Hirose S, Kim S, Miyazaki H, Park Y S, Murakami K
J Biol Chem. 1985 Dec 25;260(30):16400-5.
The biosynthesis and post-translational modifications, including proteolytic processing and core glycosylation, of the human renin precursor have been studied in vitro in a cell-free system. For this purpose, highly enriched renin mRNA was isolated from a renin-producing juxtaglomerular cell tumor and translated in rabbit reticulocyte lysate containing [35S]methionine in the presence or absence of dog pancreas microsomal membranes. Fluorographic analysis of the radioactive translation products, immunoprecipitated and then resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that the primary translation product, preprorenin (Mr = 45,000), is initially processed to glycosylated prorenin (Mr = 47,000) during or shortly after its sequestration into the lumen of the microsomal membranes. The vectorial translocation across the membrane was confirmed by the observation that the proform was resistant to digestion with trypsin while preprorenin was sensitive. Radiosequencing and the use of prorenin-specific antibodies established the cleavage points of the pre- and profragment and showed that the in vitro precursor of human renin contains a 23-residue signal peptide and a 43-residue prosegment. The post-translational modification which, despite the removal of signal peptide, resulted in an increase in apparent Mr, reflects the glycosylation as examined using Xenopus oocytes microinjected with renin mRNA in the presence of tunicamycin, an inhibitor of protein glycosylation. Four anti-peptide antibodies which specifically recognize the NH2 terminus (Pro 1), two middle parts (Pro 2A and Pro 2B), and COOH terminus (Pro 3) of the prosegment, respectively, have been raised and used to characterize plasma prorenin. Renin precursors (pre- and prorenin) synthesized in vitro or in the kidney reacted with these antibodies (anti-Pro 1, anti-Pro 2A, anti-Pro 2B, and anti-Pro 3). However, quite unexpectedly, human plasma prorenin was recognized only by anti-Pro 3, indicating that plasma prorenin is a truncated version of intact prorenin, which lacks a large portion of the NH2 terminus of the prosegment and may represent an activation intermediate. This somewhat surprising result may lead to a better understanding of the exact roles and activation mechanisms of plasma prorenin existing in a relatively large amount.
人类肾素前体的生物合成及翻译后修饰，包括蛋白水解加工和核心糖基化，已在无细胞体系中进行了体外研究。为此，从产生肾素的近球细胞肿瘤中分离出高度富集的肾素mRNA，并在含有[35S]甲硫氨酸的兔网织红细胞裂解液中，于有无犬胰腺微粒体膜存在的情况下进行翻译。对放射性翻译产物进行荧光显影分析，这些产物经免疫沉淀后再在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上进行分离，结果显示，初级翻译产物前肾素原（Mr = 45,000）在被隔离到微粒体膜腔中时或之后不久，最初会被加工成糖基化肾素原（Mr = 47,000）。通过观察到肾素原形式对胰蛋白酶消化有抗性而前肾素原敏感，证实了其跨膜的向量转运。放射性测序以及使用肾素原特异性抗体确定了前体片段和前肽片段的切割点，并表明人类肾素的体外前体含有一个23个残基的信号肽和一个43个残基的前肽。尽管信号肽被去除，但翻译后修饰导致表观Mr增加，这反映了糖基化，这是通过在存在蛋白质糖基化抑制剂衣霉素的情况下，用肾素mRNA显微注射非洲爪蟾卵母细胞来检测的。分别制备了四种特异性识别前肽NH2末端（Pro 1）、两个中间部分（Pro 2A和Pro 2B）以及COOH末端（Pro 3）的抗肽抗体，并用于鉴定血浆肾素原。体外或在肾脏中合成的肾素前体（前肾素原和肾素原）与这些抗体（抗Pro 1、抗Pro 2A、抗Pro 2B和抗Pro 3）发生反应。然而，非常出乎意料的是，人类血浆肾素原仅被抗Pro 3识别，这表明血浆肾素原是完整肾素原的截短形式，它缺少前肽NH2末端的很大一部分，可能代表一种激活中间体。这一有点令人惊讶的结果可能会使人们更好地理解相对大量存在的血浆肾素原的确切作用和激活机制。
