7
46
TIONG ET AL.
Functional significance of some alleles such as CYP2A6*15 (K194E),
TABLE 1
CYP2A6*16 (R203S), CYP2A6*21 (K476R), and CYP2A6*22 (D158E and
L160I) has yet to be investigated in detail. The occurrence of these
four alleles in the human population was first revealed in 2002 and
Specifically designed oligonucleotide primers that have been used in the
generation of CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22
mutant cDNAs
The underlined regions denote restriction sites that have been created or abolished in the
primer sequences.
2
005 with variant CYP2A6*15 found in the Korean and Japanese
populations at frequencies of approximately 1.2 and 1.5 to 2.2%,
respectively (Kiyotani et al., 2002; Nakajima et al., 2006). Con-
versely, the frequency of CYP2A6*16 was prominent among whites
Primer
Primer Sequencea
CYP2A6*15
EcoRI: 5Ј CGC TTT GAC TAT GAG GAC AAA
GAA TTC CTG TCA CTG 3Ј
FspI: 5Ј TTC CTG TCA CTG TTG AGC ATG
ATG CTA GGA ATC 3Ј
PmlI: 5Ј GAC GTG TCC CCC AGA CAT GTG
GGC TTT GCC 3Ј
Hpy99I: 5Ј GGC TTC CTC ATC GAG GCC
ATC CGG GGC ACT GGC 3Ј
(
0.3–3.6%) and African Americans (1.7%), whereas this allele re-
CYP2A6*16
CYP2A6*21
CYP2A6*22
mained undetected in the Asian population (Kiyotani et al., 2002;
Nakajima et al., 2006). Allelic variant CYP2A6*21 also has a higher
occurrence in white subjects at 0.5 to 7.0% compared with Chinese
(
3.4%) and black subjects (0.6%), whereas Japanese subjects re-
mained unaffected (Haberl et al., 2005; Al Koudsi et al., 2006;
Nakajima et al., 2006). Comprehensive evaluation of CYP2A6 poly-
morphic alleles by Nakajima et al. (2006) in four ethnic populations
did not detect any occurrence of CYP2A6*22 within the populations
studied. So far, the variant was only reported at low frequency (0.3%)
among whites (Haberl et al., 2005). Although no data are available for
CYP2A6*15 and CYP2A6*16, haplotype analyses on CYP2A6*21 and
CYP2A6*22 revealed that haplotypes carrying the two alleles oc-
curred at low frequencies (at 0.6 and 0.3%, respectively) in whites
a
Sequences shown are those of the forward primers, the reverse primers contained sequences
opposite to those of the forward primers and annealed to the same sequences on opposite strands
of the plasmid template. Nucleotides that were changed to make the desired mutation are shown
in bold.
respectively, in the expressed bacterial membrane fragment of CYP2A6*15,
CYP2A6*16, and CYP2A6*21 proteins. Likewise, primer possessing both
GAC to GAG and CTC to ATC substitutions has permitted expression of
mutant CYP2A6*22 with concurrent D158E and L160I substitutions on the
primary sequence. Before full nucleotide sequencing of the entire cDNA
coding frame of each clone, each mutant construct was subjected to restriction
analyses by several endonucleases (EcoRI, FspI, PmlI, and Hpy99I). The
preliminary analysis with restriction endonucleases was necessary as an indi-
(
Haberl et al., 2005). Haplotype frequencies of these four alleles in
other populations as well as their phenotypic association with protein
level and activity, however, remain unknown at this stage.
Despite the existence of these four CYP2A6 variants in the human cation that desired mutations had successfully taken place on the nucleotide
populations, functional characterization of the polymorphisms manifested strand of CYP2A6 cDNA. The full nucleotide sequencing of all four mutant
has yet to be determined in detail. With one or more amino acid mutations cDNAs was further verified by a capillary-based sequencing method, which
in their primary sequences, it is likely that these mutations would have a was outsourced to AITBIOTECH Pte Ltd. (Singapore).
Heterologous Expression of CYP2A6 Wild-Type and Mutant Con-
structs in Bacterial Expression System. CYP2A6 plasmid constructs harbor-
ing the desired mutations, pCW-CYP2A6*15, pCW-CYP2A6*16, pCW-
certain degree of effects on their structural stability and catalytic activity.
Structural and functional characterization of these polymorphic alleles is
necessary because it contributes to our better understanding of the con-
CYP2A6*21, and pCW-CYP2A6*22 were individually cotransformed into E.
sequences of mutation and hence aids in defining the pharmacological
coli DH5␣ cells together with the pACYC-OxR plasmid, the essential
and toxicological importance of CYP2A6 polymorphisms in humans.
