Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system.
Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system.
复制标题
使用细菌胶原蛋白系统定义了胶原蛋白III胶原蛋白酶裂解的要求。
DOI：
10.1074/jbc.m112.348979
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发表时间：
2012-06-29
期刊：
影响因子：
--
通讯作者：
Brodsky B
中科院分区：
文献类型：
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作者：
Yu Z;Visse R;Inouye M;Nagase H;Brodsky B
背景：三螺旋胶原蛋白的结构要求，胶原蛋白溶解尚未完全理解。结果：重组细菌胶原蛋白与人胶原蛋白III序列插入定义的最小序列切割的人胶原酶。结论：细菌-人胶原嵌合体对胶原酶的敏感性与人胶原III相似。意义：该重组系统可用于研究三螺旋背景下胶原片段的生物学功能。纤维状胶原的降解在许多生理和病理事件中是重要的。这些胶原蛋白由于紧密堆积的三螺旋结构而对大多数蛋白酶具有抗性，但容易在特定位点被胶原酶（基质金属蛋白酶（MMP）的选定成员）切割。为了研究胶原溶解的结构要求，将来自胶原酶切割位点周围的人III型胶原的不同数量的GXY三联体插入Scl 2细菌胶原蛋白的两个三螺旋结构域之间。原始细菌CL结构域不被MMP-1（胶原酶1）或MMP-13（胶原酶3）切割。两种胶原酶切割所需的最小III型序列为5个GXY三联体，包括切割位点前4个残基和切割位点后11个残基（P4-P11′）。这些嵌合底物的切割不能通过MMP-1或MMP-13的催化结构域实现，也不能通过全长MMP-3实现。嵌合体的动力学分析表明，含有6个III型胶原三联体（P7-P11′）的嵌合体的MMP-1裂解速率与天然III型胶原的裂解速率相似。胶原酶敏感的嵌合体被胰蛋白酶切割得非常缓慢，这也是天然胶原III的一种特性，支持胶原酶切割位点附近三螺旋的局部结构松弛。本研究所建立的重组细菌-人胶原蛋白系统是研究胶原酶的特异性和作用机制的良好模型。
Background: Structural requirements of triple-helical collagen for collagenolysis are not fully understood. Results: Recombinant bacterial collagens with human collagen III sequence insertions defined the minimum sequence for cleavage by human collagenases. Conclusion: Susceptibility of bacterial-human collagen chimeras to collagenases mimicked that of human collagen III. Significance: This recombinant system is useful to investigate biological functions of collagen segments in a triple-helical context. Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11′). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11′) of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.