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Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells
Journal of Pharmacological and Toxicological Methods 64 (2011) 258–263
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Original article
Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells Christian Helvig a, Mohammed Taimi a, Don Cameron b, d, Glenville Jones b, Martin Petkovich a, b, c, d,⁎ a
Cytochroma, Inc., 100 Allstate Parkway, Suite 600, Markham, Ontario, Canada L3R 6H3 Department of Biochemistry, Queen's University, Kingston, Ontario, Canada Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada d Cancer Research Institute, Queen's University, Kingston, Ontario, Canada b c
a r t i c l e
i n f o
Article history: Received 29 April 2011 Accepted 24 August 2011 Keywords: Retinoic Acid Metabolism CYP26 Baculovirus Assay
a b s t r a c t Introduction: The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9-cis isomer of RA. Methods: We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes. Results: We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all-trans-RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC50 values of 16, 27, and 15 nM for CYP26A1, B1, and C1 respectively for all-trans-RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans-RA could also be effectively competed with 9-cis RA, with IC50 of 62 nM, and was sensitive to ketoconazole inhibition. Discussion: CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9-cis-RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The vitamin A derivative, all-trans retinoic acid (atRA), plays an essential role in vertebrate embryogenesis and is an important regulator of cell differentiation and proliferation in the adult (Niederreither & Dolle, 2008; Pennimpede, Cameron, & Petkovich, 2006). The biological effects of retinoids are mediated by two classes of nuclear receptors, retinoic acid receptors (RARα, β, and γ), and retinoid X receptors (RXRα, β, and γ) which function as ligand-dependent transcription factors by binding to specific response elements in promoters of retinoids target genes (Chambon, 1996; Pennimpede et al., 2006). RARs are
Abbreviations: RA, retinoic acid; atRA, all-trans retinoic acid; CYP, cytochrome P450; hb5, human cytochrome b5; hOR, cytochrome P450-NADPH reductase; RT, Reverse Transcriptase; RALDH, Retinaldehyde dehydrogenase; BSA, bovine serum albumin. ⁎ Corresponding author at: Cancer Research Institute, Department of Biochemistry, Queen's University 10 Stuart St., Botterell Hall Room 354, Kingston, Ontario, Canada K7L 3N6. Tel.: + 1 613 533 6791; fax: + 1 613 533 6830. E-mail address: [email protected] (M. Petkovich). 1056-8719/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2011.08.005
activated by atRA and 9-cis- isomer of retinoic acid (9-cis-RA), and RXRs are selectively activated by 9-cis-RA. Retinoic acid dispersal in tissues is finely controlled by the tightly regulated expression of atRA synthetic retinaldehyde dehydrogenase (RALDH) and cytochrome P450 (CYP) catabolic enzymes (Abu-Abed et al., 2001, 2002; MacLean et al., 2001; Niederreither, Fraulob, Garnier, Chambon, & Dolle, 2002; Tahayato, Dolle, & Petkovich, 2003). Several classes of RALDH genes have been cloned from several species, including human, and are characterized by their ability to form atRA and 9-cis-RA from their aldehyde precursors (Brodeur, Gagnon, Mader, & Bhat, 2003; Duester, 2000; Lin & Napoli, 2000; Montplaisir et al., 2002; Niederreither, Fraulob, et al., 2002). Retinoic acid catabolism is mediated primarily by the CYP26 family of cytochrome P450 enzymes (CYP26A1, −B1 and −C1). The expression patterns of CYP26 genes in embryos and adults suggest that they play distinct physiological roles in protecting tissues from inappropriate exposure to atRA (Pennimpede et al., 2010). Both genetic and biochemical studies support a catabolic role for CYP26 enzymes. Loss of the Cyp26a1 gene in mice results in embryonic lethality, and knockout animals revealed that CYP26A1 is essential to protect tailbud and hindbrain regions of the embryo from exposure to endogenous atRA. Cyp26a1−/−
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animals exhibit spina bifida and sirenomelia (mermaid syndrome) resembling animals exposed to teratogenic doses of atRA (Abu-Abed et al., 2001; Sakai et al., 2001). Similarly, genetic deletion of Cyp26b1, which is predominantly expressed in developing limbs, results in limb defects similar to those found in animals exposed to high to excess atRA in utero (Pennimpede, Cameron, Maclean, & Petkovich, 2010; Yashiro et al., 2004). Furthermore, it is possible to rescue Cyp26a1−/− animals from lethality by intercrossing them with animals containing a single null allele for the atRA synthesizing enzyme, RALDH2; thereby reducing the endogenous levels of RA to which caudal and hindbrain tissues are exposed (Niederreither, Abu-Abed, et al., 2002). This study supports the view that atRA and not its metabolites are important for embryonic development. Although the enzymes exhibit a comparatively high degree of amino acid sequence similarity in some specific regions of the protein such as the Heme binding region, and ETLR and PERF conserved P450 domains, their overall similarity is less striking (Taimi et al., 2004). CYP26A1 and CYP26B1 share 42% amino acid identity (White et al., 2000), while CYP26C1 shares 43% identity, and 51% identity at the amino acid level with CYP26A1 and CYP26B1 respectively (Taimi et al., 2004). Discerning the functional differences between these enzymes may help to distinguish the specific enzymatic roles these enzymes play and guide the development of sub-type selective inhibitors. The CYP26A1-subtype was the first enzyme of this family to be discovered (White et al., 1996, 1997). Analysis of CYP26A1 enzymatic activity indicates that atRA is the preferred natural substrate for this enzyme. CYP26A1 rapidly converts atRA to hydroxylated forms, 4OH and 4-oxo being the predominant metabolic products. In cell culture systems, these primary metabolites can be further metabolized to multiple-hydroxylated forms (Chithalen, Luu, Petkovich, & Jones, 2002). It is not presently known whether these further metabolites are generated under physiological conditions or whether they play any specific biological role. Two additional members of this family, CYP26B1 and CYP26C1, have also been isolated and characterized. CYP26B1 displays substrate specificity similar to that of CYP26A1 (White et al., 2000). In contrast, CYP26C1 exhibits a substrate specificity profile somewhat different from those observed for the other two enzymes (Taimi et al., 2004). In addition to having a preference for atRA, CYP26C1 can also efficiently catabolize 9-cis-RA, a stereoisomer which is a comparatively poor substrate for CYP26A1 and B1. In the present study, we have expressed recombinant CYP26 cDNAs in baculovirus/Sf9 insect cell system. This approach facilitates the production of enzymes for comparison of biochemical properties in a non-mammalian background, and exploration of the utility of substrate binding as a means of identifying potential substrates or inhibitors for this family of enzymes. This approach will also enable the generation of ample quantities of CYP26 enzymes for the structural analysis of substrate/ inhibitor-enzyme interactions.
