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Vol. 302, Issue 2, 416-423, August 2002


Genetic Polymorphisms of the Human CYP2A13 Gene: Identification of Single-Nucleotide Polymorphisms and Functional Characterization of an Arg257Cys Variant

Xiuling Zhang, Ting Su, Qing-Yu Zhang, Jun Gu, Michele Caggana, Hongming Li and Xinxin Ding

Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, New York

    Abstract
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References

Human cytochrome P450 2A13 (CYP2A13), which is highly efficient in the metabolic activation of a major tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), may play important roles in xenobiotic toxicity and tobacco-related tumorigenesis in the respiratory tract. The aim of this study was to identify any genetic polymorphisms of the CYP2A13 gene, which may alter the metabolic capacities of the enzyme. Polymerase chain reaction (PCR) single-strand conformational polymorphism analysis was used to identify single-nucleotide polymorphisms (SNPs) in all of the exons and at the exon-intron boundaries, and PCR-restriction fragment length polymorphism analysis and DNA sequencing were used to determine the frequencies of the newly identified variant alleles in the four major ethnic groups. Blood spot DNA from more than 100 individuals was used for these analyses. Seven variant alleles were found, but only one SNP was detected in the coding region, in exon 5, leading to an Arg257Cys amino acid change. The frequencies of the Arg257Cys allele in white, black, Hispanic, and Asian individuals are 1.9%, 14.4%, 5.8%, and 7.7%, respectively. Functional analysis of the variant protein was performed following its heterologous expression. The Arg257Cys variant was 37 to 56% less active than the wild-type Arg-257 protein toward all substrates tested. With NNK, Cys-257 had higher Km and lower Vmax values than did Arg-257, with a >2-fold decrease in catalytic efficiency. The Arg257Cys mutation could provide some protection against xenobiotic toxicity in the respiratory tract to individuals who are homozygous for the Cys-257 allele.

    Introduction
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References

Two functional genes have been identified in the human CYP2A gene subfamily. They are CYP2A6 and CYP2A13 (Fernandez-Salguero and Gonzalez, 1995; Su et al., 2000), which share a 93.5% identity in deduced amino acid sequences. Both enzymes are active toward a number of toxicants and carcinogens. CYP2A6 is mainly expressed in the liver and is the major hepatic coumarin 7-hydroxylase and nicotine C-oxidase (Fernandez-Salguero and Gonzalez, 1995; Messina et al., 1997). It is also active in the metabolic activation of such compounds as aflatoxin B1, NNK, N-nitrosodiethylamine, N-nitrosonornicotine, 2,6-dichlorobenzonitrile, and hexamethylphosphoramide (Fernandez-Salguero and Gonzalez, 1995; Liu et al., 1996; Patten et al., 1997). Although the substrate specificity of CYP2A13 has not been studied as extensively, this enzyme, which is predominantly expressed in the respiratory tract, is the most efficient P450 enzyme known in the metabolic activation of the tobacco-specific nitrosamine NNK (Su et al., 2000). In addition, CYP2A13 is also more active than CYP2A6 in the metabolic activation of hexamethylphosphoramide, N,N-dimethylaniline, and N-nitrosomethylphenylamine (Su et al., 2000).

Both the CYP2A6 and CYP2A13 genes are located in a P450 gene cluster on chromosome 19 (Fernandez-Salguero et al., 1995, Hoffman et al., 2001). A number of genetic polymorphisms have been described for the CYP2A6 gene, including SNPs in the coding region that lead to inactivation or decreases in enzymatic function, such as Gly479Val in CYP2A6.5 (Oscarson et al., 1999a) and Arg128Gln in CYP2A6.6 (Kitagawa et al., 2001), as well as large deletions of the gene (CYP2A6*4) that lead to a nonfunctional allele (e.g., Nunoya et al., 1998; Oscarson et al., 1999b). These genetic polymorphisms in the CYP2A6 gene are responsible for large interindividual variation observed in hepatic metabolism of nicotine and other drugs that are CYP2A6 substrates (e.g., Nunoya et al., 1999a; Inoue et al., 2000). However, conflicting results have been reported on the proposed role of CYP2A6 genotypes in lung cancer risk in different ethnic populations (Miyamoto et al., 1999; Loriot et al., 2001; Tan et al., 2001). Notably, these studies did not examine the potential contributions of polymorphisms in extrahepatic P450 genes involved in the metabolic activation of tobacco-associated carcinogens. In that respect, little is known of the extent of individual differences in the expression or function of the CYP2A13 gene, which is believed to play important roles in xenobiotic toxicity and tobacco-related tumorigenesis in the human respiratory tract (Su et al., 2000) because of its selective expression in the target tissue and the high efficiency of CYP2A13 in NNK activation.

The aim of this study was to identify potential genetic polymorphisms of the CYP2A13 gene, which may alter the metabolic activities of the enzyme toward xenobiotic compounds. Human genomic DNA samples were obtained from newborn blood spots randomly sampled from the New York State Newborn Screening Program. PCR-SSCP was used to identify SNPs in all the exons and at the exon-intron boundaries. PCR primers were designed according to intron sequences, when possible, to avoid amplification of the other CYP2A sequences. PCR-RFLP was also used in some cases to determine the frequencies of distribution of the newly identified variant alleles in the four major ethnic groups. Finally, the function of a frequent variant allele (Arg257Cys) was determined following heterologous expression of the enzyme in Sf9 cells.

    Experimental Procedures
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References

DNA Samples. Anonymous human genomic DNA samples were isolated from newborn blood spots using a protocol described recently (Sheng et al., 2000). Approximately equal numbers of random samples were obtained from each of the four major ethnic groups (white, black, Hispanic, and Asian) in the New York State Newborn Screening Program.

PCR-SSCP Analysis. PCR primers (Table 1) were designed according to the sequence in clone U22028 (Fernandez-Salguero et al., 1995) to amplify each of the exons and exon-intron boundaries in the CYP2A13 gene. The CYP2A13 sequence from the completed human genome data base (accession no. AC008962) was used as a reference for documenting the location of the PCR primers and the identified SNPs. According to the recommended nomenclature system for human gene mutations (Antonarakis, 1998), the A of the ATG start codon is designated as +1. PCR amplification was carried out in a PerkinElmer thermal cycler 9600 instrument (Applied Biosystems, Foster City, CA) in a total volume of 50 µl, with Taq polymerase (Promega, Madison, WI) and 25 µl of an aqueous solution of the genomic DNA from a blood spot. PCR conditions are summarized in Table 2. A CYP2A13 genomic clone (number 27292; obtained from Dr. Harvey Mohrenweiser of Lawrence Livermore National Laboratory, Livermore, CA) was used as a positive control. An H2O blank (no template) control was routinely used to detect potential contamination of reagents. All PCR products were gel-purified using the QIAquick Gel Extraction Kit (QIAGEN, Chatsworth, CA).


                              
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  		TABLE 1
Primers used for PCR-SSCP analysis of the CYP2A13 coding region

The forward (F) and reverse (R) primers for the amplification of each exon, plus exon-intron boundaries, are shown. The CYP2A13 sequence from the completed human genome data base (accession no. AC008962) was used as a reference for documenting the location of the PCR primers, with the A of the ATG start codon (nucleotide 66807 in AC008962) designated as +1.


                              
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  		TABLE 2
Conditions of PCR amplification and restriction enzymes used before SSCP analysis

All reactions were initiated by a denaturation step at 95°C for 3 min. The programs were carried out for 35 cycles, following by a 10-min extension at 72°C or 68°C (for exon 8 only). PCR mixtures contained 1× magnesium-free DNA polymerase buffer (Promega, M190A), 2 mM MgCl2, 0.1 mM each of dNTPs, 0.4 µM of each primer, 3 units of Taq DNA polymerase, and approximately 50 ng of DNA template (about 80% of the estimated amount of total DNA present in a single blood spot of 1-mm diameter) in a total volume of 50 µl.

Before SSCP analysis, 65 to 150 ng of purified PCR products were labeled at the 5' end in 5-µl reaction mixtures that contained 1.25 µCi of [gamma -32P]ATP (3000 Ci/mM) and 1.25 units of T4 polynucleotide kinase (Promega). After end labeling, PCR products of 200 bp or smaller were directly used for SSCP, whereas those bigger than 200 bp were digested, after labeling, with an appropriate restriction enzyme (Table 2) before SSCP.

SSCP analysis of the purified and end-labeled PCR fragments was performed after optimization of multiple parameters, including electrophoresis temperature, concentration of acrylamide, ratio of acrylamide to bis-acrylamide, and addition of glycerol. DNA samples were mixed with 1 volume of 95% formamide, containing 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol. The mixture was heated at 97°C for 5 min, followed immediately by chilling on ice. Then, 2.5 to 3.0 µl of the mixture was loaded on a 6% nondenaturing polyacrylamide gel (31 cm wide, 38.5 cm long, and 0.35 cm thick) containing 2.5% glycerol and with a ratio of acrylamide to bis-acrylamide of 99:1. Electrophoresis was carried out in 1× Tris/borate/EDTA buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA; pH 8.0), with a sequencing gel electrophoresis apparatus from Invitrogen (Carlsbad, CA) (model S2001), at 3 W constant power for about 13 h at room temperature with a cooling fan. After electrophoresis, the gel was transferred to filter paper, dried, and subjected to autoradiography at -80°C for 4 to 24 h. When a fragment was found to display a shifted band, compared with the fragments from the CYP2A13 genomic clone, the PCR-SSCP procedure was repeated to eliminate potential PCR errors. For identification of the sequence change in a variant allele, the remaining samples of purified PCR products with a confirmed band shift were subjected to direct sequencing, in both directions, with use of the primers shown in Table 1, with an automated DNA sequencer from Applied Biosystems (model 373A) at the Molecular Genetics Core of the Wadsworth Center.

PCR-RFLP Analysis. PCR-RFLP assays, which were more convenient than SSCP, were developed to analyze additional DNA samples for a better estimate of the frequencies of the newly identified exon 3 and exon 5 variant alleles in different ethnic groups. No attempts were made to design similar assays for the other variants. For exon 3, the purified 173-bp PCR products were digested with HaeII, which cuts the wild-type allele to give two bands (57 and 116 bp) and the 1662G>C allele to give three bands (42, 57, and 74 bp). The digestion products were analyzed on a 12% polyacrylamide gel and stained with ethidium bromide. For exon 5, the purified PCR products were digested with HhaI, which cuts the wild-type allele to give two bands (99 and 233 bp), but does not cut the 3375C>T allele, thus producing only one band (332 bp). In addition, all samples were also digested with ApalI, which does not cut the wild-type allele but cuts the 3375C>T allele to give two bands (101 and 231 bp). The digestion products were analyzed on a 2% agarose gel.

Site-Directed Mutagenesis. The 3375C>T point mutation found in exon 5 of the CYP2A13 gene was introduced into the wild-type CYP2A13 cDNA clone (Su et al., 2000) in pCR-Script vector (Stratagene, La Jolla, CA), using the Transformer site-directed mutagenesis kit (BD Biosciences Clontech, Palo Alto, CA). Two mismatched oligonucleotide primers were used: one containing the intended site of mutation in exon 5 of CYP2A13 (5'-gagcacaaccagtgcacgctggatc-3'), and the other containing mutations in the unique EcoRI site in the vector (5'-gggctgcaggatatcgatatcaagc-3'). The introduced single-nucleotide mutation was confirmed by restriction digestion with ApalI and by sequencing.

Heterologous Expression of the Arg257Cys CYP2A13 Variant in Sf9 Cells. The mutated CYP2A13 cDNA was released using NotI and ClaI and reinserted into a pCR-Script vector at the NotI and SmaI sites to add a unique EcoRI site at the 3' end of the cDNA. The NotI-EcoRI fragment was then inserted into the multiple cloning site of the baculoviral transfer vector pVL1392 (BD Biosciences PharMingen, San Diego, CA). The integrity of the cloning sites as well as the entire cDNA insert was confirmed by sequencing. Recombinant viruses were made by cotransfecting insect Sf9 cells with the P450-encoding transfer plasmid and linearized BaculoGold viral DNA (BD Biosciences PharMingen). The preparation and titering of virus stocks and the detection of P450 expression were performed according to the procedures described previously for the expression of CYP2A6 and CYP2A13 (Liu et al., 1996; Su et al., 2000). Cells were harvested at 72 h postinfection and resuspended in 100 mM Tris-acetate buffer (pH 7.4) containing 1 mM EDTA and 150 mM potassium chloride. Microsomal fractions were prepared as described previously (Liu et al., 1996) and stored at -85°C until use. P450 expression was confirmed by immunoblot analysis of microsomal preparations with a rabbit anti-mouse CYP2A5 antibody (Gu et al., 1998).

