Women's and Children's Health Research Institute, Women's and Children's Hospital, North Adelaide, SA 5006, Australia.
Gene. 2012 Sep 15;506(2):423-8. doi: 10.1016/j.gene.2012.06.024. Epub 2012 Jul 1.
Australian marsupials are unique fauna that have evolved and adapted to unique environments and thus it is likely that their detoxification systems differ considerably from those of well-studied eutherian mammals. Knowledge of these processes in marsupials is therefore vital to understanding the consequences of exposure to xenobiotics. Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of both xenobiotics and endogenous substrates. In this study we have cloned and characterized CYP3A70, the first identified member of the CYP3A gene subfamily from Eastern gray kangaroo (Macropus giganteus). A 1665 base pair kangaroo hepatic CYP3A complete cDNA, designated CYP3A70, was cloned by reverse transcription-polymerase chain reaction approaches, which encodes a protein of 506 amino acids. The CYP3A70 cDNA shares approximately 71% nucleotide and 65% amino acid sequence homology to human CYP3A4 and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Transfection of the CYP3A70 cDNAs into 293T cells resulted in stable cell lines expressing a CYP3A immuno-reactive protein that was recognized by a goat anti-human CYP3A4 polyclonal antibody. The anti-human CYP3A4 antibody also detected immunoreactive proteins in liver microsomes from all test marsupials, including the kangaroo, koala, wallaby, and wombat, with multiple CYP3A immunoreactive bands observed in kangaroo and wallaby tissues. Relatively, very low CYP catalytic activity was detected for the kangaroo CYP3A70 cDNA-expressed proteins (19.6 relative luminescent units/μg protein), which may be due to low protein expression levels. Collectively, this study provides primary molecular data regarding the Eastern kangaroo hepatic CYP3A70 gene and enables further functional analyses of CYP3A enzymes in marsupials.
澳大利亚有袋类动物是独特的动物群，它们已经进化并适应了独特的环境，因此它们的解毒系统可能与经过充分研究的真兽类哺乳动物有很大的不同。了解这些过程对于理解外来物质暴露的后果至关重要。细胞色素 P450（CYPs）在多种外源物质和内源性底物的氧化代谢中起着至关重要的作用。在这项研究中，我们从东部灰袋鼠（Macropus giganteus）克隆并鉴定了 CYP3A70，这是 CYP3A 基因亚家族的第一个鉴定成员。通过逆转录聚合酶链反应方法克隆了一个 1665 个碱基对的袋鼠肝 CYP3A 全长 cDNA，命名为 CYP3A70，它编码一个 506 个氨基酸的蛋白质。CYP3A70 cDNA 与人类 CYP3A4 的核苷酸和氨基酸序列同源性分别约为 71%和 65%，并与其他已发表的来自人类、猴子、牛、猪、狗、大鼠、兔、鼠、仓鼠和豚鼠的哺乳动物 CYP3A 具有高度的序列相似性。将 CYP3A70 cDNA 转染 293T 细胞后，稳定表达出一种 CYP3A 免疫反应性蛋白的细胞系，该蛋白被山羊抗人 CYP3A4 多克隆抗体识别。抗人 CYP3A4 抗体还在所有测试的有袋动物的肝微粒体中检测到免疫反应性蛋白，包括袋鼠、考拉、沙袋鼠和袋熊，在袋鼠和沙袋鼠组织中观察到多个 CYP3A 免疫反应性条带。相对而言，从袋鼠 CYP3A70 cDNA 表达蛋白中检测到的 CYP 催化活性相对较低（19.6 相对发光单位/μg 蛋白），这可能是由于蛋白表达水平较低。总的来说，这项研究提供了东部袋鼠肝 CYP3A70 基因的主要分子数据，并使我们能够进一步分析有袋类动物 CYP3A 酶的功能。
