Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

YTAAP-13364; No of Pages 11 Toxicology and Applied Pharmacology xxx (2014) xxx–xxx

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Article history: Received 9 February 2015 Revised 11 April 2015 Accepted 13 April 2015 Available online xxxx

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Keywords: CYP1A1 CYP1A1*12 Variant α-helix Heme Protein structure Enzyme activity

College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Korea School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea College of Medicine, Chung-Ang University, Seoul, Republic of Korea d College of Agriculture of Life Science, Seoul National University, Seoul, Republic of Korea b c

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Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. © 2014 Published by Elsevier Inc.

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Introduction

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The cytochrome P450 (CYP)-dependent monooxygenases constitute a large family of heme-containing enzymes responsible for the oxidoreductive metabolism of a variety of endogenous and exogenous compounds. CYPs are integral endoplasmic reticulum membrane-anchored proteins, with their N-terminus embedded in the membrane and their catalytic domain exposed to the cytosol. CYP1A1 was one of the first CYP enzymes to be characterized (Nebert et al., 1991). It belongs to

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Seung Heon Lee a, Sukmo Kang a, Mi Sook Dong b, Jung-Duck Park c, Jinseo Park d, Sangkee Rhee d, Doug-Young Ryu a,⁎

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Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

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Abbreviations: ANF, α-naphthoflavone; BaP, benzo[a]pyrene; CHO, Chinese Hamster Ovary; CYP, cytochrome P450; dALA, δ-aminolevulinic acid; EDTA, ethylenediaminetetraacetic acid; EROD, 7-ethoxyresorufin O-dealkylation; EV, empty control vector; ME, βmercaptoethanol; NPR, NADPH-cytochrome P450 reductase; NTA, nitrilotriacetic acid; PBS, phosphate buffered saline; PDB, Protein Data Bank; PMSF, phenylmethanesulfonyl fluoride; WT, wild-type. ⁎ Corresponding author at: College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul, South Korea. Fax: +82 2 878 2360. E-mail address: [email protected] (D.-Y. Ryu).

the CYP1 family along with CYP1A2 and CYP1B1, and plays a role in bioactivation of several pro-carcinogens to generate reactive metabolites and also in detoxification of various environmental compounds (Nelson et al., 1996). Endogenous substrates of CYP1A1 include inflammatory mediators such as arachidonic acid (Schwarz et al., 2004), and hormones such as 17β-estradiol (Lee et al., 2003) and melatonin (Ma et al., 2005). CYP1A1 is mainly localized in extra-hepatic tissues such as lung and intestine (Shimada et al., 1992; van de Kerkhof et al., 2008). Determination of benzo[a]pyrene (BaP) hydroxylation (Nebert and Gelboin, 1968) and 7-ethoxyresorufin O-dealkylation (EROD; Burke et al., 1977) activities principally represent CYP1A1 activity. A three-dimensional structure of human CYP1A1 (Protein Data Bank (PDB) code: 4I8V), which was co-crystallized with its inhibitor αnaphthoflavone (ANF), indicates the characteristic CYP fold observed for other members of the CYP superfamily, with canonical helices, denoted A through L, and three of the four canonical β-sheets (Walsh et al., 2013). Its active site is narrow and has large hydrophobic surfaces suitable for binding polycyclic aromatic hydrocarbons, as shown for CYP1A2 (Sansen et al., 2007) which shares 72% amino acid sequence

http://dx.doi.org/10.1016/j.taap.2015.04.010 0041-008X/© 2014 Published by Elsevier Inc.

Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

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Bacterial constructs. Open reading frame cDNA clones for CYP1A1 WT, and a variant containing the G184C substitution, were prepared using total RNA from lymphocytes of an individual having the substitution (Park et al., 2004). The cDNA sequences were modified for expression in E. coli according to Guo et al (1994). The second N-terminal residue of CYP1A1, Leu, was replaced with Ala and the nucleotide sequences encoding residues 3–9 were changed to AT-rich sequence (5′-ATGGCT TTTCCAATTTCAATGTCAGCA-3′) without substitution of residues. The sequences encoding CYP1A2 and variants were also modified according to Barnes et al (1991); the nucleotide sequence GCA, encoding the second N-terminal residue Ala, was replaced with GCT without amino acid replacement. Each cDNA fragment was inserted into the NdeI and XbaI restriction sites of pCW-NPR, a human NADPH-cytochrome P450

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Site-directed mutagenesis. Site-directed mutagenesis was performed to generate sequence variants of CYPs using an EZchange site-directed mutagenesis kit (Enzynomics, Daejeon, South Korea) according to the manufacturer’s instructions. Primers used for mutagenesis are listed in Supplementary Table S1.

