Visual Overview
Abstract
UDP-Glucuronosyltransferases (UGTs) are classified into three subfamilies in mice: Ugt1a, 2b, and 2a. In the Ugt1a subfamily, Ugt1a1 and 1a6 appear to correspond to human UGT1A1 and 1A6. The mouse is an important animal for its use in investigations, but the substrate specificities of Ugt isoforms belonging to the 2b subfamily in mice remain largely unknown. To address this issue, we characterized the substrate specificity of all isoforms of the Ugt2b subfamily expressed in the mouse liver. The cDNAs of Ugt1a1, Ugt2a3, and all the Ugt2b isoforms expressed in the liver were reverse-transcribed from the total RNA of male FVB-mouse livers and then amplified. A baculovirus-Sf9 cell system for expressing each Ugt was established. Of all the Ugts examined, Ugt2b34, 2b36, and 2b37 exhibited the ability to glucuronidate morphine with Ugt2b36, the most active in this regard. Ugt1a1, but also Ugt2b34, 2b36, and 2b37 to a lesser extent, preferentially catalyzed the glucuronidation of 17β-estradiol on the 3-hydroxyl group (E3G). With these isoforms, E3G formation by Ugt1a1 was efficient; however, Ugt2b5 exhibited a preference for the 17β-hydroxyl group (E17G). Ugt2b1 and Ugt2a3 formed comparable levels of E3G and E17G. Ugt2b1 and 2b5 were the only isoforms involved in chloramphenicol glucuronidation. As Ugt2b36 is highly expressed in the liver, it is most likely that Ugt2b36 is a major morphine Ugt in mouse liver. Regarding E3G formation, Ugt1a1, like the human homolog, seems to play an important role in the liver.
Introduction
Following administration, drugs are absorbed, distributed, metabolized, and then eliminated. Usually, a fraction of the drugs administered is eliminated in unchanged form. In many cases, however, drugs are metabolized to increase their hydrophilic properties before elimination. Drug metabolism reactions are categorized into phase I and II reactions. Oxidation, reduction, and hydrolysis are categorized as phase I reactions. On the other hand, conjugations are categorized as phase II reactions. In phase I reactions, P450 cytochromes (P450s) play a key role (Guengerich, 2006). Regarding conjugation reactions, however, glucuronidation contributes to approximately 35% of phase 2 reactions (Evans and Relling, 1999). UDP-Glucuronosyltransferases (UGTs, Ugts) catalyze glucuronidation, and the classic view that P450-catalyzed hydroxylations precede glucuronidation of the metabolites formed by P450 holds true for many drugs; however, compounds such as morphine are cleared primarily by direct glucuronidation without requiring P450 enzymes (Yoshimura et al., 1969). Furthermore, UGTs occupy second place after P450 in terms of drug clearance (Williams et al., 2004). Thus, it is important to investigate UGT-catalyzed reactions.
The use of morphine to treat pain associated with cancer is widely recommended (World Health Organization, 1996); however, there is also an accompanying issue of morphine overdosing, and considerable numbers of patients medicated with morphine die (Okie, 2010). Morphine is glucuronidated in vivo to form a main metabolite, morphine-3-glucuronide (M-3-G), and a minor metabolite, morphine-6-glucuronide (M-6-G) (Yoshimura et al., 1969). Although M-3-G is a nonanalgesic metabolite, M-6-G is a more potent metabolite than the parent morphine (Shimomura et al., 1971). It is known that there are species differences in M-6-G formation. A relatively large amount of M-6-G is formed in humans and guinea pigs after morphine treatment, whereas only a small amount of M-6-G is formed in rats and mice (Oguri et al., 1970; Svensson et al., 1982; Kuo et al., 1991). In humans, UGT2B7 catalyzes mainly the formation of M-3-G and M-6-G (Coffman et al., 1997; Stone et al., 2003), whereas in guinea pigs, UGT2B21 catalyzes M-3-G formation (Ishii et al., 2001). In rats, UGT2B1 catalyzes M-3-G formation (Pritchard et al., 1994). In mice, Ugt2b1 is considered orthologous to rat UGT2B1 on the basis of sequence similarity (Mackenzie et al., 2005); however, its function has yet to be characterized. Although it is reasonable to suppose that the mouse Ugt involved in morphine glucuronidation probably belongs to the Ugt2b subfamily, mouse Ugt isform(s) involved in morphine glucuronidation are so far unknown. Since the mouse is an important experimental animal, it is expected that characterization of the Ugt isoform(s) involved in morphine metabolism will help in the extrapolation of preclinical trial data to humans.
