Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display
Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display
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DOI：
10.1074/jbc.m304436200
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发表时间：
2003-07-25
影响因子：
4.8
通讯作者：
Albright, CF
中科院分区：
文献类型：
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作者：
Pan, WJ;Arnone, M;Albright, CF
MMP-11蛋白酶，也被称为基质溶素-3，可能在人类癌症中起重要作用，因为MMP-11在人类肿瘤中经常过度表达，而MMP-11水平影响小鼠的肿瘤发生。然而，与其他MMPs不同，人MMP-11不切割细胞外基质蛋白，如胶原蛋白、层粘连蛋白、纤维连接蛋白和弹性蛋白。为了帮助鉴定生生性MMP-11底物，利用噬菌体展示文库寻找MMP-11的肽底物。一类包含26个成员的肽具有一致的序列A(A/Q)（N/A）向下箭头（L/Y）（T/V/M/R）（R/K），其中向下箭头表示裂解位点。这一共识序列与其他MMP相似，它们也切割含有3号位置Ala， 1号位置Ala和1号位置Leu/Tyr的肽，但与大多数其他MMP底物不同的是，脯氨酸很少出现在3号位置，Asn经常出现在1号位置。第二类包含四个成员的肽的一致序列为G(G/A)E向下箭头LR。虽然其他的MMPs也用这些残基切割肽，但其他MMPs更倾向于在该序列的第3位上切割脯氨酸。与其他具有首选肽底物的MMPs相比，用两类MMP-11和代表性肽进行的体外测定获得了适度的k(cat)/ k -m值。这些反应还表明，脯氨酸位于3号位置的肽是MMP-11的不良底物。与其他MMPs相比，人类MMP-11的k(cat)/ k -m值较低，并且MMP-11对3位脯氨酸底物的裂解能力较差，这为其提供了结构基础。综上所述，这些发现解释了为什么MMP-11不能切割大多数其他MMP底物，并预测MMP-11具有可能导致人类癌症的独特底物。
The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) down arrow (L/Y)(T/V/M/R)(R/K), where down arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E down arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest k(cat)/K-m values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower k(cat)/K-m values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.