Abstract
Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix (ECM) of the musculoskeletal system. Its importance is underscored by its association with several growth disorders. In this report, we investigated its interaction with aggrecan, a major component of cartilage ECM. We also tested a COMP/TSP5 mutant, designated MUT3 that accounts for 30% of human pseudoachondroplasia cases, to determine if the mutation affects function. Using a solid-phase binding assay, we have shown that COMP/TSP5 can bind aggrecan. This binding was decreased with MUT3, or when COMP/TSP5 was treated with EDTA, indicating the presence of a conformation-dependent aggrecan binding site. Soluble glycosaminoglycans (GAGs) partially inhibited binding, suggesting that the interaction was mediated in part through aggrecan GAG side chains. Using affinity co-electrophoresis, we showed that COMP/TSP5, in its calcium-replete conformation, bound to heparin, chondroitin sulfates, and heparan sulfate; this binding was reduced with EDTA treatment of COMP/TSP5. MUT3 showed weaker binding than calcium-repleted COMP/TSP5. Using recombinant COMP/TSP5 fragments, we found that the "signature domain" could bind to aggrecan, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. In summary, our data indicate that COMP/TSP5 is an aggrecan-binding protein, and this interaction is regulated by the calcium-sensitive conformation of COMP/TSP5; interaction of COMP with aggrecan can be mediated through the GAG side chains on aggrecan and the "signature domain" of COMP/TSP5. Our results suggest that COMP/TSP5 may function to support matrix interactions in cartilage ECM.
Highlights
Cartilage oligomeric matrix protein/thrombospondin 5 (COMP4/TSP5) is a pentameric extracellular matrix protein that is the fifth member of the thrombospondin (TSP) family [1, 2]
One of the major components of cartilage, have abundant GAG side chains of chondroitin sulfate (ϳ100 side chains/core protein) and keratan sulfate (ϳ50 side chains/core protein), we investigated the interaction of COMP/TSP5 with aggrecan using a solid-phase binding assay
We showed that aggrecan can bind to COMP/TSP5 in a concentration-dependent manner (Fig. 3)
Summary
Cartilage oligomeric matrix protein/thrombospondin 5 (COMP4/TSP5) is a pentameric extracellular matrix protein that is the fifth member of the thrombospondin (TSP) family [1, 2]. MED/EDM1 differ in severity with the former presenting as a severe growth deficiency affecting cartilage and bone development Both conditions are associated with gait abnormalities, and patients require hip replacement in the second or third decade of life. COMP/TSP5, type IX collagen, aggrecan, and possibly link protein have been reported to be retained in the large rER cisternae (20 –24) In this investigation, we have studied the function of MUT3, a COMP/TSP5 single amino acid deletion (D469del) mutation accounting for 30% of the PSACH patients. Cartilage ECM contains three classes of proteins: collagens, proteoglycans, and other noncollagenous proteins including COMP/TSP5 [25] Of these major components of cartilage, COMP/TSP5 can interact with type II and type IX collagens in a zinc-dependent fashion via its C-globe region (26 –28). COMP/TSP5 is thought to play a direct role in regulating collagen fibril formation and maintaining collagen network integrity
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