To date, 21 Ugt isoforms are known to be expressed in mice that belong to the Ugt1a, 2a, 2b and 3a subfamilies (Mackenzie et al., 2005). These are expressed mainly in the liver and intestine but, in some cases, in the brain and lung (Buckley and Klaassen, 2007). The gene structure of the UGT1A subfamily involves a variable exon 1 and common exons 2–5 (Iyanagi, 1991; Ritter et al., 1992). The UGT1 locus consists of variable first exons and four shared exons in a tandem array. Since such gene structures are conserved, to a degree, among the mouse, rat, and human, at least Ugt1a1 and 1a6 in mice seem orthologous to UGT1A1 and 1A6 in humans and rats, respectively (Mackenzie et al., 2005). This is supported by a study using Ugt1a locus null mice (Nguyen et al., 2008). For the mouse Ugt2b subfamily, however, all Ugt2b isoforms except Ugt2b1 have unique numbers since it is difficult to define orthologs. Many studies have investigated changes in the expression level of Ugt isoforms in mice, involving disease, chemicals, or genetically engineered model animals (Buckley and Klaassen, 2007; 2009a,b; Lu et al., 2010). Furthermore, knocking out the Ugt2 locus in mice has been reported and characterized (Fay et al., 2015); however, characterization of mouse Ugt isoforms, except 1a6 (Uchihashi et al., 2012, 2013), has not been reported, and it would be of great interest to understand the function of mouse Ugts, especially the Ugt2b subfamily.
In this study, with reference to a mouse genome database, we have cloned cDNAs encoding all Ugt isoforms belonging to the Ugt2b subfamily and established their expression system. We found that Ugt2b36 is the predominant morphine Ugt in mice.
Materials and Methods
Chemicals.
Morphine hydrochloride was purchased from Takeda Pharmaceutical Industry, Co., Ltd. (Osaka, Japan). 4-Methylumbelliferone (4-MU), estradiol 3β-D-glucuronide (E3G), estradiol 17β-D-glucuronide (E17G), and alamethicin were purchased from Sigma-Aldrich (St. Louis, MO). Chloramphenicol, UDP-glucuronic acid (UDPGA), trisodium salt, and 4-MU-β-D-glucuronide were obtained from Nakalai Tesque (Kyoto, Japan). 17β-Estradiol was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Chloramphenicol glucuronide (Sigma-Aldrich) was a gift from Dr. Yoshihisa Kato (Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Japan). M-3-G was synthesized by a method described previously (Yoshimura et al., 1968). All other reagents were of the highest grade commercially available.
Animals.
Animal experiments in this study were conducted according to the Ethics Committee for Animal Experiments of Kyushu University. Male FVB mice (7 weeks old) were obtained from CLEA Japan (Tokyo, Japan) and maintained for 1 week with free access to water and a suitable diet under a 7 AM to 7 PM light/dark cycle. For isolation of total RNA, liver tissue was quickly cut into small fragments, immediately immersed in liquid nitrogen, and stored at −80°C until required. For preparation of microsomes, the liver was removed and perfused with ice-cold saline, and the microsomes were prepared by differential centrifugation as described previously (Oguri et al., 1996). The resulting microsomes were resuspended in 0.25 M sucrose, frozen in liquid nitrogen, and stored at −80°C.
RNA Extraction and Reverse Transcription Polymerase Chain Reaction (RT-PCR).
Total RNA was extracted from male mouse-liver tissue using an RNeasy Mini Kit (QIAGEN, Hilden, Germany), and the RNA was quantified by absorbance at 260 nm. cDNA was prepared from 250 μg of total RNA in 15 μl of total volume using a Primescript RT Reagent Kit (Takara Bio, Shiga, Japan).
Amplification and Cloning of cDNAs Encoding Mouse Ugt Isoforms.
The PCR primers were designed to amplify cDNAs encoding mouse Ugt isoforms based on the genome database. The GeneBank accession numbers of the reference sequences are as follows: Ugt1a1 (AAH93516), Ugt2a3 (AAH25795), Ugt2b1 (AAH27200), Ugt2b5 (AAH28262) (Kimura and Owens, 1987), Ugt2b34 (EDL37970), Ugt2b35 (AAI13790), Ugt2b36 (AAI41233), Ugt2b37 (AAH29487), and Ugt2b38 (AAH69923). The primers used are listed in Table 1, and the PCR conditions are described (Supplemental Tables 1 and 2). The PCR products were extracted from agarose gels and purified using a QIAquick Gel Extraction Kit (QIAGEN). Purified PCR products with restriction sites at both ends were digested in NotI and XhoI and then cloned into pFastBac1 restricted with the same enzymes. The sequencing reaction was carried out using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Waltham, MA). Then, cDNA sequences were confirmed by an ABI 3130xl Genetic Analyzer (Applied Biosystems, Inc., Carlsbad, CA). There were only nonsynonymous mutations in Ugt1a1 (T915G) and Ugt2b5 (G693A) on the basis of the reference sequences (Kimura and Owens, 1987; Kong et al., 1993). The sequences of the other Ugt-cDNAs amplified were identical to each reference. Therefore, all the Ugt cDNAs amplified in this study were regarded as wild-type.
Primers used for amplification of Ugt cDNA from mouse liver
Each Ugt cDNA was amplified using the oligo-dT primed cDNA as a template that was reverse-transcribed from mouse liver total-RNA (first-round PCR). Nested PCR was carried out using two sets of primer pairs. For the first-round PCR, a primer pair without a restriction site was used. For the second-round PCR (nested PCR), each Ugt cDNA was amplified with a primer pair with a restriction site. Single and double underlines represent NotI and XhoI sites, respectively.
Expression of Recombinant Ugts.
