A Dominant Mutation to Ricin Resistance in Chinese Hamster Ovary Cells Induces UDP-G1cNAc:Glycopeptide

A biochemical basis for the LEClO mutant phenotype of Chinese hamster ovary cells has been identified. Independent LEClO mutants, originally selected for resistance to the toxicity of ricin, have been shown to exhibit reduced binding of la61-ricin at the cell surface. Although this is indicative of structural changes in cellsurface carbohydrates, labeling of plasma membranes with galactose o~idase/[~H]borohydride r vealed no significant differences between mutant and parental cells. Alterations in the carbohydrates synthesized by LEClO cells were, however, resolved by lectin-affinity chromatography of glycopeptides from the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEClO. LEClO/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 LEClO revertant (R.LEClO/ VSV) do not contain carbohydrates with these properties. High-field ‘H NMR spectroscopy of the novel LEClO/VSV carbohydrates showed that they are complex, biantennary structures containing N-acetylglucosamine in 8(1,4)-linkage to the &linked core mannose residue. The presence of these structures correlates with the xpression of the enzyme responsible for the addition of this “bisecting“ GlcNAc residue, UDPG1cNAc:glycopeptide B-4-N-acetylglucosaminyltransferase I11 (GlcNAc-TIII). Parental Chinese hamster ovary cells and the LEClO revertant possess no detectable GlcNAc-TI11 activity. The combined evidence suggests that the LEClO mutation induces the expression of the GlcNAc-TI11 enzyme in Chinese hamster ovary cells.