THE INTERGLOBULAR DOMAIN OF CARTILAGE AGGRECAN IS CLEAVED BY HEMORRHAGIC METALLOPROTEINASE HT-D (ATROLYSIN-C) AT THE MATRIX METALLOPROTEINASE AND AGGRECANASE SITES

Two primary cleavage sites have been identified within the interglobul ar domain of the cartilage aggrecan core protein: one is between amino acid residues Asn(341) and Phe(342), where many matrix metalloprotein ases (MMP) have been shown to cleave; and the other is between amino a cid residues Glu(373) and Ala(374). Although cleavage ma, the Glu(373) -Ala(374) site is believed to play a critical role in cartilage aggrec an degradation in arthritic diseases, the enzyme responsible for cleav age at this site, ''aggrecanase,'' has not been identified, Members of the ADAM (a disintegrin and metalloproteinase) family of proteins, wh ich shows structural homology to the snake venom hemorrhagic metallopr oteinases (reprolysins), have recently been demonstrated to be express ed in articular chondrocytes. Because many ADAM family members have a putative proteinase function, this raises the possibility that aggreca nase may be a member of this family of proteases. Tea examine whether reprolysins have the ability to cleave aggrecan at either the aggrecan ase site or the MMP site, the snake venom hemorrhagic toxin metallopro teinase HT-d (atrolysin C) was tested for its ability to cleave bovine aggrecan monomer. Cleavage was monitored using the BC-3 antibody, whi ch recognizes aggrecan fragments with the neu NH2 terminus ARGSV gener ated by cleavage at the aggrecanase site, and with the AF-28 antibody, which recognizes aggrecan fragments with the new NH2 terminus FFGVG g enerated lay cleavage at the MMP site, Cleavage at both the aggrecanas e and MMP sites occurred in a concentration-dependent manner with 100 net atrolysin C or greater, AF-28-reactive fragments were generated by 30 min of incubation, and levels were maximal by 8 h; BC-3-reactive f ragments were detected at 2 h and continued to increase through 48 h, thus suggesting that atrolysin C can cleave at the MMP and aggrecanase sites, NH2-terminal aggrecan fragments generated by cleavage at the a ggrecanase site were also detected using antisera recognizing the new COOH terminus, NITEGE, formed by cleavage at the Glu(373)-Ala(374) bon d, indicating that cleavage at this site does not require prior cleava ge at the MMP site, These data provide the first demonstration that a reprolysin can cleave the core protein of aggrecan and the first examp le of a specific protease that can cleave at the first example site in dependent of cleavage at the MMP cleavage site.