Shiga toxin binding alters lipid packing and the domain structure of Gb₃-containing membranes: a solid-state NMR study

Abstract

The individual response of various lipid species to shiga toxin binding to the membrane was studied by 2H NMR.

Verotoxin Receptor-Based Pathology and Therapies

“…Receptor mediated VT cell internalization is via clathrin dependent and independent mechanisms (Khine et al, 2004). The multivalent B subunit pentamer binding to cell surface raft and model membrane Gb 3 has been shown to cluster Gb 3 (Khine and Lingwood, 1994;Windschiegl et al, 2009;Pezeshkian et al, 2017) which causes subsequent energy and clathrin independent tubular invaginations (negative membrane curvature) (Römer et al, 2007;Bosse et al, 2019) (Figure 1). This effect is Gb 3 unsaturated fatty acid dependent and does not involve cytoskeletal components (Römer et al, 2007).…”

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

“…[11] Despite this difference between the natural and artificial membrane systems,a rtificial coexisting l o /l d membranes have been frequently used to analyze the partitioning of receptor lipids and proteins, [12] such as bacterial toxins,i nt he different phases. [13] Bacterial toxins are known to bind to specific glycosphingolipids embedded in the outer leaflet of the plasma membrane.Cholera toxin (CTx) produced by Vibrio cholerae and Shiga toxin (STx) produced by Shigella dysenteriae and by enterohemorrhagic strains of Escherichia coli,both belonging to the class of AB 5 toxins, [14] bind specifically to monosialotetrahexosylganglioside (G M1 ) [15] and globotriaosyl ceramide (Gb 3 ), [16,17] respectively.W hile the head groups of the glycosphingolipids indeed define the specificity of protein binding,not much attention has been drawn to the variability of the ceramide backbone harboring different fatty acids.I n various cell types (human colon Caco-2, HCT-8 epithelial cells,h uman endothelial cell lines,p rimary human umbilical vein endothelial cells,primary human endothelial cells of the brain and the kidney, [18] and references therein), aconserved repertoire of Gb 3 species was found carrying saturated C 16:0 , C 22:0 ,orC 24:0 fatty acids as well as the unsaturated C 24:1 fatty acid. Results of Lingwood and co-workers [19] suggest that the pathogenic outcome of Shiga toxin producing E. coli (STEC) infections is related to the different Gb 3 species.T og ather more molecular information, artificial membranes doped with Gb 3 were employed.…”

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

“…Reduction of the azide moiety by Staudinger reaction and direct coupling with the fatty acids 36-39 under common peptide coupling conditions (e.g., HATU or HOBt/EDCI) afforded four protected glycosphingolipids with different fatty acid chains. Global deprotection was performed under Zemplén conditions resulting in the glycosphingolipids 49-52.This modular procedure readily allowed us to also synthesize Gb 3 derivatives with deuterated fatty acids (Bosse et al 2019). Here, commercially available completely deuterated fatty acids were used for the amidation procedure ( Fig.…”

“…To illuminate the aspect of membrane organisation in more detail, we performed fluorescence and atomic force microscopy imaging on lipid bilayers resembling the outer leaflet of the plasma membrane of eukaryotic cells (van Meer and Kroon 2011), doped with the different Gb 3 sphingolipids. Table 1 Dissociation constants obtained by SPR measurements (Bosse et al 2019;Schütte et al 2015) STxB (pentamer) was bound to Gb 3 doped lipid monolayers. Fatty acid composition of Gb 3porc : C 16:0 (21%), C 18:0 (4%), C 18:1 (2%), C 22:0 (14%), C 23:0 (1%), C 24:0 (19%), C 24:1 (11%), C 22:0 2-(R)-OH (4%), C 24:0 2-(R)-OH (5%) and C 24:1 2-(R)-OH (17%) Lipid composition K D /nM DOPC/Gb 3porc (95:5) 3 ± 2/5 ± 1 DOPC/49 (Gb 3 C 24:0 ) (95:5) 4 ± 1 DOPC/50 (Gb 3 C 24:0 2-(R)-OH ) (95:5) 7 ± 2 DOPC/51 (Gb 3 C 24:1 ) (95:5) 12 ± 1 DOPC/52 (Gb 3 C 24:1 2-(R)-OH ) (95:5) 8 ± 2 DOPC/Chol/52 (75:20:5)…”

“…As the packing behaviour of Gb 3 appears to be an important aspect of its partitioning, we illuminated this aspect using 2 H solid-state NMR spectroscopy (Bosse et al 2019). To perform these experiments, perdeuterated Gb 3 lipid acyl chains were synthesized (Fig.…”