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described previously (Taimi et al., 2004; White et al., 1997, 2000). Full-length human cytochrome b5 (hb5) and cytochrome P450NADPH reductase (hOR) cDNAs were isolated from thymus total RNA by RT-PCR with specific primers. The forward primers, 5′-ATC CCG GGA TGG CAG AGC AGT CGG ACG AG-3′ and 5′-ATA GAT CTG AAA TGG GAG ACT CCC TGG AC-3′ and the reverse primers, 5′-ATG GAT CCT CAG TCC TCT GCC ATG TAT AGG-3′ and 5′-ATG AAT TCC TAG CTC CAC ACG TCC AGG G-3′ were used for PCR amplification of hb5 and hOR, respectively. The cDNAs were then subcloned together into a pAcDB3 transfer vector (BD Pharmingen, Mississauga, ON). Cultured Sf9 insect cells were co-transfected with each of the expression vectors using the BaculoGold Baculovirus Expression System according to manufacturer's instructions (BD Pharmingen, Mississauga, ON). As a control, the linearized wild-type BaculoGold viral DNA was used. Recombinant viruses were purified and the presence of CYP26A1, CYP26B1, and CYP26C1, hb5 cDNA and hOR cDNAs was confirmed by PCR analysis. Cultured Sf9 insect cells (Invitrogen, Carlsbad, CA) were grown in spinner flasks in TNM-FH media. Cultures were supplemented with hemin, δ-aminolevulinic acid and ferric citrate (final concentration 2 μg/ml, 100 μM and 100 μM, respectively) just prior to infection with recombinant baculovirus. Cells were harvested 72 h after transfection, washed with ice-cold PBS, counted, then homogenized and sonicated in lysis buffer (100 mM Tris–HCl pH 7.4, 1 mM EDTA, and 0.5 M Sucrose) containing a protease inhibitor cocktail (Boehringer Mannheim, Laval, QC). The microsomes were isolated by differential centrifugations for 10 min at 800 ×g and 10 min at 10,000 ×g. Finally the post-mitochondrial supernatant was centrifuged at 100,000 ×g for 60 min using ultracentrifugation using a Beckman, L8-55M ultracentrifuge. Microsomal pellets were isolated and homogenized gently in the storage buffer (10 mM Tris–HCl pH 7.4, 1 mM EDTA, 5 mM MgCl2, 150 mM KCl, and 10% glycerol) containing a protease inhibitor cocktail. Protein concentration in the microsomal preparation was determined using the Bradford reaction assay kit and bovine serum albumin as standard (Pierce, Rockford, IL). Microsomes were aliquoted at 1 mg/ml in storage buffer and were stored in liquid nitrogen. CYP26 protein expression was verified by Western blot. Total Cytochrome P450 content in the microsomal preparation was measured by reduced carbon-monoxide difference spectroscopic analysis as described in Omura and Sato (1964).
2.3. Immunoblot analysis of expressed CYP26A1, CYP26B1 and CYP26C1 Sf9 insect cells and CYP26-transfected Sf9 cells were lysed and analyzed using 10% SDS-PAGE gels (under reducing conditions). Western blots were performed according to the manufacturer's chemiluminescence detection system instructions (Amersham Pharmacia, Piscataway, NJ) and hybridized with anti-His6 mAb (Invitrogen, Carlsbad, CA).
2. Materials and methods 2.4. Analysis of retinoic acid metabolic activity 2.1. Reagents Retinoic acid (atRA, 13-cis-RA, 9-cis-RA), ketoconazole, and NADPH were purchased from Sigma (St. Louis, MO). Radiolabeled [ 3H]-atRA and [ 3H]-9-cis-RA were purchased from NEN PerkinElmer (Boston, MA) and Amersham Biosciences (Piscataway, NJ) respectively. 2.2. Heterologous expression of recombinant CYP26A1, CYP26B1 and CYP26C1 in insect cells The PCR-generated cDNA coding region of the histidine (His6)tagged CYP26A1, CYP26B1 and CYP26C1 open reading frame were sub-cloned into pVL1392 baculovirus transfer vector (BD Pharmingen, Torrey Pines, CA). Isolation of cDNAs encoding CYP26s has been
An appropriate amount (10 μg) of Sf9 microsomal suspension was assayed in triplicate and preincubated at 37 °C in storage buffer containing 0.5% BSA (total volume of 200 μl) with radiolabeled atRA (0.1 μCi/ml [ 3H]-atRA, at 2 nM concentration). Following a 10 min pre-incubation period at 37 °C, the reaction was initiated by addition of NADPH at final concentration of 10 mM. All procedures involving retinoids were conducted under yellow lighting to protect compounds from photochemical isomerization. After incubation for 1 h at 37 °C, the reaction was stopped by acidification with 5 μl of 10% acetic acid. Extraction of the total retinoid metabolites was conducted by Bligh–Dyer procedure as described previously (Bligh & Dyer, 1959) and the upper phase containing the water-soluble polar atRA metabolites was counted in a scintillation counter.
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2.5. Competition assay The competition assay was conducted in the microsomes storage buffer containing 0.5% BSA using a mixture (total volume of 200 μl) composed of an appropriate amount (10 μg) of Sf9 microsomal suspension incubated in triplicate with radiolabeled atRA (0.1 μCi/ml [ 3H]atRA, at 2 nM concentration) in the presence or absence of increasing concentrations of each unlabeled retinoid (atRA, 9-cis-RA, 13-cis-RA), or ketoconazole. Following a 10 min pre-incubation period at 37 °C, the reaction was initiated by addition of NADPH to a final concentration of 10 mM. All procedures involving retinoids were conducted under yellow lighting to protect compounds from photochemical isomerization. After incubation for 1 h at 37 °C, the reaction was stopped by acidification with 5 μl of 10% acetic acid. Extraction of samples was conducted using Bligh–Dyer procedure as modified in (White et al., 2000), and the upper phase containing the water-soluble polar metabolites was counted in a scintillation counter. The IC50 values which represent the concentrations of competitor required to inhibit 50% of atRAmetabolism were derived by interpolation of log-transformed data. The data represent the mean± SD of triplicate samples of a representative of three independent experiments. 2.6. CYP26 binding assay The binding assay for CYP26 enzymes to atRA and 9-cis-RA were performed in a 1.0 mm glass fiber 96-well filter plate. Incubations of microsomal fractions (50 μg) of insect cells transfected with the empty vector (negative control) or expressing either CYP26A1, CYP26B1 or CYP26C1, were carried out in triplicate in the storage buffer containing 0.5% BSA (total volume of 100 μl) with increasing concentrations (1 to 1000 nM) of either [3H]-atRA or [ 3H]-9-cis-RA. All procedures involving retinoids were conducted under yellow lighting to protect compounds from photochemical isomerization. After incubating at room temperature for one hour, separation of bound and unbound ligands was achieved by washing the filter plate three times with storage buffer. The filter plates were dried and radioactivity remaining in the glass fiber membrane was measured by β-scintillation using a liquid scintillation counter. The net binding was calculated as “bound”, minus “free”, counts, and subtracted from the non-specific binding to the microsome of insect cell infected with empty vector. The saturation curves and Scatchard plots were generated using GraphPad Prism software. The data represent the mean± SD of triplicate samples of a representative of three independent experiments. 3. Results 3.1. CYP26 expression in baculovirus infected Sf9 cells The CYP26 enzymes were expressed in Sf9 cells using the baculovirus expressing vector PACDB3 system. The inclusion of hb5 and hOR
