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Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells
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 endoglycosi...
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Published in: | Biochimica et biophysica acta. General subjects 2023-05, Vol.1867 (5), p.130331-130331, Article 130331 |
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Main Authors: | , , , , , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites |
Online Access: | Get full text |
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Summary: | 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.
•BFA induced expression of unique hybrid and complex-type free N-glycans (FNGs).•Structures of unique FNGs were determined by enzyme-assisted approach.•Temporal and subcellular glycomic analyses were performed.•Unique Gn2 FNGs were thought to be generated from glycoproteins in the cytosol by cytosolic PNGase.•Unique Gn1 FNGs were thought to be generated from Gn2 FNGs by cytosolic ENGase. |
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ISSN: | 0304-4165 1872-8006 |
DOI: | 10.1016/j.bbagen.2023.130331 |