Cloning and Tissue Distribution of a Novel Human Cytochrome P450 of the CYP3A Subfamily, CYP3A43

Biochemical and Biophysical Research Communications 281, 1349 –1355 (2001) doi:10.1006/bbrc.2001.4505, available online at http://www.idealibrary.com on

Cloning and Tissue Distribution of a Novel Human Cytochrome P450 of the CYP3A Subfamily, CYP3A43 Anna Westlind,* Sarah Malmebo,* Inger Johansson,* Charlotta Otter,† Tommy B. Andersson,‡ Magnus Ingelman-Sundberg,* ,1 and Mikael Oscarson* *Division of Molecular Toxicology, IMM, Karolinska Institutet, SE-171 77 Stockholm, Sweden; and ‡AstraZeneca Mo¨lndal R & D, DMPK & BAC, and †Molecular Biology, AstraZeneca R & D, S 431 83 Mo¨lndal, Sweden

Received February 1, 2001

On the basis of the detection of an expressed sequence tag (‘EST’) similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism. © 2001 Academic Press Key Words: CYP3A4; drug metabolism; testis; HEK 293 cells; hepatoma cells; RACE.

The cytochrome P450 (CYP) enzymes in the 1–3 families are the principal hepatic enzymes responsible for A preliminary presentation of part of this paper has been made in an abstract at the 13th International meeting on Microsomes and Drug Oxidations, Stresa, Italy, July 10 –14, 2000, Abstract No 354, p. 198. The sequence reported in this paper has been deposited in GenBank under Accession No. AF337813. Abbreviations used: CYP, cytochrome P450; bp, base pair(s); PCR, polymerase chain reaction; EST, expressed sequence tag; huPO, human acidic ribosomal phosphoprotein; RACE, rapid amplification of cDNA ends. 1 To whom correspondence should be addressed at Division of Molecular Toxicology, IMM, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden. Fax: ⫹46-8-33 73 27. E-mail: [email protected].

the metabolism of xenobiotics. Among them, CYP2C9, CYP2D6, and CYP3A4 are of major importance for metabolism of clinically used drugs (1). CYP3A4 is the most abundant of all cytochrome P450s in the human liver (2). Three functional members of the CYP3A subfamily have been identified, namely CYP3A4, CYP3A5, and CYP3A7 and the corresponding genes are localized in tandem on the long arm of chromosome 7. The CYP3A7 protein appears to be expressed mainly in fetal liver (3) but also in adult endometrium and placenta (4). CYP3A5 shares an amino acid sequence identity of 83% with CYP3A4 and is polymorphically expressed in only 10 –30% of human livers (5–7) and then at a much lower level than CYP3A4. It appears, however, that CYP3A5 is the main CYP3A form in human kidney (8). The CYP3A enzymes have very broad and overlapping substrate specificities. They catalyze, e.g., the 6␤hydroxylation of a number of steroids including testosterone, progesterone, and cortisol. Furthermore, CYP3A is involved in the metabolism of more than 50% of all drugs in use today (1, 9), including macrolide antibiotics, calcium channel blockers and the immunosuppressive agent cyclosporin A. Because of the broad substrate spectrum coupled with their inducibility, CYP3As are important factors for drug– drug interactions observed clinically in patients undergoing multidrug therapy (10 –12). For further understanding of drug response and mechanisms of interaction and drug metabolism in general, the identification of all human members of the cytochrome P450 superfamily is of great importance. Additionally, information of expressed homologous genes (with or without active protein counterpart) is vital in order to ensure the quality of future research studies. We therefore searched for yet unidentified P450s homologous to known P450s in EST (expressed sequence tag) databases. A human EST was identified that encodes a fragment of a protein related to the CYP3A subfamily. This EST formed the basis for cDNA