in these vectors described previously (Chuang et al., 2004), was to optimize enzymatic activity of the CYP26 enzymes. Each of the enzymes was tagged with a histidine hexamer (His-6) at the C-terminus to facilitate enzyme detection by anti-His antibodies using Western blot analysis. Baculovirus infected Sf9 insect cells were grown for three days, after which time, cells were fractionated and microsome isolates were prepared. Western blot analysis using anti-His-6 tag monoclonal antibody indicated strong expression of a protein which is approximately 54 kDa in size, corresponding to the predicted molecular weight of these enzymes (Fig. 1A). To determine the integrity of the protein yielded, we performed a differential spectrum analysis in the absence and presence of carbon-monoxide of the CYP26A1, CYP26B1 and CYP26C1 preparations. Fig. 1B shows a carbon monoxide/reduced difference spectroscopic analysis for CYP26B1 isolated microsomes and parent vector expressing cells. Microsomes from CYP26B1-expressing cells exhibit a strong peak of absorbance at 450 nm, typical of carbon monoxide saturated cytochrome P450s, whereas those from parent vector infected cells do not display a measurable absorbance at this wavelength. Similar results were obtained with CYP26A1 and CYP26C1 expressing cells. Using this method of carbon monoxide/reduced spectrum comparison (Omura & Sato, 1964) it was estimated that the concentration of the recombinant CYP26A1, CYP26B1 and CYP26C1 proteins in these microsomal preparations was 1.04, 0.86, and 0.64 nmol/mg of proteins, respectively. 3.2. Enzymatic activity of CYP26 enzymes Microsomal preparations were tested for their enzymatic ability to metabolize radiolabeled 3[H]-atRA using the Bligh–Dyer method as previously modified for CYP26 enzymes (Taimi et al., 2004; White et al., 2000). As shown in Fig. 2A, only background levels of radioactivity were detected in the no-cell control (buffer) and in microsomes isolated from Sf9-WT cells transfected with the parent vector. In contrast, microsomal preparations from CYP26 expressing cells, converted atRA to more polar aqueous soluble metabolites. Fig. 2B, shows that the ability of microsomal fractions to convert atRA into aqueous soluble metabolites was dependent on the presence of NADPH; NADPH depleted samples were inactive as demonstrated for CYP26C1. Similar data were obtained with CYP26A1 and CYP26B1 (data not shown). The insect microsomes expressing the active enzyme were incubated with 2 nM radiolabeled 3[H]-atRA, in presence of NADPH and increasing concentrations of either unlabeled atRA, 9-cis-RA, 13-cisRA, or ketoconazole (a general non-specific cytochrome P450 inhibitor). As shown in Fig. 3, the competition profile for the three enzymes was comparable to those previously described for the mammalian expressed proteins. Table 1, summarizes the IC50 values for atRA, 9-cis-RA, 13-cis-RA, and ketoconazole. As previously demonstrated, 9-cis-RA competed with atRA only when CYP26C1 was used as enzymatic system. In addition, CYP26C1 is not efficiently inhibited by ketoconazole (Table 1).
Fig. 1. A, Detection of recombinant CYP26 enzymes. Cell lysates from Sf9 cells transfected with empty vector (U), pVL-CYP26B1-His6 vector (1), pVL-CYP26A1-His6 vector (2), and pVL-CYP26C1-His6 vector (3) were analyzed by Western blot using anti-His6 monoclonal antibody and revealed a 54 kDa protein under reducing conditions. B, carbon monoxide/reduced spectrum of microsomes prepared from Sf9 insect cells transfected with empty vector and pcVL-CYP26 vectors.
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Fig. 2. CYP26-mediated metabolism of atRA. A, microsomal suspension from Sf9 insect cells transfected with empty vector and CYP26A1, CYP26B1, and CYP26C1 vectors were incubated with 2 nM radiolabeled atRA and 10 mM NADPH for one hour at 37 °C. The reaction was stopped by acidification, and total retinoid metabolites were extracted by the Bligh–Dyer procedure. The upper phase containing the water-soluble polar atRA metabolites was counted in a scintillation counter. B, microsomal fractions from Sf9-cell expressing CYP26C1 were tested for their atRA-metabolism in presence and absence of NADPH. All microsome samples were assayed in triplicate, and data represent the mean ± SD.
3.3. Substrate binding parameters for CYP26 enzymes We next established conditions for the protein binding assay. As the binding substrate, we used either 3[H]-atRA or 3[H]-9-cis-RA. In order to prevent the substrate from being metabolized, NADPH was not added to microsome preparations. Glass fiber 96-filter plates were used to separate bound and free substrate. The saturation analysis with 3[H]-atRA showed a saturation level comparable for all three enzymes (Fig. 4A). The B50 values (concentration at which 50% binding occurs) for atRA were determined to be 56 nM, 49 nM, and 36 nM for CYP26A1, CYP26B1, and CYP26C1, respectively (Table 2). Scatchard analysis of binding for atRA showed linear plots (Fig. 4B–D), and a similar Kd values of 100 nM for the three CYP26 enzymes. Similarly, we examined binding parameters for 3[H]-9-cis-RA. CYP26A1 and CYP26B1 preparation of enzymes did not exhibit significant specific binding (Fig. 5A). In contrast, 9-cis-RA binding to CYP26C1 exhibited characteristics of the high affinity binding as demonstrated for atRA for all three enzymes with a B50 value of 55.5 nM (Table 2). Scatchard analysis of the binding of 9-cis-RA to CYP26C1 resulted in a linear plot with a Kd value of 100 nM (Fig. 5B). These data produced a similar binding affinity constant of 9-cis-RA and atRA to CYP26C1. Thus, the binding properties of atRA and 9-cis-RA to CYP26 enzymes are congruous with their substrate specificity observed in enzymatic assays.
Fig. 3. Substrate competition assay in microsomal preparations of Sf9 cells expressing CYP26 enzymes. Microsomes from Sf9 cell transfected with CYP26A1 (A), CYP26B1 (B), or CYP26C1 (C) were incubated with radiolabeled atRA and NADPH in the presence or absence of increasing concentrations of each unlabeled retinoid (atRA, 9-cis-RA, 13cis-RA), or ketoconazole. After incubation for 1 h at 37 °C, the reaction was stopped and atRA metabolites were extracted using the Bligh–Dyer procedure. The upper phase containing the water-soluble polar metabolites was counted in a scintillation counter. The data shown represents the mean ± SD of triplicate samples. Curves in A and B from left to right, are ATRA, ketoconazole, 13-cis-RA, 9-cis-RA. Curves in C from left to right, are ATRA, 9-cis-RA, 13-cis-RA, ketoconazole.
4. Discussion The presence and evolutionary conservation of the three CYP26 family members suggests that they may have distinct physiological roles. We have previously demonstrated that CYP26 enzymes are expressed in different tissues at different times during embryogenesis, and furthermore have shown that all three family members can efficiently metabolize atRA (Abu-Abed et al., 2002; MacLean et al., 2001; Tahayato et al., 2003; Taimi et al., 2004; White et al., 2000). Using metabolic assays, we have shown that CYP26C1 activity is distinct from that of either CYP26A1 or CYP26B1, and that this enzyme can also efficiently metabolize 9-cis-RA (Taimi et al., 2004). Because the CYP26 enzymes exhibit considerable differences in primary amino acid sequences, we developed a new approach to
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Table 1 Summary of IC50 values of retinoids and ketoconazole in the competition assays. The IC50 values represent the concentrations of competitor required to inhibit 50% of atRA-metabolism and were derived by interpolation of log-transformed data. These values were derived from the data shown in Fig. 3.
