Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase

Abstract

Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients.

Fig. 1

Effect of ambroxol on GCase activity. Skin fibroblasts from normal and five GD patients (type of disease appears in parenthesis) were treated with increasing concentrations of ambroxol for 20 h at 37 °C. Samples containing 40 μg of protein were tested for GCase activity using 1.5 mM of the artificial substrate 4-MUG. Data are expressed as fold increase of GCase activity in the presence of ambroxol in comparison to untreated cells. The results represent the mean ± SEM, of three to five independent experiments.

Fig. 2

Effect of Ambroxol on total GCase amount and the lysosomal fraction of GCase. Skin fibroblasts from an unaffected individual (A) and five GD patients (B–F) were treated with increasing concentrations of ambroxol for 20 h at 37 °C. Samples containing 100 μg of protein, were subjected to Endo-H digestion overnight, electrophoresed through SDS-PAGE and the corresponding blot was interacted with anti GCase and anti erk antibodies. To normalize the results the blots were scanned and the intensity of each band was measured. To determine the amount of total GCase, the intensity of the GCase band at each lane was divided by the intensity of the erk at the same lane. The value obtained for the normal untreated cells was considered as 100%. To determine the Endo-H resistant fraction, the intensity of GCase resistant fraction was divided by the intensity of the entire amount of GCase in the same lane. The amount of GCase in lysosomes was calculated by multiplying the Endo-H resistant fraction with the total GCase in the same lane (compared to normal, which was considered 100), divided by 100. The results represent the mean of three to five independent experiments.