Bacmid of the recombinant Ugts was prepared from Escherichia coli DH10Bac strain (Life Technologies) transformed with recombinant pFastBac1. Recombinant baculovirus was prepared using the Bac-to-Bac Baculovirus Expression System (Life Technologies) as described previously (Ishii et al., 2014). The amplification of baculovirus was usually repeated two to three times to obtain the virus with a titer of more than 1.0 × 107 plaque-forming units/ml. NucleoSpin Blood (Macherey-Nagel, Düren, Germany) was used for purification of the viral DNA, and their titers were determined using a BacPAK qPCR Kit (Clontech, Mountain View, CA). For expression of recombinant enzymes, Sf9 cells (2.0 × 106 cells/ml, 200 ml) were infected with recombinant baculovirus with a multiplicity of infection (MOI = 0.01 PFU/cell) in an Erlenmeyer flask (Corning, NY) and harvested 40 hours after infection by low-speed centrifugation. Microsomes were prepared from the transfected cells as described by Ishii et al. (2014). Preparation of recombinant UGT2B7 baculovirus has been described previously (Jin et al., 1993; Miyauchi et al., 2015). The UGT2B7 baculosomes used in this study were prepared similarly to the mouse Ugt2b isoforms as described here.
Immunoblotting.
Microsomes separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were electroblotted on a polyvinyldene difluoride (PVDF) membrane (Millipore, Bedford, MA). Each of the Ugts was detected using goat anti-mouse low-pI formed UGT antibody (Mackenzie et al., 1984) or rabbit anti-UGT2B antibody (Kasai et al., 2004). As secondary antibodies, horseradish peroxidase (HRP)-rabbit anti-goat IgG (MP Biomedicals, Santa Ana, CA) and HRP-donkey anti-rabbit IgG (GE Healthcare, Little Chalfont, UK) were used. Clarity Western ECL substrate (Bio-Rad, Hercules, CA) was used as the substrate of HRP, and the chemiluminescence emitted was analyzed using a ChemiDoc MP System (Bio-Rad).
Endoglycosidase H Treatment.
Treatment of baculosomes with endoglycoside H (EndoH) was performed as described previously (Nakamura et al., 2016).
Glucuronidation Assay.
The glucuronidation of morphine at the 3-hydroxyl group was carried out by a method reported previously (Nurrochmad et al., 2010) with slight modifications. Microsomes were pretreated with alamethicin (50 μg/mg protein) on ice for 30 minutes. The method used 200 μg of protein in a 0.3-ml reaction except for kinetic analysis (100 μg of protein). For mouse liver microsomes (MLM), 100 μg of protein was used. For the kinetic study, the substrate concentration ranged from 0.1 to 6 mM. Incubation was performed at 37°C for 120 minutes, but a 90-minute incubation was used in the kinetic analysis. M-3-G was detected by high-performance liquid chromatography (HPLC). A D-2000 Elite HPLC system equipped with an automatic sampler (model L-2200), pump (L-2130), column oven (L-2300), and fluorescence detector (L-2485) (Hitachi High Technologies Co., Tokyo, Japan) were used for the chromatographic analysis. Separation was achieved using a Nova-Pak C18 column (4 μm, 8 × 100 mm; Waters, Milford, MA) housed in a column heater that was set at 25°C. The mobile phase was a mixture of eluent A (20 mM sodium phosphate, pH 2.2) and eluent B (eluent A: acetonitrile = 80:20) at a ratio of 90:10, and this condition was maintained for 35 minutes. The flow rate was set at 0.8 ml/min. M-3-G was quantified by fluorescence detection (excitation wavelength 210 nm and emission wavelength 350 nm). Under these conditions, the retention time of M-3-G was 13.6 minutes.
The 4-MU glucuronidation activity and glucuronidation of 17β-estradiol at the 3- and 17-hydroxyl groups were determined by the method described previously (Nishimura et al., 2007) with slight modifications. Unless otherwise stated, the incubation mixture consisted of 50 mM potassium phosphate buffer (pH 7.4), 5 mM MgCl2, 2 mM UDPGA, 100 μg each recombinant Ugt baculosomes, and 200 μM 4-MU or 17β-estradiol (1 mM or 10 μM). Substrates were dissolved in methanol, and the final concentration of methanol in the incubation mixture was 0.5%. Incubation was performed at 37°C for 1 hour, and the glucuronides were detected by HPLC. The eluted solution of 4-MU was prepared by mixing eluent A (20 mM sodium phosphate, pH 2.2) and eluent B (acetonitrile); the concentration of acetonitrile was 20% for 9.9 minutes and then increased to 50% for 0.1 minute, held at 50% for 3 minutes, and finally reduced to 20% and held there for 7 minutes. The flow rate was set at 0.8 ml/min. 4-MU-glucuronide was quantified by fluorescence detection (excitation 315 nm, emission 375 nm). Under these conditions, the retention time of 4-MU-glucuronide was 5.1 minutes. For detection of 17β-estradiol-3-glucuronide (E3G) and 17-glucuronide (E17G), a solution mixing eluent A (20 mM sodium phosphate, pH 2.2) and eluent B (acetonitrile) was prepared; the concentration of acetonitrile was 35% for 19.9 minutes, then increased to 60% for 0.1 minute, held at 60% for 5 minutes, and finally reduced to 35% and held there for 5 minutes. Then the column was equilibrated with the initial condition before analysis of the new sample. The flow rate was set at 1 ml/min. E3G and E17G were quantified by fluorescence detection (excitation 210 nm, emission 300 nm). Under these conditions, the retention times of E3G and E17G were 3.9 and 4.6 minutes, respectively. In kinetic assays for estradiol, the substrate concentration was varied, ranging from 1 to 100 μM. For MLM and Ugt1a1-expressing microsomes, 100 μg of protein was used. Incubation was performed at 37°C for 1 hour.