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Bacterial expression. Bacterial harvest and membrane preparation were performed as described previously (Gillam et al., 1993; Guengerich and Martin, 2006). E. coli DH5α cells were transformed with the expression constructs and grown overnight at 37 °C in Luria–Bertani broth containing 50 μg/ml ampicillin. The overnight culture was inoculated 1:1000 into Terrific Broth medium containing 50 μg/ml ampicillin and 1 mM thiamine. Cultures were incubated at 37 °C with shaking at 200 rpm until they attained an OD600 of 0.5–0.7, then supplemented with 1 mM isopropyl-β-D-thiogalactopyranoside and 0.5 mM δaminolevulinic acid (dALA), a heme precursor, and cultured for 24 h at 30 °C with shaking at 200 rpm. Culture was then chilled on ice and centrifuged at 3,800 ×g for 20 min. The cell pellets were washed with phosphate buffered saline (PBS), and the cells were weighed and resuspended in 100 mM Trisacetate buffer, pH 7.6, containing 500 mM sucrose and 0.5 mM ethylenediaminetetraacetic acid (EDTA). Lysozyme was added to 0.2 mg/ ml, and the suspensions were diluted two-fold with distilled H2O before incubation on ice for 30 min. The resulting spheroplasts were sedimented at 3,800 ×g at 4 °C for 20 min, and resuspended in 100 mM potassium phosphate buffer, pH 7.6, containing 6 mM magnesium acetate, 20% glycerol (v/v), and 10 mM β-mercaptoethanol (ME). Suspensions of spheroplasts were sonicated four times for 20 s each, on ice, and centrifuged at 10,000 ×g at 4 °C for 20 min. Supernatants were centrifuged at 100,000 ×g at 4 °C for 75 min. Sedimented membrane fractions were resuspended in 100 mM potassium phosphate buffer, pH 7.6, containing 6 mM magnesium acetate, 20% glycerol (v/v), and 10 mM ME. The membrane preparation was stored at −70 °C until use.

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CYP and heme contents. CYP content was determined by reduced CO minus reduced difference spectra (Omura and Sato, 1964). Sodium dithionite was added to reduce ferric CYPs. Ferrous-CO CYP complexes were generated by passing CO gas through solutions of the ferrous CYPs. The spectra were collected on a spectrophotometer at room temperature. Heme content in CYP proteins was quantified using a pyridine hemochromogen assay (Schenkman and Jansson, 2006; Sinclair et al., 2001). Heme content was calculated from the difference in absorption between 557 and 575 nm.

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Immunoblots. Immunoblottings were performed using primary antiCYP1A1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (GenDepot, Barker, TX). Blots were developed using a chemiluminescence detection reagent kit. Protein concentrations were determined with a BCA protein assay kit (Pierce Biotechnology, Rockford, lL) using bovine serum albumin as a standard.

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Purification of His-tagged proteins. Membrane fractions of E. coli were solubilized with either 1% CHAPS (w/v) or 1.2% Emulgen 911 (v/v; Karlan Research, Cottonwood, AZ)/0.6% sodium cholate (w/v). However, only proteins solubilized with Emulgen 911/sodium cholate were used for protein purification. The detergent was dissolved in 100 mM

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Chemicals. All chemicals used were of analytical grade or higher and were purchased from Sigma-Aldrich (St. Louis, MO) unless specified.

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reductase (NPR)-containing bicistronic expression vector (Parikh et al., 1997). For purification of WT CYP1A1 and Ala62Pro variant proteins, six-His codons were added just before the stop codon of the modified cDNAs. The resulting fragments were inserted into monocistronic vector pCW using the NdeI and HindIII restriction sites (Gillam et al., 1995).

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homology and exhibits overlapping substrate specificities (Kim and Guengerich, 2005). The crystallized structure of a CYP1A1 closely resembles that of CYP1A2. The heme prosthetic group plays an important role in electron transfer during catalysis and is anchored within CYPs by hydrogen-bonding and other interactions. Analysis of different CYPs indicates that they contain a highly conserved sequence motif, FxxGxRxCxG, which is important for heme binding. In CYP1A1, the heme group is bound to residues including I449, F450, R455, C457, I458, G459 and A463 that lie within and around this motif. Other residues such as R106, S122, W131, A317, T321, F381, V382, T385, I386, H388 and 411Q are also involved in heme binding (PDB code: 4I8V). Synthesis of CYP apoprotein and its incorporation into endoplasmic reticulum membranes are tightly-coupled processes (Sakaguchi et al., 1987). In contrast, the insertion of heme is likely to occur only after the synthesis of the CYP apoprotein. When heme is supplied insufficiently, heme saturation of the CYP apoprotein is incomplete and the apoprotein can accept exogenously-supplied heme to form the holoprotein (Correia and Meyer, 1975; Sadano and Omura, 1983, 1985). Many allelic variants and several sub-variants have been described for the CYP1A1 gene (http://www.cypalleles.ki.se/). To date, there are more than 120 single nucleotide polymorphisms described for CYP1A1 in NCBI dbSNP (http://www.ncbi.nlm.nih.gov/, accessed January 2015). Among them, two non-synonymous polymorphisms, Ile462Val and Thr461Val, have been most widely studied using various heterologous expression systems (Chernogolov et al., 2003; Kisselev et al., 2005; Zheng et al., 2010). A variant allele of CYP1A1 containing a G to C transition at nucleotide 184 (G184C, dbSNP: rs143070677), resulting in a substitution of Ala62Pro, has been identified (Park et al., 2004; http://www.1000genomes.org/) and designated as CYP1A1*12 (http:// www.cypalleles.ki.se/). However, the effect of the Ala62Pro substitution on the enzymatic properties of CYP1A1 has not been characterized thus far. In the present study, we characterized a CYP1A1 variant harboring Ala62Pro by using heterologous expression in Escherichia coli and mammalian cells. Our findings suggest that the variant apoprotein has a low affinity for its prosthetic group heme and that the variant has decreased enzymatic activity compared with wild-type (WT) CYP1A1. Based upon molecular modeling, the variant’s reduced binding affinity for heme may be due to Ala62Pro-induced structural disturbances in α-helix A that contains Ala62. The α-helix A-containing region putatively forms interaction networks with adjacent structural elements and may be indirectly involved in binding of heme to the apoprotein. Some residues within the interaction networks were determined by site-directed mutagenesis to have important structural roles in heme binding to the apoprotein.

