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Keywords:

  • cytochrome P450 3A subfamily;
  • genomic organization;
  • horse;
  • human;
  • polymorphism

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflicts of interest
  9. References
  10. Supporting Information

Cytochrome P450 enzymes (CYP450s) represent a superfamily of haem–thiolate proteins. CYP450s are most abundant in the liver, a major site of drug metabolism, and play key roles in the metabolism of a variety of substrates, including drugs and environmental contaminants. Interaction of two or more different drugs with the same enzyme can account for adverse effects and failure of therapy. Human CYP3A4 metabolizes about 50% of all known drugs, but little is known about the orthologous CYP450s in horses. We report here the genomic organization of the equine CYP3A gene cluster as well as a comparative analysis with the human CYP3A gene cluster. The equine CYP450 genes of the 3A family are located on ECA 13 between 6.97–7.53 Mb, in a region syntenic to HSA 7 99.05–99.35 Mb. Seven potential, closely linked equine CYP3A genes were found, in contrast to only four genes in the human genome. RNA was isolated from an equine liver sample, and the approximately 1.5- kb coding sequence of six CYP3A genes could be amplified by RT-PCR. Sequencing of the RT-PCR products revealed numerous hitherto unknown single nucleotide polymorphisms (SNPs) in these six CYP3A genes, and one 6- bp deletion compared to the reference sequence (EquCab2.0). The presence of the variants was confirmed in a sample of genomic DNA from the same horse. In conclusion, orthologous genes for the CYP3A family exist in horses, but their number differs from those of the human CYP3A gene family. CYP450 genes of the same family show high homology within and between mammalian species, but can be highly polymorphic.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflicts of interest
  9. References
  10. Supporting Information

Cytochrome P450 enzymes (CYP450) constitute a large superfamily of membrane-bound haem–thiolate containing monooxygenases, so called because they catalyse the incorporation of one oxygen atom from molecular oxygen into a substrate. CYP450 can be found in virtually all living organisms, from bacteria, in which cytochromes are soluble enzymes, to mammalian species, where cytochromes are bound to the smooth endoplasmic reticulum (Fink-Gremmels 2008). Members of the CYP450 family diverged from each other as early as 2 billion years ago, resulting in <40% similarity in amino acid sequences within the same CYP450 family. The CYP450 subfamilies emerged more than 400 million years ago, and within a subfamily the amino acid similarities are usually >55% (Nelson et al. 1996). To date, in humans, 57 functional genes and 58 pseudogenes have been sequenced (http://drnelson.uthsc.edu/cytochromeP450.html). CYP450s have diverse biological functions that include biosynthesis and regulation of cellular effectors such as synthesis of steroids and other endogenous compounds. Within the complex group of CYP450s, the enzyme families 1–3 are primarily involved in the biotransformation of drugs and toxins and are therefore referred to as drug-metabolizing enzymes. The pharmaceutical industry has developed an increasing interest in the structure and function of CYP450s, as they may also represent an important locus of drug–drug interactions. Determination of drug-metabolizing CYP450s has become a routine step in the drug development process and is required by the U.S. Food and Drug Administration (FDA) (Guengerich 2003).

The human CYP3A subfamily includes CYP3A4, CYP3A5, CYP3A7 and CYP3A43 encoded by a gene cluster on human chromosome 7. This is one of the most versatile biotransformation systems and facilitates the metabolism of about half of the known drugs. CYP3A4 is abundant in both the intestinal epithelium and the liver, and probably represents the most important of all drug-metabolizing enzymes. It accounts for nearly 50% of the CYP450 enzymes in humans (Wilkinson 2005). A high inter-individual variability in expression levels of CYP3A genes has been found, as well as an abundance of polymorphisms within the subfamily. This variability is further enhanced by induction and inhibition of the CYP3A enzymes by certain drugs and dietary constituents (Qiu et al. 2008).

