The iminosugar isofagomine increases the activity of N370S mutant acid beta-glucosidase in Gaucher fibroblasts by several mechanisms

Abstract

Gaucher disease is a lysosomal storage disorder caused by deficiency in lysosomal acid beta-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. One of the most prevalent disease-causing mutations, N370S, results in an enzyme with lower catalytic activity and impaired exit from the endoplasmic reticulum. Here, we report that the iminosugar isofagomine (IFG), an active-site inhibitor, increases GlcCerase activity 3.0 +/- 0.6-fold in N370S fibroblasts by several mechanisms. A major effect of IFG is to facilitate the folding and transport of newly synthesized GlcCerase in the endoplasmic reticulum, thereby increasing the lysosomal pool of the enzyme. In addition, N370S GlcCerase synthesized in the presence of IFG exhibits a shift in pH optimum from 6.4 to 5.2 and altered sensitivity to SDS. Although IFG fully inhibits GlcCerase in the lysosome in an in situ assay, washout of the drug leads to partial recovery of GlcCerase activity within 4 h and full recovery by 24 h. These findings provide support for the possible use of active-site inhibitors in the treatment of some forms of Gaucher disease.

Fig. 1.

Effect of IFG on N370S GlcCerase biosynthesis and trafficking. (A) Molecular structure of IFG. (B) Cells were cultured for 5 days with or without 30 μM IFG before labeling with 750 μCi of [³⁵S]methionine/[³⁵S]cysteine for 1 h. After labeling medium was replaced with normal growth medium, cells were harvested at the indicated times, and the GlcCerase was immunoprecipitated and analyzed by SDS/PAGE and autoradiography, as described in Methods. (C) Cells cultured in the absence of IFG were labeled with 750 μCi of [³⁵S]methionine/[³⁵S]cysteine for 1 h. After the labeling medium was replaced with normal growth medium, the cells were chased for various times. In lanes 5–7, IFG was added to a final concentration of 30 μM at the chase times indicated. Newly synthesized GlcCerase molecules were immunoprecipitated and treated with (lanes 2 and 8) or without peptide:N-glycosidase F (PNGase F) (lanes 1, 3–7) to remove all N-glycans before SDS/PAGE and autoradiography. The asterisks in B and C denote nonspecific bands.

Fig. 2.

IFG increases the lysosomal pool of N370S GlcCerase. (A) Wild-type and N370S fibroblast cultures were treated with or without 100 μM IFG for 5 days followed by fractionation of homogenized lysates on Percoll gradients. Fractions were collected, and β-N-acetylhexosaminidase activity was measured. The percent of total activity in each fraction is plotted. (B) Fractions were combined into three pools and subjected to SDS/PAGE and Western blot analysis. GCase, GlcCerase.

Fig. 3.

Effect of IFG on the relative specific activity of N370S GlcCerase (GCase). (A) Representative Western blot. (B) Cells were grown for 5 days with or without 30 μM IFG. Lysates were prepared, and equivalent protein aliquots were subjected to SDS/PAGE and Western blot analysis (see Methods). GlcCerase levels were obtained by densitometry analysis, and the mean amount of GlcCerase ± SD is plotted relative to wild-type levels (n = 8). (C) GlcCerase activity in lysates from treated and untreated N370S fibroblasts was measured in triplicate. The mean activity ± SD is plotted relative to the GlcCerase activity in wild-type cells (n = 8). Activity measurements were normalized to total protein.

Fig. 4.

The pH optimum of GlcCerase activity from IFG-treated N370S cells is altered. Wild-type and N370S fibroblasts were cultured for 5 days with or without 30 μM IFG. Before harvesting, IFG was washed out of treated cells as described in Methods. Cell lysates were prepared at various pH values, and activity was measured in triplicate. The bar graphs represent the mean of four (wild-type) or seven (N370S) experiments with the activity at pH 5.2 set at 1.0.

Fig. 5.

GlcCerase (GCase) inhibition by IFG recovers after drug washout. Cells were cultured for 5 days with 30 or 100 μM IFG before analysis. In situ GlcCerase activity was assessed in triplicate at various washout times. The mean normalized N370S GlcCerase activity ± SD at each washout time is shown. Time constants for exponential fits to the data are indicated in parentheses.