Complementary DNA cloning, functional expression and characterization of a novel cytochrome P450, CYP2D50, from equine liver
Introduction
Cytochrome P450 monooxygenases (CYP450s) are a superfamily of hemeproteins responsible for metabolizing a vast array of drugs, environmental pollutants and endogenous compounds. CYP450 enzymes enable a functional group to be introduced or unmasked in a compound, generating a more polar intermediate, which is more readily eliminated. CYP450s have been well characterized in many laboratory animal species as well as in humans. In humans, more than 50 individual CYP450s have been identified. Of these 50, only six have been shown to play a major role in metabolism and subsequent clearance of drugs. These include members of the CYP1, CYP2 and CYP3 families. CYP2D enzymes are particularly important in drug metabolism as they are responsible for metabolism of 20–25% of commonly prescribed drugs, including antiarrythmics, β-adrenoreceptor antagonists, neuroleptics and tricyclic antidepressants and because there are several functionally significant polymorphisms in the human CYP2D6 gene [1].
The most common variants of human CYP2D6 result in very high activity (as a result of gene duplication), in altered affinity for some substrates or in no activity which results from inactive splice variants or point mutations that result in an unstable product. Some of the variants have low penetrance in the human population (1–2%) while others such as some of the point mutations are present in more than half of Asians studied. The functional importance of these variants combined with the relatively high penetrance in the human population means that doses of those therapeutic agents with a narrow therapeutic index must be adjusted to obtain appropriate therapeutic levels. Polymorphic expression of CYP2D is of particular importance because of the wide array of therapeutic compounds metabolized by this enzyme. A decrease or complete inability to metabolize CYP2D6 substrates presents the potential for adverse drug reactions as a result of an inability to metabolize and clear the administered compound from the body.
Although there is considerable experimental evidence supporting the functional importance of CYP2D6 and its variants in both humans and animals used for human drug development, little is known about this protein in other species. Therapeutic agents used in equine medicine are those that are effective in other species and dose regimens are established on a trial and error basis. The studies reported here are part of a long term effort to systematically evaluate the metabolic capabilities of the drug metabolizing enzymes in equids.
Although there have been a limited number of studies using equine liver microsomes [2], [3], [4], these provide little information about individual equine CYP450s, specifically which enzymes are responsible for the metabolism of specific drugs. Human CYP450s have been well characterized and various in-vitro systems, such as recombinant cytochrome P450 enzymes expressed in E. coli or insect cells, have been developed to study the metabolism of therapeutic agents. To date, there are no reports of equine recombinant CYP450 enzymes.
Studies with equine liver microsomes have suggested the presence of a member of the CYP2D family in the horse, based on metabolic activity with the human isoform selective substrate dextromethorphan [2]. In this study, equine liver microsomes demonstrated more than 20 times the metabolic turnover of dextromethorphan as compared to human liver microsomes. However, the only definitive way to determine whether CYP2D is responsible for metabolism of dextromethorphan in the horse, or if there are multiple members of this family playing a role in its metabolism, is through the use of a recombinant enzyme. Accordingly, in this study, a member of the CYP2D family was cloned, sequenced, expressed and characterized with respect to human CYP2D6 selective substrates.
Section snippets
Chemicals
Superscript III One Step RT-PCR Kit, Novex mini-gel system, 10% Bis-Tris precast gels, NuPage loading dye and NuPage transfer buffer (20×) were from Invitrogen (Carlsbad, CA). BsrGI restriction enzyme was purchased from New England Biosciences (Ipswich, MA). All sequencing primers were synthesized by IDT (Coralville, IA). TOPO and pDEST8 cloning vectors, Cellfectin reagent, Spodoptera frugiperda (Sf9) and High Five cells and human recombinant CYP2D6 were from Invitrogen (Carlsbad, CA).
Cloning and sequencing of equine CYP2D50
RT-PCR products from two different horses were generated. The consensus sequence was 1503 base pairs. The sequence was submitted to the CYP450 Nomenclature Committee and was given the novel designation of CYP2D50. The deduced amino acid sequence of CYP2D50 (accession no. EU0190996) showed a homology of 80% to cattle CYP2D14 and CYP2D43. The amino acid sequence alignment of CYP2D50 with that of human CYP2D6 (Fig. 1) revealed that CYP2D50 is 77% homologous to CYP2D6.
