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Cloning and Characterization of the CYP2D1-Binding Protein, Retinol Dehydrogenase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 353, No. 2, May 15, pp. 331–336, 1998 Article No. BB980644
Cloning and Characterization of the CYP2D1-Binding Protein, Retinol Dehydrogenase Susumu Imaoka,*,1 Jie Wan,* Toshio Chow,* Toyoko Hiroi,* Reiko Eyanagi,† Hidenari Shigematsu,† and Yoshihiko Funae* *Laboratory of Chemistry, Osaka City University Medical School, 1-4-54 Asahimachi, Abeno-ku, Osaka 545, Japan; and †Daiichi College of Pharmaceutical Sciences, Fukuoka-shi, Japan
Received January 5, 1998, and in revised form February 18, 1998
INTRODUCTION A CYP2D1-binding protein, 29 k-protein (p29), has been isolated and its N-terminal amino acid sequence has been reported (Ohishi et al. (1993) Biochim. Biophys. Acta 1158, 227–236). In this study, p29 cDNA was isolated by PCR with oligonucleotide probes designed from the N-terminal amino acid sequence and p29 was found to be a microsomal retinol dehydrogenase, a member of the short-chain alcohol dehydrogenase family which metabolize hydroxysteroids and prostaglandins. CYP2D1 and p29 were expressed in Saccharomyces cerevisiae to characterize these proteins. CYP2D1 had an absorption maximum at 448 nm in a CO-reduced form. Expressed p29 in yeast cells was detected with anti-p29 antibody. Solubilized CYP2D1 and p29 from yeast microsomes were mixed and applied to an anti-CYP2D1 antibody-binding column. Both proteins were retained in the column and eluted with glycine buffer (pH 2.8). However, when applied alone, p29 was not retained in the column. The findings indicated that CYP2D1 bound tightly with p29. Catalytic activities of p29 expressed in yeast were investigated. p29 had retinal reductase activity in the presence of NADPH. Addition of CYP2D1 and NADPH-P450 reductase increased the retinal reductase activity of p29. These findings suggest that the complex of CYP2D1, p29, and NADPH-P450 reductase has an important role in the metabolism of retinoids. © 1998 Academic Press Key Words: cytochrome P450; CYP2D1; retinol dehydrogenase; short-chain alcohol dehydrogenase.
CYP2D forms are extensively studied P450. These P450s metabolize several drugs including b-blockers and antiarrhythmics (1). CYP2D forms are found most abundantly in the liver but are also present in the kidney and the brain, in rat and human (2, 3). CYP2D forms can metabolize endogenous substrates including testosterone, estrogen, and cortisol as well as exogenous drugs (1, 4 – 6). CYP2D1 (originally P-450UT-H) was first purified by Larrey et al. (7) Gonzalez et al. (4) have cloned CYP2D1 and 2D2 cDNAs. We also have purified rat CYP2D1 and 2D2 (originally UT-7 and UT-7b, respectively) (8). During the purification of these P450s, we found a protein that tightly bound with CYP2D1 and 2D2 and named it 29 k-protein (p29) as its molecular weight was calculated at 29 kDa based on SDS–polyacrylamide gel electrophoresis. Larrey et al. (7) and Gonzalez et al. (4) also found a protein that bound with CYP2D1 but they did not characterize it. We tried to characterize p29 and found that it did not separate from CYP2D1 and 2D2 on ion-exchange and hydroxylapatite chromatography. However, p29 was successfully purified with a reversed-phase HPLC on denaturing with TFA2 and acetonitrile. The N-terminal amino acid sequence of purified p29 was determined but did not correspond to any known protein (8). In this study, we isolated p29 cDNA using oligonucleotides based on the amino acid sequences of p29 and found that the protein is a member of the short chain alcohol dehydrogenase family and a microsomal retinol dehydrogenase (9). Short chain alcohol dehydrogenase has an important role in the metabolism of hydroxy2
To whom correspondence should be addressed. Fax: 181-6-6463922. 1
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Abbreviations used: CYP and P450, cytochrome P450; PCR, polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; TFA, trifluoroacetic acid. 331
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steroid, hydroxyprostaglandin, and retinoid (10). P450 also is an important enzyme for metabolism of steroid, prostaglandin, and retinoid. P450 usually introduces a hydroxyl group to these substrates. P450 as a hydroxylase bound with p29, implying the complex between CYP2D1 and p29 has an important biological role in metabolizing endogenous substrate such as steroids and retinoids. The cDNA was introduced to yeast cells and expressed p29 was further characterized. We found the p29 had retinal reductase activity and this activity was increased by binding of CYP2D1 and NADPH-P450 reductase. MATERIALS AND METHODS Chemicals. Bufuralol and 19-hydroxybufuralol were obtained from Daiichi Pure Chemicals Co. (Tokyo, Japan). Lidocaine and monoethylglycinexylidide were supplied by the Fujisawa Pharmaceuticals Co. (Osaka, Japan). All-trans-retinol and all-trans-retinal were obtained from Sigma Chemical Co. (St. Louis, MO). NADPH was obtained from Oriental Yeast Co. (Tokyo, Japan). Restriction enzymes such as HindIII were obtained from Nippon Gene (Toyama, Japan). Other reagents were obtained from Wako Pure Chemical Industries (Tokyo, Japan). Cloning of p29 cDNA. We originally reported 15 N-terminal amino acid residues of p29 (8). The N-terminal amino acid sequence of this protein was analyzed again and determined to