Abstract
Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin 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.
Highlights
Aggrecan is a large chondroitin sulfate proteoglycan that accounts for about 10% of the dry weight of cartilage (11, 12)
We recently demonstrated that deglycosylation of aggrecan decreases the ability of aggrecanase activity in culture media to generate aggrecan fragments formed by cleavage at the Glu373/Ala374 bond (30),3 which is consistent with this hypothesis
Evaluation of these reaction mixtures for general cleavage by Western analysis using the MAB2035 antibody showed no decrease in intensity of the band representing intact aggrecan and no appearance of smaller molecular mass bands when GAG-free aggrecan was used as the substrate (Fig. 7D). These data confirm lack of cleavage of GAG-free aggrecan at these sites and indicate that aggrecanase-1 did not cleave GAG-free aggrecan at any other sites within the molecule over this time period. These studies clearly demonstrate that the TSP-1 motif located within the C terminus of aggrecanase-1 is important for aggrecan binding and cleavage
Summary
Aggrecan is a large chondroitin sulfate proteoglycan that accounts for about 10% of the dry weight of cartilage (11, 12) It consists of three globular domains: G1, through which it interacts with hyaluronan (HA); G2; and G3 at the C terminus of the molecule. It has been reported that the TSP-1 motifs of murine ADAMTS-1 bind to heparin and the extracellular matrix (9, 29) This opens the possibility that this region in aggrecanase-1 may serve to bind the enzyme to the glycosaminoglycans of the aggrecan molecule. The goal of the studies reported was to determine whether aggrecanase-1 recognizes aggrecan through binding of the TSP-1 motif to the aggrecan glycosaminoglycans, chondroitin sulfate and keratan sulfate, and whether binding of the enzyme to these glycosaminoglycans is important for cleavage of the core protein
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