The effects of pH and iminosugar pharmacological chaperones on lysosomal glycosidase structure and stability.
Human lysosomal enzymes acid-beta-glucosidase (GCase) and acid-alpha-galactosidase (alpha-Gal A) hydrolyze, respectively, the sphingolipids glucosyl- and globotriaosyl- ceramide, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease. We have investigated the structure and stability of GCase and alpha-Gal A at the neutral-pH environment reflective of the endoplasmic reticulum and the 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, 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 pH 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 GCase/IFG complex stability 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.
PMID: 19374450 [PubMed – as supplied by publisher]
I had the same conclusions! 🙂
Oh, it would be so amazing if they came out with a chaperone therapy for Hannah!!! She takes the cerenzyme so well that this would be easy! Chaperone therapy stays in the system longer than a traditional enzyme therapy (so she may only need to go in like once a month) but with the added benefit of crossing the blood-brain barrier! Will they be starting a clinical trial soon?! I’ve got my fingers crossed that this could be a near-term treatment that preserves the brain and keeps Hannah-banana whole for when we find the cure! 🙂
Cristina’s last blog post..CT scan, swallow study and more questions