Chemical Chaperones and Permissive Temperatures Alter the Cellular Localization of Gaucher Disease Associated Glucocerebrosidase Variants

Abstract

Point mutations in the lysosomal hydrolase, glucocerebrosidase (GC), can cause Gaucher disease, a common lysosomal storage disease. Several clinically important GC mutations impede folding in the endoplasmic reticulum (ER) and target these enzymes for ER-associated degradation (ERAD). The removal of these misfolded proteins decreases the lysosomal concentration of GC, which results in glucosylceramide accumulation. The most common GC variant, N370S, and other clinically relevant variants, G202R and L444P, exhibit different cellular localization patterns in patient-derived fibroblasts. We show that these distributions can be altered by manipulation of the ER folding environment, either by chemical chaperones or by temperature shifts. N370S, L444P, and G202R GC are destabilized in the neutral pH environment of the ER, rendering them prone to ERAD. Fibroblasts harboring the G202R and L444P GC mutations grown at 30 degrees C localize the mutant proteins to the lysosome, and this increases total GC activity. Both of these temperature-sensitive mutants appear to be stable at 37 degrees C once they are trafficked to the low pH environment of the lysosome. Chemical chaperones correct the ER instability and significant ER retention of G202R GC. N370S is also destabilized under ER simulating conditions, a deficiency that is corrected by chemical chaperone binding. These data clearly show manipulating the ER environment with chemical chaperones increases the lysosomal concentration of partially active GC variants and suggest that small molecules could be used to treat Gaucher disease.

X-ray and Biochemical Analysis of N370S Mutant Human Acid β-Glucosidase

“…Our results showing that the effect on imiglucerase and N370S GCase is comparable in magnitude to that observed using patient cells suggest that the major (but not necessarily the only) mechanism by which competitive inhibitors increase lysosomal levels of GCase is by reducing degradation within the lysosome rather than an effect on protein folding and trafficking. A reduction in lysosomal degradation in the presence of unaltered transport to the lysosome is consistent with many of the reports demonstrating an increased ratio of lysosomal to nonlysosomal protein levels in the presence of competitive inhibitors (18,21).…”

“…To evaluate this possibility, we investigated the effect of the competitive inhibitor NN-DNJ on the intracellular stability of purified N370S and imiglucerase in macrophages. It is interesting that the 2-3-fold increase in GCase levels we observed after 24 h in the presence of NN-DNJ is comparable with the increase originally reported using Gaucher patient fibroblasts (20) as well as subsequent studies in patient fibroblasts using several different competitive inhibitors (17,21), suggesting that the mechanism(s) of action is similar in all cases. In our experiments, both proteins were already folded and targeted to lysosome via the mannose receptor, so an effect on protein folding or transport from the ER to lysosome could be ruled out in this case (36).…”

“…Biochemical Characterization of N370S GCase Mutant-N370S GCase was produced in a baculovirus expression system, purified, and characterized as described previously (21). To measure the K m and V max values, ϳ2 g ml Ϫ1 of enzyme was incubated with 0.2, 0.4, 0.6, 1, 2, 4, 6, and 8 mM p-nitrophenyl-␤-D-glucopyranoside (Sigma) in 0.1 M potassium phosphate, 0.1% BSA, 0.125% sodium taurocholate, 0.162% Triton X-100, 0.02% sodium azide, pH 5.9, at 37°C for 15 min.…”

“…This is based on the observation that binding of competitive inhibitors can stabilize both normal and N370S GCase against thermal denaturation and in cell culture lead to an increase in lysosomal levels of the enzymes. This has resulted in the development of several competitive inhibitors as "chemical chaperones" with the goal of promoting folding of the mutant protein in the ER and facilitating transport to the lysosome where, under lysosomal pH conditions, the inhibitor dissociates and the enzyme regains activity (17,18,(21)(22)(23).…”

Glucocerebrosidase, a new player changing the old rules in Lewy body diseases

“…Some of the mutant GCase proteins are structurally unstable (Sawkar et al , 2006 ;Lieberman et al , 2007 ;Bendikov -Bar et al, 2011 ), and this may directly affect α -synuclein aggregation. In a study of brain samples from patients with PD and DLB, samples with a homozygous GBA1 mutation showed that > 80 % of the LBs were co-localized with GCase, and those with a heterozygous GBA1 mutation showed a 75 % GCase co-localization rate (range, 32 -90 % ) with LBs, compared to a mean of 4 % in patients with PD and DLB without a GBA1 mutation (Goker -Alpan et al, 2010 ).…”