A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates

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DOIResolve DOI: http://doi.org/10.1093/glycob/cwp183
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TypeArticle
Journal titleGlycobiology
Volume20
Issue3
Pages356365; # of pages: 10
SubjectAB-variant GM2 gangliosidosis; enzyme; enhancement therapy; Pyrimethamine; Tay–Sachs disease
AbstractEnzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the ?-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis–Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay–Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant ?-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.
Publication date
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedYes
NPARC number17401014
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Record identifier533e32fe-1b2e-4014-9960-69becf8a54ed
Record created2011-03-25
Record modified2016-05-10
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