Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing.

Journal article

ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu(373)-Ala(374) bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu(1480)-Gly(1481) bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1alpha-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.

Authors: Kashiwagi, M; Enghild, J; Gendron, C; Hughes, C; Caterson, B

• Publication status: Published
• Journal: Journal of biological chemistry More from this journal
• Volume: 279
• Issue: 11
• Pages: 10109-10119
• Publication date: 2004-03-01
• DOI: 10.1074/jbc.m312123200
• EISSN: 1083-351X
• ISSN: 0021-9258
• Language: English
• Keywords: Molecular Sequence Data; Blotting, Western; Mutation; Interleukin-1; Epitopes; Transferrin; Cell Membrane; Substrate Specificity; Decorin; Time Factors; Catalytic Domain; Hydrolysis; Protein Binding; DNA, Complementary; Recombinant Proteins; Metalloendopeptidases; Cartilage, Articular; ADAM Proteins; Cattle; Proteoglycans; Cloning, Molecular; Alanine; Glutamic Acid; Gene Deletion; Binding Sites; Extracellular Matrix Proteins; Carrier Proteins; Humans; Protein Structure, Tertiary; Genetic Vectors; Procollagen N-Endopeptidase; Animals; Swine; Plasmids; Tissue Inhibitor of Metalloproteinase-1; Chondroitin Sulfates; Cell Line, Tumor; Amino Acid Sequence; Electrophoresis, Polyacrylamide Gel; Sequence Homology, Amino Acid