CYP3A paralogs have been identified among others in rat, mouse, rabbit, dog, minipig, cow and monkey. In rats there are seven genes, in mouse eight genes, and there are only three isoforms in dogs and a single isoform in the rabbit (http://drnelson.uthsc.edu/cytochromeP450.html).

A direct extrapolation between experimental animal species and humans is not always possible, for reasons such as differences in enzyme activity, abundance, specificity and regulation (Nelson 1999; Spatzenegger et al. 2007). For example, CYP2C enzymes share important functional similarities in laboratory animal species, such as testosterone-6β hydroxylation, which are not present in CYP2C of humans. It was demonstrated that small sequence changes can produce large effects on activity and function; e.g. the canine CYP3A26 displays a much lower testosterone-6β hydroxylase activity than the canine CYP3A12, although they share 96% amino acid identity (Fraser et al. 1997).

Horses have been subject to more and more refined drug treatments during recent years, with treatment regimes being based on extrapolation from human medicine or clinical experience. Identification of CYP450s responsible for the metabolism of a drug is important with respect to clinical drug–drug interactions, genetic polymorphism and toxicity. Knowledge of CYP450s involved in drug metabolism and of individual variations in the metabolism of certain drugs is still missing for the horse. The first horse CYP450s were recombinantly expressed in insect cells only very recently (Maio Knych & Stanley 2008; Maio Knych et al. 2009). As the horse genome sequence was recently completed, much faster progress in CYP450 research is now possible.

The objective of this study was to obtain the genomic structure and DNA sequence of the equine CYP3A subfamily, including existing polymorphisms, to facilitate further functional investigations of the equine CYP3A subfamily.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflicts of interest
  9. References
  10. Supporting Information

DNA sequences

The human sequence investigated covered the region 99.2–99.5 Mb of the build 37.1 HSA 7 sequence. The corresponding equine sequence contained the region 6.97–7.53 Mb of the build 2.0 on ECA 13.

Sequence analysis

BLAST searching was used to identify homologous sequence regions between species (Altschul et al. 1997). Repetitive sequences were analysed with RepeatMasker (Smit, A.F.A., Hubley R. and Green P.; RepeatMasker Open-3.0, 1996–2004 http://www.repeatmasker.org).

Dot plot and percent identity plot (pip) analyses were performed with the programs PipMaker and MultiPipMaker (Schwartz et al. 2000). For detailed comparisons, local and global pairwise alignments were calculated with the program Lalign (Huang & Miller 1991).

DNA and RNA extraction

Genomic DNA was isolated from liver tissue of a ten-year-old female Arabian horse using the Nucleon BACC 2 genomic DNA extraction kit (GE Healthcare). Additionally, total RNA was isolated from a liver sample of the same horse using TRIzol™ reagent (Invitrogen, Karlsruhe, Germany).

RT-PCR and determination of the CYP3A cDNAsequences

Aliquots of 1 μg total RNA were reverse transcribed to cDNA using 20 pmol (T)24V primer and SuperScript™III reverse transcriptase (Invitrogen). On the basis of a multiple alignment, primers were designed to distinguish between the different paralogs of the equine CYP3A subfamily (Table S1). One microlitre of the cDNA was used as template in a polymerase chain reaction. The reaction was performed in a total volume of 20 μl using TopTaq DNA polymerase (Qiagen), and products were inspected for yield and purity on agarose gels. Direct sequencing of the PCR products was performed after shrimp alkaline phosphatase (Roche) and exonuclease I treatment (N.E.B.). PCR products were sequenced on an ABI 3730 capillary sequencer (Applied Biosystems) using the PCR primers and additional internal sequencing primers. The sequences of the primers are listed in Tables S2 and S3 of the supporting information. Sequencing data were assembled with Sequencher 4.9 (GeneCodes).

