Ethanol-induced impairment in the biosynthesis of N-linked glycosylation.
Deficiency in N-linked protein glycosylation is a long-known characteristic of alcoholic liver disease and congenital disorders of glycosylation. Previous investigations of ethanol-induced glycosylation deficiency demonstrated perturbations in the early steps of substrate synthesis and in the final steps of capping N-linked glycans in the Golgi. The significance of the biosynthesis of N-glycan precursors in the endoplasmic reticulum, however, has not yet been addressed in alcoholic liver disease. Ethanol-metabolizing hepatoma cells were treated with increasing concentrations of ethanol. Transcript analysis of genes involved in the biosynthesis of N-glycans, activity assays of related enzymes, dolichol-phosphate quantification, and analysis of dolichol-linked oligosaccharides were performed. Upon treatment of cells with ethanol, we found a decrease in the final N-glycan precursor Dol-PP-GlcNAc(2) Man(9) Glc(3) and in C95- and C100-dolichol-phosphate levels. Transcript analysis of genes involved in N-glycosylation showed a 17% decrease in expression levels of DPM1, a subunit of the dolichol-phosphate-mannose synthase, and an 8% increase in RPN2, a subunit of the oligosaccharyl transferase. Ethanol treatment decreases the biosynthesis of dolichol-phosphate. Consequently, the formation of N-glycan precursors is affected, resulting in an aberrant precursor assembly. Messenger RNA levels of genes involved in N-glycan biosynthesis are slightly affected by ethanol treatment, indicating that the assembly of N-glycan precursors is not regulated at the transcriptional level. This study confirms that ethanol impairs N-linked glycosylation by affecting dolichol biosynthesis leading to impaired dolichol-linked oligosaccharide assembly. Together our data help to explain the underglycosylation phenotype observed in alcoholic liver disease and congenital disorders of glycosylation.
Institute of Physiology, University of Zürich, Zürich, Switzerland.
Michael
M
Welti
Andreas J
AJ
Hülsmeier
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D002236:Carbohydrate Conformation; D002478:Cells, Cultured; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism; D004288:Dolichol Phosphates / Q000378:metabolism; D004286:Dolichols / Q000096:biosynthesis* / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006031:Glycosylation / Q000187:drug effects*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D006602:Hexosyltransferases; D006801:Humans; D008658:Inactivation, Metabolic; D008364:Mannosyltransferases / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism
D004288:Dolichol Phosphates; D004286:Dolichols; D009844:Oligosaccharides; D011134:Polysaccharides; D014168:Transferrin; D000431:Ethanol; D000426:Alcohol Dehydrogenase; D019392:Cytochrome P-450 CYP2E1; D006602:Hexosyltransferases; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; C517590:RPN2 protein, human; D046988:Proteasome Endopeptidase Complex
ALCOHOLIC LIVER DISEASE; CONGENITAL DISORDER OF GLYCOSYLATION; DOLICHOL; DOLICHOL-LINKED OLIGOSACCHARIDES; N-LINKED GLYCOSYLATION