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

Abstract

Enzyme 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 beta-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 beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.

Crystal Structure of β-Hexosaminidase B in Complex with Pyrimethamine, a Potential Pharmacological Chaperone

“…This has recently been demonstrated in late onset Tay-Sachs cells loaded with a fluorescent derivative of G M2 ganglioside. 10 Several molecules that act as inhibitors of HexA and HexB have been identified as potential pharmacological chaperones (PC) for late-onset G M2 gangliosidosis, including known inhibitors of Hex such as N-acetylglucosamine thiazoline (NGT). 11 Additionally, two libraries have been screened, the Maybridge library 12 (50 000 compounds) and the NINDs library 13 (1040 FDA approved compounds).…”

Pyrimethamine Derivatives: Insight into Binding Mechanism and Improved Enhancement of Mutant β-N-acetylhexosaminidase Activity

“…Removal of one (cf. 8,9) or both (cf. 11) amino groups from the pyrimidine ring resulted in a >10-fold increase in IC 50 values ( Table 1).…”

“…Cell feeding experiments using a fluorescent GM2 ganglioside analogue were used to directly measure intralysosomal hydrolysis of GM2 ganglioside in live ATSD patient cells (i.e., an in cellulo assay). 8 Fibroblasts from an ATSD patient a Hex isozymes were purified from human placenta. b The Hex A isozyme, wild-type (WT), or mutant (G269S) was isolated from lysates of normal or ATSD fibroblasts, respectively, by ion exchange chromatography.…”

“…Patient cells treated with 5h (33 μg/mL) demonstrated a greater intracellular hydrolysis of the fluorescent derivative of GM2 compared to cells treated with PYR (11 μg/mL), as indicated by the higher levels of the band corresponding to GM3. Quantification of the intensity of all bands corresponding to the downstream products of GM3 hydrolysis, lactosylceramide (LacCer) and glucosylceramide (GlcCer) (further hydrolysis was inhibited by the inclusion of an irreversible inhibitor of glucocerebrosidase in the cell feeding assay 8 ), in both phases clearly shows increased levels of the products, indicative of increased lysosomal Hex A activity in response to treatment with either compound. However, 5h treatment resulted in an additional ∼1.5-fold increase in GM2 hydrolysis in comparison to that of PYR-treated cells ( Figure 6B).…”

“…8 However, the chaperoning efficacy of PYR is mutation dependent. 2 The structure of Hex B in complex with PYR has only recently been elucidated.…”