The Distribution of Repeating [Gal@1,4GlcNAc@l,3] Sequences in Asparagine-linked Oligosaccharides of the Mouse Lymphoma

The occurrence and distribution of the repeating disaccharide [Gal/31,4GlcNAc~1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5 147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-’H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin ASepharose, pea lecitn-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 Nacetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichiu freundii endo-&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 a1,3 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 (PHAR 2.1) that is deficient in UDP-G1cNAc:a-mannoside @1,6-Nacetylglucosaminyltransferase. 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 complextype Asn-linked oligosaccharides in BWS147 cells but is confined primarily to triand teraantennary species.