Glucuronidation of chloramphenicol was assayed according to Kato et al. (2000) with slight modifications. Unless otherwise stated, the incubation mixture consisted of 50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl2, 2 mM UDPGA, each recombinant Ugt baculosome (300 μg protein), and 5 mM chloramphenicol. Microsomes were pretreated with alamethicin as described already. Chloramphenicol was dissolved in methanol, and the final concentration of methanol in the incubation mixture was 0.5%. Incubation was performed at 37°C for 2 hours. The chloramphenicol glucuronide produced was quantified by HPLC coupled to a UV detector (model L-2400) (Hitachi High Technologies) at 278 nm. The eluted solution of chloramphenicol was prepared by mixing eluent A (20 mM sodium phosphate, pH 2.2) and eluent B (acetonitrile); the concentration of acetonitrile was 20% for 9.9 minutes, then increased to 50% for 0.1 minute, held at 50% for 7 minutes, and finally reduced to 20% and held there for 3 minutes. The flow rate was set at 0.8 ml/min, and the retention time of chloramphenicol glucuronide under these conditions was 11.6 minutes.
Construction of a Phylogenetic Tree of Mouse Ugts and the UGTs in Other Species.
The amino acid sequences of mouse Ugts examined in this study were compared with those of human UGT amino acids. In addition, rat (Rattu norvegicus) UGT2B1 (Mackenzie, 1987) and guinea pig (Cavia porcellus) UGT2B21 (Ishii et al., 2001), which are known to glucuronidate morphine, were used for comparison of the amino acids. The following GenBank accession numbers were used: human UGT1A1 (AAI28415) (Ritter et al., 1991), human UGT2A3 (AAI30534) (Court et al., 2008), human UGT2B7 (AAH30974) (Ritter et al., 1990), human UGT2B10 (AAI13650) (Jin et al., 1993), human UGT2B17 (EAW55622) (Beaulieu et al., 1996), human UGT2B28 (NP_001064) (Lévesque et al., 2001), rat UGT2B1 (NP_775417), and guinea pig UGT2B21 (BAB82476). A distance-based neighbor-joining phylogenetic tree was generated (Saitou and Nei, 1987). The reliability of the phylogeny was tested by bootstrap analysis with 1000 replicates. The bootstrap values are indicated in the neighbor-joining consensus tree, and the phylogenetic tree was constructed by ClustalW (http://clustalw.ddbj.nig.ac.jp/).
Data Analysis.
Data analysis for the kinetics was carried out using GraphPad Prism 5.04 software (GraphPad Software, La Jolla, CA). Kinetic data were fitted to a Michaelis-Menten model using eq. 1:
(1)where V is the reaction rate, Vmax is the maximum enzyme velocity, S is the substrate concentration, and Km is the substrate concentration giving half Vmax.
In the case of UGT2B7, the kinetic data were fitted to a sigmoidal model defined by eq. 2:
(2)where S50 is the substrate concentration giving half Vmax, and n is the Hill coefficient.
Other Method.
Protein concentrations were determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard.
Results
The substrate specificities of the Ugt2b subfamily mouse isoforms were studied. cDNAs encoding all the mouse Ugt2b subfamily isoforms were amplified by nested RT-PCR from mouse liver mRNA. Ugt1a1 and 2a3 cDNA were also cloned and expressed by the baculovirus-Sf9 system. Their microsomes were prepared, and expression of these Ugt proteins was confirmed by Western blotting using an antibody toward a low-pI mouse Ugt (Fig. 1A). Although we carried out the expression of Ugts under the same conditions, the intensity of bands varied, possibly owing to differences in immunoreactivity between isoforms. Thus, we compared the Ugt content in microsomes by use of an antipeptide antibody that recognizes the same epitope in all the mouse Ugts examined in this study (Fig. 1B). The antibody was originally produced against a common epitope (KWIPQNDLLGHPK; residues 355–367 of UGT2B7) of human UGT2B isoforms (Supplemental Table 2) (Kasai et al., 2004). The corresponding sequence of Ugt2b1 is completely identical to this epitope but differs to that of Ugt2b5 by a single substitution at residue 358 from Ile (Ugt2b1) to Leu (Ugt2b5). Despite this difference, the band intensities of Ugt2b1 and Ugt2b5 in Fig. 1B are comparable. The Ugt2b5 epitope is shared among the other mouse Ugt2b isoforms and Ugt1a1 except for Ugt2b1 and Ugt2b34. Ugt2b34 shares the sequence of Ugt2b1 but for a single amino acid difference from Pro (Ugt2b1) to Ser (Ugt2b34) at residue 369. Further, Ugt2a3 shares the epitope sequence of Ugt2b1 except for a single amino acid substitution at residue 355 from Lys (Ugt2b1) to Asn (Ugt2a3). Hence, it seems reasonable to suppose that the antibody can recognize all mouse Ugt2b subfamily isoforms, Ugt1a1 and Ugt2a3, examined in this study to the same degree. With the exception of Ugt2b35, all mouse Ugt2b forms, as well as Ugt1a1 and Ugt2a3, appeared as a single band on SDS-PAGE (Fig. 1A). In case of Ugt2b35, the microsomes were treated with EndoH before SDS-PAGE was performed (Fig. 1B). Thus, it was possible to normalize the relative Ugt content in the microsomes expressing each mouse Ugt isoform when evaluating their catalytic property.
