Publications

• Cholesterol glucosylation is catalyzed by transglucosylation reaction of beta-glucosidase 1.

  Akiyama H., Kobayashi S., Hirabayashi Y., Murakami-Murofushi K.

  Cholesteryl glucoside (β-ChlGlc), a monoglucosylated derivative of cholesterol, is involved in the regulation of heat shock responses. β-ChlGlc, which is rapidly induced in response to heat shock, activates heat shock transcription factor 1 (HSF1) leading to the expression of heat shock protein 70 ... >> More

  Cholesteryl glucoside (β-ChlGlc), a monoglucosylated derivative of cholesterol, is involved in the regulation of heat shock responses. β-ChlGlc, which is rapidly induced in response to heat shock, activates heat shock transcription factor 1 (HSF1) leading to the expression of heat shock protein 70 (HSP70) in human fibroblasts. Identification and biochemical characterization of the enzyme responsible for β-ChlGlc formation is important for a complete understanding of the molecular mechanisms leading to HSP70-induction following heat shock. Recently, we demonstrated that β-ChlGlc synthesis is not dependent on UDP-Glucose but glucosylceramide (GlcCer) in animal tissue and human fibroblasts. In this study, we examined the possibility of glucocerebrosidase, a GlcCer-degrading glycosidase, acting as β-ChlGlc-synthesizing enzyme. Overexpression of β-glucosidase 1 (GBA1, lysosomal acid β-glucocerebrosidase) led to an increase in cholesterol glucosylation activity in human fibroblasts. Using a cell line generated from type 2 Gaucher disease patients with severe defects in GBA1 activity, we found that cholesterol glucosylation activity was very low in the cells and the overexpression of GBA1 rescued the activity. In addition, purified recombinant GBA1 exhibits conduritol B-epoxide-sensitive cholesterol glucosylation activity. The optimum pH and temperature for cholesterol glucosylation by GBA1 were at about 5.3 and 43 °C, respectively. Short chain C8:0-GlcCer was the most effective donor for cholesterol glucosylation activity among GlcCer containing saturated fatty acid (C8:0 to C18:0) tested. GlcCer containing mono-unsaturated fatty acid was more preferred substrate for cholesterol glucosylation when compared with GlcCer containing same chain length of saturated fatty acid. These results demonstrate, for the first time, a novel function of GBA1 as a β-ChlGlc-synthesizing enzyme. Therefore, our results also reveal a new pathway for glycolipid metabolism in mammals. << Less

  Biochem. Biophys. Res. Commun. 441:838-843(2013) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular beta-glucosidases.

  Marques A.R., Mirzaian M., Akiyama H., Wisse P., Ferraz M.J., Gaspar P., Ghauharali-van der Vlugt K., Meijer R., Giraldo P., Alfonso P., Irun P., Dahl M., Karlsson S., Pavlova E.V., Cox T.M., Scheij S., Verhoek M., Ottenhoff R., van Roomen C.P., Pannu N.S., van Eijk M., Dekker N., Boot R.G., Overkleeft H.S., Blommaart E., Hirabayashi Y., Aerts J.M.

  The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able ... >> More

  The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-β-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through β-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer. << Less

  J. Lipid Res. 57:451-463(2016) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• THE METABOLISM OF GLUCOCEREBROSIDES. I. PURIFICATION AND PROPERTIES OF A GLUCOCEREBROSIDE-CLEAVING ENZYME FROM SPLEEN TISSUE.

  BRADY R.O., KANFER J., SHAPIRO D.

  J Biol Chem 240:39-43(1965) [PubMed] [EuropePMC]

• Use of fluorescent substrates for characterization of Gaucher disease mutations.

  Ron I., Dagan A., Gatt S., Pasmanik-Chor M., Horowitz M.

  Gaucher disease results from impaired activity of the lysosomal enzyme beta-glucocerebrosidase. More than 200 mutations within the glucocerebrosidase gene have been associated with this disease. In this study we tested the effect of several mutations (K157Q, D140H, E326K, D140H+E326K, V394L and R4 ... >> More

