Mucopolysaccharidosis II (Hunter disease), a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), has variable clinical phenotypes. Nearly 300 different mutations have been identified in the IDS gene from patients with Hunter disease, but the correlation between the genotype and phenotype has remained unclear. We studied the characteristics of 11 missense mutations, which were detected in the patients or artificially introduced, using stable expression experiments and structural analysis. The mutants found in the attenuated phenotype showed considerable residual activity (0.2-2.4% of the wild-type IDS activity) and those in the severe phenotype had no activity. In immunoblot analysis, both the 73-75 kDa precursor and processed forms were detected in the expression of 'attenuated' mutants (R48P, A85T and W337R) and the artificial active site mutants (C84S, C84T). The 73-75 kDa initial precursor was detected in the 'severe' mutants (P86L, S333L, S349I, R468Q, R468L). The truncated 68 kDa precursor form was synthesized in the Q531X mutant. The results of immunoblotting indicated rapid degradation and/or insufficiency in processing as a result of structural alteration of the IDS protein. A combination of analyses of genotype and molecular phenotypes, including enzyme activity, protein processing and structural analysis with an engineered reference protein, could provide an avenue to understanding the molecular mechanism of the disease and could give a useful tool for the evaluation of possible therapeutic chemical compounds.
We investigated mutations of the iduronate-2-sulfatase (I2S) gene and structural characteristics of I2S to clarify genotype/phenotype relationships in 18 Japanese patients with mucopolysaccharidosis type II. The I2S gene was analyzed in five patients with a severe phenotype and in 13 patients with an attenuated phenotype. The tertiary structural model of the human I2S was constructed by homology modeling using the arylsulfatase structure as a template. We identified four missense mutations and a nonsense mutation in the severe phenotype; four missense, two nonsense, three frame shifts, and one each of splice and amino acid deletion in the attenuated phenotype. Seven of them (L73del, Q75X, G140R, C171R, V401 fs, C422 fs, and H441 fs) were novel mutations. Structural analysis indicated that the residues of the mutations found in the severe phenotype would have direct interactions with the active site residues or should break the hydrophobic core domain of I2S, whereas residues of the missense mutations found in the attenuated phenotype were located in the peripheral region. In addition, effects by deletion or frameshift mutations could also be interpreted by the structure. Structural analysis of mutant proteins would help in understanding the genotype/phenotype relationships of Hunter disease.
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