1349

0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Vol. 281, No. 5, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cloning of a novel cytochrome P450 3A gene and its sequence, heterologous expression and expression in human tissues are described. MATERIALS AND METHODS Bioinformatics. The BLASTN and TBLASTN search algorithms (13) were used to search the dbEST database (14) for novel sequences related to the CYP3A4 cDNA. cDNA cloning of CYP3A43. Information from the human EST (IMAGE ID 731237) with an insert of approximately 900 bp, originating from a testis library and encoding a fragment related to CYP3A4, was used to design primers for 5⬘-RACE in order to generate a full-length cDNA sequence. A first strand cDNA was constructed from a mix of human liver RNA from three different individuals (HL 38, HL 39, and HL 41, see (15)) using the SMARTRACE cDNA amplification kit (Clontech, Palo Alto, CA). The full-length cDNA was obtained performing a 5⬘-RACE PCR reaction with the universal primer mix (UPM) and the gene specific primer 5⬘-CAGTTTCGGGGTCCAGCTCCAAAAGGTA-3⬘ followed by a nested PCR using the nested primer NUP and 5⬘-TAATGAAGGGGAGAGTGGTGCTAGTTGTG-3⬘ or 5⬘-CCAGTTCATACATAATGAAGGGGAGAGTGG-3⬘. For 5⬘-RACE and nested PCRs, the Advantage2 PCR kit (Clontech) was used. The PCR product was cloned directly into the T/A-type cloning vector pAdv (Clontech). Positive clones were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA) and analyzed on an ABI PRISM 377 DNA sequencer (Applied Biosystems). Cloning of the CYP3A43 cDNA into the V60 (pYeDP60) expression vector. The full-length CYP3A43 cDNA was first subcloned into the vector Bluescript KS. The insert was generated by PCR amplification using human liver cDNA, the forward primer 5⬘-AGAAAAACTCAGAAGACAGAGCTG-3⬘ and the reverse primer 5⬘-ATACAGCTTTCTTGAACAAAGTG-3⬘. The resulting fragment served as template for a second PCR amplifying the coding region with the forward primer 5⬘-GATCTCTAGAAGATCTAAAATGGATCTCATTCCAAACTTTGCCA-3⬘ containing an XbaI/BglII linker sequence and introducing an AAA sequence in front of the translation start, as this has previously been shown to increase expression efficiency in yeast (16), and the reverse primer 5⬘-GATCGGTACCTCAGGGTCCACTTGTAATC-3⬘. The PCR product was subcloned into Bluescript KS using the restriction enzymes XbaI and KpnI and subjected to partial digestion with BglII and full digestion with KpnI to enable subsequent cloning into the expression vector V60 using the restriction enzyme sites BamHI and KpnI. Two separate cloning experiments were done in parallel using this cloning procedure. The sequence of the insert was examined by automated DNA sequencing as described above. Heterologous expression of CYP3A43 and CYP3A4 cDNA in yeast. The yeast strain W(R), engineered to overexpress the yeast NADPH cytochrome P450 reductase (17, 18), was transfected with the V60CYP3A43 (see above) and V60-CYP3A4 (19) plasmids. The V60 vector without insert was used in control experiments. Heterologous expression of CYP3A43 and CYP3A4 was carried out as previously described (18, 20) except that yeast microsomes were prepared by differential centrifugation (20,000g, 10 min plus 100,000g, 60 min at 4°C). The total protein content and the cytochrome P450 content was estimated as described elsewhere (21, 22). Heterologous expression of CYP3A43 and 3A4 in COS-1, HEK 293 and H2.35 cells. The CYP3A43 BglII-KpnI fragment described above was subcloned into the mammalian expression vector pCMV5 (23) digested with the same enzymes. A CYP3A4 cDNA was amplified using the forward primer 5⬘-CAGGGTACCATGGCTCTCATCCCAGACTTGGCCA-3⬘ and the reverse primer 5⬘-CAGTCTAGATCAGGCTCCACTTACGGTGCCAT-3⬘. After digestion with the KpnI and XbaI restriction enzymes, the CYP3A4 fragment was subcloned

