title: Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX.
abstract: The multisubunit leucine-rich glycoprotein (GP) Ib-IX-V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard-Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse-chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbalpha undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbalpha and surface expression of the complex. Biosynthetic studies were then performed on a Bernard-Soulier variant based on previous description of abnormal GPIbalpha size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbalpha. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments ('ERGIC') and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.
authors.affiliation: INSERM U.311, Etablissement Français du Sang-Alsace, 10 rue Spielmann, BP 36, 67065 Strasbourg Cédex, France.
authors.forename: P
authors.initials: P
authors.lastname: Ulsemer
authors.forename: C
authors.initials: C
authors.lastname: Strassel
authors.forename: M J
authors.initials: MJ
authors.lastname: Baas
authors.forename: J
authors.initials: J
authors.lastname: Salamero
authors.forename: S
authors.initials: S
authors.lastname: Chasserot-Golaz
authors.forename: J P
authors.initials: JP
authors.lastname: Cazenave
authors.forename: C
authors.initials: C
authors.lastname: De La Salle
authors.forename: F
authors.initials: F
authors.lastname: Lanza
mesh_terms: D000818:Animals; D016466:CHO Cells; D050260:Carbohydrate Metabolism; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D018613:Microscopy, Confocal; D009154:Mutation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D021381:Protein Transport
chemical_list: D019038:Platelet Glycoprotein GPIb-IX Complex; D021122:Protein Subunits; D007930:Leucine