Abstract
Glucocerebrosidase (GBA) is a lysosomal β‐glucosidase‐degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD). Conduritol B epoxide (CBE), a mechanism‐based irreversible inhibitor of GBA, is used to generate cell and animal models for investigations on GD and PD. However, CBE may have additional glycosidase targets besides GBA. Here, we present the first in vivo target engagement study for CBE, employing a suite of activity‐based probes to visualize catalytic pocket occupancy of candidate off‐target glycosidases. Only at significantly higher CBE concentrations, nonlysosomal glucosylceramidase (GBA2) and lysosomal α‐glucosidase were identified as major off‐targets in cells and zebrafish larvae. A tight, but acceptable window for selective inhibition of GBA in the brain of mice was observed. On the other hand, cyclophellitol, a closer glucose mimic, was found to inactivate with equal affinity GBA and GBA2 and therefore is not suitable to generate genuine GD‐like models.EnzymesGlucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html), nonlysosomal β‐glucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html); cytosolic β‐glucosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/21.html); α‐glucosidases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/20.html); β‐glucuronidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/31.html).
Highlights
The lysosomal enzyme glucocerebrosidase (GBA, EC 3.2.1.45) is a retaining β-glucosidase that degrades the glycosphingolipid, glucosylceramide
Competition of activitybased probe (ABP) labelling by Conduritol B epoxide (CBE) and CP was compared to the loss of GBA activity measured using 3.75 mM 4methylumbelliferyl-β-glucoside as substrate[40]
IC50 values were determined and found to be 26.6 μM at 30 min CBE preincubation, and 2.30 μM at 180 min pre-incubation (Fig. 2A, Table 1). These values match the ones determined by measurement of residual enzymatic activity of GBA assay (Fig. 2A lower right panel, Table 1), validating the competitive activity-based protein profiling (ABPP) methodology
Summary
The lysosomal enzyme glucocerebrosidase (GBA, EC 3.2.1.45) is a retaining β-glucosidase that degrades the glycosphingolipid, glucosylceramide. Inherited deficiency of GBA is the cause of autosomal recessive Gaucher disease (GD) [1]. Most GD patients display heterogeneous symptoms including spleen and liver enlargement, bone deterioration, anaemia, leukopenia, and thrombocytopenia. Some patients develop fatal neurological symptoms[2]. The GBA genotype poorly predicts the onset and severity of disease in individual GD patients, even in monozygotic twins[3,4]. Carriers of a GBA defect do not develop GD but show a markedly increased risk for Parkinson’s disease (PD) and Lewy-body dementia[5,6]. The molecular basis for this risk is unknown and a subject of research
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