Chemical chaperone therapy: Luciferase assay for screening of β-galactosidase mutations Linjing Li a,b , Katsumi Higaki a, ⁎, Haruaki Ninomiya c , Zhuo Luan b , Masami Iida d , Seiichiro Ogawa e , Yoshiyuki Suzuki f , Kousaku Ohno b , Eiji Nanba a a Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, 86 Nishi-cho, Yonago, 683-8503, Japan b Division of Child Neurology, Tottori University Faculty of Medicine, 36-1 Nishi-cho, Yonago, 683-8504, Japan c Department of Biomedical Regulation, School of Health Science, Faculty of Medicine, 86 Nishi-cho, Yonago, 683-8503, Japan d Central Research Laboratories, Seikagaku Corporation, 3-1253, Tateno, Higashi-Yamato, Tokyo, 270-0021, Japan e Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan f International University of Health and Welfare Graduate School, 2600-1 Kita Kanemaru, Otawara, 324-8501, Japan abstract article info Article history: Received 12 August 2010 Accepted 12 August 2010 Available online 18 August 2010 Keywords: Chemical chaperone therapy Lysosomal storage disease Neurodegeneration G M1 -gangliosidosis β-galactosidase Luciferase 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. © 2010 Elsevier Inc. All rights reserved. 1. Introduction Lysosomal acid β-galactosidase (β-gal, EC 3.2.1.23), encoded by GLB1 (3p21.33) in humans, hydrolyzes the terminal β-galactosyl residues from ganglioside G M1 and related glycoconjugates such as oligosaccharides derived from glycoproteins and keratan sulfate in somatic cells [1]. Allelic mutations of the gene result in excessive storage of the substrates in various cells and tissues. Genetic β-galactosidase deficiency (or β-galactosidosis) is a group of disorders including two clinically distinct diseases: G M1 -gangliosidosis (OMIM 230500) and Morquio B (OMIM 253010). Until now, more than 130 GLB1 gene mutations are collected [1–3].G M1 -gangliosidosis is a generalized neurosomatic disease in children (infantile form, juvenile form), and rarely in adults (adult form). The abnormal storages of ganglioside G M1 , mucopolysaccharide keratan sulfate and glycoprotein-derived oligosaccharides are widely distributed in the central nervous system (CNS), skeletal system, and visceral organs. Morquio B disease (OMIM 253010) is a generalized skeletal dysplasia without neurological involvement. At present only symptomatic and supportive therapy is available for the brain damage in human G M1 -gangliosidosis patients. Different strategies have been explored to treat this disease including allogenic bone marrow transplantation [4], gene therapy [5] and substrate reduction therapy [6]. These approaches are promising but still far from clinical application. The enzyme replacement therapy conducted for Gaucher disease and other lysosomal storage diseases is not available at present for β-galactosidosis [7]. Based on the study on mutant α-galactosidase A in Fabry disease [8], we proposed chemical chaperone therapy for brain pathology in G M1 -gangliosidosis, using an in vitro enzyme inhibitor N-octyl-4-epi- β-valienamine (NOEV), a chemical compound newly produced by organic synthesis, as a potent stabilizer of mutant β-gal in somatic cells Molecular Genetics and Metabolism 101 (2010) 364–369 Abbreviations: β-gal, β-galactosidase; NOEV, N-octyl-4-epi-β-valienamine; Dluc, dinoflagellate luciferase; DMEM, Dulbecco's modified Eagle's medium; 4-MU, 4-methylumbelliferone. ⁎ Corresponding author. Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, 86 Nishi-cho, Yonago, 683-8503, Japan. Fax: +81 859 38 6470. E-mail address: kh4060@med.tottori-u.ac.jp (K. Higaki). 1096-7192/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2010.08.012 Contents lists available at ScienceDirect Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme