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Glycobiology, 2000, Vol. 10, No. 11 1235-1242
© 2000 Oxford University Press

Sulfation of the N-linked oligosaccharides of influenza virus hemagglutinin: temporal relationships and localization of sulfotransferases

Mary Jane Spiro and Robert G. Spiro1

Elliott P. Joslin Research Laboratory, Joslin Diabetes Center and the Departments of Medicine and Biological Chemistry, Harvard Medical School, Boston, MA 02215, USA

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-ß-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/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 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 ß-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.

1 To whom correspondence should be addressed at: Joslin Research Laboratory, One Joslin Place, Boston, MA 02215


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