Functional Characterization of 34 CYP2A6 Allelic Variants by Assessment of Nicotine C-Oxidation and Coumarin 7-Hydroxylation Activities

Hiroki Hosono; Masaki Kumondai; Masamitsu Maekawa; Hiroaki Yamaguchi; Nariyasu Mano; Akifumi Oda; Noriyasu Hirasawa; Masahiro Hiratsuka

doi:10.1124/dmd.116.073494

Article Figures & Data

Figures

Tables

Download figure
Open in new tab
Download powerpoint
Fig. 1.
Metabolic pathways for nicotine showing the first reaction, catalyzed by CYP2A6, and the second reaction, catalyzed by aldehyde oxidase (upper). Primary metabolic pathway of coumarin (lower).
Download figure
Open in new tab
Download powerpoint
Fig. 2.
Western blots showing immunoreactive CYP2A6 proteins (upper panel) and calnexin (lower panel). Microsomal protein fraction (4 μg) was loaded in each lane. Microsomal proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels and then subjected to Western blotting according to standard procedures. The CYP2A6 variant proteins were detected using a polyclonal antibody against CYP2A6, and calnexin was detected using a polyclonal antibody against calnexin. Numbers correspond to CYP2A6 variants.
Download figure
Open in new tab
Download powerpoint
Fig. 3.
Correlation between the CL_int ratios (relative to the wild type CYP2A6.1) for nicotine C-oxidation and coumarin 7-hydroxylation among CYP2A6 variants. Nicotine C-oxidation CL_int ratios are plotted on the horizontal axis and coumarin 7-hydroxylation CL_int ratios are plotted on the vertical axis. Numbers correspond to CYP2A6 variants. Correlation was analyzed using weighted linear regression analysis with IBM SPSS Statistics.
Download figure
Open in new tab
Download powerpoint
Fig. 4.
Average levels of CYP2A6 proteins expressed in 293FT cells. Each bar is presented as the mean ± S.D. of three independently performed immunoblottings. Numbers correspond to CYP2A6 variants.
Download figure
Open in new tab
Download powerpoint
Fig. 5.
Diagram of a portion of the CYP2A6 crystal structure showing the Arg128Gln substitution in CYP2A6.6. Ionic bonding differs between the wild-type enzyme and the CYP2A6.6 variant.

Tables

Figures

View popup

TABLE 1

Kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation by microsomes from 293FT cells expressing CYP2A6 wild-type and variant proteins

These data represent the mean ± S.D. of three independently performed catalytic assays.

