Abstract
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 brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As 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 infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM 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 type. Methyl-β-cyclodextrin (MβCD)-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.
Highlights
Vascular damage caused by Shiga toxin (Stx)producing Escherichia coli is largely mediated by Shiga toxins (Stxs), which in particular, injure microvascular endothelial cells in the kidneys and brain
The anti-Gb3Cer antibody bound to Gb3Cer of human brain microvascular endothelial cell (HBMEC) and EA.hy 926 cells (Fig. 1A, lane b and c, respectively) where upper bands harbor Gb3Cer structures with mostly C24 and lower bands Gb3Cer with C16 fatty acid, which were verified by IR-MALDI-orthogonal time-of-flight (o-TOF)-MS
We found that Stx receptors preferentially localized to the liquid-ordered phases of detergent-resistant membrane (DRM) fractions of HBMECs and EA.hy 926 cells, where they colocalized with prevalent flotillin-2 and caveolin-1, respectively
Summary
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 brain. As 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 antiGb3Cer and anti-Gb4Cer antibodies. Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains. While numerous proteomic studies have been conducted to identify lipid raft- or caveolae-associated proteins of various cell types, including endothelial cells [10,11,12], the GSL composition of lipid rafts or caveolae, of endothelial cells, has attracted less attention, despite their outstanding role in membrane microdomain formation. The specific GSL composition of lipid rafts and/ or caveolae of many cell types remains to be elucidated
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