Ladenheim R G, Seidah N G, Rougeon F
Unité de Génétique et Biochimie du Développement, Institut Pasteur, Paris, France.
Eur J Biochem. 1991 Jun 1;198(2):535-40. doi: 10.1111/j.1432-1033.1991.tb16047.x.
Most mouse inbred strains carry two renin genes, Ren-1 and Ren-2, Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.
大多数小鼠近交系携带两个肾素基因，即Ren-1和Ren-2。肾素-2是Ren-2基因的产物，在颌下腺中高度表达。它是一种肾素同工酶，与肾皮质肾素-1有96%的相似性，但未进行糖基化修饰。为了研究前肾素的糖基化是否会影响其加工和/或分泌，我们使用定点诱变技术在前肾素-2 cDNA中引入了两个潜在的N-连接糖基化位点。表达质粒来源于野生型和突变型肾素-2 cDNA，并转染到AtT20细胞中。两种转染细胞，分别表达糖基化或未糖基化形式，通过组成型和调节型途径分泌前肾素和肾素。前肾素被正确加工成活性肾素，但在AtT20细胞中第二个成熟位点未被切割。糖基化和未糖基化肾素表达的比较表明，糖基化活性肾素的分泌减少。用衣霉素阻止糖基化可改善活性肾素的分泌。此外，糖基化前肾素在体外的胰蛋白酶激活效率降低，而当对衣霉素处理细胞分泌的突变型肾素进行激活时，该效率得以恢复。有人提出，附着在前肾素上的大量碳水化合物对成熟酶的蛋白水解构成空间位阻。