PCR and mutation analysis

Primers for the amplification of each of the exons with flanking regions were designed with the software Primer3 (Rozen & Skaletsky 2000) after masking repetitive sequences with RepeatMasker (Smit, A.F.A., Hubley R. and Green P.; RepeatMasker Open-3.0, 1996–2004 http://www.repeatmasker.org).

PCR and direct sequencing were performed as described previously. PCR primers were used as sequencing primers, with two exceptions, in which nested primers were designed for sequencing. Sequences are listed in Tables S4 and S5. For the prediction of the effect of an amino acid exchange on protein function, we used the programs Polyphen (http://genetics.bwh.harvard.edu/pph/) and pMUT (http://mmb2.pcb.ub.es:8080/PMut/).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflicts of interest
  9. References
  10. Supporting Information

We used the repeat masked human 27205 bp CYP3A4 sequence as the query in a BLASTN analysis to identify homologous sequences in the horse genome. On the basis of these initial BLASTN results, we selected a 560- kb interval on ECA 13 that included the entire equine CYP3A gene cluster.

Pairwise dot plot analyses of the human CYP3A cluster on HSA 7 and the equine 560- kb region revealed that the horse CYP3A gene cluster contains seven potential genes and one potential pseudogene, in contrast to the human region, which includes only four genes and two pseudogenes (Fig. 1). The number of exons (13) and the lengths of the internal exons were consistent between human CYP3A4 and all seven equine genes.

image

Figure 1.  Dot plot analysis of the equine CYP3A gene cluster with the corresponding sequence of the human CYP3A gene cluster. Sequence sections were chosen that contain the entire CYP3A gene cluster without flanking genes. Except for the equine CYP3A89, no other CYP450 has been annotated in build 2.0 of the equine genome sequence at the NCBI Map Viewer, but hypothetical genes (LOCs) have been annotated based on similarities with other CYP450 genes. Names for the CYP450s were extracted from the cytochrome P450 homepage (http://drnelson.uthsc.edu/cytochromeP450.html), where suggested protein sequences have been published and named.

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A search in the CYP450 nomenclature database indicated that names had already been assigned to the equine genes (http://drnelson.uthsc.edu/Nomenclature.html), although not in a continuous order (see Fig. 1). Out of all seven paralogs, the CYP3A89 mRNA and protein displayed the highest similarity with the human CYP3A4. All genes except CYP3A129 were situated on the plus strand. The pseudogene CYP3A128P, situated between CYP3A97 and CYP3A129, aligns with exons 8–13 of the human CYP3A4 gene, but exons 8 and 9 lack one base pair compared to exons 8 and 9 of the human CYP3A4, and exon 12 showed a poor alignment below 60%. We did not undertake to sequence this pseudogene on genomic DNA.

MultiPip pairwise sequence comparison of the CYP3A genes from human and horse (Fig. 2) showed that the exons were highly conserved between the two species. Many non-coding regions also exhibited a high degree of sequence conservation. The GC content in all CYP3A genes of the horse was ≤40%, and LINES constituted the main repeat type found in the CYP3A genes.

image

Figure 2.  Sequence comparisons between the different CYP3A genes. Multiple percent identity plot (MultiPiP) of the CYP3A genes of human (h) and horse (e). In the top line, a schematic representation of the human CYP3A4 gene is given. In the lower panels, the identities of the other sequences with respect to the human CYP3A4 sequence are illustrated. Note the strong sequence identity within all exons and also most introns, except intron 3.

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The 1512- bp coding sequence of six of seven potential genes could be amplified and completely sequenced from a cDNA template. Sequences were submitted to the EMBL database and assigned the following accession numbers: FN669292, FN669293, FN669294, FN669295, FN669296 and FN669297.

Sequencing revealed a total of 19 single nucleotide polymorphisms (SNPs) and one 6-bp deletion in the coding sequences of all six genes with respect to the reference sequence (Table 1). The reference sequence used in this work was constructed by joining the exons extracted from the genomic DNA sequence (EquCab 2.0).