Expression of CYP2C92 in insect cells
An entry vector containing
Discussion
Due to their large size, the expense associated with maintenance and drug administration as well as limited access to research animals, performing drug metabolism studies in the horse, metabolism data from other species must frequently be extrapolated for equine veterinary use. However, it is important to note that interspecies differences in in-vitro metabolism as well as substrate specificity have been well documented. In this study, we have cloned and identified a member of the CYP2D family
Acknowledgements
The authors would like to thank Dr. Alan R. Buckpitt, Dr. Alan J. Conley, Jo Corbin and Dexter Morin for their helpful suggestions and Dan McKemie for technical assistance.
References (13)
- et al.
Comparative expression of liver cytochrome P450-dependent monooxygenases in the horse and in other agricultural and laboratory species
Vet J
(2003) - et al.
The carbon monoxide-binding pigment of liver microsomes
J Biol Chem
(1964) - et al.
Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography
J Biol Chem
(1976) - et al.
Molecular cloning, expression and characterization of CYP2D17 from cynomolgus monkey liver
Arch Biochem Biophys
(1999) - et al.
Expression and characterization of canine cytochrome P450 2D15
Arch Biochem Biophys
(1998) - et al.
Porcine microsomal vitamin D(3) hydroxylase (CYP2D25). Catalytic properties, tissue distribution, and comparison with human CYP2D6
J Biol Chem
(2000)
Cited by (20)
Equine uridine diphospho-glucuronosyltransferase 1A1, 2A1, 2B4, 2B31: cDNA cloning, expression and initial characterization of morphine metabolism
2020, Veterinary Anaesthesia and AnalgesiaCitation Excerpt :A two-step reverse transcription polymerase chain reaction (PCR) was used to amplify PCR products of four UGT variants from equine liver RNA. Complementary DNA (cDNA) was amplified from isolated total liver RNA as described previously (DiMaio Knych & Stanley 2008) using the QuantiTect Reverse Transcription kit (Qiagen, Germany). The UGT enzymes UGT1A1, UGT2A1, UGT2B4 and UGT2B31 were selected from the National Center for Biotechnology Information (NCBI) equine genome database based on greater than 60% homology to human UGT2B7 (Gene ID:7364; Table 1) and amplified with primers generated from sequences obtained from GenBank (Table 2).
Testosterone metabolism of equine single CYPs of the 3A subfamily compared to the human CYP3A4
2017, Toxicology in VitroCitation Excerpt :Chauret et al. (1997) reported a 7-times slower 6β-hydroxylase activity in horses, dogs, and cats. In addition, a similar pattern was seen for diclofenac and dextromethorphan in the CYP2C/D equine subfamilies (DiMaio Knych and Stanley, 2008). Due to the production of androstenedione in native insect cells, the formation of this metabolite in transduced cells with the equine CYPs was not quantifiable.
Pharmacokinetics of mirtazapine and its main metabolites after single oral administration in fasting/fed horses
2013, Journal of Equine Veterinary ScienceCitation Excerpt :Recently, a new equine CYP isoform (CYP2D50) was characterized. It was shown to have metabolic rate that was vastly slower than that of its human counterpart [29]. This could be evidence that N-demethylation is the more efficient metabolic route for MRZ in horses.
Equine cytochrome P450 2B6 - Genomic identification, expression and functional characterization with ketamine
2013, Toxicology and Applied PharmacologyCitation Excerpt :A few studies have examined gene expression, protein expression and catalytic activity of equine CYPs (Komori et al., 1993; Tyden et al., 2004, 2008) as well as the turnover of human marker substrates in equine liver microsomes (Chauret et al., 1997; Lakritz et al., 2000; Nebbia et al., 2003). In order to gain deeper knowledge on the drug metabolizing enzymes of the horse and their substrate specificities, it is necessary to systematically identify and characterize individual CYP isoforms by recombinant expression and functional analyses, as recently published for five equine CYPs for the first time (DiMaio Knych and Stanley, 2008; DiMaio Knych et al., 2009; Knych et al., 2010). After the recent completion of the horse genome sequence, investigations regarding equine CYP genes including the equine CYP3A subfamily gene cluster (Schmitz et al., 2010a) have been significantly facilitated.
Determination of oral tramadol pharmacokinetics in horses
2010, Research in Veterinary Science