Molecular Cloning and Expression of a Novel Cytochrome P450

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Chem. Res. Toxicol. 2006, 19, 30-37

Articles Molecular Cloning and Expression of a Novel Cytochrome P450 from Turkey Liver with Aflatoxin B1 Oxidizing Activity Shirley S. M. Yip and Roger A. Coulombe, Jr.* Graduate Program in Toxicology, Department of Veterinary Sciences, Utah State UniVersity, Logan, Utah 84322-4620 ReceiVed August 22, 2005

Cytochromes P450 are members of a superfamily of oxidative hemoprotein enzymes that metabolize a variety of endogenous and exogenous compounds. Previous studies in our laboratory have shown that efficient P450-mediated activation underlies the extreme sensitivity of poultry, specifically turkeys, to the toxic effects of the mycotoxin aflatoxin B1 (AFB1). Using 3′- and 5′-rapid amplification of cDNA ends (RACE), we amplified from turkey liver RNA a full-length 1.73 kb cDNA predicted to be 528 amino acids with 94.7% sequence identity to a CYP1A5 from chicken liver. A truncated construct of the turkey CYP1A5 gene with 29 amino acids deleted from the hydrophobic NH2-terminal region was cloned and heterologously expressed in Escherichia coli. The expressed protein from E. coli membranes had a CO-binding spectrum typical of P450s, and it catalyzed the O-dealkylation of the CYP1A prototype substrates ethoxyresorufin and methoxyresorufin. CYP1A5-mediated O-dealkylation of methoxyresorufin was completely inhibited by R-naphthoflavone, a specific CYP1A inhibitor. Inhibitors to other mammalian P450s (3A4, 2D, 2E, and 3A1) either slightly inhibited this activity or not at all. CYP1A5 oxidized AFB1 to form two metabolites: the reactive intermediate, AFB1-8,9-epoxide (AFBO), and aflatoxin M1 (AFM1). Because of the importance of AFBO and AFM1 in the toxicity of AFB1, we conclude that this P450 probably plays some role in the well-known hypersensitivity of turkeys to AFB1. To our knowledge, this is the first P450 cloned and sequenced from turkeys, the species in which the toxicity of AFB1 was first discovered. Introduction Cytochromes P450 (P450)1 are a superfamily of hemoproteins that catalyze the oxidation of a large number of endogenous and exogenous substrates, including steroids, eicosanoids, pharmaceuticals, pesticides, pollutants, and carcinogens (1). Much research has focused on the role of P450 enzymes in the formation of carcinogenic and mutagenic electrophilic intermediates from naturally occurring dietary compounds (2). One important example is the mycotoxin aflatoxin B1 (AFB1) produced by strains of Aspergillus flaVus and parasiticus, which is a potent liver toxin and carcinogen in humans and animals (3). AFB1 was discovered in the early 1960s as the etiological agent of “Turkey X Disease”, responsible for widespread deaths in turkeys and other poultry throughout Europe due to contaminated Brazilian peanut meal (4). A requisite step in the toxicity and carcinogenicity of AFB1 is P450-mediated oxidation * To whom correspondence should be addressed. Tel: 435-797-1598. Fax: 435-797-1601. E-mail: [email protected]. 1 Abbreviations: P450, cytochrome P450; AFB , aflatoxin B ; AFBO, 1 1 AFB1-8,9-epoxide; AFBO-GSH, AFB1-8,9-epoxide glutathione conjugate; AFG1, aflatoxin G1; AFM1, aflatoxin M1; BHA, butylated hydroxyanisole; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; endo-AFBO, AFB1 endo-8,9-epoxide; exo-AFBO, AFB1 exo-8,9-epoxide; GST, glutathione S-transferase; hNPR, human NADPH-P450 reductase; IPTG, isopropyl-β-D-thiogalactopyranoside; RNF, R-naphthoflavone; NCBI, National Center for Biotechnology Information; RACE, rapid amplification of cDNA ends; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; UTR, untranslated region.

to the reactive and electrophilic AFB1-8,9-epoxide (AFBO), which binds to DNA, proteins, and other critical cellular nucleophiles. In human liver and lung, low, environmentally relevant concentrations of AFB1 are activated primarily by CYP1A2, whereas higher concentrations are catalyzed by CYP3A4 (5-8). Toxicity caused by AFB1 is primarily in the liver, although other organs are also affected (3). In addition to its public health concern, AFB1 is also nearly universally present in corn-based animal feeds, where even small amounts of this toxin have deleterious effects on animals. As was graphically demonstrated by the “Turkey X” incident, poultry, especially turkeys, are extremely sensitive to the toxic effects of AFB1 resulting in reductions in growth rate, feed efficiency, increased susceptibility to viral and bacterial diseases, and severe hepatotoxicosis (9). We have shown that this hypersensitivity is associated with both efficient P450-mediated metabolic activation and deficient detoxification by glutathione S-transferase (GST) (10, 11). Using specific P450 inhibitors, we have previously demonstrated that AFB1 is metabolized in turkey liver microsomes primarily by a CYP1A and to a lesser extent by a CYP3A homologue (11). Because of the postulated role of the turkey CYP1A homologue in the sensitivity of this species toward AFB1, the purpose of this study was to clone, heterologously express, and partially characterize this gene product from turkey liver. Here, we report on the presence of a novel P450 from turkey liver that has AFB1 oxidizing activity. To our

10.1021/tx050233+ CCC: $33.50 © 2006 American Chemical Society Published on Web 11/18/2005

Turkey Hepatic CYP1A5

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Figure 1. Expression constructs of turkey CYP1A5 showing nucleic acid and amino acid sequences at the N- and C-terminals. Nucleotides and amino acids different from the native sequence are bolded. The wild-type construct (wtCYP1A5) has 1605 bp encoding 534 amino acids. The truncated construct (tCYP1A5) has 1518 bp encoding 505 amino acids. Six histidines are added at the C-terminal of both constructs to facilitate detection and purification.

knowledge, this is the first P450 cloned and sequenced from turkeys.

Experimental Procedures Chemicals and Reagents. Liver tissues of 1 day old male white turkeys were kindly provided by Dr. David Frame, Utah State University Extension (Ephraim, UT). QIAprep spin miniprep kit, QIAquick PCR purification kit, and gel extraction kit were obtained from Qiagen (Valencia, CA). TOPO TA cloning kit and maximum efficiency DH5RF′IQ Escherichia coli cells were from Invitrogen (Carlsbad, CA). Restriction enzymes and T4 DNA ligase were from Fermentas (Hanover, MD). Exo- and endo-AFB1-8,9-epoxide glutathione conjugate (AFBO-GSH) standard were a generous gift from Dr. F. Peter Guengerich, Vanderbilt University School of Medicine (Nashville, TN). Anti-His tag monoclonal antibody was from Novagen (San Diego, CA). The Phototope-HRP Western Blot Detection System was from Cell Signaling (Beverley, MA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Rapid Amplification of cDNA Ends (RACE). Primers used in 3′-RACE were designed based on the sequence alignments of CYP1A2 in human, rabbit, mouse, rat, and a partial clone of chicken2 using ClustalW (www.ebi.ac.uk). Primers used in 5′-RACE were based on the alignments plus the sequences found in 3′-RACE. The gene-specific primer sequence for 3′-RACE was 5′-CCCTTCACCATCCCCCACAGCAC-3′ (forward), and for 5′-RACE, it was 5′-GCGGCGATGGAGAAGTTCTTCAGGG-3′ (reverse). Both reactions used a mixture of two universal primers (long primer, 5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′, and short primer, 5′-CTAATACGACTCACTATAGGGC-3′) provided in the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto, CA). The full-length gene was cloned using forward (5′-ATGGGGCCGGAGGAAGTGATGG-3′) and reverse (5′-GTTTGAGCTCTTCATGGAGAAGCGTTTC-3′) primers, which were designed based on the sequence information obtained from 3′- and 5′-RACE. Freshly isolated turkey liver segments stored in RNAlater (Ambion, Austin, TX) were homogenized, and RNA was isolated by Poly(A)Pure mRNA purification kit (Ambion, Austin, TX). RNA was run on a 1% agarose gel to check for integrity, as determined by the presence of intact 18S and 28S ribosomal RNA, before being used as RACE templates. First-strand synthesis was performed as described in the SMART RACE Kit handbook (BD Biosciences Clontech). RACE2 GenBank accession numbers for the CYP1A enzymes cited in this article are as follows: chicken CYP1A5 (X99454), chicken CYP1A4 (X99453), rabbit CYP1A2 (X13853), human CYP1A2 (AF182274), mouse CYP1A2 (BC018298), rat CYP1A2 (NM_012541), human CYP1A1 (NM_000499), and chicken CYP1A2 (partial clone, M64537).

