The Thrombospondin Motif of Aggrecanase-1 (ADAMTS-4) Is Critical for Aggrecan Substrate Recognition and Cleavage

• Published in: The Journal of biological chemistry Vol. 275; no. 33; pp. 25791 - 25797
• Main Authors: Tortorella, Micky, Pratta, Michael, Liu, Rui-Qin, Abbaszade, Ilgar, Ross, Harold, Burn, Timothy, Arner, Elizabeth
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.08.2000; American Society for Biochemistry and Molecular Biology
• Subjects: ADAM Proteins; ADAMTS4 Protein; Aggrecans; Amino Acid Motifs; Amino Acid Sequence; Animals; Antibodies, Monoclonal - metabolism; Cattle; Cell Line; Dose-Response Relationship, Drug; Drosophila; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Glycosylation; Inhibitory Concentration 50; Kinetics; Lectins, C-Type; Metalloendopeptidases - chemistry; Molecular Sequence Data; Peptides - metabolism; Procollagen N-Endopeptidase; Protein Binding; Proteoglycans - metabolism; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Thrombospondin 1 - chemistry; Thrombospondins - chemistry; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M001065200

Aggrecanase-1 (ADAMTS-4) is a member of theadisintegrin andmetalloprotease withthrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., and Arner, E.C. (1999) Science 284, 1664–1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem. 274, 23443–23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.