1521-0111/88/4/800–812$25.00 http://dx.doi.org/10.1124/mol.115.098582MOLECULAR PHARMACOLOGY Mol Pharmacol 88:800–812, October 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Suppression of Cytochrome P450 3A4 Function byUDP-Glucuronosyltransferase 2B7 through a Protein-ProteinInteraction: Cooperative Roles of the CytosolicCarboxyl-Terminal Domain and the Luminal Anchoring Region s

Yuu Miyauchi, Kiyoshi Nagata, Yasushi Yamazoe, Peter I. Mackenzie, Hideyuki Yamada,and Yuji IshiiLaboratory of Molecular Life Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan (Y.M.,H.Y., Y.I.); Tohoku Pharmaceutical University, Sendai, Japan (K.N.); Food Safety Commission, Cabinet Office, Government ofJapan, Tokyo, Japan (Y.Y.); and Department of Clinical Pharmacology, Flinders Medical Center and Flinders University, Adelaide,Australia (P.I.M.)

Received February 23, 2015; accepted July 31, 2015

ABSTRACTThere is a large discrepancy between the interindividual differencein the hepatic expression level of cytochrome P450 3A4 (CYP3A4)and that of drug clearance mediated by this enzyme. However, thereason for this discrepancy remains largely unknown. BecauseCYP3A4 interactswithUDP-glucuronosyltransferase 2B7 (UGT2B7)to alter its function, the reverse regulation is expected to modulateCYP3A4-catalyzed activity. To address this issue, we investigatedwhether protein-protein interaction between CYP3A4 and UGT2B7modulates CYP3A4 function. For this purpose, we coexpressedCYP3A4, NADPH-cytochrome P450 reductase, and UGT2B7 usinga baculovirus-insect cell system. The activity of CYP3A4 wassignificantly suppressed by coexpressing UGT2B7, and thissuppressive effect was lost when UGT2B7 was replaced withcalnexin (CNX). These results strongly suggest that UGT2B7

negatively regulates CYP3A4 activity through a protein-proteininteraction. To identify the UGT2B7 domain associated withCYP3A4 suppression we generated 12 mutants including chime-ras with CNX. Mutations introduced into the UGT2B7 carboxyl-terminal transmembrane helix caused a loss of the suppressiveeffect on CYP3A4. Thus, this hydrophobic region is necessary forthe suppression of CYP3A4 activity. Replacement of the hydro-philic end of UGT2B7 with that of CNX produced a similarsuppressive effect as the native enzyme. The data using chimericprotein demonstrated that the internal membrane-anchoring re-gion of UGT2B7 is also needed for the association with CYP3A4.These data suggest that 1) UGT2B7 suppresses CYP3A4 function,and 2) both hydrophobic domains located near the C terminus andwithin UGT2B7 are needed for interaction with CYP3A4.

IntroductionCytochrome P450 (P450) 3A4 is one of the major drug-

metabolizing enzymes involved in the metabolism of 50% ofclinical drugs (Thummel and Wilkinson, 1998; Guengerich,1999). There are marked interindividual differences in theexpression level of hepatic CYP3A4, and the difference amongindividuals may be as much as 40-fold (Shimada et al., 1994;

Lamba et al., 2002). In contrast, the variance in drug clearancecatalyzed by CYP3A4 is less than 10-fold (Lamba et al., 2002).Thus, there is a large discrepancy between the expression level ofCYP3A4 and drug clearance mediated by this P450. In manycases, such a discrepancy can be explained by a single-nucleotidepolymorphism. However, the frequencies of CYP3A4 single-nucleotide polymorphisms, which are able to suppress enzyme

This study was supported in part by the Japan Research Foundation forClinical Pharmacology; a Grant-in-Aid for Scientific Research (B) [Grant25293039] from the Japanese Society of Promotion of Science; and a Grant-in-Aid for Scientific Research (C) [Grant 19590147] from the Ministry ofScience, Education, Sports and Technology to Y.I.

This work was presented in part at the 132nd Annual Meeting of thePharmaceutical Society of Japan, Sapporo, Japan, March 2012 (Miyauchi Y,Ishii Y, Oizaki T, Nagata K, Yamazoe Y, Mackenzie PI, Yamada H,Suppression of cytochrome P450 3A4 function by UDP-glucuronosyltransferase2B7: Role of C-terminal cytosolic region of UGT2B7); the 19th Microsomes andDrug Oxidation Meeting/12th European Regional Meeting of the InternationalSociety for the Study of Xenobiotics, Noordwijk ann Zee, The Netherlands, June2012 (Miyauchi Y, Ishii Y, Nagata K, Yamazoe Y, Mackenzie PI, Yamada H,UDP-Glucuronosyltransferase (UGT) 2B7 and 1A9 suppress cytochrome P4503A4 function: evidence for the involvement of the cytosolic tail of UGT in thesuppression); the 28th Annual Meeting of the Japanese Society of the Study ofXenobiotics, Tokyo, Japan, October 2013 (Miyauchi Y, Nagata K, Yamazoe Y,Mackenzie PI, Yamada H, Ishii Y, Suppression of cytochrome P450 3A4activity by UDP-glucuronosyltransferase 2B7: Crucial role of the length of

UGT2B7 cytosolic tail in the suppression); the 134th Annual Meeting of thePharmaceutical Society of Japan, Kumamoto, Japan, March 2014 (MiyauchiMiyauchi Y, Ishii Y, Nagata K, Yamazoe Y, Mackenzie PI, Yamada H,Alteration of cytochrome P450 3A4 activity through a protein-proteininteraction with UDP-glucuronosyltransferase 2B7: Cooperation of the luminaldomain and cytosolic tail of UGT2B7 is required in the suppression of CYP3A4function); 19th North American Regional Meeting of the International Societyfor the Study of Xenobiotics/29th Annual Meeting of the Japanese Society of theStudy of Xenobiotics, San Francisco, October, 2014 (Miyauchi Y, Ishii Y,Nagata K, Yamazoe Y, Mackenzie PI, Yamada H, Suppression of cytochromeP450 3A4 activity by UDP-glucuronosyltransferase (UGT) 2B7: the role ofcharged residue(s) in the cytosolic tail of UGT2B7); and the 20th Microsomesand Drug Oxidation Meeting, Stuttgart, Germany, May, 2014 (Ishii Y,Miyauchi Y, Yamada H, Functional interactions between cytochrome P450 andUDP-glucuronosyltransferase: a new insight into the inter-individual variationof drug metabolism).

dx.doi.org/10.1124/mol.115.098582.s This article has supplemental material available at molpharm.

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function, are rare and seem to be unable to explain theaforementioned discrepancy (Hirota et al., 2004; Lakhmanet al., 2009). It is also unconvincing that micro-RNA–dependent downregulation underlies the polymorphic functionof CYP3A4 (Takagi et al., 2008; Pan et al., 2009; Singh et al.,2011). Because CYP3A4 plays a central role in drugmetabolism,it is important to understand the reason why the variations infunction of this enzyme cannot be simply explained by theexpression level.It is well established that P450 needs electrons supplied

from redox partners, i.e., NADPH-cytochrome P450 reductase(CPR) and cytochrome b5 (b5). Therefore, it stands to reasonthat P450 function varies depending on the strength ofP450-CPR/b5 coupling. In this context, early studies examinedthe conditions required for protein-protein interactions betweenP450 and CPR/b5 mainly in a reconstituted system (Lu et al.,1969; Hildebrandt and Estabrook, 1971; Miwa and Lu, 1984).However, to the best of our knowledge, the interindividualvariation in P450 function has not been successfully explainedby interactions with the redox partners. The catalytic functionof P450s can be altered by homo- and hetero-oligomerization(Davydov, 2011; Reed and Backes, 2012). In addition, P450can bind to different sorts of proteins: for instance, ourlaboratory has provided evidence that CYP1A1 associateswith UDP-glucuronosyltransferase (UGT) and microsomalepoxide hydrolase (Taura et al., 2000; Ishii et al., 2005).Although many pairs of P450-UGT complexes can be formed(Ishii et al., 2007), we have particularly focused on a CYP3A4-UGT2B7 interaction, because these enzymes are involved inthe metabolism of many drugs. The association of CYP3A4and UGT2B7 has been confirmed by several biochemicaltechniques including coimmunoprecipitation and cross-linking(Takeda et al., 2005, 2009; Ishii et al., 2010, 2014). BecauseCYP3A4 alters the regio-selectivity of UGT2B7-catalyzedmorphine glucuronidation (Takeda et al., 2005) the bindingof CYP3A4 andUGT2B7 is undoubtedly a functional interaction.However, the reverse effect—whether UGT2B7 can modulateCYP3A4 activity—has not been investigated. If this interactionreally takes place, such a post-translational regulation of P450activity would be important for understanding why thereis a large discrepancy in the magnitude of interindividualdifferences between the expression level of CYP3A4 and drugclearance catalyzed by this P450.Our previous work has suggested that the J-helix of

