title: Shiga toxin glycosphingolipid receptors and their lipid membrane ensemble in primary human blood-brain barrier endothelial cells.
abstract: Shiga toxin (Stx)-mediated injury to microvascular endothelial cells in the brain significantly contributes to the pathogenesis of the hemolytic-uremic syndrome caused by enterohemorrhagic Escherichia coli (EHEC). Stxs are AB5toxins and the B-pentamers of the two major Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) expressed by human endothelial cells. Here we report on comprehensive structural analysis of the different lipoforms of Gb3Cer (Galα4Galβ4Glcβ1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1Cer, the less effective Stx receptor) of primary human brain microvascular endothelial cells and their association with lipid rafts. Detergent-resistant membranes (DRMs), obtained by sucrose density gradient ultracentrifugation, were used as lipid raft-analogous microdomains of the liquid-ordered phase and nonDRM fractions were employed as equivalents for the liquid-disordered phase of cell membranes. Structures of the prevalent lipoforms of Gb3Cer and Gb4Cer were those with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0) determined by electrospray ionization mass spectrometry that was combined with thin-layer chromatography immunodetection using anti-Gb3Cer and anti-Gb4Cer antibodies as well as Stx1a and Stx2a subtypes. Association of Stx receptor GSLs was determined by co-localization with lipid raft-specific membrane protein flotillin-2 and canonical lipid raft marker sphingomyelin with Cer (d18:1, C16:0) and Cer (d18:1, C24:1/C24:0) in the liquid-ordered phase, whereas lyso-phosphatidylcholine was detectable exclusively in the liquid-disordered phase. Defining the precise microdomain structures of primary endothelial cells may help to unravel the initial mechanisms by which Stxs interact with their target cells and will help to develop novel preventive and therapeutic measures for EHEC-mediated diseases.
authors: Institute for Hygiene, University of Münster, Robert-Koch-Str. 41, D-48149 Münster, Germany.
authors: Nadine
authors: N
authors: Legros
authors: Institute for Food Chemistry, University of Münster, Corrensstr. 45, D-48149 Münster, Germany.
authors: Stefanie
authors: S
authors: Dusny
authors: Institute for Food Chemistry, University of Münster, Corrensstr. 45, D-48149 Münster, Germany.
authors: Hans-Ulrich
authors: HU
authors: Humpf
authors: Institute for Hygiene, University of Münster, Robert-Koch-Str. 41, D-48149 Münster, Germany.
authors: Gottfried
authors: G
authors: Pohlentz
authors: Institute for Hygiene, University of Münster, Robert-Koch-Str. 41, D-48149 Münster, Germany.
authors: Helge
authors: H
authors: Karch
authors: Institute for Hygiene, University of Münster, Robert-Koch-Str. 41, D-48149 Münster, Germany jm@uni-muenster.de.|Interdisciplinary Center for Clinical Research (IZKF), University of Münster, Albert-Schweitzer-Campus 1, D-48149 Münster, Germany.
authors: Johannes
authors: J
authors: Müthing
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001812:Blood-Brain Barrier / Q000737:chemistry / Q000378:metabolism; D002855:Chromatography, Thin Layer; D042783:Endothelial Cells / Q000737:chemistry; D004926:Escherichia coli / Q000472:pathogenicity; D005915:Globosides / Q000737:chemistry* / Q000235:genetics; D006028:Glycosphingolipids / Q000737:chemistry / Q000235:genetics; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000235:genetics; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics; D022622:Shiga Toxin 1 / Q000737:chemistry* / Q000235:genetics; D022641:Shiga Toxin 2 / Q000737:chemistry* / Q000235:genetics; D014281:Trihexosylceramides / Q000737:chemistry* / Q000235:genetics
chemical_list: D000906:Antibodies; D005915:Globosides; D006028:Glycosphingolipids; D011956:Receptors, Cell Surface; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014281:Trihexosylceramides; C045390:shigella toxin receptor; C024032:globotetraosylceramide; C018549:globotriaosylceramide
keywords: detergent-resistant membranes; glycolipids; lipid rafts; lyso-phosphatidylcholine; primary human brain microvascular endothelial cells; sphingomyelin