Abstract
Fabry disease is a lysosomal storage disorder caused by loss of α-galactosidase function. More than 500 Fabry disease mutants have been identified, the majority of which are structurally destabilized. A therapeutic strategy under development for lysosomal storage diseases consists of using pharmacological chaperones to stabilize the structure of the mutant protein, thereby promoting lysosomal delivery over retrograde degradation. The substrate analog 1-deoxygalactonojirimycin (DGJ) has been shown to restore activity of mutant α-galactosidase and is currently in clinical trial for treatment of Fabry disease. However, only ∼65% of tested mutants respond to treatment in cultured patient fibroblasts, and the structural underpinnings of DGJ response remain poorly explained. Using computational modeling and cell culture experiments, we show that the DGJ response is negatively affected by protein aggregation of α-galactosidase mutants, revealing a qualitative difference between misfolding-associated and aggregation-associated loss of function. A scoring function combining predicted thermodynamic stability and intrinsic aggregation propensity of mutants captures well their aggregation behavior under overexpression in HeLa cells. Interestingly, the same classifier performs well on DGJ response data of patient-derived cultured lymphoblasts, showing that protein aggregation is an important determinant of chemical chaperone efficiency under endogenous expression levels as well. Our observations reinforce the idea that treatment of aggregation-associated loss of function observed for the more severe α-galactosidase mutants could be enhanced by combining pharmacological chaperone treatment with the suppression of mutant aggregation, e.g. via proteostatic regulator compounds that increase cellular chaperone expression.
Highlights
Deficiency in ␣-galactosidase activity leads to Fabry disease, for which treatment employing pharmacological chaperones is being developed
Size Exclusion Chromatography (SEC)—For the analysis of the ␣-Gal aggregation state by SEC, transfected HeLa cells were lysed in radioimmune precipitation assay (RIPA) buffer (1% octylphenoxypolyethoxyethanol (IGEPAL), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) (Pierce) supplemented with protease inhibitors (Roche Applied Science), centrifuged for 5 min at 3000 rpm, and 400 l of the supernatant was subsequently loaded onto a Superdex S200 HR10/30 column (GE Healthcare) equilibrated in hypotonic buffer (20 mM HEPES, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, pH 7.5)
The results showed that the mutants found in patients with the classic form of Fabry disease (FD) were spread through the aggregation spectrum and had high TANGO values, whereas the ones responsible for the variant form were mainly lying on the x axis
Summary
Deficiency in ␣-galactosidase activity leads to Fabry disease, for which treatment employing pharmacological chaperones is being developed. The same classifier performs well on DGJ response data of patient-derived cultured lymphoblasts, showing that protein aggregation is an important determinant of chemical chaperone efficiency under endogenous expression. By binding to its active site, DGJ chaperones unstable enzyme variants in the ER This interaction increases the thermodynamic stability of the mutant enzyme and rescues it from misfolding in the neutral environment of the endoplasmic reticulum, allowing mutant ␣-Gal to pass through ER quality control, Golgi maturation, and being sorted into the lysosomes. A recent study addressing DGJ response of 75 ␣-Gal missense mutations in cultured lymphoblasts of Fabry disease patients [13] revealed that overall about 65% of mutants showed improved ␣-Gal activity and reduced Gb3 levels. It suggests that the most severe Fabry disease mutants will benefit [23] but probably even require a therapeutic approach combining both pharmacological chaperones that stabilize the native fold and proteostatic regulators that can modulate the aggregation propensity of disease mutants in vivo [24]
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