Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals.

ABSTRACT: Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, ?-galactosylceramide (GalCer) and ?-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 ?-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-MS² method 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 ?-GlcCer, ?-GalCer, and ?-GalCer isomers without additional derivatization steps. Thereby, we show a selective decrease of ?-GlcCers versus ?-GalCers in cell-specific models of GlcCer synthase-deficiency and an increase of specific ?-GlcCers due to loss of ?-glucoceramidase 2 activity. Vice versa, ?-GalCer increased specifically when cerebroside sulfotransferase (Gal3st1) was deleted. We further confirm ?-GalCer as substrate of globotriaosylceramide synthase for galabiaosylceramide synthesis and identify additional members of the human gut microbiome to contain immunogenic ?-GalCers. Finally, this method is shown to separate corresponding hexosylsphingosine standards, promoting its applicability in further investigations.

SUBMITTER: von Gerichten J 

PROVIDER: S-EPMC5454501 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature