Interaction of recombinant subdomains of the procollagen C-proteinase with procollagen I provides a quantitative explanation for functional differences between the two splice variants, mammalian tolloid and bone morphogenetic protein 1
Interaction of recombinant subdomains of the procollagen C-proteinase with procollagen I provides a quantitative explanation for functional differences between the two splice variants, mammalian tolloid and bone morphogenetic protein 1
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DOI：
10.1021/bi060228k
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发表时间：
2006-05-30
期刊：
影响因子：
2.9
通讯作者：
Stöcker, Walter
中科院分区：
文献类型：
--
作者：
Hintze, Vera;Höwel, Markus;Stöcker, Walter
前胶原C蛋白酶（PCP）是一种属于虾红素家族和甲锌素超家族的锌肽酶。该酶去除纤维原胶原的C-末端前肽并激活其他基质蛋白。除了其催化蛋白酶结构域，前胶原C蛋白酶还含有几个C-末端CUB模块（以补体因子C1 r和C1 s、海胆UEGF蛋白和BMP-1命名）和EGF样结构域。C-蛋白酶的两种主要剪接形式在其总体结构域组成上不同。较长的变体，称为哺乳动物tolloid（mTld，即，PCP-2）具有蛋白酶-CUB 1-CUB 2-EGF 1-CUB 3-EGF 2-CUB 4-CUB 5组成，而较短形式称为骨形态发生蛋白1（BMP-1，即，PCP-1）在CUB 3结构域之后结束。两个相关基因编码类似于人类mTld的蛋白酶，并被称为哺乳动物tolloid样-1和-2（分别为mTll-1和mTll-2）。对于mTll-1，已证明其具有C-蛋白酶活性。我们证明，重组EGF 1-CUB 3，CUB 3，CUB 3-EGF 2，EGF 2-CUB 4，和CUB 4-CUB 5模块的前胶原C蛋白酶可以在细菌中表达，并采取功能性反平行β折叠构象。如通过表面等离子体共振分析所示，模块以1：1的化学计量比与原胶原I结合，解离常数（K-D）范围为622.0至1.0 nM。它们与成熟胶原蛋白I的结合弱至少1个数量级。含有EGF结构域的构建体比仅由CUB结构域组成的构建体结合更强。这表明CUB和EGF结构域的组合作为最小功能单位。含EGF的模块对前胶原的结合亲和力以EGF 1-CUB 3 <CUB 3-EGF 2 <EGF 2-CUB 4的顺序增加。在全长PCP的情况下，这意味着给定模块具有的亲和力持续增加，该模块位于PCP内的C末端越多。最紧密的结合模块EGF 2-CUB 4（K-D = 1.0 nM）仅存在于mTld中，这可能为BMP-1和mTld在前胶原C蛋白酶活性中的不同效率提供定量解释。
The procollagen C-proteinase (PCP) is a zinc peptidase of the astacin family and the metzincin superfamily. The enzyme removes the C-terminal propeptides of fibrillar procollagens and activates other matrix proteins. Besides its catalytic protease domain, the procollagen C-proteinase contains several C-terminal CUB modules (named after complement factors C1r and C1s, the sea urchin UEGF protein, and BMP-1) and EGF-like domains. The two major splice forms of the C-proteinase differ in their overall domain composition. The longer variant, termed mammalian tolloid (mTld, i.e., PCP-2), has the protease-CUB1- CUB2-EGF1-CUB3-EGF2-CUB4-CUB5 composition, whereas the shorter form termed bone morphogenetic protein 1 (BMP-1, i.e., PCP-1) ends after the CUB3 domain. Two related genes encode proteases similar to mTld in humans and have been termed mammalian tolloid like-1 and -2 (mTll-1 and mTll-2, respectively). For mTll-1, it has been shown that it has C-proteinase activity. We demonstrate that recombinant EGF1-CUB3,CUB3, CUB3-EGF2, EGF2-CUB4, and CUB4-CUB5 modules of the procollagen C-proteinase can be expressed in bacteria and adopt a functional antiparallel beta-sheet conformation. As shown by surface plasmon resonance analysis, the modules bind to procollagen I in a 1:1 stoichiometry with dissociation constants (K-D) ranging from 622.0 to 1.0 nM. Their binding to mature collagen I is weaker by at least 1 order of magnitude. Constructs containing EGF domains bind more strongly than those consisting of CUB domains only. This suggests that a combination of CUB and EGF domains serves as the minimal functional unit. The binding affinities of the EGF-containing modules for procollagen increase in the order EGF1-CUB3 < CUB3-EGF2 < EGF2-CUB4. In the context of the full length PCP, this implies that a given module has an affinity that continues to increase the more C-terminally the module is located within the PCP. The tightest binding module, EGF2-CUB4 (K-D = 1.0 nM), is only present in mTld, which might provide a quantitative explanation for the different efficiencies of BMP-1 and mTld in procollagen C-proteinase activity.