CYP3A4 is a candidate for the domain contributing toCYP3A4-UGT2B7 association (Takeda et al., 2009). How-ever, the region of UGT2B7 involved in the interaction withCYP3A4 remains largely unknown. In the present study, wefocused on this issue, and tried to identify the UGT2B7domain capable of interacting with CYP3A4. To this end,we simultaneously expressed CYP3A4, CPR, and UGT2B7using a baculovirus-insect cell system, and examined whethercoexpression of UGT2B7 alters CYP3A4 activity. We alsosearched for UGT2B7 domains necessary for functional interac-tion with CYP3A4 by constructing a series of deletion mutants,substitution mutants, and chimeric proteins with calnexin(CNX), which is a nondrug-metabolizing protein having thesame membrane topology as UGT.

Materials and MethodsSynthetic oligonucleotides were purchased from Life Technolo-

gies (Carlsbad, CA). Restriction enzymes and other DNA-modifyingenzymes were purchased from Takara Bio (Shiga, Japan). Emulgen911 was kindly gifted by Kao Chemicals, Ltd. (Tokyo). A metab-olite standard in high-performance liquid chromatography anal-ysis, 6b-hydroxytestosterone, was purchased from Sigma-Aldrich(St. Louis, MO). Pooled human liver microsomes (HLMs) preparedfrom 50 donors were purchased from Corning Gentest (Woburm,MA). All other reagents were of the highest quality commerciallyavailable.

Subcloning of cDNAs into a Baculoviral Vector for Coex-pression. To prepare recombinant baculoviruses, a Bac-to-BacBaculovirus Expression System (Life Technologies) was used. The openreading frame of UGT2B7 was amplified by polymerase chain reaction(PCR) using UGT2B7 cDNA (Jin et al., 1993) as the template. PfuTurbo DNA polymerase (Agilent Technologies, Santa Clara, CA) wasused for this amplification. The PCR product was digested with KpnI,and cloned into pFastBac1 restricted with the same enzyme. HumanCPR cDNA was subcloned from a construct provided by us into theEcoRI site of pFastBac1. We also subcloned CNX cDNA into thepFastBacl vector. Because CNX is a chaperone protein expressing onthe endoplasmic reticulum (ER) membrane with the same membranetopology as UGT (David et al., 1993; Bergeron et al., 1994), this studyused CNX as a reference protein. Human CNX cDNA was purchasedfrom OriGene Technologies (Rockville, MD), and the mRNA-codingregion sandwiched between the PstI-cleaving sites was amplified.After PstI treatment, CNX cDNAwas also cloned into pFastBac1. Theconstruction of pFastBac1-CYP3A4 was carried out as previouslydescribed (Ishii et al., 2014). For the pull-down assay, CYP3A4 havinga carboxyl terminus tagged with hexahistidine (His-CYP3A4) wasconstructed by PCR. All the primers used for subcloning are listedin the Supplemental Material (see Supplemental Tables 1–4). Thenucleotide sequences of the constructs were confirmed by an ABI3130xl Genetic analyzer, using a BigDye Terminator Cycle Sequenc-ing Kit, version 3.1 (Life Technologies). Recombinant pFastBac1vectors were transfected into the competentEscherichia coliDH10Bacstrain (Life Technologies). After positive clones were selected accord-ing to the user’s manual, recombinant bacmids (i.e., baculoviral DNAsfor transfection) were prepared.

Culture of Sf9 Cells and Expression of Recombinant Enzymes.Sf9 insect cells were grown in a 500 ml plastic Erlenmeyer flask(screwed cap) containing Grace’s medium (Life Technologies) supple-mented with 10% fetal bovine serum, 10 mg/ml gentamycin, 0.25 mg/mlFungizone, and 1% CD Lipid Concentrate (Life Technologies). To obtainrecombinant baculovirus, Sf9 cells (2� 107 cell) were seeded once ina 175 cm2 culturing flask, and then transfected with recombinantbacmids in the presence of CellfectinII reagent (Life Technologies)diluted with Grace’s medium without any additives. After 5-hourincubation, the medium was replaced with fresh medium, andthe cells continued to be cultured. The control bacmids, obtainedfrom transfection of pFastBac1 carrying no passenger DNA (mockpFastBac1), were also used for transformation as the control. Primaryvirus was collected 1 week after the first transfection. The baculovirusDNA was purified by NucleoSpinBlood (Macherey-Nagel, Düren, Ger-many) fromprimary viruses in 200ml culturemedium, and their titerwasdetermined using a BacPAK qPCR Titration Kit (Clontech, MountainView, CA). The transfection of Sf9 cells with baculoviral DNA wasrepeated usually 4 to 5 times until a titer of over 1.0� 107 plaque-formingunits/ml was obtained. For expression of recombinant enzymes, Sf9 cells(2� 106 cells/ml, 200 ml) were transfected with recombinant baculovirusin an Erlenmeyer flask, and collected after 72 hours by low-speed centri-fugation. In the case of expression of CYP3A4, 1 mg/ml hemin/1.25%albumin complex was added to the medium at a concentration of

ABBREVIATIONS: b5, cytochrome b5; CNX, calnexin; CPR, NADPH-cytochrome P450 reductase; ER, endoplasmic reticulum; HLM, human livermicrosomes; HRP, horseradish peroxidase; P450, cytochrome P450; PCR, polymerase chain reaction; UGT, UDP-glucuronosyltransferase.

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1.4 mM hemin 24 hours after transfection. Microsomes were preparedfrom the transfected cells according to the protocols described previously(Ishii et al., 2014).

Pull-Down Assay. Microsomes were diluted to 2 mg protein/mlwith 100 mM sodium phosphate (pH 7.4) containing 20% glycerol (PGbuffer). Solubilization of the microsomes with sodium cholate wasperformed by a method described elsewhere (Takeda et al., 2005). Theresulting solution was centrifuged at 105,000g for 1 hour, and thesupernatant was collected as solubilized microsomes. An aliquot(125 ml) of the solubilized microsomes was diluted four times withPG buffer containing 200 mM sodium chloride, 40 mM imidazole,0.05% Emulgen 911, and 0.2% bovine serum albumin. This solution(500 ml) was then gently mixed at 4°C for 1 hour with magneticagarose beads conjugated with nickel-nitrilotriacetic acid (Qiagen,Hilden, Germany). The beads gathered by a magnet were washedthree times with 20 mM Tris-HCl (pH 7.4 at 4°C) containing 200 mMsodium chloride, 40 mM imidazole, 20% glycerol, and 0.05%Emulgen 911. The proteins associated with His-CYP3A4 wereeluted from the beads with 10 mM sodium phosphate (pH 7.4)containing 300 mM sodium chloride, 250 mM imidazole, 20%glycerol, and 0.05% Emulgen 911.

Immunoblotting. Proteins separated by SDS-PAGEwere electro-blotted onto a polyvinylidene difluoride membrane (Millipore, Bed-ford, MA). UGT2B7 was detected either by goat anti-mouse low pIform UGT antibody (Mackenzie et al., 1984) or rabbit anti-UGT2B7antibody (Corning Gentest). A WB-MAB-3A Human CYP3A WesternBlotting Kit (Corning Gentest) was used to detect CYP3A4. Rabbitanti-rat CPR (Enzo Life Sciences, Farmingdale, NY) and Rabbit anti-CNX (GeneTex, Irvine, CA) were also purchased from the sourcesindicated. They were diluted 2000-fold when used. Immunochemicaldetection was conducted either with horseradish peroxidase (HRP)–conjugated secondary antibodies, HRP-rabbit anti-goat IgG (MPBiomedicals, Santa Ana, CA), or HRP-donkey anti-rabbit IgG (GEHealthcare, Piscataway, NJ). These were diluted 10,000- and 40,000-fold before use, respectively. ClarityWesternECLSubstrate (Bio-Rad,Hercules, CA) was used as the substrate of HRP, and the chemilumi-nescence emitted was analyzed by a ChemiDoc MP System (Bio-Rad).

Assessment of the UGT2B7 Effect on CYP3A4-CatalyzedOxidation. The activity of CYP3A4 was measured by two methods.In the first method, pentafluorobenzyl ether coupled with luciferin(Luciferin-PFBE, Promega, Madison, WI) was used as the substrate,and CYP3A4 activity was determined by measuring chemilumines-cence according to the manufacturer’s protocol. In kinetics using theabove substrate, the substrate concentration was varied from 6.25 to100–200 mM. In the second method, CYP3A4 activity was measuredusing testosterone, a traditional CYP3A substrate, and an incubationmixture (250 ml) consisting of 100 mM potassium phosphate (pH 7.4),200 mM testosterone, 1 mM NADPH, and 50 nM recombinantCYP3A4. After preincubation at 37°C for 10 minutes, the reactionwas started by adding NADPH. The incubation was continued for10minutes and terminated with 100ml of 1M trichloroacetic acid. Thesolution was kept on ice for 1 hour, and then centrifuged at 15,000 rpmfor 10 minutes. After the supernatant (300 ml) was collected, pro-gesterone was added to the solution as an internal standard (final130 mM), and a portion (20 ml) of the solution was subjected to high-performance liquid chromatography for the analysis of 6b-hydroxy-testosterone. The instrument used for this analysis was a D-2000Elite high-performance liquid chromatography system (Hitachi High-Technologies, Tokyo) consisting of an L-2200 autosampler, L-2130pump, L-2300 column oven, and L-2400 UV detector. The operationconditions were as follows: 1) column, Nova-Pak C18 column (4 mm,8 � 100 mm; Waters, Milford, MA); 2) column temperature, 25°C;3) elution program (percentage of acetonitrile in water), 20% for5.0 minutes, and then increased to 80% for 20 minutes, held at 80%for 5 minutes, and followed by a stepwise reduction to 20% for0.1 minute (this condition was maintained for 5 minutes for the nextsample); 4) flow rate, 1.2 ml/min; and 5) detection, UV absorbance at

240 nm. Under these conditions, 6b-hydroxytestosterone and pro-gesterone were eluted at retention times of 14.0 and 27.0 minutes,respectively. Data were stored and processed using the D-2000 EliteSystemManager software, version 3.0 (Hitachi High-Technologies).

CYP3A4-dependent NADPH consumption and H2O2 generationwere analyzed according to the method used by Locuson et al. (2007)with slight modifications. The reaction mixture was the same as thatused in testosterone hydroxylation except for the NADPH concentra-tion (300 mM). The reaction was started by addition of NADPH, anda reduction in absorbance at 340 nm was measured for 20 minutes.The reaction rate was calculated using an extinction coefficient of6.23 mM21/cm21. The NADPH consumption was measured in thepresence and absence of substrate, 200 mM testosterone. The amountof H2O2 produced was measured by a Pierce Quantitative PeroxideAssay Kit obtained from Thermo Scientific (Rockford, IL) after in-cubating under the same conditions as the assay of CYP3A4-catalyzedtestosterone hydroxylation. Peroxide-driven oxidation (shunt pathway)(Chefson et al., 2006; Omura, 2011) in the catalytic cycle of CYP3A4was also conducted under the common conditions for testosteronehydroxylation, except that the reaction was initiated by adding cumenehydroperoxide (final 1 mM) instead of NADPH (Chefson et al., 2006).

Generation of UGT2B7 Mutants to Identify the DomainsInteracting with CYP3A4. A series of UGT2B7 mutants wasdesigned to clarify the crucial domain(s) involved in the CYP3A4-UGT2B7 interaction (the sequences of mutants are listed in Tables 2–4). In the case of deletionmutants lackingC-terminal areas (D519-529,D511-529, and D493-529) (Table 2), the cDNA was prepared by PCRusing a reverse primer in which the stop codon was set at the desiredposition. TheKpnI sites were attached to both ends of the PCRproductso as to clone into pFastBac1. In the case of the mutant containinga one-point alanine substitution (K518A) and a truncated mutant(D511-518), cDNAs were prepared by site-directed mutagenesis(Agilent Technologies) using the primers listed in SupplementalTable 2. In contrast, several mutants inserted with alanine substitu-tions (A7, 3CA, and A8), and chimeric mutants with CNX (chimera 1–3) were generated by the megaprimer method using the primers andtemplates listed in Supplemental Tables 3 and 4. In each PCR, pfuTurbo DNA polymerase was used and the primer concentration, ifa synthetic oligonucleotide was used, was set at 300 nM. The PCRconditions for each step are described in Supplemental Tables 5 and 6.The summarized sequences of these chimeras are listed in Table 4.The sequences of all mutants were verified, and they were transfectedin a similar fashion to the wild-type construct. To quantifymicrosomalUGT2B7, Sf9 microsomes expressing UGT2B7 alone were used as astandard and the relative expression level/mg of microsomal proteinswas estimated by immunoblotting as a percentage of the standard.The ratio of UGT/P450 was calculated by dividing the UGT2B7 level(percentage of standard) by the CYP3A4 content (pmol/mg protein).

Analytical Methods. The hydrophobic region of UGT2B7 buriedin or attached to the ER membrane was predicted using an opensoftware system, TMHMMServer, version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) (Möller et al., 2001). The secondary structurelocations were estimated by Jpred3 Server (http://www.compbio.dundee.ac.uk/www-jpred/) (Cole et al., 2008). Kinetic and statisticalanalyses were carried out using GraphPad Prism 5.04 software(GraphPad software, La Jolla, CA). More specifically, kinetic datawere fitted to a sigmoidal model defined by the following equation:

V5Vmax � Sn��Sn50 1Sn�

where V is the reaction rate; S is the substrate concentration; Vmax isthe maximum enzyme velocity; S50 is the substrate concentrationgiving half Vmax; and n is the Hill coefficient.

Other Methods. Protein concentrations were determined bythe method of Lowry et al. (1951) with bovine serum albumin asa standard. The microsomal content of P450 was quantitated bymonitoring difference spectra between the reduced form and its CO

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complex (Omura and Sato, 1964). The activity of CPR was determinedby measuring cytochrome c reduction (Imai, 1976). The substrate-binding differential spectrum of P450 was measured as describedusing testosterone as the substrate (Schenkman and Jansson, 2006).A 1 ml aliquot of reaction mixture containing 100 mM sodiumphosphate (pH 7.4), 20% glycerol, and 1 mM microsomal CYP3A4 wasplaced in both the sample and reference cuvettes in a double-beamspectrophotometer. After 10 minutes of preincubation at 25°C,a baseline was calibrated. Then, testosterone and methanol (solvent)were added into the sample and reference cuvettes, respectively, andthe absorbance from 300 to 500 nm was measured. Spectra wererecorded at three different concentrations of testosterone (100, 200,and 400 mM). To quantify the degree of substrate binding to P450, thedifference in absorbance at the maximal point (390 nm) and minimalpoint (417 nm) was calculated and defined as DAbsorbance390-417. Tocheck the repeatability in the analysis, microsomes without UGT2B7(expressing CYP3A4/CPR) and with UGT2B7 (expressing CYP3A4/CPR/UGT2B7) were independently prepared three times and sixDAbsorbance390-417 values were obtained from spectra recordings underthe condition of testosterone 200 mM. When we calculated the micro-somal content of CPR, we used the reported value of specific activity,i.e., 3.0 mmol cytochrome c reduced/min per nmol CPR (Yasukochi andMasters, 1976).

