The distribution of repeating [Gal beta 1,4GlcNAc beta 1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1.
The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] 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 fractionated 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-beta-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 alpha 1,3 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-GlcNAc:alpha-mannoside beta 1,6-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 teraantennary species.
D000117:Acetylglucosamine / Q000032:analysis*; D000818:Animals; D001216:Asparagine; D002240:Carbohydrate Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D005690:Galactose / Q000032:analysis*; D005944:Glucosamine / Q000031:analogs & derivatives*; D006020:Glycopeptides / Q000302:isolation & purification; D006023:Glycoproteins / Q000302:isolation & purification*; D006026:Glycoside Hydrolases; D008223:Lymphoma / Q000032:analysis*; D008745:Methylation; D051379:Mice; D009844:Oligosaccharides / Q000302:isolation & purification*; D010835:Phytohemagglutinins / Q000302:isolation & purification
D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D010835:Phytohemagglutinins; D001216:Asparagine; D006026:Glycoside Hydrolases; D005944:Glucosamine; D000117:Acetylglucosamine; D005690:Galactose