be MWLYLLALVGLWNLLRLFRERKVVSHLQDKWVF. A cDNA library of male Sprague–Dawley rats obtained from Clea Japan (Tokyo) was prepared with Lambda-Zap cDNA synthesis kit (Stratagene, La Jolla, CA) as described (11, 12). The sense oligonucleotide primer for PCR was based on the N-terminal amino acid sequence of p29 underlined, 59-CA(A/G)GA(T/C)AA(A/ G)TA(T/C)GT(T/C/A/G)TT-39. The antisense primer was selected from the T7 promoter region of the pBluescript vector used in the cDNA library, 59-TAATACGACTCACTATAGGG-39. PCR was done with AmpliTaq gold PCR kit (Perkin–Elmer, Norwalk, CT) according to the manufacturer’s instructions using 35 cycles of 1 min at 94°C, 15 s at 50°C, and 4 min at 60°C. The PCR product was separated by agarose gel electrophoresis, extracted, and directly analyzed with an autosequencer (373A, Perkin–Elmer) after fluorescent labeling. The nucleotide sequences obtained were used for homology searches in the data base of the GenBank. The sequence was identical with that of the retinol dehydrogenase found by Chai et al. (13). Expression of CYP2D1 and p29 in Saccharomyces cerevisiae. The coding region of CYP2D1 cDNA was amplified by PCR with the sense primer, 59-ATCGCTGGACTTCTCGCTAC, nucleotide position 140 –159 (2) and antisense primer, 59-GTCTTCTGACCTTGGAAGAC, nucleotide position 778 –797 (2). PCR was done with AmpliTaq gold PCR kit (Perkin–Elmer) according to the manufacturer’s instructions using 35 cycles of 1 min at 94°C, 15 s at 50°C, and 4 min at 60°C. The PCR product was separated by agarose gel electrophoresis. Full-length CYP2D1 cDNA was isolated from the rat cDNA library with the PCR product as a probe. The nucleotide sequence of CYP2D1 cDNA was confirmed by nucleotide sequence analysis (2). CYP2D1 cDNA was introduced into pGYR1 vector with HindIII site as described previously (14). pGYR1 vector was supplied by Dr. Yabusaki of Sumitomo Chemical Co. Full-length cDNA of p29 (retinol dehydrogenase) was amplified by PCR with Pfu DNA polymerase (Stratagene) as two fragments. One fragment was amplified using two primers, sense primer, 59AAAAAAGCTTAAAAAAATGTGGCTCTACCTGCTGGCACTGG and antisense primer 59-GACTTCCCTGACTTCTGAGC. The sense primer has a HindIII site, six adenines for expression in
yeast cells (11), and an ATG, initiation codon. The other fragment was amplified using two primers, sense primer 59-TCAACGTTGCCAGCATCGCA and antisense primer 59-AGCCTTGAGTAGTAGCACAG. These fragments were amplified with 30 cycles, 1 min at 94°C, 1 min at 50°C, and 2.5 min at 75°C. Two fragments were directly subcloned into pBluescript with HindIII, SacI, and BamHI sites. Then, the full-length p29 cDNA was introduced into pGYR1 vector. The plasmids containing CYP2D1 or p29 cDNA were transformed to S. cerevisiae AH22 strain as described (11). Purification of CYP2D1 and p29 was done by a method reported previously (11) with DEAE-5PW (Tosoh, Tokyo, Japan) and hydroxylapatite columns (Koken, Tokyo, Japan). Binding study of p29 with CYP2D1. Anti-CYP2D1 IgG was bound with protein A–Sepharose 4B as described (8). Preparation and characterization of anti-CYP2D1 and anti-p29 antibodies has been described previously (8). Yeast microsomes expressing CYP2D1 or p29 were solubilized in 0.1 M potassium phosphate buffer, pH 7.2, containing 20% glycerol, 1 mM dithiothreitol, 1 mM EDTA, and 0.25 mM PMSF in the presence of 1% sodium cholate on ice. Equal amounts of yeast microsomes of CYP2D1 and p29 were mixed on ice for 30 min. Solubilized p29 only and the mixture of CYP2D1 and p29 were applied onto a CYP2D1-antibody column. After a wash with 0.2 M sodium phosphate buffer containing 0.05% sodium cholate, the columns were washed with 0.1 M glycine–HCl buffer (pH 2.8). The pass-through fraction and the fractions eluted with glycine–HCl buffer were collected and analyzed with SDS–polyacrylamide gel electrophoresis followed by immunoblotting with a mixture of CYP2D1 and p29 antibodies which detect CYP2D1 and p29 at the same time. Other methods. Immunoblotting with CYP2D1 and p29 antibodies was carried out as described (8). Lidocaine and bufuralol metabolic activities were measured by methods reported previously (15, 16). Retinal reduction activity was assayed as described with some modification (17). A 0.5-ml aliquot of incubation mixture containing microsomes (0.5 mg), 1 mM EDTA, 2 mM MgCl2, 0.15 M KCl, and 1 mM NADPH in 0.1 M Tris–HCl buffer, pH 7.4, was preincubated at 37°C for 3 min, after which the reaction was started by adding retinal (20 mM). The retinal was added following incubation in a dark room to prevent photoisomerization. The incubation continued with gentle shaking at 37°C for 15 min and the reaction was stopped by adding 0.5 ml of cold ethanol. The metabolites were extracted with hexane (3 ml) containing butylhydroxytoluene (1 mg/ml). The organic layer was transferred to a new tube and dried in vacuo. The residue was dissolved in 0.25 ml of 2-propanol:water (9:1) and 30 ml was analyzed by HPLC with a reverse-phase column (ODS-80Ts, 4.63 250 mm, Tosoh). The mobile phase consisted of solvent A (acetonitrile:water:2 M ammonium acetate:acetic acid, 500:495:5:0.5, v/v/v/v) and solvent B (acetonitrile:water:2 M ammonium acetate:acetic acid, 900:95:5: 0.5, v/v/v/v). The column was developed at 45°C with a linear gradient from 65 to 90% solvent B in 10 min at a flow rate of 1 ml/min. The metabolites were detected at 325 nm with a spectrophotometer (PD-8020, Tosoh).
