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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.

Original publication

DOI

10.1074/jbc.m312123200

Type

Journal article

Journal

The Journal of biological chemistry

Publication Date

03/2004

Volume

279

Pages

10109 - 10119

Addresses

Kennedy Institute of Rheumatology Division, Imperial College London, United Kingdom.

Keywords

Cartilage, Articular, Cell Line, Tumor, Cell Membrane, Animals, Cattle, Swine, Humans, Metalloendopeptidases, Procollagen N-Endopeptidase, Chondroitin Sulfates, Proteoglycans, Alanine, Glutamic Acid, Transferrin, Carrier Proteins, Recombinant Proteins, Extracellular Matrix Proteins, Tissue Inhibitor of Metalloproteinase-1, DNA, Complementary, Interleukin-1, Epitopes, Blotting, Western, Electrophoresis, Polyacrylamide Gel, Cloning, Molecular, Gene Deletion, Binding Sites, Amino Acid Sequence, Catalytic Domain, Protein Structure, Tertiary, Protein Binding, Sequence Homology, Amino Acid, Substrate Specificity, Hydrolysis, Mutation, Genetic Vectors, Plasmids, Time Factors, Molecular Sequence Data, ADAM Proteins, Decorin, Fibromodulin, ADAMTS4 Protein