Ricin-resistant mutants of baby-hamster-kidney cells deficient in α-mannosidase-II-catalyzed processing of asparagine-linked oligosaccharides

Authors


  • This paper is dedicated to Professor Luis Leloir on the occasion of his 80th birthday.

Correspondence to R. C. Hughes, National Institute for Medical Research, The Ridgeway, Mill Hill, London, England NW7 1AA

Abstract

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.

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