Oligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST hydrolyzes the oligosaccharide donor, releasing free N-glycans (FNGs) into the lumen of the endoplasmic reticulum (ER). Here, we established a purification method for mutated OSTs using a high-affinity epitope tag attached to the catalytic subunit Stt3, from yeast cells co-expressing the WT OST to support growth. The purified OST protein with mutations is useful for wide-ranging biochemical experiments. We assessed the effects of mutations in the Stt3 subunit on the two enzymatic activities in vitro, as well as their effects on the N-glycan attachment and FNG content levels in yeast cells. We found that mutations in the first DXD motif increased the FNG generation activity relative to the oligosaccharyl transfer activity, both in vitro and in vivo, whereas mutations in the DK motif had the opposite effect; the decoupling of the two activities may facilitate future deconvolution of the reaction mechanism. The isolation of the mutated OSTs also enabled us to identify different enzymatic properties in OST complexes containing either the Ost3 or Ost6 subunit and to find a 15-residue peptide as a better-quality substrate than shorter peptides. This toolbox of mutants, substrates, and methods will be useful for investigations of the molecular basis and physiological roles of the OST enzymes in yeast and other organisms.

中文翻译：

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通过酵母寡糖转移酶的点突变将脂联寡糖的水解与寡糖转移反应解偶联。


寡糖转移酶 (OST) 负责 N 连接糖基化的第一步，将寡糖链转移到天冬酰胺残基上以产生糖蛋白。在没有受体天冬酰胺的情况下，OST 会水解寡糖供体，将游离 N-聚糖 (FNG) 释放到内质网 (ER) 的腔中。在这里，我们建立了一种使用附着在催化亚基 Stt3 上的高亲和力表位标签从共表达 WT OST 以支持生长的酵母细胞中纯化突变 OST 的方法。纯化的带有突变的 OST 蛋白可用于广泛的生化实验。我们评估了 Stt3 亚基突变对体外两种酶活性的影响，以及它们对酵母细胞中 N-聚糖附着和 FNG 含量水平的影响。我们发现，在体外和体内，第一个 DXD 基序的突变相对于寡糖转移活性增加了 FNG 生成活性，而 DK 基序的突变则具有相反的效果。两种活动的解耦可能有助于未来反应机制的解卷积。突变 OST 的分离还使我们能够识别包含 Ost3 或 Ost6 亚基的 OST 复合物中的不同酶特性，并找到 15 个残基的肽作为比短肽质量更好的底物。这个包含突变体、底物和方法的工具箱将有助于研究 OST 酶在酵母和其他生物体中的分子基础和生理作用。