Hintze Vera, Höwel Markus, Wermter Carsten, Grosse Berkhoff Eva, Becker-Pauly Christoph, Beermann Bernd, Yiallouros Irene, Stöcker Walter
Department I, Cell and Matrix Biology, Institute of Zoology, Johannes Gutenberg-University, D-55099 Mainz, Germany.
Biochemistry. 2006 May 30;45(21):6741-8. doi: 10.1021/bi060228k.
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.
前胶原C蛋白酶（PCP）是虾红素家族和金属锌蛋白酶超家族的一种锌肽酶。该酶可去除原纤维前胶原的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外，前胶原C蛋白酶还包含几个C末端CUB结构域（以补体因子C1r和C1s、海胆UEGF蛋白以及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整体结构域组成上有所不同。较长的变体称为哺乳动物类Tolloid（mTld，即PCP-2），其结构组成为蛋白酶-CUB1-CUB2-EGF1-CUB3-EGF2-CUB4-CUB5，而较短的形式称为骨形态发生蛋白1（BMP-1，即PCP-1），在CUB3结构域后结束。两个相关基因在人类中编码与mTld相似的蛋白酶，分别被称为哺乳动物类Tolloid样-1和-2（mTll-1和mTll-2）。对于mTll-1，已证明它具有C蛋白酶活性。我们证明，前胶原C蛋白酶的重组EGF1-CUB3、CUB3、CUB3-EGF2、EGF2-CUB4和CUB4-CUB5结构域可在细菌中表达，并采用功能性反平行β折叠构象。表面等离子体共振分析表明，这些结构域以1:1的化学计量比与I型前胶原结合，解离常数（K(D)）范围为622.0至1.0 nM。它们与成熟I型胶原的结合至少弱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结构域的组合是最小功能单元。含EGF的结构域与前胶原的结合亲和力按EGF1-CUB3 < CUB3-EGF2 < EGF2-CUB4的顺序增加。在全长PCP的背景下，这意味着给定的结构域在PCP中越靠近C末端，其亲和力就会持续增加。结合最紧密的结构域EGF2-CUB4（K(D) = 1.0 nM）仅存在于mTld中，这可能为BMP-1和mTld在前胶原C蛋白酶活性方面的不同效率提供定量解释。
