Effect of trimming inhibitors on the secretion and biological activity of a murine IgE monoclonal antibody.
Since secretion of IgE antibodies is known to be blocked by tunicamycin, the first aim of the present study was to determine at which step of glycosylation or processing secretion was restored. For this purpose, murine hybridoma cells secreting an anti beta-lactoglobulin IgE were incubated either in the presence of inhibitors of glucosidase I (castanospermine or N-methyl-1-deoxynojirimycin), or of an inhibitor of Golgi mannosidase II (swainsonine). Terminal galactoses predominate on the native IgE N-linked carbohydrate chains. The action of the trimming inhibitors, which results in changes in these terminal galactose residues, was monitored through detecting binding modifications to Concanavalin A and to the lectin of Ricinus communis. The antibody activity was also evaluated by a radioimmunoassay. It was shown that neither secretion nor anti beta-lactoglobulin activity of the IgE antibody are modified in the presence of any of the trimming inhibitors, whereas secretion is blocked in the presence of tunicamycin. Other biological activities of this IgE were investigated: no difference was observed in the binding of the carbohydrate-modified IgE molecules to normal mouse mast cells, nor to RBL-1 cells, as demonstrated by passive cutaneous anaphylaxis and in vitro binding tests respectively. However, traces of unglycosylated epsilon chain (mol. wt 61,000) found in tunicamycin treated cell supernatant did not bind to RBL-1 cell Fc epsilon receptors. These findings globally suggest that secretion occurs only if the tetradecasaccharide precursor of N-linked carbohydrate chains is transferred from its lipid-carrier to the polypeptide. Further, the presence of such non-processed oligosaccharides (Glc3Man9GlcNAc2) on IgE, does not seem to modify any of the biological activities of this molecule.
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives*; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002460:Cell Line; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006031:Glycosylation; D007073:Immunoglobulin E / Q000096:biosynthesis* / Q000378:metabolism; D007212:Indolizines; D051379:Mice; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017026:Swainsonine; D014415:Tunicamycin / Q000494:pharmacology; D054836:Indolizidines
D017485:1-Deoxynojirimycin; D000470:Alkaloids; D000911:Antibodies, Monoclonal; D005944:Glucosamine; D007073:Immunoglobulin E; D007212:Indolizines; D017026:Swainsonine; D014415:Tunicamycin; C037806:castanospermine; D054836:Indolizidines; C033424:N-methyldeoxynojirimycin