  Gaucher disease results from impaired activity of the lysosomal enzyme beta-glucocerebrosidase. More than 200 mutations within the glucocerebrosidase gene have been associated with this disease. In this study we tested the effect of several mutations (K157Q, D140H, E326K, D140H+E326K, V394L and R463C) on RNA stability, protein stability and activity toward four different fluorescent substrates (LR-12-GC, Bodipy-12-GC, LR-0-PAP-glucose and 4-MUG), using the vaccinia-derived expression system. The results indicated that the K157Q mutation leads to RNA instability, causing low protein levels and a concomitant reduction in beta-glucocerebrosidase activity. All other tested mutations led to production of glucocerebrosidase RNA and protein with stabilities comparable to those of the normal counterpart. The D140H variant exhibited a high activity toward the tested substrates while the variant enzymes containing either the E326K or D140H and E326k mutations together expressed low beta-glucocerebrosidase activity. The V394L variant exhibited low activity toward the tested substrates, while a higher activity was presented by the R463C containing glucocerebrosidase variant. Our results strongly indicated that the LR-12-GC substrate distinguishes between severities of different mutant glucocerebrosidase variants overexpressed in a heterologous system. << Less

  Blood Cells Mol. Dis. 35:57-65(2005) [PubMed] [EuropePMC]

• Mutation of beta-glucosidase 2 causes glycolipid storage disease and impaired male fertility.

  Yildiz Y., Matern H., Thompson B., Allegood J.C., Warren R.L., Ramirez D.M.O., Hammer R.E., Hamra F.K., Matern S., Russell D.W.

  beta-Glucosidase 2 (GBA2) is a resident enzyme of the endoplasmic reticulum thought to play a role in the metabolism of bile acid-glucose conjugates. To gain insight into the biological function of this enzyme and its substrates, we generated mice deficient in GBA2 and found that these animals had ... >> More

  beta-Glucosidase 2 (GBA2) is a resident enzyme of the endoplasmic reticulum thought to play a role in the metabolism of bile acid-glucose conjugates. To gain insight into the biological function of this enzyme and its substrates, we generated mice deficient in GBA2 and found that these animals had normal bile acid metabolism. Knockout males exhibited impaired fertility. Microscopic examination of sperm revealed large round heads (globozoospermia), abnormal acrosomes, and defective mobility. Glycolipids, identified as glucosylceramides by mass spectrometry, accumulated in the testes, brains, and livers of the knockout mice but did not cause obvious neurological symptoms, organomegaly, or a reduction in lifespan. Recombinant GBA2 hydrolyzed glucosylceramide to glucose and ceramide; the same reaction catalyzed by the beta-glucosidase acid 1 (GBA1) defective in subjects with the Gaucher's form of lysosomal storage disease. We conclude that GBA2 is a glucosylceramidase whose loss causes accumulation of glycolipids and an endoplasmic reticulum storage disease. << Less

  J. Clin. Invest. 116:2985-2994(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Characterization of human glucosylsphingosine glucosyl hydrolase and comparison with glucosylceramidase.

  Vaccaro A.M., Muscillo M., Suzuki K.

  Properties of glucosylsphingosine (gluco-psychosine) glucosyl hydrolase were studied in detail in cultured human fibroblasts and placenta and were compared with those of glucosylceramidase. The two activities, that are deficient in tissues of Gaucher patients, showed minor but consistent differenc ... >> More

  Properties of glucosylsphingosine (gluco-psychosine) glucosyl hydrolase were studied in detail in cultured human fibroblasts and placenta and were compared with those of glucosylceramidase. The two activities, that are deficient in tissues of Gaucher patients, showed minor but consistent differences. The pH optima were 4.8 for psychosine hydrolysis and 5.3 for glucosylceramide hydrolysis. In the presence of oleic acid, taurocholate activated glucosylceramidase more than 10-fold, while it activated psychosine hydrolysis only by about 30%. Triton X-100 was stimulatory for glucosylceramidase but was strongly inhibitory for psychosine hydrolysis. Phospholipids, that increase many times glucosylceramidase activity, were moderately inhibitory to enzymatic hydrolysis of psychosine. The psychosine hydrolase activity was slightly more heat-stable than the glucosylceramidase activity. The Km values for the two substrates were similar; 1.7 X 10(-5) M for psychosine and 2.7 X 10(-5) M for glucosylceramide. The V for glucosylceramide was, however, 100-times that for psychosine hydrolysis. Psychosine acted as a potent non-competitive inhibitor (Ki = 1.8 X 10(-5) M), while glucosylceramide was a weak inhibitor against psychosine hydrolysis. Within the limit of glucosylceramide solubility, psychosine hydrolysis could not be inhibited by more than 50%. Furthermore, the Dixon plot of glucosylceramide inhibition showed an anomalous slope. The ratio of the two activities was similar in fibroblasts, in the placenta mitochondria-lysosomal fraction and in a partially purified placental preparation. These findings are best explained by the hypothesis that, although the two substrates are hydrolyzed by a single enzyme, they share an overlapping but not identical catalytic site while binding to hydrophobic sites unique for the respective substrates. << Less

  Eur J Biochem 146:315-321(1985) [PubMed] [EuropePMC]