TY - JOUR
T1 - Ultrasensitive in situ visualization of active glucocerebrosidase molecules
AU - Witte, Martin D
AU - Kallemeijn, Wouter W
AU - Aten, Jan
AU - Li, Kah-Yee
AU - Strijland, Anneke
AU - Donker-Koopman, Wilma E
AU - van den Nieuwendijk, Adrianus M C H
AU - Bleijlevens, Boris
AU - Kramer, Gertjan
AU - Florea, Bogdan I
AU - Hooibrink, Berend
AU - Hollak, Carla E M
AU - Ottenhoff, Roelof
AU - Boot, Rolf G
AU - van der Marel, Gijsbert A
AU - Overkleeft, Herman S
AU - Aerts, Johannes M F G
PY - 2010/12
Y1 - 2010/12
N2 - 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.
AB - 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.
KW - Animals
KW - Boron Compounds/chemistry
KW - Cells, Cultured
KW - Cyclohexanols/chemistry
KW - Drug Design
KW - Electrophoresis, Polyacrylamide Gel
KW - Enzyme Inhibitors/pharmacology
KW - Enzyme-Linked Immunosorbent Assay
KW - Fibroblasts/chemistry
KW - Flow Cytometry
KW - Fluorescent Dyes/chemistry
KW - Gaucher Disease/metabolism
KW - Glucosylceramidase/antagonists & inhibitors
KW - Imino Pyranoses/pharmacology
KW - Mice
KW - Microscopy, Fluorescence
KW - Molecular Chaperones/metabolism
U2 - https://doi.org/10.1038/nchembio.466
DO - https://doi.org/10.1038/nchembio.466
M3 - Article
C2 - 21079602
SN - 1552-4450
VL - 6
SP - 907
EP - 913
JO - Nature chemical biology
JF - Nature chemical biology
IS - 12
ER -