Higashi Shouichi, Miyazaki Kaoru
Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama, Japan.
J Biol Chem. 2008 Apr 11;283(15):10068-78. doi: 10.1074/jbc.M709509200. Epub 2008 Jan 31.
The extracellular domain of beta-amyloid precursor protein (APP) contains an inhibitor against matrix metalloproteinase-2 (MMP-2, gelatinase A). Our previous study ( Higashi, S. and Miyazaki, K. (2003) J Biol Chem 278, 14020-14028 ) demonstrated that the inhibitor is localized within the ISYGN-DALMP sequence of APP, and a synthetic decapeptide containing this sequence (named APP-derived inhibitory peptide, APP-IP) selectively inhibits the activity of MMP-2. To determine the region of interaction that correlates with the selective inhibition, we constructed various MMP-2 mutants. An MMP-2 mutant, which had the hemopexin-like domain and three fibronectin-like type II domains of MMP-2 deleted, and native MMP-2 showed similar affinities for APP-IP, suggesting that only the catalytic domain of MMP-2 is essential for the interaction. Studies of chimeric proteases, consisting of various parts of the MMP-2 catalytic domain and those of MMP-7 (matrilysin) or MMP-9 (gelatinase B), further revealed that Ala(88) and Gly(94) in the non-prime side and Tyr(145) and Thr(146) in the prime side of the substrate-binding cleft of MMP-2 contribute separately to the selective inhibition. Replacement of the amino acid residue at position 94 of a chimeric MMP mutant affected its interaction with the C-terminal Pro(10) of APP-IP, whereas that of residues 145-148 affected the interaction with Tyr(3) of the inhibitor, suggesting that the N to C direction of APP-IP relative to the substrate-binding cleft of MMP is analogous to that of propeptide in proMMP, and opposite to that of substrate. When the APP-IP sequence was added to the N terminus of the catalytic domain of MMP-2, the activity of the protease was intramolecularly inhibited. We speculate that the direction of interaction makes the active site-bound APP-IP resistant to cleavage, thereby supporting the inhibitory action of the peptide inhibitor.
β-淀粉样前体蛋白（APP）的细胞外结构域含有一种针对基质金属蛋白酶-2（MMP-2，明胶酶A）的抑制剂。我们之前的研究（Higashi, S.和Miyazaki, K.（2003年）《生物化学杂志》278卷，14020 - 14028页）表明，该抑制剂定位于APP的ISYGN - DALMP序列内，并且包含此序列的合成十肽（命名为APP衍生抑制肽，APP - IP）可选择性抑制MMP - 2的活性。为了确定与选择性抑制相关的相互作用区域，我们构建了各种MMP - 2突变体。一种缺失了MMP - 2的血色素结合蛋白样结构域和三个纤连蛋白样II型结构域的MMP - 2突变体，与天然MMP - 2对APP - IP表现出相似的亲和力，这表明只有MMP - 2的催化结构域对于这种相互作用是必不可少的。对由MMP - 2催化结构域的各个部分与MMP - 7（基质溶素）或MMP - 9（明胶酶B）的相应部分组成的嵌合蛋白酶的研究进一步表明，MMP - 2底物结合裂隙非切割侧的Ala(88)和Gly(94)以及切割侧的Tyr(145)和Thr(146)分别对选择性抑制有贡献。嵌合MMP突变体第94位氨基酸残基的替换影响其与APP - IP C末端Pro(10)的相互作用，而第145 - 148位残基的替换影响其与抑制剂Tyr(3)的相互作用，这表明相对于MMP的底物结合裂隙，APP - IP从N端到C端的方向类似于前MMP中前肽的方向，与底物的方向相反。当将APP - IP序列添加到MMP - 2催化结构域的N端时，蛋白酶的活性受到分子内抑制。我们推测这种相互作用的方向使结合在活性位点的APP - IP抵抗切割，从而支持肽抑制剂的抑制作用。
