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

β-Galactosidosis is a group of disorder based on heterogeneous mutations of GLB1 gene coding for the lysosomal acid β-galactosidase (β-gal). A decrease of the β-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-β-valienamine (NOEV), a chemical chaperone compound, stabilized various mutant human β-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 β-gal. This effect is mutation specific. In this study, we have established a sensitive luciferase-based assay for measuring chaperone effect on mutant human β-gal. A dinoflagellate luciferase (Dluc) cDNA was introduced to the C-terminus of human β-gal. When COS7 cells expressing the Dluc-tagged human R201C β-gal was treated with NOEV, there happened a remarkable increase of the mutant β-gal activity. In the presence of NH(4)Cl, 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 β-gal mutants, and that it will be a useful tool for finding novel chaperone compounds in the future study.