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A FRET Probe for Cell‐Based Imaging of Ganglioside‐Processing Enzyme Activity and High‐Throughput Screening
Author(s) -
Yang GuangYu,
Li Caishun,
Fischer Michael,
Cairo Christopher W.,
Feng Yan,
Withers Stephen G.
Publication year - 2015
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201411747
Subject(s) - glycosphingolipid , förster resonance energy transfer , enzyme , biochemistry , ganglioside , chemistry , high throughput screening , ceramide , cell , microtiter plate , sphingolipid , microbiology and biotechnology , biology , fluorescence , chromatography , apoptosis , physics , quantum mechanics
Gangliosides are important signaling molecules in the cell membrane and are processed by several enzymes. Deficiencies in these enzymes can cause human lysosomal storage diseases. Building an understanding of the pathways of glycosphingolipid catabolism requires methods for the analysis of these enzymatic activities A GM3‐derived FRET probe was synthesized chemoenzymatically for the detection and quantitation of a range of ganglioside‐degrading enzymes, both in cell lysates and in living cells. This is the first substrate that enables the ratiometric fluorogenic assay of sphingolipid ceramide N‐deacylase and endoglycoceramidase and can detect and localize neuraminidase activity in living cells. It is therefore a valuable tool for building a better understanding of membrane‐confined enzymology. It also enables the robust and reliable assay of ganglioside‐degrading enzymes in a microtiter plate, thus opening the door to screening for novel or engineered biocatalysts or for new inhibitors.