Table 2 Summary of B50 values for atRA and 9-cis-RA in the binding assay. The B50 value was calculated as bound minus free counts from non-linear regression saturation curves. These values were derived from the data shown in Figs. 4 and 5. Substrates
IC50 [nM] Substrates
CYP26A1
CYP26B1
CYP26C1
atRA 9-cis-RA 13-cis-RA Ketoconazole
16 N 1000 N 1000 588
27 N1000 N1000 592
15 62 N1000 33,500
investigate possible differences in the substrate binding pockets. We established a simple binding assay for the substrates of CYP26 enzymes. This assay will be a very useful tool for exploring the properties of the substrate binding pocket of CYP26A1, CYP26B1, and CYP26C1 enzymes. To this end, we established CYP26 expression in the baculovirus/Sf9 insect cell expression system to generate functionally active enzymes. We tested the microsomal preparations of the baculovirus expressed enzymes using competition assays. CYP26 enzymes are similar with respect to their atRA metabolic activity, even though these enzymes display limited sequence similarity. This suggests that the substrate pockets of these enzymes can interact with atRA in a similar manner. However, the limited sequence similarity suggests that there are significant structural differences between these enzymes with respect to the substrate binding pockets. This is clearly shown by the poor ability of CYP26A1 and CYP26B1 to metabolize 9-cis-RA in comparison to that of CYP26C1. The binding analysis in the present studies confirms these differences suggesting concordance between substrate binding and enzymatic activity. The binding activity may reflect the ability of certain structures to inhibit enzyme activity and could therefore be used to screen large libraries of compounds for potential competitive inhibitors of these enzymes. One distinct advantage in using insect cell expression
atRA 9-cis-RA a
B50 [nM] CYP26A1
CYP26B1
CYP26C1
56 NBa
49.05 NBa
36.6 55.5
NB, no binding.
is the removal of other CYP26 members from background when developing subtype selective inhibitors. Another advantage of this cell-free system is that it removes cell permeability as a confounding variable when comparing inhibitor structures to assess structure/activity relationships. This is an important feature of this system for drug design. The present studies also confirm that CYP26A1 and CYP26B1 have low affinity to 9-cis-RA. This would suggest that CYP26A1 and CYP26B1 enzymes, by metabolizing atRA, can block the atRA signaling without affecting other retinoid signaling pathways such as that for 9-cis-RA, a putative ligand for RXR, which is a co-receptor of other nuclear hormone receptor signaling pathways. In addition, these studies confirm that ketoconazole is less effective in blocking CYP26C1 activity compared to CYP26A1 and CYP26B1. These findings suggest that there are structural differences in the substrate binding pockets between CYP26C1 and that the other members of the CYP25 family can discriminate substrates based on the stereo specific arrangement of the isoprenic chain. Interestingly, 13-cis-RA, another isoprenic chain based stereoisomer, binds only weakly to CYP26 family members. This suggests that the different pharmacological profile of 13-cis-RA (Zouboulis et al., 1991) may be due to its resistance to CYP26 based metabolism. In summary, we have developed a simple novel method to explore substrate binding characteristics of CYP26, the cytochrome P450 enzymes involved in metabolism of retinoic acid. Furthermore, this
Fig. 4. Binding of CYP26 enzymes to atRA. A, microsomal fractions of insect cells expressing either CYP26A1, CYP26B1 or CYP26C1 enzymes, were incubated with increasing concentrations (1 to 1000 nM) of radiolabeled [3H]-atRA. After incubating for one hour at room temperature, the unbound ligand was washed out, and the remaining radioactivity in the membrane was measured by β-scintillation using a liquid scintillation counter. The data represent the mean ± SD of triplicate samples of a representative of three independent experiments. Scatchard plot analysis is shown of the atRA binding to the CYP26A1 (B), CYP26B1 (C), and CYP26C1 (D) enzymes.
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Fig. 5. Binding of CYP26 enzymes to 9-cis-RA. A, microsomal fractions of insect cells expressing CYP26A1, CYP26B1 or CYP26C1 proteins, were incubated with increasing concentrations (1 to 1000 nM) of radiolabeled [3H]-9-cis-RA. After incubating at room temperature for one hour, unbound ligand was washed out, and the remaining radioactivity in the membrane was measured by β-scintillation using a liquid scintillation counter. The data represent the mean ± SD of triplicate samples of a representative of three independent experiments. B, Scatchard plot analysis of the 9-cis-RA binding to the CYP26C1 enzyme.
approach can be widely adapted to other enzymes such as cytochrome P450s from other families; it can be used to identify substrates for orphan enzymes for which substrates have yet to be identified. Also, this approach could be easily adapted to high throughput screening to identify inhibitors for medically important cytochrome P450's. Acknowledgments The authors would like to thank members of the Cytochroma R&D team for insightful suggestions during the course of this work. This work was supported by the Cytochroma, Inc., Markham, Ontario. References Abu-Abed, S., Dolle, P., Metzger, D., Beckett, B., Chambon, P., & Petkovich, M. (2001). The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures. Genes & Development, 15(2), 226–240. Abu-Abed, S., MacLean, G., Fraulob, V., Chambon, P., Petkovich, M., & Dolle, P. (2002). Differential expression of the retinoic acid-metabolizing enzymes CYP26A1 and CYP26B1 during murine organogenesis. Mechanisms of Development, 110(1–2), 173–177. Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37(8), 911–917. Brodeur, H., Gagnon, I., Mader, S., & Bhat, P. V. (2003). Cloning of monkey RALDH1 and characterization of retinoid metabolism in monkey kidney proximal tubule cells. Journal of Lipid Research, 44(2), 303–313. Chambon, P. (1996). A decade of molecular biology of retinoic acid receptors. The FASEB Journal, 10(9), 940–954. Chithalen, J. V., Luu, L., Petkovich, M., & Jones, G. (2002). HPLC–MS/MS analysis of the products generated from all-trans-retinoic acid using recombinant human CYP26A. Journal of Lipid Research, 43(7), 1133–1142. Chuang, S. S., Helvig, C., Taimi, M., Ramshaw, H. A., Collop, A. H., Amad, M., et al. (2004). CYP2U1, a novel human thymus- and brain-specific cytochrome P450, catalyzes omega- and (omega-1)-hydroxylation of fatty acids. Journal of Biological Chemistry, 279(8), 6305–6314. Duester, G. (2000). Families of retinoid dehydrogenases regulating vitamin A function: Production of visual pigment and retinoic acid. European Journal of Biochemistry, 267(14), 4315–4324. Lin, M., & Napoli, J. L. (2000). cDNA cloning and expression of a human aldehyde dehydrogenase (ALDH) active with 9-cis-retinal and identification of a rat ortholog, ALDH12. Journal of Biological Chemistry, 275(51), 40106–40112. MacLean, G., Abu-Abed, S., Dolle, P., Tahayato, A., Chambon, P., & Petkovich, M. (2001). Cloning of a novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development. Mechanisms of Development, 107(1–2), 195–201. Montplaisir, V., Lan, N. C., Guimond, J., Savineau, C., Bhat, P. V., & Mader, S. (2002). Recombinant class I aldehyde dehydrogenases specific for all-trans- or 9-cis-retinal. Journal of Biological Chemistry, 277(20), 17486–17492.