Immunoblots of the expression of Ugt1a1, 2a3, and all the Ugt2b subfamily isoforms in Sf-9 cells by an antibody toward a mouse low-pI form Ugt (A) and anti-UGT2B antibody (B). To obtain microsomes expressing each mouse Ugt, Sf-9 cells were transfected with recombinant baculovirus for the corresponding Ugt isoform. (A) The lane labeled shows the microsomal samples of the transfected cells. Baculosomes (5 μg of protein) from Ugt-expressing cells were electrophoresed (10% SDS-PAGE) and electrophoretically transferred to PVDF membrane. Then Ugts were detected by anti-mouse low-pI form UGT antibody (Mackenzie et al., 1984) as the primary antibody. Pooled MLM (n = 5, 10 μg of protein) was used as a positive control. Mock represents microsomes obtained from Sf9 cells transfected with control baculovirus. (B) The lane labeled shows the microsomal samples of the transfected cells. Baculosomes (10 µg of protein) from Ugt2b5-expressing Sf-9 cells were electrophoresed (7.5% SDS-PAGE). For other baculosomes, amounts of protein were used that were comparable to the Ugt content in the Ugt2b5. For Ugt2b35, the sample was treated with EndoH according to Nakamura et al. (2016) before being subjected to the SDS-PAGE. Then those were electrophoretically transferred to PVDF membrane. Ugts were detected by anti-UGT2B antibody (Kasai et al., 2004) as the primary antibody. Details are described in Materials and Methods.
Using 4-MU as a substrate, all the Ugt isoforms examined exhibited activity; however, substrate preferences differed among isoforms. With 4-MU, very high activity was observed for Ugt2b5, Ugt2b34, 2b36, and Ugt1a1, whereas Ugt2b1 and 2b35 showed intermediate activity, and Ugt2b37, 2b38, and 2a3 exhibited much lower activity (Fig. 2). Glucuronidation of steroids is typical of UGT isoforms. We used 17β-estradiol, which possesses both an aromatic 3-hydroxyl group and a 17β-hydroxyl group as a substrate. Keeping the substrate concentration at 1 mM, we examined which isoform(s) is(are) mainly involved in the glucuronidation of this steroid. Of the Ugt isoforms examined in this study, Ugt1a1 is the main catalyst for glucuronidation at the 3-hydroxyl group (Fig. 3). Although the other isoforms showed measurable activities, these were at far lower levels than that of Ugt1a1. In addition, their specificities for the 17β-hydroxyl group were significantly different (Fig. 3). At 1 mM 17β-estradiol, Ugt2b5 exhibited high activity, followed by Ugt2b1, which also exhibited considerable activity, whereas Ugt2b34, 2b35, 2b38, and 2a3 exhibited lower activity; however, the activities of Ugt2b36, 2b37, and 1a1 were below the detection limit. Also, at 10 μM 17β-estradiol, Ugt2b34, 2b35, 2b38, and 2a3 did not have any substantial activity with regard to the 17-hydroxyl group. Ugt2b5 exhibited high activity, followed by Ugt2b1 at a low substrate condition as well.
Comparison of the glucuronidation activity of each mouse Ugt isoform toward 4-methylumbelliferone. Baculosomes (100 µg of protein) from each Ugt single-expressing Sf-9 cell were used. The concentrations of 4-MU and UDPGA were fixed at 200 μM and 2 mM, respectively. Each bar represents the mean ± S.D. of a triplicate assay. The activity was calculated based on the relative Ugt level and then normalized to the Ugt level by Ugt2b36-expressing microsomes. Details are described in Materials and Methods.
Comparison of the glucuronidation activity of each mouse Ugt isoform toward 17β-estradiol. Baculosomes (100 µg of protein) from each Ugt single-expressing Sf-9 cell were used. The concentrations of 17β-estradiol were fixed at 1 mM (A, B) and 10 μM (C), respectively. UDPGA was fixed at 2 mM. E3G and E17G formed were simultaneously determined by HPLC. (A) E3G and E17G activities are shown in the same scale. (B) E17G activity in (A) is shown in an enlarged scale. (C) E17G activity at the low substrate (10 μM) is shown. Each bar represents the mean ± S.D. of a triplicate assay. The activity was normalized as described in the legend to Fig. 2. Details are described in Materials and Methods.
For glucuronidation of morphine, Ugt2b36 exclusively catalyzed this reaction, whereas Ugt2b34, 2b35, and 2b37 exhibited very low activity (Fig. 4A). Also, no activity was detected for Ugt2b1, 2b5, 2b38, 1a1, and 2a3. Under the assay conditions used, Ugt2b36 catalyzed only glucuronidation at the 3-hydroxyl group of morphine (M-3-G formation) but not at the 6-hydroxyl group (M-6-G formation). Glucuronidation of chloramphenicol was catalyzed only by Ugt2b1 and 2b5 (Fig. 4B), with Ugt2b5 exhibiting greater activity. The other isoforms examined in this study showed no substantial activity with regard to chloramphenicol.
