It is known that oligosaccharyltransferase (OST) has hydrolytic activity towards dolichol-linked oligosaccharides, which results in the formation of free N-glycans (FNGs), i.e. unconjugated oligosaccharides with structural features similar to N-glycans. The functional importance of this hydrolytic reaction, however, remains unknown. In this study, the hydrolytic activity of OST was characterized in yeast. It was shown that the hydrolytic activity of OST is enhanced in ubiquitin ligase mutants that are involved in endoplasmic reticulum-associated degradation (ERAD). Interestingly, this enhanced hydrolysis activity is completely suppressed in asparagine-linked glycosylation (alg) mutants, bearing mutations related to the biosynthesis of dolichol-linked oligosaccharides, indicating that the effect of ubiquitin ligase on OST-mediated hydrolysis is context-dependent. The enhanced hydrolysis activity in ubiquitin ligase mutants was also found to be canceled upon treatment of the cells with dithiothreitol, a reagent that potently induces protein unfolding in the endoplasmic reticulum (ER). Our results clearly suggest that the hydrolytic activity of OST is enhanced under conditions in which the formation of unfolded proteins is promoted in the endoplasmic reticulum in yeast. The possible role of free N-glycans on protein folding is discussed.

中文翻译：

---

当错误折叠的蛋白质在内质网中积累时，酵母寡糖转移酶的水解活性增强

已知寡糖基转移酶(OST)对多醇连接的寡糖具有水解活性，这导致游离N-聚糖(FNG)的形成，即具有与N-聚糖相似的结构特征的非缀合寡糖。然而，这种水解反应的功能重要性仍然未知。在本研究中，对酵母中 OST 的水解活性进行了表征。结果表明，参与内质网相关降解 (ERAD) 的泛素连接酶突变体中 OST 的水解活性得到增强。有趣的是，这种增强的水解活性在天冬酰胺连接的糖基化 ( alg)突变体中被完全抑制，该突变体具有与多醇连接的寡糖生物合成相关的突变，表明泛素连接酶对 OST 介导的水解的影响是上下文相关的。还发现，用二硫苏糖醇处理细胞后，泛素连接酶突变体中增强的水解活性被取消，二硫苏糖醇是一种有效诱导内质网 (ER) 中蛋白质解折叠的试剂。我们的结果清楚地表明，在酵母内质网中未折叠蛋白的形成得到促进的条件下，OST 的水解活性得到增强。讨论了游离N-聚糖对蛋白质折叠的可能作用。