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 NeuNAcα2→3Galβ1→4GlcNAcβ1→2 linked to the α→3 arm mannose of the core pentasaccharide. Analysis of extracts of normal or mutant cells has shown in the mutants a deficiency in α-mannosidase activity measured with p-nitrophenyl α-mannoside. This activity is swainsonine-sensitive and exhibits a pH optimum at about 6–6.5. Assays using a specific substrate for α-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.
- BHK cells
baby hamster kidney cell line
- CHO cells
Chinese hamster ovary cell line
Phaseolous vulgaris agglutinin
PHA-resistant. All sugars are of the d-configuration unless otherwise stated
α-Mannosidase (EC 18.104.22.168)
UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyl transferases I and II (EC 22.214.171.124 and 126.96.36.199)