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

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, Glc_lMan₈GlcNAc₂ and Glc_lMan₇GlcNAc₂, 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.