into the pCMV5 vector. Both constructs were sequenced to exclude any PCR artefacts. The expression vectors were transfected into three different cell lines: the SV40 transformed African green monkey kidney cell line COS-1 (CRL-1650, ATCC, Manassas, VA), the adenovirus 5 transformed human embryonic kidney cell line 293 (HEK 293; CRL-1573, ATCC) and the SV40 transformed mouse hepatocytes H2.35 (CRL-1995, ATCC). COS-1 cells were maintained in Dulbecco’s modified Eagle medium (with 1000 mg/l glucose) containing 10% FBS, 100 ␮g/ml streptomycin and 100 U/ml penicillin, and transfected using the DEAE-dextran method as previously described (24, 25). HEK 293 cells were maintained in modified Eagle medium containing 10% FBS, 100 ␮g/ml streptomycin, 100 U/ml penicillin and Non Essential Amino Acids (Life Technologies, Rockville, MD). When the cells had reached ⬎90% confluency, they were transfected using 3 ␮l/ml Lipofectamin 2000 (Life Technologies) and 12 ␮g of plasmid DNA per 10 cm plate for 6 h according to the manufacturer’s instructions. H2.35 cells were maintained in Dulbecco’s modified Eagle medium (with 1000 mg/l glucose) containing 4% FBS, 100 ␮g/ml streptomycin, 100 U/ml penicillin (Life Technologies) and 0.2 ␮M dexamethasone (Sigma, St Louis, MO). When the cells had reached 70% confluency, they were transfected using 3 ␮l/ml DMRIE-C (Life Technologies) and 18 ␮g of plasmid DNA per 10 cm plate for 5 h according to the manufacturer’s instructions. After 24 (H2.35) or 60 – 65 (COS-1 and HEK 293) h, the cells were harvested in 0.1 M sodium phosphate buffer (pH 7.4) and cell homogenates were prepared by sonication for 20 ⫻ 2 s. The protein concentration was determined by the method of Lowry et al. (22). Western blotting. Cell homogenates corresponding to 40 ␮g of protein (COS-1) or 30 ␮g of protein (HEK 293 and H2.35) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 8.5% gels. Human Lymphocyte-Expressed CYP3A4 (1 ␮g, Gentest, Woburn, MA) was used as positive control. The proteins were transferred to a Hybond-C Nitrocellulose filter (Amersham Pharmacia Biotech, Uppsala, Sweden), incubated with anti-CYP3A antibody and with a corresponding horseradish peroxidaseconjugated secondary antibody/Protein A (Gentest; Dako, Carpinteria, CA; Bio-Rad Laboratories, Hercules, CA), and visualized by the SuperSignal West Pico Chemiluminiscence method (Pierce, Rockford, IL). The filters were scanned using LAS-1000 (Fujifilm, Fuji, Stockholm, Sweden). The following different CYP3A antibodies were used: Gentest Human CYP3A4/3A7 (polyclonal raised against a peptide specific for CYP3A4, Cat No A234), Gentest Human CYP3A4/ 3A5/3A7 (monoclonal, Cat. No. A254), Gentest Human CYP3A4/3A5 (polyclonal, Cat. No. 242496) and a polyclonal antibody raised against rat CYP3A1 (18). Multiple tissue cDNA panel. PCR was carried out on Human Multiple Tissue cDNA (MTC) Panels I and II, Human Tumor MTC Panel (Clontech), and cDNA synthesized from human liver total RNA, serving as positive control. A reaction without cDNA was run as a negative control in each experiment. The following gene specific primers were used: 5⬘-TGTTTCCAAAAGATGTTACCC-3⬘/5⬘-TTCTGCAGTGCTCTAATGAC-3⬘ (CYP3A43), 5⬘-CCATTCCTCATCCCAATTC-3⬘/5⬘-GAAGAAGTCCTCCTAAGCT-3⬘ (CYP3A4) and G3PDH Control Primers (Clontech). A total reaction volume of 25 ␮l contained 0.5 ng Clontech First-Strand cDNA or 0.5 ng human liver cDNA, 0.2 mM of each dNTP, 0.25 ␮M of each primer, 2.5 mM MgCl 2, 1⫻ Reaction Buffer IV and 0.625 U Taq DNA Polymerase (Life Technologies). The PCR reaction was performed by an initial denaturation step at 95°C for 1 min, after which the amplification was carried out for indicated number of cycles, with denaturation at 95°C for 15 s; annealing at 57°C for 20 s; and extension at 72°C for 1 min, ending with a final extension at 72°C for 7 min. The PCR products (12.5 ␮l) were separated on a 1% agarose gel stained with ethidium bromide. The PCR products generated with the CYP3A4 and CYP3A43 primer pairs were sequenced in order to ensure specificity of the primers.

