Globotriaosylceramide (Gb3) content in HeLa cells is correlated to Shiga toxin-induced cytotoxicity and Gb3 synthase expression.

Globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) are the proposed functional receptors for Shiga toxin (Stx). To elucidate the effect of Gb3 content on Stx-induced cytotoxicity in HeLa cells, we cloned HeLa cells and determined the correlation between glycolipids content and Stx-induced cytotoxicity. The 29 HeLa cell clone (HLCC) lines used showed a wide range of sensitivity to Stx, compared to Gb3-rich cells which were more sensitive, showing as little as 20% viability to 100 pg/ml Stx. In contrast, Gb3-deficient cells proved resistant as they were more than 80% viable to 100 ng/ml Stx. Gb3 content in the HLCC lines corresponded with Stxs-induced cytotoxicity as well as Gb3 synthase expression, but no correlation with Gb4 content was noted. These data show that Gb3 content, which is regulated by the expression of Gb3 synthase, determines the sensitivity of HeLa cells toward Stx.

Histidine-tagged shiga toxin B subunit binding assay: simple and specific determination of gb3 content in mammalian cells.

A two-step binding assay for globotriaosylceramide (Gb3) content was developed by histidine-tagging strategy, which is a well-established method for the purification of recombinant proteins. The complete binding of the recombinant His-tagged Shiga toxin 1B subunit (1B-His) (1 microg/ml) to the standard Gb3 adsorbed on a multi-well H type plate was observed within 30 min at 37 degrees C; and its binding could be visualized by the following applications of HisProbe-HRP (8 microg/ml) and tetramethylbenzidine (TMB) peroxidase substrate. The 1B-His binding assay was linear over the range of 1 to 100 ng of Gb3 per well. The binding of 1B-His was specific to Gb3 separated from HeLa cells, and no major cross-reactivity of other glycolipids in Folch's lower fractions extracted from HeLa cells was detected. The glycolipids in Folch's lower fractions from HeLa cells, human fibroblasts and mouse heart were suitable for this assay, but the further purification was needed for glycolipids from human plasma, thus sample preparation is critical factor for the reliable determination of Gb3 content. The 1B-His binding to Gb3 was inhibited by the addition of galactose, but not mannose. This 1B-His binding assay will be useful not only for the determination of Gb3 content, but also for screening for the compounds which inhibit the toxin-binding to Gb3. The strategy of our present method may be applicable for other binding assay, such as Cholera toxin B-subunit for ganglioside GM1.