12. Each sample contained two major aggrecan core protein populations with apparent molecular masses of approximately 90 kD and 150 kD. For all samples, NH2-terminal analysis of both populations gave a single major sequence beginning ARGSV. This NH2 terminus results from cleavage of the human aggrecan core protein at the Glu 373-Ala 374 bond within the interglobular domain between the G1 and G2 domains. Cleavage at this site also occurs during control and interleukin-1 stimulated aggrecan catabolism in bovine cartilage explant cultures (Sandy, J., P. Neame, R. Boynton, and C. Flannery. 1991. J. Biol. Chem. 266:8683-8685). These results indicate that the major aggrecan fragments present in both osteoarthritic human synovial fluid and in normal bovine synovial fluid are large, being composed of a short NH2-terminal stretch of the interglobular domain, the G2 domain, the keratan sulfate domain, and variable lengths of the chondroitin sulfate domain(s). We conclude that the release of aggrecan fragments from articular cartilage into the synovial fluid seen at all stages of human osteoarthritis (Lohmander, L. S. 1991. Acta Orthop. Scand. 62:623-632) is promoted by the action of a normal cartilage proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain.