Materials.

Oligonucleotide primers were obtained from Cybergene AB (Huddinge, Sweden) and the University of Texas Medical Branch Recombinant DNA Laboratory, National Institute of Environmental Health Sciences Center (Galveston, TX). Restriction enzymes, Luria-Bertani Broth, and Terrific Broth were purchased from Life Technologies (Grand Island, NY). The pGEM5 vector was purchased from Promega (Madison, WI). The Expand High Fidelity PCR kit and Rapid Ligation kit were purchased from Roche (Indianapolis, IN). The GeneClean II kit was obtained from Bio101 (Carlsbad, CA). CHAPS, DLPC, DOPC, phosphatidylserine, tetracycline, ampicillin, δ-aminolevulinic acid, isopropyl β-d-thiogalactoside, and MOPS were purchased from Sigma (St. Louis, MO). 7-BFC was purchased from Gentest (Woburn, MA). Acrylamide was obtained from National Diagnostics (Atlanta, GA). Nitrocellulose, ammonium persulfate, and TEMED were purchased from Bio-Rad (Hercules, CA). Talon metal affinity resin and Marathon ready cDNA from human liver were purchased from CLONTECH (Palo Alto, CA), and Qiagen Ni-NTA from Qiagen (Valencia, CA). [¹⁴C]Progesterone was purchased from New England Nuclear (Boston, MA), and [¹⁴C]testosterone was obtained from Amersham Pharmacia (Piscataway, NJ). CYP3A5 cDNA was kindly provided by Dr. Frank Gonzalez (National Cancer Institute, Bethesda, MD). The cDNA was cloned into the expression plasmid pKK233–2 (obtained from Amersham Pharmacia) with an N-terminal modification described previously (Gillam et al., 1995) and a C-terminal 4X histidine tag to produce pKK3A5H. CYP3A5 was expressed in E. coli and purified as described previously for CYP3A4 (Domanski et al., 1998). CYP3A4 was purified previously (Domanski et al., 1998). Silica thin-layer chromatography plates were purchased from J.T. Baker (Phillipsburg, NJ), and other reagents and supplies were obtained from standard sources.

cDNA Cloning.

Human liver cDNA was prepared as described before (Finta and Zaphiropoulos, 2000a). RACE amplification (Frohman et al., 1988) on Marathon ready cDNA from human liver was performed in a two-step PCR approach using an initial and subsequently various nested primers, essentially following the recommendations of the kit, with Expand polymerase. A SalI site overhang was present in each of the nested primers, allowing cloning of the resulting products into a SalI-NotI digested pGEM5 vector. The primers used for PCR amplification were as follows: 3A4 exon 1 forward (position 30–48 in GenBank accession number M18907); 3A43 exon 1 forward, 5′ AAC TCA GAA GAC AGA GCT GAA A; 3A43 exon 1 forward, nested, 5′ GCG TCG ACT TTG CCA TGG AAA CAT GGG TTC; 3A43 exon 3 forward, nested, 5′ GCG TCG ACT TTG GAA TTT TGA CAG AGA ATG TAA TG; 3A43 exon 10 forward, nested, 5′ GCG TCG ACT GAT CTG GAG CTT GTG GCC CAG.

Full Length cDNA Construct.

The following forward and reverse primers were used with NotI and SalI overhangs, respectively: exon 1 forward, 5′ GCG CGG CCG CGA TGG ATC TCA TTC CAA ACT TT; exon 13 reverse, 5′ GCG TCG ACA AAG TCA GGG TCC ACT TGT AA. Human liver cDNA was amplified for thirty cycles using Expand polymerase for 1 min at 94°C, 1 min at 58°C and 1 min at 72°C with sequential 4 sec increases at each of the extension steps.

Analysis of CYP3A43 Tissue Distribution.