Niederreither, K., Abu-Abed, S., Schuhbaur, B., Petkovich, M., Chambon, P., & Dolle, P. (2002). Genetic evidence that oxidative derivatives of retinoic acid are not involved in retinoid signaling during mouse development. Nature Genetics, 31(1), 84–88. Niederreither, K., & Dolle, P. (2008). Retinoic acid in development: Towards an integrated view. Nature Reviews. Genetics, 9(7), 541–553. Niederreither, K., Fraulob, V., Garnier, J. M., Chambon, P., & Dolle, P. (2002). Differential expression of retinoic acid-synthesizing (RALDH) enzymes during fetal development and organ differentiation in the mouse. Mechanisms of Development, 110(1–2), 165–171. Omura, T., & Sato, R. (1964). The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. Journal of Biological Chemistry, 239, 2370–2378. Pennimpede, T., Cameron, D. A., MacLean, G. A., Li, H., Abu-Abed, S., & Petkovich, M. (2010). The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis. Birth Defects Research. Part A, Clinical and Molecular Teratology, 88(10), 883–894. Pennimpede, T., Cameron, D. A., Maclean, G. A., & Petkovich, M. (2010). Analysis of Cyp26b1/Rarg compound-null mice reveals two genetically separable effects of retinoic acid on limb outgrowth. Developmental Biology, 339(1), 179–186. Pennimpede, T., Cameron, D., & Petkovich, M. (2006). Regulation of murine embryonic patterning and morphogenesis by retinoic acid signaling. Advances in Developmental Biology, 16, 66–104. Sakai, Y., Meno, C., Fujii, H., Nishino, J., Shiratori, H., Saijoh, Y., et al. (2001). The retinoic acid-inactivating enzyme CYP26 is essential for establishing an uneven distribution of retinoic acid along the anterio-posterior axis within the mouse embryo. Genes & Development, 15(2), 213–225. Tahayato, A., Dolle, P., & Petkovich, M. (2003). Cyp26C1 encodes a novel retinoic acid-metabolizing enzyme expressed in the hindbrain, inner ear, first branchial arch and tooth buds during murine development. Gene Expression Patterns, 3(4), 449–454. Taimi, M., Helvig, C., Wisniewski, J., Ramshaw, H., White, J., Amad, M., et al. (2004). A novel human cytochrome P450, CYP26C1, involved in metabolism of 9-cis and all-trans isomers of retinoic acid. Journal of Biological Chemistry, 279(1), 77–85. White, J. A., Beckett-Jones, B., Guo, Y. D., Dilworth, F. J., Bonasoro, J., Jones, G., et al. (1997). cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450. Journal of Biological Chemistry, 272 (30), 18538–18541. White, J. A., Guo, Y. D., Baetz, K., Beckett-Jones, B., Bonasoro, J., Hsu, K. E., et al. (1996). Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase. Journal of Biological Chemistry, 271(47), 29922–29927. White, J. A., Ramshaw, H., Taimi, M., Stangle, W., Zhang, A., Everingham, S., et al. (2000). Identification of the human cytochrome P450, P450RAI-2, which is predominantly expressed in the adult cerebellum and is responsible for all-trans-retinoic acid metabolism. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6403–6408. Yashiro, K., Zhao, X., Uehara, M., Yamashita, K., Nishijima, M., Nishino, J., et al. (2004). Regulation of retinoic acid distribution is required for proximodistal patterning and outgrowth of the developing mouse limb. Developmental Cell, 6(3), 411–422. Zouboulis, C. C., Korge, B., Akamatsu, H., Xia, L. Q., Schiller, S., Gollnick, H., et al. (1991). Effects of 13-cis-retinoic acid, all-trans-retinoic acid, and acitretin on the proliferation, lipid synthesis and keratin expression of cultured human sebocytes in vitro. The Journal of Investigative Dermatology, 96(5), 792–797.
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Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox
Original article
Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells Christian Helvig a, Mohammed Taimi a, Don Cameron b, d, Glenville Jones b, Martin Petkovich a, b, c, d,⁎ a
Cytochroma, Inc., 100 Allstate Parkway, Suite 600, Markham, Ontario, Canada L3R 6H3 Department of Biochemistry, Queen's University, Kingston, Ontario, Canada Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada d Cancer Research Institute, Queen's University, Kingston, Ontario, Canada b c
a r t i c l e
i n f o
Article history: Received 29 April 2011 Accepted 24 August 2011 Keywords: Retinoic Acid Metabolism CYP26 Baculovirus Assay
a b s t r a c t Introduction: The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9-cis isomer of RA. Methods: We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes. Results: We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all-trans-RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC50 values of 16, 27, and 15 nM for CYP26A1, B1, and C1 respectively for all-trans-RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans-RA could also be effectively competed with 9-cis RA, with IC50 of 62 nM, and was sensitive to ketoconazole inhibition. Discussion: CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9-cis-RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The vitamin A derivative, all-trans retinoic acid (atRA), plays an essential role in vertebrate embryogenesis and is an important regulator of cell differentiation and proliferation in the adult (Niederreither & Dolle, 2008; Pennimpede, Cameron, & Petkovich, 2006). The biological effects of retinoids are mediated by two classes of nuclear receptors, retinoic acid receptors (RARα, β, and γ), and retinoid X receptors (RXRα, β, and γ) which function as ligand-dependent transcription factors by binding to specific response elements in promoters of retinoids target genes (Chambon, 1996; Pennimpede et al., 2006). RARs are
Abbreviations: RA, retinoic acid; atRA, all-trans retinoic acid; CYP, cytochrome P450; hb5, human cytochrome b5; hOR, cytochrome P450-NADPH reductase; RT, Reverse Transcriptase; RALDH, Retinaldehyde dehydrogenase; BSA, bovine serum albumin. ⁎ Corresponding author at: Cancer Research Institute, Department of Biochemistry, Queen's University 10 Stuart St., Botterell Hall Room 354, Kingston, Ontario, Canada K7L 3N6. Tel.: + 1 613 533 6791; fax: + 1 613 533 6830. E-mail address: [email protected] (M. Petkovich). 1056-8719/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2011.08.005
activated by atRA and 9-cis- isomer of retinoic acid (9-cis-RA), and RXRs are selectively activated by 9-cis-RA. Retinoic acid dispersal in tissues is finely controlled by the tightly regulated expression of atRA synthetic retinaldehyde dehydrogenase (RALDH) and cytochrome P450 (CYP) catabolic enzymes (Abu-Abed et al., 2001, 2002; MacLean et al., 2001; Niederreither, Fraulob, Garnier, Chambon, & Dolle, 2002; Tahayato, Dolle, & Petkovich, 2003). Several classes of RALDH genes have been cloned from several species, including human, and are characterized by their ability to form atRA and 9-cis-RA from their aldehyde precursors (Brodeur, Gagnon, Mader, & Bhat, 2003; Duester, 2000; Lin & Napoli, 2000; Montplaisir et al., 2002; Niederreither, Fraulob, et al., 2002). Retinoic acid catabolism is mediated primarily by the CYP26 family of cytochrome P450 enzymes (CYP26A1, −B1 and −C1). The expression patterns of CYP26 genes in embryos and adults suggest that they play distinct physiological roles in protecting tissues from inappropriate exposure to atRA (Pennimpede et al., 2010). Both genetic and biochemical studies support a catabolic role for CYP26 enzymes. Loss of the Cyp26a1 gene in mice results in embryonic lethality, and knockout animals revealed that CYP26A1 is essential to protect tailbud and hindbrain regions of the embryo from exposure to endogenous atRA. Cyp26a1−/−
