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

Ivan U Kouzel,Martina Bielaszewska,Helge Karch,Johannes Müthing,Christoph Cichon,Gottfried Pohlentz,Wiebke Storck,Lena Radamm,Michael Mormann,Andreas Bauwens,Petra Hoffmann,M Alexander Schmidt

doi:10.1194/jlr.m031781

Abstract

Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively. The presence of Stx-receptors in GSL preparations of Raji and THP-1 cells and the absence in Jurkat and HL-60 cells revealed high compliance of solid-phase immunodetection assays with the expression profiles of receptor-related glycosyltransferases, performed by qRT-PCR analysis, and Stx2-caused cellular damage. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin.

Highlights

Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains
Because leukocytes may act as transporter and transfer cells in the blood, probably exploited by Stxproducing E. coli (STEC) for the delivery of Stxs to endothelial target cells, we investigated four leukocyte-derived cell lines representing B- and T-cell descendants (Raji and Jurkat cells, respectively) as well as cells of the monocyte and granulocyte lineage (THP-1 and HL-60 cells, respectively) with respect to the occurrence of globo-series GSLs
Stx2 was found to bind to its high-affinity receptor Gb3Cer detected in Raji and THP-1 cells (Fig. 1C) and exhibited similar relative TLC overlay assay binding intensities for both cell lines compared with those obtained for Gb3Cer (d18:1, C24:1/ C24:0) and Gb3Cer (d18:1, C16:0) with the anti-Gb3Cer antibody

Summary

Introduction

Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin.—Kouzel, I. Lipid rafts in living cells are characterized as dynamic nanoscale assemblies composed of cholesterol, sphingomyelin, phosphatidylcholine (PC), glycosphingolipids (GSLs), and certain proteins such as glycosylphosphatidylinositolanchored proteins and transmembrane proteins. This article is available online at http://www.jlr.org obtained from DRMs as the ruling method to assigning lipid raft association [10]

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