Mechanism of inhibition of protein glycosylation by the antiviral sugar analog 2-deoxy-2-fluoro-D-mannose: inhibition of synthesis of Man(GlcNAc)2-PP-Dol by the guanosine diphosphate ester

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Abstract
2-Deoxy-2-fluoro-D-mannose (2FMan), an antiviral mannose analogue, inhibited the dolichol cycle of protein glycosylation. To specifically inhibit oligosaccharide-lipid synthesis, and not (viral) protein synthesis in influenza virus infected cells, the addition of guanosine to the 2FMan-treated cells was required. Under these conditions an early step in the assembly of the oligosaccharide-lipid was inhibited, and as a consequence, the glycosylation of proteins was strongly inhibited. Low-molecular-weight, lipid-linked oligosaccharides accumulated in cells treated with 2FMan plus guanosine, although dolichol phosphate (Dol-P) and GDP-Man were still present in the treated cells, and membranes from these cells were not defective in assembly of lipid-linked oligosaccharides. Thus, the presence of a soluble inhibitor of oligosaccharide-lipid assembly in these cells was postulated, and GDP-2FMan and UDP-2FMan, two metabolites found in 2FMan-treated cells, were synthesized and used to study in cell-free systems the inhibition of oligosaccharide-lipid assembly. GDP-2FMan inhibited the synthesis of Man(GlcNAc)2-PP-Dol from (GlcNAc)2-PP-Dol and GDP-Man, and in addition, it caused a trapping of Dol-P as 2FMan-P-Dol, whereas UDP-2FMan only inhibited Glc-P-Dol synthesis. However, it is probable that neither trapping of Dol-P nor inhibition of Glc-P-Dol synthesis by UDP-2FMan contributed to inhibition of protein glycosylation in cells treated with 2FMan. Incorporation of 2FMan from GDP-2FMan or UDP-2FMan into dolichol diphosphate linked oligosaccharides and interference of GDP-2FMan with the latter steps of assembly of the dolichol diphosphate linked oligosaccharide could not be shown. It is concluded that 2FMan, via GDP-2FMan, inhibits protein glycosylation by blocking formation of Man(GlcNAc)2-PP-Dol and, thus, further assembly of the oligosaccharide-lipid.