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Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

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BaP hydroxylation. BaP hydroxylation activity was assayed using modifications of a previously reported method (Nebert and Gelboin, 1968). The reaction mixture contained 100 pmol CYP reductase (Invitrogen), 20 μg of dilaurylphosphatidyl choline, 3 mM MgCl2, 80 μM BaP (dissolved in acetone), and E. coli membrane fractions (containing 50 pmol CYP) in a total volume of 1 mL of 100 mM potassium phosphate buffer, pH 7.4. Following a 5 min pre-incubation at 37 °C in water bath, reactions were initiated by the addition of the NADPHgeneration system. Incubation was terminated after 10 min by the addition of 1 mL of ice-cold acetone. After addition of acetone, 3.25 mL of hexane were added and 2 mL of the organic phase was extracted with 4 mL of 1 N NaOH. The fluorescence was measured in the alkali extract using excitation and emission wavelengths of 396 nm and 522 nm, respectively. BaP hydroxylation was determined using quinine sulfate in 0.1 N H2SO4 as a standard. In case of quinine sulfate, the wavelengths were set at 350 nm for excitation and 450 nm for emission.

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EROD. EROD activities were assayed by fluorometric detection of resorufin using excitation and emission wavelengths of 544 and 595 nm, respectively (Chang and Waxman, 2006; Shimada and Yamazaki, 1998). The reaction mixture contained 50 pmol CYP reductase (Invitrogen, Carlsbad, CA), 20 μg of dilauroylphosphatidyl choline, 0.5–20 μM 7-ethoxyresorufin dissolved in dimethylsulfoxide, and 50 pmol CYP proteins (E. coli membranes fractions and purified proteins), in a total volume of 0.5 mL of 100 mM potassium phosphate buffer, pH 7.4. To measure microsomal activities, microsomes equivalent to 100 μg of microsomal protein were used. Following a 5 min preincubation at 37 °C in a water bath, reactions were initiated by the addition of the NADPH-generation system (final concentrations 0.25 mM NADP+, 2.5 mM glucose 6-phosphate, 0.25 IU of yeast glucose 6-phosphate dehydrogenase). Incubations were terminated after 10 min by the addition of 1 mL ice-cold methanol.

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Expression in mammalian cells and microsomal preparations. Full-length cDNAs for WT CYP1A1 and variants were inserted into the HindIII and XbaI restriction sites of expression vector pcDNA3.1(+) (Invitrogen). The constructs were used for the expression of WT CYP1A1 and variant proteins in Chinese Hamster Ovary (CHO) cells (CCL-61, American Type Culture Collection, Manassas, VA). CHO cells were cultured in RPMI1640 medium with L-glutamine, supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 0.1 mM MEM non-essential amino acids solution and 10% fetal bovine serum (Life Technologies, Grand Island, NY). The cells were maintained at 37 °C in a 5% CO2 incubator. Twenty-four

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Sequence alignment. To compare the amino acid sequences of CYP1A1s from various species and human CYP1A2, a multiple-sequence alignment was generated using the ESPript 3.0 alignment program (Robert and Gouet, 2014). Secondary structural elements were imported from a human CYP1A1 structure (PDB code: 4I8V).

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potassium phosphate buffer, pH 7.4, containing 20% glycerol, 1 mM EDTA, 30 μM ANF, 10 mM ME and 1 mM phenylmethanesulfonyl fluoride (PMSF). After stirring for 3 h at 4 °C, the resulting solutions were centrifuged at 100,000×g at 4 °C for 45 min to eliminate insoluble material. The supernatants were then applied to a Ni-NTA (nitrilotriacetic acid) agarose column (Qiagen, Valencia, CA). The proteins were eluted with 100 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol, 0.5 M NaCl, 300 mM imidazole and 0.5% Emulgen 911. The eluted fractions were concentrated by ultrafiltration using a YM-30 membrane (Millipore, Bedford, MA). Imidazole was removed by multiple dialyses using 100 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol and 1 mM EDTA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to assess final protein purity.

dALA supplementation. E. coli expressing CYP1A1 WT and Ala62Pro variant proteins were cultured in media supplemented with dALA, to determine the effect of heme availability on holo-CYP enzyme production (Jansson et al., 2000). In addition to 0.5 mM dALA supplementation at the onset of 24 h culture, the culture medium was supplemented up to twice more with 0.5 mM dALA, 8 h and 16 h after the onset. Harvested E. coli cells and their membrane fractions were used for analysis of CYP content.

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Molecular modeling. Computational molecular modeling was performed for α-helix A, containing Ala62, and adjacent structural elements in a human CYP1A1 structure (PDB code: 4I8V; Walsh et al., 2013). Interdomain contact residues were defined as being within 4.0 Å of the partner domain, identified using the CONTACT program in the CCP4 suite (Winn et al., 2011) and confirmed by manual inspection. The structural representation was prepared with PyMol (Schrödinger Inc., Portland, OR).

