Chemically synthesized Gb(3) glycosphingolipids: tools to access their function in lipid membranes

Gb(3) glycosphingolipids are the specific receptors for bacterial Shiga toxin. Whereas the trisaccharidic head group of Gb(3) defines the specificity of Shiga toxin binding, the lipophilic part composed of sphingosine and different fatty acids is suggested to determine its localization within membranes impacting membrane organisation and protein binding eventually leading to protein internalisation. While most studies use Gb(3) extracts, chemical synthesis provides a unique tool to access different tailor-made Gb(3) glycosphingolipids. In this review, strategies to synthesize these complex glycosphingolipids are presented. Special emphasis is put on the preparation of Gb(3) molecules differing only in their fatty acid part (saturated, unsaturated, α-hydroxylated and both, unsaturated and α-hydroxylated). With these molecules in hand, it became possible to investigate the phase behaviour of liquid ordered/liquid disordered supported membranes doped with the Gb(3) species by means of fluorescence and atomic force microscopy. The results clearly highlight the influence of the different fatty acids of the Gb(3) sphingolipids on the phase behaviour and the binding properties of Shiga toxin B subunits, even though the membranes were only doped with 5 mol% of the receptor lipid. To obtain fluorescent Gb(3) derivatives, either fatty acid labelled Gb(3) molecules or head group labelled ones were synthesized. These molecules enabled us to address the question, where the Gb(3) sphingolipids are localized prior protein binding by means of fluorescence microscopy on giant unilamellar vesicles. The results again demonstrate that the fatty acid of Gb(3) plays a pivotal role for the overall membrane organisation.