Synthesis of Gb3 Glycosphingolipids with Labeled Head Groups: Distribution in Phase‐Separated Giant Unilamellar Vesicles

Jeremias Sibold,Claudia Steinem,Daniel B Werz,Loan Vuong,Katharina Kettelhoit,Fangyuan Liu

doi:10.1002/ange.201910148

Abstract

AbstractThe 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 (lo)/liquid‐disordered (ld) giant unilamellar vesicles (GUVs), and STx binding was verified by fluorescence microscopy. Gb3 with the C24:0 fatty acid partitioned mostly in the lo phase, while the unsaturated C24:1 fatty acid distributes more into the ld phase. The α‐hydroxylation does not influence its partitioning.

Highlights

The eukaryotic plasma membrane of animals is a heterogeneous structure with a plethora of different lipids
Our results clearly demonstrate that the fatty acid of Gb3 influences its partitioning into the lo phase
GM1 and Gb3 detection by fluorescently labeled Cholera toxin B subunits (CTxB) and Shiga toxin B subunits (STxB), respectively is a well-established tool for monitoring lo membrane domains[58] and implies that these glycosphingolipids are localized in the lo phase

Summary

Introduction

The eukaryotic plasma membrane of animals is a heterogeneous structure with a plethora of different lipids. In coexisting lo/ld supported lipid membranes, Gb3 species differing in their fatty acid gave rise to a different phase behavior before and after binding of the B subunits of STx (STxB) as well as differences in the protein organization on the membrane surface.[20,21] In giant unilamellar vesicles (GUVs), Gb3 species with an unsaturated acyl chain caused the formation of tubular invaginations upon STxB binding, in contrast to Gb3 with a saturated acyl chain.[22] In all these studies, it became evident that STxB binds exclusively to the lo phase, which implies that the receptor Gb3 is localized in the lo phase after protein binding It remains unclear how Gb3 is distributed in coexisting lo/ld membranes prior protein binding. This approach in turn allows us to alter the fatty acid of the Gb3 molecules

Results and Discussion

Conclusion

Conflict of interest