title: β-Glucosidase 2 (GBA2) activity and imino sugar pharmacology.
abstract: β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher KI) and a lower rate of enzyme inactivation (k(inact)). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5-6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.
authors.affiliation: From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Ridley
authors.affiliation: From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Thur
authors.affiliation: From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Jessica
authors.initials: J
authors.lastname: Shanahan
authors.affiliation: the Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Nagendra Babu
authors.initials: NB
authors.lastname: Thillaiappan
authors.affiliation: From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Ann
authors.initials: A
authors.lastname: Shen
authors.affiliation: From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Karly
authors.initials: K
authors.lastname: Uhl
authors.affiliation: the Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom, and.
authors.forename: Charlotte M
authors.initials: CM
authors.lastname: Walden
authors.affiliation: the Gene Transfer Technology Group, Institute of Women's Health, University College London, London WC1E 6HX, United Kingdom.
authors.forename: Ahad A
authors.initials: AA
authors.lastname: Rahim
authors.affiliation: the Gene Transfer Technology Group, Institute of Women's Health, University College London, London WC1E 6HX, United Kingdom.
authors.forename: Simon N
authors.initials: SN
authors.lastname: Waddington
authors.affiliation: the Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Frances M
authors.initials: FM
authors.lastname: Platt
authors.affiliation: From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada,. Electronic address: spoela@dal.ca.
authors.forename: Aarnoud C
authors.initials: AC
authors.lastname: van der Spoel
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002524:Cerebellar Ataxia / Q000188:drug therapy / Q000201:enzymology; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics* / Q000494:pharmacology; D005962:Glucosylceramidase; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D051379:Mice; D015419:Spastic Paraplegia, Hereditary / Q000188:drug therapy / Q000201:enzymology; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D017485:1-Deoxynojirimycin; D007294:Inositol; C090092:migalastat; D001617:beta-Glucosidase; C517538:beta-glucosidase 2, mouse; C517537:GBA2 protein, human; D005962:Glucosylceramidase; C011423:conduritol epoxide
keywords: Conduritol B Epoxide; Enzyme Inhibitors; Glucosylceramide; Glycolipids; Glycoside Hydrolases; Mechanism-based Enzyme Inhibitor; Membrane Lipids; Miglustat; Nonlysosomal Glucosylceramidase; Sphingolipid