Fig. 1. Expression of CYP1A1 Ala62Pro variant in E. coli. (A) CYP content and CYP1A1 total protein expression in E. coli expressing wild-type (WT) CYP1A1 and its Ala62Pro variant. (B) CYP content and CYP1A1 total protein expression in the membrane fractions of E. coli corresponding to panel A. (C) Heme content in the membrane fractions of E. coli corresponding to panel A. E. coli were transformed either with pCW-NPR vector harboring CYP1A1 WT or Ala62Pro variant cDNAs, or with empty control vector (EV). Upper right insets in panels A and B show reduced CO minus reduced difference spectra. Detection of CYP1A1 protein was performed with immunoblots. Each bar represents the mean ± SD of six independent samples. Different lower-case letters above the bars indicate a significant difference among the groups (P b 0.05, Tukey’s post hoc test).

Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

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Fig. 2. Enzyme activities of CYP1A1 Ala62Pro variant and WT proteins. (A) EROD and (B) BaP hydroxylation activities of E. coli membrane fractions expressing the variant and WT proteins. (C) EROD and (D) BaP hydroxylation activities of microsomes of CHO cells expressing the variant and WT proteins. Detection of CYP1A1 protein was performed with immunoblots. Each bar represents the mean ± SD of four independent samples. “a” above the bars indicates a significant difference from the WT (P b 0.05, unpaired Student’s t-test).

Fig. 3. Purification of CYP1A1 Ala62Pro variant. (A) Detection of CYP1A1 protein was performed with immunoblots in spheroplasts and membrane fractions of E. coli, and in proteins solubilized from the membrane fractions with either 1% CHAPS, or 1.2% Emulgen 911/0.6% sodium cholate. (B) The proteins solubilized from the membrane fractions with 1.2% Emulgen 911/ 0.6% sodium cholate were subjected to Ni-NTA column chromatography. (C) SDS-PAGE showing purified CYP1A1 WT and Ala62Pro proteins. (D) CYP content of the purified proteins. The upper right inset shows reduced CO minus reduced difference spectra. (E) Heme content of the purified proteins. Each bar represents the mean ± SD of four independent samples. “a” above the bars indicates a significant difference from the WT (P b 0.05, unpaired Student’s t-test).

Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

S.H. Lee et al. / Toxicology and Applied Pharmacology xxx (2014) xxx–xxx Table 1 EROD activities of CYP1A1 WT and the Ala62Pro variant purified from E. coli. Kinetic parameters were determined by using Lineweaver–Burk plots and represent the means ±

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SD of four independent experiments. “a” indicates a significant difference from the WT (unpaired Student’s t-test, P b 0.05).

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hours after being plated, the cells were transfected using a Lipofectamine LTX reagent (Invitrogen) following the manufacturer’s instructions. The cells were harvested 24 h post-transfection, washed with PBS, and resuspended in 100 mM phosphate buffer containing 20% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol and 1 mM PMSF. The cell resuspension was sonicated on ice for 40 s and centrifuged at 10,000×g at 4 °C for 20 min to remove cell debris. Supernatants were then centrifuged at 100,000×g at 4 °C for 60 min. The pelleted microsomal fractions were resuspended in 100 mM phosphate buffer containing 10% glycerol and 1 mM EDTA, and stored at −70 °C until use.

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Expression in E. coli

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Based upon reduced CO minus reduced difference spectra, the cellular CYP (i.e. holoenzyme) content in E. coli expressing the Ala62Pro variant of CYP1A1 was about 8.6% of that in cells expressing the WT (P b 0.05; Fig. 1A). However, based on the immunoblot analysis, the total level of CYP1A1 apoprotein in E. coli expressing the variant was similar to that in cells expressing the WT. E. coli transformed with the empty control vector (EV) was used as a negative control, for which CYP content and CYP1A1 protein expression were undetectable. The CYP content was also about 10.0-times lower in the membrane fraction of E. coli expressing the Ala62Pro variant than in cells expressing the WT (P b 0.05; Fig. 1B), while the total CYP1A1 protein levels were comparable. The CYP1A1 protein was not detected in the membrane fraction of E. coli transformed with EV, and CYP content was also undetectable.

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Membrane fractions of E. coli expressing Ala62Pro variant and WT proteins were used for two enzymes assays. EROD and BaP hydroxylation activities of the variant-expressing membranes were 2.10% and 3.44% of those of the WT counterparts, respectively (P b 0.05; Figs. 2A and B). In addition, microsomal preparations of CHO cells expressing the variant and WT proteins were used for those assays. The expression levels of total CYP1A1 protein were similar among the microsomal preparations, suggesting that the expression of the apoproteins and their incorporation into microsomal membranes were not affected by the substitution. However, EROD and BaP hydroxylation activities for the variant-expressing microsomes were 20.8% and 20.2% of those of the WT-expressing microsomes, respectively (P b 0.05; Figs. 2C and D).

