title: Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood-brain barrier.
abstract: Shiga toxin (Stx) 2e, released by certain Stx-producing Escherichia coli, is presently the best characterized virulence factor responsible for pig edema disease, which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Although Stx2e-mediated brain vascular injury is the key event in development of neurologic signs, the glycosphingolipid (GSL) receptors of Stx2e and toxin-mediated impairment of pig brain endothelial cells have not been investigated so far. Here, we report on the detailed structural characterization of Stx2e receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which make up the major neutral GSLs in primary porcine brain capillary endothelial cells (PBCECs). Various Gb3Cer and Gb4Cer lipoforms harboring sphingenine (d18:1) or sphinganine (d18:0) and mostly a long-chain fatty acid (C20-C24) were detected. A notable batch-to-batch heterogeneity of primary endothelial cells was observed regarding the extent of ceramide hydroxylation of Gb3Cer or Gb4Cer species. Gb3Cer, Gb4Cer and sphingomyelin preferentially distribute to detergent-resistant membrane fractions and can be considered lipid raft markers in PBCECs. Moreover, we employed an in vitro model of the blood-brain barrier (BBB), which exhibited strong cytotoxic effects of Stx2e on the endothelial monolayer and a rapid collapse of the BBB. These data strongly suggest the involvement of Stx2e in cerebral vascular damage with resultant neurological disturbance characteristic of edema disease.
authors: Institute for Hygiene, Robert-Koch-Str. 41, Germany.
authors: Iris
authors: I
authors: Meisen
authors: Regina
authors: R
authors: Rosenbrück
authors: Hans-Joachim
authors: HJ
authors: Galla
authors: Sabine
authors: S
authors: Hüwel
authors: Ivan U
authors: IU
authors: Kouzel
authors: Michael
authors: M
authors: Mormann
authors: Helge
authors: H
authors: Karch
authors: Johannes
authors: J
authors: Müthing
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000276:immunology / Q000473:pathology*; D001921:Brain / Q000473:pathology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D017097:Electric Impedance; D042783:Endothelial Cells / Q000276:immunology / Q000378:metabolism*; D004727:Endothelium / Q000276:immunology / Q000503:physiopathology; D005915:Globosides / Q000737:chemistry / Q000378:metabolism*; D006017:Glycolipids / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D061251:Primary Cell Culture; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism; D022641:Shiga Toxin 2 / Q000494:pharmacology; D034421:Sus scrofa; D014281:Trihexosylceramides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005915:Globosides; D006017:Glycolipids; D011956:Receptors, Cell Surface; D022641:Shiga Toxin 2; C068556:Shiga-like toxin receptor; D014281:Trihexosylceramides; C024032:globotetraosylceramide; C018549:globotriaosylceramide
keywords: TLC immunostaining; edema disease; glycolipids; lipid rafts; mass spectrometry