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

TitleEffects of pH and iminosugar pharmacological chaperones on lysosomal glycosidase structure and stability.
Publication TypeJournal Article
Year of Publication2009
AuthorsLieberman RL, J D'Aquino A, Ringe D, Petsko GA
Date Published2009 Jun 9
Keywordsalpha-Galactosidase, beta-Glucosidase, Crystallography, X-Ray, Drug Delivery Systems, Enzyme Activation, Enzyme Stability, Humans, Hydrogen-Ion Concentration, Imino Pyranoses, Lysosomes, Molecular Chaperones, Protein Binding, Recombinant Proteins, Thermodynamics

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.

Alternate JournalBiochemistry
PubMed ID19374450
PubMed Central IDPMC2699628
Grant ListF32 AG027647 / AG / NIA NIH HHS / United States
F32 AG027647-01 / AG / NIA NIH HHS / United States
F32 AG027647-02 / AG / NIA NIH HHS / United States
F32 AG027647-03 / AG / NIA NIH HHS / United States
F32AG027647 / AG / NIA NIH HHS / United States
GM68762 / GM / NIGMS NIH HHS / United States
R37 GM032415-29 / GM / NIGMS NIH HHS / United States
RR007707 / RR / NCRR NIH HHS / United States
Y1-CO-1020 / CO / NCI NIH HHS / United States
Y1-GM-1104 / GM / NIGMS NIH HHS / United States