Sulphation of N-linked oligosaccharides of vesicular stomatitis and influenza virus envelope glycoproteins: host cell specificity, subcellular localization and identification of substituted saccharides

摘要

pThe presence of sulphate groups on various saccharide residues of N-linked carbohydrate units has now been observed in a number of glycoproteins. To explore the cell specificity of this post-translational modification, we evaluated sulphate incorporation into virus envelope glycoproteins by a variety of cells, since it is believed that assembly of their N-linked oligosaccharides is to a large extent dependent on the enzymic machinery of the host. Employing the vesicular stomatitis virus (VSV) envelope glycoprotein (G protein) as a model, we noted that the addition of [sup35/supS]sulphate substituents into its complex carbohydrate units occurred in Madin-Darby canine kidney (MDCK), Madin-Darby bovine kidney, LLC-PKsub1/sub and BHK-21 cell lines but was not detectable in BRL 3A, BW5147.3, Chinese hamster ovary, HepG2, NRK-49F, IEC-18, PtK1 or 3T3 cells. The sulphate groups were exclusively located on C-3 of galactose [Gal(3-SOsub4/sub)] and/or C-6 of iN/i-acetylglucosamine [GlcNAc(6-SOsub4/sub)] residues in the iN/i-acetyllactosamine sequence of the branch chains. Moreover, we observed that the pronounced host-cell-dependence of the terminal galactose sulphation was reflected by the 3?-phosphoadenosine 5?-phosphosulphate:Gal-3-iO/i-sulphotransferase activity assayed iin iv/iitro/i. Comparative studies carried out on the haemagglutinin of the influenza virus envelope formed by MDCK and LLC-PKsub1/sub cells indicated that sulphate in this glycoprotein was confined to its complex N-linked oligosaccharides where it occurred as Gal(3-SOsub4/sub) and GlcNAc(6-SOsub4/sub) on peripheral chains as well as on the mannose-substituted iN/i-acetylglucosamine of the core. Since sulphation in both internal and peripheral locations of the virus glycoproteins was found to be arrested by the α1 → 2 mannosidase inhibitor, kifunensine, as well as by the intracellular migration block imposed by brefeldin A, it was concluded that this modification is a late biosynthetic event which most likely takes place in the itrans/i-Golgi network./p