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. 2017 Jun;58(6):1247-1258.
doi: 10.1194/jlr.D076190. Epub 2017 Apr 3.

Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals

Johanna von Gerichten  1   2 , Kerstin Schlosser  3 , Dominic Lamprecht  1   4 , Ivan Morace  5 , Matthias Eckhardt  6 , Dagmar Wachten  7   8 , Richard Jennemann  5 , Hermann-Josef Gröne  5 , Matthias Mack  3 , Roger Sandhoff  9   4
Affiliations

Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals

Johanna von Gerichten et al. J Lipid Res. 2017 Jun.

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-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 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.

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.

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Conflict of interest statement

The authors declare that they have no conflicts of interest related to the contents of this article.

The authors declare that they have no conflicts of interest related to the contents of this article.

Figures

Fig. 1.
Fig. 1.
A: Stereochemical structures of β-GlcCer, β-GalCer, and α-GalCer containing a C18-sphingosine and an N-linked palmitic acid [HexCer(d18:1/16:0)]. B: Product ion spectra of corresponding HexCers obtained at 25 eV collision energy by ultra-performance LC-ESI-triple quadrupole MS2. Note, abundant product ions are detected in all three compounds. C: Extracted ion chromatograms from reversed phase chromatography (CSH C18) for the detection of HexCer(d18:1/16:0) from the three diastereomers, β-GlcCer, β-GalCer, and α-GalCer. Note the identical retention time of all three compounds.
Fig. 2.
Fig. 2.
A: HILIC separation of a synthetic α/β-anomeric mixture of GlcCer(d18:1/24:1) (dark cyan) and a synthetic α/β-anomeric mixture of GalCer(d18:1/24:1) (red), each with approximately 15% α-content. B: Dilution of β-GlcCer(d18:1/24:1) in the presence of constant amounts of β-GalCer(d18:1/24:1). C: Dilution of β-GalCer(d18:1/24:1) in the presence of constant amounts of β-GlcCer(d18:1/24:1). D: Constant amounts of α-GalCer(d18:1/24:1) in the presence of increasing amounts of β-GalCer(d18:1/24:1). E: Retention times of standard HexCers marked with an asterisk and endogenous β-HexCers, which have been described in literature and were identified based on the behavior of standard compounds and on the molecular ion size. Note the relatively strong shift from nonhydroxy (NS) to α-hydroxy fatty acid containing compounds (AS), while introduction of phytosphingosine (NP) instead of sphingosine (NS) as well as decreasing acyl chain length contributed in a minor way to later elution. Additional double bonds as in HexCer[d18:1/24:1(15Z)] did not contribute to a significant retention time shift.
Fig. 3.
Fig. 3.
Relative distribution of β-GlcCer and β-GalCer in various mouse organs. NS-, AS-, NP-, and AP-HexCers were determined, which contained a C18-sphingoid base and N-bound fatty acids with the chain length C16 up to C26 (as annotated). Special structures with ultra-long acyl chains as they occur in epidermis (ultra-long omega hydroxyl fatty acids) or in male germ cells (ultra-long polyunsaturated fatty acids) as well as with different sphingoid bases (e.g., C20-sphingosine in brain or kidney papillae or C17-sphingosine in epidermis) were not considered in this study. Note, nervonic acid is mainly incorporated into brain cerebrosides, whereas corresponding glucocerebrosides are enriched in stearic acid, which is the typical acyl chain of neuronal gangliosides.
Fig. 4.
Fig. 4.
HILIC-MS2-based quantification of β-GlcCers and β-GalCers in liver of WT, Gba2−/−, and Ugcgf/fAlbCre mice (A); kidney of WT and Gba2−/− mice (B); small intestine of WT, and Gba2−/− mice (C); kidney of WT, Ugcgf/fPax8Cre, CSTf/fPax8Cre, (Ugcg and CST)f/fPax8Cre mice (D); and in kidney cortex of WT and Gb3s−/− mice. Note the specific increase of NS-β-GlcCer in liver, kidney and small intestine of Gba2−/− mice, the selective decrease of NS-GlcCer in liver and of NS- and AS-GlcCer in kidney of Ugcgf/fAlbCre and Ugcgf/fPax8Cre [and (Ugcg and CST)f/fPax8Cre] mice, respectively (E). Vice versa, NS- and AS-β-GalCer accumulates in kidneys with CST-deficiency [CSTf/fPax8Cre and (Ugcg and CST)f/fPax8Cre] as well as NS-β-GalCer in cortex of Gb3s−/− mice. n ≥ 3, except for (D) WT = 1.
Fig. 5.
Fig. 5.
HILIC-MS2-based separation of AS-type β-GalCers with 2R- and 2S-hydroxy stearic acid. Extracted ion chromatogram (EIC) for AS-HexCer(d18:1/h18:0) and (d18:1/h24:0) from a purified mixture of brain AS-type β-GalCers [phrenosin (A, E)], the synthetic standards β-GalCer[d18:1/(2S)h18:0] and β-GalCer[d18:1/(2R)h18:0] (D), a mouse brain lipid extract enriched in neutral GSLs (B, F), and a stomach lipid extract from WT (C, G) and Fa2h−/− (H) mice. The intensity in (H) is normalized to that of the corresponding WT signal in (G). In compliance with a report that AS-type β-GalCer from brain contain 2R-hydroxy fatty acids, the AS-HexCer(d18:1/h18:0) from phrenosin and from mouse brain migrate together with the β-GalCer[d18:1/(2R)h18:0] standard. Note, the decrease of β-GalCer(d18:1/h24:0) with 2R-hydroxy configuration in Fa2h−/− stomach.
Fig. 6.
Fig. 6.
HILIC-MS2-based detection of α-GalCers from B. fragilis and identification of equivalent compounds in B. vulgatus and P. copri. A: Comparison of the determined retention times of α-GalCers from B. fragilis with those of synthetic α-GalCer and β-GalCer standards and predicted values for AS-type α-GalCers. B: Extracted ion chromatograms for individual HexCer from B. fragilis. C: Quantification of the three α-GalCers in B. fragilis, B. vulgatus, and P. copri. Note the 100-fold amount of α-GalCers in B. fragilis. n = 3.
Fig. 7.
Fig. 7.
HILIC-MS2-based detection of diastereomeric HexSph standards with a C18-sphingosine (d18:1). β-GlcSph elutes first and separates from β-GalSph with Δt = 0.12 ± 0.005 min. After these compounds, α-GalSph elutes with Δt = 0.07 ± 0.005 min significantly behind β-GalSph.
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