N-linked oligosaccharides of the murine transferrin receptor from a plasmacytoma cell line. Comparison with total cellular N-glycans.
The N-linked oligosaccharides synthesised by the murine plasmacytoma cell line NS-1 have been analysed by lectin affinity chromatography on columns of immobilised concanavalin A (Con A), Lens culinaris (lentil), Ricinus communis agglutinin (RCA) and leuko-phytohemagglutinin (L-PHA). The majority of complex N-glycans in this transformed cell line were branched structures with only a low level of biantennary complex chains detected. The analysis showed the major complex N-glycan fraction consisted of a minimum sialylated triantennary structure. [3H]Mannose-labelled transferrin receptor was isolated from NS-1 cells by immunoprecipitation followed by electroelution from SDS polyacrylamide gels. The isolated receptor was digested with Pronase and the 3H-labelled glycopeptides analysed by lectin affinity chromatography. Analysis by Con A-Sepharose indicated that approx. 50% of the labelled glycopeptides were branched complex N-glycans (unbound fraction) while the remainder were oligomannose structures (strongly bound). The presence of tri and/or tetraantennary structures in the Con A unbound fraction was further suggested by the interaction of 61% of the fraction with L-PHA. The lectin profiles obtained for the complex N-glycans of the transferrin receptor glycopeptides were similar to those for the total cellular glycopeptides of NS-1 cells. Reverse-phase HPLC analysis of tryptic glycopeptides of the isolated [3H]mannose-labelled transferrin receptor gave three 3H-labelled peaks, indicating that all three potential N-glycosylation sites on the receptor are utilised. The Con A-Sepharose profiles of the three fractions indicated the presence of branched complex N-glycans and high mannose chains at each site. The profiles of two of the tryptic glycopeptide fractions were very similar, while the third had a higher content of oligomannose oligosaccharides.
Gleeson
P A
PA
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Australia.
Dias
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D000818:Animals; D002846:Chromatography, Affinity; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D037102:Lectins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009844:Oligosaccharides / Q000032:analysis*; D010954:Plasmacytoma / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis*; D011990:Receptors, Transferrin / Q000032:analysis* / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D006020:Glycopeptides; D037102:Lectins; D009844:Oligosaccharides; D011134:Polysaccharides; D011990:Receptors, Transferrin