A dominant mutation to ricin resistance in Chinese hamster ovary cells induces UDP-GlcNAc:glycopeptide beta-4-N-acetylglucosaminyltransferase III activity.
A biochemical basis for the LEC10 mutant phenotype of Chinese hamster ovary cells has been identified. Independent LEC10 mutants, originally selected for resistance to the toxicity of ricin, have been shown to exhibit reduced binding of 125I-ricin at the cell surface. Although this is indicative of structural changes in cell-surface carbohydrates, labeling of plasma membranes with galactose oxidase/[3H]borohydride revealed no significant differences between mutant and parental cells. Alterations in the carbohydrates synthesized by LEC10 cells were, however, resolved by lectin-affinity chromatography of glycopeptides from the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC10. LEC10/VSV glycopeptides contain a fraction which is not bound to concanavalin A-Sepharose but is strongly retarded on E-PHA (erythroagglutinin from Proteus vulgaris)-agarose. In contrast, CHO/VSV glycopeptides or those from a LEC 10 revertant (R.LEC 10/VSV) do not contain carbohydrates with these properties. High-field 1H NMR spectroscopy of the novel LEC10/VSV carbohydrates showed that they are complex, biantennary structures containing N-acetylglucosamine in beta(1,4)-linkage to the beta-linked core mannose residue. The presence of these structures correlates with the expression of the enzyme responsible for the addition of this "bisecting" GlcNAc residue, UDP-GlcNAc:glycopeptide beta-4-N-acetylglucosaminyltransferase III (GlcNAc-TIII). Parental Chinese hamster ovary cells and the LEC10 revertant possess no detectable GlcNAc-TIII activity. The combined evidence suggests that the LEC10 mutation induces the expression of the GlcNAc-TIII enzyme in Chinese hamster ovary cells.
D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D004790:Enzyme Induction; D005260:Female; D005799:Genes, Dominant; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases; D010053:Ovary; D010641:Phenotype; D012276:Ricin / Q000494:pharmacology*
D002241:Carbohydrates; D012276:Ricin; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C029391:alpha-D-mannose-beta-1,2-N-acetylglucosaminyltransferase