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Adamalysin-like metalloproteinases with thrombospondin (TS) motifs (ADAMTS)-5 is the multi-domain metalloproteinase that most potently degrades aggrecan proteoglycan in the cartilage and its activity is implicated in the development of osteoarthritis (OA). To generate specific exosite inhibitors for it, we screened a phage display antibody library in the presence of the zinc-chelating active site-directed inhibitor GM6001 (Ilomastat) and isolated four highly selective inhibitory antibodies. Two antibodies were mapped to react with exosites in the catalytic/disintegrin domains (Cat/Dis) of the enzyme, one in the TS domain and one in the spacer domain (Sp). The antibody reacting with the Sp blocked the enzyme action only when aggrecan or the Escherichia coli-expressed aggrecan core protein were substrates, but not against a peptide substrate. The study with this antibody revealed the importance of the Sp for effective aggrecanolytic activity of ADAMTS-5 and that this domain does not interact with sulfated glycosaminoglycans (GAGs) but with the protein moiety of the proteoglycan. An antibody directed against the Cat/Dis of ADAMTS-5 was effective in a cell-based model of aggrecan degradation; however, the anti-Sp antibody was ineffective. Western blot analysis of endogenous ADAMTS-5 expressed by human chondrocytes showed the presence largely of truncated forms of ADAMTS-5, thus explaining the lack of efficacy of the anti-Sp antibody. The possibility of ADAMTS-5 truncation must then be taken into account when considering developing anti-ancillary domain antibodies for therapeutic purposes.

Original publication

DOI

10.1042/BJ20150758

Type

Journal article

Journal

Biochem J

Publication Date

01/11/2015

Volume

471

Pages

391 - 401

Keywords

adamalysin-like metalloproteinases with thrombospondin motifs (ADAMTS)-5, aggrecan, exosite inhibition, osteoarthritis, phage display, substrate selectivity, ADAM Proteins, ADAMTS5 Protein, Aggrecans, Antibodies, Binding Sites, Cartilage, Catalytic Domain, Cell Surface Display Techniques, Chondrocytes, Dipeptides, Humans, Osteoarthritis, Protein Binding, Protein Structure, Tertiary, Substrate Specificity