RESULTS
Amplification of a DNA Fragment of p29 cDNA by PCR From the amino acid sequence of the N-terminal region of p29 and the T7 promoter region of the pBluescript vector, two oligonucleotides were synthesized. With the two primers and a liver cDNA library, PCR was conducted. The main band amplified by PCR (Fig. 1) was directly sequenced. The nucleotide sequence obtained was used for homology
EXPRESSION OF RETINOL DEHYDROGENASE
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FIG. 1. PCR product from rat liver cDNA library. The PCR product and DNA marker were analyzed by electrophoresis with 2% agarose gel.
searches of the database in GenBank. Surprisingly, its sequence was identical with a microsomal retinol dehydrogenase cDNA sequence found by Chai et al. (13). There are three types of microsomal retinol dehydrogenases. The N-terminal amino acid sequence of p29 was identical with that deduced from retinol dehydrogenase type II cDNA (9). Therefore, we synthesized the oligonucleotides to amplify fulllength retinol dehydrogenase type II cDNA. cDNA of retinol dehydrogenase type II was amplified from a rat liver cDNA library. The nucleotide sequence of the cDNA amplified by PCR was identical with that reported by Chai et al. (9).
FIG. 3. Changes in the p29 expression levels with age. Hepatic microsomes (2 mg) of male and female rats of various ages were analyzed by immunoblotting with p29 antibody. Values were expressed as means 6 SD.
ing protein plays a role in the regulation of retinoid metabolism. The protein level changes markedly with development (18). However, p29 expression did not change with development and p29 was expressed in the rat liver of both sexes at similar level (Fig. 3). The development profile of p29 in rat liver was also similar with that of CYP2D1 (11). Expression of CYP2D1 and p29 in S. cerevisiae
Localization and Age-Dependent Expression of p29 We investigated the localization of p29 and sex- or age-dependent expression of p29 by Western blotting. p29 was dominantly expressed in liver and also expressed in the kidney but its expression level in the lung and brain was very low (Fig. 2). Distribution of p29 in organs was similar with CYP2D1. Cellular retinol-bind-
FIG. 2. Localization of p29 in various tissues. Purified CYP2D1 (0.25 pmol), purified p29 (30 ng), liver (2 mg), kidney (20 mg), lung (20 mg), and brain microsomes (20 mg) were analyzed by immunoblotting with CYP2D1 and p29 antibodies.
CYP2D1 and p29 cDNAs were introduced into yeast cells with pGYR1 vector. Yeast microsomes were analyzed by Western blotting (Fig. 4). Both microsomes gave a single staining band at similar mobility with hepatic microsomes and purified proteins. The CO-reduced absorption spectrum of CYP2D1 in yeast microsomes was also investigated (Fig. 5). CYP2D1 expressed in yeast gave an absorption maximum at 448 nm and was calculated to be 0.079 nmol/mg of microsomal protein. From compar-
FIG. 4. Western blotting of yeast microsomes expressing CYP2D1 and p29. Purified CYP2D1 (lane 1, 0.25 pmol), purified p29 (lane 4, 30 ng), hepatic microsomes (lanes 2 and 5, 5 mg), and yeast microsomes (lanes 3 and 6, 10 mg) were analyzed with immunoblotting with CYP2D1 and p29 antibodies.
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FIG. 5. Absorption spectra of CYP2D1 expressed in yeast cells. CO-reduced different spectrum of CYP2D1 of yeast microsomes (1.2 mg/ml) was measured in the presence of 0.4% Emulgen 911.
ison of the intensity of purified p29 with expressed p29, p29 content in yeast microsomes was calculated at 30 mg/mg of microsomal protein. Binding of p29 with CYP2D1 To investigate whether p29 binds to CYP2D1 in vitro, we used antibody column chromatography. CYP2D1 antibody was bound with protein A–Sepharose 4B. CYP2D1 and p29 expressed in yeast microsomes were solubilized with 1% sodium cholate and mixed. p29 only and the mixture of CYP2D1 and p29 were applied onto CYP2D1-antibody binding columns. The pass-through fraction and the fraction eluted with glycine buffer (pH 2.8) were analyzed by immunoblotting with CYP2D1 and p29 antibodies (Fig. 6). When applied alone, p29 was not retained on the column and eluted in the passthrough fraction. However, when applied together with CYP2D1, p29 was retained on the CYP2D1 antibody binding col-
FIG. 6. Binding of p29 with CYP2D1 using antibody column. p29 only and a mixture of p29 and CYP2D1 were applied onto CYP2D1 antibody-binding protein A–Sepharose column. Eluted fraction was analyzed by immunoblotting with p29 and CYP2D1 antibodies. Lanes 1 and 3 were fractions from the column applied by mixture of p29 and CYP2D1 and lanes 2 and 4 were fractions from the column applied by p29 alone. Lanes 1 and 2 are the passthrough fraction and lanes 3 and 4 are the fraction eluted with glycine buffer, pH 2.8.
FIG. 7. Chromatographic profile of retinal and retinol produced by p29. Authentic retinol and retinal (1 nmol) were separated by HPLC in the condition described under Material and Methods. p29 (10 mg) purified from yeast cells was reacted with retinal in presence or absence of NADPH.
umn. These results indicated that p29 bound tightly with CYP2D1 even in the presence of 1% sodium cholate. Catalytic Activities of CYP2D1 and p29 Expressed in Yeast Cells The catalytic activities of p29 and CYP2D1 in yeast microsomes were investigated. CYP2D1 metabolized bufuralol, a typical substrate for CYP2D forms, efficiently and also metabolized lidocaine. The bufuralol 19-hydroxylation and lidocaine N-deethylation activities of CYP2D1 expressed in yeast were 0.11 nmol/ min/mg of yeast microsomes (1.4 nmol/min/nmol of P450) and 0.23 nmol/min/mg of yeast microsomes (2.9 nmol/min/nmol of P450), respectively. Yeast microsomes expressing p29 had no activity toward these substrates. Furthermore, these catalytic activities of CYP2D1 were not changed by addition of p29 (data not shown). However, p29 had retinal reductase activity in the presence of NADPH (Fig. 7). Purified p29 expressed in yeast cells produced retinol from retinal in the presence of NADPH. Furthermore, when purified NADPHP450 reductase and CYP2D1 were added to purified p29, retinal reductase activity was increased twofold (Fig. 8). Native p29 purified from rat liver gave the same results. In addition, retinol and p29 were incubated in the presence of NADP but production of retinal was very low.
