Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells.

Kanako Sugiura, Yuho Kawai, Arisa Yamamoto, Hiroki Yoshioka, Yuika Kiyohara, Ayaka Iida, Yurika Ozawa, Mai Nishikawa, Nobuaki Miura, Hisatoshi Hanamatsu, Jun-Ichi Furukawa, Yasuro Shinohara

Biochimica et biophysica acta. General subjects 2023 May

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This study determined the effect of brefeldin A (BFA) on the free N-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 free N-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 and N-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-bound N-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 cytosolic endo-β-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs. Copyright © 2023 Elsevier B.V. All rights reserved.

Citation

Kanako Sugiura, Yuho Kawai, Arisa Yamamoto, Hiroki Yoshioka, Yuika Kiyohara, Ayaka Iida, Yurika Ozawa, Mai Nishikawa, Nobuaki Miura, Hisatoshi Hanamatsu, Jun-Ichi Furukawa, Yasuro Shinohara. Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells. Biochimica et biophysica acta. General subjects. 2023 May;1867(5):130331