1350

Vol. 281, No. 5, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1.

Nucleotide and predicted amino acid sequence of the CYP3A43 cDNA.

Real-time quantitative PCR detection. CYP3A4, CYP3A5, and CYP3A43 mRNA expression was quantified in 10 different livers, 6 obtained from the previously described liver bank (15) and 4 obtained from the International Institute for the Advancement of Medicine (Exton, PA). Total RNA was prepared as previously described (15). In order to eliminate amplification of contaminating chromosomal DNA in the real-time quantification, the isolated total RNA was treated with DNase using RQ1 RNAase-Free DNase (Promega, Madison, WI). Reverse transcriptase reactions were performed using the SuperScript Preamplification System for First Strand cDNA Synthesis (Life Technologies). Quantitative real-time PCR assay of transcripts was carried out with the use of gene specific double fluorescent labeled probes and

the TaqMan Universal PCR Mix in a 7700 Sequence Detector (Applied Biosystems, Norwalk, CT). VIC was used as the 5⬘-fluorescent reporter while tetramethylrhodamine (TAMRA) was added to the 3⬘end as a quencher. The following primers and probe sequences were used: CYP3A4 forward primer, 5⬘-CATTCCTCATCCCAATTCTTGAAGT-3⬘; CYP3A4 reverse primer, 5⬘-CCACTCGGTGCTTTTGTGTATCT-3⬘; CYP3A4 probe, 5⬘-VIC-CGAGGCGACTTTCTTTCATCCTTTTTACAGATTTTC-TAMRA-3⬘; CYP3A5 forward primer, 5⬘-GCTCGCAGCCCAGTCAATA-3⬘; CYP3A5 reverse primer, 5⬘AGGTGGTGCCTTATTGGGC-3⬘; CYP3A5 probe, 5⬘-VIC-TGAAACCACCAGCAGTGTTCTTTCCTTCAC-TAMRA-3⬘; CYP3A43 forward primer, 5⬘-AAT ACG AAC ATT GCT ATC TCC AGC T-3⬘; CYP3A43 reverse primer, 5⬘-GCT TCT CAC CAA CAT ATC TCC ACA T-3⬘;

1351

Vol. 281, No. 5, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 2. Amino acid sequence alignment of the cytochrome P450s in the CYP3A family. The predicted substrate recognition sites (SRSs) are indicated on top of the sequences (27, 29).

CYP3A43 probe, 5⬘-VIC-TTC ACC AGT GTA AAA TTC AAG GAA ATG GTC CC-TAMRA-3⬘. The primers or the probe were designed to span exon junctions in the fully processed mRNA in order to prevent detection of any possible contaminating genomic DNA. All primers and probes are purchased from Applied Biosystems. Standard curves were constructed with the use of serial 10-fold dilutions ranging from 1.0 fg to 10 pg, of an accurately determined concentration of a plasmid containing the cDNA of interest. The use of the endogenous reference huPO, as described in the TaqMan Human Endogenous Control Plate manual (Applied Biosystems), permitted normalisation for variation in reverse transcriptase efficiency in the cDNA reactions and differences in the amount of RNA added to each reaction. To ensure that the efficiency of the formation of the CYP3A4, 3A5, and 3A43 products within the samples were equal, standard curves with serial dilutions of the samples were made. Since the amplification of each target template, both when using the plasmid and the diluted liver samples, exhibited an equivalent rate of fluorescent emission intensity change per amount of target, plasmids were selected for standardization to determine the

