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ADAMTS-4, also referred to as 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 (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M r ϳ68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M r ϳ53,000 and M r ϳ40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAGbinding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the Cterminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/ spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.Extracellular metalloproteases play a pivotal role in the proteolytic processing and turnover of the component molecules of a variety of tissues. Although a number of matrix metalloproteinases (MMPs) 1 may participate in such events, evidence for the involvement of A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases in these processes is increasing. For example, ADAMTS-2, ADAMTS-3, and ADAMTS-14 can function as procollagen N-proteinases (1-3), and ADAMTS-13 has been identified as a von Willebrand factor-cleaving protease (4 -6).Within the extracellular matrix of cartilage, ADAMTS-4 (7), ADAMTS-5 (8), and ADAMTS-1 (9 -11) may all potentially function as aggrecanases, glutamyl endopeptidases that cleave specific Glu-Xaa bonds of the aggrecan core protein (reviewed in (21)), indicating that regulation of the proteolytic activities of ADAMTS family members is likely to be important for maintenance of homeostasis in a variety of extracellular matrices. Unlike most of the MMPs, which are secreted in a state of latency conferred by the cysteine switch region of the retained propeptide (22), ADAMTS proteases can be cleaved N-terminally by furin or related pro-protein convertase(s) within the trans-Golgi, resulting in secretion of mature, potentially active enzymes lacking the propeptide region. Interestingly, however, ADAMTS family members such as ADAMTS-1 and AD-AMTS-12 have been shown to undergo proteolytic processing within their C-terminal regions, resulting in removal of domains that can bin...

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.Keywords: protein structure; enzymes; metalloproteins; aggrecanases Supplemental material: see www.proteinscience.org Osteoarthritis (OA) is a progressive disease that results in degradation of articular cartilage and chronic pain. The extracellular matrix is composed of two major components, aggrecan and collagen. Aggrecan is a large multidomain proteoglycan that provides cartilage with compressibility and elasticity by swelling and hydrating the collagen network (Vertel and Ratcliffe 2000). Loss of aggrecan is considered a critical early event in OA, occurring initially at the joint surface and progressing to the deeper zones. This is followed by degradation of collagen fibrils and mechanical failure of the tissue (Nagase and Kashiwagi 2003). Aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) gene family, cleave aggrecan at a unique site termed the ''aggrecanase site Tortorella et al. 1999). ADAMTS4 and ADAMTS5 are expressed in human normal and OA cartilage (Yamanishi et al. 2002) and in OA synovium, and contribute to the structural damage that characterizes human OA (Powell et al. 2007;Song et al. 2007). However, there is no consensus in the literature as to which aggrecanase is the most important in human OA. In mice, ADAMTS5 (but not ADAMTS4) is responsible for disease progression in a surgically induced model of OA (Glasson et al. 2004(Glasson et al. , 2005. ADAMTS4/ADAMTS5 double knockout mice are physiologically normal (Majumdar et al. 2007) and also protected from developing OA. Given the normal phenotype of the double knockout mice, dual inhibition Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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