Many lysosomal glycosidases are retaining enzymes, employing a double-displacement mechanism involving covalent binding of sugar to the catalytic nucleophile of the enzyme . Cyclophellitol is natural gluco-mimetic that irreversibly binds to the catalytic nucleophile of glucocerebrosidase (lysosomal β-glucosidase; GBA). Based on this scaffold a fluorescent activity-based probe (ABP) was designed allowing labeling of active GBA in vitro and in vivo (MDW933) . Subsequently appropriate ABPs were designed for many lysosomal glycosidases, including acid α-glucosidase (GAA) and α-galactosidase (GLA) [3-7].
The available ABPs can be used in diagnosis of corresponding lysosomal storage disorders. Cells, or cell extracts, can be incubated with ABPs, and following SDS-PAGE and fluorescence scanning active enzyme molecules can be visualized and quantified. In this manner a deficiency of active lysosomal glycosidases such as, GBA, GAA and GLA can be demonstrated in cells from individuals suffering from Gaucher disease, Pompe disease and Fabry disease, respectively [2-6]. We also extended the procedure to urine and developed a sensitive method to visualize active GBA, GAA, GLA and other lysosomal enzymes in urine samples. The method is remarkably sensitive, requiring only a few ml of urine. The practical application of this procedure for diagnostic screening is discussed.
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