Characterization of CYP2C9-splicing variant isolated from human liver

本研究的目的是表征一种新的cyp2c9剪接变体，这是我们实验室最近从人肝脏中克隆出来的，并阐明生理因素，如肝脏脂肪变性，是否通过剪接方式的改变参与了个体间和/或个体内药物代谢能力的差异。cyp2c9剪接变体包含内含子1的开头，但丢失了外显子2的开头。然而，有趣的是，没有观察到由于intron1部分的插入和exon2部分的删除而引起的移码。因此，该剪接变异体被认为是由482个氨基酸残基组成的蛋白质。由于CYP2C9的完整（原始）形式拥有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-昆虫细胞将该剪接变异体与原剪接变异体以及其中的多态性蛋白CYP2C9.3构建异体表达系统，进一步研究翻译蛋白的各种特性。Western blotting结果显示，剪接变体确实被翻译成一种蛋白，该蛋白被cyp2c9特异性抗体识别。表观分子量略小于原CYP2C9 （CYP2C9.1）和CYP2C9.3。最奇怪的是，翻译的剪接变体显示co -差异光谱是典型的细胞色素P450。因此，我们试图测量甲苯丁酰胺和双氯芬酸的催化活性，这两种物质都是众所周知的CYP2C9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。为了探讨8种氨基酸的缺失是否会改变底物特异性，我们使用分别是CYP2D6和CYP3A4特异性底物的丁呋罗和三唑仑进行了代谢研究。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至少对外源化合物（异种生物）没有活性。为了明确剪接方式是否会因肝脏脂肪变性而改变，我们采用实时荧光定量PCR技术对原始CYP2C9和剪接变体的mRNA编码进行了定量。结果，在三分之二的肝脂肪变性患者中，剪接变体mRNA比编码原始CYP2C9的mRNA更丰富。少

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