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animals exhibit spina bifida and sirenomelia (mermaid syndrome) resembling animals exposed to teratogenic doses of atRA (Abu-Abed et al., 2001; Sakai et al., 2001). Similarly, genetic deletion of Cyp26b1, which is predominantly expressed in developing limbs, results in limb defects similar to those found in animals exposed to high to excess atRA in utero (Pennimpede, Cameron, Maclean, & Petkovich, 2010; Yashiro et al., 2004). Furthermore, it is possible to rescue Cyp26a1−/− animals from lethality by intercrossing them with animals containing a single null allele for the atRA synthesizing enzyme, RALDH2; thereby reducing the endogenous levels of RA to which caudal and hindbrain tissues are exposed (Niederreither, Abu-Abed, et al., 2002). This study supports the view that atRA and not its metabolites are important for embryonic development. Although the enzymes exhibit a comparatively high degree of amino acid sequence similarity in some specific regions of the protein such as the Heme binding region, and ETLR and PERF conserved P450 domains, their overall similarity is less striking (Taimi et al., 2004). CYP26A1 and CYP26B1 share 42% amino acid identity (White et al., 2000), while CYP26C1 shares 43% identity, and 51% identity at the amino acid level with CYP26A1 and CYP26B1 respectively (Taimi et al., 2004). Discerning the functional differences between these enzymes may help to distinguish the specific enzymatic roles these enzymes play and guide the development of sub-type selective inhibitors. The CYP26A1-subtype was the first enzyme of this family to be discovered (White et al., 1996, 1997). Analysis of CYP26A1 enzymatic activity indicates that atRA is the preferred natural substrate for this enzyme. CYP26A1 rapidly converts atRA to hydroxylated forms, 4OH and 4-oxo being the predominant metabolic products. In cell culture systems, these primary metabolites can be further metabolized to multiple-hydroxylated forms (Chithalen, Luu, Petkovich, & Jones, 2002). It is not presently known whether these further metabolites are generated under physiological conditions or whether they play any specific biological role. Two additional members of this family, CYP26B1 and CYP26C1, have also been isolated and characterized. CYP26B1 displays substrate specificity similar to that of CYP26A1 (White et al., 2000). In contrast, CYP26C1 exhibits a substrate specificity profile somewhat different from those observed for the other two enzymes (Taimi et al., 2004). In addition to having a preference for atRA, CYP26C1 can also efficiently catabolize 9-cis-RA, a stereoisomer which is a comparatively poor substrate for CYP26A1 and B1. In the present study, we have expressed recombinant CYP26 cDNAs in baculovirus/Sf9 insect cell system. This approach facilitates the production of enzymes for comparison of biochemical properties in a non-mammalian background, and exploration of the utility of substrate binding as a means of identifying potential substrates or inhibitors for this family of enzymes. This approach will also enable the generation of ample quantities of CYP26 enzymes for the structural analysis of substrate/ inhibitor-enzyme interactions.
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described previously (Taimi et al., 2004; White et al., 1997, 2000). Full-length human cytochrome b5 (hb5) and cytochrome P450NADPH reductase (hOR) cDNAs were isolated from thymus total RNA by RT-PCR with specific primers. The forward primers, 5′-ATC CCG GGA TGG CAG AGC AGT CGG ACG AG-3′ and 5′-ATA GAT CTG AAA TGG GAG ACT CCC TGG AC-3′ and the reverse primers, 5′-ATG GAT CCT CAG TCC TCT GCC ATG TAT AGG-3′ and 5′-ATG AAT TCC TAG CTC CAC ACG TCC AGG G-3′ were used for PCR amplification of hb5 and hOR, respectively. The cDNAs were then subcloned together into a pAcDB3 transfer vector (BD Pharmingen, Mississauga, ON). Cultured Sf9 insect cells were co-transfected with each of the expression vectors using the BaculoGold Baculovirus Expression System according to manufacturer's instructions (BD Pharmingen, Mississauga, ON). As a control, the linearized wild-type BaculoGold viral DNA was used. Recombinant viruses were purified and the presence of CYP26A1, CYP26B1, and CYP26C1, hb5 cDNA and hOR cDNAs was confirmed by PCR analysis. Cultured Sf9 insect cells (Invitrogen, Carlsbad, CA) were grown in spinner flasks in TNM-FH media. Cultures were supplemented with hemin, δ-aminolevulinic acid and ferric citrate (final concentration 2 μg/ml, 100 μM and 100 μM, respectively) just prior to infection with recombinant baculovirus. Cells were harvested 72 h after transfection, washed with ice-cold PBS, counted, then homogenized and sonicated in lysis buffer (100 mM Tris–HCl pH 7.4, 1 mM EDTA, and 0.5 M Sucrose) containing a protease inhibitor cocktail (Boehringer Mannheim, Laval, QC). The microsomes were isolated by differential centrifugations for 10 min at 800 ×g and 10 min at 10,000 ×g. Finally the post-mitochondrial supernatant was centrifuged at 100,000 ×g for 60 min using ultracentrifugation using a Beckman, L8-55M ultracentrifuge. Microsomal pellets were isolated and homogenized gently in the storage buffer (10 mM Tris–HCl pH 7.4, 1 mM EDTA, 5 mM MgCl2, 150 mM KCl, and 10% glycerol) containing a protease inhibitor cocktail. Protein concentration in the microsomal preparation was determined using the Bradford reaction assay kit and bovine serum albumin as standard (Pierce, Rockford, IL). Microsomes were aliquoted at 1 mg/ml in storage buffer and were stored in liquid nitrogen. CYP26 protein expression was verified by Western blot. Total Cytochrome P450 content in the microsomal preparation was measured by reduced carbon-monoxide difference spectroscopic analysis as described in Omura and Sato (1964).
2.3. Immunoblot analysis of expressed CYP26A1, CYP26B1 and CYP26C1 Sf9 insect cells and CYP26-transfected Sf9 cells were lysed and analyzed using 10% SDS-PAGE gels (under reducing conditions). Western blots were performed according to the manufacturer's chemiluminescence detection system instructions (Amersham Pharmacia, Piscataway, NJ) and hybridized with anti-His6 mAb (Invitrogen, Carlsbad, CA).
2. Materials and methods 2.4. Analysis of retinoic acid metabolic activity 2.1. Reagents Retinoic acid (atRA, 13-cis-RA, 9-cis-RA), ketoconazole, and NADPH were purchased from Sigma (St. Louis, MO). Radiolabeled [ 3H]-atRA and [ 3H]-9-cis-RA were purchased from NEN PerkinElmer (Boston, MA) and Amersham Biosciences (Piscataway, NJ) respectively. 2.2. Heterologous expression of recombinant CYP26A1, CYP26B1 and CYP26C1 in insect cells The PCR-generated cDNA coding region of the histidine (His6)tagged CYP26A1, CYP26B1 and CYP26C1 open reading frame were sub-cloned into pVL1392 baculovirus transfer vector (BD Pharmingen, Torrey Pines, CA). Isolation of cDNAs encoding CYP26s has been
An appropriate amount (10 μg) of Sf9 microsomal suspension was assayed in triplicate and preincubated at 37 °C in storage buffer containing 0.5% BSA (total volume of 200 μl) with radiolabeled atRA (0.1 μCi/ml [ 3H]-atRA, at 2 nM concentration). Following a 10 min pre-incubation period at 37 °C, the reaction was initiated by addition of NADPH at final concentration of 10 mM. All procedures involving retinoids were conducted under yellow lighting to protect compounds from photochemical isomerization. After incubation for 1 h at 37 °C, the reaction was stopped by acidification with 5 μl of 10% acetic acid. Extraction of the total retinoid metabolites was conducted by Bligh–Dyer procedure as described previously (Bligh & Dyer, 1959) and the upper phase containing the water-soluble polar atRA metabolites was counted in a scintillation counter.
