A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase,the Enzyme Deficient in Krabbe Disease |
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| Authors: | Dr. André R. A. Marques Dr. Lianne I. Willems Daniela Herrera Moro Dr. Bogdan I. Florea Saskia Scheij Roelof Ottenhoff Cindy P. A. A. van Roomen Marri Verhoek Dr. Jessica K. Nelson Dr. Wouter W. Kallemeijn Dr. Anna Biela‐Banas Prof. Dr. Olivier R. Martin Dr. M. Begoña Cachón‐González Nee Na Kim Prof. Dr. Timothy M. Cox Dr. Rolf G. Boot Prof. Dr. Herman S. Overkleeft Prof. Dr. Johannes M. F. G. Aerts |
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| Affiliation: | 1. Department of Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;2. Present address: Institute of Biochemistry, Christian-Albrechts-University of Kiel, Kiel, Germany;3. Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands;4. Present address: Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada;5. Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands;6. Institute of Organic and Analytical Chemistry, Université D'Orléans, Orléans, France;7. Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK |
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| Abstract: | Galactosylceramidase (GALC) is the lysosomal β‐galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity‐based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a β‐galactopyranose‐configured cyclophellitol‐epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra‐cerebroventricular infusion of the ABP. In conclusion, this GALC‐specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease. |
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| Keywords: | beta-galactosidase fluorescent probes galactosylceramidase hydrolase Krabbe disease |
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