n-octyl-beta-valienamine and Mucopolysaccharidosis-IV

GM1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal β-galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human β-Gal(WT) and two representative mutations in GM1 gangliosidosis Japanese patients, β-Gal(R201C) and β-Gal(I51T). We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of β-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.

Topics: 1-Deoxynojirimycin; beta-Galactosidase; Catalytic Domain; Crystallography, X-Ray; Cyclohexenes; Enzyme Inhibitors; Enzyme Stability; Gangliosidosis, GM1; Hexosamines; Humans; Hydrogen Bonding; Hydrogen-Ion Concentration; Imino Sugars; Inositol; Kinetics; Models, Molecular; Molecular Structure; Mucopolysaccharidosis IV; Mutation; Protein Structure, Tertiary; Static Electricity; Structure-Activity Relationship

β-Galactosidase deficiency is a group of lysosomal lipid storage disorders with an autosomal recessive trait. It causes two clinically different diseases, G(M1) -gangliosidosis and Morquio B disease. It is caused by heterogeneous mutations in the GLB1 gene coding for the lysosomal acid β-galactosidase. We have previously reported the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on mutant β-galactosidase proteins. In this study, we performed genotype analyses of patients with β-galactosidase deficiency and identified 46 mutation alleles including 9 novel mutations. We then examined the NOEV effect on mutant β-galactosidase proteins by using six strains of patient-derived skin fibroblast. We also performed mutagenesis to identify β-galactosidase mutants that were responsive to NOEV and found that 22 out of 94 mutants were responsive. Computational structural analysis revealed the mode of interaction between human β-galactosidase and NOEV. Moreover, we confirmed that NOEV reduced G(M1) accumulation and ameliorated the impairments of lipid trafficking and protein degradation in β-galactosidase deficient cells. These results provided further evidence to NOEV as a promising chaperone compound for β-galactosidase deficiency.

Topics: Animals; beta-Galactosidase; Cells, Cultured; Enzyme Stability; Fibroblasts; Gangliosidosis, GM1; Gene Expression; Genetic Vectors; Hexosamines; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidosis IV; Mutation, Missense; Protein Structure, Tertiary; Structure-Activity Relationship

The patient is a 24-year-old woman who first came for consultation at age 10 years. Based on clinical phenotype and thin-layer chromatography of urinary oligosaccharides, peripheral leukocytes were sent for beta-galactosidase assay. This testing showed a deficiency in enzyme activity, and gene mutation analysis identified a previously reported mutation p.H281Y (875C > T) and a novel mutation p.W273R (817T > C). Unlike previously reported patients, mutant enzymes in this patient's cultured skin fibroblasts did not respond to treatment with a chaperone compound, N-octyl-4-epi-beta-valienamine.

Topics: Adult; Amino Acid Substitution; beta-Galactosidase; Female; Fibroblasts; Hexosamines; Humans; In Vitro Techniques; Mucopolysaccharidosis IV; Phenotype; Point Mutation; Skin