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

Session: 
PS1 Poster session 1 Odd numbers
Code: 
P123
Location (hall): 
Foyer
Start/end time: 
Monday, July 1, 2019 - 15:45 to 17:15
Kassiani
Kytidou

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. 

References: 
  1. Kallemeijn WW, Witte MD, Wennekes T, Aerts JM. Mechanism-based inhibitors of glycosidases: design and applications. Adv Carbohydr Chem Biochem. 2014;71:297-338. 
  2. Witte MD, et al. Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat Chem Biol. 2010;6(12):907-13. 
  3. Kallemeijn WW, et al. Novel activity-based probes for broad-spectrum profiling of retaining β-exoglucosidases in situ and in vivo. Angew Chem Int Ed Engl. 2012;51(50):12529-33. 
  4. Jiang J, et al. Detection of Active Mammalian GH31 α-Glucosidases in Health and Disease Using In-Class, Broad-Spectrum Activity-Based Probes. ACS Cent Sci. 2016;2(5):351-8. 
  5. Willems LI, et al. Potent and selective activity-based probes for GH27 human retaining α-galactosidases. J Am Chem Soc. 2014;136(33):11622-5. 
  6. Kytidou K, Beenakker TJM, Westerhof LB, et al. Human Alpha Galactosidases Transiently Produced in Nicotiana benthamiana Leaves: New Insights in Substrate Specificities with Relevance for Fabry Disease. Front Plant Sci. 2017; 8:1026. 
  7. Wu L et al. Activity-based probes for functional interrogation of retaining β-glucuronidases. Nat Chem Biol. 2017 Aug;13(8):867-873. 

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