Table 1. CYP450 Gene Polymorphisms.
CYP450 GenePolymorphism (cDNA)Polymorphism (genomic DNA)Position within GeneProtein
  1. 1Published by Broad Institute.

CYP3A93c.17G>Cg.6996581G>CExon 1p.Ser6Thr
c.26C>Tg.6996590C>TExon 1p.Thr9Met
c.64A>Gg.6996628A>GExon 1p.Ile122Val
c.66T>Cg.6996630T>CExon 1Silent
c.87G>Ag.7000744G>AExon 2Silent
c.173G>Cg.7002936G>CExon 3p.Trp58Ser1
c.748G>Ag.7014254G>AExon 8p.Ala250Thr1
CYP3A89c.1010T>Cg.7079070T>CExon 10p.Val337Ala1
c.1095G>Ag.7081607G>AExon 11Silent1
CYP3A94c.17G>Ag.7133787G>AExon 1p.Ser6Asn
c.558T>Cg.7145876 T>CExon 7Silent1
CYP3A95c.640C>Gg.7229727C>GExon 7p.His214Asp
c.718G>Cg.7234134G>CExon 8p.Val240Leu
c.1174A>Tg.7241704A>TExon 11p.Thr392Ser
c.1254G>Ag.7243321G>AExon 12Silent
CYP3A96c.437T>Ag.7316374T>AExon 6p.Phe146Tyr
c.1496_1501delCCGTGAg.7334175_7334180delExon 13p.Thr499_Val500del
CYP3A97c.126T>Ag.7380650T>AExon 2Silent
c.356C>Tg.7387399C>TExon 5p.Thr119Ile1
c.1498G>Ag.7413137G>AExon 13p.Val500Met1

Seven of the SNPs that we detected had already been entered in the SNP database from the Broad Institute at MIT and Harvard (http://www.broadinstitute.org/ftp/distribution/horse_snp_release/v2). We did not confirm the established SNPs on the genomic DNA of this horse as we did for the formerly unknown SNPs (Table 1). CYP3A93 exhibited the highest variability, with 7 SNPs in the coding sequence, four of which were located in exon 1.

The majority of SNPs were found to be either synonymous or to code for conserved amino acid exchanges which are not expected to influence the protein function. This was calculated by using the programs Polyphen and pMUT. The only non-conserved amino acid exchange we found was the exchange of tryptophan with serine in exon 3 of CYP3A93. The replacement of the large side chain of tryptophan 58 with the much smaller side chain of a serine (p.Trp58Ser) was predicted to be ‘possibly damaging’ (Polyphen) or ‘pathological’ (pMUT) to the protein. Amplification of CYP3A95 rendered, apart from the expected product of 1512 bp, a second product of about 900 bp.

Sequencing of the gel-extracted 900- bp product provided three sequences that covered 695 bp in total. The three sequences between 200 and 300 bp comprised bases of exons 1, 2, exons 4, 5, 6 and exons 11, 12. Thus, our data suggest the existence of an alternatively spliced CYP3A95 mRNA isoform. We did not pursue the effort of obtaining a full sequence of the hypothetic splice variant.

In CYP3A96, a 6- bp deletion (p.Thr499_Val500del) was detected in exon 13, 8 bp before the stop codon, rendering a protein with only 501 amino acids instead of 503. All SNPs and the 6-bp deletion were confirmed on genomic DNA of the same horse. It is interesting to note that even if the SNPs investigated on the genomic level were rated to be heterozygous, in most cases the mutated variant was clearly predominating on the cDNA level, indicating potential allelic imbalances in the mRNA expression. We were unable to amplify the mRNA of CYP3A129 from a liver cDNA template, which may be because of low or absent expression of this gene in liver.

Alignment of the coding sequences and deduced protein sequences with the homologous sequences of the human CYP3A subfamily (Table 2) revealed high identity between human and horse CYP3A subfamilies (79–86% for the coding sequence and 68–81% for the amino acid sequence), but also very high identity between the members of the equine CYP3A subfamily (88–91.7% for the coding sequence and 83.9–88.5% on the protein level).