EXPRESSION OF RETINOL DEHYDROGENASE
FIG. 8. Retinal reduction activity of p29. CYP2D1* and p29* indicate activities of yeast microsmes expressing CYP2D1 and p29, respectively, and activities are expressed as nmol/min/mg of yeast microsomes. ND means that the activity was not detectable (less than 0.01 nmol/min/mg). ‘‘Purified p29’’ is p29 purified from yeast microsomes. The assay system included purified p29 (10 mg), dilauroylphosphatidylcholine (5 mg), retinal (20 mM), and NADPH (1 mM). ‘‘p29 1 CYP2D1 1 red’’ is an equimolar mixture of purified p29, CYP2D1, and NADPH-P450 reductase, and dilauroylphosphatidylcholine. ‘‘native p29’’ which includes native CYP2D1 is p29 purified from rat liver. Assay conditions were the same as for p29 from yeast. ‘‘native p29 1 red’’ is a mixture of native p29 and NADPH-P450 reductase. Activities other than CYP2D1* and p29* are expressed as nmol/min/mg of p29 estimated by Western blotting.
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(ADH) and microsomal retinol dehydrogenase catalyze the reversible interconversion of retinol/retinal. Retinol dehydrogenase catalyzes retinol oxidation with NADP and retinal reduction with NADPH (20). In vivo, retinal reduction is important because NADPH is more abundant than NADP and the level of retinal reduction is five times higher than that of retinol oxidation (20). In the present study, retinol oxidation activity of p29 was very low. In step 2, two families of enzymes including aldehyde dehydrogenase (ALDH) and P450 catalyze the irreversible oxidation of retinal to retinoic acid. It is interesting that an enzyme in the step 1, p29, bound with an enzyme in the step 2, P450, although CYP2D1 did not metabolize retinal. p29 (retinol dehydrogenase) is closely related to corticosteroid 11b-dehydrogenase isolated from rat hepatic microsomes on the basis of nucleotide sequence homology (21). Corticosteroid 11b-dehydrogenase is very important to the metabolism of cortisol; absence of this enzyme leads to high renal concentrations of cortisol and causes hypernatremia hypertension in humans (22). CYP2D forms of P450 contribute to steroid metabolism and can hydroxylate testosterone, estrogen, and cortisol (5, 6). These findings suggest that the binding between p29 and CYP2D1 or NADPH-P450 reductase is an important event in the metabolism of bioactive substances such as retinoid and steroid. ACKNOWLEDGMENTS This work was supported by the Fund for Medical Research from Osaka City University Medical Research Foundation. We thank Miss Atsuko Tominaga for her technical assistance.
REFERENCES DISCUSSION
In this study, we discovered that CYP2D1-binding protein purified previously in our laboratory (8) was microsomal retinol dehydrogenase type II isolated by Chai et al. (9). During the purification of retinol dehydrogenase, Boerman et al. (19) found a 54-kDa protein tightly bound with retinol dehydrogenase. They did not identify the 54-kDa protein but speculated that it modifies retinol dehydrogenase activity or participates in retinol transport or metabolism. From the results of this study, we believe that the 54-kDa protein is a CYP2D isoform. We proved binding of p29 with CYP2D1 and reductase increased retinal reduction activity of p29. Retinol undergoes metabolic activation in two steps: dehydrogenation into retinal (step 1), followed by oxidation into retinoic acid (step 2) (20). In step 1, two families of enzymes which include alcohol dehydrogenase
1. Gonzalez, F. J. (1996) Cytochromes P450 (Ionnides, C., Ed.), pp. 183–210. CRC Press, London. 2. Matsunaga, E., Zanger, U. M., Hardwick, J. P., Gelboin, H. V., Meyer, U. A., and Gonzalez, F. J. (1989) Biochemistry 28, 7349 – 7355. 3. Wyss, A., Gustafsson, J. Å, and Warner, M. (1995) Mol. Pharmacol. 47, 1148 –1155. 4. Gonzalez, F. J., Matsunaga, T., Nagata, K., Meyer, U. A., Nebert, D. W., Pastewka, J., Kozak, C. A., Gillette, J., Gelboin, H. V., and Hardwick, J. P. (1987) DNA 6, 149 –161. 5. Sugita, O., Sassa, S., Miyairi, S., Fishman, J., Kubota, I., Noguchi, T., and Kappas, A. (1988) Biochemistry 27, 678 – 686. 6. Jiang, Q., Huang, Y., Voigt, J. M., DeBolt, K. M., Kominami, S., Takemori, S., Funae, Y., and Colby, H. D. (1996) Endocrinology 137, 4811– 4816. 7. Larrey, D., Distlerath, L. M., Dannan, G. A., Wilkinson, G. R., and Guengerich, F. P. (1984) Biochemistry 23, 2787– 2795. 8. Ohishi, N., Imaoka, S., Suzuki, T., and Funae, Y. (1993) Biochim. Biophys. Acta. 1158, 227–236.