Variants	Nucleotide Changes	Amino Acid Changes	Nicotine C-Oxidation			Coumarin 7-Hydroxylation
Variants	Nucleotide Changes	Amino Acid Changes	K_m	V_max	CL_int (V_max/K_m)	K_m	V_max	CL_int (V_max/K_m)
			μM	pmol/min per pmol CYP2A6	nl/min per pmol CYP2A6  (% of wild-type)	μM	pmol/min  per pmol CYP2A6	μl/min per pmol CYP2A6 (% of wild-type)
CYP2A61*			33.3 ± 5.44	5.91 ± 1.26	176 ± 11.2	1.94 ± 0.06	5.89 ± 0.34	3.04 ± 0.08
CYP2A62*^a^,^c	1799T>A	Leu160His	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
CYP2A65*^a^,^c	6582G>T	Gly479Val	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
CYP2A66*^a^,^c	1703G>A	Arg128Gln	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
CYP2A67*	6558T>C	Ile471Thr	267 ± 59.9	2.36 ± 0.41	9.14 ± 2.44* (5%)	4.55 ± 1.94	3.33 ± 0.62	0.80 ± 0.17* (26%)
CYP2A68*	6600G>T	Arg485Leu	103 ± 16.3	2.10 ± 0.24	20.6 ± 2.44* (12%)	3.13 ± 0.30	2.22 ± 0.16*	0.71 ± 0.06*** (23%)
CYP2A610*^b^,^d	6558T>C	Ile471Thr	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
CYP2A610*^b^,^d	6600G>T	Arg485Leu	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
CYP2A611*	3391T>C	Ser224Pro	142 ± 31.3	4.31 ± 0.34	31.6 ± 5.88* (18%)	3.30 ± 0.37	2.57 ± 0.03*	0.79 ± 0.09*** (26%)
CYP2A613*	13G>A	Gly5Arg	30.8 ± 0.80	3.94 ± 0.20	128 ± 5.36 (73%)	1.37 ± 0.03*	2.61 ± 0.19	1.90 ± 0.17 (63%)
CYP2A614*	86G>A	Ser29Asn	28.8 ± 0.65	7.01 ± 0.12	243 ± 5.67 (138%)	1.77 ± 0.15	8.02 ± 0.43	4.53 ± 0.16* (149%)
CYP2A615*	2134A>G	Lys194Glu	23.4 ± 0.74	9.03 ± 0.09	385 ± 8.46*** (219%)	1.27 ± 0.03*	9.10 ± 0.31*	7.17 ± 0.28* (236%)
CYP2A616*	21611C>A	Arg203Ser	39.0 ± 2.52	4.58 ± 0.26	118 ± 3.60 (67%)	1.93 ± 0.15	4.72 ± 0.14	2.46 ± 0.21 (81%)
CYP2A617*	5065G>A	Val365Met	25.9 ± 6.16	1.70 ± 0.21	68.3 ± 12.2* (39%)	1.72 ± 0.03	2.76 ± 0.08*	1.60 ± 0.02** (53%)
CYP2A618*	5668A>T	Tyr392Phe	82.1 ± 7.42	7.94 ± 0.63	97.1 ± 6.86 (55%)	2.77 ± 0.18	8.03 ± 0.43	2.90 ± 0.05 (95%)
CYP2A619*	5668A>T	Tyr392Phe	405 ± 118	2.29 ± 0.46	5.80 ± 0.53* (3%)	4.75 ± 0.14***	2.18 ± 0.08*	0.46 ± 0.01*** (15%)
CYP2A619*	6558T>C	Ile471Thr	405 ± 118	2.29 ± 0.46	5.80 ± 0.53* (3%)	4.75 ± 0.14***	2.18 ± 0.08*	0.46 ± 0.01*** (15%)
CYP2A621*	6573A>G	Lys476Arg	40.3 ± 3.69	6.15 ± 0.33	153 ± 9.35 (87%)	2.34 ± 0.29	6.20 ± 0.35	2.67 ± 0.20 (88%)
CYP2A622*	1794C>G	Asp158Glu	53.8 ± 1.70	6.26 ± 0.36	116 ± 6.49 (66%)	2.40 ± 0.10	5.28 ± 0.20	2.20 ± 0.06* (72%)
CYP2A622*	1798C>A	Leu160Ile	53.8 ± 1.70	6.26 ± 0.36	116 ± 6.49 (66%)	2.40 ± 0.10	5.28 ± 0.20	2.20 ± 0.06* (72%)
CYP2A623*	2161C>T	Arg203Cys	41.1 ± 3.50	3.73 ± 0.32	91.1 ± 8.08* (52%)	1.88 ± 0.04	4.86 ± 0.17	2.59 ± 0.04 (85%)
CYP2A624*	594G>A	Val110Leu	52.4 ± 8.56	4.79 ± 0.30	93.4 ± 12.1 (53%)	2.22 ± 0.18	3.23 ± 0.11	1.46 ± 0.07*** (48%)
CYP2A624*	6458A>T	Asn438Tyr	52.4 ± 8.56	4.79 ± 0.30	93.4 ± 12.1 (53%)	2.22 ± 0.18	3.23 ± 0.11	1.46 ± 0.07*** (48%)
CYP2A625*^d	1672T>C	Phe118Leu	59.9 ± 5.94	1.67 ± 0.09	28.0 ± 2.48* (16%)	N.D.	N.D.	N.D.
