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.
Highlights
GBA2 and GBA are both -glucosidases that degrade glucosylceramide
Having established that GBA is not affected by NB-DGJ, these results indicate that in various wild-type mouse tissues and cell lines a proportion of GBA2 activity was sensitive to inhibition by CBE
To fully appreciate the physiological and pathological roles of GBA2, as well as its pharmacology, it is fundamental to characterize the -glucosidase activity exerted by this enzyme and to distinguish its activity from that of GBA
Summary
GBA2 and GBA are both -glucosidases that degrade glucosylceramide. Results: Conduritol B epoxide inactivates both GBA and GBA2, whereas the imino sugar NB-DGJ selectively inhibits GBA2. Considering the biochemical activity of GBA2, its involvement in various diseases, and the clinical application and development of pharmacological agents that have overlapping effects on UGCG, GBA, and GBA2 (Fig. 1; Table 1), the characterization of the -glucosidase activity of GBA2 is of fundamental importance Both GBA and GBA2 contribute to the total level of -glucosidase activity that can be measured in tissue/cell homogenates and membrane preparations using the artificial substrate 4-methylumbelliferyl--D-glucoside and the fluorescent GlcCer analog C12NBD-GlcCer. Even though GBA requires detergents to be maximally active in vitro, it is active under the same conditions as used for measuring GBA2 activity, i.e. in detergent-free membrane preparations [34, 62]. We have established that the GBA2 -glucosidase activity in mouse tissues and cultured cells is considerably higher than the CBE-resistant -glucosidase activity, and the sensitivity of GBA2 to inhibition by NB-DNJ and NB-DGJ differs from previous estimates
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