title: Glycosphingolipid requirements for endosome-to-Golgi transport of Shiga toxin.
abstract: Shiga toxin binds to globotriaosylceramide (Gb3) receptors on the target cell surface. To enter the cytosol, Shiga toxin is dependent on endocytic uptake, retrograde transport to the Golgi apparatus and further to the endoplasmic reticulum before translocation of the enzymatically active moiety to the cytosol. Here, we have investigated the importance of newly synthesized glycosphingolipids for the uptake and intracellular transport of Shiga toxin in HEp-2 cells. Inhibition of glycosphingolipid synthesis by treatment with either PDMP or Fumonisin B(1) for 24-48 h strongly reduced the transport of Gb3-bound Shiga toxin from endosomes to the Golgi apparatus. This was associated with a change in localization of sorting nexins 1 and 2, and accompanied by a protection against the toxin. In contrast, there was no effect on transport or toxicity of the plant toxin ricin. High-resolution mass spectrometry revealed a 2-fold reduction in Gb3 at conditions giving a 10-fold inhibition of Shiga toxin transport to the Golgi. Furthermore, mass spectrometry showed that the treatment with PDMP (DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and Fumonisin B(1) among other changes of the lipidome, affected the relative content of the different glycosphingolipid species. The largest depletion was observed for the hexosylceramide species with the N-amidated fatty acid 16:0, whereas hexosylceramide species with 24:1 were less affected. Quantitative lipid profiling with mass spectrometry demonstrated that PDMP did not influence the content of sphingomyelins, phospholipids and plasmalogens. In contrast, Fumonisin B(1) affected the amount and composition of sphingomyelin and glycolipids and altered the profiles of phospholipids and plasmalogens.
authors.affiliation: Centre for Cancer Biomedicine, Faculty Division Norwegian Radium Hospital, University of Oslo, 0316 Oslo, Norway.
authors.forename: Hilde
authors.initials: H
authors.lastname: Raa
authors.forename: Stine
authors.initials: S
authors.lastname: Grimmer
authors.forename: Dominik
authors.initials: D
authors.lastname: Schwudke
authors.forename: Jonas
authors.initials: J
authors.lastname: Bergan
authors.forename: Sébastien
authors.initials: S
authors.lastname: Wälchli
authors.forename: Tore
authors.initials: T
authors.lastname: Skotland
authors.forename: Andrej
authors.initials: A
authors.lastname: Shevchenko
authors.forename: Kirsten
authors.initials: K
authors.lastname: Sandvig
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037341:Fumonisins / Q000494:pharmacology; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D022621:Shiga Toxin / Q000378:metabolism*; D014281:Trihexosylceramides / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D004791:Enzyme Inhibitors; D037341:Fumonisins; D006028:Glycosphingolipids; D051907:Lysosomal Membrane Proteins; D009025:Morpholines; D022621:Shiga Toxin; D014281:Trihexosylceramides; D033921:Vesicular Transport Proteins; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C093846:early endosome antigen 1; C056933:fumonisin B1; C018549:globotriaosylceramide; C033110:RV 538