Chemical chaperones increase the cellular activity of N370S beta -glucosidase: a therapeutic strategy for Gaucher disease

Abstract

Gaucher disease is a lysosomal storage disorder caused by deficient lysosomal beta-glucosidase (beta-Glu) activity. A marked decrease in enzyme activity results in progressive accumulation of the substrate (glucosylceramide) in macrophages, leading to hepatosplenomegaly, anemia, skeletal lesions, and sometimes CNS involvement. Enzyme replacement therapy for Gaucher disease is costly and relatively ineffective for CNS involvement. Chemical chaperones have been shown to stabilize various proteins against misfolding, increasing proper trafficking from the endoplasmic reticulum. We report herein that the addition of subinhibitory concentrations (10 microM) of N-(n-nonyl)deoxynojirimycin (NN-DNJ) to a fibroblast culture medium for 9 days leads to a 2-fold increase in the activity of N370S beta-Glu, the most common mutation causing Gaucher disease. Moreover, the increased activity persists for at least 6 days after the withdrawal of the putative chaperone. The NN-DNJ chaperone also increases WT beta-Glu activity, but not that of L444P, a less prevalent Gaucher disease variant. Incubation of isolated soluble WT enzyme with NN-DNJ reveals that beta-Glu is stabilized against heat denaturation in a dose-dependent fashion. We propose that NN-DNJ chaperones beta-Glu folding at neutral pH, thus allowing the stabilized enzyme to transit from the endoplasmic reticulum to the Golgi, enabling proper trafficking to the lysosome. Clinical data suggest that a modest increase in beta-Glu activity may be sufficient to achieve a therapeutic effect.

Fig 1.

The influence of selected alkylated DNJs on β-Glu activity in N370S fibroblasts cultured for 5 days with butyl (⧫), octyl (▪), 7-oxadecyl (○), nonyl (▴), and dodecyl (×) DNJ. β-Glu activity was evaluated at pH 4.0 by using 4-methyllumbelliferyl β-d-glucoside as a substrate in intact cells. Enzyme activity is normalized to untreated cells, assigned a relative activity of 1. Mean values ± SD are shown for quadruplicate experiments.

Fig 2.

The dependence of time of incubation with NN-DNJ on the activity of N370S β-Glu in intact fibroblasts. (A) N370S fibroblasts were cultured for 1 (▴), 2 (□), or 4 (•) days with NN-DNJ. (B) N370S fibroblasts were pulsed with NN-DNJ for 4 days and then chased with fresh media for 0 (•), 4 (□), or 6 (▴) days. The intact cells were assayed at pH 4.0 by using 4-methyllumbelliferyl β-d-glucoside as a substrate. Enzyme activity is normalized to untreated cells, assigned a relative activity of 1. Mean values ± SD are shown for quadruplicate experiments.

Fig 3.

The effect of NN-DNJ on β-Glu activity in three different cell lines. NN-DNJ was added to the culture medium of N370S (•), WT (□), or L444P (▴) fibroblasts for 4 days. The intact cells were assayed at pH 4.0 by using 4-methyllumbelliferyl β-d-glucoside as a substrate. Enzyme activity is normalized to untreated cells, assigned a relative activity of 1. The actual activity of L444P β-Glu ≪ N370S β-Glu ≪ WT β-Glu. Numbers were measured in triplicate, and SDs were <10%.

Fig 4.

Stabilization of isolated β-Glu by NN-DNJ evaluated in vitro by using heat inactivation. Ceredase (alglucerace injection) aliquots were incubated with 0 μM (•), 50 μM (□), or 100 μM (▴) NN-DNJ at pH 7.4. The samples were heated at 48°C for the indicated amount of time and then assayed for activity at pH 5.0 with 0.1% Triton X-100 and 0.2% taurodeoxycholic acid. Enzyme activity is reported relative to unheated enzyme. Mean values ± SD are shown for duplicate experiments.