Newly Identified CYP2C93 Is a Functional Enzyme in Rhesus Monkey, but Not in Cynomolgus Monkey

Yasuhiro Uno,Sakae Kohara,Koichiro Fukuzaki,Hiroshi Yamazaki,Kazuhide Iwasaki,Masahiro Utoh,Norie Murayama,Ryoichi Nagata,Shotaro Uehara,Ulrich Zanger

doi:10.1371/journal.pone.0016923

Yasuhiro Uno, Sakae Kohara + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0016923

Copy DOI

Abstract

Cynomolgus monkey and rhesus monkey are used in drug metabolism studies due to their evolutionary closeness and physiological resemblance to human. In cynomolgus monkey, we previously identified cytochrome P450 (P450 or CYP) 2C76 that does not have a human ortholog and is partly responsible for species differences in drug metabolism between cynomolgus monkey and human. In this study, we report characterization of CYP2C93 cDNA newly identified in cynomolgus monkey and rhesus monkey. The CYP2C93 cDNA contained an open reading frame of 490 amino acids approximately 84–86% identical to human CYP2Cs. CYP2C93 was located in the genomic region, which corresponded to the intergenic region in the human genome, indicating that CYP2C93 does not correspond to any human genes. CYP2C93 mRNA was expressed predominantly in the liver among 10 tissues analyzed. The CYP2C93 proteins heterologously expressed in Escherichia coli metabolized human CYP2C substrates, diclofenac, flurbiprofen, paclitaxel, S-mephenytoin, and tolbutamide. In addition to a normal transcript (SV1), an aberrantly spliced transcript (SV2) lacking exon 2 was identified, which did not give rise to a functional protein due to frameshift and a premature termination codon. Mini gene assay revealed that the genetic variant IVS2-1G>T at the splice site of intron 1, at least partly, accounted for the exon-2 skipping; therefore, this genotype would influence CYP2C93-mediated drug metabolism. SV1 was expressed in 6 of 11 rhesus monkeys and 1 of 8 cynomolgus monkeys, but the SV1 in the cynomolgus monkey was nonfunctional due to a rare null genotype (c.102T>del). These results suggest that CYP2C93 can play roles as a drug-metabolizing enzyme in rhesus monkeys (not in cynomolgus monkeys), although its relative contribution to drug metabolism has yet to be validated.

Highlights

The cytochrome P450 (P450 or CYP) superfamily contains a large number of genes, functional genes and pseudogenes in human
For cloning of this potential cDNA, Reverse transcription (RT)-polymerase chain reaction (PCR) was performed with liver total RNA of rhesus monkey using the primers that were designed to amplify the putative open reading frame (ORF), which led to the successful identification of the cDNA for this novel CYP2C named CYP2C93 (Fig. 1A)
Amino acids deduced from this CYP2C93 cDNA had relatively low sequence identity (77-81%) to human, cynomolgus monkey, and rhesus monkey CYP2C proteins (Table 1)

Summary

Introduction

The cytochrome P450 (P450 or CYP) superfamily contains a large number of genes, functional genes and pseudogenes in human (see http://drnelson.uthsc.edu/cytochromeP450). The number of CYP2Cs differs, indicating the difficulty in determining orthologous relationships of CYP2Cs between the two species [2]. This suggests that the data from rodent must be cautiously interpreted and extrapolated to human. The investigation of CYP2C76 revealed that CYP2C76 is not orthologous to human genes [6] and that CYP2C76 is at least partly responsible for the difference in drug metabolism between macaque and human [9]. Identification and characterization of the macaquespecific genes are important for understanding the differences of drug metabolism in the two species

Methods

Results

Conclusion