arbitrary units of CYP3A4, 3A5, and 3A43. As the efficiency in each PCR amplification of the targets in the different livers was approximately equal and close to 1, no standard curves were needed for the relative quantification using the comparative C T method (⌬C T) as described in ABI PRISM 7700 User Bulletin #2. The relative abundance of the targets within the livers was the same using the standard curve or the comparative C T method. Each sample was analyzed in triplicate at two occasions, and the ratio of the amount of reporter dye emission to the amount of quenching dye emission (⌬Rn) and average threshold cycle (Ct) values were calculated for each reaction. Data was analyzed using the Sequence Detector V1.6 program (Applied Biosystems). Testosterone 6␤-hydroxylation assay. Yeast microsomes (500 ␮g) were incubated at 37°C for 30 min in 0.5 ml reaction mixtures with 100 mM potassium phosphate buffer (pH 7.4), in the presence of 200 ␮M testosterone (added in 5 ␮l ethanol). The incubations were done in duplicate or triplicate, with externally added cytochrome b 5 (Gentest), and with or without human reductase (Gentest) at about 120

1352

Vol. 281, No. 5, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pmol/mg of protein. The reactions were initiated with NADPH (1 mM) and terminated by removal to ice and addition of 2.5 ml of ethylacetate. Zero time incubations served as blanks, and blanks spiked with 6␤-hydroxytestosterone (Steraloids Inc, Newport, RI) served as external standards. After extraction, centrifugation and separation, the organic phase was removed and evaporated under nitrogen. The residues were dissolved in 150 ␮l of mobile phase. Twenty microliters of the samples were injected and analyzed on a ProStar HPLC system equipped with a ProStar 410 autosampler (Varian, Walnut Creek, CA). Testosterone and metabolites were resolved on a LiChrospher100 RP-18 prepacked column (Merck, Darmstadt, Germany) at ambient temperature and eluted with a mobile phase consisting of a mixture of acetonitrile: 0.5% acetic acid (33:67 v/v) at a flow rate of 1 ml/min. Both the product 6␤hydroxytestosterone and the substrate testosterone were monitored at 242 nm using a ProStar 310 UV-VIS detector (Varian) and quantified by comparison of their peak areas with those of the standards. The approximate retention times for 6␤-hydroxy-testosterone and testosterone were 3.7 and 20.7 min, respectively.

RESULTS AND DISCUSSION Using bioinformatic search algorithms, we detected a human EST clone that encodes part of a protein, closely related to CYP3A4, 3A5, and 3A7. This sequence was extended in the 5⬘-direction by RACE experiments in order to obtain the full-length cDNA sequence of this novel human CYP3A form. From the products of the 5⬘-extension reaction, a fragment of 1000 –1100 bp was cloned. Its sequence overlapped the EST and a final human cDNA sequence of 1785 nucleotides consisting of 103 bp 5⬘-untranslated region, followed by an open reading frame of 1512 bp could be assembled (Fig. 1). The start of the 5⬘-untranslated sequence corresponds exactly to the region where the transcription initiation site has been mapped in the CYP3A4 and CYP3A7 genes (26), indicating that transcription initiation is well-conserved within the CYP3A subfamily. The sequence generated by the 5⬘-RACE experiments together with sequencing of the EST, was confirmed in cloning experiments when the novel CYP3A was amplified in its full-length from reverse transcribed liver mRNA. The fact that two separate cloning experiments, done in parallel, produced identical nucleotide sequences verified that an authentic and accurate sequence had been identified. The novel CYP3A sequence shares 84, 83, and 82% sequence identity on the DNA level with CYP3A4, 3A5, and 3A7 respectively. After completion of the cDNA cloning, the genomic sequence has been generated (GenBank Accession No. AC069294) and the gene has been designated CYP3A43 (http://drnelson.utmem.edu/ CytochromeP450.html). Sequence comparison (Fig. 2) revealed a corresponding amino acid identity between the novel CYP3A sequence and CYP3A4, CYP3A5 and CYP3A7 of 76, 76, and 72%, respectively. Variability in the sequence of these members of the CYP3A subfamily is mainly seen in four regions of the protein, whereas in the CYP3A43 sequence additional nonconserved substitutions are observed in regions where the

