title: Identification of phosphorylated oligosaccharides in cells of patients with a congenital disorders of glycosylation (CDG-I).
abstract: Protein N-glycosylation is initiated by the dolichol cycle in which the oligosaccharide precursor Glc(3)Man(9)GlcNAc(2)-PP-dolichol is assembled in the endoplasmic reticulum (ER). One critical step in the dolichol cycle concerns the availability of Dol-P at the cytosolic face of the ER membrane. In RFT1 cells, the lipid-linked oligosaccharide (LLO) intermediate Man(5)GlcNAc(2)-PP-Dol accumulates at the cytosolic face of the ER membrane. Since Dol-P is a rate-limiting intermediate during protein N-glycosylation, continuous accumulation of Man(5)GlcNAc(2)-PP-Dol would block the dolichol cycle. Hence, we investigated the molecular mechanisms by which accumulating Man(5)GlcNAc(2)-PP-Dol could be catabolized in RFT1 cells. On the basis of metabolic labeling experiments and in comparison to human control cells, we identified phosphorylated oligosaccharides (POS), not found in human control cells and present evidence that they originate from the accumulating LLO intermediates. In addition, POS were also detected in other CDG patients' cells accumulating specific LLO intermediates at different cellular locations. Moreover, the enzymatic activity that hydrolyses oligosaccharide-PP-Dol into POS was identified in human microsomal membranes and required Mn(2+) for optimal activity. In CDG patients' cells, we thus identified and characterized POS that could result from the catabolism of accumulating LLO intermediates.
authors.affiliation: Laboratory for Molecular Diagnosis, Center for Human Genetics, University of Leuven, B-3000 Leuven, Belgium.
authors.forename: Wendy
authors.initials: W
authors.lastname: Vleugels
authors.forename: Sandrine
authors.initials: S
authors.lastname: Duvet
authors.forename: Romain
authors.initials: R
authors.lastname: Peanne
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Mir
authors.forename: René
authors.initials: R
authors.lastname: Cacan
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Michalski
authors.forename: Gert
authors.initials: G
authors.lastname: Matthijs
authors.forename: François
authors.initials: F
authors.lastname: Foulquier
mesh_terms: D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D018981:Congenital Disorders of Glycosylation / Q000378:metabolism* / Q000473:pathology; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics; D006801:Humans; D008364:Mannosyltransferases / Q000235:genetics; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics; D008861:Microsomes / Q000378:metabolism; D020125:Mutation, Missense; D009844:Oligosaccharides / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases) / Q000235:genetics; D011755:Pyrophosphatases / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D009844:Oligosaccharides; C486729:RFT1 protein, human; D005964:Glucosyltransferases; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D010770:Phosphotransferases; D011755:Pyrophosphatases; D017875:Phosphotransferases (Phosphomutases); C519582:phosphomannomutase 2, human