Purification of Arg-257 and Cys-257 CYP2A13. Procedures for CYP2A13 purification were modified from those used previously for the purification of other microsomal P450s (Ding et al., 1991; Gu et al., 1998). Sf9 cell microsomes (about 90-130 nmol of total P450; at about 3 mg of protein/ml) were solubilized with Tergitol-NP-10 (Sigma-Aldrich, St. Louis, MO) and sodium cholate (0.5% and 1.0%, respectively; w/v) and fractionated by polyethylene glycol (PEG) precipitation. The 6% PEG 8000 (Sigma-Aldrich) supernatant fraction, to which 20% glycerol and 10% NP-10 were added to make the final concentration of PEG about 2% and that of NP-10, 0.5%, was loaded onto a 25- to 40-ml HTP column (Bio-Rad, Hercules, CA) previously equilibrated with 10 mM phosphate buffer, pH 7.4, containing 0.5% NP-10 and 20% glycerol (buffer A). After washing with 10 volumes of buffer A, the HTP column was eluted with a linear gradient of potassium phosphate (10-300 mM, pH 7.4) in buffer A. CYP2A13 was recovered at about 240 mM phosphate, as determined by immunoblot analysis. The P450-containing fraction was dialyzed overnight with two changes of 10 mM phosphate buffer, pH 6.4, containing 1 mM EDTA, 0.5% NP-10, and 20% glycerol (buffer B), and loaded onto an 8- to 12-ml S Sepharose column (Sigma-Aldrich) previously equilibrated with buffer B. The column was washed with buffer B and eluted with a pH gradient from 6.4 (buffer B) to 7.4 (200 mM phosphate in buffer A). A nearly homogeneous preparation of CYP2A13 was recovered at about pH 6.9, dialyzed overnight against 10 mM phosphate buffer, pH 7.4, 0.2% NP-10, and 20% glycerol (buffer C), and applied to a second HTP column (3-ml) to remove NP-10, as described previously (Ding and Coon, 1988). The detergent-free CYP2A13 was eluted and dialyzed as described for the purification of CYP2A5 (Gu et al., 1998). The final preparations, which showed CYP2A13 as the predominant band upon SDS-polyacrylamide gel electrophoretic analysis, had specific P450 contents of 5.0 to 9.5 nmol/mg as determined by CO-difference spectroscopy using an absorption coefficient of 91 mM-1cm-1 (Omura and Sato, 1964).

Determination of Catalytic Activity. Formaldehyde formed from hexamethylphosphoramide, 2'-methoxyacetophenone, N,N-dimethylaniline, and N-nitrosomethylphenylamine was measured with CYP2A13 in Sf9 microsomes as described recently (Su et al., 2000), according to the method of Nash (1953). The rates of product formation were corrected for zero time blanks that were quenched before the addition of NADPH. Reactions were carried out at 37°C for 10 to 30 min. The enzyme activities were linear with incubation time under the conditions used. NNK metabolism was assayed by high-pressure liquid chromatography with an on-line radioactivity detector as described previously (Su et al., 2000), except that purified and reconstituted CYP2A13 was used and that cytochrome b5 was not included. Reactions were carried out at 37°C and terminated with 50 µl each of 25% zinc sulfate and saturated barium hydroxide. The contents of individual reaction mixtures are described in the legends to the tables.

Other Methods and Materials. [5-3H]NNK (1.9 Ci/mmol; purity > 98%) and unlabeled NNK were obtained from Chemsyn Science Laboratories (Lenexa, KS). CO-difference spectra of microsomal P450 were recorded at room temperature using a Varian model Cary 3E spectrometer, according to the procedure described by Omura and Sato (1964). Protein concentrations were determined by the bicinchoninic acid method (Pierce Chemical, Rockford, IL) using bovine serum albumin as a standard. Polyclonal rabbit antibodies to CYP2A5 have been described previously (Gu et al., 1998). Immunoblot analysis was performed with an enhanced chemiluminescence kit from Amersham Biosciences (Piscataway, NJ). Rat and rabbit NADPH-cytochrome P450 reductase was obtained as described elsewhere (Ding and Coon, 1994; Zhang et al., 1998). Chi-square (uncorrected) or Fisher's exact test was used to examine the significance of differences in allele frequency between two different ethnic groups and the significance of linkage disequilibrium between SNP pairs, with use of SigmaStat software (SPSS Science, Chicago, IL). Student's t test was used to examine the significance of differences in metabolic activity between wild-type and variant CYP2A13.

    Results
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Abstract
Introduction
Experimental Procedures
Results
Discussion
References

Identification of SNPs in the CYP2A13 Gene and Determination of Allele Frequency in Major Population Groups. Variant alleles were first detected in the CYP2A13 coding region and exon-intron boundaries by PCR-SSCP or direct DNA sequencing of PCR products. Primers for exons 1, 2, 3, 5, 6, 7, and 8 (Table 1) did not amplify either CYP2A6 or CYP2A7, although those for exons 1, 2, and 7 generated an additional unidentified band (not shown), which was removed during gel purification prior to SSCP analysis. The primers for exons 4 and 9 produced a single PCR band, which, nevertheless, contained both CYP2A6 and CYP2A13 sequences as revealed by SSCP analysis and DNA sequencing.