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Purification

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The Ala62Pro variant and WT proteins were purified from the membrane fractions of E. coli. The total CYP1A1 protein level of the variant was similar to that of WT in the spheroplasts and membrane fractions of the E. coli (Fig. 3A), in agreement with the findings in Fig. 1. However, the level of the variant was lower than that of the WT in protein samples solubilized from the membrane fractions. These findings were consistently observed in two different detergent conditions, either 1% CHAPS or 1.2% Emulgen 911/0.6% sodium cholate, suggesting resistance of the membrane-bound variant protein to detergent-mediated solubilization. Emulgen 911/sodium cholate was found to be more effective in the solubilization of both the WT and variant, so it was used for further protein purification procedures.

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Enzyme activities of the E. coli membrane fractions and mammalian 313 microsomes 314

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Statistics. All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed with SPSS 19.0 for Windows (SPSS, Inc., Chicago, IL). Differences between sample groups were analyzed using the unpaired Student t-test and one-way ANOVA followed by Tukey’s post hoc test. A P-value of b 0.05 was considered significant.

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The peaks at about 420 nm in CO-spectra of E. coli and membrane fractions expressing the Ala62Pro variant may represent P420, a denatured heme-containing fraction of CYP1A1. However, the peak intensities were very low when normalized with those for EV controls and were regarded as negligible. Additionally, the total heme level in the membrane fraction of E. coli expressing the Ala62Pro variant was about 38% of that in E. coli expressing the WT (P b 0.05; Fig. 1C), and was not significantly different from that in the EV group. These findings suggest that the Ala62Pro substitution leads to a decrease in holo-CYP1A1 production by lowering the heme content of the protein without affecting the expression of the apoprotein. We therefore hypothesize that the Ala62Pro substitution disrupts the ordered framework of the protein structure and results in a loss of heme incorporation.

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Fig. 4. Effect of dALA supplementation. (A) CYP content in E. coli expressing CYP1A1 WT protein or its Ala62Pro variant after culture in medium supplemented with dALA in a repeated manner. (B) CYP content in the membrane fractions of E. coli cells corresponding to panel A. ×1, 0.5 mM dALA added to medium at the onset of 24 h culture; ×2, 0.5 mM dALA added to medium at the onset of culture and 8 h after the onset; ×3, 0.5 mM dALA added to medium at the onset of culture, and 8 and 16 h after the onset. Each circle represents the mean ± SD of four independent samples. Different lower-case letters indicate a significant difference among the groups (P b 0.05, Tukey’s post hoc test).

Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

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EROD activities of the purified proteins

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For functional characterization of the purified Ala62Pro variant, EROD activities at different substrate concentrations were fitted to the Michaelis–Menten equation to obtain kinetic parameters. The kinetic parameters for the variant were compared with those obtained for the purified WT protein (Table 1). The Vmax value for the variant was about 21% of that for the WT (P b 0.05), and the KM value for the variant was 1.84-fold higher than that for the WT. The catalytic activity of the variant, Vmax/KM, was 11% of the WT activity. Thus the Ala62Pro variant has a markedly reduced catalytic activity for EROD compared with the WT enzyme.

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Effect of repeated dALA supplementation

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The effect of increased heme availability on CYP content in E. coli expressing the WT and Ala62Pro variants was investigated. CYP content of E. coli expressing the WT protein became higher after repeated supplementation with dALA during culture. Cells supplemented once or twice during growth (in addition to supplementation at the onset of culture) had cellular CYP content 1.77- and 2.03-fold higher, respectively, than cells supplemented only at the onset of culture (P b 0.05; Fig. 4A). The membrane fractions of E. coli supplemented once or twice during the culture had 1.49- and 1.65-fold higher levels of CYP content than those from cells given dALA only at the onset of culture (P b 0.05; Fig. 4B). In contrast, the same treatments did not affect the CYP content either in whole cells or in membranes of E. coli expressing the Ala62Pro variant.

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When solubilized fractions containing the same amount of total protein were applied to Ni-NTA agarose columns, the columns turned reddish for both the WT and variant, but much less so for the latter (Fig. 3B). Because hemoproteins tend to be red/brown, this may be due to lower heme content in the variant fraction. The yields of the WT and variant from chromatography were approximately 4.3% and 2.3%, respectively, expressed as total amount of purified protein eluted as a percentage of total amount of protein loaded. The CYP and heme contents of the purified variant protein (Fig. 3C) were about 8.9% and 14% of those for the purified WT protein, respectively (P b 0.05; Figs. 3D and E).

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Molecular modeling of α-helix A and its adjacent structural elements was undertaken for CYP1A1 (Fig. 5). Based on a crystal structure of CYP1A1 (PDB code: 4I8V), α-helix A corresponds to residues 59–70. Residues 53–65 were designated Region A, shown in green. Region A does not appear to be directly involved in heme binding, but putatively interacts with three adjacent regions. Those structural segments are designated Region B (residues 381–388; shown in yellow), Region C (residues 409–413; shown in blue), and Region D (residues 493–498; shown in cyan). The major features of the inter-domain interaction networks among the four regions are a few hydrogen bonds and other interactions, defined as distances of 4 Å or less (Fig. 5C). The Pro59, His60, Leu61, Leu63, and Ser64 residues of Region A appear to mediate most of interactions with Regions B, C and D. His60 has putative contacts with all three regions. Region B is located C-terminal to α-helix K (residues 366–379), encompasses part of βstrand 1–4 (residues 388 and 389) and maintains direct interactions with heme via residues Phe381, Val382, Thr385, Ile386 and His388. Residues in Regions C and D make putative contacts with those in Region B. However, no direct contact was identified between Regions C and D.

