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

Ole M. Schütte, Annika Ries, Alexander Orth, Lukas J. Patalag, Winfried Römer, Claudia Steinem, Daniel B. Werz
2014 Chemical Science  
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,
more » ... thesized 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. † Electronic supplementary information (ESI) available: Experimental details of the synthesis and analytical data of 27-30; detailed experimental procedures on the preparation and analysis of solid supported bilayers; fatty acid distribution of natural Gb 3 and bovine brain SM; additional uorescence and AFM images of phase-separated lipid bilayers; calculation of STxB surface coverage and receptor coverage; ternary phase diagram to estimate phase compositions. See Scheme 2 Preparation of a-galactosyl building block 3. Scheme 3 Assembly of the globotriaosyl trichloroacetimidate 17. Scheme 4 Syntheses of hydroxylated fatty acids 22 and 24. 3106 | Chem. Sci., 2014, 5, 3104-3114 This journal is
doi:10.1039/c4sc01290a fatcat:fz4hhrs5und4rc3tyjcue7tkny