Use of Bmp1/Tll1 doubly homozygous null mice and proteomics to identify and validate in vivo substrates of bone morphogenetic protein 1/Tolloid-like metalloproteinases
Use of Bmp1/Tll1 doubly homozygous null mice and proteomics to identify and validate in vivo substrates of bone morphogenetic protein 1/Tolloid-like metalloproteinases
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DOI：
10.1128/mcb.23.13.4428-4438.2003
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发表时间：
2003-07-01
影响因子：
5.3
通讯作者：
Greenspan, DS
中科院分区：
文献类型：
--
作者：
Pappano, WN;Steiglitz, BM;Greenspan, DS
骨形态发生蛋白1（BMP-1）和哺乳动物Tolloid（mammalian Tolloid），两种由Bmp 1编码的蛋白酶，提供前胶原C-蛋白酶（pCP）活性，其将前胶原I至III转化为哺乳动物细胞外基质（ECM）的主要纤维组分。然而，虽然Bmp 1（-/-）小鼠有异常的胶原纤维，它们有残留的pCP活性，表明遗传冗余。哺乳动物具有两种结构上类似于BMP-1和mTLL的蛋白酶：遗传上不同的哺乳动物Tolloid样I（mTLL-1）和mTLL-2。缺乏mTLL-1基因Tll 1的小鼠是胚胎致死的，但具有与野生型相似的pCP活性水平，这表明mTLL-1可能不是体内pCP。体外研究表明BMP-1和mTLL-1能够切割Chordin，一种BMP信号传导的细胞外拮抗剂，表明这些蛋白酶也可能用于调节BMP信号传导并协调后者与ECM形成。然而，缺乏BMP-1和mTLL-1在哺乳动物中BMP信号传导中的作用的体内证据。为了消除模糊BMP-1相关蛋白酶在哺乳动物体内功能的功能冗余，我们在这里表征Bmp 1 Tll 1双无效小鼠胚胎。尽管这些在形态上与Tll 1（-/-）胚胎无法区分，但对来自双无效胚胎的细胞的生化分析显示功能冗余被去除到一定程度，使我们能够证明（i）Bmp 1和Tll 1的产物负责哺乳动物体内的脊索蛋白切割，（ii）mTLL-1是提供在Bmp 1（-/-）胚胎中观察到的残留活性的体内pCP。功能冗余的去除还使得能够在蛋白质组学方法中使用Bmp 1（-/-）Tll 1（-/-）细胞来鉴定Bmp 1和Tll 1产物的新底物。
Bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD), two proteinases encoded by Bmp1, provide procollagen C-proteinase (pCP) activity that converts procollagens I to III into the major fibrous components of mammalian extracellular matrix (ECM). Yet, although Bmp1(-/-) mice have aberrant collagen fibrils, they have residual pCP activity, indicative of genetic redundancy. Mammals possess two additional proteinases structurally similar to BMP-1 and mTLD: the genetically distinct mammalian Tolloid-like I (mTLL-1) and mTLL-2. Mice lacking the mTLL-1 gene Tll1 are embryonic lethal but have pCP activity levels similar to those of the wild type, suggesting that mTLL-1 might not be an in vivo pCP. In vitro studies have shown BMP-1 and mTLL-1 capable of cleaving Chordin, an extracellular antagonist of BMP signaling, suggesting that these proteases might also serve to modulate BMP signaling and to coordinate the latter with ECM formation. However, in vivo evidence of roles for BMP-1 and mTLL-1 in BMP signaling in mammals is lacking. To remove functional redundancy obscuring the in vivo functions of BMP-1-related proteases in mammals, we here characterize Bmp1 Tll1 doubly null mouse embryos. Although these appear morphologically indistinguishable from Tll1(-/-) embryos, biochemical analysis of cells derived from doubly null embryos shows functional redundancy removed to an extent enabling us to demonstrate that (i) products of Bmp1 and Tll1 are responsible for in vivo cleavage of Chordin in mammals and (ii) mTLL-1 is an in vivo pCP that provides residual activity observed in Bmp1(-/-) embryos. Removal of functional redundancy also enabled use of Bmp1(-/-) Tll1(-/-) cells in a proteomics approach for identifying novel substrates of Bmp1 and Tll1 products.