Degradative fragments of cartilage oligomeric matrix protein (COMP) have been observed in arthritic patients. The physiological enzyme(s) that degrade COMP, however, remain unknown. We performed a yeast two-hybrid screen (Y2H) to search for proteins that associate with COMP to identify an interaction partner that might degrade it. One screen using the epidermal growth factor (EGF) domain of COMP as bait led to the discovery of ADAMTS-7. Rat ADAMTS-7 is composed of 1595 amino acids, and this protein exhibits higher expression in the musculoskeletal tissues. COMP binds directly to ADAMTS-7 in vitro and in native articular cartilage. ADAMTS-7 selectively interacts with the EGF repeat domain but not with the other three functional domains of COMP, whereas the four C-terminal TSP motifs of ADAMTS-7 are required and sufficient for association with COMP. The recombinant catalytic domain and intact ADAMTS-7 are capable of digesting COMP in vitro. The enzymatic activity of ADAMTS-7 requires the presence of Zn 2+ and appropriate pH (7.5-9.5), and the concentration of ADAMTS-7 in cartilage and synovium of patients with rheumatoid arthritis is significantly increased as compared to normal cartilage and synovium. ADAMTS-7 is the first metalloproteinase found to bind directly to and degrade COMP.-Liu, C., Kong, W., Ilalov, K., Yu, S., Xu, K., Prazak, L., Fajardo, M., Sehgal, B., Di Cesare, P. E. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. The extracellular matrix (ECM) of cartilage consists of several types of collagens, proteoglycans, and other noncollagenous macromolecules, all of which interact to form a highly specialized connective tissue (1). Early extracellular cartilage matrix degeneration in arthritis is the result of the action of degradative enzymes. As the severity of arthritis progresses, the synthesis and secretion of matrix-degrading enzymes markedly increase (2). The control 1 These authors contributed equally to this work.
Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factorlike repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn 2؉ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.More than 15% of the world population older than 18 years are affected by arthritic disorders, including osteoarthritis (OA) 3 and rheumatoid arthritis (RA) (1). Accumulating evidence suggests that proteases perform an important function in the breakdown of the extracellular matrix in OA and RA (2). Cartilage oligomeric matrix protein (COMP), a prominent noncollagenous component of cartilage, accounts for ϳ1% of the wet weight of articular tissue (3, 4). COMP is a 524-kDa pentameric, disulfide-bonded, multidomain glycoprotein composed of approximately equal subunits (ϳ110 kDa each) (5, 6). Several studies suggest that monitoring of COMP levels (in both joint fluid and serum) can be used to assess the presence and progression of arthritis (7-11). Synovial fluid COMP levels were found to be higher in individuals with knee pain or injury (12), anterior cruciate ligament or meniscal injury (9, 12), OA (8, 12), and RA (7, 13) than in healthy individuals.Fragments of COMP have been detected in the cartilage, synovial fluid, and serum of patients with post-traumatic and primary OA and RA (7,8,13). The molecular mechanism of COMP degradation and the enzyme (s) responsible for it, however, remain largely unknown. Theoretically, inhibition of degradative enzymes can slow down or block the initiation and progression of arthritic diseases. The isolation of cartilage degradative enzymes is therefore of great interest from both a pathophysiological and a therapeutic standpoint. The ADAMTS family (ADAMTS: (a disintegrin and metalloprotease with thrombospondin motifs) consists of secreted zinc metalloproteinases with a precisely ordered modular organization that includes at least o...
Objective-As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (adisintegrin and metalloprotease with thrombospondin motifs) family, degrade COMP in vitro and are significantly induced in the cartilage and synovium of arthritic patients. The purpose of this study was to determine 1) whether cleavage activity of ADAMTS-7 and-12 of COMP are associated with COMP degradation in osteoarthritis; 2) whether a2M is a novel substrate for ADAMTS-7 and-12; and 3) whether a2M inhibits ADAMTS-7 or-12 cleavage of COMP. Methods-An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with TNF-α or IL-1β with or without blocking antibodies; and in human chondrocytes treated with specific siRNA to knock down ADAMTS-7 or/and-12. Digestion of alpha-2-macroglobulin (a2M) by ADAMTS-7 and-12 in vitro and the inhibition of ADAMTS-7 or-12-mediated digestion of COMP by α2M were also analyzed. Results-The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-α-or IL-1β-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-α-or IL-1β-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave α 2 M, giving rise to 180 and 105 kDa cleavage products, respectively. Furthermore, α 2 M inhibited both ADAMTS-7-and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. Conclusion-Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, α 2 M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, α 2 M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.
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