NADPH-cytochrome P450 oxidoreductase (OxR) coenzyme. CYP2A6 and
OxR protein expression in bacterial cells and the subsequent membrane prep-
aration was determine as described previously (Singh et al., 2008). The
Materials and Methods
membrane fragments of E. coli were stored at Ϫ80°C in a 1:1 mixture of pH
Materials. The QuikChange site-directed mutagenesis system was pur-
chased from Stratagene (La Jolla, CA), and endonuclease restriction enzymes
were obtained from New England Biolabs (Ipswich, MA). Mouse anti-human
cytochrome P450 CYP2A6 monoclonal antibody and goat anti-mouse IgG-
horseradish peroxidase were purchased from Santa Cruz Biotechnology, Inc.
7
.6 TES buffer (100 mM Tris, 0.5 mM EDTA, and 500 mM sucrose) and
ice-cold distilled water before analysis in the enzyme assay reaction.
Coumarin 7-Hydroxylase Assay. Enzyme kinetic activities of wild-type
CYP2A6 and mutants were assessed by a fluorescence-based coumarin 7-hy-
(
Santa Cruz, CA). 5-Bromo-4-chloro-3-indolyl--D-galactopyranoside, isopro- droxylase assay with slight modifications based a published protocol (Ghosal
pyl--D-thiogalactopyranoside, and Tris base were acquired from Promega
et al., 2003; Donato et al., 2004). Coumarin 7-hydroxylation was measured in
(
Madison, WI). Escherichia coli DH5␣ competent cells, oligonucleotide primers, a reaction mixture consisting of 50 g of expressed CYP2A6, an NADPH-
and Luria-Bertani and Teriffic broth media were purchased from Invitrogen
generating system (1.3 mM NADP, 3.5 mM glucose 6-phosphate, 2 IU of
glucose-6-phosphate dehydrogenase, and 5 mM MgCl ) and 0.313 to 40 M
coumarin in 100 mM Tris-HCl buffer (pH 7.5). Coumarin was dissolved in
(Carlsbad, CA). All other chemicals and reagents were obtained from Sigma-
2
Aldrich (St. Louis, MO).
In Vitro Site-Directed Mutagenesis of CYP2A6 cDNA. Site-directed acetonitrile with the final concentration of the organic solvent in each incu-
mutagenesis on CYP2A6 cDNA was performed using the QuikChange site- bation mixture at 1% (v/v) or less. Reactions were initiated by addition of 50
directed mutagenesis system according to the manufacturer’s instructions.
Basically, in this a supercoiled double-stranded DNA vector with an insert of min and later were terminated by 50 l of 500 mM Tris base after 25 min of
interest and two synthetic oligonucleotide primers containing the desired
incubation at 37°C. Quantification of 7-hydroxycoumarin was performed im-
mutation are used. The DNA template was a pCW-CYP2A6 vector, which was mediately by using an Infinite 200 series microplate reader (Tecan, M a¨ nner-
previously constructed in our laboratory (C. E. Ong, unpublished data). It
dorf, Switzerland) at the excitation wavelength of 365 nm and emission
contained N-terminal sequence modification, a P450 17␣-derived MALL- wavelength of 450 nm. Standard curves of 7-hydroxycoumarin were con-
g of CYP2A6 protein after prewarming at 37°C in a metabolic shaker for 10
LAVF sequence as reported by Barnes et al. (1991) and the full coding
structed in the range of 15.63 to 2000 pmol/well, and the metabolite formation
sequence of human CYP2A6. The sequence has 100% identity to that of the
rate was calculated based on the curves. All samples and standards were
reported wild-type CYP2A6 in GenBank (GenBank accession number incubated in duplicate.
NM_000762). Mutagenic primers that were exclusively designed for generat-
Kinetic Analysis. Enzyme kinetic data were analyzed by the nonlinear
ing mutant alleles of CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22 least-squares regression analysis software EZ-Fit (Perrella Scientific, Anherst,
are listed in Table 1. Single nucleotide substitution of AAG to GAG, CGC to NH), and the kinetic parameters, Michaelis-Menten constant (K ), and max-
m
AGC, and AAA to AGA (nucleotides that were changed to make the desired
imum velocity (Vmax) were determined over the substrate range studied.
mutation are shown in bold) on the CYP2A6 primary sequence had each
Statistical analyses were performed by using the SPSS statistical program
contributed to the amino acid substitution of K194E, R203S, and K476R, (SPSS Inc., Chicago, IL).