Man2, a1 and Ricin Resistance in ES Cells Figure 4. N-linked glycosylation pathway analysis. A, diagram indicating steps and key inhibitors (numbered 1–8) involved in processing core sugar structures. B1 assay demonstrating neutral red viability data after na¨ıve ES cells were treated with glycosylation inhibitors deoxynojirmycin (#3 in fig. 4A), deoxymannojirimycin (#6 in fig. 4A) or Swainsonine (#7 in fig. 4A) at 1, 10 or 100 mM 4 hours prior to, and during, exposure to 10 pM ricin for 3 days. B2, a comparison of neutral red viability data after na¨ıve ES cells were treated with glycosylation inhibitors either 24 hours or 4 hours prior to, and during, ricin exposure. doi:10.1371/journal.pone.0022993.g004 three different mannose specific lectins (Concanavalin A, Pisum sativum agglutinin and Lens culinaris agglutinin). Mannosidase 2, alpha 1 is known to cleave the N-linked GIcNAcMan5GlcNAc2 intermediate to complete the mannose trimming reactions and gives rise to the oligosaccharide, GIcNAc- Man3GlcNAc2. The enzyme is mostly located in the medial-cisternae of cells but its distribution can vary with cell type [23]. The results from our glycomic analyses demonstrate that there are alterations in the hybrid and complex N-glycans released from our ricin-resistant clones. The high levels of residual complex glycans, still present in the F10 cells, indicates some redundancy in the complex N-glycan biosynthetic pathway. There is evidence of a complementary enzyme, alpha mannosidase llX [24] which also resides in the golgi and which can perform similar trimming but not to the extent of Man2a1. Mice deficient in both enzymes are mostly non-viable beyond the 18.5 d embryonic stage indicating the importance of N-linked glycosylation in embryo development [24,25]. A recent publication has described an isolate, Lec36, of the human embryonic kidney HEK293T cell line, which has been identified as ricin resistant following ethyl methanesulfonate mutagenesis [26]. By nucleotide sequencing, the causal defect was linked to separate mutations on both alleles of Man2a1, one due to a point mutation at the active site and the second an in- frame deletion of twelve nucleotides. However, chemical muta- genesis can generate an array of unlinked and unmapped mutations that can contribute to a mutant phenotype. These data strongly support the results demonstrated in our homozygous gene-trap mutagenesis system and confirms the usefulness of screening mutation libraries in undifferentiated ES cells. Immuno-gold imaging for ricin binding in the Man2a1-deficient ES cell clones failed to identify ricin bound to these cells. This suggests that the mechanism of resistance is related to the structure of the N-linked glycoproteins on the cell surface, which could be caused by loss of specific or general ricin-binding moieties on the cell surface. In conclusion, we emphasise the usefulness of the PiggyBac gene- trap transposon system in Blm-deficient ES cells to generate mutants and directly identify genes required for action of toxins. PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e22993