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

人二二二醇脱氢酶的四个同工酶（DD1-DD4）具有83-97％的氨基酸序列身份，属于Aldo-Keto降低的含量，但在内源性底物的特异性和调节剂的特异性方面有所不同。此外，最近已经报道了与四个同工酶相似的九种cDNA，这表明各自同工酶的遗传多态性存在。进行了这项研究以阐明四个同工酶之间功能差的结构决定因素，各自同工酶的遗传多态性以及变体基因的临床意义。获得的结果总结如下：1。重组DD3和AKRLC3的动力学分析与DD3不同的氨基酸不同，表明它们在功能上是相同的，并且3个固定量与活动位点无关。底物特异性研究表明，该酶在生理上充当前列腺素D2 1 1-酮核酶，而不是羟基固醇脱氢酶……更多的rongenase.2。同工酶的位置定向诱变和嵌合酶的形成表明几种氨基酸与底物和调节剂相互作用，并负责辅酶和激活剂的结合和/或方向。3。抗杂化血管固定衍生物和抗炎2-芳基丙酸衍生物被鉴定为特异性DD4的激活剂。另外，发现甲状腺激素是该同工酶的物理激活剂。这些化合物的激活机制是在动力学上阐明的，并保留在同工酶的激活剂结合位点中，通过位置定向的诱变建议。4。我们建立了RT-PCR和RFLP方法，以区分二氢二醇脱氢酶同工酶报告的13种CDNA。在50种人体组织样品中，分别仅检测到DDL，DD2和DD3的mRNA物种。结果表明，DDI，DD2和DD3的CDNA是三个基因的主要等位基因，而报道的其他CDNA源自相应的同工酶基因的稀有变体，或者可能是测序误差。另一方面，大约20％的样品显示了DD4 mRNA的杂合表达，而一种与DD4不同的变体不同。对137个血液样本的基因组DNA的分析证实了该变体DD4基因，其中一个样品显示了该变体基因的纯合表达。日语中这种变体等位基因的频率约为9％。目前尚不清楚该变体基因的物理作用及其与肝病的关系尚不清楚。较少的