Mannosidase 2 , alpha 1 Deficiency Is Associated with Ricin Resistance in Embryonic Stem (ES) Cells Wei Wang1., Christine Hale1*., Dave Goulding1, Stuart M. Haslam2, Be´ range` re Tissot2, Christopher Lindsay3, Stephen Michell4, Rick Titball4, Jun Yu5, Ana Luisa Toribio1, Raffaella Rossi1, Anne Dell2, Allan Bradley1, Gordon Dougan1 1 The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom, 2 Division of Molecular Biosciences, Imperial College London, South Kensington Campus, South Kensington, London, United Kingdom, 3 Cellular Toxicity Team, Biomedical Sciences Department, Porton Down, Salisbury, Wiltshire, United Kingdom, 4 School of Biosciences, University of Exeter, Exeter, Devon, United Kingdom, 5 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Royal College, Glasgow, Scotland, United Kingdom 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 (Man2a1) 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 Man2a1 (2/2) ricin resistant cells revealed an increase in the levels of hybrid glycan structures and a reduction in terminal b-galactose moieties, potential target receptors for ricin. Furthermore, na¨ıve 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. Citation: Wang W, Hale C, Goulding D, Haslam SM, Tissot B, et al. (2011) Mannosidase 2, alpha 1 Deficiency Is Associated with Ricin Resistance in Embryonic Stem (ES) Cells. PLoS ONE 6(8): e22993. doi:10.1371/journal.pone.0022993 Editor: Je´roˆ me Nigou, French National Centre for Scientific Research - Universite´ de Toulouse, France Received November 3, 2010; Accepted July 11, 2011; Published August 23, 2011 Copyright: 2011 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Biotechnology and Biological Sciences Research Council (BBSRC, www.bbsrc.ac.uk) grant number BBF0083091 (awarded to AD, SMH) and the Wellcome Trust (www.wellcome.ac.uk) grant numbers 077187 and 076962 (awarded to AB and GD respectively). Ricin was supplied by the Biomedical Sciences Department, Dstl Porton Down, Salisbury, Wiltshire SP4 0JQ. The funding providers had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cbh@sanger.ac.uk . These authors contributed equally to this work. Introduction 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. The identification and study of host factors which interact with toxins is important for a detailed understanding of how they disturb normal cellular physiology and to identify host cell components as potential targets for mitigating their effects. 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. This toxin is extremely toxic and enters mammalian cells through the interaction of the ricin B chain with surface exposed b1–4 linked terminal galactose residues [1,2]. Once inside the cell, ricin holotoxin is taken into early endosomes and a small fraction of the intoxicating protein then undergoes retrograde transport to the cytosol, via the endosomes, trans-golgi network (TGN) and endoplasmic reticulum (ER) [3–6], whilst the majority recycles to the cell surface. Once in the ER, the disulphide bridge between the A and B chain is reduced and the free A chain can be transported into the cytosol where it inactivates the 28S subunit of the ribosome. The process of crossing the ER is thought to involve the Sec61 translocon [7] in a fashion similar to that of misfolded proteins. Many steps in the process of uptake and retrograde transport of ricin are unknown and therefore of scientific interest. Blm-deficient mouse ES cells have been developed as a platform for conducting phenotype-driven recessive screens. These cells have an elevated rate of loss of heterozygosity (LOH) approxi- mately 20 fold higher compared with wild type ES cells. Following mutagenesis, the Blm-deficient background greatly facilitates the segregation of homozygous mutant clones, enabling genome wide recessive screens in the diploid mammalian genome. Using this genetic background we have successfully conducted screens for DNA mismatch repair genes and retroviral resistance [8,9]. Here we describe a recessive screen designed to identify host cell components required for ricin toxicity. In contrast to our previous screens, PiggyBac transposon gene-trap vectors were used as insertional mutagens in Blm-deficient ES cells in place of retroviral PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e22993