# Protection against Shiga Toxins
## 1 Introduction
### Gb3 and Its Interaction with Stx  
Globotriaosylceramide (Gal-\(\alpha\)1\(\rightarrow\)4 Gal-\(\beta\)1\(\rightarrow\)4 Glc-\(\beta\)1\(\rightarrow\)Cer, Gb3; Figure 2) is a glycosphingolipid expressed on the surface of certain cell types. Gb3 is formed by the addition of one galactose residue to lactoseylceramide (LacCer), which is a common precursor for different classes of glycosphingolipids, and the reaction is catalyzed by Gb3 synthase (lactosylceramide \(\alpha\)-1,4-galactosidaseosyltransferase). Gb3 is the first glycosphingolipid in the globo-series and thus serves as a precursor for the synthesis of more complex globo-series glycosphingolipids, such as globotetraosylceramide (Gb4). Gb4 is formed after addition of \(N\)-acetylgalactosamine (GalNAc) to the terminal galactose of Gb3. Although Gb3 is the primary receptor for all Stxs, it has been suggested that Gb4 might facilitate Stx2 binding to colon epithelium cells, which normally have no Gb3 or very low levels of Gb3 [41]. The sphingosine chain in the ceramide part of Gb3 is relatively invariable (most often it is monounsaturated with 18 carbon atoms, i.e., d18:1), but the \(N\)-amidated fatty acyl chain varies both in length (most common are 16-24 carbon atoms) and saturation resulting in multiple Gb3 species present in cells. Importantly, the receptor function of Gb3 has been shown to depend on its species composition [42; 43; 44; 45], which in turn depends on cell type [46] and growth conditions [47], and might change in response to certain treatments, like exposure to butyric acid and cytokines [43; 48; 49; 50; 51; 52]. It has been suggested that the production of butyric acid by the bacterial flora in the intestine may affect  
Figure 1: The schematic and structural models of Shiga toxins (Stxs). (**A**) Stxs consist of two non-covalently linked moieties: an A-moiety of \(\sim\)32 kDa (shown in red), and a B-moiety (shown in green), comprised of five 7.7 kDa B-chains [27; 28]. During intracellular toxin transport, the A-moiety is cleaved by the protease furn [39] into two fragments: an enzymatically active A\({}_{1}\) fragment (–27 kDa) and a carboxyl terminal A\({}_{2}\) fragment, which remain linked by a disulfide bond until arrival to the endoplasmic reticulum (ER) [40]. (**B**) The structure of the holotoxin as determined by \(X\)-ray crystallography [28] (PDB ID:1 DM0); (**C**) The receptor-binding surface of the B-pentamer based on the structure of Stx1 complexed with the Gb3 analogue MCO-PK (methoxycarbonylyl glycoside of P\({}^{\text{k}}\) trisaccharide) [33] (PDB ID:1 BOS); the sugar moieties of MCO-PK are shown in black. Structure images were prepared using PDB ProteinWorkshop 4.2. Figure 2: Chemical structure and biosynthesis of the Stx receptor globotriaosylceramide (Gb3). Sphingosine most often contains 18 carbon atoms, whereas the fatty acyl chain of ceramide varies both in length and saturation (here shown as C16\(\leftrightarrow\)0). Gb3 is synthesized from LacCer by the addition of one galactose, and the reaction is catalyzed by Gb3 synthase (lactosylceramide \(\alpha\)-1,4-galactosidaseyltransferase). The sugar chain for Gb3 is: Gal-\(\alpha\)1\(\rightarrow\)4 Gal-\(\beta\)1\(\rightarrow\)4 Glc-\(\beta\)1\(\rightarrow\)Ceramide. the expression and composition of Gb3 in the target cells and in turn lead to different susceptibility to the toxin between individuals [53]. In addition, the turnover time of Gb3 in the cells depends on its species, with longer fatty acyl chain-containing species having a longer half-life than the species with short fatty acyl chain [54]. Thus, inhibition of Gb3 synthesis will primarily lead to changes in Gb3 species composition in cells [43; 50]. Studies based on artificial systems, where Gb3 was immobilized on thin layer chromatography (TLC) or ELISA plates, have shown that Stx1 and Stx2 have different binding preferences for different Gb3 species [44; 55], although a mixture of various Gb3 species was required for the highest binding affinity [45].