title: Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals.
abstract: Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, β-galactosylceramide (GalCer) and β-glucosylceramide (GlcCer), which are involved in several diseases, such as sphingolipidosis, diabetes, chronic kidney diseases, or cancer. In contrast,Bacteroides fragilis, a member of the human gut microbiome, and the marine sponge,Agelas mauritianus, produce α-GalCer, one of the most potent stimulators for invariant natural killer T cells. To dissect the contribution of these individual stereoisomers to pathologies, we established a novel hydrophilic interaction chromatography-based LC-MS2method and separated (R> 1.5) corresponding diastereomers from each other, independent of their lipid anchors. Testing various bacterial and mammalian samples, we could separate, identify (including the lipid anchor composition), and quantify endogenous β-GlcCer, β-GalCer, and α-GalCer isomers without additional derivatization steps. Thereby, we show a selective decrease of β-GlcCers versus β-GalCers in cell-specific models of GlcCer synthase-deficiency and an increase of specific β-GlcCers due to loss of β-glucoceramidase 2 activity. Vice versa, β-GalCer increased specifically when cerebroside sulfotransferase (Gal3st1) was deleted. We further confirm β-GalCer as substrate of globotriaosylceramide synthase for galabiaosylceramide synthesis and identify additional members of the human gut microbiome to contain immunogenic α-GalCers. Finally, this method is shown to separate corresponding hexosylsphingosine standards, promoting its applicability in further investigations.
authors.affiliation: Lipid Pathobiochemistry Group German Cancer Research Center, Heidelberg, Germany.|Instrumental Analytics and Bioanalytics, Mannheim University of Applied Sciences, Mannheim, Germany.
authors.forename: Johanna
authors.identifier: 0000-0002-9224-5296
authors.initials: J
authors.lastname: von Gerichten
authors.affiliation: Department of Biotechnology, Institute for Technical Microbiology, Mannheim University of Applied Sciences, Mannheim, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Schlosser
authors.affiliation: Lipid Pathobiochemistry Group German Cancer Research Center, Heidelberg, Germany.|Center for Applied Research in Biomedical Mass Spectrometry (ABIMAS), Mannheim University of Applied Sciences, Mannheim, Germany.
authors.forename: Dominic
authors.initials: D
authors.lastname: Lamprecht
authors.affiliation: Department of Molecular and Cellular Pathology, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Ivan
authors.initials: I
authors.lastname: Morace
authors.affiliation: Institute of Biochemistry and Molecular Biology and Center for Rare Diseases University of Bonn, Bonn, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Eckhardt
authors.affiliation: Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany.|Institute of Innate Immunity, University Hospital, University of Bonn, Bonn, Germany.
authors.forename: Dagmar
authors.identifier: 0000-0003-4800-6332
authors.initials: D
authors.lastname: Wachten
authors.affiliation: Department of Molecular and Cellular Pathology, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Jennemann
authors.affiliation: Department of Molecular and Cellular Pathology, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Hermann-Josef
authors.identifier: 0000-0003-3613-2649
authors.initials: HJ
authors.lastname: Gröne
authors.affiliation: Department of Biotechnology, Institute for Technical Microbiology, Mannheim University of Applied Sciences, Mannheim, Germany.
authors.forename: Matthias
authors.identifier: 0000-0002-7753-2422
authors.initials: M
authors.lastname: Mack
authors.affiliation: Lipid Pathobiochemistry Group German Cancer Research Center, Heidelberg, Germany r.sandhoff@dkfz.de.|Center for Applied Research in Biomedical Mass Spectrometry (ABIMAS), Mannheim University of Applied Sciences, Mannheim, Germany.
authors.forename: Roger
authors.initials: R
authors.lastname: Sandhoff
mesh_terms: D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D002518:Ceramides / Q000737:chemistry* / Q000378:metabolism*; D000069196:Gastrointestinal Microbiome; D006801:Humans; D051379:Mice; D013237:Stereoisomerism
chemical_list: D002518:Ceramides
keywords: Bacteroides fragilis; KRN7000; cerebroside; electrospray ionization-tandem mass spectrometry; fatty acid 2-hydroylase; galactosylceramide; galactosylsphingosine; globotriaosylceramide synthase; glucocerebroside; glucopsychosine; glucosidase beta 2; glucosylceramide; glucosylceramide synthase; glucosylsphingosine; hydrophilic interaction chromatography; kidney; liver; microbiota; psychosine; α-galactosylceramide