Institute of Molecular, Cell and Systems Biology, College of Medical Veterinary and Life Sciences, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, UK.
Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, Headington, Oxford OX3 7LD, UK.
J Cell Sci. 2021 Nov 15;134(22). doi: 10.1242/jcs.259340. Epub 2021 Nov 25.
N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here, we used an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could be mimicked by the addition of a membrane-impermeable reducing agent. We identified a hypoglycosylated acceptor site that is adjacent to a cysteine involved in a short-range disulfide. We show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A- and STT3B-dependent glycosylation. Our results provide further insight into the important role of the endoplasmic reticulum redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.
蛋白质进入分泌途径时的 N 连接糖基化是蛋白质稳定性和功能所必需的修饰。先前已经表明，蛋白质折叠和糖基化之间存在时间关系，这会影响特定糖基化位点的占据。在这里，我们使用了一种体外翻译系统，该系统在定义的氧化还原条件下再现了分泌蛋白易位、折叠和糖基化的初始阶段。我们发现，触珠蛋白的糖基化效率取决于强大的 NADPH 依赖性细胞质还原途径，该途径可以通过添加不可渗透膜的还原剂来模拟。我们鉴定了一个邻近涉及短程二硫键的半胱氨酸的低聚糖接受位点。我们表明，该位点的有效糖基化受作用于 STT3A 和 STT3B 依赖性糖基化的细胞质还原途径的影响。我们的结果进一步深入了解了内质网氧化还原条件在糖基化位点占据中的重要作用，并证明了细胞质中氧化还原条件与糖基化效率之间的联系。
