The molecular basis of pharmacological chaperoning in human α-galactosidase

Abstract

Fabry disease patients show a deficiency in the activity of the lysosomal enzyme α-galactosidase (α-GAL or α-Gal A). One proposed treatment for Fabry disease is pharmacological chaperone therapy, where a small molecule stabilizes the α-GAL protein, leading to increased enzymatic activity. Using enzyme kinetics, tryptophan fluorescence, circular dichroism, and proteolysis assays, we show that the pharmacological chaperones 1-deoxygalactonojirimycin (DGJ) and galactose stabilize the human α-GAL glycoprotein. Crystal structures of complexes of α-GAL and chaperones explain the molecular basis for the higher potency of DGJ over galactose. Using site-directed mutagenesis, we show the higher potency of DGJ results from an ionic interaction with D170. We propose that protonation of D170 in acidic conditions leads to weaker binding of DGJ. The results establish a biochemical basis for pharmacological chaperone therapy applicable to other protein misfolding diseases.

Figure 1

Pharmacological chaperones slow the unfolding kinetics of α-GAL (measured by Trp fluorescence). Rate of unfolding of α-GAL at pH 6.5 (a and b) and pH 4.5 (c and d) in the absence (open symbols) and presence (filled symbols) of 50 μM DGJ (a and c) and 50 mM galactose (b and d). See also Figure S2.

Figure 2. Increased apparent melting temperature T_m(app) of α-GAL (monitored by CD)

DGJ (a, c, e) and galactose (b, d, f) were tested at pH 7.2 (a, b), pH 6.5 (c, d), and pH 4.5 (e, f) in the absence (white) and presence of 50 μM (red) or 50 mM (blue) DGJ or galactose. In (c) and (e), the D170A mutant is also shown in the absence (black) and presence (green) of 1.4 or 2 mM DGJ. The D170A mutant does not respond to even 30- or 40- fold higher concentrations of DGJ.

Figure 3. DGJ and galactose confer protease resistance upon α-GAL

Thermolysin (a) and pepsin (b) digestion of wild type α-GAL (open symbols) and D170A α-GAL (filled symbols) in urea after incubation with DGJ (black) and galactose (grey) respectively. Wild type and D170A α-GAL band intensities were quantitated at multiple chaperone concentrations. The D170A mutant responds only to high concentrations of chaperone. See also Figures S3 and S4, and Table S2.

Figure 4. Crystal structures of human α-GAL bound to pharmacological chaperones

(a) and (b) show σ_A-weighted 2Fo-Fc electron density maps of DGJ- and galactose-soaked crystals of wild-type human α-GAL, and (c) shows the D170A mutant α-GAL with DGJ bound. All maps are contoured at 1.8σ with the ligand density colored red for clarity. (d) shows a superposition of the (a) and (b), highlighting the key interaction between the ligand and the D170 carboxylate. See also Table S3.