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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




Journal article


J biol chem

Publication Date





10109 - 10119


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