CHARACTERIZATION OF HELICAL CLEAVAGES IN TYPE-II COLLAGEN GENERATED BY MATRIXINS

Several vertebrate collagenases have been reported to cleave type II c ollagen, leading to irreversible tissue destruction in osteoarthritis. We have investigated the action of MMP-1 and MMP-13 on type II collag en by use of neoepitope antibodies and N-terminal sequencing. Previous studies have suggested that the initial cleavage of type II collagen by MMP-13 is followed by a second cleavage, three amino acids carboxy- terminal to the primary cleavage site. We show here that this cleavage is also produced by APMA-activated MMP-1 in combination with MMP-3 (i .e. fully activated MMP-1). The use of a selective inhibitor of MMP-3 has shown that it is this enzyme, rather than interstitial collagenase which had been exposed to MMP-3, which makes the second cleavage. In addition we have identified, through N-terminal sequencing, a third cl eavage site, three residues carboxy-terminal to the secondary site. Si nce MMP-2 is thought to be responsible for gelatinolytic action on typ e TI collagen we have investigated the effect of MMP-2 after the initi al helical cleavage made by either MMP-1 or MMP-13. A combination of M MPs-1, -2 and -3 results in both the second and third cleavage sites; adding MMP-2 to MMP-13 did not alter the cleavage pattern produced by MMP-13 on its own. We conclude that none of the three cleavage sites w ill provide information about the specific identity of the collagenoly tic enzymes involved in collagen cleavage in situ. Staining of cartila ge sections of osteoarthritis patients with the neoepitope antibodies revealed type II collagen degradation starting at or near the articula r surface and extending into the mid and deep zones with increasing de generation of the cartilage.