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9. Chai, X., Zhai, Y., Popescu, G., and Napoli, J. L. (1995) J. Biol. Chem. 270,28408 –28412. 10. Krozowski, Z. (1994) J. Steroid. Biochem. Mol. Biol. 51, 125– 130. 11. Imaoka, S., Yamada, T., Hiroi, T., Hayashi, K., Sakaki, T., Yabusaki, Y., and Funae, Y. (1996) Biochem. Pharmacol. 51, 1041–1050. 12. Imaoka, S., Hiroi, T., Tamura, Y., Yamazaki, H., Shimada, T., Komori, M., Degawa, M., and Funae, Y. (1995) Arch. Biochem. Biophys. 321, 255–262. 13. Chai, X., Boerman, M. H., Zhai, Y., and Napoli, J. L. (1995) J. Biol. Chem. 270, 3900 –3904. 14. Murakami, H., Yabusaki, Y., Sakaki, T., Shibata, M., and Ohkawa, H. (1990) J. Biochem. 108, 859 – 865. 15. Kronbach, T., Mathys, D., Gut, J., Catin, T., and Meyer, U. A. (1987) Anal. Biochem. 162, 24 –32.
16. Imaoka, S., Enomoto, K., Oda, Y., Asada, A., Fujimori, M., Shimada, T., Fujita, S., Guengerich, F. P., and Funae, Y. (1990) J. Pharmacol. Exp. Ther. 255, 1385–1391. 17. Leo, M. A., and Lieber, C. S. (1990) Methods Enzymol. 189, 520 –524. 18. Ong, D. E., MacDonald, P. N., and Gubitosi, A. M. (1988) J. Biol. Chem. 263, 5789 –5796. 19. Boerman, M. H., and Napoli, J. L. (1995) Biochemistry 34, 7027– 7037. 20. Duester, G. (1996) Biochemistry 35, 12221–12227. 21. Agarwal, A. K., Monder, C., Eckstein, B., and White, P. C. (1989) J. Biol. Chem. 264, 18939 –18943. 22. Monder, C., Shackleton, C. H., Bradlow, H. L., New, M. I., Stoner, E., Iohan, F., and Lakshmi, V. (1986) J. Clin. Endocrinol. Metab. 63, 550 –557.
Vol. 353, No. 2, May 15, pp. 331–336, 1998 Article No. BB980644
Cloning and Characterization of the CYP2D1-Binding Protein, Retinol Dehydrogenase Susumu Imaoka,*,1 Jie Wan,* Toshio Chow,* Toyoko Hiroi,* Reiko Eyanagi,† Hidenari Shigematsu,† and Yoshihiko Funae* *Laboratory of Chemistry, Osaka City University Medical School, 1-4-54 Asahimachi, Abeno-ku, Osaka 545, Japan; and †Daiichi College of Pharmaceutical Sciences, Fukuoka-shi, Japan
Received January 5, 1998, and in revised form February 18, 1998
INTRODUCTION A CYP2D1-binding protein, 29 k-protein (p29), has been isolated and its N-terminal amino acid sequence has been reported (Ohishi et al. (1993) Biochim. Biophys. Acta 1158, 227–236). In this study, p29 cDNA was isolated by PCR with oligonucleotide probes designed from the N-terminal amino acid sequence and p29 was found to be a microsomal retinol dehydrogenase, a member of the short-chain alcohol dehydrogenase family which metabolize hydroxysteroids and prostaglandins. CYP2D1 and p29 were expressed in Saccharomyces cerevisiae to characterize these proteins. CYP2D1 had an absorption maximum at 448 nm in a CO-reduced form. Expressed p29 in yeast cells was detected with anti-p29 antibody. Solubilized CYP2D1 and p29 from yeast microsomes were mixed and applied to an anti-CYP2D1 antibody-binding column. Both proteins were retained in the column and eluted with glycine buffer (pH 2.8). However, when applied alone, p29 was not retained in the column. The findings indicated that CYP2D1 bound tightly with p29. Catalytic activities of p29 expressed in yeast were investigated. p29 had retinal reductase activity in the presence of NADPH. Addition of CYP2D1 and NADPH-P450 reductase increased the retinal reductase activity of p29. These findings suggest that the complex of CYP2D1, p29, and NADPH-P450 reductase has an important role in the metabolism of retinoids. © 1998 Academic Press Key Words: cytochrome P450; CYP2D1; retinol dehydrogenase; short-chain alcohol dehydrogenase.