The human I, II, and fetal human cDNA panels were subjected to 25, 30, 35, and 40 cycles of PCR amplification for 5 s at 92°C, 20 sec at 52°C and 30 sec at 72°C using the following primers: exon 1 forward, 5′ ACA GAG CTG AAA AAG AAA AC; Exon 2 reverse, 5′ TAG AAC AAA ATA GTT CCC AG. Products started to be detected at cycle 35 and were more clearly visible after 40 cycles. The PCR products were cloned and sequenced to ensure that they represent 3A43 and not misprimed sequences.

Cloning CYP3A43 cDNA into the pSE380 Expression Vector.

For efficient expression in E. coli, PCR was used to modify the N terminus of CYP3A43. Amino acid residues 2 to 12 were deleted and residues 13 to 18 were modified to coincide with the corresponding sequence described by Barnes et al. for P450 17α (Barnes et al., 1991; Gillam et al., 1993). In addition, a 6X histidine tag was added at the C terminus and an NcoI site that interfered with cloning was removed (C to A at bp 540 of cDNA). A two-step PCR methodology was used. Plasmid pGEM5 containing the entire coding region of CYP3A43 was used as the template. In the first step, primer 5′NcoI, incorporating an NcoI site and the 17α sequence (5′ GCC GGC CCA TGG CTC TGT TAT TAG CAG TTT TTC TGG TAC TCC TCT ATA TTT ATG GG), and primer Δ NcoI, to remove an additional NcoI site (5′ GTG ATT ACA TCC ATT GTG TAG GCC CC), were used to produce a fragment containing 532 bp. This product, A, and primer 6X his (5′ CCG GGC ACT AGT TCA ATG GTG ATG GTG ATG GTG GGG TCC ACT TGT AAT CCC), were used in the second PCR, again using the 3A43-containing pGEM5 clone. The 1520-bp product of the second PCR was digested with NcoI and SpeI. The desired fragment was purified with the GeneClean II kit, ligated to pSE380 treated similarly, and transformed into DH5α cells. Resulting colonies were checked for the presence of the appropriately sized pS3A43H. DNA was purified with the Qiagen Midi kit and verified by sequencing (Protein Chemistry Laboratory, University of Texas Medical Branch, Galveston, TX).

Heterologous Expression and Purification of CYP3A43.

CYP3A43 was expressed in E. coli as described previously (Domanski et al., 1998) with the following modifications. Plasmid pS3A43H was transformed into TOPP3 cells. Single colonies were inoculated into 2 ml of Luria-Bertani broth containing ampicillin (50 μg/ml) and tetracycline (15 μg/ml). The cultures were grown overnight at 37°C. A 1/1000 dilution of culture was inoculated into 20 ml of Luria-Bertani media containing ampicillin (50 μg/ml) and tetracycline (15 μg/ml) and was grown as described above. Fifteen microliters of this culture was added to 250 ml of TB media containing ampicillin (50 μg/ml). The cultures were grown 2 to 2.5 h at 37°C at 250 rpm, after which δ-aminolevulinic acid (80 mg/L) and isopropyl β-d-thiogalactoside (1 mM) were added. The flasks were incubated at 30°C for 72 h at 190 rpm.

Solubilized membranes were prepared as described previously (John et al., 1994; Richardson et al., 1995; Harlow and Halpert, 1997). For preparations that were used for purification, the procedure was performed with the modifications outlined previously (Domanski et al., 1998). His-tagged CYP3A43 was purified by column chromatography usng Qiagen Ni-NTA Agarose. Resin (1 ml) was equilibrated with 5 ml of buffer A (100 mM MOPS, pH 8.0, 10% glycerol) plus 0.5% CHAPS and 0.5 M KCl. Solubilized membrane preparations of CYP3A43 from 12 250-ml cultures were loaded onto the column at 9 ml/h. The column was then washed with 10 bed volumes of buffer A plus 0.5% CHAPS and 0.5 M KCl followed by 10 bed volumes of buffer A alone. CYP3A43 was eluted with buffer A containing 0.5 M imidazole. The P450-containing fraction was dialyzed overnight in two 1-l changes of MOPS buffer (100 mM MOPS, pH 7.3, 10% glycerol, 0.2 mM dithiothreitol, 1 mM EDTA). The P450 content was determined by reduced carbon monoxide difference spectra.

