Inhibition of MMP-2 gelatinolysis by targeting exodomain-substrate interactions.
Inhibition of MMP-2 gelatinolysis by targeting exodomain-substrate interactions.
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DOI：
10.1042/bj20070591
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发表时间：
2007-08
期刊：
影响因子：
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通讯作者：
Xiaoping Xu;Zhihua Chen;Yao Wang;L. Bonewald;B. Steffensen
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文献类型：
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作者：
Xiaoping Xu;Zhihua Chen;Yao Wang;L. Bonewald;B. Steffensen
基质金属蛋白酶2(MMP2)含有一个胶原结合结构域(CBD)，该结构域对于明胶底物分子相对于切割催化部位的定位是必不可少的。CBD的缺失或CBD介导的明胶结合的破坏会抑制基质金属蛋白酶-2对明胶的分解。为了确定I型胶原和胶原肽上的CBD结合部位，使其与明胶竞争CBD结合，从而抑制明胶对明胶的降解，我们筛选了以重组CBD为诱饵的单珠单肽组合多肽文库。对CBD结合肽的序列分析表明，人α1(I)胶原链上的715-721残基是CBD的结合部位。合成了含有该胶原蛋白片段的多肽(P713)进行分析。在SPR(表面等离子共振)分析中，CBD和MMP2(E404A)，一个催化失活的MMP2突变体，都以浓度依赖的方式结合固定化的P713，而不是一个杂乱的控制肽。此外，P713还与CbD和MMP2(E404A)竞争结合明胶。在对照实验中，非胶原结合的烷基化CBD或缺失CBD的基质金属蛋白酶-2(MMP-2DeltaCBD)均未与P713结合。与CBD的外域功能一致，P713抑制了大约90%的MMP-2明胶切割，但不到20%的MMP-2在不需要CBD进行切割的多肽底物(NFF-1)上的活性。P713不改变MMP2DeltaCBD或MMP8的活性，证实了抑制的特异性。这些实验确定了I型胶原上的CBD结合部位，并证明了相应的合成肽可以通过竞争CBD介导的明胶与基质金属蛋白酶-2的结合来抑制I型和IV型胶原的水解。
MMP-2 (matrix metalloproteinase 2) contains a CBD (collagen-binding domain), which is essential for positioning gelatin substrate molecules relative to the catalytic site for cleavage. Deletion of the CBD or disruption of CBD-mediated gelatin binding inhibits gelatinolysis by MMP-2. To identify CBD-binding sites on type I collagen and collagen peptides with the capacity to compete CBD binding of gelatin and thereby inhibit gelatinolysis by MMP-2, we screened a one-bead one-peptide combinatorial peptide library with recombinant CBD as bait. Analyses of sequences from the CBD-binding peptides pointed to residues 715-721 in human alpha1(I) collagen chain as a binding site for CBD. A peptide (P713) including this collagen segment was synthesized for analyses. In SPR (surface plasmon resonance) assays, the CBD and MMP-2(E404A), a catalytically inactive MMP-2 mutant, both bound immobilized P713 in a concentration-dependent manner, but not a scrambled control peptide. Furthermore, P713 competed gelatin binding by the CBD and MMP-2(E404A). In control assays, neither of the non-collagen binding alkylated CBD or MMP-2 with deletion of CBD (MMP-2DeltaCBD) bound P713. Consistent with the exodomain functions of the CBD, P713 inhibited approximately 90% of the MMP-2 gelatin cleavage, but less than 20% of the MMP-2 activity on a peptide substrate (NFF-1) which does not require the CBD for cleavage. Confirming the specificity of the inhibition, P713 did not alter MMP-2DeltaCBD or MMP-8 activities. These experiments identified a CBD-binding site on type I collagen and demonstrated that a corresponding synthetic peptide can inhibit hydrolysis of type I and IV collagens by competing CBD-mediated gelatin binding to MMP-2.