title: The compartmentalisation of phosphorylated free oligosaccharides in cells from a CDG Ig patient reveals a novel ER-to-cytosol translocation process.
abstract: BACKGROUND
  Biosynthesis of the dolichol linked oligosaccharide (DLO) required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man(5)GlcNAc(2)-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc(3)Man(9)GlcNAc(2)-PP-dolichol). After transfer of Glc(3)Man(9)GlcNAc(2) onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P). Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation.
  
  
  RESULTS
  In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man(7)GlcNAc(2)-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man(7)GlcNAc(2)-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man(7)GlcNAc(2)-P appears in the cytosol without detectable generation of ER luminal Man(7)GlcNAc(2)-P.
  
  
  CONCLUSIONS
  The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P. The kinetics of cytosolic fOSGN2-P generation from a luminally-generated DLO intermediate demonstrate the presence of a previously undetected ER-to-cytosol translocation process for either fOSGN2-P or DLO.
authors.affiliation: INSERM U773 CRB3, Paris, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Peric
authors.forename: Christelle
authors.initials: C
authors.lastname: Durrant-Arico
authors.forename: Christophe
authors.initials: C
authors.lastname: Delenda
authors.forename: Thierry
authors.initials: T
authors.lastname: Dupré
authors.forename: Pascale
authors.initials: P
authors.lastname: De Lonlay
authors.forename: Hélène Ogier
authors.initials: HO
authors.lastname: de Baulny
authors.forename: Cécile
authors.initials: C
authors.lastname: Pelatan
authors.forename: Brigitte
authors.initials: B
authors.lastname: Bader-Meunier
authors.forename: Olivier
authors.initials: O
authors.lastname: Danos
authors.forename: Isabelle
authors.initials: I
authors.lastname: Chantret
authors.forename: Stuart E H
authors.initials: SE
authors.lastname: Moore
mesh_terms: D000818:Animals; D002239:Carbohydrate Metabolism, Inborn Errors / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D006031:Glycosylation; D006801:Humans; D008223:Lymphoma / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009844:Oligosaccharides / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology
chemical_list: D009844:Oligosaccharides