Western Blot Analysis.

Purified samples of CYP3A4, CYP3A5, and CYP3A43 (1 pmol per lane of each) were run separately, in pairs, or in a mix of all three on an 8.5% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose and probed with anti-3A12 IgG as described previously (Ciaccio and Halpert, 1989; Kedzie et al., 1991). Human anti-3A4 IgG, raised against a C-terminal peptide, was obtained from Gentest and immunoblotting was performed according to the manufacturer's instructions.

Steroid Hydroxylase Assays.

CYP3A43 was assayed under a number of conditions. All assays were carried out with 10 pmol of P450 in 100 μl with a final methanol concentration of 1% for substrate delivery. Except for the cumene hydroperoxide-dependent assays, the reactions were started by the addition of 1 mM NADPH. All incubations were carried out for 5 to 10 min, the reactions were stopped with 50 μl of tetrahydrofuran, and the metabolites were resolved by thin-layer chromatography as described previously (Domanski et al., 1998). Steroid hydroxlyase assays contained 50 to 150 μM [¹⁴C]progesterone or 50 to 200 μM [¹⁴C]testosterone. First, some reactions were carried out in 50 mM HEPES, pH 7.6, containing 15 mM MgCl₂, 0.1 mM EDTA, and 0.04% CHAPS (Domanski et al., 1998). These reconstitutions contained a range of molar ratios of P450 to rat NADPH-P450 reductase (1:2 to 1:8), varying molar ratios of P450 to cytochrome b ₅ (1:0 to 1:2), and 0.1 mg/ml DOPC. A second set of reactions was run in 50 mM HEPES, pH 7.6, plus 30 mM MgCl₂, 0.1 mM EDTA, and 0.02% sodium cholate (Ueng et al., 1997). These reconstitutions included P450 to rat NADPH-P450 reductase molar ratios of 1:2 and 1:4, molar ratios of P450 to cytochrome b ₅ of 1:0 and 1:2, and 0.1 mg/ml of a 1/1/1 mix of DOPC, DLPC, and phosphatidylserine. Third, other reactions were conducted in 0.1 M KPO₄buffer, pH 7.4, containing 0.04% CHAPS (Imaoka et al., 1992). These reconstitutions contained a range of molar ratios of P450 to rat NADPH-P450 reductase (1:4 to 1:8), varying molar ratios of P450 to cytochrome b₅ (1:0 to 1:2), and 0.1 mg/ml of a 1/1/1 phospholipid mix. Fourth, cumene hydroperoxide-dependent assays were conducted. The reactions contained either 50 mM HEPES, pH 7.6, 30 mM MgCl₂, and 0.1 mM EDTA (Ueng et al., 1997) or 0.1 M KPO₄, pH 7.4, and 0.1 mg/ml 1/1/1 phospholipid mix (Imaoka et al., 1992). The reactions were started by the addition of 150 μM cumene hydroperoxide in acetone (1% final concentration).

cDNA Cloning.

Using a primer from the 5′ untranslated region of the 3A4 cDNA (see Experimental Procedures) in RT/PCR amplification from human liver RNA, a PCR product was obtained that encompassed the complete coding sequence of an exon 1 and differed from the known sequences of 3A4, 3A5, and 3A7. Because this exonic sequence had a methionine codon at the equivalent position as the known CYP3As and an open reading frame, it was likely to represent part of a novel 3A P450. To obtain the remaining sequence, RACE amplification was performed using primers designed from this exon 1. Twenty cloned products were analyzed from human liver but none extended beyond exon 3 of this new 3A P450. To obtain additional sequence information, RACE was performed using a nested primer from the newly identified exon 3. The longest clones obtained extended up to exon 10, and this sequence information was used to design an additional nested primer that allowed the cloning of the remaining sequences, including the last exon, exon 13. The coding sequence of this P450 and its comparison with 3A4, 3A5, and 3A7 are shown in Fig. 1. This sequence is identical with a putative cytochrome P450 genomic sequence recently identified within the High Throughput Genomic Sequence Database (http://drnelson.utmem.edu/Nomenclature.html). According to the standardized P450 nomenclature, the name assigned for this P450 is 3A43.

