Galabiosylceramide is present in human cerebrospinal fluid.

Cerebrospinal fluid (CSF) contains glycosphingolipids, including lactosylceramide (LacCer, Galß(1,4)Glcß-ceramide). LacCer and its structural isomer, galabiosylceramide (Gb2, Galα(1,4)Galß-ceramide), are classified as ceramide dihexosides (CDH). Gb2 is degraded by α-galactosidase A (GLA) in lysosomes, and genetic GLA deficiency causes Fabry disease, an X-linked lysosomal storage disorder. In patients with Fabry disease, Gb2 accumulates in organs throughout the body. While Gb2 has been reported to be in the liver, kidney, and urine of healthy individuals, its presence in CSF has not been reported, either in patients with Fabry disease or healthy controls. Here, we isolated CDH fractions from CSF of patients with idiopathic normal pressure hydrocephalus. Purified CDH fractions showed positive reaction with Shiga toxin, which specifically binds to the Galα(1,4)Galß structure. The isolated CDH fractions were analyzed by hydrophilic interaction chromatography (HILIC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS). HILIC-ESI-MS/MS separated LacCer and Gb2 and revealed the presence of Gb2 and LacCer in the fractions. We also found Gb2 in CSF from neurologically normal control subjects. This is the first report to show Gb2 exists in human CSF.

Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol.

ß-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form ß-cholesterylglucoside (ß-GlcChol) in vitro ß-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate ß-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (ß-GalChol), in addition to ß-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for ß-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for ß-GalChol formation. Liquid chromatography-tandem MS revealed that ß-GlcChol and ß-GalChol are present throughout development from embryo to adult in the mouse brain. We found that ß-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of ß-GalChol biosynthesis appeared to be during myelination. We also found that ß-GlcChol and ß-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form ß-GalChol. This is the first report of the existence of ß-GalChol in vertebrates and how ß-GlcChol and ß-GalChol are formed in the brain.