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

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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.