N-glycosylation plays an important role in the endoplasmic reticulum quality control (ERQC). N-glycan biosynthesis pathways have been engineered in yeasts and fungi to enable the production of therapeutic glycoproteins with human-compatible N-glycosylation, and some glycoengineering approaches alter the synthesis of the lipid-linked oligosaccharide (LLO). Because the effects of LLO engineering on ERQC are currently unknown, we characterized intracellular processing of IgG in glycoengineered Δalg3 Δalg11 Saccharomyces cerevisiae strain and analyzed how altered LLO structures affect endoplasmic reticulum-associated degradation (ERAD). Intracellular IgG light and heavy chain molecules expressed in Δalg3 Δalg11 strain are ERAD substrates and targeted to ERAD independently of Yos9p and Htm1p, whereas in the presence of ALG3 ERAD targeting is dependent on Yos9p but does not require Htm1p. Blocking of ERAD accumulated ER and post-Golgi forms of IgG and increased glycosylation of matα secretion signal but did not improve IgG secretion. Our results show ERAD targeting of a heterologous glycoprotein in yeast, and suggest that proteins in the ER can be targeted to ERAD via other mechanisms than the Htm1p-Yos9p-dependent route when the LLO biosynthesis is altered.

中文翻译：

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研究ERAD在糖工程化酿酒酵母中对抗体加工的作用。

N-糖基化在内质网质量控制（ERQC）中起着重要作用。N-聚糖的生物合成途径已经在酵母和真菌中进行了工程改造，从而能够产生具有人类相容性N-糖基化的治疗性糖蛋白，并且一些糖工程改造方法改变了脂质连接的寡糖（LLO）的合成。因为目前尚不清楚LLO工程对ERQC的影响，所以我们表征了糖工程Δalg3Δalg11酿酒酵母菌株中IgG的胞内加工，并分析了改变的LLO结构如何影响内质网相关降解（ERAD）。在Δalg3Δalg11菌株中表达的细胞内IgG轻链和重链分子是ERAD底物，独立于Yos9p和Htm1p靶向ERAD，而在存在ALG3的情况下，ERAD的定位取决于Yos9p，但不需要Htm1p。阻断ERAD会积聚ER和高尔基后形式的IgG，并增加matα分泌信号的糖基化，但并不能改善IgG的分泌。我们的结果显示ERAD靶向酵母中的异源糖蛋白，并表明当LLO生物合成发生改变时，ER中的蛋白可以通过Htm1p-Yos9p依赖途径以外的其他机制靶向ERAD。