Chemical chaperone therapy: Luciferase assay for screening of β-galactosidase mutations

beta-Galactosidosis is a group of disorder based on heterogeneous mutations of GLB1 gene coding for the lysosomal acid beta-galactosidase (beta-gal). A decrease of the beta-gal enzyme activity results in progressive accumulation of substrates in somatic cells, particularly in neurons, leading to severe neuronal dysfunction. We have previously reported that N-octyl-4-epi-beta-valienamine (NOEV), a chemical chaperone compound, stabilized various mutant human beta-gal proteins and increased residual enzyme activities in cultured fibroblasts from human patients. These data proved a potential therapeutic benefit of chemical chaperone therapy for patients with missense beta-gal. This effect is mutation specific. In this study, we have established a sensitive luciferase-based assay for measuring chaperone effect on mutant human beta-gal. A dinoflagellate luciferase (Dluc) cDNA was introduced to the C-terminus of human beta-gal. When COS7 cells expressing the Dluc-tagged human R201C beta-gal was treated with NOEV, there happened a remarkable increase of the mutant beta-gal activity. In the presence of NH4Cl, luciferase level in the medium increased in parallel with the enzyme activity in cell lysates. We also found that proteasome inhibitors enhance chaperone effect of NOEV. These results demonstrate that the luciferase-based assay is a reliable and convenient method for screening and evaluation of chaperone effects on human beta-gal mutants, and that it will be a useful tool for finding novel chaperone compounds in the future study. (C) 2010 Elsevier Inc. All rights reserved.