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The amino acid sequence for Region A and its vicinity is highly conserved among CYP1A1s from various animal species and in human CYP1A2 (Fig. 6A). Two exceptions are the residues equivalent to Ala62 and Gln69 of human CYP1A1. Ala62 is replaced with Val in cow (B. taurus) and with Ser in zebrafish (D. rerio), mouse (M. musculus) and rat (R. norvegicus). The three residues that occupy the position equivalent to human Ala62, Ala, Ser, and Val, have small residue masses. Gln69 of human CYP1A1 is replaced with residues having polar side chains such as Arg and Cys in some species and with Arg in human CYP1A2. Residues in Region B and its vicinity are highly conserved among CYP1A1s from various species, and in human CYP1A2 (Fig. 6B), suggesting essential roles in various CYPs. In Region C and its vicinity, the residue equivalent to Arg405 in human CYP1A1 is substituted with residues such as Thr, His, and Cys in other species and with Cys in human

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Fig. 5. A three-dimensional structure of CYP1A1 (PDB code: 4I8V). (A) A ribbon model and (B) a magnified view of heme and Regions A, B, C and D. (C) A schematic representation of interactions among residues of Regions A, B, C, and D, and with heme. Red lines indicate hydrogen bonds, blue lines indicate other interactions (defined as distances of 4 Å or less). Heme is shown in magenta; Regions A, B, C and D are shown in green, yellow, blue and cyan, respectively.

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Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

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Fig. 6. Sequence comparison for (A) Region A, (B) Region B, (C) Region C, and (D) Region D of CYP1A1 from various species and human CYP1A2. The sequence of PDB 4I8V molecule A (Fig. 5) is identical to that of human CYP1A1 in the four regions. The accession numbers for human CYP1A1 and CYP1A2 are NP_000490.1 and NP_000752.2, respectively. The animal species and associated accession numbers are: B. taurus (XP_002696681.1), C. lupus (XP_003433938.1), D. rerio (NP_571954.1), M. mulatta (NP_001035328.1), M. musculus (NP_001129531.1) and R. norvegicus (NP_036672.2). Conserved residues are in blue boxes; identical residues are shown with a red background, similar residues in red typeface. Ala62 of human CYP1A1 and corresponding residues are indicated by a blue triangle. Secondary structure elements from 4I8V are displayed on the top of the alignment. Residue numbers are shown on the right hand side.

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CYP1A2. Human CYP1A1 Gln418 is substituted with Pro and Arg in other species and with Pro in human CYP1A2 (Fig. 6C). Residues equivalent to those from 487 to 505 of human CYP1A1, including Region D, are well conserved among the vertebrate species (Fig. 6D). Two exceptions include residues equivalent to Ile494 and Cys502; Ile494 is substituted by Val, Glu, Thr and Ala in the other species analyzed, while Arg replaces C502 in CYP1A1 from the non-primate species and in human CYP1A2.

Substitutions of CYP1A1 Ala62 and CYP1A2 Ala64 residues

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CYP content was measured in E. coli expressing some single-residue variants of Ala62 (Fig. 7A). Substitution to Gly, like Pro an α-helixincompatible residue, decreased the CYP content by about 14.3-fold as compared with the WT expressing cells (P b 0.05). Ala62 was also substituted with α-helix-compatible residues such as Ser and Val, which replace Ala62 in equivalent positions in CYP1A1s from other

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Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

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Fig. 7. CYP content in E. coli expressing (A) CYP1A1 WT and single-residue Ala62 variants and (B) CYP1A2 WT and single-residue Ala64 variants. E. coli cells were transformed either with pCW-NPR vector harboring cDNAs or with EV. Each bar represents the mean ± SD of six independent samples. Different lower-case letters above the bars indicate a significant difference among the groups (P b 0.05, Tukey’s post hoc test).

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variant had CYP content that was almost equivalent to the WT group. The expression levels of CYP1A2 apoprotein were similar among the WT and variant groups. The EV controls exhibited no detectable levels of CYP1A2 apoprotein and CYP content.

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animal species (Fig. 6A). E. coli expressing Ala62Ser and Ala62Val variants had CYP contents that were lower by only 1.68- and 1.59-fold, respectively, than cells expressing the WT (P b 0.05), and were higher by at least 8.52-fold than E. coli expressing the Ala62Gly variant (P b 0.05). The expression levels of CYP1A1 total protein (holo + apo) were equivalent among the WT and variant groups. The EV controls exhibited no detectable levels of CYP1A1 apoprotein or CYP content. Additionally, the Ala64 residue of CYP1A2 was replaced with Pro and Ser (Fig. 7B). Ala64 of CYP1A2 is structurally equivalent to Ala62 of CYP1A1 (Fig. 6A). E. coli expressing the Ala64Pro variant had a decreased CYP content by about 2.91-fold (P b 0.05), as compared with the CYP1A2 WT group. By contrast, E. coli expressing the Ala64Ser

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Fig. 8. EROD activities in microsomal fractions of CHO cells expressing CYP1A1 WT and single-residue Ala62 variants. CHO cells were transfected with either pcDNA3.1(+) vector harboring cDNAs of CYP1A1 WT or its variants, or with EV. Each bar represents the mean ± SD of three independent samples. Expression of CYP1A1 protein was analyzed with immunoblots. Different lower-case letters above the bars indicate a significant difference among the groups (P b 0.05, Tukey’s post hoc test).

