Ricin-resistant mutants of baby-hamster-kidney cells deficient in alpha-mannosidase-II-catalyzed processing of asparagine-linked oligosaccharides.
Previous work has shown that two ricin-resistant mutants of baby hamster kidney (BHK) cells, RicR15 and RicR19, synthesize only hybrid and oligomannose-type asparagine-linked oligosaccharides [Hughes, R. C. and Mills, G. (1985) Biochem. J. 226, 487-498]. In the present report glycopeptides were released from disrupted cells by exhaustive digestion with pronase, fractionated by chromatography on concanavalin-A--Sepharose, DEAE-Sephacel and lentil-lectin--Sepharose and characterized by 500-MHz 1H-NMR spectroscopy. The major hybrid structure identified in both cell lines contains five mannose residues and the sequence NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----2 linked to the alpha 1----3 arm mannose of the core pentasaccharide. Analysis of extracts of normal or mutant cells has shown in the mutants a deficiency in alpha-mannosidase activity measured with p-nitrophenyl alpha-mannoside. This activity is swainsonine-sensitive and exhibits a pH optimum at about 6-6.5. Assays using a specific substrate for alpha-mannosidase II, a terminal processing glycosidase in conversion of penta-mannose hybrid intermediates to complex N-glycans, reveals a reduced activity in RicR15 cells. Analysis of glycopeptides obtained from cells labelled with [3H]fucose or [3H]galactose revealed a small proportion of branched complex N-glycans of normal structure in mutant cells.
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006224:Cricetinae; D005690:Galactose / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000032:analysis / Q000172:deficiency* / Q000494:pharmacology; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D012276:Ricin / Q000494:pharmacology*; D014316:Tritium; D043323:alpha-Mannosidase
D006020:Glycopeptides; D009844:Oligosaccharides; D011134:Polysaccharides; D014316:Tritium; D001216:Asparagine; D012276:Ricin; D008361:Mannosidases; D043323:alpha-Mannosidase; D008358:Mannose; D005690:Galactose