Influence of Gb3 glycosphingolipids differing in their fatty acid chain on the phase behaviour of solid supported membranes: chemical syntheses and impact of Shiga toxin binding

Abstract

The Shiga toxin B subunit (STxB), which is involved in cell membrane attachment and trafficking of Shiga holotoxin, binds specifically to the glycosphingolipid Gb 3. In biological membranes, Gb 3 glycosphingolipids differ in their fatty acid composition and there is strong evidence that the fatty acid alters the binding behaviour of STxB as well as the intracellular routing of the Shiga toxin/Gb 3 complex. To analyse the binding of STxB to different Gb 3 s, we chemically synthesized saturated, unsaturated, a-hydroxylated Gb 3 s and a combination thereof, all based on a C 24-fatty acid chain starting from monosaccharide building blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb 3 s were inserted into solid supported phase-separated lipid bilayers composed of DOPC/sphingomyelin/cholesterol as a simple mimetic of the outer leaflet of animal cell membranes. By fluorescenceand atomic force microscopy the phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty acid of Gb 3 significantly alters the ratio between the ordered and disordered phase and induces a third intermediate phase in the presence of unsaturated Gb 3. The lateral organization of STxB on the membranes varies significantly. While STxB attached to membranes with Gb 3 s with saturated fatty acids forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb 3 s. Large interphase lipid redistribution is observed for a-hydroxylated Gb 3 doped membranes. Our results clearly demonstrate that the fatty acid of Gb 3 strongly influences the lateral organization of STxB on the membrane and impacts the overall membrane organization of phase-separated lipid membranes.

The effects of globotriaosylceramide tail saturation level on bilayer phases

“…For lipid bilayers containing DOPC, sphingomyelin, cholesterol and Gb 3 -C24 : 0, which are phase separated at room temperature, no change in the area percentage of the l o phase was observed upon STxB binding experimentally: 13 we have shown that S-Gb 3 tends to remain phase separated in such a DOPC bilayer. Thus, SGb 3 is probably already in a phase separated state in the mixed bilayer even before STxB binds.…”

“…Phase behaviour of such a bilayer is strongly inuenced by STxB clustering as well as the saturation levels and length of the Gb 3 fatty acyl chain. 11,13,15 Despite the important cellular roles of Gb 3 and its signicant impact on the physical behaviour of the lipid bilayers, it has not been studied by simulations before. Therefore, we use all-atom MD simulations to investigate a mixed bilayer of DOPC and Gb 3 with two different acyl chain compositions, Gb 3 -22 : 0 (Gb 3 with a saturated acyl chain with 22 carbon atoms) and Gb 3 -22 : 1 (Gb 3 with an unsaturated acyl chain with 22 carbon atoms and a trans-double bound in C 13 -C 14 ).…”

Plasma membrane reorganization: A glycolipid gateway for microbes

“…They observed the existence of several laterally segregated phases, containing regions of hexagonally packed saturated hydrocarbon chains separated by cholesterol and unsaturated hydrocarbon-rich interstitial regions. Watkins [26,27]. Windschiegl et al used GUVs and planar SLBs comprising a lipid mixture of DOPC/cholesterol/Gb3 to study Shiga toxin-induced Gb3 clustering [28].…”

“…This proved that sites I and II lead to high affinity receptor binding. However, site III played a role in scanning additional Gb3 receptor epitopes available for toxin binding [27].…”

Influence of length and conformation of saccharide head groups on the mechanics of glycolipid membranes: Unraveled by off-specular neutron scattering

“…Using standard carbohydrate protecting group chemistry, 33,34 bare Gb3 was perbenzoylated, followed by anomeric deprotection and trichloroacetimidate formation. 35 In the following glycosylation reaction, globotriaosyl trichloroacetimidate ( Fig. 1(a)) and either 1,2-di-O-hexadecyl-sn-3-glycerol ( Fig.…”