CYP2D forms are extensively studied P450. These P450s metabolize several drugs including b-blockers and antiarrhythmics (1). CYP2D forms are found most abundantly in the liver but are also present in the kidney and the brain, in rat and human (2, 3). CYP2D forms can metabolize endogenous substrates including testosterone, estrogen, and cortisol as well as exogenous drugs (1, 4 – 6). CYP2D1 (originally P-450UT-H) was first purified by Larrey et al. (7) Gonzalez et al. (4) have cloned CYP2D1 and 2D2 cDNAs. We also have purified rat CYP2D1 and 2D2 (originally UT-7 and UT-7b, respectively) (8). During the purification of these P450s, we found a protein that tightly bound with CYP2D1 and 2D2 and named it 29 k-protein (p29) as its molecular weight was calculated at 29 kDa based on SDS–polyacrylamide gel electrophoresis. Larrey et al. (7) and Gonzalez et al. (4) also found a protein that bound with CYP2D1 but they did not characterize it. We tried to characterize p29 and found that it did not separate from CYP2D1 and 2D2 on ion-exchange and hydroxylapatite chromatography. However, p29 was successfully purified with a reversed-phase HPLC on denaturing with TFA2 and acetonitrile. The N-terminal amino acid sequence of purified p29 was determined but did not correspond to any known protein (8). In this study, we isolated p29 cDNA using oligonucleotides based on the amino acid sequences of p29 and found that the protein is a member of the short chain alcohol dehydrogenase family and a microsomal retinol dehydrogenase (9). Short chain alcohol dehydrogenase has an important role in the metabolism of hydroxy2
To whom correspondence should be addressed. Fax: 181-6-6463922. 1
0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Abbreviations used: CYP and P450, cytochrome P450; PCR, polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; TFA, trifluoroacetic acid. 331
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steroid, hydroxyprostaglandin, and retinoid (10). P450 also is an important enzyme for metabolism of steroid, prostaglandin, and retinoid. P450 usually introduces a hydroxyl group to these substrates. P450 as a hydroxylase bound with p29, implying the complex between CYP2D1 and p29 has an important biological role in metabolizing endogenous substrate such as steroids and retinoids. The cDNA was introduced to yeast cells and expressed p29 was further characterized. We found the p29 had retinal reductase activity and this activity was increased by binding of CYP2D1 and NADPH-P450 reductase. MATERIALS AND METHODS Chemicals. Bufuralol and 19-hydroxybufuralol were obtained from Daiichi Pure Chemicals Co. (Tokyo, Japan). Lidocaine and monoethylglycinexylidide were supplied by the Fujisawa Pharmaceuticals Co. (Osaka, Japan). All-trans-retinol and all-trans-retinal were obtained from Sigma Chemical Co. (St. Louis, MO). NADPH was obtained from Oriental Yeast Co. (Tokyo, Japan). Restriction enzymes such as HindIII were obtained from Nippon Gene (Toyama, Japan). Other reagents were obtained from Wako Pure Chemical Industries (Tokyo, Japan). Cloning of p29 cDNA. We originally reported 15 N-terminal amino acid residues of p29 (8). The N-terminal amino acid sequence of this protein was analyzed again and determined to be MWLYLLALVGLWNLLRLFRERKVVSHLQDKWVF. A cDNA library of male Sprague–Dawley rats obtained from Clea Japan (Tokyo) was prepared with Lambda-Zap cDNA synthesis kit (Stratagene, La Jolla, CA) as described (11, 12). The sense oligonucleotide primer for PCR was based on the N-terminal amino acid sequence of p29 underlined, 59-CA(A/G)GA(T/C)AA(A/ G)TA(T/C)GT(T/C/A/G)TT-39. The antisense primer was selected from the T7 promoter region of the pBluescript vector used in the cDNA library, 59-TAATACGACTCACTATAGGG-39. PCR was done with AmpliTaq gold PCR kit (Perkin–Elmer, Norwalk, CT) according to the manufacturer’s instructions using 35 cycles of 1 min at 94°C, 15 s at 50°C, and 4 min at 60°C. The PCR product was separated by agarose gel electrophoresis, extracted, and directly analyzed with an autosequencer (373A, Perkin–Elmer) after fluorescent labeling. The nucleotide sequences obtained were used for homology searches in the data base of the GenBank. The sequence was identical with that of the retinol dehydrogenase found by Chai et al. (13). Expression of CYP2D1 and p29 in Saccharomyces cerevisiae. The coding region of CYP2D1 cDNA was amplified by PCR with the sense primer, 59-ATCGCTGGACTTCTCGCTAC, nucleotide position 140 –159 (2) and antisense primer, 59-GTCTTCTGACCTTGGAAGAC, nucleotide position 778 –797 (2). PCR was done with AmpliTaq gold PCR kit (Perkin–Elmer) according to the manufacturer’s instructions using 35 cycles of 1 min at 94°C, 15 s at 50°C, and 4 min at 60°C. The PCR product was separated by agarose gel electrophoresis. Full-length CYP2D1 cDNA was isolated from the rat cDNA library with the PCR product as a probe. The nucleotide sequence of CYP2D1 cDNA was confirmed by nucleotide sequence analysis (2). CYP2D1 cDNA was introduced into pGYR1 vector with HindIII site as described previously (14). pGYR1 vector was supplied by Dr. Yabusaki of Sumitomo Chemical Co. Full-length cDNA of p29 (retinol dehydrogenase) was amplified by PCR with Pfu DNA polymerase (Stratagene) as two fragments. One fragment was amplified using two primers, sense primer, 59AAAAAAGCTTAAAAAAATGTGGCTCTACCTGCTGGCACTGG and antisense primer 59-GACTTCCCTGACTTCTGAGC. The sense primer has a HindIII site, six adenines for expression in
