title: Endoplasmic reticulum retention and prolonged association of a von Willebrand's disease-causing von Willebrand factor variant with ERp57 and calnexin.
abstract: We have previously identified a mutation (R273W) in the von Willebrand factor (VWF) propeptide that results in quantitative deficiency of plasma VWF and a loss of high molecular weight VWF multimers. Recombinant VWF having the R273W mutation (rVWFR273W) expressed in COS-7 cells demonstrated severely impaired secretion and degradation in an intracellular location [Allen, S., et al. (2000) Blood 96, 560-568]. In this report we used pulse-chase analysis and endoglycosidase H digestion of wild-type rVWF and rVWFR273W immunoprecipitated from COS-7 cells to show that rVWFR273W was retained in the endoplasmic reticulum (ER). We demonstrate for the first time that wild-type rVWF and rVWFR273W interacted with the thiol-dependent oxidoreductase ERp57 during biosynthesis in the ER. Pulse chase analysis demonstrated that the interactions of rVWFR273W with ERp57 and calnexin were prolonged compared to wild-type rVWF. In contrast there was no apparent difference between rVWFR273W and wild-type rVWF in their time-courses of interaction with calreticulin.
authors.affiliation: Division of Clinical Sciences, Royal Hallamshire Hospital, University of Sheffield, United Kingdom. simon.allen@sheffield.ac.uk
authors.forename: S
authors.initials: S
authors.lastname: Allen
authors.forename: A C
authors.initials: AC
authors.lastname: Goodeve
authors.forename: I R
authors.initials: IR
authors.lastname: Peake
authors.forename: M E
authors.initials: ME
authors.lastname: Daly
mesh_terms: D000818:Animals; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D014644:Genetic Variation / Q000235:genetics; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D021381:Protein Transport; D014162:Transfection; D014842:von Willebrand Diseases / Q000235:genetics*; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D006360:Heat-Shock Proteins; D014841:von Willebrand Factor; D037281:Calnexin; D006596:Hexosaminidases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human