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Figure 1

Amino acid sequence comparison of 3A43 with 3A4 (GenBank accession number M18907), 3A5 (GenBank accession numberJ04813), and 3A7 (GenBank accession number D00408). The underlined sequence shows the region that was modified to ALLLAVF for bacterial expression. The underlined and shaded residues are those that have previously been studied and found to play a role in CYP3A4 substrate selectivity. The bold, italicized residues in the CYP3A43 amino acid sequence are those that differ from CYP3A4 at such sites.

Amino Acid Sequence Alignment of CYP3A43 with CYP3A4, 3A5, and 3A7.

An amino acid alignment of CYP3A43 with the other members of the human CYP3A subfamily, illustrates that although the four P450s show significant similarity, there are notable differences. For example, CYP3A43 differs significantly from the other forms at the N terminus (underlined sequence in Fig. 1). Structure-function analyses of CYP3A4 have so far identified 17 residues that are important for substrate specificity/regioselectivity (Fig. 1, shaded, underlined text) (Harlow and Halpert, 1997; He et al., 1997; Domanski et al., 1998; Wang et al., 1998). Of these, CYP3A43 differs from CYP3A4 at six sites (Fig. 1, italic, bold text). Of these 17 sites of known importance, CYP3A43 contains unique residues at two sites conserved in the other members of the human CYP3A subfamily (V370 and V373) (Fig.1). CYP3A also differs from CYP3A4 and/or CYP3A5 at four additional sites that are important for regioselectivity of steroid hydroxylation (L108, V369, D478, and N479) (Wang et al., 1998). In addition, a number of other putative substrate recognition site residues vary between CYP3A43 and the other CYP3A members.

Tissue Distribution of CYP3A43.

The expression level of3A43 in human liver is not very high, making it difficult to obtain clear signals in a dot blot assay with mRNA from human liver and from other tissues. Specifically, the Human Multiple Tissue Expression Array (Clontech) was negative when probed with sequences from 3A43. However when cDNA panels from adult and fetal human tissues were subjected to PCR amplification, 3A43 expression was detected in adult liver, kidney, pancreas, and prostate, as well as in fetal liver and skeletal muscle. The results of this tissue distribution analysis are shown in Fig. 2.

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Figure 2

Distribution of CYP3A43 mRNA in various (a and b) adult and (c) fetal tissues. The products of a 40-cycle PCR amplification with CYP3A43 primers (see Experimental Procedures) on adult and fetal human cDNA panels (CLONTECH) were analyzed by a 3% agarose gel electrophoresis.

cDNA Products Generated by the Use of Exons 1 and 13 PCR Primers.

The 3A43 sequence obtained from the RACE analysis was not represented in a single clone, because three sequential RACE amplifications had been performed. To obtain a single cDNA clone containing the contiguous coding sequence of 3A43, PCR amplification was performed on human liver cDNA using primers encompassing the methionine and the termination codons (see Experimental Procedures). Fourteen clones were analyzed, and one was identified with no nucleotide misincorporation that would alter the reading frame. The single base change present at codon 243, AAG instead of AAA, does not alter the corresponding amino acid, Lys. Interestingly, eight of the analyzed clones did not encompass the contiguous coding sequence of 3A43. Instead, each of these revealed one or more splicing events that distinguished them from the canonical 3A43 mRNA. Two clones were found to lack exon 12 and are therefore likely to code for a nonfunctional P450, because the conserved cysteine residue would be absent and the change in the reading frame in exon 13 would result in premature termination. Exon 4 was found to be skipped in four clones, and this event would also change the reading frame. The complete intron five was found in two clones, as judged by the identity with GenBank accession number AC011904 that contains the complete 3A43 gene. The 58 bases positioned at the 5′ end of intron 5 (a sequence followed by GT intronic dinucleotides in GenBank accession number AC011904) were present in three clones. A 121-base Alu-like sequence from intron 7 (position 30765–30885 in GenBank accession number AC011904, flanked by AG and GT intronic dinucleotides) was found in two clones. Collectively these observations provide evidence that at a high frequency (8 of 14), transcripts distinct form the canonical 3A43 mRNA are generated from the 3A43 locus.

