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

R. D. Cummings, S. Kornfeld

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Abstract

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.

Original languageEnglish
Pages (from-to)6253-6260
Number of pages8
JournalJournal of Biological Chemistry
Volume259
Issue number10
StatePublished - 1984

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