Comparison of the glucuronidation activity of each mouse Ugt isoform toward morphine and chloramphenicol. Baculosomes (200 µg of protein) from each Ugt single-expressing Sf-9 cell were used. The concentrations of morphine and chloramphenicol used were 5 mM. The UDPGA concentration was fixed at 2 mM. Formation of M-3-G (A) and chloramphenicol glucuronide (B) were determined by HPLC. Each bar represents the mean ± S.D. of a triplicate assay. The activity was normalized as described in the legend to Fig. 2. Details are described in Materials and Methods.
In the kinetic study, Ugt2b36 microsome-catalyzed morphine glucuronidation was fitted to a Michaelis-Menten equation similar to that of MLM (Fig. 5A; Table 2), whereas human UGT2B7, the predominant isoform participating in hepatic morphine glucuronidation, exhibited sigmoidal kinetics (Fig. 5C; Table 2). Again, the Vmax of MLM was greater than that of Ugt2b36, but they cannot be simply compared because the Ugt2b36 content was not normalized between MLM and the Ugt2b36-expressing microsomes; however, the Km values of Ugt2b36 and MLM for morphine were comparable. The Km of Ugt2b36 was lower than that of UGT2B7, whereas the intrinsic clearance of Ugt2b36 was greater than that of UGT2B7. It is reasonable to suppose that Ugt2b36 is a representative isoform involved in morphine glucuronidation in the mouse liver. On the basis of the expressed enzymes in this study, mouse Ugt2b36 exhibits greater activity than human UGT2B7. Regarding the kinetics of estradiol 3-glucuronidation activity, Ugt1a1 and MLM were examined (Fig. 6; Table 3). Again, the Vmax of MLM was greater than that of Ugt1a1, but it cannot be simply compared because the Ugt1a1 content was not normalized between MLM and the Ugt1a1-expressing microsomes; however, the Km of Ugt1a1 for the 3-hydroxyl group of estradiol was lower than that of MLM. This finding suggested that glucuronidation at the 3-hydroxyl group of estradiol was catalyzed by Ugt1a1.
Comparison of Michaels-Menten plots of morphine-3-glucuronidation activity among MLM and Sf-9 microsomes expressing mouse Ugt2b36 and human UGT2B7. Morphine-3-glucuronidation activity was compared among MLM (A), microsomes expressing Ugt2b36 (B), and UGT2B7 (C). Baculosomes (100 µg of protein) from each Ugt-expressing Sf-9 cell or MLM were used. Each plot represents the mean ± S.D. of a triplicate assay. Data were fitted to a Michaelis-Menten equation or a sigmoidal equation. The morphine concentrations ranged from 0.1 to 6 mM. The kinetic parameters obtained are listed in Table 2.
Kinetic parameters for morphine-3-glucuronidation catalyzed by MLM, microsomes of Sf-9-cells expressing Ugt2b36 and UGT2B7: kinetics by varying the morphine concentration
Comparison of Michaels-Menten plots of estradiol-3-glucuronidation activity between MLM and Sf-9 microsomes expressing mouse Ugt1a1. E3G activity was compared between MLM (A) and microsomes expressing Ugt1a1 (B). MLM (100 µg of protein) or baculosomes (100 µg of protein) from Ugt1a1-expressing Sf-9 cell were used. Each plot represents the mean ± S.D. of a triplicate assay. Data were fitted to a Michaelis-Menten equation. The 17β-estradiol concentrations ranged from 1 to 100 μM. The kinetic parameters obtained are listed in Table 3.
Kinetic parameters for estradiol-3-glucuronidation catalyzed by MLM and microsomes of Sf-9-cells expressing Ugt1a1: kinetics by varying 17β-estradiol
Discussion
A number of reports have described changes in the level of mouse Ugt isoforms, depending on gender, disease, or chemicals (Buckley and Klaassen, 2007, 2009a,b). Although there is a study of the substrate specificity of mouse Ugt1a6a and 1a6b (Uchihashi et al., 2012), the substrate specificities of all mouse Ugts, except Ugt1a6, remain to be clarified. In this study, we comprehensively characterized the substrate specificity of Ugt1a1, Ugt2a3, and all the Ugt2b subfamily isoforms expressed in the mouse liver. We demonstrated for the first time that Ugt2b36 is the predominant Ugt isoform involved in morphine glucuronidation in the mouse liver. Anti-mouse low-pI from UGT antibody (Mackenzie et al., 1984) recognized all the mouse Ugts examined in this study. Of these Ugts, Ugt2b36 was strongly recognized by this antibody. Since the purified low-pI form mouse Ugt catalyzed morphine glucuronidation (Mackenzie et al., 1984), it is reasonable to suppose that the purified preparation contained Ugt2b36. Although the infection of Sf9 with baculovirus encoding each Ugt isoform was carried out with the same MOI, the intensity of the Ugt bands varied (Fig. 1A). Therefore, we evaluated relative Ugt levels with an anti-UGT2B antibody that recognizes all the mouse Ugt2b subfamily isoforms, Ugt1a1, and Ugt2a3 to a comparable extent (Fig. 1B). This enabled normalization of the Ugt activity on the basis of relative Ugt protein levels. The level of each recombinant Ugt was within the range of the Ugt level observed in the MLM. Based on relative UGT levels, the glucuronidation of morphine was catalyzed almost exclusively by Ugt2b36. Following Ugt2b36, morphine glucuronidation activity was exhibited by Ugt2b34, 2b35, and 2b37, but this activity was quite low compared with