Characterization of CYP2C9-splicing variant isolated from human liver

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15390171
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied pharmacology
• Research Institution: Chiba University
• Principal Investigator: ARIYOSHI Noritaka Chiba Univ., Sch.Med., Div.Pharmacy, Univ.Hosp., associate Prof., 医学部附属病院, 助教授 (00243957)
• Co-Investigator(Kenkyū-buntansha): ISHII Itsuko Chiba Univ., Graduate Sch of Pharmacy, associate Prof., 大学院・薬学研究院, 助教授 (00202929) ; KITADA Mitsukazu Chiba Univ., Sch.Med., Div.Pharmacy, Univ.Hosp., Prof., 医学部附属病院, 教授 (90110345)
• Project Period (FY): 2003 – 2004
• Keywords: Interindividual difference in ability of drug metabolism / CYP2C9 / alternative splicing / hepatic steatosis

Research Abstract

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 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.