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Microsomal fractions from CHO cells expressing CYP1A1 WT and single-residue variants, Ala62Gly and Ala62Val, were used for EROD assays (Fig. 8). The expression levels of total CYP1A1 protein were similar among the three microsomal preparations, suggesting that the expression of the apoproteins and their incorporation into microsomal membranes were not affected by the substitutions. However, the microsomes containing CYP with the Ala62Gly substitutions exhibited lower EROD activities, 14.2% of that for the WT group (P b 0.05). In contrast, on substitution to Val, the EROD activity was 73.7% of that for the WT group (P b 0.05). Microsomes from EV-transformed cells exhibited no detectable CYP1A1 apoprotein or EROD activity.

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Substitution of residues in Regions A, B, C and D

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We determined cellular CYP content in E. coli expressing CYP1A1 with single-residue variations in residues in Regions A, B, C and D (Fig. 9). The expression levels of CYP1A1 total protein were comparable among the sample groups analyzed. E. coli expressing Gly substitution variants for residues in Region A had CYP content 61% or less than that in cells expressing the WT (P b 0.05; Fig. 9A). For residues from Lys57 to Leu63, the CYP content was 10.0% or less than that of the WT group (P b 0.05); these levels were more than 3.1-fold lower than those for substitution of the surrounding residues Leu53, Thr54, Leu55, Ser64, and Arg65. These findings suggest a critical role for residues Lys57 to Leu63 in formation of holoCYP1A1. Residues Pro59 and Leu63 of Region A were also replaced with Ala (Fig. 9B). The Pro59Ala and Leu63Ala substitutions partially recovered

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Fig. 9. CYP content in E. coli expressing single-residue variants in Regions A, B, C or D of CYP1A1. E. coli cells were transformed with pCW-NPR vector harboring cDNAs. (A) Gly variants of residues from 53 to 65 in Region A; (B) Ala variants of residues 59 and 63 in Region A; (C) Gly variants of residues from 381 to 388 in Region B; (D) Ala variant of residue 384 in Region B; (E) Gly variants of residues from 409 to 413 in Region C; (F) Gly variants of residues 493–498 in Region D; (G) Ala variants of residues 494 and 498 in Region D. Residues 56 and 495 are naturally Gly and thus not included. Each bar represents the mean ± SD of six independent samples. Different lower-case letters above the bars indicate a significant difference between or among the groups (P b 0.05, unpaired Student’s t-test for Panel D, Tukey’s post hoc test for the other panels).

Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

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Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

Interactions between functional groups within CYPs are essential for the protein’s stability and ability to bind the heme group (Kitagawa et al., 2001; Klein et al., 2007; Lam et al., 2001; Palma et al., 2010; Zheng et al., 1998). Based upon the findings in this study, it is proposed that an Ala62Pro substitution in CYP1A1 disrupts the ordered framework of the protein structure and results in a loss of heme incorporation in a major fraction of the variant proteins (Figs. 1 and 3). The hemedeficient pool of the variant is unlikely to have oxidative enzymatic activity due to loss of electron transfer capacity. The remaining hemecarrying pool of variant appears to have altered enzymatic characteristics, as shown in decreased enzyme activities (Fig. 2; Table 1). Pro is a potent breaker of α-helix structure. Nevertheless, Pro occurs in 8% of helices analyzed, especially in the first turn, acting as an N-capping residue (O'Neil and DeGrado, 1990; Richardson and Richardson, 1988; Strehlow et al., 1991). Ala62 is the fourth residue in an α-helix of CYP1A1, designated α-helix A. It is thus possible, in principle, that Ala62Pro substitution occurred at a location where Pro is compatible with an α-helix structure. However, a decrease and partial recovery of CYP content in E. coli were demonstrated by substitution of Ala62 to α-helix-incompatible and -compatible residues, respectively (Fig. 7). That was also the case for CYP1A2. Mammalian microsomes harboring Ala62 variants carrying substitutions to α-helixincompatible and -compatible residues also exhibited decreased and partially recovered catalytic activities, respectively (Fig. 8). These findings suggest that the Ala62Pro substitution-induced alterations in the structure and function of CYP1A1 are due to disturbance in the structure of α-helix A. Proper folding of apo-CYP is essential for heme insertion, and the heme plays an essential role as a template for protein folding (Correia et al., 2011). Enhanced availability of a heme precursor during culture did not increase the CYP content of Ala62Pro-expressing bacteria, which was in contrast to the WT group (Fig. 4). These findings lead us to propose that the variant protein has structural characteristics that disfavor heme binding. However, a small fraction of heme-containing

