Cleavage of Cartilage Oligomeric Matrix Protein (COMP) by ADAMTS4 generates a neoepitope associated with osteoarthritis and other forms of degenerative joint disease

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.