Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
Department of Chemistry, Sapporo Medical University Center for Medical Education, Sapporo 060-8556, Japan.
Biochemistry. 2021 Jun 1;60(21):1708-1721. doi: 10.1021/acs.biochem.0c00969. Epub 2021 May 13.
Endoplasmic reticulum (ER) stress has been reported in a variety of diseases. Although ER stress can be detected using specific markers, it is still difficult to quantitatively evaluate the degree of stress and to identify the cause of the stress. The ER is the primary site for folding of secretory or transmembrane proteins as well as the site where glycosylation is initiated. This study therefore postulates that tracing the biosynthetic pathway of asparagine-linked glycans (-glycans) would be a reporter for reflecting the state of the ER and serve as a quantitative descriptor of ER stress. Glycoblotting-assisted mass spectrometric analysis of the HeLa cell line enabled quantitative determination of the changes in the structures of -glycans and degraded free oligosaccharides (fOSs) in response to tunicamycin- or thapsigargin-induced ER stress. The integrated analysis of neutral and sialylated -glycans and fOSs showed the potential to elucidate the cause of ER stress, which cannot be readily done by protein markers alone. Changes in the total amount of glycans, increase in the ratio of high-mannose type -glycans, increase in fOSs, and changes in the ratio of sialylated -glycans in response to ER stress were shown to be potential descriptors of ER stress. Additionally, drastic clearance of accumulated -glycans was observed in thapsigargin-treated cells, which may suggest the observation of ER stress-mediated autophagy or ER-phagy in terms of glycomics. Quantitative analysis of -glycoforms composed of -glycans and fOSs provides the dynamic indicators reflecting the ER status and the promising strategies for quantitative evaluation of ER stress.
内质网（ER）应激已在多种疾病中被报道。虽然可以使用特定的标记物来检测 ER 应激，但定量评估应激程度并确定应激原因仍然具有挑战性。ER 是折叠分泌或跨膜蛋白的主要场所，也是糖基化起始的场所。因此，本研究假设追踪天冬酰胺连接聚糖（-聚糖）的生物合成途径将是反映 ER 状态的报告，并作为 ER 应激的定量描述符。通过对 HeLa 细胞系进行糖基印迹辅助质谱分析，能够定量测定乳胞素或他普西龙诱导的 ER 应激下 -聚糖结构和降解游离寡糖（fOS）的变化。中性和唾液酸化 -聚糖和 fOS 的综合分析显示出阐明 ER 应激原因的潜力，这是仅使用蛋白质标记物无法轻易做到的。总糖量的变化、高甘露糖型 -聚糖比例的增加、fOS 的增加以及 ER 应激下唾液酸化 -聚糖比例的变化被证明是 ER 应激的潜在描述符。此外，在他普西龙处理的细胞中观察到积累的 -聚糖大量清除，这可能表明从糖组学的角度观察到了 ER 应激介导的自噬或 ER 吞噬。-聚糖和 fOS 组成的 -糖型的定量分析提供了反映 ER 状态的动态指标，并为 ER 应激的定量评估提供了有前景的策略。
