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Comparison of the glycolipid receptor specificities of Shiga-like toxin type II and Shiga-like toxin type II variants
J E Samuel1, L P Perera, S Ward
1Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Abstract:
The antigenically distinct Shiga-like toxins (SLTs) SLT-1 and SLT-II are cytotoxic for both Vero and HeLa cells and use Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb3) molecules as functional receptors. SLT-II-related variants SLT-IIvp and SLT-IIvh, produced by a porcine isolate and a human isolate, respectively, are cytotoxic for Vero but not HeLa cells. To investigate the basis for these differences in cytotoxic specificity among SLTs, the nature of the receptor for the SLT-II variants was examined. First, the patterns of binding of SLT-II and the SLT-II variants to Gb3 receptor analogs Gal alpha 1-4Gal-bovine serum albumin and Gal alpha 1-4Gal beta 1-4Glc-bovine serum albumin were compared. SLT-IIvp bound the trisaccharide neoglycoprotein preferentially, while SLT-IIvh bound both analogs equally but with less affinity than did SLT-II. Next, the glycolipids to which the SLT-II variants bound in Vero and HeLa cells were identified by thin-layer chromatography. SLT-IIvp bound to Gb3, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb4), and Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb5) in Vero cells but only Gb3 in HeLa cells. However, SLT-IIvh bound to Gal alpha 1-4Gal beta 1-1Cer (Gb2) and Gb3 in HeLa cells but only Gb3 in Vero cells. In addition, hybrid toxins (SLT-IIvp subunit A with SLT-II subunit B or SLT-II subunit A with SLT-IIvp subunit B) were used to show that the receptor specificities of the SLTs was B subunit specific. These differences in receptor specificities are important in vivo, as evidenced by a 400-fold difference in the 50% lethal doses of purified SLT-IIvp and SLT-II (200 versus 0.5 ng, respectively) for mice. These data indicate that SLT-II-cytotoxic variants can occur as a consequence of differences in receptor specificity and affinity.
Insights
Shiga-like toxins (SLTs) exhibit varied cell cytotoxicity due to differences in their binding to specific glycolipid receptors. These receptor specificities, determined by the B subunit, influence toxin potency and are crucial for in vivo effects.
Area of Science:
- Microbiology
- Molecular Biology
- Toxicology
Background:
- Shiga-like toxins (SLTs) are cytotoxic agents with distinct variants, including SLT-I and SLT-II.
- SLT-I and SLT-II utilize globotriaosylceramide (Gb3) as their functional receptor.
- SLT-II variants, SLT-IIvp and SLT-IIvh, display differential cytotoxicity against Vero and HeLa cells.
Purpose of the Study:
- To investigate the molecular basis for the differing cytotoxic specificities of SLT-II variants.
- To characterize the receptor binding properties of SLT-IIvp and SLT-IIvh.
- To determine the role of the B subunit in SLT receptor specificity.
Main Methods:
- Comparison of SLT-II and its variants' binding to Gb3 receptor analogs (neoglycoproteins).
- Thin-layer chromatography to identify glycolipids bound by SLT-II variants in different cell lines.
- Construction and analysis of hybrid toxins using SLT-II and SLT-IIvp subunits.
Main Results:
- SLT-IIvp preferentially bound to a trisaccharide neoglycoprotein, while SLT-IIvh showed less affinity for Gb3 analogs compared to SLT-II.
- SLT-IIvp bound to Gb3, Gb4, and Gb5 in Vero cells, but only Gb3 in HeLa cells; SLT-IIvh bound to Gb2 and Gb3 in HeLa cells, and Gb3 in Vero cells.
- Hybrid toxin experiments confirmed that the B subunit dictates the receptor specificity of SLTs.
Conclusions:
- Differences in receptor specificity and affinity among SLT-II variants contribute to their distinct cytotoxic profiles.
- The B subunit is the primary determinant of SLT receptor binding and specificity.
- These variations in receptor interaction have significant implications for the in vivo pathogenicity of SLTs, as demonstrated by differences in lethal doses.