CYP1D1, pseudogenized in human, is expressed and encodes a functional drug-metabolizing enzyme in cynomolgus monkey

doi:10.1016/j.bcp.2010.11.003

Biochemical Pharmacology

Volume 81, Issue 3, 1 February 2011, Pages 442-450

https://doi.org/10.1016/j.bcp.2010.11.003 Get rights and content

Abstract

Cytochrome P450 (P450 or CYP) 1 family consists of the CYP1A, CYP1B, CYP1C, and CYP1D subfamilies. In the human genome, CYP1A1, CYP1A2, and CYP1B1 are expressed and encode functional enzymes, whereas CYP1D1P (formerly known as CYP1A8P) is present as a pseudogene due to five nonsense mutations in the putative coding region. In this study, we identified CYP1D1 cDNA, highly identical (nearly 95%) to human CYP1D1P sequence, in cynomolgus monkey, a species frequently used in drug metabolism studies due to its evolutionary closeness to human. The amino acid sequence deduced from cynomolgus monkey CYP1D1 cDNA shared the high sequence identity (91%) with human CYP1D1P (postulated from the gene sequence), and the highest sequence identity (44–45%) with CYP1A1 and CYP1A2 among cynomolgus monkey P450s. CYP1D1 mRNA was most abundantly expressed in liver, followed by kidney, and jejunum. The hepatic expression level of CYP1D1 mRNA was comparable to that of CYP1A1 mRNA and much higher than that of CYP1A2 mRNA. CYP1D1 was barely detectable in immunoblots of cynomolgus monkey liver. Cynomolgus monkey CYP1D1 mRNA was induced in primary hepatocytes with omeprazole. Cynomolgus monkey CYP1D1 protein heterologously expressed in Escherichia coli catalyzed ethoxyresorufin O-deethylation and caffeine 8-hydroxylation, which CYP1As also catalyze. Finally, no nonsense mutations, corresponding to those found in human CYP1D1P, were found in the 20 cynomolgus monkeys and 10 rhesus monkeys used in this study. These results suggest that CYP1D1 plays a role as a functional, drug-metabolizing enzyme in cynomolgus monkey liver.

Graphical abstract

Introduction

Cytochrome P450 (P450 or CYP) is a gene family, consisting of 57 functional genes and 58 pseudogenes in human [1]. The CYP1 family has diverged in each species during evolution, leading to different member genes in each species, due to gene gain and gene loss. The CYP1 family in human consists of the CYP1A and CYP1B subfamilies, including two genes (CYP1A1 and CYP1A2) and one gene (CYP1B1), respectively. Additional CYP1 subfamilies, CYP1C and CYP1D, were recently found in fish. CYP1D1, identified in killfish and zebrafish [2], shared the highest amino acid sequence identity (55–56%) with CYP1A. CYP1D1P (formerly known as CYP1A8P) found in human, contains five nonsense mutations in the potential coding region of gene sequence in the genome, and thus is not expected to encode a functional protein. Yet, discovery of functional CYP1D1 in fish raised the possibility that CYP1D1 in the genome, without nonsense mutations, could be expressed as a functional enzyme in other species.

The CYP1 family is involved in metabolism of drugs, and human CYP1A1 and CYP1A2 are known to preferentially catalyze ethoxyresorufin O-deethylation (EROD) and methoxyresorufin O-deethylation (MROD), respectively [3], [4]. Human CYP1A2 also catalyzes caffeine N-3 demethylation [5]. The CYP1 family is also involved in bioactivation of various environmental promutagens such as polycyclic aromatic hydrocarbons (PAHs), herbicides, and pesticides [6]. PAHs are metabolized into reactive intermediates, which form DNA and protein adducts that cause tumor formation and toxicity [7]. The CYP1 family is strongly induced by PAHs through the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, binding to xenobiotic response elements (XREs) at upstream regulatory regions of CYP1 genes.

Macaques, including cynomolgus monkey (Macaca fascicularis) and rhesus monkey (Macaca mulatta), are frequently used in biomedical studies such as drug metabolism due to their evolutionary closeness and physiological resemblance to humans. However, some differences in drug metabolism are occasionally noted, which could be partly due to divergence in the genes encoding drug-metabolizing enzymes such as P450s. Indeed, cynomolgus monkey CYP2C76, which we recently identified, has no ortholog in human, and is responsible for differences in pitavastatin metabolism between cynomolgus monkey and human [8]. Furthermore, a part of the differences in drug metabolism could be attributable to genes, such as CYP1D1P, that are pseudogenized in human, but might be expressed and functional in macaques.

To assess this possibility, in this study, we identified CYP1D1 cDNA in cynomolgus monkey. The CYP1D1 was characterized by sequence analysis, phylogeny, tissue expression patterns, genome organization, protein expression, and drug-metabolizing assays. The induction profile was also examined using typical human P450 inducers.

Section snippets

Materials

Pooled hepatic microsomes from male cynomolgus monkeys and humans were purchased from BD-GENTEST (Woburn, MA). Recombinant human CYP1A enzymes were prepared as described [9]. Oligonucleotides were synthesized by Invitrogen (Tokyo, Japan), and fluorescent probes were synthesized by Applied Biosystems (Foster City, CA) and Biosearch Technology Japan (Tokyo, Japan). Caffeine, dexamethazone, 7-ethoxyresorufin, omeprazole, rifampicin, and all other reagents were purchased from Sigma–Aldrich (St.

Identification of CYP1D1 sequence in the macaque genome

To identify gene sequences highly identical to human CYP1D1P, the genome data of rhesus macaque, a species closely related to cynomolgus monkey, was analyzed using BLAT, since the genome sequencing has not been completed in cynomolgus monkey. We successfully found a CYP1D1P sequence in macaque chromosome 15, located along with TMC1, ALDH1A1, and ANX1, coinciding well in location and direction with their respective orthologs in human chromosome 9 (Fig. 1). Interestingly, in zebrafish, ALDH1A1

Discussion

Human CYP1D1P, a pseudogene, does not encode a functional protein due to five nonsense mutations in the potential coding region. By analyzing rhesus monkey genome data, we found a gene sequence highly identical to human CYP1D1P. Using this sequence information, we isolated CYP1D1 cDNA from cynomolgus and rhesus monkey livers. The deduced amino acid sequences of these macaque CYP1D1s were highly identical to amino acid sequences predicted from the human CYP1D1P sequence (without nonsense

Acknowledgements

We greatly thank Mr. Masahiro Utoh, Dr. Koichiro Fukuzaki, and Dr. Ryoichi Nagata for their support of this work. We also appreciate Mr. Patrick Gray for reviewing the paper and Mr. Ryo Koizumi for technical assistance.

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CYP1D1, pseudogenized in human, is expressed and encodes a functional drug-metabolizing enzyme in cynomolgus monkey

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Identification of CYP1D1 sequence in the macaque genome

Discussion

Acknowledgements

Aquat Toxicol

Biochem Pharmacol

Biochem Pharmacol

Lancet

J Biol Chem

Drug Metab Pharmacokinet

Biochem Pharmacol

Protein Expr Purif

J Biol Chem

Arch Biochem Biophys

Chem Biol Interact

Biochem Pharmacol

Arch Biochem Biophys

Arch Biochem Biophys

J Biol Chem

Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants

Pharmacogenetics