The SSCP patterns of six of the seven allelic variants detected are shown in Fig. 1. For 1662G>C, 3375C>T, and 3441C>A, the variant and wild-type alleles were well separated. For 523C>T and 6404C>G, the two bands representing two different alleles were distinguishable, although the resolution is not apparent in Fig. 1. The 6424C>T + 6432C>T allele was initially identified by SSCP; the two bands can only be distinguished, with difficulty, by comparison of homozygous individuals. Sequence analysis revealed that the band shift was a result of the double mutation, since individuals with only the 6424C>T mutation did not show a band shift (not shown) and those with only the 6432C>T mutation were not detected.


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  		Fig. 1.   Detection of variant alleles by PCR-SSCP in the CYP2A13 gene. PCR-SSCP was performed as described under Experimental Procedures. For each panel, the shifted band, representing a variant allele, is indicated by an arrow. The genotypes of the individuals analyzed are shown at the bottom of each lane. The band shift in the 6424C>T + 6432C>T panel can only be detected between individuals homozygous for the T-T allele (T/Tstar ) and those homozygous for the C-C allele (C/Cstar ).

The frequency of the seven variant alleles was determined in the four major ethnic groups in New York state. As shown in Table 3, three of these variants represent rare mutations, with allele frequencies lower than 0.5%. Found in an Asian newborn, 523C>T is in exon 2 and does not correspond to a change in predicted amino acid sequence. Found in an Hispanic newborn, 1662G>C is in exon 3; it causes a G144R change in the predicted amino acid sequence. An additional 129 samples (42 from white, 24 from black, 25 from Hispanic, and 38 from Asian people) were subsequently screened for this allele by PCR-RFLP, as described under Experimental Procedures, but none were found to have this allele. Found in an Hispanic newborn, 3441C>A is in intron 5, at the fourth nucleotide downstream of the junction with exon 5. 

                              
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  		TABLE 3
Variant alleles detected in the CYP2A13 gene

About 100 individuals were analyzed for each exon by PCR-SSCP (or by direct sequencing of PCR products, as indicated). No band shift was detected in exons 1, 4, 6, 7, 8, and 9. The identities of all shifted bands were confirmed by sequencing. DNA from blood spots was used for PCR as described under Experimental Procedures. PCR product of the expected size was obtained with all blood spots. No other nucleotide was detected at the variant position. The CYP2A13 sequence from the completed human genome data base (accession no. AC008962) was used as a reference for documenting the location of the SNPs, with the A of the ATG start codon (nucleotide 66807 in AC008962) designated as +1. The number of samples analyzed were slightly different for each position because there was insufficient isolated DNA from certain subjects.

The other four variants represent SNPs, including 3375C>T, 6404C>G, 6424C>T, and 6432C>T (Table 3). The 3375C>T allele is in exon 5 and corresponds to an Arg257Cys change in the predicted amino acid sequence. Of 104 individuals initially analyzed by SSCP, there were 10 heterozygotes and one homozygote for the 3375C>T allele. An additional 104 individuals were subsequently genotyped for this allele by PCR-RFLP. The combined results from the 208 samples are shown in Table 4. The frequency of this allele was highest in blacks (14.4%) and lowest in whites (1.9%). The allelic frequency in black people was significantly higher than that in white (P = 0.001) and Hispanic (P = 0.038) people. No significant frequency difference was found between male and female populations (male, 7.2%; female, 7.7%). Furthermore, the observed genotype frequencies did not deviate significantly from that expected by Hardy-Weinberg equilibrium.

                              
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  		TABLE 4
Frequencies of the exon 5 3375C>T allele (Arg257Cys) in major ethnic groups

One-half (104) of the samples were analyzed by PCR-SSCP, whereas the other half were genotyped by PCR-RFLP as described under Experimental Procedures. Results from males and females were not significantly different.

