The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotea se with thrombospondin motifs (ADAMTS) protein family that was recently ide ntified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes pur ified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. Ri., Liu, R., Ros enfeld, S. A,, Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A, Yang , F., Duke, J. L., Solomon, K., George, R, Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Boss, 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., Boss, O. H., Link, J. R., Ell is, 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., Cop eland, 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 fou nd in cartilage cultures stimulated with cytokines and in synovial fluid fr om patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the t hrombospondin type-1 (TSP-1) motif located within the C terminus of aggreca nase-1 binds to the glycosaminoglycans of aggrecan. Data from several studi es indicate that this binding of aggrecanase-1 to aggrecan through the TSP- 1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated for m of aggrecanase-1 lacking the TSP-I motif was not effective in cleaving ag grecan. 2) Several peptides representing different regions of the TSP-1 mot if effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective i n cleaving glycosaminoglycan-free aggrecan. Taken together, these data sugg est that the TSP-1 motif of aggrecanase-1 is critical for substrate recogni tion and cleavage.