PCRs were done by using BD Advantage 2 PCR Kit (BD Bioscience Clontech). PCR products were cloned into pCR 4-TOPO vector and sent to Center for Integrated BioSystems (CIB), Utah State University (Logan, UT) for sequencing. Sequences were analyzed by Lasergene SeqMan II software (DNASTAR Inc., Madison, WI). The final sequence was confirmed by at least three clones in any segment, with at least one sequenced from either direction. Construction of Expression Plasmid. The original pCW1A2/ hNPR bicistronic vector developed by Dr. Guengerich (1) was kindly provided by Dr. Gary Yost, University of Utah (Salt Lake City, UT). This plasmid was derived from the pCWOri+ expression vector, which contains two tac promoter cassettes upstream of an NdeI restriction enzyme cloning site coincident with the initiation ATG codon. Bicistronic constructs consisting of the coding sequence of CYP1A5 followed by that of the human NADPH-P450 reductase (hNPR), a redox partner, were constructed in the pCW1A2/hNPR expression vector (1). Two variants of CYP1A5 sequence were cloned as follows: the wild-type sequence (wtCYP1A5) and an N-terminal truncated sequence (tCYP1A5) (Figure 1). Because N-terminal modifications were shown before to improve bacterial expression of P450s (12-14), we engineered the tCYP1A5 sequence such that it had a G-to-A mutation at the second codon and lacked codons 3-31. In addition, the nucleotide sequence of codons 2-8 was modified to enhance P450 protein expression according to Barnes et al. (15). Both constructs had a PCR-introduced NdeI site at the 5′-end and an XbaI site at the 3′end for subcloning; a 6× His tag was also added to the C-terminal to facilitate immunodetection and purification. The sequences for the forward primers used to clone the expression constructs wtCYP1A5 and tCYP1A5 were 5′-CTACACAAGCATATGGGGCCGGAGGAAGTG-3′ and 5′-CTACACAAGCATATGGCTTTATTATTAACTCAAACTCGCCGGCAGCACACACCC-3′ respectively, with the NdeI sites underlined. The reverse primer sequence for both constructs was 5′-TGGGCGTCTAGATCAGTGATGGTGATGGTGATGGTTTGAGCTCTTCATGGAG-3′, with the XbaI site underlined. The expression constructs were created by digesting the original pCW1A2/hNPR vector with NdeI and XbaI to excise the CYP1A2 coding sequence. The gel-purified vector fragment was ligated with the wild-type or truncated CYP1A5 NdeI- and XbaI-digested, PCR-amplified fragment using T4 DNA ligase. The resulting ligation mix was transformed into DH5RF′IQ cells. Individual clones were screened by colony PCR and diagnostic restriction digest before being submitted to CIB for sequencing. Sequences in the entire reading frame of both constructs as well as the 5′- and 3′-junctions of the inserts to the vectors were verified before individual clones were used in expression. Bacterial Expression. Expression in E. coli was performed as described by Jenkins et al (16). Five milliliters of Luria-Bertani ampicillin medium was inoculated with a single colony of the

