gms | German Medical Science

Objectives: During progression of osteoarthritis (OA) joint cartilage tissue is continuously degraded by types of proteases including matrix metalloproteinases (MMPs). Besides collagens and proteoglycans, extracellular matrix (ECM) proteins like cartilage oligomeric matrix protein (COMP) and thrombospondin-4 (TSP-4) play an important role in cartilage homeostasis. During OA, both these proteins are proteolytically cleaved and resulting fragments can be detected in synovial fluid and serum. COMP is already widely used as marker for OA and its levels correlates with both OA severity and number of affected joints. TSP-4 is upregulated in later stages of OA but its exact function is still elusive. As it is still not yet completely clear which proteases are able to cleave COMP and TSP-4, we performed a systematic digestion with recombinant substrates and four OA-relevant MMPs in vitro.

Methods: Human COMP and TSP-4 as well as MMPs (MMP-2,-8,-9,-13) were recombinantly expressed in HEK-293EBNA cells and purified by affinity chromatography using a StrepII-tag. MMPs were activated with 1mM 4-aminophenylmercuric acetate and incubated with recombinant substrates for up to 24 hours. In addition to a kinetic analysis, MMPs were used in a broad range of concentrations including those that were detected in synovial fluid of OA patients. The digests were separated up by SDS-PAGE and the fragmentation was visualized by Coomassie staining and by immunoblots.

Results and Conclusion: We successfully established an in vitro assay to analyze the cleavage of cartilage matrix proteins by MMPs. This assay can now be applied to any other matrix component that is available as purified protein. We could show that COMP and TSP-4 are both cleaved by all MMPs tested in a time- and concentration dependent manner. Interestingly, incubation with different MMPs resulted in a slightly different fragment pattern. In all substrate-enzyme combinations, we were able to identify stable fragments of varying molecular size that seem to be MMP-resistant. Preliminary experiments revealed that similar fragments can be found in synovial fluid from OA patients as well as in tissue cultures that were kept under inflammatory conditions. Future studies will focus on the biological activity of these fragments.

The degeneration of the cartilage ECM is a hallmark of OA. Our results shed some light on the MMPs that might be involved in the cleavage of distinct ECM components. Using MMP concentrations close to the (patho)physiological range, we identified several MMP-specific and MMP-resistant fragments. The identification of the exact cleavage sites is necessary and will provide a better understanding of cartilage degeneration at the molecular level. Earlier studies have demonstrated that protein fragments could even have cartilage-protecting properties. Therefore, the biological activity of such fragments has to be carefully investigated. Only then, the use of specific MMP inhibitors can be considered as a treatment option for OA patients.