OBJECTIVE: To determine the proteolytic fragmentation patterns and N-terminal sequence of aggrecan fragments in human synovial fluid from patients with inflammatory arthritides, joint injury, or osteoarthritis (OA). METHODS: Knee synovial fluid was obtained from patients with joint injury, OA, acute pyrophosphate arthritis (pseudogout), reactive arthritis, psoriatic arthritis, or juvenile rheumatoid arthritis. Chondroitin sulfate-substituted aggrecan fragments present in the fluid were purified by cesium chloride gradient centrifugation and enzymatically deglycosylated. Core protein species were determined by N-terminal analysis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electroblotting and detection with monoclonal antibody 3B3. RESULTS: Samples from patients with joint injury, OA, and inflammatory joint disease all showed a similar 3-band pattern, with core sizes of approximately 200 kd, 170 kd, and 135 kd. In all samples, diffuse immunoreactive products were also seen, with an apparent size of > 250 kd. N-terminal analysis of core preparations of all samples showed a consistent single predominant sequence, beginning at alanine 374 of the human aggrecan core protein. CONCLUSION: The aggrecan fragments present in joint fluids from patients with various inflammatory arthritides, joint injury, or OA result from a predominant cleavage of the human aggrecan core protein at the glutamate 373-alanine 374 bond within the interglobular domain, between the G1 and G2 domains. The consistent pattern of fragments seen on SDS-PAGE and the single predominant N-terminal sequence suggest a common degradative mechanism of aggrecan in these different joint conditions. The identity of the proteolytic agent (aggrecanase), however, remains unknown. These results appear to have important implications with regard to the development of therapies to protect cartilage from degradation in patients with joint disease.
OBJECTIVE: To determine the proteolytic fragmentation patterns and N-terminal sequence of aggrecan fragments in human synovial fluid from patients with inflammatory arthritides, joint injury, or osteoarthritis (OA). METHODS: Knee synovial fluid was obtained from patients with joint injury, OA, acute pyrophosphatearthritis (pseudogout), reactive arthritis, psoriatic arthritis, or juvenile rheumatoid arthritis. Chondroitin sulfate-substituted aggrecan fragments present in the fluid were purified by cesium chloride gradient centrifugation and enzymatically deglycosylated. Core protein species were determined by N-terminal analysis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electroblotting and detection with monoclonal antibody 3B3. RESULTS: Samples from patients with joint injury, OA, and inflammatory joint disease all showed a similar 3-band pattern, with core sizes of approximately 200 kd, 170 kd, and 135 kd. In all samples, diffuse immunoreactive products were also seen, with an apparent size of > 250 kd. N-terminal analysis of core preparations of all samples showed a consistent single predominant sequence, beginning at alanine 374 of the humanaggrecan core protein. CONCLUSION: The aggrecan fragments present in joint fluids from patients with various inflammatory arthritides, joint injury, or OA result from a predominant cleavage of the humanaggrecan core protein at the glutamate 373-alanine 374 bond within the interglobular domain, between the G1 and G2 domains. The consistent pattern of fragments seen on SDS-PAGE and the single predominant N-terminal sequence suggest a common degradative mechanism of aggrecan in these different joint conditions. The identity of the proteolytic agent (aggrecanase), however, remains unknown. These results appear to have important implications with regard to the development of therapies to protect cartilage from degradation in patients with joint disease.
Authors: Natasha Baker; Paul Sharpe; Kirsty Culley; Miguel Otero; Damon Bevan; Peter Newham; Wendy Barker; Kristen M Clements; Caroline J Langham; Mary B Goldring; Jelena Gavrilović Journal: Arthritis Rheum Date: 2012-07
Authors: M W Lark; E K Bayne; J Flanagan; C F Harper; L A Hoerrner; N I Hutchinson; I I Singer; S A Donatelli; J R Weidner; H R Williams; R A Mumford; L S Lohmander Journal: J Clin Invest Date: 1997-07-01 Impact factor: 14.808