Abstract
Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus region of glutamate receptor, ionotropic, N-methyl-D-asparate-associated protein 1 (GRINA), a member of the transmembrane BAX inhibitor motif containing (TMBIM) family. Overexpression of the truncated form, named GRINA-C, and some members of the full-length TMBIM family, including FAS inhibitory molecule 2 (FAIM2), reduced Gb3, and lactosylceramide was accumulated instead. The change of glycolipid composition was restored by overexpression of Gb3 synthase, suggesting that the synthase is affected by GRINA-C and FAIM2. Interestingly, the mRNA level of Gb3 synthase was unchanged. Rather, localization of the synthase as well as TGN46, a trans-Golgi network marker, was perturbed to form punctate structures, and degradation of the synthase in lysosomes was enhanced. Furthermore, GRINA-C was associated with Gb3 synthase. These observations may demonstrate a new type of posttranscriptional regulation of glycosyltransferases.
Highlights
Glycosphingolipids (GSLs)2 are composed of an oligosaccharide chain linked to ceramide and are ubiquitously expressed in animal cells
One gene was ganglioside GM3 synthase (ST3GalV), which competes with Gb3 synthase for the common precursor LacCer, and the other was a truncated cDNA of GRINA, glutamate receptor, ionotropic, N-methyl-Dasparate-associated protein 1, which encodes a C-terminal hydrophobic polypeptide of the hypothetical full-length, and the expression of the polypeptide reduces the biosynthesis of Gb3 posttranscriptionally
Viability was estimated by MTT assay, and expressed as the percentage of the value (OD570) in the absence of Shiga Toxin 1 (Stx1): mean percentage Ϯ S.D. obtained from three independent experiments
Summary
Cell Culture, Antibodies, and Reagents—HeLa cells (ATCC CCL-2) were maintained in DMEM containing 10% fetal bovine serum (FBS). For the preparation of radiolabeled standards by in vitro galactose labeling, HeLa cells were sonicated in sonication buffer (10 mM Hepes/NaOH (pH 7.4), 1 mM EDTA, 15 mM MnCl2, 0.5% Triton X-100, 0.25 M sucrose, protease inhibitor mixture), which was used as an enzyme source. For real time PCR, the LightCycler system with the LightCycler-FastStart DNA master SYBR Green I kit (Roche Diagnostics) was used according to the manufacturer’s protocol [19] Primers used in these experiments were: hGb3S sense 1 described above and hGb3S antisense 1 (5Ј-CGAACTTCCACATGAGTGCGATCC-3Ј), GAPDH sense 1 (5Ј-GAGTCAACGGATTTGGTCGT-3Ј), and GAPDH antisense 1 (5ЈTTGATTTTGGAGGGATCTCG-3Ј). To see the effect of transiently expressed proteins on the expression of Stx receptors, HeLa-mCAT#8 cells (2.5 ϫ 104 cells in 12-well plates) were co-transfected with 0.25 g of plasmids containing the objective genes and 0.05 g of EGFP-N3 (Clontech, Mountain View, CA). After a 3-day incubation, the MTT assay was performed as described previously [23]
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