Ugt2b36. Thus, it is reasonable to conclude that Ugt2b36 is the predominant isoform participating in morphine glucuronidation in the mouse liver. On the basis of the phylogenetic tree of the selected UGT isoforms constructed by the Neighbor-Joining-method (Supplemental Fig. 1), mouse Ugt2b36 is suggested to branch from a common ancestor of guinea pig UGT2B21, another morphine-conjugating UGT. Also, mouse Ugt2b1 has been considered an ortholog of rat UGT2B1 (Mackenzie, 1987; Mackenzie et al., 2005), which catalyzes morphine glucuronidation (Pritchard et al., 1994). Furthermore, this is also supported by the phylogenetic tree in this study (Supplemental Fig. 1). It is reasonable to suppose that mouse Ugt2b1 and rat UGT2B1 are orthologs; however, mouse Ugt2b1 did not show any substantial activity toward morphine (Fig. 4). Therefore, care should be taken in discussing the function of Ugt isoforms, especially a candidate ortholog such as mouse Ugt2b1, before characterizing function. For human UGT2B7, it has been shown that Asp99 is important for the binding of morphine (Coffman et al., 2003). In rat UGT2B1, the residue is conserved as Asp100, but it was substituted to Glu100 in mouse Ugt2b1. Of the other UGT isoforms catalyzing morphine glucuronidation, guinea pig UGT2B21 (Ishii et al., 2001) and mouse Ugt2b36 have Thr98 and Glu100 at the corresponding position, respectively. Although mouse Ugt2b36 carries the acidic amino acid Glu instead of Asp, it is unlikely to be definitive for the catalysis of morphine glucuronidation. The amino acid identity of mouse Ugt2b1 and rat UGT2B1 is 86%, and there are 37 amino acid differences between them. Therefore, it is assumed that these differences resulted in the different substrate specificity of mouse Ugt2b1 from rat UGT2B1. The amino acid sequences in the N-terminal half of UGTs have been considered important for substrate binding, but the details remain to be clarified.
Data from the kinetic analysis showed that the Km of mouse Ugt2b36 is lower than that of UGT2B7, which is a predominant UGT catalyzing morphine glucuronidation in human liver. Thus, it seems that Ugt2b36 exhibits a greater affinity for morphine than UGT2B7. Furthermore, the Vmax of Ugt2b36 was approximately five times greater than that of UGT2B7. Thus, the intrinsic clearance of Ugt2b36 was 17 times greater than that of UGT2B7, although the expression level of UGT2B7 in Sf9 microsomes used in this study has not been accurately determined. The Km of UGT2B7 in COS cell microsomes (Takeda et al., 2005) was comparable to that of Ugt2b36 in this study, suggesting that Ugt2b36 exhibits greater glucuronidation activity toward morphine than UGT2B7; however, since there are differences in the expression systems, we cannot conclude that there are definitive differences. Also, there is a report that the relative morphine-glucuronidation activities of human liver microsomes and MLM were 1:64 (Higashi et al., 2014). Therefore, this report strongly suggests that both Ugt2b36 and UGT2B7 are predominant UGT isoforms in mouse and human livers, respectively. Mouse Ugt2b36 is expressed mainly in the liver, but the level is quite low in other tissues (Buckley and Klaassen, 2007). The hepatic ontogeny of Ugt mRNA of mice has been reported (Lu et al., 2013). Mouse Ugt2b36 is expressed as one of the major isoforms in the fetus and then reduced at birth, but it is increased to be a predominant isoform again in the liver at puberty and in adulthood (Lu et al., 2013). Taking these findings into consideration, mouse Ugt2b36 is a predominant isoform participating in morphine glucuronidation in the liver.
There were multiple bands in the Ugt2b35 preparation examined in this study, whereas the other Ugts examined in this study were expressed as an apparent single band (Fig. 1A). Therefore, we carried out the EndoH digestion on Ugt2b35 (Supplemental Fig. 2) to determine the cause of this multiple banding. It is evident that Ugt2b35 in the preparation is glycosylated. There are three potential N-glycosylation-sites in Ugt2b35. The lower and higher bands (Fig. 1A) are probably differently glycosylated forms. Ugt2b35 exhibited considerable activity toward the universal substrate 4-MU. Since the Ugt2b35 preparation used in this study consisted of a relatively large amount of the lesser glycosylated form, we cannot exclude the possibility that the multiple-glycosylated form shows greater activity. In addition, glycosylation of Ugt2b36 was not obvious (Supplemental Fig. 2). We carried out the expression of Ugt carefully, and most Ugt isoforms except Ugt2b35 are expressed as a single band on SDS-PAGE gels. A previous report suggested that transfection with high amounts of virus results in the production of inactive UGT (Zhang et al., 2012). In consideration of this information, we used low MOI and shorter durations of transfection so that it is reasonable to suppose that the Ugt isoforms examined in this study are expressed properly as demonstrated by their substantial glucuronidation activity toward the universal substrate 4-MU. Again, Ugt2b37 also exhibits substantial activity toward 4-MU. Better substrates for Ugt2b35 and 2b37 are yet to be identified.
