Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glycosyl ceramide in lysosomes and the development of the lysosomal storage disease known as Gaucher’s disease.  More recently, mutations in the GBA1 gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinson’s disease (PD).  Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of live cells.  Indeed, selectively visualizing the activity of endogenous levels of any intracellular glycoside hydrolases, including GCase, has proven problematic in live mammalian cells.  Here we describe the modular design and synthesis of fluorescence-quenched substrates for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched fluorescent substrates permit assessment of GCase inhibition in live cells by either confocal microscopy or high content imaging. Such substrates should enable improved understanding of GCase in situ and optimization of molecular chaperones for this enzyme. These findings also suggest routes to generate fluorescence-quenched substrates for other mammalian glycoside hydrolases. Recent advances on related studies will also be discussed.

Fluorescence-quenched substrates for live cell imaging of endogenous human glucocerebrosidase activity

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.