Ohba Yohsuke, Motohashi Mizuki, Arita Makoto
Division of Physiological Chemistry and Metabolism, Graduate School of Pharmaceutical Sciences, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan.
Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
J Biochem. 2025 Feb 5;177(2):141-152. doi: 10.1093/jb/mvae084.
Monogalactosyl diacylglycerol (MGDG) is a major membrane lipid component in plants and is crucial for proper thylakoid functioning. However, MGDG in mammals has not received much attention, partly because of its relative scarcity in mammalian tissues. In addition, the biosynthetic pathway of MGDG in mammals has not been thoroughly analysed, although some reports have suggested that UGT8, a ceramide galactosyltransferase, has the potential to catalyse MGDG biosynthesis. Here, we successfully captured the endogenous levels of MGDG in HeLa cells using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS)-based lipidomics. Cellular MGDG was completely depleted in CRISPR/Cas9-mediated UGT8 knockout (KO) HeLa cells. Transient overexpression of UGT8 enhanced MGDG production in HeLa cells, and the corresponding cell lysates displayed MGDG biosynthetic activity in vitro. Site-directed mutagenesis revealed that His358 within the UGT signature sequence was important for its activity. UGT8 was localized in the endoplasmic reticulum and activation of the unfolded protein response by membrane lipid saturation was impaired in UGT8 KO cells. These results demonstrate that UGT8 is an MGDG synthase in mammals and that UGT8 regulates membrane lipid saturation signals in cells.
单半乳糖基二酰基甘油（MGDG）是植物中的一种主要膜脂成分，对类囊体的正常功能至关重要。然而，哺乳动物体内的MGDG尚未受到太多关注，部分原因是其在哺乳动物组织中相对稀少。此外，尽管一些报告表明，一种神经酰胺半乳糖基转移酶UGT8有可能催化MGDG的生物合成，但哺乳动物中MGDG的生物合成途径尚未得到彻底分析。在这里，我们使用基于液相色谱四极杆飞行时间质谱（LC-QTOF-MS）的脂质组学技术成功捕获了HeLa细胞中MGDG的内源性水平。在CRISPR/Cas9介导的UGT8基因敲除（KO）的HeLa细胞中，细胞内的MGDG完全耗尽。UGT8的瞬时过表达增强了HeLa细胞中MGDG的产生，并且相应的细胞裂解物在体外显示出MGDG生物合成活性。定点诱变显示，UGT特征序列中的His358对其活性很重要。UGT8定位于内质网，在UGT8基因敲除细胞中，膜脂饱和度对未折叠蛋白反应的激活受到损害。这些结果表明，UGT8是哺乳动物中的一种MGDG合酶，并且UGT8调节细胞中的膜脂饱和度信号。
