Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells

Abstract

Membrane microdomain association of the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the highly and less effective receptors, respectively, for Shiga toxins (Stxs), is assumed as a functional requirement for Stx-mediated cytotoxicity. In a previous study, we demonstrated predominant localization of Stx receptors in cholesterol-enriched membrane microdomains of moderately Stx-sensitive human brain microvascular endothelial cells (HBMECs) by means of detergent-resistant membranes (DRMs). Here we report a different preferential distribution of Stx receptors in non-DRM fractions of human glomerular microvascular endothelial cells (GMVECs), the major targets of Stxs in the human kidney. Full structural characterization of Stx receptors using electrospray ionization (ESI) mass spectrometry revealed Gb3Cer and Gb4Cer lipoforms with ceramide moieties mainly composed of C24:0/C24:1 or C16:0 fatty acid and sphingosine (d18:1) in GMVECs comparable to those previously found in HBMECs. Thin-layer chromatography immunostaining demonstrated an approximately 2-fold higher content of Gb3Cer and a 1.4-fold higher content of Gb4Cer in GMVECs than in HBMECs. However, this does not explain the remarkable higher cytotoxic action of Stx1 and Stx2 toward GMVECs as compared with HBMECs. Our finding opens new questions on the microdomain association of Stx receptors and the functional role of GSLs in the membrane assembly of GMVECs.

Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment

“…Minor quantities of GSLs harbored Cer (d18:1, C22:0) and Cer (d18:1, C18:0) as the lipid anchor. This receptor repertoire closely resembles those of human brain and glomerular microvascular endothelial cells ( 62,78 ), which represent the bottom fraction F7 (highlighted by grayed boxes in the mass spectrum shown in Fig. 9B ).…”

“…Molybdenum blue-colored phospholipid, dark brownish cholesterol, and deep purpled orcinol bands were scanned at wavelengths of = 700 nm, = 365 nm, and = 544 nm, respectively, and immunopositive GSL bands were densitometrically quantifi ed at a wavelength of = 630 nm ( 62,70,78 ). All scans were performed in triplicate from three independent experiments.…”

Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells

“…After ingestion, STEC colonize the gut and release Stx1 and/or Stx2, the two main types of Stxs, into the intestinal lumen. Stxs enter the blood stream and target the microvascular endothelial cells of the kidneys and the brain endowed with high-affi nity Gb3Cer receptors (44)(45)(46), resulting in HUS, a severe systemic complication ( 47 ). However, the absorption of Stxs into the blood circulation and their delivery to endothelial cells, most likely conducted by polymorphonuclear leukocytes, has been (and is still) greatly debated due to contradictory results obtained from different research groups ( 48,49 ) and the lack of mechanistic information.…”

“…Dark brownish-colored cholesterol, molybdenum blue-stained phospholipid, and blue BCIP-immunopositive GSL bands were scanned at wavelengths of = 365 nm, =720 nm, and = 630 nm, respectively ( 44,45,79 ). All scans were performed in triplicate from three independent approaches.…”

“…DRMs were prepared according to Brown and Rose ( 8 ) as previously described in detail ( 44,45 ). Briefl y, the cell suspensions were harvested at cell densities of approximately 1 × 10 6 cells/ml by centrifugation (200 g for 5 min) at room temperature and washed with PBS.…”