OverviewProtein N-glycosylation occurs in the lumen of the ER. The oligosaccharide moiety from the glycolipid dolichol-diphosphate-oligosaccharide (the 'dolichol-linked N-glycan precursor' or 'dolichol-linked oligosaccharide (DLO)') is transferred by oligosaccharyltransferase to specific asparagine residues of secretory proteins as they emerge from the protein translocation channel into the ER lumen. DLO synthesis is initiated on the cytoplasmic face of the ER and after seven glycosyltransfer reactions, the resulting glycolipid is flipped across the ER membrane to be further elaborated before being used for protein N-glycosylation (Figure 1). The optimal N-glycan precursor oligosaccharide in yeast and mammals has a terminal cap of three glucose residues sourced from the glycolipid glucosyl-phosphoryl dolichol (GPD) in the ER lumen. Paradoxically, this lipid is synthesized on the cytoplasmic face of the ER and must be flipped across the ER membrane to provide a source of lumenal glucose. As the Figure 1. Role of GPD scramblase in the synthesis of Glc3Man9GlcNAc2-PP-dolichol (G3M9-DLO) GPD is synthesized from dolichyl-P and UDP-glucose on the cytoplasmic face of the ER by GPD synthase (yeast Alg5) (bottom left), and then flipped into the lumen by GPD scramblase. In the lumen, the glucosyltransferases Alg6, Alg8 and Alg10 transfer glucose residues from GPD to M9-DLO to generate Glc3Man9GlcNAc2-PP-dolichol (G3M9-DLO) that provides the oligosaccharide used by oligosaccharyltransferase (OST) to N-glycosylate proteins. The synthesis, scrambling and consumption of GPD likely form a co-evolving metabolic unit. This hypothesis underlies our phylogenetic profiling identification of GPD scramblase candidates. Please see references 1-3). Note that the seven glycosyltransfer reactions that generate M5-DLO from dolichyl phosphate on the cytoplasmic face of the ER are not shown. Only M5-DLO scramblase is depicted, as this protein transfers cytoplasmically generated M5-DLO to the luminal leaflet for conversion to M9-DLO and G3M9-DLO.84Molecular mechanism of glucosylation ofthe dolichol-linked N-glycan precursorAnant K. MenonWeill Cornell Medical College, Department of Biochemistry
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