Hybrid, sialylated N-glycans accumulate in a ricin-resistant mutant of baby hamster kidney BHK cells.
Glycoproteins synthesized in a ricin-resistant mutant of BHK cells, clone RICR21, were labelled by growth of the cells in radioactive D-mannose, D-glucosamine, or L-fucose. Glycopeptides obtained from disrupted cells by exhaustive digestion with Pronase were fractionated into components binding to concanavalin A-Sepharose and nonbinding components. The binding components eluted with methyl alpha-D-mannopyranoside were separated by gel filtration on Bio-Gel P-4 into two main subfractions: an oligomannosidic fraction that was susceptible to Jack bean alpha-D-mannosidase and a fraction that became totally degraded only in the additional presence of neuraminidase, beta-D-galactosidase, and N-acetyl-beta-D-glucosaminidase. Further analysis of the latter fraction by exoglycosidase digestion together with consideration of the known pathways for the biosynthesis of asparagine-linked sugar chains of glycoproteins was consistent with a "hybrid" structure containing a NeuAc leads to Gal leads to GlcNAc sequence linked to the alpha-D-mannosyl-(1 leads to 3) residue of the core sequence, and a terminal alpha-D-mannosyl group linked to the alpha-(1 leads to 6) branch of the core sequence. The hybrid fraction was labelled after growth of the cells in radioactive L-fucose and was adsorbed to a lentil lectin-Sepharose column indicating the presence of core fucosylation. The novel structure represented about 30-35% of the total cellular glycopeptides of RICR21 cells and was not present in the glycopeptides of normal, ricin-sensitive BHK cells. Conversely, double-branched (biantennary) complex N-glycans, a prominent constituent of BHK cell glycoproteins, were absent in RICR21 cells, and analysis of the nonbinding fraction obtained from concanavalin A-Sepharose indicated that triple- and quadruple-branched (tri- and tetra-antennary), complex N-glycans present in normal BHK cell glycoproteins were also absent.
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D004351:Drug Resistance; D006020:Glycopeptides / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis; D007668:Kidney; D009154:Mutation; D011134:Polysaccharides / Q000235:genetics / Q000302:isolation & purification*; D012276:Ricin / Q000494:pharmacology*; D012794:Sialic Acids / Q000032:analysis
D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D012794:Sialic Acids; D012276:Ricin