The variants 6404C>G, 6424C>T, and 6432C>T are all located in intron 7, at about 84, 64, and 56 nucleotides, respectively, upstream of the junction with exon 8. The frequency of the 6404C>G allele was 16% for white, 8.7% for black, 2.1% for Hispanic, and 0% for Asian people. The frequency in white people was significantly higher than in Hispanic (P = 0.031) and Asian (P = 0.006) people. The 6404C>G mutation was not linked to 6424C>T or 6432C>T, since only 3 of the 13 individuals with 6404C>G carried either 6424C>T or 6432C>T (not shown). On the other hand, the 5 individuals homozygous for 6432C>T were also homozygous for 6424C>T, and the 15 individuals heterozygous for 6432C>T also had at least one 6424C>T allele (Table 5). Statistical analyses using Fisher's exact test indicated that there is significant linkage disequilibrium between the 6424C>T and 6432C>T alleles (P < 0.001). Further analysis of genotype distribution indicated that there are only three possible allele combinations or haplotypes among the individuals examined: 6424C>T and 6432C>T (T-T), 6424C and 6432C (C-C), and 6424C>T and 6432C (T-C). The frequency of the T-T allele was 0% for white, 40.9% for black, 8.7% for Hispanic, and 7.5% for Asian people. The frequency in blacks was significantly higher than in other groups (P < 0.001). The frequency of the T-C allele was 10.9% for white, 2.3% for black, 19.6% for Hispanic, and 5.0% for Asian people. The frequency in Hispanic people was significantly higher than in black (P = 0.015) and Asian (P = 0.044) people.

                              
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  		TABLE 5
Frequencies of the 6424C>T + 6432C>T and 6424C>T + 6432C alleles in major ethnic groups

The following genotypes were not detected: T-C/T-C (6424C>T + 6432C/6424C>T + 6432C), C-T/C-T, C-C/C-T, and T-T/C-T.

Functional Characterization of the Arg257Cys Variant. A cDNA for the Arg257Cys variant was generated by site-directed mutagenesis of the wild-type CYP2A13 plasmid (Su et al., 2000) and cloned into a baculoviral transfer vector for expression in insect Sf9 cells. The sequence of the Arg257Cys cDNA insert in the baculoviral expression vector and the sequences at the cloning sites were confirmed. The yield of P450 in lysate from recombinant virus-infected Sf9 cells ranged from 10 to 30 nmol/l in different batches of cells cultured with addition of hemin. The level of the cytochrome in the microsomal fraction ranged from 0.20 to 0.39 nmol/mg of protein.

A typical P450 CO-difference spectrum with an absorbance maximum at about 450 nm was recorded with detergent-solubilized microsomal preparations from Arg257Cys-expressing Sf9 cells (not shown). In other experiments not presented, the two cytochromes, when partially purified, had essentially identical absolute spectra. Furthermore, the Cys-257 protein migrated to the same position as did Arg-257 upon immunoblot analysis, indicating that it was not selectively degraded or post-translationally modified.

Initial studies indicated that the Arg257Cys protein was less active than the Arg-257 protein in coumarin 7-hydroxylation in a reconstituted system, with turnover numbers of 0.14 and 0.21 nmol/min/nmol P450, respectively, at a substrate concentration of 0.1 mM. Subsequently, the activities of Arg257Cys toward several other known CYP2A13 substrates, including HMPA, N,N-dimethylaniline, 2'-methoxyacetophenone, and N-nitrosomethylphenylamine, were examined. As shown in Table 6, the Arg257Cys protein was about 37 to 56% less active than the Arg-257 protein with each of the substrates analyzed. The differences in turnover number, although small, are statistically significant.

                              
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  		TABLE 6
Catalytic activities of wild-type CYP2A13 and the Arg257Cys variant toward xenobiotic compounds

Assays were performed as described under Experimental Procedures. Reaction mixtures contained 50 mM phosphate buffer, pH 7.4, 1 mM NADPH, 1 mM substrate, a reconstituted P450 system containing 0.1 µM recombinant P450 in Sf9 microsomes and 0.4 µM purified rabbit NADPH-P450 reductase. The reactions were carried out at 37°C for 10 min, during which product formation was linear with time. Values presented are means ± S.D. of three duplicate determinations.

Kinetic parameters for HMPA metabolism were also determined for the two enzymes, with use of Sf9 microsomal fractions. The assays were performed essentially as described in Table 6, with HMPA at 0.25 to 2 mM. The results indicated that the Arg257Cys protein had a higher Km (0.65 versus 0.34 mM) and a lower Vmax (5.1 versus 8.2 nmol/min/nmol P450) than did the Arg-257 protein, with a three-fold difference in catalytic efficiency (7.8 versus 24.1).

The ability of the Arg257Cys protein to activate NNK was also examined, in comparison to the Arg-257 protein. These experiments were performed with purified P450 proteins to avoid potential interference by other proteins in the Sf9 microsomal fraction. As shown in Table 7, the Arg257Cys protein had significantly higher Km and lower Vmax values than did the Arg-257 protein in the formation of keto-aldehyde and keto-alcohol, which resulted in 2- to 3-fold decreases in catalytic efficiency. No additional product was detected by radiometric high-pressure liquid chromatography. In additional experiments with Sf9 microsomal preparations, the two proteins also had similar differences in turnover numbers for NNK metabolism (data not shown).

