Multiple bone morphogenetic protein 1-related mammalian metalloproteinasesprocess pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis, Pro-lysyl oxida se is processed by procollagen C-proteinase activity, which also removes th e C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollage n C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD), Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinc t BMP-l-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare p ro-lysyl oxidase processing by these four related proteinases. In vitro ass ays with purified recombinant enzymes show that all four proteinases produc tively 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 lysy l oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Till-null, and Bmp2/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL I, or all three enzymes, respectively, were assayed for l ysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature similar to 30-kDa lysyl oxidase, Wild type cells or cells singly nul l for Bmp1 or Till all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processin g, consistent with enzyme activity data, In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa prolysyl oxidase and had l ysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp 1-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 activ ation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro st udies identify pro-lysyl oxidase as the first known substrate for mTLL-2.