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Saito S.,Hokkaido Information University | Ohno K.,NPO for the Promotion of Research on Intellectual Property Tokyo | Ohno K.,Astellas Pharm Inc | Maita N.,Tokushima University | Sakuraba H.,Meiji Pharmaceutical University
Molecular Genetics and Metabolism | Year: 2014

Allelic mutations, predominantly missense ones, of the α- l-iduronidase (IDUA) gene cause mucopolysaccharidosis type I (MPS I), which exhibits heterogeneous phenotypes. These phenotypes are basically classified into severe, intermediate, and attenuated types. We previously examined the structural changes in IDUA due to MPS I by homology modeling, but the reliability was limited because of the low sequence identity. In this study, we built new structural models of mutant IDUAs due to 57 amino acid substitutions that had been identified in 27 severe, 1 severe-intermediate, 13 intermediate, 1 attenuated-intermediate and 15 attenuated type MPS I patients based on the crystal structure of human IDUA, which was recently determined by us. The structural changes were examined by calculating the root-mean-square distances (RMSD) and the number of atoms influenced by the amino acid replacements. The results revealed that the structural changes of the enzyme protein tended to be correlated with the severity of the disease. Then we focused on the structural changes resulting from amino acid replacements in the immunoglobulin-like domain and adjacent region, of which the structure had been missing in the IDUA model previously built. Coloring of atoms influenced by an amino acid substitution was performed in each case and the results revealed that the structural changes occurred in a region far from the active site of IDUA, suggesting that they affected protein folding. Structural analysis is thus useful for elucidation of the basis of MPS I. © 2013 Elsevier Inc. Source

Hossain M.A.,Osaka University | Higaki K.,Tottori University | Saito S.,Hokkaido Information University | Ohno K.,NPO for the Promotion of Research on Intellectual Property Tokyo | And 6 more authors.
Journal of Human Genetics | Year: 2015

Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on mutant G M1 β-galactosidase proteins, and in a murine G M1 -gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations. © 2015 The Japan Society of Human Genetics. Source

Matsuoka K.,Tokushima University | Matsuoka K.,Japan National Institute of Biomedical Innovation | Tamura T.,Tokushima University | Tsuji D.,Tokushima University | And 10 more authors.
Molecular Therapy | Year: 2011

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of Β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human Β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB. © The American Society of Gene &Cell Therapy. Source

Saito S.,University of Tokyo | Ohno K.,NPO for the Promotion of Research on Intellectual Property Tokyo | Ohno K.,Astellas Pharma Inc. | Sese J.,Ochanomizu University | And 2 more authors.
Journal of Human Genetics | Year: 2010

Fabry disease is a genetic disorder caused by a deficiency of α-galactosidase, exhibiting a wide clinical spectrum, from the early-onset severe classic form to the late-onset mild variant one. Recent screening of newborns revealed that the incidence of Fabry disease is unexpectedly high, and that the genotypes of patients with this disease are quite heterogeneous and many novel mutations have been identified in them. This suggests that a lot of Fabry patients will be found in an early clinical stage when the prognosis is obscure and a proper therapeutic schedule for them cannot be determined. Thus, it is significant to predict the clinical phenotype of this disease resulting from a novel mutation. Herein, we proposed a phenotype prediction model based on sequential and structural information. As far as we know, this is the first report of phenotype prediction for Fabry disease. First, we investigated the sequential and structural changes in the α-galactosidase molecule responsible for Fabry disease. The results showed that there are quite large differences in several properties between the classic and variant groups. We then developed a phenotype prediction model involving the decision tree technique. The accuracy of this prediction model is high (86%), and Matthew's correlation coefficient is also high (0.49). The phenotype predictor proposed in this paper may be useful for determining a proper therapeutic schedule for this disease. © 2010 The Japan Society of Human Genetics All rights reserved. Source

Tsukimura T.,Meiji Pharmaceutical University | Nakano S.,Tokyo Metropolitan Institute of Medical Science | Nakano S.,Synthera Technologies Co. | Togawa T.,Meiji Pharmaceutical University | And 5 more authors.
Molecular Genetics and Metabolism Reports | Year: 2014

Fabry disease is an X-linked genetic disorder characterized by deficient activity of α-galactosidase A (GLA) and accumulation of glycolipids, and various GLA gene mutations lead to a wide range of clinical phenotypes from the classic form to the later-onset one. To investigate the biochemical heterogeneity and elucidate the basis of the disease using available clinical samples, we measured GLA activity, GLA protein and accumulated globotriaosylsphingosine (Lyso-Gb3), a biomarker of this disease, in plasma samples from Fabry patients. The analysis revealed that both the enzyme activity and the protein level were apparently decreased, and the enzyme activity was well correlated with the protein level in many Fabry patients. In these cases, a defect of biosynthesis or excessive degradation of mutant GLAs should be involved in the pathogenesis, and the residual protein level would determine the accumulation of Lyso-Gb3 and the severity of the disease. However, there are some exceptional cases, i.e., ones harboring p.C142Y, p.R112H and p.M296I, who exhibit a considerable amount of GLA protein. Especially, a subset of Fabry patients with p.R112H or p.M296I has been attracted interest because the patients exhibit almost normal plasma Lyso-Gb3 concentration. Structural analysis revealed that C142Y causes a structural change at the entrance of the active site. It will lead to a complete enzyme activity deficiency, resulting in a high level of plasma Lyso-Gb3 and the classic Fabry disease. On the other hand, it is thought that R112H causes a relatively large structural change on the molecular surface, and M296I a small one in a restricted region from the core to the surface, both the structural changes being far from the active site. These changes will cause not only partial degradation but also degeneration of the mutant GLA proteins, and the degenerated enzymes exhibiting small and residual activity remain and probably facilitate degradation of Lyso-Gb3 in plasma, leading to the later-onset phenotype. The results of this comprehensive analysis will be useful for elucidation of the basis of Fabry disease. © 2014 The Authors. Published by Elsevier Inc. Source

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