title: Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment.
abstract: Shiga toxins (Stxs) are produced by enterohemorrhagic Escherichia coli (EHEC), which cause human infections with an often fatal outcome. Vero cell lines, derived from African green monkey kidney, represent the gold standard for determining the cytotoxic effects of Stxs. Despite their global use, knowledge about the exact structures of the Stx receptor glycosphingolipids (GSLs) and their assembly in lipid rafts is poor. Here we present a comprehensive structural analysis of Stx receptor GSLs and their distribution to detergent-resistant membranes (DRMs), which were prepared from Vero-B4 cells and used as lipid raft equivalents. We identified globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) as the GSL receptors for Stx1a, Stx2a, and Stx2e subtypes using TLC overlay detection combined with MS. The uncommon Stx receptor, globopentaosylceramide (Gb5Cer, Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), which was specifically recognized (in addition to Gb3Cer and Gb4Cer) by Stx2e, was fully structurally characterized. Lipoforms of Stx receptor GSLs were found to mainly harbor ceramide moieties composed of sphingosine (d18:1) and C24:0/C24:1 or C16:0 fatty acid. Moreover, co-occurrence with lipid raft markers, SM and cholesterol, in DRMs suggested GSL association with membrane microdomains. This study provides the basis for further exploring the functional impact of lipid raft-associated Stx receptors for toxin-mediated injury of Vero-B4 cells.
authors.affiliation: Institutes for Hygiene University of Münster, D-48149 Münster, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Steil
authors.affiliation: Institutes for Hygiene University of Münster, D-48149 Münster, Germany.
authors.forename: Catherine-Louise
authors.initials: CL
authors.lastname: Schepers
authors.affiliation: Institutes for Hygiene University of Münster, D-48149 Münster, Germany.
authors.forename: Gottfried
authors.initials: G
authors.lastname: Pohlentz
authors.affiliation: Institutes for Hygiene University of Münster, D-48149 Münster, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Legros
authors.affiliation: Food Chemistry, University of Münster, D-48149 Münster, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Runde
authors.affiliation: Food Chemistry, University of Münster, D-48149 Münster, Germany.
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Humpf
authors.affiliation: Institutes for Hygiene University of Münster, D-48149 Münster, Germany.
authors.forename: Helge
authors.initials: H
authors.lastname: Karch
authors.affiliation: Institutes for Hygiene University of Münster, D-48149 Münster, Germany Interdisciplinary Center for Clinical Research (IZKF), University of Münster, D-48149 Münster, Germany jm@uni-muenster.de.
authors.forename: Johannes
authors.initials: J
authors.lastname: Müthing
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D004847:Epithelial Cells / Q000378:metabolism; D005915:Globosides / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D021962:Membrane Microdomains / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D022621:Shiga Toxin / Q000378:metabolism; D014281:Trihexosylceramides / Q000378:metabolism; D014709:Vero Cells
chemical_list: D005915:Globosides; D006028:Glycosphingolipids; D011956:Receptors, Cell Surface; D014281:Trihexosylceramides; C045390:shigella toxin receptor; C024032:globotetraosylceramide; C010163:Forssman glycolipid; C018549:globotriaosylceramide; D022621:Shiga Toxin
keywords: Shiga toxin-producing Escherichia coli; enterohemorrhagic Escherichia coli; globo-series; globopentaosylceramide; glycolipids; lysophospholipids; mass spectrometry; microdomains; phospholipids; sphingolipids