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variant was observed (Figs. 1 and 3). It is possible that the hemebound fraction of the variant was formed by heme insertion that occurred in the early stage of the apoprotein folding, before the polypeptide became “closed” to heme. In addition, it cannot be excluded that the structure of the variant protein is leakier, resulting in abnormally high dynamic turnover of heme and unstable heme incorporation. Site-directed mutagenesis analyses suggest important roles for the residues in Region A, the region adjacent to the N-terminus of α-helix A, and in Region B, the α-helix K/β-sheet 1-4 region, in holo-CYP1A1 formation (Fig. 9). It is not surprising that residues in Region B had a major influence on holoCYP1A1 formation, because some of those residues putatively interact directly with heme. However, the impact of substitutions in α-helix A residues was greater than expected, because these residues are rather remote from the heme group compared with the three other structural elements analyzed in this study (Fig. 5). The interactions of Region A, especially residues Pro59, His60, and Leu63, with Region B appear to be critical in Region A’s role in heme binding by CYP1A1. In support of this conclusion, the substitution to Gly of those three and surrounding residues of Region A dramatically decreased CYP content in E. coli expressing the variants (Fig. 9A). The substitution of residues Pro59 and Leu63 to Ala also exhibited a considerably negative effect on the bacterial CYP content (Fig. 9B). It is thus proposed that Ala62Pro substitution disrupts the interaction of Region A with Region B by distorting the structure of the former, especially with respect to the structural integrity of α-helix A conformation, and reduces the variant’s heme-binding capability. A putative interaction was identified between Asp61 and Arg372 in a three-dimensional structure of CYP3A4 (Lewis et al., 1996; Szklarz and Halpert, 1997); the interaction was shown to play a critical role in heme binding by the isozyme (He et al., 1997). Interestingly, residues Asp61 and Arg372 of CYP3A4 appear to be located in regions corresponding to Region A and Region B of CYP1A1. Asp61 is the fifth residue of α-helix A, and Arg372 is located between α-helix K and β strand 1-3 of CYP3A4 (Johnson and Stout, 2005). In addition to CYP1A1 and CYP3A4, interactions of α-helix A with a heme-binding region adjacent to the C-terminal of α-helix K have been demonstrated in many human CYPs belonging to families 1, 2, and 3 (Supplementary Table S2), suggesting that the interaction is essential in the structure and function of various CYPs. Amino acid substitutions in Region C appeared to have relatively minor effects on CYP content in variant-expressing E. coli (Fig. 9) compared with those for Regions A and B, which may suggest that Region C has only a limited role in holo-CYP expression. However, it cannot be excluded that dense interactions between Regions B and C, including four putative H-bond interactions, are robust enough to resist those single-residue substitutions. Structural details of the CYP protein-membrane interaction are not well understood. In a computer-based modeling study, however, the α-helix A of membrane-bound CYP2C9 was observed to be in contact with the lipid bilayer (Cojocaru et al., 2011). Based upon wellconserved structures of various CYP proteins (Sirim et al., 2010), it is possible that the α-helix A of CYP1A1 is located adjacent to, or in contact with, the membrane. Altered solubility of the variant (Fig. 3) suggests aberrant integration into the bacterial membrane or formation of insoluble aggregates due to improper folding of the protein. In addition, it cannot be excluded that a portion of the variant proteins were degraded upon extraction from the membrane. Taken together, our findings suggest the Ala62Pro substitution distorts the structural integrity of CYP1A1, and results in the formation of protein that has a decreased heme-binding affinity and holoenzyme with altered enzymatic characteristics. Thus, the G184C nucleotide polymorphism responsible for this amino acid variation may have a critical impact on affected individuals’ phenotype in metabolism of endogenous and environmental substrates of CYP1A1. Future studies are warranted to show the influence of the polymorphism on

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CYP content in E. coli expressing these variants, relative to their Gly counterparts. However, those expressing the two Ala variants still had lower CYP content than the WT group by 45% and 24%, respectively (P b 0.05). Residues from Phe381 to His388 in Region B were also substituted with Gly (Fig. 9C). E. coli expressing those variants contained less than 46% of the CYP level of the WT group (P b 0.05). Protein with substitutions in residues Phe381, Val382, Pro383, Phe384 and His388 had no detectable CYP content (P b 0.05), which supports the finding that some of them make direct interactions with heme (Fig. 5). The Phe384Ala group had CYP content that was 18.9% of the level of the WT group (P b 0.05), exhibiting a partial recovery in CYP content compared with the Gly substitution variant (Fig. 9D). Gly substitution of residues Asn410 to Gln413 in Region C reduced CYP content by 16% to 29% in E. coli expressing the corresponding variants, relative to the WT group (P b 0.05; Fig. 9E). Cells expressing the Val409Gly variant had CYP content comparable to the WT group. Gly substitutions of residues Ile493, Tyr494, Thr497 and Met498 in Region D decreased CYP content of E. coli expressing the variants by 57%, 23.0%, 68.0% and 92%, respectively, relative to the WT group (P b 0.05; Fig. 9F). In contrast, the Leu496Gly variant-expressing E. coli had CYP content comparable to the WT group (Fig. 9F), as did cells expressing the Tyr494Ala variant (Fig. 9G). Cells expressing the Met498Ala variant exhibited 59% lower CYP content than those expressing the WT (P b 0.05), but had about five-fold higher CYP content than those expressing Met498Gly. Overall, substitution of several specific residues in Regions A and B decreased the CYP content most profoundly among the residues analyzed.

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the individuals’ susceptibility to various related diseases such as environmentally-associated cancers. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.taap.2015.04.010.

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This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0022822).

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Please cite this article as: Lee, S.H., et al., Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2015.04.010

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