Sulphation of N-linked oligosaccharides of vesicular stomatitis and influenza virus envelope glycoproteins: host cell specificity, subcellular localization and identification of substituted saccharides.
The 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 [35S]sulphate substituents into its complex carbohydrate units occurred in Madin-Darby canine kidney (MDCK), Madin-Darby bovine kidney, LLC-PK1 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-SO4)] and/or C-6 of N-acetylglucosamine [GlcNAc(6-SO4)] residues in the N-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-O-sulphotransferase activity assayed in vitro. Comparative studies carried out on the haemagglutinin of the influenza virus envelope formed by MDCK and LLC-PK1 cells indicated that sulphate in this glycoprotein was confined to its complex N-linked oligosaccharides where it occurred as Gal(3-SO4) and GlcNAc(6-SO4) on peripheral chains as well as on the mannose-substituted N-acetylglucosamine of the core. Since sulphation in both internal and peripheral locations of the virus glycoproteins was found to be arrested by the alpha1-->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 trans-Golgi network.
Karaivanova
V K
VK
Department of Biological Chemistry, Harvard Medical School, Joslin Diabetes Center, Boston, MA 02215, USA.
Spiro
R G
RG
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D020126:Brefeldin A; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D004285:Dogs; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins; D051379:Mice; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000378:metabolism; D013552:Swine; D014407:Tumor Cells, Cultured; D014721:Vesicular stomatitis Indiana virus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D000998:Antiviral Agents; D003517:Cyclopentanes; C028391:G protein, vesicular stomatitis virus; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D013431:Sulfates; D014759:Viral Envelope Proteins; D020126:Brefeldin A; C022321:PAPS sulfotransferase; D015238:Sulfotransferases