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,Claudia Steinem,Daniel B Werz,Annika Ries,Lukas J Patalag,Alexander Orth,Winfried Römer

doi:10.1039/c4sc01290a

Ole M Schütte, Claudia Steinem + Show 5 more

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https://doi.org/10.1039/c4sc01290a

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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.

Highlights

Glycosphingolipids are the cellular receptors of a number of viruses and bacterial toxins
Romer et al.[6] showed that the pentameric B-subunits, which can bind up to 15 Gb3 molecules, induce a clathrin- and caveolin-independent tubule formation upon attachment to HeLa-cells and that invaginations were even found in arti cial membranes, when STxB binds to giant unilamellar vesicles (GUVs)
From a retrosynthetic point of view a glycosphingolipid can be divided into three major parts: (i) the carbohydrate head group as the hydrophilic and the ceramide as the lipophilic part that can be further subdivided into (ii) a sphingosine such as D-erythro-sphingosine and (iii) a fatty acid being attached to the amine of the sphingosine (Scheme 1)

Summary

Introduction

Glycosphingolipids are the cellular receptors of a number of viruses and bacterial toxins. A er endocytosis, Shiga toxin leaves the endocytic pathway at the level of early endosomes following the retrograde transport route.[5] Romer et al.[6] showed that the pentameric B-subunits, which can bind up to 15 Gb3 molecules, induce a clathrin- and caveolin-independent tubule formation upon attachment to HeLa-cells and that invaginations were even found in arti cial membranes, when STxB binds to giant unilamellar vesicles (GUVs) These results suggest a signi cant lipid contribution to the earliest steps of toxin uptake into cells and there are several lines of evidence that the cytotoxicity of Shiga toxin is strongly associated with Gb3 density and binding to cholesterol-containing lipid ra s.5,7,8.

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