|Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
Four isozymes (DD1-DD4) of human dihydrodiol dehydrogenase share 83-97% amino acid sequence identity and belong to the aldo-keto reductase fhaiily, but differ in specificity for endogenous substrates and effects by modulators. In addition, nine cDNAs similar to that for the four isozymes have recently been reported, which suggests the existence of genetic polymorphism of the respective isozymes. This study was performed to elucidate structural determinants for the functional difference among the four isozymes, genetic polymorphism of the respective isozymes, and clinical significance of the variant gene. The results obtained are summarized as follows :
1. Kinetic analyses of recombinant DD3 and AKRlC3, which differs by 3 amino acids from DD3, indicated that they are functionally identical and the 3 residues are not involved in the active site. Substrate specificity study suggested that the enzymes physiologically act as prostaglandin D2 1 1-ketoreductase rather than hydroxysteroid dehyd
2. Site-directed mutagenesis of the isozymes and formation of chimeric enzymes suggested several amino acid residues, which interact with substrates and modulators, and are responsible for the binding and/or orientation of the coenzyme and activators.
3. Anti-hyperlipidemic clofibrate derivatives and anti-inflammatory 2-arylpropionic acid derivatives were identified as activators specific for DD4. In addition, thyroxin, a thyroid hormone, was found to be the physiological activator of this isozyme. The mechanism of activation by these compounds was kinetically elucidated and residues in the activator-binding site of the isozyme were suggested by site-directed mutagenesis.
4. We established the RT-PCR and RFLP methods to discriminate the 13 cDNAs reported for the dihydrodiol dehydrogenase isozymes. Only one mRNA species for DDl, DD2 and DD3, respectively, were detected in 50 human tissue samples. The result suggested that the cDNAs for DDI, DD2 and DD3 are the principal alleles of the three genes, and the other cDNAs reported are derived from rare variants of the respective isozyme genes or could be sequencing errors. On the other hand, about 20% of the samples showed heterozygous expression of mRNAs for DD4 and a variant that differs by 2 amino acids from DD4. Analysis of genomic DNA for 137 blood samples confirms this variant DD4 gene, and one of the samples showed homozygous expression of this variant gene. The frequency of this variant allele in Japanese was about 9%. The physiological effect of this variant gene and its relationship to hepatic diseases are not unclear at present. Less