Structures and clinical significance of polymorphism of human dihydrodiol dehydrogenase

人二氢二醇脱氢酶多态性的结构及临床意义

基本信息

批准号：
09672240
负责人：
HARA Akira
金额：
$ 2.05万
依托单位：
Gifu Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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 … More rogenase.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

人二氢二醇脱氢酶的四种同工酶(DD1-DD4)具有83-97%的氨基酸序列同一性，并且均属于醛酮还原酶，但对内源底物的特异性和调节剂的作用不同。此外，还有九种与之相似的cDNA。最近报道了四种同工酶的遗传多态性，这表明各自同工酶的遗传多态性的存在。四种同工酶功能差异的决定因素、各自同工酶的遗传多态性以及变异基因的临床意义所得结果总结如下： 1.重组DD3和AKR1C3的动力学分析，其与3个氨基酸不同。 DD3，表明它们在功能上是相同的，并且 3 个残基不参与活性位点，底物特异性研究表明这些酶在生理上充当前列腺素 D2。 1 1-酮还原酶而不是羟基类固醇脱水酶... 更多同工酶的定点诱变和嵌合酶的形成表明有几个氨基酸残基，它们与底物和调节剂相互作用，并负责结合和/或定向。 3．抗高脂氯贝特衍生物和抗炎2-芳基丙酸衍生物。此外，甲状腺素（一种甲状腺激素）被发现是该同工酶的生理激活剂，这些化合物的激活机制已通过动力学阐明，并且按位点提示了同工酶激活剂结合位点中的残基。 -定向诱变4。我们建立了RT-PCR和RFLP方法来区分报道的二氢二醇脱氢酶的13个cDNA。在50个人体组织样品中分别仅检测到DD1、DD2和DD3的一种mRNA。结果表明DDI、DD2和DD3的cDNA是这三个基因的主要等位基因，并且报道的其他cDNA是。源自各自同工酶基因的罕见变异或可能是测序错误。另一方面，约 20% 的样品显示 DD4 和 a 的 mRNA 杂合表达。与 DD4 有 2 个氨基酸不同的变体。对 137 个血液样本的基因组 DNA 分析证实了该变体 DD4 基因，其中一个样本显示该变体基因的纯合表达。该变体等位基因在日本人中的频率约为 9%。该变异基因的生理作用及其与肝脏疾病的关系目前尚不清楚。