Contributions of the MMP-2 collagen binding domain to gelatin cleavage. Substrate binding via the collagen binding domain is required for hydrolysis of gelatin but not short peptides.
Contributions of the MMP-2 collagen binding domain to gelatin cleavage. Substrate binding via the collagen binding domain is required for hydrolysis of gelatin but not short peptides.
复制标题
DOI：
10.1016/j.matbio.2004.05.002
复制
发表时间：
2004-06
期刊：
影响因子：
--
通讯作者：
Xiaoping Xu;Yao Wang;Janelle L. Lauer-Fields;G. Fields;B. Steffensen
中科院分区：
文献类型：
--
作者：
Xiaoping Xu;Yao Wang;Janelle L. Lauer-Fields;G. Fields;B. Steffensen
两种基质金属蛋白酶，MMP-2和MMP-9，分别含有三种纤维连接蛋白ii型样模块，形成它们的胶原结合域（CBDs）。CBD底物相互作用对这些明胶酶的催化活性的贡献引起了特别的兴趣。重组(r) CBDs保留胶原结合特性，这些MMPs中CBDs的缺失会降低胶原和弹性蛋白的活性。我们进一步表征了CBD对明胶的MMP-2裂解的要求。该分析使用完整的rMMP-2和rCBD来消除因缺失约20 kDa的内部CBD而引起的rMMP-2结构扰动可能导致的任何混杂效应。在蛋白结合试验中，2% DMSO破坏了rCBD和rMMP-2的明胶相互作用。在这个浓度下，DMSO还使明胶溶解活性降低了约70%，这表明在MMP-2裂解明胶过程中，cbd -底物相互作用发挥了核心作用。随后，可溶性rCBD被确定为竞争性地抑制未修饰的rMMP-2与明胶结合73%，并将明胶的MMP-2降解率降低70-80%。即使摩尔过量的rCBD也不能抑制残余明胶的裂解，这可以用短底物分子的降解来解释。事实上，rCBD抑制rMMP-2切割11个氨基酸的胶原样肽底物（NFF-1）的幅度小于10%。这些观察结果在小鼠实验肿瘤的酶提取物中得到证实。在rCBD存在的情况下，约65%的mmp源性明胶溶解活性被消除。总之，这些结果表明，CBD是MMP-2切割全长胶原α-链所必需的，而不是由明胶水解产生的短蛋白片段所必需的。
Two matrix metalloproteinases, MMP-2 and MMP-9, contain each three fibronectin type II-like modules, which form their collagen binding domains (CBDs). The contributions of CBD substrate interactions to the catalytic activities of these gelatinases have attracted special interest. Recombinant (r) CBDs retain collagen binding properties and deletions of CBDs in these MMPs reduce activities on collagen and elastin. We have characterized further the requirement of the CBD for MMP-2 cleavage of gelatin. The analyses used intact rMMP-2 and rCBD to eliminate any confounding effects that might result from structural perturbations in rMMP-2 induced by deletion of the ∼20 kDa internal CBD. In protein-protein binding assays, 2% DMSO disrupted gelatin interactions of both rCBD and rMMP-2. At this concentration, DMSO also reduced the gelatinolytic activity by ∼70%, pointing to a central role of CBD-substrate interactions during MMP-2 cleavage of gelatin. Subsequently, soluble rCBD was determined to competitively inhibit gelatin binding of unmodified rMMP-2 to gelatin by 73% and to reduce the MMP-2 degradation of gelatin by 70–80%. The residual gelatin cleavage that was not inhibited even by molar excess rCBD could be accounted for by degradation of short substrate molecules. Indeed, rCBD inhibited rMMP-2 cleavage of an 11 amino acid collagen-like peptide substrate (NFF-1) by less than 10%. These observations were confirmed with enzyme extracts from experimental tumors in mice. In the presence of rCBD, ∼65% of the MMP-derived gelatinolytic activity was eliminated. Together, these results demonstrate that the CBD is absolutely required for MMP-2 cleavage of full-length collagen α-chains, but not for short protein fragments such as those generated by hydrolysis of gelatin.