Altered Proteolytic Activities of ADAMTS-4 Expressed by C-terminal Processing

• Published in: The Journal of biological chemistry Vol. 279; no. 11; pp. 10109 - 10119
• Main Authors: Kashiwagi, Masahide, Enghild, Jan J., Gendron, Christi, Hughes, Clare, Caterson, Bruce, Itoh, Yoshifumi, Nagase, Hideaki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.03.2004; American Society for Biochemistry and Molecular Biology
• Subjects: ADAM Proteins; ADAMTS4 Protein; aggrecanase; Alanine - chemistry; Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Carrier Proteins - chemistry; Cartilage, Articular - metabolism; Catalytic Domain; Cattle; Cell Line, Tumor; Cell Membrane - metabolism; Chondroitin Sulfates - chemistry; Cloning, Molecular; Decorin; DNA, Complementary - metabolism; Electrophoresis, Polyacrylamide Gel; Epitopes - chemistry; Extracellular Matrix Proteins; Fibromodulin; Gene Deletion; Genetic Vectors; Glutamic Acid - chemistry; Humans; Hydrolysis; Interleukin-1 - metabolism; Metalloendopeptidases - chemistry; Metalloendopeptidases - metabolism; Molecular Sequence Data; Mutation; Plasmids - metabolism; Procollagen N-Endopeptidase; Protein Binding; Protein Structure, Tertiary; Proteoglycans - chemistry; Recombinant Proteins - chemistry; Sequence Homology, Amino Acid; Substrate Specificity; Sus scrofa; Swine; Time Factors; Tissue Inhibitor of Metalloproteinase-1 - metabolism; Transferrin - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M312123200

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 Glu373-Ala374 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 Glu1480-Gly1481 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 Glu373-Ala374 and Glu1480-Gly1481 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-1α-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.