Expression and Purification of CYP3A43.

The N terminus of CYP3A43 was truncated (amino acids 2–12) and modified to MALLLAVF to correspond to that found in CYP17α (Barnes et al., 1991; Gillam et al., 1993). This sequence has been found to significantly increase the heterologous expression of a number of mammalian cytochromes P450 (Gillam et al., 1993; John et al., 1994; Richardson et al., 1995;Harlow and Halpert, 1997). When CYP3A43 was expressed in E. coli TOPP3 cells, the protein expression level was relatively low (4–28 nmol of P450/liter of culture) compared with the expression typically observed with CYP3A4 (100–300 nmol P450/liter of culture). The absorbance peak of the Fe²⁺-CO complex was found to be at 450 nm (data not shown).

For purification of CYP3A43, solubilized preparations from 12 separate 250-ml cultures were combined to produce sufficient protein (18–20 nmol) to undergo column purification. In a first attempt, Talon Metal Affinity Resin was used. In this instance, more than 80% of the protein bound to the column but would not elute, although a final concentration of up to 1 M imidazole was added. Instead, Qiagen Ni-NTA was used to purify CYP3A43 (see Experimental Procedures). CYP3A43 was eluted in buffer containing 500 mM imidazole and dialyzed in MOPS buffer, pH 7.3. The total yield of CYP3A43 was 18.7%. CYP3A43 was analyzed by SDS-polyacrylamide gel electrophoresis to ensure a similar level of purity compared with CYP3A4 (Fig.3a).

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Figure 3

Analysis of CYP3A43 protein. (a) SDS-polyacrylamide gel electrophoresis. Metal affinity-purified samples of CYP3A4 and CYP3A43 (1 pmol) were run on an 8.5% polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue. (b) Immunoblot analysis of CYP3A4, CYP3A5 and CYP3A43. Each P450 (1 pmol) was run on an 8.5% polyacrylamide gel separately, in pairs, or all mixed. Proteins were transferred to nitrocellulose and probed with anti-3A12 IgG.

Western Blot Analysis of CYP3A43.

As reported by others, CYP3A4 and CYP3A5 have different electrophoretic mobilities on polyacrylamide gels and in Western blot analysis (Aoyama et al., 1989). In this study, equal amounts of CYP3A4, CYP3A5, and CYP3A43 were run together, in pairs, and singly on a SDS-PAGE. After transfer to nitrocellulose and probing with an anti-3A12 polyclonal antibody, CYP3A43 separated from CYP3A5, but CYP3A4 and CYP3A43 were found to comigrate (Fig. 3b). Both CYP3A4 and CYP3A43 were recognized by anti-3A4 IgG that was raised against a C-terminal peptide (data not shown).

Functional Characterization of CYP3A43.

Neither solubilized membrane preparations nor purified samples of CYP3A43 displayed appreciable testosterone or progesterone hydroxylase activity when reconstituted according to our standard procedure for CYP3A4 and 3A5 (Domanski et al., 1998) (data not shown). Consequently, a number of additional assay conditions were tested. Each condition was derived from previous studies (Imaoka et al., 1992; Ueng et al., 1997) (seeExperimental Procedures) and is similar to methods used by others for CYP3A4, CYP3A5, and CYP3A7 (Gillam et al., 1993, 1995,1997). Conditions that resulted in a relatively low, but reproducible level of testosterone 6β-hydroxylase activity by CYP3A43 are presented in Table 1. When similar conditions were used in progesterone hydroxylase and 7-BFC debenzylase assays, significant levels of CYP3A43 activity were not observed (data not shown).