Imbalanced substrate specificity of mutant (beta)-galactosidase in patients with Morquio B disease

GM1-gangliosidosis and Morquio B disease are distinct in clinical and biochemical features, but both disorders are caused by genetic defects of the same enzyme, acid (beta)-galactosidase ((beta)-Gal). We analyzed the kinetic properties of mutant (beta)-Gals from patients with GM1-gangliosidosis and Morquio B disease to examine the clinical and biochemical differences between both disorders. Five skin fibroblast lines from patients with GM1-gangliosidosis (2 cases; R201C/R201C and I51T/I51T), Morquio B disease (2 cases; W273L/W273L and Y83H/R482C), and galactosialidosis (1 case; Y395C/S90L) were used as enzyme sources. Residual enzyme activity in the cells was subjected to kinetic analysis. Substrate analogs including Gal(beta)1-3GalNAc, as an analog for GM1-ganglioside, and Gal(beta)1-4GlcNAc, as an analog for keratan sulfate, were used to determine IC50 and Ki for (beta)-Gals with an artificial substrate (4-methylumbelliferyl (beta)-D-galactopyranoside). Enzymatic assay method was established to examine the hydrolytic activity with the mutant (beta)-Gal for the substrate analogs. The mutant (beta)-Gal activities were inhibited by Gal(beta)1-3GalNAc and Gal(beta)1-4GlcNAc in a concentrationdependent manner. Remarkable increase in IC50 ratio and Ki ratio (Gal(beta)1-4GlcNAc/Gal(beta)13GalNAc) was observed in Morquio B disease. Relative hydrolytic activity (Gal(beta)1-4GlcNAc/Gal(beta)13GalNAc) was markedly decreased in Morquio B disease as compared with other subjects; controls (means +- SD, n=4), 1.00+-0.02; galactosialidosis, 1.03; GM1-gangliosidosis, 1.15 and 1.00; and Morquio B disease, 0.27 and 0.32. The mutant (beta)-Gals from the patients with Morquio B disease exhibited lower affinity and lower hydrolytic activity toward Gal(beta)1-4GlcNAc rather than Gal(beta)13GalNAc. These findings suggest that imbalanced substrate specificity of the mutant (beta)-Gals induces predominant accumulation of keratan sulfate and a rationale for performing differential diagnostic analysis for both disorders.