Effects of pH and iminosugar pharmacological chaperones on lysosomal glycosidase structure and stability

Abstract

Human lysosomal enzymes acid-beta-glucosidase (GCase) and acid-alpha-galactosidase (alpha-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and alpha-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using alpha-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of alpha-Gal A with DGJ. Both GCase and alpha-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in alpha-Gal A are not seen. Thermodynamic parameters obtained from alpha-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and alpha-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological chaperones for lysosomal storage disorders.

Fig. 1

Cartoon representation of (a) α-Gal A with DGJ (this work) (b) GCase with IFG (PDB code 2NSX). DGJ and IFG are pictured as ball-and-stick, and their chemical structures are depicted below their respective cartoons. (c) Activity profile of α-Gal A as a function of pH and (d) activity profile of GCase as a function of pH.

Fig. 2

Structures of apo GCase grown under different conditions. Overlay of two monomers in asymmetric unit exhibiting distinct loop 1 structures (α-helical or extended). (A) Left C2, pH 5.5; middle C3, pH 4.5; right C4, pH 4.5 (soaked). Ribbon diagrams are colored using a rainbow corresponding to increasing thermal factor from blue (low B-factor) to red (high B-factor). (B) Superposition of structures in (A)-(C) in loop 1, loop 2 region. Red: α-helical loop 1, blue: extended loop 1conformation.

Fig. 3

GCase structures at various pHs and in complex with IFG. (A) Overlay of GCase structure with IFG at pH 4.5 (2NSX, green) and pH 7.5 (C1, this work, blue). (B) Overlay of IFG-bound C1 (blue) with with apo GCase at pH 5.5 (C2, beige) exhibiting α-helical loop 1. (C) Superposition of active site residues from overlay shown in (A). Fo-Fc difference density from initial rigid body refinement is contoured at 3 •. (D) Schematic diagram of hydrogen bonding interactions involved in stabilizing IFG in the active site of C1. Distances are in Å. (E) Active-site stereo view of glycerol-bound monomer of C1 superimposed with corresponding monomer from apo pH 7.5 (2NT1). (F) Active-site stereo view of IFG-bound monomer of C1 superimposed with corresponding monomer from apo GCase pH 7.5 (2NT1). For (E)-(F), the bound ligand, nucleophilic (Glu 340) and basic (Glu 235) residues, and two key loop 1 residues (Tyr 313, Asp 315) are shown in ball-and-stick representation.

Fig. 4

α-Gal A structures. (A) Superposition of apo α-Gal A (beige), galactose-bound α-Gal A (silver), and DGJ-bound α-Gal A (purple). (B) Active site region of DGJ-bound α-Gal A. Fo-Fc difference density immediately following original molecular replacement solution is contoured at 2.5 •. (C) Active site region of galactose-bound α-Gal A. Fo-Fc difference density immediately following original molecular replacement solution at 3 •. (D) Schematic diagram of hydrogen bonding interactions involved in stabilizing galactose in the active site of DGJ.

Fig. 5

Differential scanning calorimetry. (A) Thermal denaturation of GCase upon addition of IFG at pH 7.4 (red) and pH 5.2 (black). (B) Thermal denaturation for α-Gal A upon addition of DGJ at pH 7.4 (red), and pH 5.2 (black).