The distribution of repeating [Galβ1,4GlcNAcβ1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHA(R) 2.1. Binding of oligosaccharides containing these sequences to immobilized Datura stramonium agglutinin

The occurrence and distribution of the repeating disaccharide [Galβ1,4GlcNAcβ1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-³H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractioned by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin-Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-β-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked α1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line (PHA(R) 2.1) that is deficient in UDP-GlcNAc:α-mannoside β1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and tetraantennary species.