Autocatalytic Cleavage of ADAMTS-4 (Aggrecanase-1) Reveals Multiple Glycosaminoglycan-binding Sites

• Published in: The Journal of biological chemistry Vol. 277; no. 45; pp. 42775 - 42780
• Main Authors: Flannery, Carl R, Zeng, Weilan, Corcoran, Chris, Collins-Racie, Lisa A, Chockalingam, Priya S, Hebert, Tracy, Mackie, Stewart A, McDonagh, Thomas, Crawford, Tara K, Tomkinson, Kathy N, LaVallie, Edward R, Morris, Elisabeth A
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 08.11.2002
• Subjects: ADAM Proteins; ADAMTS4 Protein; Amino Acid Sequence; Binding Sites; Catalysis; Cysteine; Glycosaminoglycans - chemistry; Humans; Hydrolysis; Isoenzymes - chemistry; Isoenzymes - metabolism; Kinetics; Metalloendopeptidases - chemistry; Metalloendopeptidases - metabolism; Molecular Sequence Data; Molecular Weight; Procollagen N-Endopeptidase; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M205309200

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 ∼68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms ( M r ∼53,000 and M r ∼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.