TABLE 1

Expression of CYP3A4 and CYP3A43 in Different Human Tissues as Revealed by PCR Analysis Using Gene Specific Primers and MTC Panels (Clontech) Tissue

3A43

3A4

Brain Colon Heart Kidney Leukocytes Liver Lung Ovary Pancreas Placenta Prostate Skeletal muscle Small intestine Spleen Stomach Testis Thymus Breast carcinoma Colon adenocarcinoma Lung carcinoma Pancreatic adenocarcinoma Prostatic adenocarcinoma

36–40 nd 36–40 31–35 nd 21–25 36–40 nd 31–35 36–40 36–40 36–40 nd nd 36–40 26–30 36–40 36–40 nd nd 36–40 31–35

36–40 nd 36–40 26–30 nd 11–15 36–40 nd 31–35 36–40 nd 36–40 31–35 nd 31–35 31–35 36–40 36–40 36–40 36–40 36–40 36–40

Approximate ratio 3A43/3A4 1 — 1 0.05 — 0.001 1 — 1 1 ⬎100 1 ⬍0.001 — 0.05 20 1 1 ⬍0.01 ⬍0.01 1 20

Note. The number of cycles required to obtain a visible product band on an ethidium-bromide stained gel is given in the columns as well as an approximate ratio between the expression levels of CYP3A43 and CYP3A4 mRNA. nd, not detected, i.e., no product band could be visualized after 40 cycles of amplification.

other members of the CYP3A family have a conserved sequence. Many of the differences are in fact found in the predicted substrate-recognition sites (SRSs; (27)), suggesting that CYP3A43 might have a substrate specificity different from the other human CYP3A enzymes. Interestingly, CYP3A43 has a valine at position 370, whereas a conserved alanine is seen in the other enzymes. Alanine-370 has been shown to be important for the interaction of CYP3A4 with many of its substrates (28). Furthermore it appears that the sequence corresponding to exon 6, which includes the D-helix, is relatively unique compared to the other human CYP3As. The tissue distribution of CYP3A43 and CYP3A4 was analyzed using PCR, gene specific primers and cDNAs from various human tissues. These samples consist of cDNA pooled from several individuals and have been normalised to the levels of at least four different house-keeping genes, which allows for a quantitative comparison. As seen in Table 1 and Fig. 3, the highest relative levels of CYP3A43 mRNA were present in testis and liver but the mRNA was also seen at quite low levels in most of the tissues investigated including brain, lung, kidney, skeletal muscle, heart, pancreas, stomach, thymus, prostate, and placenta. Interestingly, the CYP3A43 EST clone was also found

1353

Vol. 281, No. 5, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 3. Relative expression levels of CYP3A43 (667-bp fragment) and CYP3A4 (772-bp fragment) in different human tissues as assessed by gene specific PCR amplification for 40 cycles.

in a testis library. In prostate, CYP3A43, but not CYP3A4 was detected, whereas only CYP3A4 was observed in small intestine. From the tumor samples analyzed, it appeared that CYP3A43 was expressed in breast carcinoma, pancreatic adenocarcinoma, and prostatic adenocarcinoma. Real time PCR was used to quantify the relative levels of CYP3A43 in 10 different livers as compared to the expression of CYP3A4 and CYP3A5. As seen in Table 2, CYP3A43 was significantly expressed in all but three livers. On average, the amount of CYP3A43 mRNA was approximately 0.1% of the CYP3A4 and 2% of the CYP3A5 level. The CYP3A5 expression level was about 34% of that of CYP3A4. The CYP3A43 expression did not significantly correlate to the levels of any of the other gene products, although the highest correlation was found to CYP3A4 mRNA (r ⫽ 0.34). CYP3A43 cDNA was subjected to heterologous expression in yeast, COS-1 cells, mouse hepatic H2.35 cells, and human embryonic kidney 293 (HEK 293) cells using CYP3A4 cDNA as a control. In yeast, CYP3A4 was well expressed and 122 pmol P450/mg microsomal protein was obtained, estimated by spectral analysis of the reduced CO-bound form, whereas microsomes from CYP3A43 transfected cells did not contain any measurable P450 in any of four independent transfection experiments. Incubations with testosterone showed that microsomes from CYP3A4 transfected yeast exhibited a catalytic activity of 84 ⫾ 3 pmol/min/mg when only external cytochrome b 5 was added and a rate of 675 ⫾ 62 pmol/min/mg when both external cytochrome b 5 and human cytochrome P450 reductase was added. In contrast, no testosterone 6␤-

