TIMP-3 Inhibition of ADAMTS-4 (Aggrecanase-1) Is Modulated by Interactions between Aggrecan and the C-terminal Domain of ADAMTS-4

Abstract

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...

Emerging roles of ADAM and ADAMTS metalloproteinases in cancer

“…The activity of ADAM-8 and -9 is not controlled by any TIMP [30]. TIMP-3 has the ability to inhibit ag-grecanase activity by targeting ADAMTS-4 while TIMP-1 and TIMP-2 display such an effect at higher range of concentrations [31].…”

The Role of Synovial Macrophages and Macrophage-Produced Mediators in Driving Inflammatory and Destructive Responses in Osteoarthritis

“…The main endogenous inhibitor of ADAMTS4 and ADAMTS5 is tissue inhibitor of metalloproteinases (TIMP)-3, with K i values in the subnanomolar range (Kashiwagi et al, 2001). This inhibition may well be modulated by interactions between aggrecan and the Cterminal domain of ADAMTS4 (Wayne et al, 2007). Interestingly, reactive-site mutants of the N-terminal inhibitory domain of TIMP-3, also inhibit ADAMTS4 (Lim et al, 2010).…”

ADAMTS-5: The story so far

“…In vivo, there is increased aggrecan loss and increased NITEGE 373 neoepitope, concomitant with mild cartilage degradation in the TIMP-3 null mouse (Sahebjam et al, 2007), suggesting that TIMP-3 is indeed an endogenous aggrecanase inhibitor. TIMP-3 binds tightly to negatively charged glycosaminoglycans (Yu et al, 2000) and TIMP-3 potency against ADAMTS-4 is enhanced by ADAMTS-4 binding to aggrecan (Wayne et al, 2007); it appears that TIMP-3 has a greater affinity for ADAMTS-4 complexed with aggrecan, than it does for ADAMTS-4 alone (Wayne et al, 2007). To date there are no studies comparing the effect of aggrecan or CS on the affinity of TIMP-3 for ADAMTS-5.…”