Structural analysis and molecular designing of enzyme proteins : Its application to clarification of pathology of inherited metabolic diseases and development of therapy

1.α-半乳糖苷酶与Fabry病我们在巴斯德毕赤酵母中表达了重组人α-半乳糖苷酶，并测定了其2.6A分辨率的晶体结构。人的α-半乳糖苷酶由一个催化结构域和一个包括β-链片的亚结构域组成，催化结构域形成桶状结构。结构分析将有助于法布里病治疗的发展。保护蛋白和半唾液酸中毒我们鉴定了一例新发现的具有K453E突变的缺陷保护蛋白基因产物，该突变发生在一例半唾液酸中毒患者中。免疫细胞化学、表达和代谢研究表明，前体保护蛋白被合成，但在突变体中很难加工成成熟的形式并被降解。通过替换野生型保护蛋白前体晶体结构上的氨基酸残基，构建了突变型保护蛋白的结构模型。结果表明，K453E突变位于保护蛋白的二聚体界面，减少了二聚体中氢键的形成。这种结构变化可能导致保护性蛋白二聚体的不稳定。GM2激活剂和GM2神经节苷脂AB变异型我们已经确定了一例日本GM2神经节苷脂AB变异症患者的临床特征和生化基础，患者细胞中未检测到成熟的GM2激活剂，GM1神经节苷脂的分解代谢被阻断在GM2神经节苷脂的水平。

1. alpha-Galactosidase and Fabry diseaseWe have expressed recombinant human alpha -galactosidase in Pichia pastoris and determined its crystal structure at 2.6A resolution. The human alpha-galactosidase consists of a catalytic domain, which forms barrel-like structure, and a subdomain including beta-stranded sheets . The structural analysis would facilitate development of therapy for Fabry disease.2. Protective protein and galactosialidosisWe characterized a defective protective protein gene product with a K453E mutation newly found in a patient with galactosialidosis. Immunocytochemical, expression and metabolic studies revealed that the precursor protective protein was synthesized but it hardly processed to the mature form and degraded in the mutant. Structural model of the mutant protective protein was constructed by replacement of the amino acid residue on the crystal structure of the wild type protective protein precursor reported. The result showed that the K453E mutation would locate at the dimer interface of the protective protein and reduce the hydrogen bond formation in the dimer. The structural change might cause instability of the protective protein dimer.3. GM2 activator and GM2 gangliosidosis AB variantWe have determined clinical features and biochemical basis of a Japanese patient with GM2 gangliosidosis AB variant, In the patient's cells no mature GM2 activator was detected and the catabolism of GM1 ganglioside was blocked at the level of GM2 ganglioside.