Activity-Based Labeling and Detection of Active Lysosomal Glycosidases: Application in Diagnostic Screening of Urine Samples

S2.4 Sugar biosynthesis
Location (hall): 
Start/end time: 
Monday, July 1, 2019 - 15:20 to 15:25

Kassiani Kytidou1, Ethan Kuo, Marta Artola, Ivanna Denysiuk, Hermen Overkleeft, Hans Aerts

1Medical Biochemistry & Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

Many lysosomal glycosidases are retaining enzymes, employing a double-displacement mechanism involving covalent binding of sugar to the catalytic nucleophile of the enzyme [1]. 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) [2]. 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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