Abstract
Host gene products required for mediating the action of toxins are potential targets for reversing or controlling their pathogenic impact following exposure. To identify such targets libraries of insertional gene-trap mutations generated with a PiggyBac transposon in Blm-deficient embryonic stem cells were exposed to the plant toxin, ricin. Resistant clones were isolated and genetically characterised and one was found to be a homozygous mutant of the mannosidase 2, alpha 1 (Man2α1) locus with a matching defect in the homologous allele. The causality of the molecular lesion was confirmed by removal of the transposon following expression of PB-transposase. Comparative glycomic and lectin binding analysis of the Man2α1 (−/−) ricin resistant cells revealed an increase in the levels of hybrid glycan structures and a reduction in terminal β-galactose moieties, potential target receptors for ricin. Furthermore, naïve ES cells treated with inhibitors of the N-linked glycosylation pathway at the mannosidase 2, alpha 1 step exhibited either full or partial resistance to ricin. Therefore, we conclusively identified mannosidase 2, alpha 1 deficiency to be associated with ricin resistance.
Highlights
Toxins are poisonous substances produced by a wide range of organisms in a variety of different forms including small molecules, peptides, proteins and larger order complexes which bind, enter and interfere with target cell functions by different mechanisms
A selective dose of ricin for Embryonic Stem (ES) cells was established for the wild type parental cell line (AB2.2), the Blm-deficient feeder-dependent line (NGG5. 3) and a Blmdeficient mutant line adapted for growth in feeder-free conditions (NN5)
A series of PiggyBac (PB) transposon-based gene-trap vectors (PBGTVs; Fig. 1B) containing an adenovirus splice acceptor or a mouse gene En2 splice acceptor (En2SA) and a b-geo gene-trap cassette flanked by the 59 and 39 PB terminal DNA repeats were used as the gene-trap vectors in this study
Summary
Toxins are poisonous substances produced by a wide range of organisms in a variety of different forms including small molecules, peptides, proteins and larger order complexes which bind, enter and interfere with target cell functions by different mechanisms. While many toxins mediate physiological affects by targeting specific cellular components, the binding, entry and transport through a cell to their site of action is dependent on interactions with a variety of other cellular components. Ricin is a heterodimeric lectin produced in the seeds of the castor oil plant, Ricinus communis. It is a type-2 toxin, having two chains, A and B. Many steps in the process of uptake and retrograde transport of ricin are unknown and of scientific interest
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