Abstract:
It is known that oligosaccharyltransferase (OST) has hydrolytic activity toward dolichol-linked oligosaccharides (DLO), 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. Interestingly, this enhanced hydrolysis activity is completely suppressed in asparagine-linked glycosylation (alg) mutants, bearing mutations related to the biosynthesis of DLO, 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 ER in yeast. The possible role of FNGs on protein folding is discussed.
摘要:
已知寡糖基转移酶(OST)具有水解活性,可以水解与多角醇连接的寡糖,这导致游离N-聚糖(FNG)的形成,即具有类似于N-聚糖的结构特征的未缀合的寡糖。这种水解反应的功能重要性,然而,仍然未知。在这项研究中,在酵母中表征了OST的水解活性。研究表明,在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,携带与dolichol连接的寡糖的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依赖性的。在用二硫苏糖醇处理细胞后,还发现泛素连接酶突变体中增强的水解活性被取消,一种在内质网(ER)中有效诱导蛋白质解折叠的试剂。我们的结果清楚地表明,在酵母内质网中促进未折叠蛋白质形成的条件下,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。