Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells
Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells
复制标题
暴露于布雷菲德菌素 A 会诱导 HepG2 细胞中杂合型和复合型游离 N-聚糖的异常表达
DOI：
10.1016/j.bbagen.2023.130331
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Shinohara Yasuro
中科院分区：
文献类型：
--
作者：
Sugiura Kanako;Kawai Yuho;Yamamoto Arisa;Yoshioka Hiroki;Kiyohara Yuika;Iida Ayaka;Ozawa Yurika;Nishikawa Mai;Miura Nobuaki;Hanamatsu Hisatoshi;Furukawa Jun-ichi;Shinohara Yasuro
本研究测定了灯盏花素A(BFA)对HepG2细胞游离糖谱的影响，以更好地了解阻断细胞内小泡形成和蛋白质从内质网运输到高尔基体的作用。一系列外糖苷酶和内糖苷酶辅助的分析阐明了糖代谢改变的复杂性质。BFA介导的GN2型多糖改变的一个关键特征是表达不寻常的混合型、单触角型和复合型游离多糖(FNG)。BFA介导的Gn1型糖链改变的特征是不寻常的混合和单触角FNGs的表达，而没有显著的复合型FNGs的表达。时程分析表明，唾液酸化的混合型和复合型GN2型FNGs的形成晚于ASIALO-GN2型FNGs，并且GN2型FNGs和N-糖链的表达谱相似，提示FNGs的代谢通量与蛋白质结合的N-糖链相同。亚细胞血糖分析显示，几乎所有的FNGs都在胞浆中检测到。我们的数据表明，混合型、单触角型和复合型GN2型FNG被胞浆中的糖蛋白裂解，然后被胞质β-N-乙酰氨基葡萄糖苷酶(ENGase)消化，产生Gn1型FNG。ENGase的底物特异性解释了复杂的Gn1型FNGs的有限表达。
This study determined the effect of brefeldin A (BFA) on the freeN-glycomic profile of HepG2 cells to better understand the effect of blocking intracellular vesicle formation and transport of proteins from the endoplasmic reticulum to the Golgi apparatus. A series of exoglycosidase- and endoglycosidase-assisted analyses clarified the complex nature of altered glycomic profiles. A key feature of BFA-mediated alterations in Gn2-type glycans was the expression of unusual hybrid-, monoantennary- and complex-type freeN-glycans (FNGs). BFA-mediated alterations in Gn1-type glycans were characterized by the expression of unusual hybrid- and monoantennary-FNGs, without significant expression of complex-type FNGs. A time course analysis revealed that sialylated hybrid- and complex-type Gn2-type FNGs were generated later than asialo-Gn2-type FNGs, and the expression profiles of Gn2-type FNGs andN-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-boundN-glycans. Subcellular glycomic analysis revealed that almost all FNGs were detected in the cytoplasmic extracts. Our data suggest that hybrid-, monoantennary- and complex-type Gn2-type FNGs were cleaved from glycoproteins in the cytosol by cytosolic PNGase, and subsequently digested by cytosolicendo-β-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs.