Characterization of CYP2C9-splicing variant isolated from human liver

从人肝脏中分离的 CYP2C9 剪接变体的表征

• 批准号: 15390171
• 负责人: ARIYOSHI Noritaka
• 金额: $ 6.46万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390171/
• 关键词: Interindividual difference in ability of drug metabolism; CYP2C9; alternative splicing; hepatic steatosis; 遺伝的多型; スプライシング; 異種細胞発現系

The aim of this study was to characterize a novel CYP2C9-splicing variant, which has recently been cloned from the human liver in our laboratory, and clarify whether or not physiological factors such as hepatic steatosis were involved in inter-and/or intraindividual differences in an ability of drug metabolism via alteration of splicing manner.The CYP2C9-splicing variant contained the beginning of intron 1, but lost the beginning of exon 2. Interestingly, however, no frameshift due to insertion of the part of intron1 and deletion of the part of exon2 was observed. Thus, the splicing variant assumed to be expressed as protein consisted by 482 amino acid residues. Since the intact (original) form of CYP2C9 possesses 490 amino acids, the lack of 8 amino acids might change or lose function of the monooxygenase. The heterologous expression system using bacurovirus-insect cells of this splicing variant together with original form and CYP2C9.3, which is one of the polymorphic proteins were co … More nstructed to investigate various properties of translated protein. Western blotting revealed that the splicing variant was indeed translated to a protein, which was recognized by CYP2C9-specific antibody. Apparent molecular weight was slightly smaller than that of either original CYP2C9 (CYP2C9.1) or CYP2C9.3. The most curiously, translated splicing variant showed CO-difference spectrum being typical for cytochrome P450. Therefore, we tried to measure catalytic activity of tolbutamide and diclofenac, both of which are well known to be specific substrate of CYP2C9. However, the translated splice variant had no ability to metabolize both drugs. To explore the possibility that the lack of 8 amino acids alters the substrate specificity, metabolic study using bufurarol and triazolam, which are specific substrate for CYP2D6 and CYP3A4, respectively, was conducted. Again, no catalytic activity was shown for either drugs. According to the results described above, translated splicing variant seemed to have no activity to at least exogenous compounds (xenobiotics). To clarify whether the splicing manner can be altered by hepatic steatosis, mRNA encoding original CYP2C9 and splicing variant were quantified by real-time PCR. As a results, splicing variant mRNA was more abundant compared to the mRNA encoding original CYP2C9 in two out of three individuals with hepatic steatosis. Less

本研究的目的是描述一种新的CYP 2C 9剪接变异体，它是我们实验室最近从人肝脏中克隆的，并阐明是否生理因素如肝脂肪变性通过剪接方式的改变参与药物代谢能力的个体间和/或个体内差异。CYP 2C 9剪接变异体包含内含子1的开始，但缺失外显子2的起始部分。然而，有趣的是，没有观察到由于内含子1的部分插入和外显子2的部分缺失而引起的移码。因此，假定剪接变体表达为由482个氨基酸残基组成的蛋白质。由于CYP 2C 9的完整（原始）形式具有490个氨基酸，缺少8个氨基酸可能会改变或失去单加氧酶的功能。利用杆状病毒-昆虫细胞异源表达系统，将该剪接变体与原始形式和CYP2C9.3（多态性蛋白之一）一起进行异源表达， ...更多信息 旨在研究翻译蛋白的各种性质。Western blotting结果显示，该剪接变异体确实被翻译成蛋白质，并能被CYP 2C 9特异性抗体识别。表观分子量略小于原始CYP 2C 9（CYP2C9.1）或CYP2C9.3。最奇怪的是，翻译的剪接变体显示出细胞色素P450典型的CO差异光谱。因此，我们尝试测量甲苯磺丁脲和双氯芬酸的催化活性，这两种药物都是众所周知的CYP 2C 9的特异性底物。然而，翻译的剪接变体没有代谢这两种药物的能力。为了探索缺乏8个氨基酸改变底物特异性的可能性，使用丁呋洛尔和三唑仑（分别是CYP 2D 6和CYP 3A 4的特异性底物）进行了代谢研究。同样，两种药物都没有显示出催化活性。根据上述结果，翻译的剪接变体似乎对至少外源化合物（异生物质）没有活性。为了阐明剪接方式是否可以被肝脂肪变性改变，通过实时PCR定量编码原始CYP 2C 9和剪接变体的mRNA。结果，在三分之二的肝脂肪变性个体中，剪接变体mRNA比编码原始CYP 2C 9的mRNA更丰富。少