title: Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle.
abstract: Oligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co(2+)-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [(3)H]OSP release from [(3)H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc2-PP-solanesol is hydrolyzed to yield GlcNAc2-P and inhibits [(3)H]OSP release from [(3)H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction.
authors.affiliation: INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France Université Pierre et Marie Curie, Paris 6, Paris, France.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Massarweh
authors.affiliation: Université Paris Descartes, CICB-Paris, CNRS UMR8601, LCBPT, Paris, France.
authors.forename: Michaël
authors.initials: M
authors.lastname: Bosco
authors.affiliation: INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France.
authors.forename: Soria
authors.initials: S
authors.lastname: Iatmanen-Harbi
authors.affiliation: INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France.
authors.forename: Clarice
authors.initials: C
authors.lastname: Tessier
authors.affiliation: Université Paris Descartes, CICB-Paris, CNRS UMR8601, LCBPT, Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Auberger
authors.affiliation: Université Paris Descartes, CICB-Paris, CNRS UMR8601, LCBPT, Paris, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Busca
authors.affiliation: INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Chantret
authors.affiliation: Université Paris Descartes, CICB-Paris, CNRS UMR8601, LCBPT, Paris, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Gravier-Pelletier
authors.affiliation: INSERM U1149, Paris, France Université Denis Diderot, Paris 7, Paris, France stuart.moore@inserm.fr.
authors.forename: Stuart E H
authors.initials: SE
authors.lastname: Moore
mesh_terms: D004288:Dolichol Phosphates / Q000737:chemistry / Q000378:metabolism; D004286:Dolichols / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004288:Dolichol Phosphates; D004286:Dolichols; D009844:Oligosaccharides; D011106:Polyisoprenyl Phosphates; C028306:dolichol monophosphate; C063778:solanesyl pyrophosphate; D011755:Pyrophosphatases; C057833:oligosaccharide-diphosphodolichol pyrophosphatase
keywords: EC 3.6.1.44; Golgi apparatus; N-linked glycosylation; dolichol-linked oligosaccharide; endoplasmic reticulum; isoprenoids; oligosaccharide-diphosphodolichol; oligosaccharide-diphosphodolichol diphosphatase; oligosaccharyl phosphates; phosphatases; phospholipids/trafficking