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.

R D Cummings,S Kornfeld

doi:10.1016/s0021-9258(20)82134-6

Abstract

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.

Highlights

The Distribution of Repeating [Gal@1,4GlcNAc@l,3] Sequencines Asparagine-linked Oligosaccharidesof the Mouse Lymphoma Cell Lines BW5147 and PHAR2.1
[6-’H]galactose, and the bi, tri, and tetraantennary sugar sequences manifest an enormous variety of structures, Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A
Particular.attention has been directed recently to outer sequences containing the repeating disaccharide [Gal~1,4GlcNAc/31,3]. Oligosaccharides containing these structuresare present in embryonal [7], erythrocyte [8,9]Chinese hamster ovary, Ehrlich ascites mannose residue substituted at C-2 and C-6 by N- tumor [11], and lymphocyte [12] cell surface glycoproteins, acetyllactosamine

Summary

The occurrence anddistribution of therepeating

The complex-type Asn-linked oligosaccharides of cell surdisaccharide [Gal/31,4GlcNAc~1,3]inthedifferent face glycoproteinsvary both in the substitution pattern of the types of Asn-linked oligosaccharides in mouse lym- common trimannosyl core and in the sequences of sugars phoma BW5147cells have been studied. The biosynthesis of these structures, we have examined the As thedatainTable I indicate, only 15% of the total outer sugar sequences in Asn-linked oligosaccharides from a incorporated radiolabeled galactose from the mutant cells is leukoagglutinating phytohemagglutinin-resistant cell line contained in oligosaccharides which bind to D. stramonium (PHAR 2.1), which synthesizes altered Asn-linked oligosac- agglutinin-agarose. These observations are consistent with the results of Yoshima et al [19] who examined the Asn-linked oligosaccharides

DISCUSSION

- RESULTS

Findings

Cell Llne