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.

Figure 1

Specificity of NDB-GM2 hydrolysis by the Hex A/B isozymes in the presence or absence of rGM2AP. Following hydrolysis of NBD-GM2, substrate and products were resolved by HPTLC. Fluorescent bands corresponding to NBD-GM2 (substrate) and NBD-GM3 (product) are labeled as GM2 and GM3, and were visualized by scanning of the plates using Molecular Devices Storm Imager. The far left lane labeled “Ctrl” is the substrate blank. The concentrations (μg/mL) of the Hex isozyme and the rGM2AP included in each 50 μL reaction mix are given below each sample lane.

Figure 2

MS/MS profile of the extracted bands from a HPTLC separation of the substrate (NBD-GM2) and product (NBD-GM3) generated by incubating overnight NBD-GM2 with Hex A and rGM2AP. In NBD-GM2 the fatty acid of the naturally occurring ganglioside has been replaced with a fluorescent short acyl chain NBD fatty acid, the C6-NBD group. In the presence of Hex A and rGM2AP, the terminal GlcNAc (dashed oval) is released resulting in the product NBD-GM3. The predicted and experimentally determined sizes of the derivatives are shown alongside the product ion spectra of the precursor ions 1393.6 (698.8⁻²) and 1189.6, which correspond to NBD-GM2 (top) and NBD-GM3 (bottom), respectively. The loss of the terminal sialic acid moiety, 290.1, from either compound result in peaks of 1102.5 NBD-GM2 (top) and 898.5 NBD-GM3 (bottom). The product ion spectrum of NBD-GM2 has been reconstructed in the form of singly charged ions. Both NBD-GM2 and NBD- asialo-GM2 were present as double charged ions in the original spectrum (698.8 and 550.3, respectively). To aid in comparison with NBD-GM3, the doubly charged peaks produced from NBD-GM2 are also shown reconstructed as singly charged ions.

Figure 3

Kinetic characterization of NBD-GM2 hydrolysis by Hex A with rGM2AP acting as a co-factor. A) Comparison of the hydrolysis rate of NBD-GM2 (pmoles NBD-GM3/U MUGS) when presented as simple micelles (open squares), in neutral (filled circle) or acidic (open upward triangles) liposomes, or acidic liposomes that had been extruded through a 100 nm filter (filled downward triangle). B) Michaelis-Menten plot of the hydrolysis rates of NBD-GM2 incorporated into acidic liposomes in the presence of increasing concentrations (μg/mL) of rGM2AP. C) Michaelis-Menten plot of the hydrolysis rates of increasing amounts of NBD-GM2 substrate (mM) contained in acidic liposomes in the presence of saturating levels of rGM2AP.

Figure 4

Differential diagnosis of fibroblast lysates from Normal and AB-variant patients using the NBD-GM2 substrate. Lysates from unaffected (Normal) and AB-variant patient cells in presence (+) or absence (−) of CBE were incubated with NBD-GM2 liposomes with (+) or without (−) rGM2AP. Arrows point to the positions of the step-wise products arising from the catabolic pathway of the substrate. The lane labeled “Ctrl” is a sample containing only substrate, CBE and rGM2AP (no cell lysate).

Figure 5

Validation of the specificity of an anti-human Hex A isozyme (α-subunit) obtained after absorption of a polyclonal rabbit anti-Hex IgG with purified Hex B. Fibroblasts from a patient with infantile TSD (ITSD, a–c), unaffected (WT, d–f) or ATSD (g–i) were incubated with the prepared Hex α-subunit specific rabbit IgG (a,d,g) and a mouse Lamp-1 IgG (b,e,h), followed by fluorescently labeled secondary antibodies against rabbit (green) or mouse (red) IgG. Merged images are shown in the right most panels with co-localization of the signals appearing as yellow-orange (c,f,i).

Figure 6

Increased colocalization of αG269S Hex A with lysosomal NBD-GM2 in Pyr-treated ATSD fibroblasts. Wild-type fibroblasts were treated with NBD-GM2 (WT, a) and Lysotracker (WT, b). The merged images for the two fluorescents molecules show virtually total overlap of the NBD-GM2 signal (green) with that of lysosomal marker (Lysotracker, red), as denoted by the yellow-orange punctate pattern (WT, c). Fibroblasts from mock-treated (ATSD; d–f) and PYR-treated (ATSD+PYR; g–i) ATSD patient were loaded with NBD-GM2 (visualized in green in d & g) and incubated with our Hex A-specific primary IgG (visualized in red in e & h). Merged images of the two fluorophores (f, i) show an increased degree of overlap of the two probes (denoted by the yellow-orange color) in the treated fibroblasts (ATSD+Pyr).

Figure 7

Hydrolysis of NBD-GM2 is increased in ATSD patient cells pre-treated with the PC Pyr. Differential Folch extraction was performed simultaneously on CBE and NBD-GM2 loaded ATSD fibroblasts treated with Pyr (P1-P3) or untreated (C1-C3). Partitioned NDB-ganglioside derivatives in the aqueous (upper) phase (A) and NBD-neutral glycolipid derivatives in the organic (lower) phase (B) were resolved by HPTLC (samples from each of the three replicate pairs are shown). Standards (S) consisting of GM2 and GM3 are included in the right most lane of each plate. Arrows denote the positions of the relevant ganglioside/glycolipid derivatives.