Uzel M I, Scott I C, Babakhanlou-Chase H, Palamakumbura A H, Pappano W N, Hong H H, Greenspan D S, Trackman P C
Division of Oral Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118, USA.
J Biol Chem. 2001 Jun 22;276(25):22537-43. doi: 10.1074/jbc.M102352200. Epub 2001 Apr 19.
Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.
赖氨酰氧化酶催化细胞外基质生物合成中胶原蛋白和弹性蛋白交联所需的最后一步酶促反应。前赖氨酰氧化酶由前胶原C蛋白酶活性进行加工处理，该酶还能去除前胶原I - III的C端前肽。Bmp1基因编码两种前胶原C蛋白酶：骨形态发生蛋白1（BMP - 1）和哺乳动物类 tolloid蛋白（mTLD）。哺乳动物类 tolloid样蛋白（mTLL）- 1和 - 2是两种基因不同的与BMP - 1相关的蛋白酶，且已证明mTLL - 1具有前胶原C蛋白酶活性。本研究首次直接比较了这四种相关蛋白酶对前赖氨酰氧化酶的加工处理情况。用纯化的重组酶进行的体外实验表明，所有这四种蛋白酶均能在正确的生理位点有效切割前赖氨酰氧化酶，但BMP - 1的效率分别比mTLL - 1、mTLL - 2和mTLD高3倍、15倍和20倍。为了更直接地评估BMP - 1和mTLL - 1在结缔组织细胞激活赖氨酰氧化酶中的作用，分别对从Bmp1基因缺失、Tll1基因缺失以及Bmp1/Tll1双基因缺失的小鼠胚胎中培养的成纤维细胞进行了检测，这些细胞分别缺乏BMP - 1/mTLD、mTLL - 1或所有这三种酶，检测内容包括赖氨酰氧化酶的酶活性以及前赖氨酰氧化酶和成熟的约30 kDa赖氨酰氧化酶的积累情况。野生型细胞或单独缺失Bmp1或Tll1基因的细胞产生前赖氨酰氧化酶和加工后的赖氨酰氧化酶的水平相似，表明加工水平明显正常，这与酶活性数据一致。相比之下，双基因缺失的Bmp1/Tll1细胞主要产生未加工的50 kDa前赖氨酰氧化酶，其赖氨酰氧化酶活性与野生型、Bmp1基因缺失和Tll1基因缺失的细胞相比降低了70%。因此，BMP - 1/mTLD和mTLL - 1的组合被证明是小鼠胚胎成纤维细胞激活赖氨酰氧化酶的主要加工过程的原因，而体外研究确定前赖氨酰氧化酶是mTLL - 2已知的首个底物。