CYP2A626*^a^,^c	1672T>C	Phe118Leu	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
	1703G>T	Arg128Leu
	1711T>G	Ser131Ala
CYP2A628*	5745A>G	Asn418Asp	35.0 ± 4.19	6.30 ± 0.73	181 ± 14.7 (103%)	1.43 ± 0.12	5.05 ± 0.25	3.55 ± 0.23 (117%)
CYP2A628*	5750G>C	Glu419Asp	35.0 ± 4.19	6.30 ± 0.73	181 ± 14.7 (103%)	1.43 ± 0.12	5.05 ± 0.25	3.55 ± 0.23 (117%)
CYP2A631*	16A>C	Met6Leu	35.4 ± 6.15	6.30 ± 0.30	184 ± 36.3 (105%)	1.61 ± 0.07	5.64 ± 0.13	3.50 ± 0.12 (115%)
CYP2A635*	6458A>T	Asn438Tyr	75.7 ± 14.9	3.98 ± 0.51	53.5 ± 5.31* (30%)	2.50 ± 0.28	4.63 ± 0.35	1.86 ± 0.08*** (61%)
CYP2A636*^b^,^d	6458A>T	Asn438Tyr	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
CYP2A636*^b^,^d	6558T>C	Ile471Thr	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
CYP2A637*^b^,^c	6458A>T	Asn438Tyr	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
	6558T>C	Ile471Thr
	6600G>T	Arg485Leu
CYP2A638*	5023T>C	Tyr351His	108 ± 9.63*	2.37 ± 0.12	22.2 ± 2.84* (13%)	1.51 ± 0.08	2.07 ± 0.02*	1.37 ± 0.07*** (45%)
CYP2A639*	468G>A	Val68Met	93.7 ± 20.4	1.91 ± 0.03	21.4 ± 4.72* (12%)	1.50 ± 0.20	1.85 ± 0.02*	1.26 ± 0.16* (41%)
CYP2A640*	1767C>G	Ile149Met	168 ± 72.0	1.97 ± 0.31	13.0 ± 2.93* (7%)	1.34 ± 0.08*	1.33 ± 0.08*	0.99 ± 0.01** (33%)
CYP2A641*	3515G>A	Arg265Gln	263 ± 82.3	2.15 ± 0.43	8.43 ± 0.95* (5%)	2.32 ± 0.23	1.26 ± 0.04*	0.54 ± 0.04*** (18%)
CYP2A642*	3524T>C	Ile268Thr	450 ± 72.9	2.72 ± 0.37	6.07 ± 0.24* (3%)	3.25 ± 0.44	0.63 ± 0.02*	0.20 ± 0.02*** (6%)
CYP2A643*	4406C>T	Thr303Ile	1049 ± 683	4.76 ± 2.00	5.33 ± 1.28* (3%)	8.55 ± 1.47	1.34 ± 0.09*	0.16 ± 0.02*** (5%)
CYP2A644*^d	5661G>A	Glu390Lys	1097 ± 284	2.39 ± 0.43	2.22 ± 0.16* (1%)	N.D.	N.D.	N.D.
	5745A>G	Asn418Asp
	5750G>C	Glu419Asp
CYP2A645*	6531C>T	Leu462Pro	55.1 ± 11.3	1.19 ± 0.12	22.0 ± 2.24* (13%)	1.65 ± 0.16	1.07 ± 0.02*	0.65 ± 0.05*** (21%)

N.D., Not determined.
* P < 0.05, **P < 0.01, and ***P < 0.005 compared with CYP2A6.1.
↵^a The kinetic parameters for nicotine C-oxidation of CYP2A6.2, CYP2A6.5, CYP2A6.6, and CYP2A6.26 could not be determined because the enzymatic activity of P450 was not detected at the highest substrate concentration (1000 μas not detec
↵^b The kinetic parameters for nicotine C-oxidation of CYP2A6.10, CYP2A6.36, and CYP2A6.37 could not be determined because the amount of product, cotinine, produced by these variants was below the quantification limit at low substrate concentrations.
↵^c The kinetic parameters of CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.26, and CYP2A6.37 could not be determined because the enzymatic activity of P450 was not detected at the highest substrate concentration (25 μas not detec
↵^d The kinetic parameters for nicotine C-oxidation of CYP2A6.10, CYP2A6.25, CYP2A6.36, and CYP2A6.44 could not be determined because the amount of product, 7-hydroxycoumarin, produced by these variants was below the quantification limit at low substrate concentrations.