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2.5. Competition assay The competition assay was conducted in the microsomes storage buffer containing 0.5% BSA using a mixture (total volume of 200 μl) composed of an appropriate amount (10 μg) of Sf9 microsomal suspension incubated in triplicate with radiolabeled atRA (0.1 μCi/ml [ 3H]atRA, at 2 nM concentration) in the presence or absence of increasing concentrations of each unlabeled retinoid (atRA, 9-cis-RA, 13-cis-RA), or ketoconazole. Following a 10 min pre-incubation period at 37 °C, the reaction was initiated by addition of NADPH to a final concentration of 10 mM. All procedures involving retinoids were conducted under yellow lighting to protect compounds from photochemical isomerization. After incubation for 1 h at 37 °C, the reaction was stopped by acidification with 5 μl of 10% acetic acid. Extraction of samples was conducted using Bligh–Dyer procedure as modified in (White et al., 2000), and the upper phase containing the water-soluble polar metabolites was counted in a scintillation counter. The IC50 values which represent the concentrations of competitor required to inhibit 50% of atRAmetabolism were derived by interpolation of log-transformed data. The data represent the mean± SD of triplicate samples of a representative of three independent experiments. 2.6. CYP26 binding assay The binding assay for CYP26 enzymes to atRA and 9-cis-RA were performed in a 1.0 mm glass fiber 96-well filter plate. Incubations of microsomal fractions (50 μg) of insect cells transfected with the empty vector (negative control) or expressing either CYP26A1, CYP26B1 or CYP26C1, were carried out in triplicate in the storage buffer containing 0.5% BSA (total volume of 100 μl) with increasing concentrations (1 to 1000 nM) of either [3H]-atRA or [ 3H]-9-cis-RA. All procedures involving retinoids were conducted under yellow lighting to protect compounds from photochemical isomerization. After incubating at room temperature for one hour, separation of bound and unbound ligands was achieved by washing the filter plate three times with storage buffer. The filter plates were dried and radioactivity remaining in the glass fiber membrane was measured by β-scintillation using a liquid scintillation counter. The net binding was calculated as “bound”, minus “free”, counts, and subtracted from the non-specific binding to the microsome of insect cell infected with empty vector. The saturation curves and Scatchard plots were generated using GraphPad Prism software. The data represent the mean± SD of triplicate samples of a representative of three independent experiments. 3. Results 3.1. CYP26 expression in baculovirus infected Sf9 cells The CYP26 enzymes were expressed in Sf9 cells using the baculovirus expressing vector PACDB3 system. The inclusion of hb5 and hOR
in these vectors described previously (Chuang et al., 2004), was to optimize enzymatic activity of the CYP26 enzymes. Each of the enzymes was tagged with a histidine hexamer (His-6) at the C-terminus to facilitate enzyme detection by anti-His antibodies using Western blot analysis. Baculovirus infected Sf9 insect cells were grown for three days, after which time, cells were fractionated and microsome isolates were prepared. Western blot analysis using anti-His-6 tag monoclonal antibody indicated strong expression of a protein which is approximately 54 kDa in size, corresponding to the predicted molecular weight of these enzymes (Fig. 1A). To determine the integrity of the protein yielded, we performed a differential spectrum analysis in the absence and presence of carbon-monoxide of the CYP26A1, CYP26B1 and CYP26C1 preparations. Fig. 1B shows a carbon monoxide/reduced difference spectroscopic analysis for CYP26B1 isolated microsomes and parent vector expressing cells. Microsomes from CYP26B1-expressing cells exhibit a strong peak of absorbance at 450 nm, typical of carbon monoxide saturated cytochrome P450s, whereas those from parent vector infected cells do not display a measurable absorbance at this wavelength. Similar results were obtained with CYP26A1 and CYP26C1 expressing cells. Using this method of carbon monoxide/reduced spectrum comparison (Omura & Sato, 1964) it was estimated that the concentration of the recombinant CYP26A1, CYP26B1 and CYP26C1 proteins in these microsomal preparations was 1.04, 0.86, and 0.64 nmol/mg of proteins, respectively. 3.2. Enzymatic activity of CYP26 enzymes Microsomal preparations were tested for their enzymatic ability to metabolize radiolabeled 3[H]-atRA using the Bligh–Dyer method as previously modified for CYP26 enzymes (Taimi et al., 2004; White et al., 2000). As shown in Fig. 2A, only background levels of radioactivity were detected in the no-cell control (buffer) and in microsomes isolated from Sf9-WT cells transfected with the parent vector. In contrast, microsomal preparations from CYP26 expressing cells, converted atRA to more polar aqueous soluble metabolites. Fig. 2B, shows that the ability of microsomal fractions to convert atRA into aqueous soluble metabolites was dependent on the presence of NADPH; NADPH depleted samples were inactive as demonstrated for CYP26C1. Similar data were obtained with CYP26A1 and CYP26B1 (data not shown). The insect microsomes expressing the active enzyme were incubated with 2 nM radiolabeled 3[H]-atRA, in presence of NADPH and increasing concentrations of either unlabeled atRA, 9-cis-RA, 13-cisRA, or ketoconazole (a general non-specific cytochrome P450 inhibitor). As shown in Fig. 3, the competition profile for the three enzymes was comparable to those previously described for the mammalian expressed proteins. Table 1, summarizes the IC50 values for atRA, 9-cis-RA, 13-cis-RA, and ketoconazole. As previously demonstrated, 9-cis-RA competed with atRA only when CYP26C1 was used as enzymatic system. In addition, CYP26C1 is not efficiently inhibited by ketoconazole (Table 1).
Fig. 1. A, Detection of recombinant CYP26 enzymes. Cell lysates from Sf9 cells transfected with empty vector (U), pVL-CYP26B1-His6 vector (1), pVL-CYP26A1-His6 vector (2), and pVL-CYP26C1-His6 vector (3) were analyzed by Western blot using anti-His6 monoclonal antibody and revealed a 54 kDa protein under reducing conditions. B, carbon monoxide/reduced spectrum of microsomes prepared from Sf9 insect cells transfected with empty vector and pcVL-CYP26 vectors.
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Fig. 2. CYP26-mediated metabolism of atRA. A, microsomal suspension from Sf9 insect cells transfected with empty vector and CYP26A1, CYP26B1, and CYP26C1 vectors were incubated with 2 nM radiolabeled atRA and 10 mM NADPH for one hour at 37 °C. The reaction was stopped by acidification, and total retinoid metabolites were extracted by the Bligh–Dyer procedure. The upper phase containing the water-soluble polar atRA metabolites was counted in a scintillation counter. B, microsomal fractions from Sf9-cell expressing CYP26C1 were tested for their atRA-metabolism in presence and absence of NADPH. All microsome samples were assayed in triplicate, and data represent the mean ± SD.
