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.