yeast cells (11), and an ATG, initiation codon. The other fragment was amplified using two primers, sense primer 59-TCAACGTTGCCAGCATCGCA and antisense primer 59-AGCCTTGAGTAGTAGCACAG. These fragments were amplified with 30 cycles, 1 min at 94°C, 1 min at 50°C, and 2.5 min at 75°C. Two fragments were directly subcloned into pBluescript with HindIII, SacI, and BamHI sites. Then, the full-length p29 cDNA was introduced into pGYR1 vector. The plasmids containing CYP2D1 or p29 cDNA were transformed to S. cerevisiae AH22 strain as described (11). Purification of CYP2D1 and p29 was done by a method reported previously (11) with DEAE-5PW (Tosoh, Tokyo, Japan) and hydroxylapatite columns (Koken, Tokyo, Japan). Binding study of p29 with CYP2D1. Anti-CYP2D1 IgG was bound with protein A–Sepharose 4B as described (8). Preparation and characterization of anti-CYP2D1 and anti-p29 antibodies has been described previously (8). Yeast microsomes expressing CYP2D1 or p29 were solubilized in 0.1 M potassium phosphate buffer, pH 7.2, containing 20% glycerol, 1 mM dithiothreitol, 1 mM EDTA, and 0.25 mM PMSF in the presence of 1% sodium cholate on ice. Equal amounts of yeast microsomes of CYP2D1 and p29 were mixed on ice for 30 min. Solubilized p29 only and the mixture of CYP2D1 and p29 were applied onto a CYP2D1-antibody column. After a wash with 0.2 M sodium phosphate buffer containing 0.05% sodium cholate, the columns were washed with 0.1 M glycine–HCl buffer (pH 2.8). The pass-through fraction and the fractions eluted with glycine–HCl buffer were collected and analyzed with SDS–polyacrylamide gel electrophoresis followed by immunoblotting with a mixture of CYP2D1 and p29 antibodies which detect CYP2D1 and p29 at the same time. Other methods. Immunoblotting with CYP2D1 and p29 antibodies was carried out as described (8). Lidocaine and bufuralol metabolic activities were measured by methods reported previously (15, 16). Retinal reduction activity was assayed as described with some modification (17). A 0.5-ml aliquot of incubation mixture containing microsomes (0.5 mg), 1 mM EDTA, 2 mM MgCl2, 0.15 M KCl, and 1 mM NADPH in 0.1 M Tris–HCl buffer, pH 7.4, was preincubated at 37°C for 3 min, after which the reaction was started by adding retinal (20 mM). The retinal was added following incubation in a dark room to prevent photoisomerization. The incubation continued with gentle shaking at 37°C for 15 min and the reaction was stopped by adding 0.5 ml of cold ethanol. The metabolites were extracted with hexane (3 ml) containing butylhydroxytoluene (1 mg/ml). The organic layer was transferred to a new tube and dried in vacuo. The residue was dissolved in 0.25 ml of 2-propanol:water (9:1) and 30 ml was analyzed by HPLC with a reverse-phase column (ODS-80Ts, 4.63 250 mm, Tosoh). The mobile phase consisted of solvent A (acetonitrile:water:2 M ammonium acetate:acetic acid, 500:495:5:0.5, v/v/v/v) and solvent B (acetonitrile:water:2 M ammonium acetate:acetic acid, 900:95:5: 0.5, v/v/v/v). The column was developed at 45°C with a linear gradient from 65 to 90% solvent B in 10 min at a flow rate of 1 ml/min. The metabolites were detected at 325 nm with a spectrophotometer (PD-8020, Tosoh).
RESULTS
Amplification of a DNA Fragment of p29 cDNA by PCR From the amino acid sequence of the N-terminal region of p29 and the T7 promoter region of the pBluescript vector, two oligonucleotides were synthesized. With the two primers and a liver cDNA library, PCR was conducted. The main band amplified by PCR (Fig. 1) was directly sequenced. The nucleotide sequence obtained was used for homology
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FIG. 1. PCR product from rat liver cDNA library. The PCR product and DNA marker were analyzed by electrophoresis with 2% agarose gel.
searches of the database in GenBank. Surprisingly, its sequence was identical with a microsomal retinol dehydrogenase cDNA sequence found by Chai et al. (13). There are three types of microsomal retinol dehydrogenases. The N-terminal amino acid sequence of p29 was identical with that deduced from retinol dehydrogenase type II cDNA (9). Therefore, we synthesized the oligonucleotides to amplify fulllength retinol dehydrogenase type II cDNA. cDNA of retinol dehydrogenase type II was amplified from a rat liver cDNA library. The nucleotide sequence of the cDNA amplified by PCR was identical with that reported by Chai et al. (9).
FIG. 3. Changes in the p29 expression levels with age. Hepatic microsomes (2 mg) of male and female rats of various ages were analyzed by immunoblotting with p29 antibody. Values were expressed as means 6 SD.
ing protein plays a role in the regulation of retinoid metabolism. The protein level changes markedly with development (18). However, p29 expression did not change with development and p29 was expressed in the rat liver of both sexes at similar level (Fig. 3). The development profile of p29 in rat liver was also similar with that of CYP2D1 (11). Expression of CYP2D1 and p29 in S. cerevisiae
Localization and Age-Dependent Expression of p29 We investigated the localization of p29 and sex- or age-dependent expression of p29 by Western blotting. p29 was dominantly expressed in liver and also expressed in the kidney but its expression level in the lung and brain was very low (Fig. 2). Distribution of p29 in organs was similar with CYP2D1. Cellular retinol-bind-
FIG. 2. Localization of p29 in various tissues. Purified CYP2D1 (0.25 pmol), purified p29 (30 ng), liver (2 mg), kidney (20 mg), lung (20 mg), and brain microsomes (20 mg) were analyzed by immunoblotting with CYP2D1 and p29 antibodies.
CYP2D1 and p29 cDNAs were introduced into yeast cells with pGYR1 vector. Yeast microsomes were analyzed by Western blotting (Fig. 4). Both microsomes gave a single staining band at similar mobility with hepatic microsomes and purified proteins. The CO-reduced absorption spectrum of CYP2D1 in yeast microsomes was also investigated (Fig. 5). CYP2D1 expressed in yeast gave an absorption maximum at 448 nm and was calculated to be 0.079 nmol/mg of microsomal protein. From compar-
FIG. 4. Western blotting of yeast microsomes expressing CYP2D1 and p29. Purified CYP2D1 (lane 1, 0.25 pmol), purified p29 (lane 4, 30 ng), hepatic microsomes (lanes 2 and 5, 5 mg), and yeast microsomes (lanes 3 and 6, 10 mg) were analyzed with immunoblotting with CYP2D1 and p29 antibodies.
