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.