Synthesis of Gb₃ Glycosphingolipids with Labeled Head Groups: Distribution in Phase-Separated Giant Unilamellar...

The receptor lipid Gb₃ is responsible for the specific internalization of Shiga toxin (STx) into cells. The head group of Gb₃ defines the specificity of STx binding, and the backbone with different fatty acids is expected to influence its localization within membranes impacting membrane organization and protein internalization. To investigate this influence, a set of Gb₃ glycosphingolipids labeled with a BODIPY fluorophore attached to the head group was synthesized. C₂₄ fatty acids, saturated, unsaturated, α-hydroxylated derivatives, and a combination thereof, were attached to the sphingosine backbone. The synthetic Gb₃ glycosphingolipids were reconstituted into coexisting liquid-ordered (l_o )/liquid-disordered (l_d ) giant unilamellar vesicles (GUVs), and STx binding was verified by fluorescence microscopy. Gb₃ with the C_24:0 fatty acid partitioned mostly in the l_o phase, while the unsaturated C_24:1 fatty acid distributes more into the l_d phase. The α-hydroxylation does not influence its partitioning.