ADAMTS-4 (aggrecanase-1) is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In this study, we developed a sensitive fluorescence resonance energy transfer peptide assay with a K m in the 10 M range and utilized this assay to demonstrate that inhibition of full-length ADAMTS-4 by full-length TIMP-3 (a physiological inhibitor of metalloproteinases) is enhanced in the presence of aggrecan. Our data indicate that this interaction is mediated largely through the binding of glycosaminoglycans (specifically chondroitin 6-sulfate) of aggrecan to binding sites in the thrombospondin type 1 motif and spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. The results of this study therefore indicate that the cartilage environment can modulate the function of enzyme-inhibitor systems and could have relevance for therapeutic approaches to aggrecanase modulation.
The disintegrin metalloproteinases with thrombospondin motifs (ADAMTS)3 are a novel family of extracellular proteases forming an integral part of the extracellular matrix itself. Along with serine proteases, matrix metalloproteinases, bone morphogenetic protein-1/tolloid metalloproteinases, and ADAM (a disintegrin and metalloproteinase) proteins, ADAMTS proteins play a pivotal role in the proteolytic processing and turnover of the component molecules of the extracellular matrix of a broad range of tissues and have potential roles in the turnover of cell-surface proteins. ADAMTS proteins share the characteristic protease, disintegrin-like, and cysteine-rich domains in common with ADAM proteins, but differ in being soluble rather than membrane-bound and by the presence of thrombospondin type 1 (TSP-1) repeats (1, 2). ADAMTS-2, -3, and -14 are potential procollagen N-proteinases, and ADAMTS-13 has been identified as a von Willebrand factor-cleaving protease. ADAMTS-4 is a member of the "angiogenesis/aggrecanase" group of ADAMTS proteases (which also includes ADAMTS-1, -5, -8, -9, and -15) and is unique among the currently known ADAMTS proteases in containing only a single TSP-1-like motif, located between its disintegrin-like and cysteine-rich domains, and lacking any C-terminal TSP-1-like repeats (see Fig. 1). Like the other members of the angiogenesis/ aggrecanase group and ADAMTS-9, ADAMTS-4 has been demonstrated to act as an aggrecanase in vitro. In common with other aggrecanases, ADAMTS-4 is able to cleave aggrecan at multiple sites (five in total) (see Fig. 1) (3); however, it is one of only four that cleave aggrecan at the Glu 373 -Ala 374 "interglobulin domain" cleavage site (the others being ADAMTS-1, -5, and -8, which cleave with varying affinities). Aggrecan...