                              
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  		TABLE 7
Kinetic parameters of NNK metabolism by purified wild-type CYP2A13 and the Arg257Cys variant

Reaction mixtures contained 100 mM sodium phosphate, pH 7.4, 1 mM EDTA, an NADPH-generating system (5 mM glucose 6-phosphate, 3 mM MgCl2, 1 mM NADP+, and 1.5 units of glucose-6-phosphate dehydrogenase), 2 to 100 µM NNK (containing 1 µCi [5-3H]NNK), 5 mM sodium bisulfite, and 10 pmol of purified, reconstituted CYP2A13 in a total volume of 0.4 ml. CYP2A13 was reconstituted with rat NADPH-P450 reductase, at a ratio of 1:5 (P450/reductase), and dilauroylphosphatidylcholine (30 µg/ml final concentration). Reactions were carried out for lengths of time that allowed the determination of initial linear rates. The Vmax (nmol/min/nmol of P450) and Km (µM) values (means ± S.D.) were obtained from three separate determinations.

    Discussion
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References

This is the first report on genetic polymorphisms in the CYP2A13 gene. Of the seven variant alleles found, only four qualify as SNPs based on the allele frequency, including the Arg257Cys variant and the three variants in intron 7. It is interesting that six of the seven changes occur at a cytosine. In contrast, the single-nucleotide changes in the CYP2A6 gene occur at either a guanine or a thymidine (home page of the Human Cytochrome P450 Allele Nomenclature Committee, http://www.imm.ki.se/cypalleles).

The 3375C>T allele was found most frequently in black (14.4%) and least frequently in white newborns (1.9%). The decreased catalytic efficiency of the corresponding Arg257Cys CYP2A13 protein in NNK bioactivation suggests that the Cys-257 genotype may contribute to a lower risk of respiratory tract xenobiotic toxicity. However, the activity differences between the Arg-257 and Cys-257 proteins are relatively small, which will make it difficult to find potential associations between the Cys-257 genotype alone and the incidence of tobacco-related tumorigenesis in the respiratory tract. On the other hand, the role of this SNP may be more apparent when analyzed in conjunction with the genotypes of other biotransformation enzymes implicated in the metabolism of tobacco-related chemical carcinogens, particularly CYP2A6. CYP2A6 is believed to play the principal role in determining systemic clearance of nicotine and, thus, the number of cigarettes smoked in addicted individuals (Tyndale and Sellers, 2001). An association of the CYP2A6 deletion genotype with reduced lung cancer incidence was found in a Japanese population (Miyamoto et al., 1999), although apparently conflicting results have been reported by others (Loriot et al., 2001; Tan et al., 2001). It would be interesting to see whether a stronger association can be found in subgroups when the data are stratified according to the CYP2A13 genotypes. A loss of hepatic CYP2A6 in conjunction with a Cys-257 genotype may further reduce the incidence of tobacco-related tumors in the respiratory tract. In this regard, due to the limited quantities of DNA available from the blood spot samples, the subjects analyzed in the present study were not screened for CYP2A6 SNPs. However, other efforts to evaluate potential linkage between CYP2A6 and CYP2A13 SNPs are under way.

Mechanistically, the decrease in activity by the Arg257Cys mutation is intriguing. The Arg at the 257 position, which is conserved in the CYP2As, seems to be located near the carboxyl end of the G-helix according to alignments based on sequence conservation (Gotoh, 1992; Hasemann et al., 1995). The changed residue corresponds to Lys253 of CYP2C5, according to an alignment and homology modeling (not shown) based on the coordinates available from the PDB for CYP2C5 (1DT6). The residue is expected to be located on the surface of the protein, away from any of the proposed substrate access channels (Williams et al., 2000). However, conformational changes that occur with substrate binding may require the various helices involved in substrate binding to rotate and move, as found in the structure of P450 BM3 with a bound substrate analog (Li and Poulos, 1997). Thus, it is possible that a mutation in the loop region impedes such changes and, consequently, alters substrate binding or product release in a substrate-independent fashion. In addition, a recent study (Lehnerer et al., 2000) indicated that an Arg253Ala mutation near the end of the G helix interfered with the interaction of rabbit CYP2B4 with the P450 reductase, leading to an approximately 50% decrease in activity. The similar extent of impact of the Arg257Cys change on the turnover by CYP2A13 of all substrates tested is consistent with the possible involvement of such general, substrate-independent mechanisms.

