Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases.
We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.
Foddy
L
L
National Institute for Medical Research, London, England.
Hughes
R C
RC
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D001216:Asparagine; D002460:Cell Line; D002850:Chromatography, Gel; D002854:Chromatography, Paper; D006224:Cricetinae; D005959:Glucosidases / Q000037:antagonists & inhibitors*; D006020:Glycopeptides / Q000096:biosynthesis* / Q000302:isolation & purification; D007212:Indolizines; D007668:Kidney; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000302:isolation & purification; D054836:Indolizidines
D000470:Alkaloids; D001216:Asparagine; D005959:Glucosidases; D006020:Glycopeptides; D007212:Indolizines; D008358:Mannose; D009844:Oligosaccharides; C037806:castanospermine; D054836:Indolizidines