32 Chem. Res. Toxicol., Vol. 19, No. 1, 2006 DH5RF′IQ E. coli transformants and allowed to grow overnight at 37 °C with vigorous shaking. The starter culture was then used to inoculate (1:100 dilution) 450 mL of modified Terrific Broth ampicillin medium with trace elements in a 2 L baffled flask. Cultures were allowed to grow to OD600 ) 0.3-0.5 at 37 °C and then equilibrated at 25 °C for 30 min before adding 0.5 mM δ-aminolevulinic acid and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to start induction. Cells were harvested after 24 h, and levels of P450 expression were determined by reduced CO/ reduced difference spectrum (17) (Spectronic GENESYS 6 UVvis spectrophotometer, Thermo Spectronic, Rochester, NY). Western blots using anti-His tag monoclonal antibody and anti-rat NADPH-P450 reductase polyclonal antibody (which cross-reacts with hNPR) were also employed to verify the expression of the recombinant proteins. E. coli membranes and cytosol were prepared as described (16). Sample aliquots were stored at -80 °C. All kinetic assays were performed with tCYP1A5 E. coli membranes, which we found to be more stable than E. coli cytosol in terms of activity when stored at -80 °C. Purification of Recombinant CYP1A5 for Polyclonal Antibody Production. Bacterial cytosol was used in the purification protocols for the ease of handling and abundance of soluble protein. Affinity chromatography (Ni-NTA resins, Qiagen) and hydroxyapatite chromatography (Econo-Pac CHT-II cartridge, BioRad, Hercules, CA) were performed as recommended by the manufacturer except that 0.1% sodium cholate and 5% glycerol were included in all buffers. Elution buffer containing purified tCYP1A5 was exchanged to PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.7) using Amicon Ultra centrifugal filter devices (Millipore, Billerica, MA) before being shipped to ProSci Inc. (Poway, CA) for rabbit polyclonal custom antibody production. Immunodetection. Turkey liver microsomes, prepared as described by Klein et al. (10), E. coli membranes expressing tCYP1A5 and pCWOri+ (control vector without P450 and reductase inserts) were run on a 4-15% Tris-HCl Ready Gel (Bio-Rad). Immunoblotting was performed using procedures as described in QIA express Detection and Assay Handbook (Qiagen). The rabbit polyclonal antiserum against the E. coli expressed and purified tCYP1A5 was first preadsorbed by pCWOri+ E. coli membranes to reduce the extraneous bands caused by the recognition of bacterial proteins (18). Briefly, 120 µL of pCWOri+ membranes (22 mg protein) was added to 1.8 mL of polyclonal antiserum (118 mg protein), incubated for 2.5 h at 4 °C with gentle rotation, and then centrifuged at 13000g for 10 min at 4 °C. The supernatant was used as the primary antibody (1:3500), and goat anti-rabbit IgG HRP-conjugated antibody was used as the secondary antibody (1:5000). Signals were detected by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce, Rockford, IL) and computer-archived using a Nucleovision E20 Imaging Workstation (Nucleotech, Hayward, CA). Quantative Western blot was performed by loading turkey microsomes samples and various amounts of tCYP1A5 E. coli membranes as standards on the same gel. Blotting procedures were described as above. The amount of CYP1A5 in turkey liver microsomes was estimated by comparing the relative band intensities of the samples and standards. Determination of AFB1 Metabolites. The oxidation activity toward AFB1 was determined by HPLC using conditions that we have described previously (7). Because of its short half-life, AFBO was indirectly measured as the GSH-trapped conjugate, which provides an accurate and quantitative measurement (19). E. coli membranes expressing tCYP1A5 (1.8 µM) were incubated in epoxide-trapping buffer (5 mM MgCl2, 25 mM KCl, 0.25 mM sucrose, and 80 mM potassium phosphate, pH 7.6) with 2 mM NADPH, 5 mM GSH, 34 µL of butylated hydroxyanisole (BHA)induced mouse cytosol as a source of GST (∼800 µg of protein) and 2-500 µM AFB1 in spectral grade dimethyl sulfoxide (DMSO), to give a final volume of 250 µL. The reactions were incubated at 37 °C for 3 or 5 min (5 min for AFB1 e 20 µM and 3 min for concentrations > 20 µM) with gentle shaking and stopped by adding 250 µL of cold methanol spiked with 24 µM aflatoxin G1 (AFG1)

Yip and Coulombe as an internal standard. When an inhibitor was used, it was added into the reaction mixture before incubation and the incubation time was extended to 10 min. The samples were stored at -20 °C overnight to facilitate protein precipitation and then centrifuged at 13000g for 10 min. The supernatant was filtered through a 0.2 µm nylon membrane before it was injected into the HPLC. Metabolites were separated on a Beckman System Gold chromatographic system (Beckman, Fullerton, CA), equipped with a model 126 pump, a model 166 UV-vis detector, and an Econosphere C18 (150 mm × 4.6 mm) column (Alltech Associates, Deerfield, IL), which was kept at 40 °C. The elution of peaks was monitored by UV absorbance (λ ) 254 nm). The mobile phases and HPLC program used in this assay were as described (11). Amounts of metabolite formation were calculated by establishing calibration curves between the peak areas in the chromatograms and the amounts of metabolites injected, using authentic AFBO-GSH and aflatoxin M1 (AFM1) HPLC standards. Other Assays. O-dealkylation of 7-ethoxyresorufin and 7-methoxyresorufin (EROD and MROD, respectively) was assayed basically as described (20) with some modifications, using a Gilford Fluoro IV spectrofluorometer (Corning, Oberlin, OH). E. coli membranes containing 165 pmol of tCYP1A5 (final concentration ) 82 nM) were incubated in reaction buffer [50 mM Tris, pH 7.5, 25 mM MgCl2, and 1.6 mg/mL bovine serum albumin (BSA)] with 0.25 mM NADPH and 5 µM substrate in a total volume of 2 mL at 37 °C. The excitation wavelength was set at 530 nm, and the emission wavelength was at 585 nm. Resorufin was used as the standard. Various selective inhibitors of mammalian P450s were evaluated for their potential inhibitory effect on MROD catalyzed by tCYP1A5. The specific inhibitors used in the assay were R-naphthoflavone (RNF) (CYP1A1, 1A2), 17R-ethynylestradiol (3A4), quinidine (2D), 4-methylpyrazole (2E), and erythromycin (3A1) (11, 21). In brief, 82 nM P450 in E. coli membranes was incubated with inhibitor with varying concentrations of inhibitor in DMSO or DMSO alone as a control, in a 37 °C water bath for 1 min before adding the rest of the components in the MROD assay as described above. The hNPR activity was determined by cytochrome c reduction as described (22), except that 75 µM cytochrome c and 0.25 mM NADPH were used. The total protein concentration was determined according to Bradford (23) using a Multiskan EX photometer (Labsystems, Helsinki, Finland). Kinetic data were analyzed by SigmaPlot Enzyme Kinetics Module software (Jandel Scientific, San Rafael, CA) and were fit using the Michaelis-Menten equation [V ) Vmax [S]/([S] + Km)] and nonlinear regression analysis to calculate the kinetic constants Vmax and Km.

