The effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins.
The influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. In order to determine what structural features of the oligosaccharide were required for fucosylation or where in the processing pathway fucosylation occurred, influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, [5,6-3H]fucose and [1-14C]mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the [14C]mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the [14C]mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the [3H]fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the [14C]mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002250:Carbon Radioisotopes; D002460:Cell Line; D002472:Cell Transformation, Viral; D004285:Dogs; D005643:Fucose / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D009980:Influenza A virus / Q000235:genetics* / Q000378:metabolism; D007668:Kidney; D008358:Mannose / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017026:Swainsonine; D014316:Tritium
D000470:Alkaloids; D002250:Carbon Radioisotopes; D006023:Glycoproteins; D014316:Tritium; D005643:Fucose; D008358:Mannose; D017026:Swainsonine