ResultsDetection of the Interaction between CYP3A4 and

UGT2B7 by Pull-Down Assay. A UGT2B7-CYP3A4 inter-action was examined by pull-down assay in which UGT2B7was simultaneously expressed with His-CYP3A4 capable ofbinding to Ni21-coupled beads. As expected, UGT2B7 wasdetected in the solubilized microsomes of Sf9 cells expressingthis UGT, independently of His-CYP3A4 coexpression (Fig.1A). However, when we analyzed the imidazole-eluted frac-tion, UGT2B7 was detectable only in microsomes expressingboth of UGT2B7 and His-CYP3A4 (Fig. 1A). When UGT2B7was replaced with a negative control, CNX, it was not detectedin the imidazole-eluted fraction even under the conditions of

His-CYP3A4 coexpression (Fig. 1B). These results clearlydemonstrated that CYP3A4 and UGT2B7 associate specifi-cally with each other, and that this association seems to befairly strong because the complex can be detected even in thepresence of detergent.Suppression of CYP3A4 Activity by UGT2B7. The

effect of UGT2B7 on CYP3A4 function was examined ina ternary expression system carrying CPR as well asUGT/P450. Conceivably, CYP3A4 function varies depending onthe expression level of CPR. Thus, it should be necessary tocompare CYP3A4 activity between the constructs theP450/CPR ratios that are comparable. Keeping this in mind,we prepared a total of 12 batches of Sf9 cells coexpressingUGT2B7, CYP3A4, and CPR. Another 13 batches were con-structed as the control expressing only CYP3A4 and CPR. Therelationship involving the CYP3A4 activity, P450/CPR ratio,andUGT/P450 score is shown in Fig. 2. The activity of CYP3A4toward the luciferin derivative in the absence of UGT2B7coexpression (shown by the open bars in Fig. 2) was increasedabout 6-fold in a CPR content–dependent fashion. However,when coexpressed with UGT2B7 (shown by the shaded bars inFig. 2), CYP3A4 activity became lower than that of UGT2B7-lacking microsomes, even though the paired constructs show-ing a close P450/CPR ratio were compared. Therefore, it is

Fig. 1. Detection of an interaction between CYP3A4 and UGT2B7 byHis-tag pull-down assay. UGT2B7 and CNX were expressed in thepresence and absence of coexpressed His-CYP3A4. Microsomes weresolubilized with sodium cholate and ultracentrifuged. The supernatantswere collected, and an aliquot was gently mixed with magnetic agarosebeads conjugated with nickel-nitrilotriacetic acid. After washing the beadsthree times, His-CYP3A4 and proteins trapped by this P450 were elutedwith buffer containing imidazole at a high concentration. Each proteinwas detected by immunoblotting with a specific antibody: (A) UGT2B7;(B) CNX. Solubilized microsomes and His-CYP3A4–trapped samples areindicated as input and imidazole-eluted fraction, respectively. The lanesshown by D represent double expression, which indicates microsomescoexpressing UGT2B7 or CNX with His-CYP3A4. On the other hand, thelanes shown by S represent single expression, which indicates microsomesexpressing UGT2B7 or CNX alone. Details are described in the section onMaterials and Methods.

Fig. 2. Batch-to-batch differences in the expression levels of CYP3A4,UGT2B7, and CPR, and their relationship to CYP3A4 activity. Two typesof microsomes were prepared: one type [13 preparations (open bars)]expressed CYP3A4 andCPR, and the other [12 preparations (shaded bars)]expressed UGT2B7 as well as CYP3A4 and CPR. CYP3A4 and CPR werequantified by spectrophotometric methods and the relative expressionlevel of UGT2B7 was determined by immunoblotting. Different levels ofexpression were achieved by transecting Sf9 cells with recombinantbaculoviruses at different titers: for instance, the following ranges ofmultiplicity of infection were used for transfection: 0.5–1.0 (CYP3A4);0.005–0.1 (CPR); and 0.0–3.0 (UGT2B7). For the calculation of the P450/CPR ratio and UGT/P450 score, refer to the section on Materials andMethods. CYP3A4 activity was measured using a luciferin-derivative asa substrate. The CYP3A4 activity in microsomes of each transfection isplotted along with the P450/CPR ratio and UGT/P450 score in this three-dimensional plot. The data for microsomes lacking UGT2B7 (UGT/P450score = 0) are shown by open bars, and UGT2B7-carrying preparations areshaded. Each bar represents the mean of a triplicate assay.

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suggested that UGT2B7 modulates CYP3A4 activity nega-tively. This suppressive effect was scarcely affected by theUGT/P450 score: for instance, even samples showing a highscore of 2.0–4.0 failed to exhibit augmented suppressioncompared with those exhibiting lower scores. The P450/CPRratio and UGT/P450 score of pooled HLMs used in this studywere estimated to be 3.19 and 1.36, respectively. These valueswere comparable to those observed in some of the ternary-expressed microsomes (Fig. 2). Therefore, it seems likely thatthe suppression of CYP3A4 by UGT2B7 can occur underphysiologic conditions.We then carried out a kinetic analysis to investigate the

suppressive mode of UGT2B7. The results of the Michaelis-Menten plots and the kinetic parameters obtained from theseplots are listed in Fig. 3 and Table 1, respectively. UGT2B7significantly reduced the Vmax value of CYP3A4, withoutaffecting the S50 value and Hill coefficient. A referenceprotein, CNX (which is a chaperone protein having the samemembrane topology as UGT toward the ER), did not exhibitany effect on the kinetic parameters. The coexpression ofUGT2B7 also suppressed CYP3A4-catalyzed testosterone6b-hydroxylation (Fig. 4A), although the activity was mea-sured only at a single substrate concentration. In addition,UGT2B7 reduced other steps in the catalytic cycle of CYP3A4;for example, NADPH consumption, H2O2 generation throughan uncoupling reaction, and cumene hydroperoxide–driventestosterone 6b-hydroxylation were all reduced to the samedegree by this UGT (Fig. 4, B, D, and E). Interestingly,UGT2B7 did not affect NADPH consumption in the absenceof a substrate (Fig. 4C). Because the effect of UGT2B7 onNADPH consumption was markedly different when the P450substrate was present, we then compared the substrate-binding

spectrum of P450 in the absence and presence of coex-pressed UGT2B7. The spectra from three independentsets of microsomal preparations were compared and thedegree of substrate-binding (DAbsorbance319-417) was quan-tified. A representative comparison of the spectra in all of theconcentrations of testosterone shown in Fig. 5, A–C suggestedthat coexpression of UGT2B7 tended to inhibit the binding ofsubstrate to CYP3A4; however, the quantified difference didnot reach statistical significance (Fig. 5D). Although thespectral change at 390–417 nm was increased by addingtestosterone in a concentration-dependent fashion (see thespectra depicted by the solid lines in Fig. 5, A–C), thepercentage of alteration in a testosterone-induced spectralchange between the absence and presence of UGT2B7 did notdiffer significantly (48, 58, and 65% at 100, 200, and 400 mMtestosterone, respectively). Therefore, unlike the kinetics dueto a change in substrate concentration, the effect of UGT2B7on a substrate-induced spectral change seems to be saturatedat the UGT2B7 concentrations used in the present study. Inaddition, UGT2B7 failed to alter the cytochrome c–reducingactivity of CPR (Fig. 6). These results demonstrate thatUGT2B7most likely interacts directly with CYP3A4 and suppressesCYP3A4 activity through inhibiting entrance of substrate intothe P450.Role of the C-Terminal Area of UGT2B7 in the