Results RACE. We used 1 day old turkey for mRNA isolation since we had previously demonstrated in our laboratory that young turkeys (9 days old) were more susceptible to AFB1 than older turkeys (10). In a separate study, we were also able to isolate the same gene from the liver of a 24 day old turkey (data not shown). Using 3′-RACE in this study, we identified a partial clone, which translated into 137 amino acids and was 98.5% identical to the C-terminal of the chicken CYP1A5 sequence (24). We then used the chicken CYP1A5 nucleotide sequence together with the CYP1A2 sequence alignment from several species2 to design primers for 5′-RACE to clone the full-length gene. Sequence analyses of 12 clones that together covered the entire open reading frame revealed a sequence of 1587 bp, coding for 528 amino acids, plus 44 bp in the 5′-untranslated region (UTR) and 101 bp in the 3′-UTR (Figure 2). The predicted molecular mass of the gene product is 60 kDa. The amino acid and nucleic acid sequence identity comparisons between this P450 gene and other CYP1A genes using EMBOSS-Align program (www.ebi.ac.uk) are shown in Table 1. This gene was remarkably close to chicken CYP1A5, with 94.7% nucleic acid and amino acid sequence identity. Therefore,

Turkey Hepatic CYP1A5

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Figure 2. Complete cDNA and predicted amino acid sequence of turkey CYP1A5, with 5′- and 3′-UTRs. The start and stop codons are bolded, the heme-binding site is underlined, and the ribosome-binding site is double underlined. The open reading frame contains 528 residues. Table 1. Nucleic Acid and Amino Acid Sequence Identity and Similarity between Turkey CYP1A5 and Other P450s in Selected Species nucleic acid

amino acid

P450

species

% identity

% identity

% similaritya

1A5 1A4 1A2 1A2 1A2 1A2 1A1

chicken chicken rabbit human mouse rat human

94.7 79.6 67.2 65.0 64.0 62.8 62.7

94.7 75.5 57.3 59.5 54.7 54.9 59.5

97.7 85.8 74.1 74.2 73.1 72.9 76.9

a Percentage similarities of amino acid sequences are determined based on EBLOSUM62 matrix.

we named this turkey P450 gene CYP1A5.3 The N-terminal sequence of the turkey CYP1A5 contained the typical endoplasmic reticulum (ER) anchoring signal in the hydrophobic region, followed by a halt-transfer signal containing several positively charged amino acids (25). The heme-binding motif, usually represented as FXXGXXXCXG, was identified in CYP1A5 as FGLGKRRCIG (amino acids 460-469). Other potential secondary modification sites found in CYP1A5 included two casein II phosphorylation sites (SVLE, amino acids 60-63; TAVE, amino acids 273-276), an N-linked glycosy3 The nucleotide sequence for the turkey CYP1A5 gene has been deposited in the GenBank database under GenBank accession number AY964644. The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number AAX73011.

lation site (NFSI, amino acids 153-156), a tyrosine kinase phosphorylation site (KQSFDPYRY, amino acids 190-198), an amidation site (DGKK, amino acids 494-497), a cAMP/ cGMP protein kinase phosphorylation site (KRFS, amino acids 520-523), and a protein kinase C phosphorylation site (SMK, amino acids 523-525) (Figure 2). Bacterial Expression. As shown previously (26), wild-type eukaryotic P450s without N-terminal modifications often fail to express in E. coli. Predictably, there was no detectable P450 expression for the wild-type construct, as measured by the reduced CO/reduced difference spectrum. However, Western blots using the anti-His tag antibody revealed a low level of expression from wtCYP1A5 (Supporting Information, Figure S1). Furthermore, E. coli cultures expressing wtCYP1A5 showed the formation of indigo, indicating the metabolism of indole by P450 (27). The hydropathy plot of the native sequence showed that approximately the first 30 amino acids were in the hydrophobic membrane-anchoring region. To increase the solubility and expression level of the protein, we truncated 29 amino acids from the N-terminal of CYP1A5 and created tCYP1A5 (as described in the Experimental Procedures). This approach resulted in a relatively high level of expression, with 200-300 nmol tCYP1A5 per liter culture, as estimated by reduced CO/reduced difference spectrum using whole cells (16). After membrane and cytosol isolation, approximately 30% of the tCYP1A5 was recovered in the membrane fraction, and 3050% was in the cytosol fraction. Membranes from E. coli expressing tCYP1A5 also expressed active hNPR, a redox

