Glycoconjugate Journal

, Volume 25, Issue 4, pp 291–304

Glycosphingolipids in vascular endothelial cells: relationship of heterogeneity in Gb3Cer/CD77 receptor expression with differential Shiga toxin 1 cytotoxicity

  • Christian H. Schweppe
  • Martina Bielaszewska
  • Gottfried Pohlentz
  • Alexander W. Friedrich
  • Heino Büntemeyer
  • M. Alexander Schmidt
  • Kwang S. Kim
  • Jasna Peter-Katalinić
  • Helge Karch
  • Johannes Müthing
Article

DOI: 10.1007/s10719-007-9091-7

Cite this article as:
Schweppe, C.H., Bielaszewska, M., Pohlentz, G. et al. Glycoconj J (2008) 25: 291. doi:10.1007/s10719-007-9091-7

Abstract

Shiga toxin (Stx) 1 binds to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer/CD77) and injures human endothelial cells. In order to gain insight into Stx1-induced cellular impairment, we analysed in detail the molecular heterogeneity of Stx1 receptors in two endothelial cell lines differing in their Stx1-sensitivity. We observed a moderate sensitivity to Stx1 of human brain microvascular endothelial cells (HBMECs, CD50 > 200 ng/ml), but a considerably higher mortality rate in cultures of EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (CD50 of 0.2 ng/ml). Immunofluorescence microscopy demonstrated the presence of Gb3Cer in both cell lines, but showed an enhanced content of Gb3Cer in EA.hy 926 cells. Solid phase overlay binding assays of isolated GSLs combined with nanoelectrospray ionization quadrupole time-of-flight mass spectrometry demonstrated a balanced proportion of Gb3Cer and globotetraosylceramide (Gb4Cer) in HBMECs, but an increase of Gb3Cer and absence of Gb4Cer in EA.hy 926 cells. Gb3Cer species with C24:1/C24:0 fatty acids were found to dominate over those with C16:0 fatty acids in EA.hy 926 cells, but were similarly distributed in HBMECs. Reverse transcriptase polymerase chain reaction indicated the concomitant presence of Gb3Cer and Gb4Cer synthases in HBMECs, whereas EA.hy 926 cells expressed Gb3Cer synthase, but completely lacked Gb4Cer synthase. This deficiency, resulting in the accumulation of Gb3Cer in EA.hy 926 cells, represents the most prominent molecular reason that underlies the different Stx1 sensitivities of HBMECs and EA.hy 926 endothelial cells.

Keywords

GlycolipidsHBMECsEA.hy 926 cellsGlycosyltransferases

Abbreviations

CID

Collision-induced-dissociation

DTAF

dichlorotriazinylamino fluorescein

EA.hy 926

HUVEC derived endothelial cell line

ESI Q-TOF-MS

electrospray ionization quadrupole time-of-flight mass spectrometry

GSL(s)

glycosphingolipid(s)

HBMECs

human brain microvascular endothelial cells

HPTLC

high-performance thin-layer chromatography

HUVECs

human umbilical vein endothelial cells

RT-PCR

reverse transcriptase polymerase chain reaction

Stx

Shiga toxin

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Christian H. Schweppe
    • 1
    • 2
  • Martina Bielaszewska
    • 1
  • Gottfried Pohlentz
    • 3
  • Alexander W. Friedrich
    • 1
    • 2
  • Heino Büntemeyer
    • 4
  • M. Alexander Schmidt
    • 5
  • Kwang S. Kim
    • 6
  • Jasna Peter-Katalinić
    • 3
  • Helge Karch
    • 1
    • 2
  • Johannes Müthing
    • 3
  1. 1.Institute for HygieneUniversity of MünsterMünsterGermany
  2. 2.Interdisciplinary Center for Clinical Research (IZKF) MünsterMünsterGermany
  3. 3.Institute for Medical Physics and BiophysicsUniversity of MünsterMünsterGermany
  4. 4.Institute for Cell Culture TechnologyUniversity of BielefeldBielefeldGermany
  5. 5.Institute of InfectiologyUniversity of MünsterMünsterGermany
  6. 6.Division of Pediatric Infectious DiseasesJohns Hopkins University School of MedicineBaltimoreUSA