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, , a member of the human gut microbiome, and the marine sponge,, 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-MS method and separated ( > 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 () 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.

Supplementary key words glycolipids • sphingolipids • mass spectrometry • immunology • bacteria • invariant natural killer T cells • Western diet • experimental colitis • influenza A virus • cluster of differentiation 1d Besides recognition of classical major histocompatibility complex class I or II molecules presenting oligopeptides by T lymphocytes, a wide variety of nonclassical nearly monomorphic major histocompatibility complex molecules, including CD1, may be recognized by  and  T cells as Abstract The glycosphingolipid, -galactosylceramide (GalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of GalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS 2 , we screened murine intestinal tracts to identify and quantify GalCers, and we investigated the GalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of GalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor GalCer. The identified GalCer contained a (R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis. Unlike -anomeric structures, but similar to GalCers from B. fragilis, the synthetic form of the murine GalCer induced iNKT cell activation in vitro. Last, we observed a decrease in GalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that GalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future

In plants, low molecular weight terpenes produced by terpene synthases (TPS) contribute to multiple ecologically and economically important traits. The present study investigates a carrot terpene synthase gene cluster on chromosome 4 associated with volatile monoterpene production. Two carrot mutants, yellow and cola, which are contrasting in the content of low molecular weight terpenes, were crossed to develop an F2 mapping population. The mapping analysis revealed overlapping QTLs on chromosome 4 for sabinene, α-thujene, α-terpinene, γ-terpinene, terpinen-4-ol and 4-carene. The genomic region of this locus includes a cluster of five terpene synthase genes (DcTPS04, DcTPS26, DcTPS27, DcTPS54 and DcTPS55). DcTPS04 and DcTPS54 displayed genotype- and tissue-specific variation in gene expression. Based on the QTL mapping results and the gene expression patterns, DcTPS04 and DcTPS54 were selected for functional characterization. In vitro enzyme assays showed that DcTPS54 is a single-product enzyme catalysing the formation of sabinene, whereas DcTPS04 is a multiple-product terpene synthase producing α-terpineol as a major product and four additional products including sabinene, β-limonene, β-pinene and myrcene. Furthermore, we developed a functional molecular marker that could discriminate carrot genotypes with different sabinene content in a set of 85 accessions.