Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: association with membrane lipid raft microdomains that differ by their stability to cholesterol depletion

Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which, in particular, injure microvascular endothelial cells in the kidneys and the brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and to a lesser extent to globotetraosylceramide (Gb4Cer). Since clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with IRMALDI mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membrane (DRM) than in non-DRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell types. Methyl-ß-cyclodextrin-mediated cholesterol depletion resulted in only partial destruction of lipid rafts accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types. Supplementary keywords: Glycolipids, caveolae, DRMs, Gb3Cer, Gb4Cer, caveolin-1, flotillin-2 3 by gest, on M arch 0, 2020 w w w .j.org D ow nladed fom

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