Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4)

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity; is one of the first matrix co mponents to undergo measurable loss in arthritic diseases. Two major sites of proteolytic cleavage have been identified within the interglobular domai n (IGD) of the aggrecan core protein, one between amino acids Asn(341)-Phe( 342) which is cleaved by matrix metalloproteinases and the other between Gl u(373)-Ala(374) that is attributed to aggrecanase. Although several potenti al aggrecanase-sensitive sites had been identified within the COOH terminus of aggrecan, demonstration that aggrecanase cleaved at these sites awaited isolation and purification of this protease. We have recently cloned human aggrecanase-1 (ADAMTS-4) (Tortorella, M. D., Burn, T. C., Pratta, M. A., A bbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., De cicco, C. P., Wynn, R., Rockwell, A, Yang, F., Duke, J. L., SoIomon, K., Ge orge, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Boss, H., Wiswa ll, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., Newton, a C., Ma golda, R L., Trzaskos, J. M, and Arner, E. C. (1999) Science 284, 1664-1666 ) and herein demonstrate that in addition to cleavage at the Glu(373)-Ala(3 74) bond, this protease cleaves at four sites within the chondroitin-sulfat e rich region of the aggrecan core protein, between G2 and G3 globular doma ins. importantly, we show that this cleavage occurs more efficiently than c leavage within the IGD at the Glu(373)-Ala(374) bond. Cleavage occurred pre ferentially at the KEEE(1667-1668)GLGS bond to produce both a 140-kDa COOH- terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavag e also occurred at the GELE(1480-1481)GRGT bond to produce a 55-kDa COOH-te rminal fragment and a G1-containing fragment of 320 hDa Cleavage of this 32 0-kDa fragment within the IGD at the Glu(373)-Ala(374) bond then occurred t o release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kD a GLGS-reactive fragment resulting from the preferential cleavage was furth er processed at two additional cleavage sites, at TAQE(1771-1172)AGEG and a t VSQE(1871-1872)LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of similar to 20 kDa. These dat a elucidate the sites and efficiency of cleavage during aggrecan degradatio n by aggrecanase and suggest potential tools for monitoring aggrecan cleava ge in arthritis.