Regarding estradiol glucuronidation, at the 3-hydroxyl group, it is seen as a marker activity of human UGT1A1 (Senafi et al., 1994; Court 2005). Similarly, in mice, Ugt1a1 exhibits a high level of activity at the 3-hydroxyl position of estradiol but not at the 17-hydroxyl group (Figs. 3 and 6). Glucuronidation at the 17-hydroxyl group was also catalyzed by Ugt2b1, 2b5, 2a3, 2b34, 2b35, and 2b38 (Fig. 3); however, Ugt2a3, 2b34, 2b35, and 2b38 did not exhibit any significant activity at the lower concentration (10 μM), which suggests that Ugt2b5 and 2b1 are important under physiologic conditions. In humans, UGT2B15 and 2B17 are predominant catalysts of 17β-hydroxysteroid glucuronidation (Beaulieu et al., 1996). Therefore, it is reasonable to suppose that, in mice, Ugt2b1 and 2b5 are functional orthologs of human UGT2B15 and 2B17. Also, mouse Ugt2b1 and 2b5 are the major isoforms expressed in adults (Lu et al., 2013). Therefore, mouse Ugt2b1 and 2b5 seem to play important roles in the glucuronidation of 17β-hydroxysteroids in the adult.
Ugt2b1 and 2b5 exclusively catalyze chloramphenicol glucuronidation (Fig. 4B), whereas the other isoforms examined did not. Chloramphenicol is known to be a substrate for rat UGT2B1 (Mackenzie, 1987). Therefore, the substrate specificity of Ugt2b1 was somewhat conserved for chloramphenicol glucuronidation. Also, a high level of Ugt2b5 is expressed in the fetus and in the newborn, and expression of Ugt2b1 is very low at the fetal and newborn stages (Lu et al., 2013); however, both Ugt2b1 and 2b5 are significantly expressed in the adult mouse liver (Lu et al., 2013). Although both Ugt2b1 and 2b5 exhibit similar substrate specificity (Figs. 3 and 4B), further studies are necessary to clarify why they are differently regulated during the developmental stages.
Although the catalytic properties of other substrates are also important, this study provides a comprehensive characterization of Ugt2b subfamily isoforms in mice using typical substrates. The data obtained suggest that Ugt2b36 is a predominant isoform involved in morphine glucuronidation in mouse liver. Furthermore, typical isoforms involved in estradiol and chloramphenicol glucuronidation were identified. Further studies are necessary to interpret the stress- and chemical-induced changes in glucuronidation activity and the expression levels of Ugt isoforms in mouse liver.
Acknowledgments
The authors thank the Research Support Center, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, for technical support.
Authorship Contributions
Participated in research design: Kurita, Miyauchi, Mackenzie, Yamada, Ishii.
Conducted experiments: Kurita, Miyauchi, Ishii.
Contributed new reagents or analytical tools: Kurita, Miyauchi, Ikushiro.
Performed data analysis: Kurita, Miyauchi, Ishii.
Wrote or contributed the writing of the manuscript: Kurita, Mackenzie, Ishii.
Footnotes
- Received January 25, 2017.
- Accepted February 14, 2017.
This study was supported in part by Grants-in-Aid for Scientific Research (B) [Grant 25293039] and Challenging Exploratory Research [Grant 16K15148] from the Japanese Society for the Promotion of Science to Y.I.
This work was previously presented in part at the following meetings: International Workshop on Conjugation 2016 Vancouver, Canada, June 2016 (Ishii Y, Kurita A, Miyauchi Y, Mackenzie PI, the late Yamada H, Comprehensive characterization of mouse UDP-glucuronosyltransferases belonging to Ugt2b subfamily: Identification of Ugt2b36 as a predominant morphine UGT in mouse); 135th Annual Meeting of the Pharmaceutical Society of Japan, Kobe, Japan, March 2015 (Kurita A, Miyauchi Y, Mackenzie PI, Yamada H, Ishii Y, Characterization of Ugt2b36 as a morphine UDP-glucuronosyltransferase in mouse liver); and 30th Annual Meeting of the Japanese Society for the Study of Xenobiotics, Tokyo, Japan, November 2015 (Kurita A, Miyauchi Y, Mackenzie PI, Yamada H, Ishii Y, Comprehensive characterization of mouse hepatic UDP-glucuronosyltransferases belonging to the 2b subfamily involved in morphine and estradiol glucuronidation).
↵
This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- 4-MU
- 4-methylumbelliferone
- E3G
- estradiol-3-glucuronide
- E17G
- estradiol-17-glucuronide
- EndoH
- endoglycosidase H
- HRP
- horseradish peroxidase
- M-3-G
- morphine-3-glucuronide
- M-6-G
- morphine-6-glucuronide
- MLM
- mouse liver microsome
- MOI
- multiplicity of infection
- P450
- cytochrome P450
- PCR
- polymerase chain reaction
- PVDF
- polyvinyldene difluoride
- SDS-PAGE
- SDS-polyacrylamide gel electrophoresis
- UDPGA
- UDP-glucuronic acid
- Ugt and UGT
- UDP-glucuronosyltransferase
- Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics
References
In this issue
Ugt2b36 as a Mouse Morphine Ugt
