Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular β-glucosidases

André R A Marques ,Mina Mirzaian,Rianne Meijer,Pilar Giraldo,Navraj S Pannu,Marri Verhoek,Paulo Gaspar,Herman S Overkleeft,Rolf G Boot,Roelof Ottenhoff,Timothy M Cox,Saskia Scheij,Maria J Ferraz,Yoshio Hirabayashi,Pilar Irún,Pilar Alfonso,Marco Van Eijk,Patrick Wisse,Hisanori Akiyama ,Karen Ghauharali‐Van Der Vlugt ,E F C Blommaart ,Natashe Lemos Dekker ,Cindy P A A Van Roomen ,Morten Dahl ,Stefán Karlsson ,Elena Pavlova ,Johannes M F G Aerts

doi:10.1194/jlr.m064923

Abstract

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 13C6-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.

Highlights

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded
The non-neuronopathic variant of Gaucher disease (GD) is presently treated by enzyme replacement therapy (ERT), implying chronic twice-weekly intravenous infusion of macrophagetargeted recombinant enzyme [17]
For quantitative analysis of GlcChol in samples of plasma, we developed a LC-MS/MS method using the multiple reaction monitoring (MRM) mode of the transitions mentioned above

Summary

Introduction

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading ␤-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Akiyama et al [10] showed that GCS does not synthesize GlcChol They noticed that GM-95 cells deficient in GCS are unable to synthesize GlcChol without the addition of exogenous GlcCer. the same researchers demonstrated that, at least in vitro, the lysosomal enzyme glucocerebrosidase (GBA; E.C.3.2.1.45) generates, through transglucosylation, 25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl) methyl] amino]-27-norcholesterol (25-NBD-cholesterol)glucoside from GlcCer and artificial 25-NBD-cholesterol [11]. Mammalian cells and tissues contain other ␤-glucosidases besides GBA that degrade GlcCer. All cells express the membrane-associated nonlysosomal glucosylceramidase, named GBA2 [21,22,23]. Some tissues express the enzyme GBA3, referred to as broad-specific cytosolic ␤-glucosidase [26] This enzyme shows relatively poor in vitro hydrolytic activity toward GlcCer and is thought to be primarily involved in detoxification of glucosylated xenobiotics [26]. Our investigation demonstrates the surprising versatility of ␤-glucosidases, a finding discussed in relation to metabolism of sphingolipids and sterols in health and disease

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Results

Conclusion