Table 2.   Percent identities at the cDNA level between the human CYP3A cluster and the equine variants found.
HorseHuman
 CYP3A4CYP3A5CYP3A7CYP3A431
% Identity at the nucleotide level
 CYP3A9385.483.783.880.5
 CYP3A8986.484.784.481.6
 CYP3A9485.383.283.680.7
 CYP3A9583.782.381.981.2
 CYP3A9684.38382.579.8
 CYP3A9783.381.981.779.6
% Identity at the amino acid level
 CYP3A9379.578.976.571.2
 CYP3A8981.380.376.373.2
 CYP3A9480.177.976.971.2
 CYP3A9579.177.375.372
 CYP3A9679.177.576.570.6
 CYP3A9775.375.573.468.7

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflicts of interest
  9. References
  10. Supporting Information

This study describes the detailed genomic organization of the equine CYP3A gene cluster for the first time. Comparison and genomic characterization of the human CYP3A gene cluster with the complete horse CYP3A gene cluster became possible through the recent completion of the horse genome sequence.

Comparative analysis of the human chromosome region containing the CYP3A gene cluster and the corresponding equine region illustrated that the horse genome contains six highly similar CYP3A genes and two potential pseudogenes, in contrast to the human genome, which has only four known functional CYP3A genes and two pseudogenes.

Our findings of six functional CYP3A genes in the horse genome versus only four in the human genome are in accordance with reports about CYP3A genes in other mammalian species such as dog, cow, mouse and rat. In an extensive research project, CYP3 loci from 16 different species were compared, and conclusions were drawn for the evolution of the CYP3A genomic loci over a period of 450 million years (Qiu et al. 2008). Among the species investigated in this paper, the horse belongs to the ones with the highest amount of CYP3A genes, a level met only by mouse and opossum. Most CYP3 genes build gene clusters within a genomic CYP3 locus that develops through independent gene duplications in distantly related species. A dominant role of tandem duplications in the development of the CYP3 clusters has been suggested (Thomas 2007; Qiu et al. 2008). Frequent gene duplications and losses have also been attributed to other so-called unstable CYP450 gene families, which all share a tendency for positive selection in amino acid sequence, especially in the substrate binding regions of the enzyme (Thomas 2007; Qiu et al. 2008; Chen et al. 2009). It was hypothesized that phylogenetically stable genes have core functions in development and physiology and metabolize mainly endogenous substrates, whereas unstable genes have accessory metabolic functions associated with unstable environmental interactions such as toxin and pathogen exposure (Thomas 2007). The high number of CYP3A genes in the horse could possibly reflect a tendency of this herbivore to metabolize a wide variety of different substrates that might be contained in the forage. It could be assumed that wild horses had to be able to digest a huge variety of plant species depending on many environmental factors in different geographical regions and climate conditions.

CYP450 genes are known to be rich in variations and there are a higher number of polymorphisms in CYP450 genes than for other investigated gene sets (Solus et al. 2004). More than 30 SNPs have been identified for the human CYP3A4 gene, out of which about 20 produce an amino acid exchange (http://www.cypalleles.ki.se/cyp3a4.htm). In general, variants in the coding regions of CYP3A4 occur at allele frequencies <5% (Lamba et al. 2002). Allelic frequency of the variants in horses and the amount of variation between different breeds still remains to be analysed using population studies.

Automatic prediction of the influence of certain variations on protein function, as performed in this study, showed that out of all 13 recorded non-synonymous SNPs in the horse CYP3A cluster, only one was predicted to affect protein function. This finding coincides well with results from studies in humans. Although a strong inter-individual variability in CYP3A-dependent clearance is reported in man, it is considered that different allelic variants only contribute to the variability to a small extent. Inter-individual differences in enzyme expression as a result of a variety of factors seem to be more relevant (Lamba et al. 2002).