hydroxylation activity was detected in microsomes from yeast transfected with CYP3A43. The absence of a P450 spectrum and testosterone hydroxylase activity in microsomes from yeast transfected with the CYP3A43 cDNA expression vector, indicates that the CYP3A43 protein does not properly fold in this expression system. Transfection of expression vectors into COS-1 cells showed strong CYP3A4 signals, but no CYP3A43 signals, when analyzed by Western-blotting using four different anti-CYP3A antibodies. Similar results were obtained in H2.35 cells and HEK 293 cells. Overall, these data indicate that CYP3A43, in contrast to CYP3A4, does not fold properly in any of the yeast and mammalian expression systems under the conditions used. The reason for this is presently unknown. The sequences of the expression plasmids were carefully analyzed and found to be correct. It might be that the enzyme is unable to incorporate heme, as seen in other cases for certain P450s in the CYP2 family, and therefore is nonfunctional. In particular the unique sequence of exon 6 in relation to other CYP3As might indicate another origin of this part of the gene by a gene conversion event, which has caused the basis for the formation of a nonfunctional gene product. Alternatively, the correct folding requires specific folding proteins or conditions that we have not been able to reproduce. Further research is directed towards this problem. In conclusion, we have cloned a new member of the human CYP3A subfamily and analyzed its expression in different tissues. It appears that the gene product is

TABLE 2

Real Time PCR-Based Quantification of CYP3A4, CYP3A5, and CYP3A43 mRNA in 10 Different Human Livers mRNA expression, arbitrary units Liver No.

3A4

3A5

Ratio a

3A43 3A5:3A4 3A43:3A4 3A43:3A5

HL 115 HL 117 HL 120 HL 121 HL 123 HL 126 HL 127 99-2864 99-2858 99-2667 99-2668

80792 2944 95982 1841 108190 13479 101726 10315 75588 5203 170456 8735 143248 6878 5846 8262 923857 8755 2450 2657 5727 4317

87 86 78 92 99 364 162 3 430 2 5

3.64 1.92 12.4 10.1 6.88 5.12 4.80 141 0.95 108 75.4

0.11 0.09 0.07 0.09 0.13 0.21 0.11 0.05 0.05 0.08 0.09

2.95 4.67 0.58 0.89 1.90 4.17 2.35 0.04 4.91 0.07 0.12

Average Std. dev.

155805 260700

128 142

33.7 50.2

0.09 0.044

2.05 1.88

6671 3630

a The ratios are the relation between the three transcripts in the individual samples which thus reflects the relation of the transcripts in each liver.

1354

Vol. 281, No. 5, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

well expressed in liver and testis, and it can be speculated that the enzyme has a physiological role in the latter tissue. We can conclude that CYP3A43, due to its relatively low expression level in the liver, would not contribute to drug metabolism at any higher extent. Functional analysis has to await the finding of a proper expression system. ACKNOWLEDGMENTS We are indebted to Dr Denis Pompon (Centre Nationale de Recherche Sientifique, Gif-sur-Yvette, France) for the Saccharomyces cerevisiae W(R) strain and the pYeDP60 expression vector. We also express our gratitude to Fredrik Vondracek for expression of CYP3A43 in HEK 293 cells, Åsa Nordling for help with Western blotting analyses, and Brith Leidvik and Anna B Johansson for help with real time PCR. This work was supported by grants from the Swedish Medical Research Council and from AstraZeneca.

