Man2, a1 and Ricin Resistance in ES Cells Figure 1. Mutagenesis and selection of ricin resistant mutants. A, establishing the selective dose of ricin. Viability as percentage of control calculated from neutral red staining of AB2.2, NN5 and NGG5.3 cells after exposure to different ricin doses. B, PBGTVs vector structure showing the position of the PiggyBac (PB) 39 and 59 inverted terminal repeats, the adenovirus (adSA) and the mouse gene En2 (En2SA) splice acceptor, the polyadenylation site (pA) and the b-geo cassette. C, methylene blue staining of NGG5.3 cells or ricin-resistant mutant F10 clones -1 and -2 before and after treatment with G418 or 20 pm ricin. D, Southern blot of NGG5.3 or F10 clones -1 and -2 genomic DNA probed with the b-geo probe, a fragment of 5.6 Kb is visible in only F10 mutant clones -1 and -2. E, splinkerette PCR of the PB 59 junction generates a fragment of 329 bp and the PB 39 junction generates a product of 129 bp. doi:10.1371/journal.pone.0022993.g001 (Fig. 4B 1). To ensure that all cells were fully exposed to the inhibitors, pre-treatment was extended to 24 hours prior to ricin intoxication and this led to full protection by Swainsonine (Fig. 4B 2). However, deoxymannojirimycin still only exhibited partial protection (35% survival above that of ricin alone level) whilst deoxynojirimycin continued to afford little protection. The former is a specific glucosidase inhibitor of the trimming glucosidases I and II, enzymes that sequentially remove the three glucose residues from the precursor Glc3Man9-GlcNAc2 in N-linked glycan biosynthesis whilst the latter is a specific a-mannosidase I inhibitor that blocks conversion of high mannose to complex oligosaccharides. If the block in processing of N-linked glycans only was the direct causality of ricin resistance, inhibition of all steps prior to the action of mannosidase 2, alpha 1 should have been effective at inducing a level of ricin resistance. The fact that only Swainsonine protected fully indicates the importance of this step in the generation of the ricin receptor. Thus, it is possible that Man2a1 is involved in other cellular processes such as the retrograde transport of mis-folded proteins, in a similar manner to mannosidase 1 alpha, potentially explaining why only Swainsonine consistently confers ricin resistance to na¨ıve cells. To confirm the impact of the mutation on surface sugars, flow cytometric analysis was conducted of cells incubated with a range of different FITC conjugated lectins. The F10 (Man2a1-deficient) line exhibited a 2.5–3 fold higher level of binding to lectins with specificity for mannose (Fig. 5A) than wild type controls. In contrast, lectins with specificity for either galactose or sialic acid, moieties added to the core sugar post mannosidase 2, alpha 1 action, were found to have reduced binding (Figs. 5B and 5C respectively), with respect to the control, in the ricin resistant F10 line. Immunogold imaging of ricin resistant cell line To investigate how the alterations in surface glycosylation patterns revealed by lectin binding might be affecting ricin resistance, anti-ricin immunogold staining was conducted on cells exposed to ricin. After treatment with 10 pM ricin for 1 hour, control NN5 cells exhibited positive staining of the toxin throughout the cell. Ricin was demonstrated to be distributed on the plasma and nuclear membranes (Figs. 6A, B and C). The F10 ricin resistant cells exhibited no detectable staining (Figs. 6D and E), indicating that reduced levels of surface exposed hybrid N- glycans may be responsible for a reduced level of ricin binding and a corresponding ricin resistance phenotype. Glycomic analysis The parental and F10 cells were subjected to glycomic analysis (N- and O-glycans, glycosphingolipids) using MALDI-TOF MS, Figure 7. Apart from the high mannose structures, the glycan profiles of mutant and control cell types are similar (Supplemen- tary Figures S1 and S2). However, two regions of these profiles PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e22993