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Yip and Coulombe

Figure 3. Purification of truncated turkey CYP1A5 (tCYP1A5) for polyclonal antiserum production and immunodetection by the antiserum. (A) Coomassie-stained SDS-PAGE gel showing final eluates (E1E3) from tCYP1A5 purification were >90% homogeneous. (B) Immunoblotting using rabbit polyclonal antiserum raised against purified tCYP1A5. The antiserum detected a major band about 60 kDa in both microsomes and tCYP1A5 E. coli membranes. The minor band that was shown at around 80 kDa was presumably a bacterial protein, which was present in both E. coli membrane samples: tCYP1A5 and pCWOri+, the empty vector control.

partner necessary for P450-mediated catalysis. The activity of hNPR, as measured by reduction of cytochrome c, was 1.24 µmol/min/mg total protein. As compared to the activity of purified hNPR expressed in E. coli from the same plasmid (50 µmol/min/mg),4 we determined that hNPR was comprised of 2.5% (w/w) of the proteins in tCYP1A5 E. coli membrane (vs 7.6% of P450). This corresponds to a hNPR-to-P450 molar ratio of about 1:4. Background cytochrome c reduction activity in membranes from the empty vector, pCWOri+, was found to contain less than 0.1% of that of the tCYP1A5 membranes. Hence, we considered nonhNPR reductase activity in this bacterial system negligible. Immunoblot using anti-rat NADPHP450 reductase polyclonal antibody also confirmed the expression of hNPR (data not shown). Purification of Recombinant Turkey CYP1A5. We purified tCYP1A5 from E. coli cytosol, which contained a substantial amount of soluble and enzymatically active protein as judged by reduced CO/reduced difference spectrum and the results of AFB1 oxidation (data not shown). The final eluate was approximately 90% homogeneous and free of degradation product (Figure 3A). The total yield was estimated to be 4.5 mg from 500 mL of culture. Immunodetection. The rabbit polyclonal antiserum against the E. coli expressed and purified tCYP1A5 detected a major band in turkey microsomes, with an apparent molecular mass of 55 kDa, and in tCYP1A5 E. coli membranes, with an apparent molecular mass of 58 kDa (Figure 3B). Quantative Western blot showed that CYP1A5 comprised of approximately 1.6% (mol/ mol) of the total amount of P450 in turkey liver microsomes (data not shown). This antiserum, however, was not immunoinhibitory against AFB1 oxidation by either bacterial expressed tCYP1A5 or turkey liver microsomes. O-Dealkylation of CYP1A Prototype Substrates and Inhibition by Specific P450 Inhibitors. Because of its sequence similarity to other CYP1As, tCYP1A5 was tested for Odealkylation activity against methoxyresorufin and ethoxyresorufin, prototype substrates for mammalian P450 1A2 and 1A, respectively. The tCYP1A5 E. coli membranes showed an 4

Aust, S. D., and Davis, T. Z. Personal communication.

Figure 4. (A) Effect of specific mammalian P450 inhibitors on MROD activity catalyzed by tCYP1A5-expressing E. coli membranes. Inhibitors used were as follows: erythromycin (b) for CYP3A1, quinidine (O) for CYP2D, 4-methylpyrazole (1) for CYP2E, 17R-ethynylestradiol (3) for CYP3A4, and RNF (9) for CYP1A1 and 1A2. (B) Inhibitory effect of RNF on MROD activity (9) and on AFB1 oxidation to form exo-AFBO (b) and AFM1 (3). Each data point represents the mean of duplicate determinations.