REFERENCES 1. Bertz, R. J., and Granneman, G. R. (1997) Clin. Pharmacokinet. 32, 210 –258. 2. Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich, F. P. (1994) J. Pharmacol. Exp. Ther. 270, 414 – 423. 3. Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K., and Kamataki, T. (1990) Biochemistry 29, 4430 – 4433. 4. Schuetz, J. D., Kauma, S., and Guzelian, P. S. (1993) J. Clin. Invest. 92, 1018 –1024. 5. Aoyama, T., Yamano, S., Waxman, D. J., Lapenson, D. P., Meyer, U. A., Fischer, V., Tyndale, R., Inaba, T., Kalow, W., Gelboin, H. V., et al. (1989) J. Biol. Chem. 264, 10388 –10395. 6. Schuetz, J. D., Beach, D. L., and Guzelian, P. S. (1994) Pharmacogenetics 4, 11–20. 7. Wrighton, S. A., Ring, B. J., Watkins, P. B., and VandenBranden, M. (1989) Mol. Pharmacol. 36, 97–105. 8. Schuetz, E. G., Schuetz, J. D., Grogan, W. M., Naray-Fejes-Toth, A., Fejes-Toth, G., Raucy, J., Guzelian, P., Gionela, K., and Watlington, C. O. (1992) Arch. Biochem. Biophys. 294, 206 –214. 9. Li, A. P., Kaminski, D. L., and Rasmussen, A. (1995) Toxicology 104, 1– 8.

10. Periti, P., Mazzei, T., Mini, E., and Novelli, A. (1992) Clin. Pharmacokinet. 23, 106 –131. 11. Pichard, L., Fabre, I., Fabre, G., Domergue, J., Saint Aubert, B., Mourad, G., and Maurel, P. (1990) Drug Metab. Dispos. 18, 595– 606. 12. Murray, M. (1999) Int. J. Mol. Med. 3, 227–238. 13. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389 –3402. 14. Boguski, M. S., Lowe, T. M., and Tolstoshev, C. M. (1993) Nat. Genet. 4, 332–333. 15. Westlind, A., Lo¨fberg, L., Tindberg, N., Andersson, T. B., and Ingelman-Sundberg, M. (1999) Biochem. Biophys. Res. Commun. 259, 201–205. 16. Krynetski, E. Y., Drutsa, V. L., Kovaleva, I. E., and Luzikov, V. N. (1995) Pharmacogenetics 5, 103–109. 17. Truan, G., Cullin, C., Reisdorf, P., Urban, P., and Pompon, D. (1993) Gene 125, 49 –55. 18. Bellamine, A., Gautier, J. C., Urban, P., and Pompon, D. (1994) Eur. J. Biochem. 225, 1005–1013. 19. Masimirembwa, C. M., Otter, C., Berg, M., Jonsson, M., Leidvik, B., Jonsson, E., Johansson, T., Backman, A., Edlund, A., and Andersson, T. B. (1999) Drug Metab. Dispos. 27, 1117–1122. 20. Oscarson, M., Hidestrand, M., Johansson, I., and IngelmanSundberg, M. (1997) Mol. Pharmacol. 52, 1034 –1040. 21. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2370 –2378. 22. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265–275. 23. Andersson, S., Davis, D. L., Dahlback, H., Jo¨rnvall, H., and Russell, D. W. (1989) J. Biol. Chem. 264, 8222– 8229. 24. Zuber, M. X., Simpson, E. R., and Waterman, M. R. (1986) Science 234, 1258 –1261. 25. Johansson, I., Oscarson, M., Yue, Q. Y., Bertilsson, L., Sjo¨qvist, F., and Ingelman-Sundberg, M. (1994) Mol. Pharmacol. 46, 452– 459. 26. Hashimoto, H., Toide, K., Kitamura, R., Fujita, M., Tagawa, S., Itoh, S., and Kamataki, T. (1993) Eur. J. Biochem. 218, 585–595. 27. Gotoh, O. (1992) J. Biol. Chem. 267, 83–90. 28. Khan, K. K., and Halpert, J. R. (2000) Arch. Biochem. Biophys. 373, 335–345. 29. Wang, H., Dick, R., Yin, H., Licad-Coles, E., Kroetz, D. L., Szklarz, G., Harlow, G., Halpert, J. R., and Correia, M. A. (1998) Biochemistry 37, 12536 –12545.

1355