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 localiz ation and temporal relationships of the addition of these anionic groups em ploying as a model system the hemagglutinin (HA) produced by influenza viru s-infected Madin-Darby canine kidney (MDCK) cells. It became apparent from a study of the HA glycoprotein in subcellular fractions resolved by Nycoden z gradient centrifugation following pulse-chase radiolabeling that sulfatio n of the complex N-linked oligosaccharides occurs only after they have been processed to an endo-beta -N-acetylglucosaminidase-resistant state and hav e reached the medial/trans Golgi and the trans Golgi network (TGN), with th e former carrying out most of the sulfation activity. Hydrazine/nitrous aci d/NaBH4 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 t he N-acetyllactosamine chains became sulfated in these post ER fractions, a s 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'-pho sphosulfate (PAPS):GlcNAc-6-O-sulfotransferase migrated in the gradient to a medial/trans Golgi position while in contrast the PAPS:Gal-3-O-sulfotrans ferase was found in both Golgi and TGN locations. In accordance with the co ncept that beta -galactosylation must precede the sulfation catalyzed by th e latter enzyme, we observed the presence of UDP-Gal:GlcNAc galactosyltrans ferase in both these sites in the MDCK cells. The presence of the Gal-3-O-s ulfotransferase in the TGN is particularly important in the influenza virus -infected cells, as it makes possible the addition of terminal anionic grou ps after removal of the sialic acid residues by the viral neuraminidase.