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Localization of Active Endogenous and Exogenous GBA by Correlative Light-Electron Microscopy in Human Fibroblasts.

著者 van Meel E , Bos E , van der Lienden MJC , Overkleeft HS , van Kasteren SI , Koster AJ , Aerts JMFG
Traffic.2019 Mar 21 ; ():.
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β-Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor-targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol-derived activity-based probes with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre-labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the activity-based probe containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme. This article is protected by copyright. All rights reserved.
PMID: 30895685 [PubMed - as supplied by publisher]
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