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Localization of active endogenous and exogenous β‐glucocerebrosidase by correlative light‐electron microscopy in human fibroblasts
Author(s) -
van Meel Eline,
Bos Erik,
van der Lienden Martijn J. C.,
Overkleeft Herman S.,
van Kasteren Sander I.,
Koster Abraham J.,
Aerts Johannes M. F. G.
Publication year - 2019
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/tra.12641
Subject(s) - biology , recombinant dna , glucocerebrosidase , enzyme , biochemistry , endogeny , fluorophore , lysosome , fluorescence microscope , mannose receptor , lysosomal storage disease , mannose , cytochemistry , receptor , microbiology and biotechnology , fluorescence , in vitro , macrophage , physics , quantum mechanics , gene
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 (ABPs) 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 ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme.