UNLABELLED: Insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) are trophic factors for cartilage and have been shown to be chondroprotective in animal models of osteoarthritis (OA). IGFBP-5 is degraded in joint fluid and inhibition of IGFBP-5 degradation has been shown to enhance the trophic effects of IGF-I. OBJECTIVE: To determine the identity of IGFBP-5 protease activity in human OA joint fluid. METHOD: OA joint fluid was purified and the purified material was analyzed by IGFBP-5 zymography. RESULTS: Both crude joint fluid and purified material contained a single band of proteolytic activity that cleaved IGFBP-5. Immunoblotting of joint fluid for complement 1s (C1s) showed a band that had the same Mr estimate, e.g., 88 kDa. In gel tryptic digestion and subsequent peptide analysis by LC-MS/MS showed that the band contained human C1s. A panel of protease inhibitors was tested for their ability to inhibit IGFBP-5 cleavage by the purified protease. Three serine protease inhibitors, FUT175 and CP-143217 and CB-349547 had IC50's between 1 and 6 microM. Two other serine protease inhibitors had intermediate activity (e.g., IC50's 20-40 microM) and MMP inhibitors had no detectible activity at concentrations up to 300 microM. CONCLUSION: Human OA fluid contains a serine protease that cleaves IGFBP-5. Zymography, immunoblotting and LC-MS/MS analysis indicate that C1s is the protease that accounts for this activity.
UNLABELLED: Insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) are trophic factors for cartilage and have been shown to be chondroprotective in animal models of osteoarthritis (OA). IGFBP-5 is degraded in joint fluid and inhibition of IGFBP-5 degradation has been shown to enhance the trophic effects of IGF-I. OBJECTIVE: To determine the identity of IGFBP-5 protease activity in human OA joint fluid. METHOD: OA joint fluid was purified and the purified material was analyzed by IGFBP-5 zymography. RESULTS: Both crude joint fluid and purified material contained a single band of proteolytic activity that cleaved IGFBP-5. Immunoblotting of joint fluid for complement 1s (C1s) showed a band that had the same Mr estimate, e.g., 88 kDa. In gel tryptic digestion and subsequent peptide analysis by LC-MS/MS showed that the band contained humanC1s. A panel of protease inhibitors was tested for their ability to inhibit IGFBP-5 cleavage by the purified protease. Three serine protease inhibitors, FUT175 and CP-143217 and CB-349547 had IC50's between 1 and 6 microM. Two other serine protease inhibitors had intermediate activity (e.g., IC50's 20-40 microM) and MMP inhibitors had no detectible activity at concentrations up to 300 microM. CONCLUSION:Human OA fluid contains a serine protease that cleaves IGFBP-5. Zymography, immunoblotting and LC-MS/MS analysis indicate that C1s is the protease that accounts for this activity.
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