It is interesting to note that 14 of the 19 SNPs we found in the horse CYP3A cluster are positioned in the first half of the coding sequence (codons 1–250), whereas only 5 SNPs were found in the second half. When comparing the distribution of SNPs within the coding sequence between human CYP3A4 and the horse CYP3As, we found that the majority of SNPs were also in the first half of the coding sequence in the human CYP3A4 (http://www.cypalleles.ki.se/cyp3a4.htm).

This might underline the typical structure of the CYP450 family, which is usually a small N-terminal predominantly beta-strand domain with the membrane-spanning part and a larger helical C-terminal domain containing the functionally important active site and the haem (Williams et al. 2004).

One characteristic feature of human CYP3A4 is the phenylalanine cluster forming a hydrophobic core above the active site (Williams et al. 2004). In the horse CYP3A cluster, the phenylalanine at positions 108, 213, 219 and 220 was found to be sometimes replaced by a leucine or isoleucine. In contrast, positions 241 and 304 were highly conserved, and all CYP3As had a phenylalanine at that position. Functional differences between the different CYP3A proteins from this cluster are not easy to predict from the sequence level. In the human CYP3A cluster, the two most widely expressed isoforms CYP3A4 and CYP3A5 had 84% identity at the amino acid level (Guengerich 1997) and share most substrate specificities, whereas the number of substrates can differ between the isoforms (Daly 2006). In contrast, the human 2C subfamily has 4 known isoforms, CYP2C9, CYP2C8, CYP2C18 and 2C19, which are more than 80% homologous to each other in the amino acid sequence but can differ markedly in function (Guengerich 1997). In extreme cases, a single amino acid exchange can change substrate specificity completely (Lindberg & Negishi 1989; Ramarao & Kemper 1995).

Our failure to amplify the coding sequence of CYP3A129 from the cDNA of horse liver can imply different situations: either the gene, although showing a complete set of exons, does not code for a functional protein and is therefore one of the numerous pseudogenes or splice variants that have been described in many other species, or as another consequence, CYP3A129 could be an enzyme not expressed predominantly in the liver and may instead be found in other tissues at the protein level. This has been reported for the human CYP3A43, which is expressed in prostate and testis rather than in liver. Sex-dependent expression of CYP450s has been predominantly described in rats and was hypothesized to be a consequence of several situations, including extensive inbreeding (Mugford & Kedderis 1998). It may be less likely that the horse shows a sex specific set of CYP450s like the strongly inbred rat. However, a male horse should also be tested for the expression of CYP3A129 to rule this out.

In humans, a correlation between CYP3A5 genotype and cancer susceptibility has been drawn. Certain combined CYP3A4/CYP3A5 haplotypes show differential susceptibility to prostate cancer (Rebbeck et al. 1998; Paris et al. 1999). CYP3A5*1 homozygotes may have higher systolic blood pressure (Givens et al. 2003; Qiu et al. 2008). More research is necessary before such conclusions can be drawn for horses.

In conclusion, orthologous genes for the CYP3A family exist in horses, but their number differs from the human CYP3A gene family. CYP genes of the same family show high homology within and between species but can be highly polymorphic. These data provide a basis for functional analyses of the equine CYP3A family and perhaps some day in the future drug treatments of horses will be adjusted to their individual CYP genotypes to increase the efficacy of pharmacological interventions.

Acknowledgement

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflicts of interest
  9. References
  10. Supporting Information

This study was supported by a grant from the Swiss National Science Foundation.

References

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflicts of interest
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflicts of interest
  9. References
  10. Supporting Information

Table S1 Primers placed in the UTR for amplification of coding sequence from the cDNA template.

Table S2 Internal sequencing primers used for several CYP450s.

Table S3 Internal sequencing primers.

Table S4 Primers for PCR on genomic DNA.

Table S5 Internal sequencing primers for genomic PCR products.

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