Cytochrome P450 oxidase (CYP) proteins oxidize a wide variety of substrates. They could metabolize not only many endogenous substrates such as steroid hormones, but also exogenous toxins for excretion . In biomedical research, CYP are widely recognized as drug-metabolizing enzymes. Probably, normal function of drug-metabolizing CYPs in natural habitats would be detoxication of xenophobic chemicals taken from foods, especially from plants .
Humans have about 60 CYP genes in the genome . Among them, CYP1A gene subfamily is one of the most major CYP subfamilies in humans. The human genome contains two CYP1A genes on chromosome 15, CYP1A1 and CYP1A2, which are tandemly aligned with 23-kb interval and head-to-head orientation . The amino acid sequence identity between the paralogs is > 80% and their protein structures are supposed to be very similar, partly accounting for overlapping substrate selectivity of these enzymes . Human CYP1A2 is constitutively expressed in livers and contributes to the hepatic metabolism of many important chemical compounds such as caffeine . In contrast, the tissue distribution of human CYP1A1 gene expression is very broad and its expression is strongly induced by exogenous compounds, indicating CYP1A2 rather than CYP1A1 is a major hepatic CYP1A gene in humans. Polymorphisms in human CYP1A genes have been intensively studied for medical benefits (e.g., ), while a few studies from an evolutionary standpoint have been conducted for human CYP1A genes [8, 9]. For example, Jorge-Nebert et al. surveyed the pattern of single nucleotide polymorphisms (SNPs) in several human populations at the CYP1A1-CYP1A2 locus and found the signature of selective sweep around the CYP1A1 untranslated region . However, the role of the selective sweep for human CYP1A1 evolution remains unclear.
Because macaque monkeys are widely used for testing drug toxicity in preclinical trials, the genetic similarity of CYP genes between humans and macaques is important not only for evolutionary research but also for biomedical applications . Unexpected phenotypic difference between divergent species is one the biggest concerns in drug development, e.g., . A previous study has suggested that drug metabolism by CYP1A1/2 was not perfectly conserved among humans, macaques, and marmosets . Elucidating the cause of phenotypic differences among primates at a genetic level is an important task.
Our previous sequence analysis showed that the region encompassing the first intron and exon 2 of macaque CYP1A2 had the highest genetic diversities among randomly selected 54 unlinked autosomal loci . Because allelic variation of CYP1A2 proteins may increase a repertory of toxic substrates metabolized by CYP1A2, it is reasonable to imagine that the high genetic diversity in macaque CYP1A2 has been maintained by balancing selection, where natural selection maintains highly diverged alleles having different functions [14, 15]. However, other studies also have shown that the gene expression pattern of CYP1A2 in macaques was quite different from that in humans. The level of gene expression of macaque CYP1A2 is very low in all Macaca fascicularis (cynomolgus macaques) livers so far examined [16, 17]. The expression level of CYP1A1 is instead much more abundant in macaque livers. Interestingly, the pattern of CYP1A1/2 protein expression appears similar between humans and marmosets , indicating a major hepatic CYP1A gene was switched at some time after the divergence of the human and macaque lineages . The result casts doubt that the balancing selection hypothesis accounts for high CYP1A2 diversity in macaques. However, if macaque CYP1A2 lost its function, the evolutionary history of CYP1A2 should resemble many other neutral loci.
In order to answer the question why macaque CYP1A2 has unusually high genetic diversity, we first investigated the long-term evolution pattern of CYP1A subfamily. Gene conversion between paralogs was examined with particular attention, because gene conversion between paralogs may create an unusual pattern of polymorphisms in paralogous genes. We found that the last gene conversion event between CYP1A1 and CYP1A2 in the lineages to Catarrhini primates occurred after the divergence of primates and rodents, and before the divergence of Catarrhini and Platyrrhini primates. We further sequenced CYP1A1/2 coding regions of 63 M. fascicularis and 28 M. mulatta individuals, and found several deletions and single nucleotide polymorphisms in macaque CYP1A2 with relatively high frequencies, which cause premature stop codons. We propose that macaque CYP1A2 is in the process of pseudogenization, and rapid degeneration of CpG sites is creating a high genetic diversity.