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 Gb₃. In biological membranes, Gb₃ 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/Gb₃ complex. To analyse the binding of STxB to different Gb₃s, we chemically synthesized saturated, unsaturated, α-hydroxylated Gb₃s and a combination thereof, all based on a C₂₄-fatty acid chain starting from monosaccharide building blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb₃s 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 fluorescence- and atomic force microscopy the phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty acid of Gb₃ significantly alters the ratio between the ordered and disordered phase and induces a third intermediate phase in the presence of unsaturated Gb₃. The lateral organization of STxB on the membranes varies significantly. While STxB attached to membranes with Gb₃s with saturated fatty acids forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb₃s. Large interphase lipid redistribution is observed for α-hydroxylated Gb₃ doped membranes. Our results clearly demonstrate that the fatty acid of Gb₃ strongly influences the lateral organization of STxB on the membrane and impacts the overall membrane organization of phase-separated lipid membranes.