Molecular Cloning and Characterization of Marmoset Aldehyde Oxidase
Molecular Cloning and Characterization of Marmoset Aldehyde Oxidase
复制标题
狨猴醛氧化酶的分子克隆和表征
DOI：
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发表时间：
2017
影响因子：
3.9
通讯作者：
H. Yamazaki
中科院分区：
文献类型：
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作者：
Shotaro Uehara;Y. Uno;Eriko Okamoto;Takashi Inoue;E. Sasaki;H. Yamazaki
常见的绒猴(Callithrix Jacchus)是一种新大陆猴，由于细胞色素P450(P450)的酶功能与人类相似，因此是一种有希望用于临床前药物代谢研究的灵长类动物模型。尽管越来越多的候选药物由非P450酶催化，但除了P450以外的药物代谢酶在绒猴中几乎没有被识别或表征。在本研究中，我们通过对绒猴基因组的分析鉴定了醛氧化酶(AOX)1基因。采用逆转录-聚合酶链式反应从猕猴肝脏中克隆了AOX1基因。推导的氨基酸序列与灵长类AOX1s有较高的同源性(92-93%)。系统进化分析表明，绒猴AOX1与灵长类动物AOX1聚在一起，不同于猪、兔、大鼠和小鼠用于药物代谢的非灵长类动物模型AOX1。实时定量逆转录聚合酶链式反应和免疫印迹分析表明，绒猴AOX1基因在肝脏中大量表达，与食蟹猴和人相似。在大肠杆菌中异源表达的绒猴AOX1催化典型的AOX1底物卡巴泽兰和二氮杂氮的氧化，类似于食蟹猴和人AOX1S。用Michaelis-Menten动力学评价，人和绒猴AOX1能有效地催化二氮杂菲的氧化，而食蟹猴AOX1则以高容量的协同动力学催化这一反应。这些结果表明AOX1酶在绒猴和人类之间的组织分布和酶功能相似，提示绒猴是临床前研究AOX依赖药物代谢的合适的灵长类动物模型。
Common marmosets (Callithrix jacchus), New World monkeys, are a promising primate model for preclinical drug metabolism studies because of the similarities of cytochrome P450 (P450) enzyme function to those of humans. Despite an increasing number of drug candidates catalyzed by non-P450 enzymes, drug metabolizing enzymes other than P450s have been hardly identified or characterized in marmosets. In this study, we identified aldehyde oxidase (AOX) 1 gene by marmoset genome analysis. AOX1 cDNA was cloned from marmoset livers by reverse transcription-polymerase chain reaction. Deduced amino acid sequences of AOX1 cDNA showed high sequence identities (92–93%) with primate AOX1s. Phylogenetic analysis showed that marmoset AOX1 was closely clustered with primate AOX1s, unlike nonprimate animal model AOX1s of pig, rabbit, rat, and mouse used in drug metabolism. The tissue expression analyses by real-time reverse-transcription polymerase chain reaction and immunoblotting showed that marmoset AOX1 mRNA and protein were abundantly expressed in livers, similar to cynomolgus monkeys and humans. Marmoset AOX1 heterologously expressed in Escherichia coli catalyzed the oxidation of carbazeran and phthalazine, typical AOX1 substrates, similar to cynomolgus monkey and human AOX1s. Human and marmoset AOX1 effectively catalyzed phthalazine oxidation when assessed with Michaelis-Menten kinetics, but cynomolgus monkey AOX1 catalyzed this reaction with cooperative kinetics with high capacity. These results indicated that tissue distribution and enzymatic function of AOX1 enzyme is similar between marmosets and humans, suggesting that marmosets are a suitable primate model for AOX-dependent drug metabolism in preclinical studies.
DOI：
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发表时间：
1994-07
期刊：
The Journal of pharmacology and experimental therapeutics
影响因子：
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作者：
T. Shimada;H. Yamazaki;M. Mimura;Y. Inui;F. Guengerich
通讯作者：
T. Shimada;H. Yamazaki;M. Mimura;Y. Inui;F. Guengerich