MROD activity of 29 pmol/min/nmol P450 and a similar EROD activity of 28 pmol/min/nmol P450. Figure 4A shows that at 10 µM inhibitor concentration, the specific CYP1A inhibitor, RNF, completely inhibited MROD activity catalyzed by tCYP1A5. At the same concentration, 17R-ethynylestradiol (3A4) had little, while quinidine (2D), 4-methylpyrazole (2E), and erythromycin (3A1) had almost no inhibitory effect. Importantly, the inhibitory effect of RNF was not limited to oxidation of prototype substrates. As shown in Figure 4B, the inhibitory effect of RNF on AFB1 oxidation by tCYP1A5 [as determined by the rates of AFB1 exo-8,9-epoxide (exo-AFBO) and AFM1 formation as compared to controls] closely followed that of the MROD activity, with an IC50 of about 1 µM. AFB1 Oxidation by Bacterial Expressed Turkey CYP1A5. The tCYP1A5 expressed in E. coli membranes possessed substantial AFB1 oxidizing activity, with the formation of two principal metabolites, exo-AFBO and AFM1, at a molar ratio of about 1:2 (Figure 5). As mentioned previously (28), mouse liver cytosol conjugated the exo-AFBO isomer almost exclusively. In our study, the formation of exo-AFBO and AFM1 from AFB1 oxidation conformed to simple Michaelis-Menten kinetics (Figure 6). The Km and Vmax values of exo-AFBO formation were 65 ( 12 µM and 0.61 ( 0.037 nmol/min/nmol P450, respectively (R2 ) 0.93), whereas those values of AFM1

Turkey Hepatic CYP1A5

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Figure 5. HPLC chromatogram showing the oxidation of AFB1 by turkey tCYP1A5 E. coli membranes to form exo-AFBO and AFM1, which were eluted at 6.59 and 13.28 min, respectively. Conditions of the experiment were as described in the Experimental Procedures except that 124 µM AFB1 and a 20 min incubation time were used. AFBO was detected as the stable, GSH-trapped conjugate (AFBO-GSH). AFG1 was injected as an internal standard. All of the unlabeled background peaks in the chromatogram were also present in the control (boiled membranes).

Figure 6. Michaelis-Menten plot for AFB1 oxidation by E. coli membranes expressing truncated turkey CYP1A5. Km and Vmax of exoAFBO formation are 65 ( 12 µM and 0.61 ( 0.037 nmol/min/nmol P450, respectively (b, R2 ) 0.93), whereas those values of AFM1 formation are 34 ( 9 µM and 0.91 ( 0.070 nmol/min/nmol P450, respectively (O, R2 ) 0.83). Each data point represents the mean of triplicate ((SE).

formation were 34 ( 9 µM and 0.91 ( 0.070 nmol/min/nmol P450, respectively (R2 ) 0.83).

Discussion Herein, we describe the cloning and heterologous expression of a P450 from turkey liver with AFB1 oxidizing activity. The CYP1A5 cDNA amplified from turkey liver had an open reading frame of 1587 bp encoding a protein of 528 amino acids and is predicted to be 60 kDa. The nucleic acid sequence of this gene was between 65 and 67% homologous with that of CYP1A1/ 1A2 genes from human, mouse, rat, and rabbit. Notably, the sequence of tCYP1A5 was 94.7% identical to that of a 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD)-inducible CYP1A5 from chicken liver (24). That membranes from E. coli expressing tCYP1A5 had activity toward prototypical CYP1A substrates and that it was completely inhibited by RNF lend further support for this P450 family designation.

