Deletion of epidermal growth factor-like domains converts mammalian tolloid into a chordinase and effective procollagen C-proteinase
Deletion of epidermal growth factor-like domains converts mammalian tolloid into a chordinase and effective procollagen C-proteinase
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DOI：
10.1074/jbc.m408134200
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发表时间：
2004-11-26
影响因子：
4.8
通讯作者：
Kadler, KE
中科院分区：
文献类型：
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作者：
Garrigue-Antar, L;François, V;Kadler, KE
骨形态发生蛋白（BMP）-1和哺乳动物tolloid（mTld）是由bmp 1基因的选择性剪接产生的Ca 2+依赖性金属蛋白酶。它们具有不同的蛋白酶活性，例如BMP-1有效地切割前胶原（一种细胞外基质蛋白）和腱蛋白（一种BMP拮抗剂），而mTld是一种差的前胶原蛋白酶，并且在没有扭曲原肠胚形成的情况下不会切割腱蛋白。这是令人困惑的，因为mTld（是较长的变体）可能被预期切割所有被BMP-1切割的底物。研究表明，前胶原蛋白酶活性的最小结构是蛋白酶-CUB1-CUB2（BMP-1 DeltaEC 3），因此缺乏被认为是BMP-1的Ca2+依赖性的表皮生长因子（EGF）样结构域。在这项研究中，我们产生了三个缺失突变体的mTld，缺乏一个或两个EGF样结构域（称为"mTld-DeltaEGF"）。突变的蛋白质从293-EBNA细胞中分泌很少，但足以用于前胶原和腱蛋白切割的体外测定。最令人惊讶的是，mTld-DeltaEGF突变体需要Ca2+来进行蛋白水解活性，从而表明EGF样结构域不能解释BMP-1/mTld的Ca2+依赖性。此外，mTld-DeltaEGF是有效的前胶原蛋白酶并切割腱蛋白。此外，BMP-1 DeltaEC 3切割脊索蛋白，并且需要Ca2+来进行活性。使用非变性凝胶的研究表明，缺乏EGF样结构域的mTld分子具有松散的构象，使得在存在Ca2+的情况下，暴露分子的"BMP-1-部分"上的脊索蛋白和前胶原的结合位点。我们提出，EGF样结构域可以保持CUB4/5结构域的位置，排除底物可裂解的BMP-1。
Bone morphogenetic protein (BMP)-1 and mammalian tolloid (mTld) are Ca2+-dependent metalloproteinases that result from alternative splicing of the bmp1 gene. They have different proteinase activities, e.g. BMP-1 effectively cleaves procollagen (an extracellular matrix protein) and chordin (a BMP antagonist), whereas mTld is a poor procollagen proteinase and will not cleave chordin in the absence of twisted gastrulation. This is perplexing because mTld (being the longer variant) might be expected to cleave all substrates cleaved by BMP-1. Studies have shown that the minimal structure for procollagen proteinase activity is proteinase-CUB1-CUB2 (BMP-1DeltaEC3) and therefore lacking the epidermal growth factor (EGF)-like domain thought to account for the Ca2+ dependence of BMP-1. In this study we generated three deletion mutants of mTld that lacked either one or both EGF-like domains (referred to as "mTld-DeltaEGF"). The mutated proteins were poorly but sufficiently secreted from 293-EBNA cells for in vitro assays of procollagen and chordin cleavage. Most surprisingly, the mTld-DeltaEGF mutants required Ca2+ for proteolytic activity, thereby showing that the EGF-like domains do not account for the Ca2+ dependence of BMP-1/mTld. Moreover, the mTld-DeltaEGFs are effective procollagen proteinases and cleave chordin. Furthermore, BMP-1DeltaEC3 cleaves chordin and requires Ca2+ for activity. Studies using nondenaturing gels showed that mTld molecules lacking EGF-like domains have a loose conformation such that in the presence of Ca2+ binding sites for chordin and procollagen on the "BMP-1-part" of the molecule are exposed. We propose that the EGF-like domains could hold CUB4/5 domains in locations that exclude substrates cleavable by BMP-1.