Suppression of CYP3A4 Function. As described previ-ously, CYP3A4 J-helix, a surface domain facing the cytosol,has been suggested to contribute to a CYP3A4-UGT2B7interaction (Takeda et al., 2009). However, the main body ofUGT is present in the luminal space of theER, and only a shortregion at the C terminus, called the cytosolic tail, is extrudedinto the cytosol. Thus, we initially focused on the cytosolic

Fig. 3. Effects of UGT2B7 and CNX on the kineticsof CYP3A4 expressed together with CPR in Sf9cells. CYP3A4 activity was compared between dou-ble expression (CYP3A4/CPR), indicated as absence,and ternary expression [(A) CYP3A4/CPR/UGT2B7;(B) CYP3A4/CPR/CNX], indicated as presence. Eachplot represents the mean 6 S.D. of triplicate assays.Data were fitted to a sigmoidal model, and thekinetic parameters obtained are listed in Table 1.An asterisk represents statistical significance in theVmax value (P , 0.017). See Table 1 for the criterionof significant difference.

TABLE 1Effect of coexpression of UGT2B7 and CNX on CYP3A4 activityKinetic parameters calculated from the results shown in Fig. 3 are listed. Statistical significance between parametersobtained in the absence and presence of UGT2B7 coexpression was determined by extra sum-of-squares F test. This testwas repeated three times for each parameter. To keep the total error under 0.05, P values below 0.017 (= 0.05/3) wereconsidered as statistically significant (*P , 0.017).

Construct (P450/CPR Ratio)

Kinetic Parameter

Vmax (P450) S50Hill

Coefficient

nmol/min per nmol mM n

Coexpression: UGT2B7Absence (1.40) 0.248 6 0.019 28.2 6 3.0 1.79 6 0.20Presence (1.58) 0.140 6 0.012* 21.4 6 2.2 2.49 6 0.53UGT/P450 score = 0.72

Coexpression: CNXAbsence (2.22) 0.211 6 0.011 48.9 6 3.5 2.41 6 0.31Presence (2.96) 0.236 6 0.011 51.0 6 3.3 2.19 6 0.21

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tail (Lys510–Asp529) (Mackenzie, 1986; Radominska-Pandyaet al., 1999) of UGT2B7 as a candidate region involved in theinteraction with CYP3A4. To estimate the role of theC-terminal domains in the suppression of CYP3A4, several

truncated mutants of UGT2B7 were prepared (see Table 2 forthe constructs produced). As can be seen in the immunoblotting(Fig. 7A), a slight decrease in molecular weight was ob-served depending on the length of the deletion introduced.

Fig. 4. Effect of UGT2B7 on P450 catalytic cycle. The effect of UGT2B7 on CYP3A4-catalyzed reaction was examined using testosterone as a substrate.The comparison was carried out in the presence (black bar) or absence (white bar) of coexpression of UGT2B7. Each bar indicates the mean 6 S.D. oftriplicate assays in (A), (D), and (E). In (B) and (C), each bar represents the mean 6 S.D. of three independent experiments. Statistical significance wascalculated by Student’s t test (***P, 0.001; ns, not significant). In (A), CYP3A4-catalyzed testosterone 6b-hydroxylation is compared. Concentrations ofCYP3A4, testosterone, andNADPHwere fixed at 50 nM, 200 mM, and 1mM in a final volume of 1ml 100mMpotassium phosphate buffer (pH 7.4). In (B),CYP3A4-dependent NADPH consumption was measured by monitoring absorbance at 340 nm in the presence of substrate. The initial NADPHconcentration was 300 mM and other conditions were the same as those described in (A). In (C), NADPH consumption was measured in the absence ofsubstrate. In (D), generation of H2O2 thoughP450 uncoupling reaction was compared. The reaction conditions were the same as described previously. Theamount of H2O2 in the reaction-quenched samples was determined with the Pierce Quantitative Peroxide Assay Kit (Thermo Scientific). In (E), Cumenehydroperoxide–driven CYP3A4-catalyzed oxidation was measured using testosterone as a substrate. The incubation conditions were the same as thosedescribed in (A), except that the reaction was started by addition of 1 mM cumene hydroperoxide instead of NADPH.

Fig. 5. Effect of UGT2B7 on insertion of substrateinto the catalytic pocket of CYP3A4. Substrate-binding difference spectra were compared in theabsence and presence of coexpressed UGT2B7.Details are described in theMaterials andMethodssection. Three batches of microsomes expressingCYP3A4/CPR and CYP3A4/CPR/UGT2B7 wereprepared and each spectrum was independentlymeasured for each spectrum. Thus, we finallyobtained six different spectra. In (A)–(C), the pairsof spectra that exhibited the most marked differ-ence in each testosterone concentration are shown.To quantify the degree of CYP3A4 substrate bind-ing, we subtracted the absorbance at the minimalpoint (417 nm) from that at the maximal point(390 nm) (DAbsorbance390-417) for every sample.Each value after DA denotes the degree. All spectrawith different substrate concentrationswere recordedusing the same microsomal preparation. In (D), eachsymbol represents the difference in absorbance be-tween 390 and 417 nm using each of the threedifferent preparations under 200 mM of testosterone.Statistical significance was determined by Student’s ttest (P , 0.05); however, there was no significantdifference between microsomes with or withoutUGT2B7.

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The effects on the Michaelis-Menten kinetics of CYP3A4 areshown in Fig. 7B and the kinetic parameters obtained arelisted in Supplemental Table 7. A mutant, UGT2B7D519-529,which lacked 11 residues from the C terminus maintainedthe ability to reduce the Vmax value of CYP3A4 similar towild-type enzyme. However, deletion of a further eight resi-dues (D511-529) failed to suppress CYP3A4 activity. TheUGT2B7D493-529 mutant lacking the whole transmembraneregion also lost any suppressing potential. From these lines ofevidence, the region between the 511 and 518th residuesseemed to play an important role in CYP3A4 suppression.However, confusingly, a D511-518 mutant retained the sup-pressive effect similar to the wild type. Conceivably, this maycome from the possibility that the membrane-spanning do-main of UGT2B7 may actually differ from that predicted bytraditional modeling.In Silico Remodeling of the UGT2B7 C-Terminal

Domain and Identification of the Region Involved inCYP3A4 Suppression. To better understand the role of theUGT2B7 C-terminal domain on the suppression of CYP3A4function, we simulated the secondary structures and hy-drophobic region of UGT2B7 by in silico modeling (see theMaterials and Methods for details). The model constructedsuggests that the UGT2B7 C-terminal domain, includingthe membrane-anchoring region, can be divided into twoparts: one is a hydrophilic region containing severalcharged residues, and the other is a hydrophobic regioncontaining a transmembrane helix (Table 3). Regarding

these regions, we initially focused on the charged residuesin the hydrophilic area, making use of information thatP450-CPR and P450/b5 associations are driven by electro-static interactions (Bridges et al., 1998; Backes and Kelley,2003; Laursen et al., 2011). While there are many hydro-philic amino acids near the C terminus of UGT2B7(Table 3), we focused on Lys518 because this is the onlycharged residue located in the 511–518th region (Table 2),and the lines of results of deleted mutants suggested theregion played some critical roles in the suppressive effect(see Fig. 7). Thus, a mutant (K518A) in which Lys518 wasreplaced with alanine was prepared to assess the role of thecationic site. In another mutant, A7, all the chargedresidues including Lys518 in the hydrophilic region weresubstituted with alanines. The expression of K518A and A7was confirmed by immunoblotting (Fig. 8A). With K518A,a reduction in the Vmax value of CYP3A4 still took place (Fig.8B; Supplemental Table 8), suggesting that the positivecharge of Lys518 is not essential for suppression. AlthoughtheVmax value did not exhibit a significant difference with andwithout A7 coexpression (Fig. 8B; Supplemental Table 8), theCYP3A4 activity appeared to be markedly lower in micro-somes coexpressing A7 than in the control preparation [see theMichaelis-Menten plot (Fig. 8B)]. Therefore, we judged thatthe A7 mutant retains the suppressive effect, and hydrophilicamino acids play only a minor role in CYP3A4 suppression.Regarding the role of the hydrophobic region, we designed twomutants, 3CA and A8. In 3CA, three cysteines at the 511, 512,

