Differential recognition of lipid domains by two Gb3-binding lectins

Abstract

The two lectins LecA from Pseudomonas aeruginosa and the B-subunit of Shiga toxin from Shigella dysenteriae (StxB) share the glycosphingolipid globotriaosylceramide (Gb3) as receptor. Counterintuitively, we found that LecA and StxB segregated into different domains after recognizing Gb3 at the plasma membrane of cells. We hypothesized that the orientation of the carbohydrate head group of Gb3 embedded in the lipid bilayer differentially influences LecA and StxB binding. To test this hypothesis, we reconstituted lectin-Gb3 interaction using giant unilamellar vesicles and were indeed able to rebuild LecA and StxB segregation. Both, the Gb3 fatty acyl chain structure and the local membrane environment, modulated Gb3 recognition by LecA and StxB. Specifically, StxB preferred more ordered membranes compared to LecA. Based on our findings, we propose comparing staining patterns of LecA and StxB as an alternative method to assess membrane order in cells. To verify this approach, we re-established that the apical plasma membrane of epithelial cells is more ordered than the basolateral plasma membrane. Additionally, we found that StxB recognized Gb3 at the primary cilium and the periciliary membrane, whereas LecA only bound periciliary Gb3. This suggests that the ciliary membrane is of higher order than the surrounding periciliary membrane.

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

“…Of note, LecA and StxB both bind to Gb3, but partially localize to different membrane domains [ 23 ] and exhibit distinct trafficking routes [ 106 ]. In our analysis, no clear difference in binding behavior to Gb3 species of LecA in comparison to StxB was observed.…”

“…These domains are enriched in GSLs, cholesterol and glycosylphosphatidylinositol (GPI)-anchored proteins and serve as sorting and signaling platforms [ 24 – 26 ]. Due to the high saturation level of the fatty acyl chains of Gb3, the lipid is preferentially but not exclusively located in lipid rafts [ 23 , 27 , 28 ].…”

“…Additionally, the local lipid and protein environment of Gb3 may affect the induced signaling events and endocytic trafficking routes. The lipid environment as well as length and saturation level of the fatty acyl chains of GSLs influence orientation and accessibility of the carbohydrate groups and affect the binding behavior of StxB to Gb3 in model membrane systems [21][22][23]. Ordered domains in the outer leaflet of the cellular plasma membrane are often termed lipid rafts.…”

How Does Liquid-Liquid Phase Separation in Model Membranes Reflect Cell Membrane Heterogeneity?

“…In synthetic and cell-derived membrane systems, glycosphingolipids with saturated fatty acyl chains incorporate in Lo, whereas GSLs with unsaturated chains prefer Ld domains. This was shown for GSLs monosialotetrahexosylganglioside (GM1), and globotriaosylceramide (Gb3) [32,68,[73][74][75]. Furthermore, introduction of a fluorophore can induce the loss of specificity to Lo domains, and similar to the phospholipids, introduction of a flexible spacer between the fluorescent moiety and GSL headgroup can improve partitioning to Lo [68].…”

Chemically synthesized Gb3 glycosphingolipids: tools to access their function in lipid membranes

“…The globotriaosyl ceramide Gb 3 , also referred to as P K blood group antigen and CD77 (Johannes and Römer 2010 ), is the major receptor lipid for a number of lectins. Just recently, Römer and co-workers have shown that Gb 3 segregates into different domains at the plasma membrane of cells, which leads to a different binding behaviour of Shiga toxin compared to the lectin LecA from Pseudomonas aeruginosa (Schubert et al 2020 ). The receptor lipid is composed of the trisaccharide α-D-galactose-(1 → 4)-β-D-galactose-(1 → 4)-β-D-glucose, bound via an acetal to a sphingosine moiety.…”