A lectin-resistant mouse lymphoma cell line is deficient in glucosidase II, a glycoprotein-processing enzyme

Glycosylation of asparagine residues of glycoproteins occurs by the transfer of a glucose₃mannose₉N-acetyl-glucosamine₂ (Glc₃Man₉GlcNAc₂) oligosaccharide from a lipid carrier to the nascent protein. Normally, this transfer is quickly followed by the stepwise removal of the glucose residues which are arranged in the sequence: Glc1→2Glc1→3Glc1→3Man. We now report studies which demonstrate that a lectin-resistant mutant of the BW5147 mouse lymphoma cell line is deficient in the enzyme which removes the two inner glucose residues. This cell line (PHA(R)2.7) was selected for resistance to the cytotoxic effects of Phaseolus vulgaris leukoagglutinating lectin. Glycopeptides prepared from cells equilibrium-labeled with either [2-³H]mannose or [6-³H]galactose were characterized using lectin affinity chromatography, treatment with specific endo- and exoglycosidases, sizing by paper chromatography, and methylation analysis. Approximately 50% of the radioactivity in [³H]mannose-labeled glycopeptides from the mutant cells is present as glucosylated high mannose-type oligosaccharides whereas parent cell glycopeptides labeled under similar conditions lack detectable amounts of these species. Using [³H]galactose labeling, the major glucosylated oligosaccharides were identified as (Glc₅Man₉GlcNAc₂) and Glc₂Man₈GlcNA₂. In vitro enzyme assays demonstrated that the mutant cells cannot remove either of the two inner 1→3-linked glucose residues. Removal of the outer 1→2-linked glucose is normal. We conclude from these data that the PHA(R)2.7 cell line is deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the oligosaccharides of newly glycosylated proteins.