Fig. 6. Effect of UGT2B7 on cytochrome c reduction mediated by CPR. CPR was expressed in the absence (2) and presence (++ or +) of UGT2B7coexpression. In microsomes coexpressing UGT2B7, the expression levels of the UGT differed between preparations: high and low UGT levels are shownby (++) and (+), respectively. A reaction mixture (final volume 1 ml) contained the same amount of CPR determined by immunoblotting [0.95 mgcytochrome c, 1mMKCN, and 3.3mMMgCl2 in 300mMpotassiumphosphate buffer (pH 7.5)]. Reactionwas started by addition of anNADPH-generatingsystem (1.3 mM NADP+, 3.3 mM glucose-6-phosphate, and 0.4 U/ml glucose-6-phosphate dehydrogenase) and incubated at 25°C. CPR reactions weredetermined bymeasuring the absorbance at 550 nm. One of the representative examples for the alteration of absorbance at 550 nm is shown in (A). In (B),each bar represents the mean 6 S.D. of three independent assays. No statistical significance (ns) was observed between CPR alone and coexpression byDunnett’s test.

TABLE 2Carboxyl-terminal sequences of deletion mutants of UGT2B7Four mutants lacking parts of the C-terminal region were constructed. The postulated transmembrane region of UGT2B7is underlined (Mackenzie, 1986; Radominska-Pandya et al., 1999), and the area adjacent to it is defined as the cytosolictail.

Protein Sequence

UGT2B7 DVIGFLLVCVATVIFIVTKCCLFCFWKFARKAKKGKND529D519-529 DVIGFLLVCVATVIFIVTKCCLFCFWK518D511-529 DVIGFLLVCVATVIFIVTK510D493-529 D493D511-518 DVIGFLLVCVATVIFIVTK—FARKAKKGKND

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and 515th positions were substituted with alanines. On theother hand, in A8, the eight residues located at the 511–518thpositions were substituted with alanines (see Table 3). Both ofthese mutants had no suppressive effect on CYP3A4 activity(Fig. 8B; Supplemental Table 8). This observation demon-strates that UGT2B7 needs part of the C-terminal region,

especially the hydrophobic region forming a transmembranehelix.UGT2B7 Domain Involved in the Suppression of

CYP3A4: Prediction Using Chimeras. For a better un-derstanding of the role of the lipophilic region in the trans-membrane helix near the C terminus in terms of a suppressive

Fig. 7. Effect of the deletion of the C-terminal region of UGT2B7 on the repression of CYP3A4 activity. (A) The deletion mutants of UGT2B7 listed inTable 2 were expressed in Sf9 cells, and the microsomes (5 mg protein) were separated by 8.8% SDS-PAGE followed by immunobloting. HLMwas used asa positive control. Mock represents control microsomes used as a negative control. The amount of protein amount in these controls was also 5 mg.Detection was carried out using goat anti-mouse UGT IgG (Mackenzie et al., 1984) and HRP-rabbit anti-goat IgG, as primary and secondary antibodies,respectively. (B) Four deletion mutants of UGT2B7 were expressed with CYP3A4, and their effects on the P450 activity were determined. The P450/CPRratios of each pair of microsomes (double and ternary expressions, respectively) are shown below. The P450/CPR ratios of microsomes are shown (in theparentheses) without or with the UGT2B7 mutant, respectively: D519-529 (2.22, 2.16); D511-529 (4.51, 4.91); D493-529 (0.28, 0.36); and D511-518 (1.22,0.73). Data were fitted to a sigmoidal model and are shown as Michaelis-Menten plots. Calculated parameters are listed in Supplemental Table 7. Eachplot represents the mean 6 S.D. of triplicate assays. An asterisk represents statistical significance in the Vmax value (P , 0.017).

TABLE 3Alanine substitution introduced into the C-terminal region of UGT2B7The C-terminal position where the residue(s) was substituted with alanine(s) is shown in italics. This study predictedtransmembrane or membrane-bound hydrophobic regions of all mutants by TMHMM Server, version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), and these regions are shown in underline. The secondary structures of mutants were alsopredicted by Jpred3 Server (http://www.compbio.dundee.ac.uk/www-jpred/) (Cole et al., 2008), and the residuesconstituting a helix are shown by brackets. The subscript numbers represent the residue number counted from the Nterminus of wild-type UGT2B7.

Protein Sequence

UGT2B7 T484[WFQYHSLDVIGFLLVCVATVIFIVTK510CCLFCFWKFA]RKAKKGKND529K518A T484[WFQYHSLDVIGFLLVCVATVIFIVTKCCLFCFWAFA]RKAKKGKNDA7 T484W[FQYHSLDVIGFLLVCVATVIFIVTKCCLFCFWAFAA]AAAAGANA3CA T484[WFQYHSLDVIGFLLVCVATVIFIVTKAALFAFWKFAR]KAKKGKNDA8 T484[WFQYHSLDVIGFLLVCVATVIFIVTKAAAAAAAAFA]RKAKKGKNDD511-518 T484[WFQYHSLDVIGFLLVCVATVIFIVTK-(lack of eight residue)-FARKAK]KGKND

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interaction with CYP3A4, we generated two chimeric proteinsof UGT2B7 and CNX by replacing their hydrophilic ends. Forinstance, one (chimera 1: CNX-UGT tail) was constructedso as to have the transmembrane helix of CNX, followed bythe hydrophilic C-terminal region of UGT2B7. In contrast,another chimera (chimera 2: UGT-CNX tail) was prepared asa fusion protein consisting of the transmembrane helix ofUGT2B7 and the hydrophilic end of CNX. Their sequencesare listed in Table 4. The expression of these chimeras was con-firmed by immunoblotting with the antibodies recognizing theluminal domains of UGT2B7 and CNX (Fig. 9A). As expected,chimeras 1 and 2 were detectable with anti-CNX antibody and

anti-UGT antibody, respectively. Since the hydrophilic end ofCNX was longer than that of UGT2B7, chimera 1 had a lowermolecular weight than the wild-type CNX. In contrast, themolecular mass of chimera 2 was greater than that of the wild-type UGT2B7. The predicted molecular weights of chimeras 1and 2 were 67,000 and 78,000, respectively, and the chimeraswere detected at the expected positions. The effects of thesechimeras on CYP3A4 activity were quite different. Whilechimera 1 failed to reduce the CYP3A4 activity, chimera 2showed a suppressive effect in the kinetic analysis (Fig. 9B;Supplemental Table 9). This observation strongly supports ourhypothesis that the hydrophobic region in the UGT2B7

Fig. 8. Effect of alanine substitutions introducedinto the C-terminal area of UGT2B7 on the sup-pression ofCYP3A4. (A)Alanine-substitutedmutants,the sequences of which are listed in Table 3, wereexpressed, and 5 mg of the microsomal protein wasseparated by 8.8% SDS-PAGE. HLM was used asa positive control. Mock represents control micro-somes applied as a negative control. The amount ofprotein in these controls was also 5 mg. (B) Sub-stitution with alanine was introduced into theC-terminal region of UGT2B7 as shown in Table3. The mutants were tested to see if they workedas a CYP3A4 suppressor, similar to wild-typeUGT2B7. Each P450/CPR ratio of the microsomesevaluated is shown as described in Fig. 7: K518A(1.22, 1.26); A7 (4.51, 3.73); 3CA (2.22, 2.41); and A8(1.39, 1.38). The results of the kinetic analyses areshown as Michaelis-Menten plots. Data were fittedto a sigmoidal model, and the calculated parame-ters are listed in Supplemental Table 8. Each plotrepresents the mean6 S.D. of triplicate assays. Anasterisk represents statistical significance in theVmax value (P , 0.017).

