A mouse lymphoma cell line resistant to the leukoagglutinating lectin from Phaseolus vulgaris is deficient in UDP-GlcNAc:α-D-mannoside β1,6 N-acetylglucosaminyltransferase

A UDP-GlcNAc:α-mannoside β1,6 N-acetylglucosaminyltransferase was identified in cell-free preparations from the mouse lymphoma cell line BW5147. A novel assay employing concanavalin A-Sepharose, lentil lectin-Sepharose, and radioiodinated glycopeptide acceptors was devised to specifically measure the activity of this enzyme. The enzyme transfers N-acetylglucosamine to the C-6 position of α-linked mannose residues and utilizes as acceptors biantennary Asn-linked oligosaccharides containing N-acetylglucosamine, but not sialic acid or galactose, at the nonreducing termini. The product of the enzyme is a triantennary glycopeptide. The level of this enzyme activity was markedly decreased in a mouse lymphoma cell line (BW5147-PHA(R)2.1), which has previously been shown to be resistant to the cytotoxic effects of the leukoagglutinating lectin (L-PHA) from Phaseolus vulgaris. The consequence of this enzyme deficiency on the biosynthesis of Asn-linked oligosaccharides was assessed by labeling the mutant cells with [2-³H]mannose and analyzing the radiolabeled oligosaccharides by serial lectin-agarose affinity chromatography. The mutant cells, when compared to the parent cell line, were found to synthesize markedly decreased amounts of tri- and tetraantennary glycopeptides with an α-linked mannose residue substituted at both positions C-2 and C-6 by N-acetylglucosamine. This change was associated with an increased amount of biantennary glycopeptides in the mutant cells compared to the parent cell line. The mutant cells also have > 80% fewer high affinity binding sites for L-PHA. We conclude that the deficiency of UDP-GlcNAc:α-mannoside β1,6 N-acetylglucosaminyltransferase activity results in a failure to synthesize tri- and tetraantennary glycopeptides, which interact with L-PHA with the highest affinity.