Multiple Bone Morphogenetic Protein 1-related Mammalian Metalloproteinases Process Pro-lysyl Oxidase at the Correct Physiological Site and Control Lysyl Oxidase Activation in Mouse Embryo Fibroblast Cultures

Abstract

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 ϳ30-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 prolysyl 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.

Osteogenesis imperfecta

“…Mutations that impair the cleavage site of the C pro peptides cause a mild osteogenesis imperfecta with normal or increased z scores (indicating normal or increased bone mineral density) measured by dual energy X ray absorptio metry. Mutations in BMP1 result in more severe forms of osteogenesis imperfecta than cleavage site mutations, as BMP1 broadly affects extra cellular matrix assembly and structure by processing the C propeptides of type I and type III collagen 48,49 , N propeptides of pro α1(V) and pro α1(XI) 50,51 , cleavage of the collagen and elastin crosslinking enzyme pro lysyl oxidase 52 and of small leucine rich proteoglycans, such as prodecorin and probiglycan 53,54 . BMP1 is also respon sible for the activation of multiple cytokines, such as TGFβ1, BMP2 and BMP4 (REF.…”

The bone morphogenetic protein 1/Tolloid-like metalloproteinases

“…In addition to processing fibrillar procollagens, the BMP1/TLD-like proteinases affect fibrillogenesis by activating the zymogen of lysyl oxidase (Panchenko et al, 1996;Uzel et al, 2001), an enzyme necessary to forming the covalent crosslinks that provide collagen and elastic fibers with most of their tensile strength. Similarly, BMP1 has been shown to activate the related cross-linking enzyme lysyl oxidase-like (Borel et al, 2001).…”

Autocrine Growth Factor Regulation of Lysyl Oxidase Expression in Transformed Fibroblasts

“…Lysyl oxidase is synthesized as a 50-kDa proenzyme, secreted into the extracellular environment, and then processed by proteolytic cleavage, resulting in a functional 30-kDa enzyme and an 18-kDa propeptide. Evidence supports that 30-kDa lysyl oxidase is active and that the 50-kDa proenzyme is enzymatically inactive (2)(3)(4). Abnormally increased lysyl oxidase expression and enzyme activity can lead to excessive accumulation of insoluble collagen fibers.…”