Our results support the well-demonstrated utility of N-terminal modifications on the expression of membrane-bound proteins in E. coli (15). With codons 3-31 deleted, cells expressing tCYP1A5 produced a substantial amount of catalytically active P450. While E. coli cultures expressing wtCYP1A5 showed formation of indigo (27) and immunoblots using the anti-His tag antibody revealed the expression of wtCYP1A5 protein, the E. coli membranes isolated from this construct exhibited neither a measurable reduced CO/reduced difference spectrum nor an AFB1-oxidizing activity. Immunoblots showed that the predicted molecular mass of the native CYP1A5 in turkey microsomes (60 kDa) was higher than that estimated by SDS-PAGE (55 kDa). This discrepancy is probably due to the binding of a greater amount of SDS by the hydrophobic residues in the native protein than in other proteins (tCYP1A5 and molecular mass standard) in the gel, resulting in a higher electrophoretic mobility and subsequent lower molecular mass estimate for the P450 in turkey microsomes (29). The minor protein band (∼80 kDa) that we detected in membranes from both the negative control (pCWOri+ vector) and that from tCYP1A5 was presumably caused by the recognition of a contaminating bacterial protein by the antiserum. To ensure that the amount of NADPH-P450 reductase in the assays was sufficient for the P450 activity, we quantified hNPR by cytochrome c reduction. Our hNPR-to-P450 ratio in the E. coli membranes was 1:4, well above the previously reported ratio of 1:5-20 in mammalian liver microsomes (1, 30-32). Therefore, we consider that hNPR is unlikely to be a limiting factor for the P450 activity. Nevertheless, because the actual ratio of hNPR to P450 in turkey liver has not been determined, there remains a small chance that hNPR is not saturating in the tCYP1A5 reactions tested. Turkey CYP1A5 closely resembles human CYP1A2 in terms of its catalytic activities toward prototype substrates and AFB1, as well as its response toward specific inhibitors. For example, CYP1A5 possessed EROD activity that was similar to that measured for E. coli-expressed human CYP1A2 (12). Furthermore, the CYP1A5-mediated MROD activity and AFB1oxidizing activity were completely inhibited by RNF, a CYP1A class inhibitor. However, these observations must be tempered with the caveat that P450 prototype substrates as well as inhibitors used in this study have been validated for mammalian (mostly human) P450s. Their specificities for avian P450s have not been extensively characterized. This lack of complete species-to-species homology probably explains our observation that 17R-ethynylestradiol (a specific inhibitor of human CYP3A4) also partially inhibited CYP1A5. We have previously shown that this compound partially inhibits conversion of AFB1 to AFBO in microsomal incubations from turkey liver. Indeed, 17R-ethynylestradiol was recently demonstrated to inhibit CYP1A activity in Atlantic cod liver (33). Previous studies from our laboratory have demonstrated that efficient P450-mediated AFB1 oxidation is associated with the extreme susceptibility of turkeys to the toxic effects of this mycotoxin (11). Membranes from CYP1A5-expressing E. coli cells oxidized AFB1 to form two principal metabolites, the exoAFBO and AFM1. The former is generally regarded as the ultimate electrophilic intermediate responsible for the chronic adverse effects of AFB1, such as liver cancer, while the latter has been postulated to be an important mediator of shorterterm toxicity (34). In the case of poultry, the acute toxicity is more relevant to the adverse health impacts of AFB1. Although AFM1 is usually considered to be a detoxification product of AFB1, it is still a potent cytotoxin and genotoxin, which is active

36 Chem. Res. Toxicol., Vol. 19, No. 1, 2006

at relatively low doses and in the absence of metabolic activation (35-37). There are striking similarities between kinetic constants of AFB1 oxidation measured from tCYP1A5-expressing E. coli membranes and those reported for human CYP1A2 cDNA-expressed microsomes, which also oxidized AFB1 into AFBO and AFM1 (at a molar ratio of 2.5:1) (8). The Km values determined for CYP1A5 were very close to those of human CYP1A2 (34 vs 36 µM for AFM1; 64 µM for exo-AFBO in our study and 41 µM for AFBO in ref 8), although caution must be taken when comparing the AFBO kinetic data as the method employed by Gallagher et al. (8) did not distinguish the endoand exo- stereoisomers. In the same way, Vmax values for AFM1 formation of CYP1A5 and human CYP1A2 were nearly identical (0.91 vs 0.92 nmol/min/nmol P450), while that for exoAFBO formation in our study was more than 4-fold lower than that the AFBO formation measured for human CYP1A2 (0.61 vs 2.63 nmol/min/nmol P450). Consequently, the catalytic efficiencies (Vmax/Km) of both enzymes for AFM1 formation are almost identical, with the values being 0.027 for turkey CYP1A5 and 0.026 for human CYP1A2. As for AFBO formation (exoisomer alone in our case), the catalytic efficiency of turkey CYP1A5 is only 0.0095, about seven times lower than that of the human CYP1A2, which is 0.064. In conclusion, we report the identification of a novel P450, which we designated CYP1A5 based on the nucleic acid and amino acid sequence similarities with chicken CYP1A5. The gene product, which was heterologously expressed in sufficient quantities in E. coli, oxidized AFB1 to form two metabolites with kinetic properties largely similar to human CYP1A2, the P450 principally responsible for AFB1 oxidation in human liver. Because of the importance of these metabolites in the toxicity of AFB1, we conclude that this P450 probably plays some role in the hypersensitivity of turkeys to AFB1, although it comprises only a small portion of the total P450 content in turkey liver microsomes. We are currently exploring the development of antipeptide antibodies to estimate the contribution of this enzyme in overall AFB1 metabolism in turkey liver microsomes. To our knowledge, this is the first P450 cloned and sequenced from turkeys, the species in which the toxicity of AFB1 was first discovered. Acknowledgment. We thank Dr. Gary Yost, University of Utah, for his generous expert advice. This work was supported by USDA-NRI competitive Grant 2002-35204-12294 and by the Utah Agricultural Experiment Station, where this paper is published as number 7707. Supporting Information Available: Western blots showing expressions of wild-type and truncated turkey CYP1A5 in E. coli cell lysate. This material is available free of charge via the Internet at http://pubs.acs.org.

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