Cole L A, Metsch L A, Grotjan H E
Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06510.
Mol Endocrinol. 1987 Sep;1(9):621-7. doi: 10.1210/mend-1-9-621.
Several reports have described the destruction of the N-linked oligosaccharides on glycoprotein hormones by hydrogen fluoride treatment and have noted the accompanying marked reduction, or complete loss, in biological activity. This has led to the concept that the oligosaccharides have an obligatory role in glycoprotein hormone steroidogenic function. Using a less radical and more complete method for removing sugar units, endoglycosidase treatment and ovine LH (oLH) and human LH (hLH) as examples, we examined the role of oligosaccharides in hormone function. Ovine LH and hLH were digested with endoglycosidase F. After treatment cleavage of oligosaccharides was demonstrated by compositional studies, greater than 87% cleavage was demonstrated and only N-acetylglucosamine or N-acetylglucosamine-Fucose shown to remain attached to the peptide, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (appropriate size change) and by chromatofocusing (appearance of a single basic peak). Biological activities and relative potencies of preparations were then assessed in an in vitro assay, in which the ability of samples to promote testosterone production by testicular interstitial cells was measured. Although endoglycosidase F treatment reduced relative potencies 2- to 3-fold in the bioassay, (possibly in part due to subunit dissociation) it did not lessen abilities to induce maximal testosterone response (that of native hLH and oLH). These findings contrast with those obtained from hydrogen fluoride studies and indicate that the oligosaccharides, per se, do not play an obligatory role in the steroidogenic activity of LH.
几份报告描述了用氟化氢处理糖蛋白激素上的N-连接寡糖的破坏情况，并指出随之而来的生物活性显著降低或完全丧失。这导致了这样一种概念，即寡糖在糖蛋白激素的类固醇生成功能中具有必不可少的作用。我们采用一种不太激进且更完整的去除糖单元的方法，以内切糖苷酶处理为例，以绵羊促黄体生成素（oLH）和人促黄体生成素（hLH）为研究对象，研究了寡糖在激素功能中的作用。用内切糖苷酶F消化绵羊促黄体生成素和人促黄体生成素。处理后，通过成分研究证实了寡糖的裂解，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（适当的大小变化）和色谱聚焦（单个碱性峰的出现）证明裂解率大于87%，并且只有N-乙酰葡糖胺或N-乙酰葡糖胺-岩藻糖显示仍附着在肽上。然后在体外试验中评估制剂的生物活性和相对效价，在该试验中测量样品促进睾丸间质细胞产生睾酮的能力。尽管内切糖苷酶F处理在生物测定中使相对效价降低了2至3倍（可能部分是由于亚基解离），但它并没有降低诱导最大睾酮反应的能力（天然hLH和oLH的反应能力）。这些发现与氟化氢研究的结果形成对比，表明寡糖本身在促黄体生成素的类固醇生成活性中并不起必不可少的作用。
