Ultrasensitive in situ visualization of active glucocerebrosidase molecules

Martin D Witte; Wouter W Kallemeijn; Jan Aten; Kah-Yee Li; Anneke Strijland; Wilma E Donker-Koopman; Adrianus M C H van den Nieuwendijk; Boris Bleijlevens; Gertjan Kramer; Bogdan I Florea; Berend Hooibrink; Carla E M Hollak; Roelof Ottenhoff; Rolf G Boot; Gijsbert A van der Marel; Herman S Overkleeft; Johannes M F G Aerts

doi:10.1038/nchembio.466

Ultrasensitive in situ visualization of active glucocerebrosidase molecules

Nat Chem Biol. 2010 Dec;6(12):907-13. doi: 10.1038/nchembio.466. Epub 2010 Oct 31.

Authors

Martin D Witte¹, Wouter W Kallemeijn, Jan Aten, Kah-Yee Li, Anneke Strijland, Wilma E Donker-Koopman, Adrianus M C H van den Nieuwendijk, Boris Bleijlevens, Gertjan Kramer, Bogdan I Florea, Berend Hooibrink, Carla E M Hollak, Roelof Ottenhoff, Rolf G Boot, Gijsbert A van der Marel, Herman S Overkleeft, Johannes M F G Aerts

Affiliation

¹ Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.

PMID: 21079602
DOI: 10.1038/nchembio.466

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.

MeSH terms

Animals
Boron Compounds / chemistry
Cells, Cultured
Cyclohexanols / chemistry
Drug Design
Electrophoresis, Polyacrylamide Gel
Enzyme Inhibitors / pharmacology
Enzyme-Linked Immunosorbent Assay
Fibroblasts / chemistry
Fibroblasts / metabolism
Flow Cytometry
Fluorescent Dyes / chemistry
Gaucher Disease / metabolism
Glucosylceramidase / antagonists & inhibitors
Glucosylceramidase / chemistry*
Glucosylceramidase / metabolism
Imino Pyranoses / pharmacology
Mice
Microscopy, Fluorescence
Molecular Chaperones / metabolism

Substances

4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
Boron Compounds
Cyclohexanols
Enzyme Inhibitors
Fluorescent Dyes
Imino Pyranoses
Molecular Chaperones
isofagomine
cyclophellitol
Glucosylceramidase

Associated data

PubChem-Substance/99376249
PubChem-Substance/99376250
PubChem-Substance/99376251
PubChem-Substance/99376252
PubChem-Substance/99376253
PubChem-Substance/99376254
PubChem-Substance/99376255
PubChem-Substance/99376256
PubChem-Substance/99376257
PubChem-Substance/99376258
PubChem-Substance/99376259
PubChem-Substance/99376260
PubChem-Substance/99376261
PubChem-Substance/99376262
PubChem-Substance/99376263