Background: Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) resulting from deficient activity of acid ß-glucocerebrosidase (GBA). Identification of small molecule pharmacological chemical chaperone (PC) acting as agents for enzyme enhancement therapy (EET) is a valuable appr...
Background: Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) resulting from deficient activity of acid ß-glucocerebrosidase (GBA). Identification of small molecule pharmacological chemical chaperone (PC) acting as agents for enzyme enhancement therapy (EET) is a valuable approach for treatment of different forms of GD. Ambroxol (ABX) has the biochemical characteristics of a safe and effective EET agent for treatment of patients with the most common GD genotypes. Here, an evaluation of the effects of a PC ABX on GBA activities in vitro, in ten groups of cells transfected with missense mutations in the GBA gene, was conducted.
Methods: Six missense mutations, including one novel mutation, were cloned into mammalian expression vectors. Following transient expression in COS-7 cells using Effectene Transfection Reagent, analysis of GBA enzyme activity and protein expression was performed using a standard fluorometric method and Western blot analysis, respectively. COS-7 cells were cultured for two days in DMEM medium with 0, 5, 10, and 30 uM Ambroxol. In order to evaluate the effects of the PC ABX, analysis of GBA enzyme activity and protein expression in mutant cultured medium containing ABX was performed.
Results: Assessment of activities of GBA variants, including G46E, L444P, F213I, R257Q, R277C, N188S, G46E/F213I, G46E/L444P, G45E/R257Q, and N188S/R257Q, in COS-7 cells lines was performed before and after incubation with PC ABX. Compared to the wild type enzyme activity, most of the mutant enzymes showing the level of low residual activity corresponded to alleles N188S (59.18%), N188S/R257Q (53.46%), R277C (37.91%), F213I (33.6%), G46E/F213I (30.77%), G46E/L444P (25.14%), G46E (21.99%), G46E/R257Q (19.48%), and L444P (12.45%) under these assay conditions. However, no residual activity of the R257Q mutant was observed. Treatment with ABX resulted in enhancement of mutant L444P enzyme activity by 86.2% (1.9-fold) in the presence of 10 μM ABX. Enhancement of GBA enzyme activity by 82.9% (1.8-fold) was observed in the G46E in the presence of 30 μM ABX. In addition, significant enhancement of enzyme activities of N188S, G46E/R257Q, F213I, R277C, G46E/F213I, R257Q, and G46E/L444P was observed in the presence of 5-30 μM ABX: 54.7% (1.6-fold), 33.8% (1.3-fold), 32.3% (1.3-fold), 31.8% (1.3-fold), 26.3% (1.3-fold), 15.8% (1.2-fold), and 11.0% (1.1-fold), respectively. However, enzyme activity of double mutant N188S/R257Q did not show significant enhancement in the presence of ABX. In Western blotting, expression of the mature GBA protein showed a remarkable increase in the presence of 5-10 μM ABX in all mutants tested, except for the G46E/L444P and L444P mutants. However, most of the mature GBA proteins showed a significant decrease in the presence of 30 μM ABX.
Conclusions: This data suggest that the use of PC ABX resulted in more effective enhancement of enzyme activity in all GBA mutants, excepting the N188S/R257Q double mutant. Therefore, these mutants will be amenable to chemical chaperoning by ABX. This result will provide fundamental data and understanding of molecular characteristics of mutation and effect of PC on GBA mutants commonly identified in Korean patients with GD.
Background: Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) resulting from deficient activity of acid ß-glucocerebrosidase (GBA). Identification of small molecule pharmacological chemical chaperone (PC) acting as agents for enzyme enhancement therapy (EET) is a valuable approach for treatment of different forms of GD. Ambroxol (ABX) has the biochemical characteristics of a safe and effective EET agent for treatment of patients with the most common GD genotypes. Here, an evaluation of the effects of a PC ABX on GBA activities in vitro, in ten groups of cells transfected with missense mutations in the GBA gene, was conducted.
Methods: Six missense mutations, including one novel mutation, were cloned into mammalian expression vectors. Following transient expression in COS-7 cells using Effectene Transfection Reagent, analysis of GBA enzyme activity and protein expression was performed using a standard fluorometric method and Western blot analysis, respectively. COS-7 cells were cultured for two days in DMEM medium with 0, 5, 10, and 30 uM Ambroxol. In order to evaluate the effects of the PC ABX, analysis of GBA enzyme activity and protein expression in mutant cultured medium containing ABX was performed.
Results: Assessment of activities of GBA variants, including G46E, L444P, F213I, R257Q, R277C, N188S, G46E/F213I, G46E/L444P, G45E/R257Q, and N188S/R257Q, in COS-7 cells lines was performed before and after incubation with PC ABX. Compared to the wild type enzyme activity, most of the mutant enzymes showing the level of low residual activity corresponded to alleles N188S (59.18%), N188S/R257Q (53.46%), R277C (37.91%), F213I (33.6%), G46E/F213I (30.77%), G46E/L444P (25.14%), G46E (21.99%), G46E/R257Q (19.48%), and L444P (12.45%) under these assay conditions. However, no residual activity of the R257Q mutant was observed. Treatment with ABX resulted in enhancement of mutant L444P enzyme activity by 86.2% (1.9-fold) in the presence of 10 μM ABX. Enhancement of GBA enzyme activity by 82.9% (1.8-fold) was observed in the G46E in the presence of 30 μM ABX. In addition, significant enhancement of enzyme activities of N188S, G46E/R257Q, F213I, R277C, G46E/F213I, R257Q, and G46E/L444P was observed in the presence of 5-30 μM ABX: 54.7% (1.6-fold), 33.8% (1.3-fold), 32.3% (1.3-fold), 31.8% (1.3-fold), 26.3% (1.3-fold), 15.8% (1.2-fold), and 11.0% (1.1-fold), respectively. However, enzyme activity of double mutant N188S/R257Q did not show significant enhancement in the presence of ABX. In Western blotting, expression of the mature GBA protein showed a remarkable increase in the presence of 5-10 μM ABX in all mutants tested, except for the G46E/L444P and L444P mutants. However, most of the mature GBA proteins showed a significant decrease in the presence of 30 μM ABX.
Conclusions: This data suggest that the use of PC ABX resulted in more effective enhancement of enzyme activity in all GBA mutants, excepting the N188S/R257Q double mutant. Therefore, these mutants will be amenable to chemical chaperoning by ABX. This result will provide fundamental data and understanding of molecular characteristics of mutation and effect of PC on GBA mutants commonly identified in Korean patients with GD.
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