Trackman P C, Bedell-Hogan D, Tang J, Kagan H M
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
J Biol Chem. 1992 Apr 25;267(12):8666-71.
The synthesis and post-translational processing of a lysyl oxidase precursor protein predicted by the cDNA sequence of rat aorta lysyl oxidase were investigated. In vitro transcription of the cloned lysyl oxidase cDNA and cell-free translation of its mRNA transcript yielded a 47-kDa protein in the absence and a 50-kDa glycosylated protein in the presence of pancreatic membranes, each of which were immunoprecipitated with antibody against the 32-kDa bovine enzyme. Similarly, an N-glycosylated, 50-kDa protein band was synthesized by and immunoprecipitated from cultured neonatal rat aorta smooth muscle cells labeled with [35S]methionine. Pulse-chase studies of proteins newly synthesized by these cells demonstrated that the glycosylated 50-kDa precursor is secreted and that it is processed to the 32-kDa molecular form of lysyl oxidase principally in the medium. The presence of an extracellular processing enzyme activity which converts the 50-kDa precursor to the 32-kDa species was demonstrated by incubating conditioned medium of neonatal rat aorta smooth muscle cell cultures as a source of processing activity with conditioned medium containing 35S-labeled precursor synthesized but not processed by a tumorigenic cell line transfected with a lysyl oxidase expression vector. In contrast to the 50-kDa species, the 32-kDa protein does not appear to be N-glycosylated consistent with the loss of N-linked oligosaccharide units during the processing to the smaller species. The modes of biosynthesis and secretion of lysyl oxidase are discussed in terms of other nonproteolytic activities required for activation of prolysyl oxidase.
对大鼠主动脉赖氨酰氧化酶cDNA序列预测的赖氨酰氧化酶前体蛋白的合成及翻译后加工进行了研究。克隆的赖氨酰氧化酶cDNA的体外转录及其mRNA转录本的无细胞翻译在无胰腺膜存在时产生了一种47 kDa的蛋白，在有胰腺膜存在时产生了一种50 kDa的糖基化蛋白，这两种蛋白均用抗32 kDa牛酶的抗体进行了免疫沉淀。同样，用[35S]甲硫氨酸标记的培养新生大鼠主动脉平滑肌细胞合成并免疫沉淀出一条N-糖基化的50 kDa蛋白条带。对这些细胞新合成的蛋白质进行脉冲追踪研究表明，糖基化的50 kDa前体被分泌出来，并且主要在培养基中加工成32 kDa分子形式的赖氨酰氧化酶。通过将新生大鼠主动脉平滑肌细胞培养物的条件培养基作为加工活性来源，与含有由转染了赖氨酰氧化酶表达载体的致瘤细胞系合成但未加工的35S标记前体的条件培养基一起孵育，证明了存在一种将50 kDa前体转化为32 kDa形式的细胞外加工酶活性。与50 kDa形式不同，32 kDa蛋白似乎没有N-糖基化，这与在加工成较小形式过程中N-连接寡糖单元的丢失一致。根据原赖氨酰氧化酶激活所需的其他非蛋白水解活性，讨论了赖氨酰氧化酶的生物合成和分泌模式。
