Abstract
Osteoarthritis (OA) is a highly prevalent joint disease, affecting millions of people worldwide and characterized by degradation of articular cartilage, subchondral bone remodeling and low-grade inflammation, leading to pain, stiffness and disability. Cartilage Oligomeric Matrix Protein (COMP) is a major structural component of cartilage and its degradation has been proposed as a marker of OA severity/progression. Several proteases cleave COMP in vitro, however, it is unclear which of these COMPase activities is prevalent in an osteoarthritic joint. Here, using purified recombinant proteins, we show that A Disintegrin And Metalloproteinase with Thrombospondin motifs 4 (ADAMTS4) is the most potent COMPase, followed by ADAMTS1. Using liquid chromatography-tandem mass spectrometry, we identified several novel cleavage sites in COMP resulting from ADAMTS4 and ADAMTS1 activity. Cleavage at S77-V78 disrupted the pentameric organization of COMP and generated a neopeptide previously identified in the synovial fluid of OA patients. Immunoblots with anti-QQS77 antibodies confirmed that ADAMTS4 efficiently cleaved this peptide bond. By analyzing five ADAMTS4 variants, we found that the C-terminal spacer domain is strictly necessary for COMPase activity and identified the specific residues involved in the interaction with COMP. An inhibitory anti-ADAMTS4 antibody significantly decreased generation of the COMP QQS77 neoepitope in human OA cartilage explants, implicating ADAMTS4 as a key protease in generating the QQS77 neopeptides in OA. Since another major ADAMTS4 substrate is aggrecan, the most abundant proteoglycan in cartilage, these findings highlight that, by cleaving both COMP and aggrecan, ADAMTS4 may play a crucial role in modulating the structural integrity of cartilage.