Man2, a1 and Ricin Resistance in ES Cells Figure 2. Insertion site of PiggyBac (PB) transposon into the mannosidase 2, alpha 1 gene. A, illustration of the insertion site in intron 21 of the Man2a1 gene on murine chromosome 17. The precise insertion site and the exon 21-b-geo spliced product is shown. B, long range PCR on DNA from F10 mutant clones -1 and -2 or NGG5.3 generates a fragment of 6.6 kb for the mutants or 428 bp for the parent confirming homozygous insertion of the PB transposon. C, triple primer PCR confirms homozygosity by the generation of junction fragments corresponding to the PB 39 end of 335 bp and the PB 59 end of 376 bp in F10 mutant clones -1 and -2 compared to the parental NGG5.3 428 bp. doi:10.1371/journal.pone.0022993.g002 exhibit some clear differences. It has been demonstrated that relative quantitation based on signal intensities of permethylated glycans analyzed by MALDI-TOF MS is a reliable method, especially when comparing signals over a small mass range within the same spectrum [16]. In the region m/z 2400 to 2700 (Fig. 7 A, B and C), the relative intensities of peaks such as m/z 2489, 2519 and 2693 which would have terminal b-linked galactose are all reduced in Mannosidase 2, alpha 1-deficient F10 cells (relative % intensities in AB2.2 cells 42/18/14, relative % intensities in NN5 cells 49/29/24 and relative % intensities in F10 cells 10/5/6). Additional F10 peaks at m/z 2407 and 2564 are also observed, which are not detectable in the profiles of the control cell lines. These correspond to hybrid type N-glycan structures on which only one mannosylated arm has been trimmed down and extended, the other arm remaining high-mannose like. In the second region of interest and at high m/z values (Fig. 8 D, E and F, Supplementary Figures S1 and S2), the F10 mutant exhibits less high molecular weight structures than the control. These data suggest that the complex binding specificity of ricin does not simply reflect the level of terminal galactose. The O-glycan profiles (Supplementary table S2) exhibit very simple patterns with major structures being mono- and disialylated core-1. The samples do not show any major differences suggesting that the Man2a1 mutation does not affect the O-glycosylation biosynthetic pathways. The polar and non polar species of glycolipids were also examined and also did not reveal any differences between the F10 Man2a1-deficient and control cell lines, revealing that this mutation did not affect the glycosphin- golipid pathways either (Supplementary table S3). Discussion The action of ricin on susceptible cells leads to rapid cell death induced by the inactivation of the 28S rRNA of the 60S ribosome subunit [17]. Many reviewers have alluded to the mechanisms of uptake of ricin but acknowledge a lack of full understanding. We have successfully conducted a genetic screen in ES cells to identify a gene, the mutation of which confers resistance to ricin intoxication. The ricin-resistant clone identified in this screen resulted from a homozygous mutation in the Mannosidase 2, alpha 1 gene, which encodes a Golgi enzyme involved in N-linked glycosylation. The enzyme hydrolysis of the terminal a1–3 and a1–6 linked mannose residues of the GIcNAcMan5GlcNAc2 intermediate gives rise to the oligosaccharide GIcNAcMan3Glc- NAc2. This represents one of the committal steps in the biosynthesis of complex N-glycans which will carry the terminal PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e22993