Vankemmelbeke M, Dekeyser P M, Hollander A P, Buttle D J, Demeester J
Institute for Bone & Joint Medicine, Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, U.K.
Biochem J. 1998 Mar 1;330 ( Pt 2)(Pt 2):633-40. doi: 10.1042/bj3300633.
Several vertebrate collagenases have been reported to cleave type II collagen, leading to irreversible tissue destruction in osteoarthritis. We have investigated the action of MMP-1 and MMP-13 on type II collagen 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 cleavage site, three residues carboxy-terminal to the secondary site. Since MMP-2 is thought to be responsible for gelatinolytic action on type II collagen we have investigated the effect of MMP-2 after the initial helical cleavage made by either MMP-1 or MMP-13. A combination of MMPs-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 will provide information about the specific identity of the collagenolytic enzymes involved in collagen cleavage in situ. Staining of cartilage sections of osteoarthritis patients with the neoepitope antibodies revealed type II collagen degradation starting at or near the articular surface and extending into the mid and deep zones with increasing degeneration of the cartilage.
据报道，几种脊椎动物胶原酶可切割Ⅱ型胶原蛋白，导致骨关节炎中组织的不可逆破坏。我们通过使用新表位抗体和N端测序研究了基质金属蛋白酶-1（MMP-1）和基质金属蛋白酶-13（MMP-13）对Ⅱ型胶原蛋白的作用。先前的研究表明，MMP-13对Ⅱ型胶原蛋白的初始切割之后，在初级切割位点的羧基末端三个氨基酸处会发生第二次切割。我们在此表明，这种切割也可由APMA激活的MMP-1与MMP-3（即完全激活的MMP-1）共同产生。使用MMP-3的选择性抑制剂表明，进行第二次切割的是这种酶，而非已暴露于MMP-3的间质胶原酶。此外，我们通过N端测序确定了第三个切割位点，位于二级位点的羧基末端三个残基处。由于MMP-2被认为负责对Ⅱ型胶原蛋白的明胶分解作用，我们研究了在由MMP-1或MMP-13进行初始螺旋切割后MMP-2的作用。MMP-1、-2和-3共同作用会产生第二个和第三个切割位点；将MMP-2添加到MMP-13中并不会改变MMP-13单独产生的切割模式。我们得出结论，这三个切割位点均无法提供有关原位胶原蛋白切割中所涉及的胶原分解酶具体身份的信息。用新表位抗体对骨关节炎患者的软骨切片进行染色显示，Ⅱ型胶原蛋白降解始于关节表面或其附近，并随着软骨退变加剧而延伸至中层和深层区域。