The functional consequence of the Gly144Arg substitution was not examined in this study. Sequence alignment and modeling suggest that it corresponds to Ser140 in CYP2C5, which is located just before helix D and also on the surface of the protein (Williams et al., 2000). The other exon mutation, 523C>T in exon 2, is silent. However, it remains to be determined whether this and the Gly144Arg and Arg257Cys mutations are within an exonic splicing enhancer or exonic splicing silencer sequence (Blencowe, 2000). Mutations in these regulatory sequences have been implicated in human genetic diseases (e.g., Liu et al., 2001).

The 3441C>A variant sequence detected in intron 5 is part of the 5' splice site (AGGTACAT); "A" is actually the consensus sequence at this position, although "C" is also found in some genes (Senapathy et al., 1990). Notably, genetic polymorphisms that affect proper splicing of human P450 transcripts have been reported previously, such as the mutations found recently in the CYP3A5 gene that lead to alternative splicing and protein truncation (Chou et al., 2001; Kuehl et al., 2001). For CYP2A13, it will be interesting to determine whether the 3441C>A variant allele is expressed at higher levels than the 3441C allele, possibly due to a more efficient splicing of the pre-mRNA.

The three SNPs in intron 7 are less likely to be involved in splicing. The changed sequences do not generate consensus splicing donor or acceptor sites. Analysis of potential binding sites for regulatory proteins in this region through a search of the TRANSFAC data base (Heinemeyer et al., 1998), using the TFSEARCH program (Y. Akiyama: "TFSEARCH: Searching Transcription Factor Binding Sites", http://www.rwcp.or.jp/papia/), indicated that the 6424C>T change would destroy a putative stress response element (score 91.5, on the minus strand) (Schuller et al., 1994) and a putative binding site for alcohol dehydrogenase gene regulator 1 (score 90.8, on the minus strand), whereas the 6404C>G and 6432C>T changes would not impact any known DNA sequences for transcription factor binding (Cheng et al., 1994).

SSCP analysis may not detect all SNPs in a given gene. Thus, it is possible that additional variant alleles occur in the CYP2A13 exons, which remain to be identified using other techniques, such as direct DNA sequencing. Although not detected in the present study (data not shown), a potential variant allele was implicated by a recently reported CYP2A13 exon 1 sequence (Nunoya et al., 1999a), which had two amino acid differences, Arg30Lys and Leu33Val, from the known CYP2A13 sequence. Notably, potential variants in the flanking regions were not examined in the present study; such variants are also likely according to the findings on other P450 genes, such as CYP2A6 (Pitarque et al., 2001). However, large deletions such as those found in CYP2A6, which appear to arise from homologous unequal crossover of the neighboring CYP2A6 and CYP2A7 genes (Nunoya et al., 1999b), may be less likely, since the CYP2A13 gene is located relatively far away from the other CYP2A genes in the CYP2 gene cluster on chromosome 19 (Fernandez-Salguero et al., 1995; Hoffman et al., 2001). It should also be noted that, although the present study suggests significant differences in allelic variant frequencies among broadly defined ethnic groups, these results are preliminary because the number of subjects examined in each group is relatively small. In any case, additional studies along these lines are warranted and may provide useful models for determining the in vivo function of CYP2A13 in xenobiotic bioactivation and toxicity in the respiratory tract.

    Acknowledgments

We thank Drs. Laurence Kaminsky and Adriana Verschoor for reading the manuscript and Drs. Eric Johnson and James Halpert for helpful discussions. We also gratefully acknowledge the use of the Molecular Genetics Core facility and the Tissue Culture Core facility of the Wadsworth Center.

    Footnotes

Accepted for publication March 25, 2002.

Received for publication February 21, 2002.

This work was supported in part by Research Grant ES07462 from the National Institute of Environmental Health Sciences, National Institutes of Health (Bethesda, MD).

Address correspondence to: Dr. Xinxin Ding, Wadsworth Center, New York State Department of Health, Empire State Plaza, Box 509, Albany, NY 12201-0509. E-mail: xding{at}wadsworth.org

    Abbreviations

NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; P450, cytochrome P450; SNP, single-nucleotide polymorphism; PCR, polymerase chain reaction; SSCP, single-strand conformational polymorphism; NP-10, Nonidet P-10; RFLP, restriction fragment length polymorphism; bp, base pair(s); HMPA, hexamethylphosphoramide; PEG, polyethylene glycol; HTP, hydroxyapatite.

    References
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Abstract
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Experimental Procedures
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