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...