TABLE 4Composition of UGT2B7/CNX chimeric proteins used in this study: replacement of hydrophilicC-terminal and internal membrane-binding regions between UGT2B7 and CNXThe sequences of the C-terminal (upper three) and internal membrane-binding (lower two) regions of the chimericproteins are listed together with that of wild-type UGT2B7. The hydrophobic region and secondary structure werepredicted using TMHMM Server, version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) and Jpred3 Server (http://www.compbio.dundee.ac.uk/www-jpred/) (Cole et al., 2008), respectively. In the C-terminal region, the hydrophobic region andhelix are shown in underline and by brackets, respectively. Chimera 1: the hydrophilic end of CNX was replaced with thatof UGT2B7. Chimera 2: the hydrophilic end of UGT2B7 was replaced with that of CNX. Chimera 3: a part of the internalmembrane-anchoring region of UGT2B7 was replaced with CNX amphiphilic helix spanning the 402–422nd residues(David et al., 1993; Bergeron et al., 1994; Ouzzine et al., 1999; Lewis et al., 2011). The superscript and subscript numbersrepresent the location of the residues of wild-type CNX and UGT2B7, respectively, counted from the N terminus. Inaddition, plain and italic characters represent UGT2B7 and CNX sequences, respectively.

Protein Sequence

UGT2B7 T484[WFQYHSLDVIGFLLVCVATVIFIVTKCCLFCFWKFA]RKAKKGKND529

Chimera 1 A458EERPWLWV[VYILT]VALPV[FLVILFCC484K510CCLFCFWKFARK]AKKGKND529

Chimera 2 T484[WFQYHSLDVIGFLLVCVATVIFIVT509S485GKKQTSGMEYKK]TDAP∼KPRRE573

UGT2B7 —EKH183SGGFIFPPSYVPVVMSE200LT—Chimera 3 —EK182A

402IGLELWSMTSDIFFDNFIIC422L201T—

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transmembrane helix plays a crucial role in the suppression ofCYP3A4 activity.The present study generated another chimera (chimera 3:

UGT-CNX-UGT tail) to examine the role of the internal domainof UGT2B7 on CYP3A4 suppression. As reported previously(Meech andMackenzie, 1998; Ouzzine et al., 1999; Lewis et al.,2011), the internal membrane-anchoring region of UGT2B7seems to be located at the 183–200th residues. This domainwasreplaced with the luminal amphiphilic helix of CNX to producechimera 3. Unlike other chimeras, this substitution showedlittle alteration in molecular weight compared with wild-typeUGT2B7 (Fig. 9A). Although chimera 3 contained the wholeC-terminal segment of UGT2B7, this chimera had no ability tosuppress CYP3A4 activity (Fig. 9B; Supplemental Table 9).This result suggests that in order to have a suppressive effect onCYP3A4 activity two regions of UGT2B7 are required, i.e., bothhydrophobic residues in the transmembrane helix and theinternal membrane-anchoring domain are needed.

DiscussionThis study provides evidence that UGT modulates P450

activity. In the pull-down assay employed in this study,

UGT2B7, but not CNX, was trapped with His-CYP3A4 (Fig.1). Such specific association between P450 and UGT agreeswith our previous reports (Taura et al., 2004; Takeda et al.,2005; Ishii et al., 2007). Although the P450/CPR andUGT/P450 ratios varied among the microsomal preparations,coexpression of UGT2B7 lowered CYP3A4 function in everypreparation. Pooled HLMs showed P450/CPR and UGT/P450ratios that are within the range of variance in transformedSf9 cells. These results strongly suggest that UGT2B7-dependentsuppression of CYP3A4 function occurs under physiologicconditions. Early studies have reported that the activity ofliver microsomal P450 is lower than that of reconstitutedsystems, even though their P450 contents are comparable(West and Lu, 1972; Kaminsky et al., 1983; Wood et al., 1983;Sonderfan et al., 1987). This may be due to the competition forCPR by different P450s and/or P450-P450 interactions (Reedand Backes, 2012). Moreover, it was also found that theCYP3A4/CPR system prepared in a baculovirus-insect cellsystem showed a higher turnover of CYP3A4 than that ofHLMs (Crespi and Miller, 1999). The data reported here areapparently consistent with the aforementioned phenomena:that is, the lower function of CYP3A4 in HLMs than inartificial expression systems could be explained, at least

Fig. 9. Effect of the chimeric proteins onCYP3A4activity. (A) Three chimeric proteins, i.e., CNX-UGT-tail (chimera 1),UGT-CNX-tail (chimera 2), andUGT-CNX-UGT-tail (chimera 3) were designed as shown in Table 4, and expressed in insect cells. The cellular microsomes (5 mg protein) were separated by SDS-PAGE followedby immunoblotting (10 and 7.5%gels for upper and lower panels, respectively). Specific antibodies to recognize luminal bodies ofUGT2B7andCNXwereusedas primary antibodies.HLMs and controlmicrosomes indicatedasMockwere loaded aspositive andnegative controls, respectively. (B) Effectof chimeric proteins on CYP3A4 activity is shown. The P450/CPR ratios of microsomes evaluated are shown below as described in Fig. 7: chimera 1 (1.11,1.23); chimera 2 (1.40, 1.58); and chimera 3 (0.68, 0.71). Each plot represents the mean 6 S.D. of triplicate assays. Kinetic parameters were calculated byfitting the curve to a sigmoidal model and are listed in Supplemental Table 9. An asterisk represents statistical significance in the Vmax value (P , 0.017).

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partially, by coexisting UGTs. However, the degree of suppres-sion was independent of the UGT/P450 score. As mentioned inthe introduction, P450 can form homo- and hetero-oligomers(Davydov, 2011; Reed and Backes, 2012), and the same is truefor UGTs (Ishii et al., 2001, 2010; Fujiwara et al., 2007; Lewiset al., 2011). If UGT-UGTandP450-UGT interactions take placeat the same domain of UGT, UGT2B7 homo-oligomerizationwould compete with the CYP3A4-UGT2B7 interaction.Such competition may produce a situation under which thesuppression of CYP3A4 activity takes place independently ofthe expression level of UGT2B7. The glycosylation status ofUGT may be another factor affecting P450 function. MostUGTs including UGT2B7 have potential N-glycosylationsites in their sequences, and N-glycosylation of UGT hasbeen reported to influence UGT conformations and functions(Mackenzie, 1990; Barbier et al., 2000; Nakajima et al.,2010). It is conceivable that defective UGT2B7 lackingcorrect modification with a sugar chain was expressed tosome extent in our system, and such an enzyme failed tointeract with CYP3A4. In this regard, it has been reportedthat, in a baculovirus-insect cell system, inactive UGT wasexpressed at a significant level whenmany viruses were usedfor transfection (Zhang et al., 2012).In the kinetic analysis, UGT2B7markedly reduced theVmax

value of CYP3A4 (Fig. 3; Table 1). Previous studies havesuggested that the conformation and function of P450s varyaccording to how they interact with the membrane (Ahn et al.,1998; Kim et al., 2003). Thus, UGT2B7 may affect CYP3A4topology in the membrane to repress CYP3A4 activity. On theother hand, the present study also suggests that UGT2B7suppresses the whole catalytic cycle of P450. This is becausecoexpression of UGT2B7 reduced not only the oxidation of thesubstrate but also NADPH consumption and the uncouplingreaction to the same degree (Fig. 4). Moreover, UGT2B7 failedto alter NADPH consumption in the absence of substrate anddid not affect the activity of CPR (Fig. 4C and 6). Additionally,coexpression of UGT2B7 tended to suppress the substrate

