Glycosylation of human chorionic gonadotropin in mRNA-dependent cell-free extracts: post-translational processing of an asparagine-linked mannose-rich oligosaccharide.
Glycosylation of human chorionic gonadotropin in mRNA-dependent cell-free extracts: post-translational processing of an asparagine-linked mannose-rich oligosaccharide.
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mRNA依赖性无细胞提取物中人绒毛膜促性腺激素的糖基化：天冬酰胺连接的富含甘露糖的寡糖的翻译后加工。
DOI：
10.1073/pnas.76.3.1208
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发表时间：
1979
影响因子：
11.1
通讯作者：
I. Boime
中科院分区：
文献类型：
--
作者：
M. Bielińska;I. Boime
胎盘RNA先前已被证明可指导小鼠腹水肿瘤细胞裂解物中人绒毛膜促性腺激素（hCG-α）α亚基的天冬酰胺连接的富含甘露糖的糖基化形式的合成。糖基化依赖于同源微粒体膜的存在，糖基化蛋白被隔离到微粒体囊泡中。在这里，我们表明，当Triton X-100的翻译60分钟后加入，并继续孵育，有一个新的离散的低分子量蛋白质的糖基化形式的转变。这些新蛋白质的形成不是蛋白水解的明显结果，因为（i）用内切糖苷酶H处理完全糖基化的蛋白质或在Triton存在下形成的蛋白质导致形成在十二烷基硫酸钠凝胶上以相同速率迁移的单一蛋白质;（ii）在从衣霉素分离的膜存在下合成的非糖基化hCG-α的迁移，预处理的腹水肿瘤细胞在Triton添加后没有改变;和（iii）Triton诱导的变化被甘露糖内酯、酵母甘露聚糖和纯化的甘露糖寡糖抑制。它也表明，很少的加工富含甘露糖的糖蛋白发生在微粒体膜单独存在。然而，将无核糖体的上清液级分添加到糖蛋白中导致加工。这些数据表明，寡糖核心的加工是一个区室化的过程，其中糖（可能是甘露糖）的去除需要糖蛋白从内质网转移到分泌级联的另一个组分。
Placental RNA has previously been shown to direct the synthesis of an asparagine-linked mannose-rich glycosylated form of the alpha subunit of human chorionic gonadotropin (hCG-alpha) in lysates derived from mouse ascites tumor cells. Glycosylation was dependent on the presence of homologous microsomal membranes, and the glycosylated protein was sequestered into the microsomal vesicles. Here we show that when Triton X-100 is added after 60 min of translation and the incubation is continued, there is a shift of this glycosylated form to new discrete lower molecular weight proteins. The formation of these new proteins was not the apparent result of proteolysis because (i) treatment of the fully glycosylated protein or the proteins formed in the presence of Triton with endoglycosidase H resulted in the formation of a single protein migrating at the same rate on sodium dodecyl sulfate gels; (ii) the migration of nonglycosylated hCG-alpha synthesized in the presence of membranes isolated from tunicamycin-pretreated ascites tumor cells was not changed upon Triton addition; and (iii) the Triton-induced change was inhibited by mannonolactone, yeast mannan, and purified mannose oligosaccharides. It was also shown that little processing of the mannose-rich glycoprotein occurred in the presence of microsomal membranes alone. However, addition of the ribosome-free supernatant fraction to the glycoprotein resulted in processing. These data suggest that processing of the oligosaccharide core is a compartmentalized process in which removal of sugar, presumably mannose, requires a transfer of the glycoprotein from the endoplasmic reticulum to another component of the secretory cascade.