3.3. Substrate binding parameters for CYP26 enzymes We next established conditions for the protein binding assay. As the binding substrate, we used either 3[H]-atRA or 3[H]-9-cis-RA. In order to prevent the substrate from being metabolized, NADPH was not added to microsome preparations. Glass fiber 96-filter plates were used to separate bound and free substrate. The saturation analysis with 3[H]-atRA showed a saturation level comparable for all three enzymes (Fig. 4A). The B50 values (concentration at which 50% binding occurs) for atRA were determined to be 56 nM, 49 nM, and 36 nM for CYP26A1, CYP26B1, and CYP26C1, respectively (Table 2). Scatchard analysis of binding for atRA showed linear plots (Fig. 4B–D), and a similar Kd values of 100 nM for the three CYP26 enzymes. Similarly, we examined binding parameters for 3[H]-9-cis-RA. CYP26A1 and CYP26B1 preparation of enzymes did not exhibit significant specific binding (Fig. 5A). In contrast, 9-cis-RA binding to CYP26C1 exhibited characteristics of the high affinity binding as demonstrated for atRA for all three enzymes with a B50 value of 55.5 nM (Table 2). Scatchard analysis of the binding of 9-cis-RA to CYP26C1 resulted in a linear plot with a Kd value of 100 nM (Fig. 5B). These data produced a similar binding affinity constant of 9-cis-RA and atRA to CYP26C1. Thus, the binding properties of atRA and 9-cis-RA to CYP26 enzymes are congruous with their substrate specificity observed in enzymatic assays.
Fig. 3. Substrate competition assay in microsomal preparations of Sf9 cells expressing CYP26 enzymes. Microsomes from Sf9 cell transfected with CYP26A1 (A), CYP26B1 (B), or CYP26C1 (C) were incubated with radiolabeled atRA and NADPH in the presence or absence of increasing concentrations of each unlabeled retinoid (atRA, 9-cis-RA, 13cis-RA), or ketoconazole. After incubation for 1 h at 37 °C, the reaction was stopped and atRA metabolites were extracted using the Bligh–Dyer procedure. The upper phase containing the water-soluble polar metabolites was counted in a scintillation counter. The data shown represents the mean ± SD of triplicate samples. Curves in A and B from left to right, are ATRA, ketoconazole, 13-cis-RA, 9-cis-RA. Curves in C from left to right, are ATRA, 9-cis-RA, 13-cis-RA, ketoconazole.
4. Discussion The presence and evolutionary conservation of the three CYP26 family members suggests that they may have distinct physiological roles. We have previously demonstrated that CYP26 enzymes are expressed in different tissues at different times during embryogenesis, and furthermore have shown that all three family members can efficiently metabolize atRA (Abu-Abed et al., 2002; MacLean et al., 2001; Tahayato et al., 2003; Taimi et al., 2004; White et al., 2000). Using metabolic assays, we have shown that CYP26C1 activity is distinct from that of either CYP26A1 or CYP26B1, and that this enzyme can also efficiently metabolize 9-cis-RA (Taimi et al., 2004). Because the CYP26 enzymes exhibit considerable differences in primary amino acid sequences, we developed a new approach to
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Table 1 Summary of IC50 values of retinoids and ketoconazole in the competition assays. The IC50 values represent the concentrations of competitor required to inhibit 50% of atRA-metabolism and were derived by interpolation of log-transformed data. These values were derived from the data shown in Fig. 3.
Table 2 Summary of B50 values for atRA and 9-cis-RA in the binding assay. The B50 value was calculated as bound minus free counts from non-linear regression saturation curves. These values were derived from the data shown in Figs. 4 and 5. Substrates
IC50 [nM] Substrates
CYP26A1
CYP26B1
CYP26C1
atRA 9-cis-RA 13-cis-RA Ketoconazole
16 N 1000 N 1000 588
27 N1000 N1000 592
15 62 N1000 33,500
investigate possible differences in the substrate binding pockets. We established a simple binding assay for the substrates of CYP26 enzymes. This assay will be a very useful tool for exploring the properties of the substrate binding pocket of CYP26A1, CYP26B1, and CYP26C1 enzymes. To this end, we established CYP26 expression in the baculovirus/Sf9 insect cell expression system to generate functionally active enzymes. We tested the microsomal preparations of the baculovirus expressed enzymes using competition assays. CYP26 enzymes are similar with respect to their atRA metabolic activity, even though these enzymes display limited sequence similarity. This suggests that the substrate pockets of these enzymes can interact with atRA in a similar manner. However, the limited sequence similarity suggests that there are significant structural differences between these enzymes with respect to the substrate binding pockets. This is clearly shown by the poor ability of CYP26A1 and CYP26B1 to metabolize 9-cis-RA in comparison to that of CYP26C1. The binding analysis in the present studies confirms these differences suggesting concordance between substrate binding and enzymatic activity. The binding activity may reflect the ability of certain structures to inhibit enzyme activity and could therefore be used to screen large libraries of compounds for potential competitive inhibitors of these enzymes. One distinct advantage in using insect cell expression
atRA 9-cis-RA a
B50 [nM] CYP26A1
CYP26B1
CYP26C1
56 NBa
49.05 NBa
36.6 55.5
NB, no binding.
is the removal of other CYP26 members from background when developing subtype selective inhibitors. Another advantage of this cell-free system is that it removes cell permeability as a confounding variable when comparing inhibitor structures to assess structure/activity relationships. This is an important feature of this system for drug design. The present studies also confirm that CYP26A1 and CYP26B1 have low affinity to 9-cis-RA. This would suggest that CYP26A1 and CYP26B1 enzymes, by metabolizing atRA, can block the atRA signaling without affecting other retinoid signaling pathways such as that for 9-cis-RA, a putative ligand for RXR, which is a co-receptor of other nuclear hormone receptor signaling pathways. In addition, these studies confirm that ketoconazole is less effective in blocking CYP26C1 activity compared to CYP26A1 and CYP26B1. These findings suggest that there are structural differences in the substrate binding pockets between CYP26C1 and that the other members of the CYP25 family can discriminate substrates based on the stereo specific arrangement of the isoprenic chain. Interestingly, 13-cis-RA, another isoprenic chain based stereoisomer, binds only weakly to CYP26 family members. This suggests that the different pharmacological profile of 13-cis-RA (Zouboulis et al., 1991) may be due to its resistance to CYP26 based metabolism. In summary, we have developed a simple novel method to explore substrate binding characteristics of CYP26, the cytochrome P450 enzymes involved in metabolism of retinoic acid. Furthermore, this
Fig. 4. Binding of CYP26 enzymes to atRA. A, microsomal fractions of insect cells expressing either CYP26A1, CYP26B1 or CYP26C1 enzymes, were incubated with increasing concentrations (1 to 1000 nM) of radiolabeled [3H]-atRA. After incubating for one hour at room temperature, the unbound ligand was washed out, and the remaining radioactivity in the membrane was measured by β-scintillation using a liquid scintillation counter. The data represent the mean ± SD of triplicate samples of a representative of three independent experiments. Scatchard plot analysis is shown of the atRA binding to the CYP26A1 (B), CYP26B1 (C), and CYP26C1 (D) enzymes.
C. Helvig et al. / Journal of Pharmacological and Toxicological Methods 64 (2011) 258–263
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Fig. 5. Binding of CYP26 enzymes to 9-cis-RA. A, microsomal fractions of insect cells expressing CYP26A1, CYP26B1 or CYP26C1 proteins, were incubated with increasing concentrations (1 to 1000 nM) of radiolabeled [3H]-9-cis-RA. After incubating at room temperature for one hour, unbound ligand was washed out, and the remaining radioactivity in the membrane was measured by β-scintillation using a liquid scintillation counter. The data represent the mean ± SD of triplicate samples of a representative of three independent experiments. B, Scatchard plot analysis of the 9-cis-RA binding to the CYP26C1 enzyme.
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