Fig 1.
The active site and the allosteric binding site in human lysosomal alpha-galactosidase.
The structure of lysosomal alpha-galactosidase is shown as a cartoon. In Panel A the active site (GLA site) is pinpointed by a blue sphere while the allosteric binding site is pinpointed by a violet sphere. Residues involved in the 2,6-dithiopurine allosteric and alpha-galactose binding site respectively are shown as sticks in Panel B or C respectively along with docked DTP. Predicted hydrogen bonds between DTP and AGAL within the allosteric binding site are shown as yellow dashed lines in Panel B. Best ranked pose of DTP in the alpha-galactose binding site area is shown in Panel C.
Fig 2.
Energetic contributions to the binding of 2–6 dithiopurine to the allosteric site and the active site of human lysosomal alpha-galctosidase.
Depicted energetic contributions are: van der Waals interactions (dark blue), volume-based free energy of solvation (green), hydrogen bonds (red), electrostatic energy (cyan), internal energy (pink), metal interactions (light yellow), energy of entropic losses associated with ligand’s rotatable bonds (dark yellow), dihedral energy (light blue), internal energy penalty (light green) and total predicted binding affinity (black). A) Decomposition for the allosteric site, and B), decomposition for the active site.
Fig 3.
2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay.
Panel A. Fabrazyme® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.
Fig 4.
2–6 dithiopurine protects human alpha-galactosidase against urea-induced unfolding.
Fabrazyme® was incubated in the presence of urea (from 0 to 5 M), with (B) or without (A) DTP 6 mM, at 10°C over night, then each sample was treated for 1 min at 37°C with thermolysin (1:5 protease to protein ratio). Folded undigested proteins were visualized by SDS-PAGE. The intensity of the bands was quantified and the relative intensity of major band of Fabrazyme® and thermolysin in each lane was plotted against urea concentration (C).
Fig 5.
2–6 dithiopurine rescues mutant human alpha-galactosidase in monotherapy or in synergy.
COS-7 cells were cultured in conventional medium and transfected with plasmid containing A230T mutant. Cells were treated with: DTP 6 mM, DGJ 1 microM, DGJ 20 microM, ambroxol 40 microM, ambroxol 40 microM plus DTP 6 mM, galactose 100 mM, galactose 100 mM plus DTP 6 mM, DGJ 1microM plus DTP 6 mM. All the molecules were dissolved in DMSO and an appropriate control was realized. After 48 h incubation, the cells were scraped and lysed then water-soluble extracts were analysed by western blotting (A and B) and enzyme assay (C). The effect of DTP 2 mM is shown in D and E. Cell extracts were treated with N-Glycosidase F and then analysed by WB (F). Standard deviations is indicated by bars and differences that are statistically significant are flagged by * when compared to the control with DMSO (p < 0.05:*; p < 0.01: **; p < 0.001:***), are flagged by § when compared to DTP (p < 0.01:§§; p < 0.001:§§§).
Fig 6.
2–6 dithiopurine promotes processing of some mutant human alpha-galactosidase.
COS-7 cells were cultured in conventional medium and transfected with plasmid containing C56Y, C63Y and E341D mutants. Cells were treated with: DTP 6 mM, DGJ 1 microM, DGJ 1 microM plus DTP 6 mM. All the molecules were dissolved in DMSO and an appropriate control was realized. After 48 h incubation, the cells were scraped and lysed then water-soluble extracts were analysed by western blotting (A, B and C) and enzyme assay (D). Standard deviations are indicated by bars.
Fig 7.
2–6 dithiopurine has no effect on mutations affecting the allosteric site.
COS-7 cells were cultured in conventional medium and transfected with plasmid containing A37T, P40S, M42T, M42V mutants. Cells were treated with DTP 6 mM dissolved in DMSO and an appropriate control was realized. After 48 h incubation, the cells were scraped and lysed then water-soluble extracts were analysed by western blotting (A and B) and enzyme assay (C). Standard deviations are indicated by bars.