MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein |
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Authors: | Michaela Durigova Hideaki NagaseJohn S Mort Peter J Roughley |
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Institution: | a Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montreal, H3G 1A6 Canadab Department of Surgery, McGill University, Montreal, Quebec, Canadac Kennedy Institute of Rheumatology, Imperial College London, London, W6 8L, United Kingdom |
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Abstract: | Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu373-374Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as “aggrecanase” (ADAMTS) cleavage sites, while cleavage between Ser341-342Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu2047-2048Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan. |
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Keywords: | ADAMTS a disintegrin and metalloproteinase with thrombospondin motifs APMA p-amino-phenyl-mercuric acetate anti-ARLEIE an antibody directed against the ARLEIE sequence within aggrecan CS-2 chondroitin sulfate-2 region ECM extracellular matrix G1 globular domain 1 G3 globular domain 3 GAG glycosaminoglycan IGD interglobular domain IL-1β interleukin-1beta MMP matrix metalloproteinase OSM oncostatin M OA osteoarthritis RA rheumatoid arthritis SDS/PAGE sodium dodecyl sulfate/ polyacrylamide gel electrophoresis |
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