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.
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Acknowledgements
Funding from The Netherlands Organization for Scientific Research (NWO-CW, to M.D.W., W.W.K., R.G.B., G.v.d.M., H.S.O. and J.M.F.G.A.) and The Netherlands Proteomics Centre (to B.I.F. and H.S.O.) is acknowledged.
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M.D.W. and W.W.K. designed the experiments. M.D.W., K.-Y.L. and A.M.C.H.v.d.N. conducted the synthesis, and W.W.K. conducted the kinetic experiments and the cell assays. J.A. conducted the microscopic analysis. A.S. conducted the in situ assays. W.E.D.-K. conducted the in vitro assays. B.B. conducted the structural modeling. G.K. and B.I.F. conducted the proteomic experiments. B.H. conducted the FACS analysis. C.E.M.H. conducted the tissue culture. R.O. conducted the animal experiments. R.G.B. supervised the cell assays. G.v.d.M. supervised the synthesis. H.S.O. and J.M.F.G.A. conceived of the idea and supervised the project.
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Supplementary Figures 1–14, Supplementary Methods and Supplementary Results (PDF 10099 kb)
Supplementary Video
Time-lapse microscopy of GBA labeling in intact fibroblasts. Cells incubated with 5 nM MDW941 6 were imaged every 5 minutes for 2 hours simultaneously using fluorescence microscopy and phase-contrast bright field microscopy. (AVI 180003 kb)
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Witte, M., Kallemeijn, W., Aten, J. et al. Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat Chem Biol 6, 907–913 (2010). https://doi.org/10.1038/nchembio.466
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DOI: https://doi.org/10.1038/nchembio.466
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