title: Ultrasensitive in situ visualization of active glucocerebrosidase molecules.
abstract: Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Witte
authors.forename: Wouter W
authors.initials: WW
authors.lastname: Kallemeijn
authors.forename: Jan
authors.initials: J
authors.lastname: Aten
authors.forename: Kah-Yee
authors.initials: KY
authors.lastname: Li
authors.forename: Anneke
authors.initials: A
authors.lastname: Strijland
authors.forename: Wilma E
authors.initials: WE
authors.lastname: Donker-Koopman
authors.forename: Adrianus M C H
authors.initials: AM
authors.lastname: van den Nieuwendijk
authors.forename: Boris
authors.initials: B
authors.lastname: Bleijlevens
authors.forename: Gertjan
authors.initials: G
authors.lastname: Kramer
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Florea
authors.forename: Berend
authors.initials: B
authors.lastname: Hooibrink
authors.forename: Carla E M
authors.initials: CE
authors.lastname: Hollak
authors.forename: Roelof
authors.initials: R
authors.lastname: Ottenhoff
authors.forename: Rolf G
authors.initials: RG
authors.lastname: Boot
authors.forename: Gijsbert A
authors.initials: GA
authors.lastname: van der Marel
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
authors.forename: Johannes M F G
authors.initials: JM
authors.lastname: Aerts
mesh_terms: D000818:Animals; D001896:Boron Compounds / Q000737:chemistry; D002478:Cells, Cultured; D003511:Cyclohexanols / Q000737:chemistry; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D005776:Gaucher Disease / Q000378:metabolism; D005962:Glucosylceramidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D050112:Imino Pyranoses / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D003511:Cyclohexanols; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D050112:Imino Pyranoses; D018832:Molecular Chaperones; C098432:isofagomine; C063285:cyclophellitol; D005962:Glucosylceramidase