Ultrasensitive in situ visualization of active glucocerebrosidase molecules

TitleUltrasensitive in situ visualization of active glucocerebrosidase molecules
Publication TypeJournal Article
Year of Publication2010
AuthorsWitte, M.D., W.W. Kallemeijn, J. Aten, K.Y. Li, A. Strijland, W.E. Donker-Koopman, A.M.C.H. van den Nieuwendijk, B. Bleijlevens, G. Kramer, B.I. Florea, B. Hooibrink, C.E.M. Hollak, R. Ottenhoff, R.G. Boot, G.A. van der Marel, H.S. Overkleeft, J.M.F.G. Aerts
JournalNature Chemical Biology
Date PublishedDec
ISBN Number1552-4450
Accession NumberISI:000284214700020
Keywordsacid beta-glucosidase, biosynthesis, chaperones increase, enzyme, fibroblasts, inhibitors, isofagomine increases, n-butyldeoxynojirimycin, replacement therapy, type-1 gaucher-disease

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.


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