Sulfation of the N-linked oligosaccharides of influenza virus hemagglutinin: temporal relationships and localization of sulfotransferases.
The occurrence of sulfate substituents on several positions of glycoprotein N-linked oligosaccharides prompted us to determine the subcellular localization and temporal relationships of the addition of these anionic groups employing as a model system the hemagglutinin (HA) produced by influenza virus-infected Madin-Darby canine kidney (MDCK) cells. It became apparent from a study of the HA glycoprotein in subcellular fractions resolved by Nycodenz gradient centrifugation following pulse-chase radiolabeling that sulfation of the complex N-linked oligosaccharides occurs only after they have been processed to an endo-beta-N-acetylglucosaminidase-resistant state and have reached the medial/trans Golgi and the trans Golgi network (TGN), with the former carrying out most of the sulfation activity. Hydrazine/nitrous acid/NaBH(4) treatment of the HA from the subcellular fractions indicated that C-3 of the galactose as well as C-6 of the N-acetylglucosamine residues of the N-acetyllactosamine chains became sulfated in these post ER fractions, as did the C-6 of the outer N-acetylglucosamine of the di-N-acetylchitobiose core. Consistent with the specificities of the stepwise assembly of the N-acetyllactosamine branches, we observed that the 3'-phosphoadenosine 5'-phosphosulfate (PAPS):GlcNAc-6-O-sulfotransferase migrated in the gradient to a medial/trans Golgi position while in contrast the PAPS:Gal-3-O-sulfotransferase was found in both Golgi and TGN locations. In accordance with the concept that beta-galactosylation must precede the sulfation catalyzed by the latter enzyme, we observed the presence of UDP-Gal:GlcNAc galactosyltransferase in both these sites in the MDCK cells. The presence of the Gal-3-O-sulfotransferase in the TGN is particularly important in the influenza virus-infected cells, as it makes possible the addition of terminal anionic groups after removal of the sialic acid residues by the viral neuraminidase.
Spiro
M J
MJ
Elliott P. Joslin Research Laboratory, Joslin Diabetes Center and the Department of Medicine, Harvard Medical School, Boston, MA 02215, USA.
Spiro
R G
RG
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004285:Dogs; D005700:Galactosyltransferases / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology / Q000821:virology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry; D009975:Orthomyxoviridae / Q000737:chemistry / Q000201:enzymology; D013431:Sulfates / Q000737:chemistry; D015238:Sulfotransferases / Q000378:metabolism
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D009844:Oligosaccharides; D013431:Sulfates; D005700:Galactosyltransferases; D015238:Sulfotransferases