Assembly of asparagine‐linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases

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Abstract
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‐l‐deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537–5441. 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 α‐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, GlclMan8GlcNAc2 and GlclMan7GlcNAc2, 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.