binding of CYP3A4 with every concentration of testosteronewe used (Fig. 5, A–C). Although no significant difference wasobserved in the quantified result (Fig. 5D), even a slightdifference may equate to a large difference in the degree ofsubstrate binding (Schenkman et al., 1967). Interestingly,coexpression of UGT2B7 reduced CYP3A4 sensitivity towardthe testosterone quarter. From these results, it would bereasonable to consider that UGT2B7 suppresses CYP3A4activity by inhibiting the insertion of substrates into thecatalytic pocket of CYP3A4. The binding of substrate toP450 causes a change in its electrical potential, and thischange drives the catalytic cycle of P450 (Isin andGuengerich,2008). If UGT2B7 inhibits this first step, CYP3A4 functionwould be suppressed in all the later catalytic steps as weobserved. Concerning the shunt pathway, P450 catalysis drivenby cumene hydroperoxide, the observation that UGT2B7 re-duced the reaction, also supports the above assumption (Fig. 4E).To identify the UGT2B7 domain involved in the suppressive

interactionwith CYP3A4, we generated a series ofmutants. Inour previous study, the J-helix, one of the CYP3A4 helicesfacing the cytosol, was suggested as the domain that isinvolved in the interaction with UGT2B7 (Takeda et al.,2009). Thus, initially, we designed mutants lacking theC-terminal region called the cytosolic tail, which is the soleregion extruded into the cytosol. A deletionmutant, D519-529,which lacked half of the tail, retained its suppressive potentialon CYP3A4; however, further truncation led to a loss of effect(Fig. 7B; Supplemental Table 7). However, the analysis usingdeletion mutants failed to identify the essential segment inthe C-terminal region. To overcome this, we made a furtherattempt to reconstruct a model for the secondary structure byin silico modeling. The in silico modeling suggested that theC-terminal transmembrane helix consisting of many hydropho-bic residues is longer than the classic one, and passes from theluminal side to the cytosolic side. In contrast, the subsequenthydrophilic region of the C terminus is shorter than thatconsidered before. Then, we introduced alanine substitutions

Fig. 10. Cooperative suppression of CYP3A4 func-tion by different domains of UGT2B7. (A) TwoUGT2B7 domains that were suggested to contributeto the interaction with CYP3A4 are schematicallyshown together with the signal peptide. (B) Thecooperative suppression of CYP3A4 by the twodomains is illustrated. The C-terminal hydrophobicdomain serves as a transmembrane helix. The helixin thismodel consists of 35 residues, and it was longerthan the previous model (Mackenzie, 1986) by beingextended at both ends. The internal membraneanchoring segment and a possible dimerization do-main are predicted on the basis of previous work(Ouzzine et al., 1999; Lewis et al., 2011).

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into both the hydrophobic and hydrophilic areas. As expected,alanine substitutions following the in silico modeling enabledus to find important regions for the suppression of CYP3A4activity. Even ifmutationswere introduced into the hydrophilicend, this mutant (A7) retained the ability to suppress CYP3A4activity. In contrast, the suppressive potential declinedwhen some residues in the hydrophobic region were replacedwith alanines (Fig. 8B; Supplemental Table 8; Table 3).These results strongly suggest the important role of thehydrophobic region constituting part of the transmembranehelix of UGT2B7 in the suppression of CYP3A4 activity. Forfurther investigation of the role of this region, some chimericproteins were generated. In chimera 1 (CNX-UGT-tail) theCNX main body, linked to its own transmembrane helix, wasfused to the hydrophilic end of UGT2B7 (Fig. 9B; Supplemen-tal Table 9). The disappearance of a suppressive effect inchimera 1 agreed with the importance of the hydrophobicregion in the transmembrane helix. Conversely, in chimera 2(UGT-CNX-tail), the UGT2B7 main body, linked to its owntransmembrane helix, was fused to the hydrophilic end ofCNX, and chimera 2 exhibited a suppressive effect onCYP3A4 activity. These results suggest that the hydrophobicresidues in the transmembrane helix of UGT2B7 but notCNX are important. These observations convincingly sup-port a view that the C-terminal hydrophobic region of UGT2B7plays an important role in the negative regulation of CYP3A4activity.In addition to the hydrophobic area near the C terminus of

UGT2B7, our data demonstrate the important role of theinternal region spanning the 183–200th residues for interac-tion with CYP3A4. In UGT1A6, the corresponding region isassumed to serve as a part of the internal membrane-anchoring region (Ouzzine et al., 1999). Furthermore, it hasbeen suggested that this region is the domain needed forhomodimerization of UGT2B7 (Lewis et al., 2011). Chimera3 was a UGT2B7-based mutant in the 183–200th region thatwas replaced with the luminal amphiphilic helix of CNX(Table 4). This chimera failed to reduce CYP3A4 activity,although it retained the wild-type UGT2B7 hydrophobicregion in the transmembrane helix (Fig. 9B; SupplementalTable 9). Therefore, it is likely that a part of the internalmembrane-anchoring domain is also necessary for interactionwith CYP3A4. However, because chimera 3 was catalyticallyinactive (unpublished data), it is conceivable that its inabilityto suppress CYP3A4 activity may result from inappropriateprotein folding. From these lines of evidence, UGT2B7 wouldneed at least two domains to exhibit its suppressive effect onCYP3A4: 1) the hydrophobic region in the transmembranehelix, and 2) part of the internal membrane-anchoring do-main. It is suggested that these domains work to negativelyregulate CYP3A4 function (Fig. 10).This study suggests that the functional interaction of

CYP3A4 and UGT2B7 is one of the reasons underlying theinterindividual differences in CYP3A4 activity. From a phys-iologic viewpoint, it may not be beneficial that CYP3A4,a major defensive mechanism protecting against hazardousxenobiotics, is inhibited by UGT. However, interaction ofCYP3A4 and UGT2B7 is expected to reduce the CYP3A4-dependent production of cytotoxic hydrogen peroxide bysuppression of the uncoupling that occurs under physiologicconditions. In other words, it is reasonable to suppose that theP450-UGT interaction prevents cells from being exposed to

undesirable stress. This hypothesis is supported by the effectof UGT2B7 on the catalytic cycle of CYP3A4 in testosteroneoxidation (Fig. 4B): that is, this UGT suppressed both theH2O2 production and the uncoupling reaction.In conclusion, this is the first report describing the modu-

lation of P450 activity by UGT. However, there are likely to bea number of P450-UGT interactions involving a variety ofP450 and UGT isoforms (Thummel andWilkinson, 1998; Ishiiet al., 2010, 2014), and their effects on P450s have not yet beenelucidated. Further studies are necessary to understandthe details and physiologic significance of these P450-UGTinteractions.

Acknowledgments

The authors thank Ayumi Furukawa and Dr. Yoshio Nishimura forconstructing the C-terminal truncated mutants of UGT2B7 in pTargeTand expression plasmid for pFastBac1-CNX, respectively. The authorsalso thank the Research Support Center, Research Center for HumanDisease Modeling, Graduate School of Medical Sciences, KyushuUniversity, for technical support.

Authorship Contributions

Participated in research design: Miyauchi, Nagata, Yamazoe,Mackenzie, Yamada, Ishii.

Conducted experiments: Miyauchi, Ishii.Contributed new reagents or analytic tools: Miyauchi, Nagata.Performed data analysis: Miyauchi, Yamada, Ishii.Wrote or contributed to the writing of the manuscript: Miyauchi,

Mackenzie, Yamada, Ishii.

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Address correspondence to: Yuji Ishii, Laboratory of Molecular LifeSciences, Graduate School of Pharmaceutical Sciences, Kyushu University,3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: ishii@phar.kyushu-u.ac.jp

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