Glycosphingolipids in vascular endothelial cells: relationship of heterogeneity in Gb3Cer/CD77 receptor expression with differential Shiga toxin 1 cytotoxicity

Abstract

Shiga toxin (Stx) 1 binds to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer/CD77) and injures human endothelial cells. In order to gain insight into Stx1-induced cellular impairment, we analysed in detail the molecular heterogeneity of Stx1 receptors in two endothelial cell lines differing in their Stx1-sensitivity. We observed a moderate sensitivity to Stx1 of human brain microvascular endothelial cells (HBMECs, CD(50) > 200 ng/ml), but a considerably higher mortality rate in cultures of EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (CD(50) of 0.2 ng/ml). Immunofluorescence microscopy demonstrated the presence of Gb3Cer in both cell lines, but showed an enhanced content of Gb3Cer in EA.hy 926 cells. Solid phase overlay binding assays of isolated GSLs combined with nanoelectrospray ionization quadrupole time-of-flight mass spectrometry demonstrated a balanced proportion of Gb3Cer and globotetraosylceramide (Gb4Cer) in HBMECs, but an increase of Gb3Cer and absence of Gb4Cer in EA.hy 926 cells. Gb3Cer species with C24:1/C24:0 fatty acids were found to dominate over those with C16:0 fatty acids in EA.hy 926 cells, but were similarly distributed in HBMECs. Reverse transcriptase polymerase chain reaction indicated the concomitant presence of Gb3Cer and Gb4Cer synthases in HBMECs, whereas EA.hy 926 cells expressed Gb3Cer synthase, but completely lacked Gb4Cer synthase. This deficiency, resulting in the accumulation of Gb3Cer in EA.hy 926 cells, represents the most prominent molecular reason that underlies the different Stx1 sensitivities of HBMECs and EA.hy 926 endothelial cells.

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

“…1B , lane c), which is in agreement with the results of the orcinol stain ( Fig. 1C ) and previous investigations ( 52 ). the steps.…”

“…Lipid raft association of GSLs has been described so far in different cell types, such as intestinal ( 45,50 ), HeLa, and Vero cells (47)(48)(49)46 ), whereas the membrane localization of GSLs of human endothelial cells has so far not been analyzed in detail and remains largely unknown. To this day, the structural characterization of the different lipoforms of Stx GSL receptors of the macrovascular HUVEC-derived EA.hy 926 cell line and human brain microvascular endothelial cells (HBMECs) has been reported by us ( 52 ), indicating that HBMECs express both Gb3Cer and Gb4Cer, whereas EA.hy 926 cells were found to synthesize Gb3Cer but not elongated Gb4Cer. luted 1:500 in PBS with 1% BSA and 0.3% Triton X-100.…”

“…Moreover, GSLs can be comparatively analyzed without any loss of analyte of particular interest in case of small GSL amounts, e.g., isolated from DRM fractions (see below). These are the major advantages of this approach compared with previous investigations, where the Stx receptors of HBMECs and EA.hy 926 cells were analyzed from pooled silica gel extracts of several TLC runs ( 52 ).…”

Identification of a Novel Streptococcal Adhesin P (SadP) Protein Recognizing Galactosyl-α1–4-galactose-containing Glycoconjugates

“…It has been shown that the carbohydrate-binding pentameric B-subunit of E. coli verotoxin causes apoptosis in susceptible endothelial cells, such as the Eahy.926 hybridoma cell line, upon binding to the GbO3 glycolipid receptor (64,65), which makes the interaction of GbO3 with endogenous and exogenous ligands a potential target for therapies. The availability of the SadP with its distinct and well characterized Gal␣1-4Gal binding specificity could be a useful tool in studies aiming at these goals.…”

Tumor-associated CD75s- and iso-CD75s-gangliosides are potential targets for adjuvant therapy in pancreatic cancer

“…Fluorochrome-labeled antibodies and DAPI-stained nuclei were evaluated under a fluorescence microscope (Axioskop; Zeiss), original magnification Â40 (objective lense Plan-Neofluar, numerical aperture 0.75), following published protocols (26). To confirm the lipid nature of the positive staining with anti-ganglioside antibodies, lipids were extracted from air-dried sections with methanol and chloroform/methanol (1:1, v/v) for 10 min each and stained by the same procedure as described above.…”