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FIG. 5. Absorption spectra of CYP2D1 expressed in yeast cells. CO-reduced different spectrum of CYP2D1 of yeast microsomes (1.2 mg/ml) was measured in the presence of 0.4% Emulgen 911.
ison of the intensity of purified p29 with expressed p29, p29 content in yeast microsomes was calculated at 30 mg/mg of microsomal protein. Binding of p29 with CYP2D1 To investigate whether p29 binds to CYP2D1 in vitro, we used antibody column chromatography. CYP2D1 antibody was bound with protein A–Sepharose 4B. CYP2D1 and p29 expressed in yeast microsomes were solubilized with 1% sodium cholate and mixed. p29 only and the mixture of CYP2D1 and p29 were applied onto CYP2D1-antibody binding columns. The pass-through fraction and the fraction eluted with glycine buffer (pH 2.8) were analyzed by immunoblotting with CYP2D1 and p29 antibodies (Fig. 6). When applied alone, p29 was not retained on the column and eluted in the passthrough fraction. However, when applied together with CYP2D1, p29 was retained on the CYP2D1 antibody binding col-
FIG. 6. Binding of p29 with CYP2D1 using antibody column. p29 only and a mixture of p29 and CYP2D1 were applied onto CYP2D1 antibody-binding protein A–Sepharose column. Eluted fraction was analyzed by immunoblotting with p29 and CYP2D1 antibodies. Lanes 1 and 3 were fractions from the column applied by mixture of p29 and CYP2D1 and lanes 2 and 4 were fractions from the column applied by p29 alone. Lanes 1 and 2 are the passthrough fraction and lanes 3 and 4 are the fraction eluted with glycine buffer, pH 2.8.
FIG. 7. Chromatographic profile of retinal and retinol produced by p29. Authentic retinol and retinal (1 nmol) were separated by HPLC in the condition described under Material and Methods. p29 (10 mg) purified from yeast cells was reacted with retinal in presence or absence of NADPH.
umn. These results indicated that p29 bound tightly with CYP2D1 even in the presence of 1% sodium cholate. Catalytic Activities of CYP2D1 and p29 Expressed in Yeast Cells The catalytic activities of p29 and CYP2D1 in yeast microsomes were investigated. CYP2D1 metabolized bufuralol, a typical substrate for CYP2D forms, efficiently and also metabolized lidocaine. The bufuralol 19-hydroxylation and lidocaine N-deethylation activities of CYP2D1 expressed in yeast were 0.11 nmol/ min/mg of yeast microsomes (1.4 nmol/min/nmol of P450) and 0.23 nmol/min/mg of yeast microsomes (2.9 nmol/min/nmol of P450), respectively. Yeast microsomes expressing p29 had no activity toward these substrates. Furthermore, these catalytic activities of CYP2D1 were not changed by addition of p29 (data not shown). However, p29 had retinal reductase activity in the presence of NADPH (Fig. 7). Purified p29 expressed in yeast cells produced retinol from retinal in the presence of NADPH. Furthermore, when purified NADPHP450 reductase and CYP2D1 were added to purified p29, retinal reductase activity was increased twofold (Fig. 8). Native p29 purified from rat liver gave the same results. In addition, retinol and p29 were incubated in the presence of NADP but production of retinal was very low.
EXPRESSION OF RETINOL DEHYDROGENASE
FIG. 8. Retinal reduction activity of p29. CYP2D1* and p29* indicate activities of yeast microsmes expressing CYP2D1 and p29, respectively, and activities are expressed as nmol/min/mg of yeast microsomes. ND means that the activity was not detectable (less than 0.01 nmol/min/mg). ‘‘Purified p29’’ is p29 purified from yeast microsomes. The assay system included purified p29 (10 mg), dilauroylphosphatidylcholine (5 mg), retinal (20 mM), and NADPH (1 mM). ‘‘p29 1 CYP2D1 1 red’’ is an equimolar mixture of purified p29, CYP2D1, and NADPH-P450 reductase, and dilauroylphosphatidylcholine. ‘‘native p29’’ which includes native CYP2D1 is p29 purified from rat liver. Assay conditions were the same as for p29 from yeast. ‘‘native p29 1 red’’ is a mixture of native p29 and NADPH-P450 reductase. Activities other than CYP2D1* and p29* are expressed as nmol/min/mg of p29 estimated by Western blotting.
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(ADH) and microsomal retinol dehydrogenase catalyze the reversible interconversion of retinol/retinal. Retinol dehydrogenase catalyzes retinol oxidation with NADP and retinal reduction with NADPH (20). In vivo, retinal reduction is important because NADPH is more abundant than NADP and the level of retinal reduction is five times higher than that of retinol oxidation (20). In the present study, retinol oxidation activity of p29 was very low. In step 2, two families of enzymes including aldehyde dehydrogenase (ALDH) and P450 catalyze the irreversible oxidation of retinal to retinoic acid. It is interesting that an enzyme in the step 1, p29, bound with an enzyme in the step 2, P450, although CYP2D1 did not metabolize retinal. p29 (retinol dehydrogenase) is closely related to corticosteroid 11b-dehydrogenase isolated from rat hepatic microsomes on the basis of nucleotide sequence homology (21). Corticosteroid 11b-dehydrogenase is very important to the metabolism of cortisol; absence of this enzyme leads to high renal concentrations of cortisol and causes hypernatremia hypertension in humans (22). CYP2D forms of P450 contribute to steroid metabolism and can hydroxylate testosterone, estrogen, and cortisol (5, 6). These findings suggest that the binding between p29 and CYP2D1 or NADPH-P450 reductase is an important event in the metabolism of bioactive substances such as retinoid and steroid. ACKNOWLEDGMENTS This work was supported by the Fund for Medical Research from Osaka City University Medical Research Foundation. We thank Miss Atsuko Tominaga for her technical assistance.
REFERENCES DISCUSSION
In this study, we discovered that CYP2D1-binding protein purified previously in our laboratory (8) was microsomal retinol dehydrogenase type II isolated by Chai et al. (9). During the purification of retinol dehydrogenase, Boerman et al. (19) found a 54-kDa protein tightly bound with retinol dehydrogenase. They did not identify the 54-kDa protein but speculated that it modifies retinol dehydrogenase activity or participates in retinol transport or metabolism. From the results of this study, we believe that the 54-kDa protein is a CYP2D isoform. We proved binding of p29 with CYP2D1 and reductase increased retinal reduction activity of p29. Retinol undergoes metabolic activation in two steps: dehydrogenation into retinal (step 1), followed by oxidation into retinoic acid (step 2) (20). In step 1, two families of enzymes which include alcohol dehydrogenase
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