Hybrid, sialylated N-glycans accumulate in a ricin-resistant mutant of baby hamster kidney BHK cells

Abstract

Glycoproteins synthesized in a ricin-resistant mutant of BHK cells, clone RIC R21, 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 α- 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 α- d-mannosidase and a fraction that became totally degraded only in the additional presence of neuraminidase, β- d-galactosidase, and N-acetyl-β- 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→Gal→GlcNAc sequence linked to the α- d-mannosyl-(1→3) residue of the core sequence, and a terminal α- d-mannosyl group linked to the α-(1→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 RIC R21 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 RIC R21 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.