Properties of baby-hamster kidney (BHK) cells treated with Swainsonine, an inhibitor of glycoprotein processing. Comparison with ricin-resistant BHK-cell mutants.
Baby-hamster kidney (BHK) cells were grown continuously in long-term monolayer culture in the presence of Swainsonine, an inhibitor of alpha-mannosidase II, a processing enzyme involved in glycoprotein biosynthesis. The asparagine-linked oligosaccharides (N-glycans) were isolated from Pronase-digested cells by gel filtration, ion-exchange chromatography and affinity chromatography on concanavalin A--Sepharose and lentil lectin--Sepharose. The major N-glycans, analysed by 500 MHz 1H-n.m.r. spectroscopy, were identified as hybrid structures containing five mannose residues and neutral high-mannose N-glycans. The major hybrid species contained a core-substituted fucose alpha(1----6) residue and a NeuNAc alpha(2----3)Gal beta(1----4)GlcNAc terminal sequence; smaller amounts of non-sialylated and non-fucosylated hybrid structures were also detected. Swainsonine-treated cells also produced neutral oligosaccharides containing a single reducing N-acetylglucosamine residue substituted with polymannose sequences. The glycopeptide composition of Swainsonine-treated BHK cells resembles closely that of the ricin-resistant BHK cell mutant, RicR21 [P. A. Gleeson, J. Feeney and R. C. Hughes (1985) Biochemistry 24, 493-503], except the hybrid structures of RicR21 cells contain three, not five, mannose residues. Like RicR21 cells, Swainsonine-treated BHK cells showed a greatly increased resistance to ricin cytotoxicity, but not to modeccin, another galactose-binding lectin. These effects were readily reversed on removal of Swainsonine and growth in normal medium.
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005643:Fucose / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D012276:Ricin / Q000494:pharmacology; D017026:Swainsonine
D000470:Alkaloids; D006020:Glycopeptides; D005643:Fucose; D012276:Ricin; D017026:Swainsonine