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) approximately 68,000) undergoes autocatalytic C-terminal truncation to generate two discrete
isoforms (M(r) approximately 53,000 and M(r) approximately 40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAG-binding
thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the C-terminal
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.