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.
Highlights
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
We report that the lectins LecA from Pseudomonas aeruginosa[27,28,29] and the B-subunit of Shiga toxin (StxB) from Shigella dysenteriae[30,31,32], which recognize the same glycosphingolipid, the globotriaosylceramide
Upon application of LecA and StxB to either the apical or basolateral side of polarized Madin-Darby canine kidney (MDCK) strain II cells, stably expressing Gb3 synthase[9], both lectins were found at higher concentration at the apical cell membranes than at the basolateral ones (Fig. 1A)
Summary
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. 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 able to rebuild LecA and StxB segregation. We report that the lectins LecA from Pseudomonas aeruginosa[27,28,29] and the B-subunit of Shiga toxin (StxB) from Shigella dysenteriae[30,31,32], which recognize the same glycosphingolipid, the globotriaosylceramide (Gb3, referred to as PK blood group antigen and CD7733), segregate into different domains at the plasma membrane of cells. We revealed that StxB has a preference for more ordered domains in comparison to LecA Based on these results we suggest that comparing the staining patterns of LecA and StxB yields a novel approach for evaluating membrane order in cells. This surprising finding suggests that the membrane of the primary cilium contains a lipid domain that has a higher order than the surrounding apical plasma membrane
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have