Department of Bioproducts and Biosystems, Aalto University, Finland, Kemistintie 1, 02150 Espoo, Finland.
FEMS Yeast Res. 2020 Feb 1;20(1). doi: 10.1093/femsyr/foaa002.
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.
N-糖基化在内质网质量控制 (ERQC) 中起着重要作用。已经在酵母和真菌中对 N-聚糖生物合成途径进行了工程改造，以能够生产具有与人相容的 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连接寡糖 (LLO) 的合成。由于LLO 工程对 ERQC 的影响目前尚不清楚，我们在糖工程化的Δalg3Δalg11酿酒酵母菌株中表征了 IgG 的细胞内加工，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，并独立于 Yos9p 和 Htm1p 靶向 ERAD，而在存在 ALG3 时，ERAD 靶向依赖于 Yos9p，但不需要 Htm1p。ERAD 的阻断积累了 ER 和 IgG 的高尔基体后形式，并增加了 matα 分泌信号的糖基化，但并没有改善 IgG 的分泌。我们的结果表明，酵母中异源糖蛋白的 ERAD 靶向，并表明当 LLO 生物合成发生改变时，蛋白质可以通过其他机制而不是 Htm1p-Yos9p 依赖性途径靶向 ERAD。
