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Mannosidase 2, alpha 1 deficiency is associated with ricin resistance in embryonic stem (ES) cells.

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 (Man2?1) 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 Man2?1 (-/-) ricin resistant cells revealed an increase in the levels of hybrid glycan structures and a reduction in terminal ?-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.

SUBMITTER: Wang W

PROVIDER: S-EPMC3160287 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Mannosidase 2, alpha 1 deficiency is associated with ricin resistance in embryonic stem (ES) cells.

Wang Wei W Hale Christine C Goulding Dave D Haslam Stuart M SM Tissot Bérangère B Lindsay Christopher C Michell Stephen S Titball Rick R Yu Jun J Toribio Ana Luisa AL Rossi Raffaella R Dell Anne A Bradley Allan A Dougan Gordon G

PloS one 20110823 8

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 ...[more]

PMID: 21886775

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Project description:The RNA binding protein, DEAD END (DND1), is essential for maintaining viable germ cells in vertebrates. It is also a testicular germ cell tumor susceptibility factor in mice. DND1 has been shown to interact with the 3'-untranslated region (3'-UTR) of mRNAs such as P27 and LATS2. Binding of DND1 to the 3'-UTRs of these transcripts blocks the inhibitory function of microRNAs (miRNA) from these transcripts and in this way DND1 helps maintain P27 and LATS2 protein expression. We found that DND1 is also expressed in embryonic stem (ES) cells. Because ES cells share similar gene expression patterns as germ cells, we utilized ES cells to identify additional candidate mRNAs that associate with DND1.ES cells are readily amenable to genetic modification and easier to culture in vitro compared to germ cells. Therefore, for the purpose of our study, we made a genetically modified, stable, human embryonic stem (hES) cell line that expresses hemagluttinin (HA)-tagged DND1 in a doxycycline (dox) regulatable manner. This line expresses modest levels of HA-DND1 and serves as a good system to study DND1 function in vitro. We used this stable cell line to identify the transcripts that physically interact with DND1. By performing ribonucleoprotein immunoprecipitation (RIP) followed by RT-PCR, we identified that transcripts encoding pluripotency factors (OCT4, SOX2, NANOG, LIN28), cell cycle regulators (TP53, LATS2) and apoptotic factors (BCLX, BAX) are specifically associated with the HA-DND1 ribonucleoprotein complex. Surprisingly, in many cases, bioinformatics analysis of the pulled-down transcripts did not reveal the presence of known DND1 interacting motifs.Our results indicate that the inducible ES cell line system serves as a suitable in vitro system to identify the mRNA targets of DND1. The RIP-RT results hint at the broad spectrum of mRNA targets that interact with DND1 in ES cells. Based on what is known about DND1 function, our results suggest that DND1 may impose another level of translational regulation to modulate expression of critical factors in ES cells.

Dataset Information

Mannosidase 2, alpha 1 deficiency is associated with ricin resistance in embryonic stem (ES) cells.

Publications

Mannosidase 2, alpha 1 deficiency is associated with ricin resistance in embryonic stem (ES) cells.

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