Materials and Methods

Materials.

Human stomach polyA⁺ RNA was obtained form OriGene Technologies (Rockville, MD). HTFMC and HTFMC glucuronide were purchased from Aldrich Chemical Co. (Milwaukee, WI) and Sigma-Aldrich (St. Louis, MO), respectively. E2 was purchased from Wako Pure Chemicals (Osaka, Japan), and 3-O- and 17-O-glucuronide were from Sigma-Aldrich.

Plasmid Construction.

Oligo(dT) primed cDNA was synthesized from human stomach polyA⁺ RNA using a SuperScript First-strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The cDNA encoding wild-type UGT1A10 was amplified from the single-stranded stomach cDNA by theattB adaptor PCR, and cloned into the pDONR201 vector (Invitrogen) by the Gateway cloning technology (Walhout et al., 2000). Briefly, the UGT1A10 cDNA was first amplified by 10-cycle PCR using the gene specific primers (5′-AAAAAGCAGGCTGCAGTTCTCTCATGGCTCGC-3′ and 5′-AGAAAGCTGGGTCTCAATGGGTCTTGGATTTGTGGG-3′). The 12-base pairs partial attB sequences at the 5-end are underlined. Then an aliquot of the reaction mixture was subjected to a second round of PCR (25 cycles) using the following attB adaptor primers; 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′ and 5′- GGGGACCACTTTGTACAAGAAAGCTGGGT-3′. The resulting attB-flanked product was cloned into pDONR201 vector using the Gateway BP reaction, a recombination between attB site andattP site. Mutations were introduced into the wild-type UGT1A10 cDNA clone in pDONR201, using a QuikChange multi site-directed mutagenesis kit (Stratagene, La Jolla, CA). Oligonucleotide primers to construct T202I (605C > T) and M59I (177G > A) variants by PCR were 5′-phospho-GGTGGTTGTAGTCATACCAGAGGTGAGTTGG-3′ and 5′-phospho-GTTCTCAGATGCCATGATTTTCAAGGAGAGAG-3′, respectively. The single base changes are underlined. To ensure no mistakes had been introduced during the amplification process, all the plasmid constructs were verified by DNA sequencing of both strands. Subcloning of each UGT1A10 fragment from pDONR201 into pcDNA-DEST40 was performed by the Gateway LR reaction, a recombination betweenattL site and attR site.

Expression of Wild-Type and Variant UGT1A10s in COS-1 Cells.

COS-1 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The day before transfection, COS-1 cells were plated in 100-mm culture dishes at a density of 5.5 × 10⁴ cells/cm². On the following day, the culture medium was replaced with 8 ml of Opti-MEM (Invitrogen), and the cells were transfected using a LipofectAMINE 2000 reagent (Invitrogen). Briefly, the plasmid DNA (14 μg in 810 μl of Opti-MEM) and LipofectAMINE 2000 reagent (48 μl in 810 μl of Opti-MEM) were combined, and incubated for 20 min at room temperature. The resulting DNA-LipofectAMINE 2000 complexes were added directly to each dish.

Forty-eight hours after transfection, the COS-1 cells were washed twice with ice-cold phosphate-buffered saline and harvested in 0.25 M sucrose-5 mM HEPES, pH 7.4 (buffered-sucrose). The cell suspensions were disrupted by sonication (three 10-s bursts) using an ultrasonic processor (USP-300; Shimadzu, Kyoto, Japan), followed by centrifugation at 105,000g for 60 min. The resulting pellets were resuspended in the buffered-sucrose and stored at −80°C.

Western Blotting.

Twenty micrograms of the membrane fraction proteins from COS-1 cells were resolved by SDS-polyacrylamide gel electrophoresis (10% gel) and electrophoretically transferred onto polyvinylidene difluoride membranes. Immunochemical detection of each UGT1A10 protein was performed by chemifluorescence using rabbit anti-human UGT1A (diluted at 1:5000; BD Gentest, Woburn, MA) and donkey anti-rabbit Ig coupled to horseradish peroxidase (diluted at 1:2000). Chemifluorescence (enhanced chemiluminescence-plus; Amersham Biosciences, Piscataway, NJ) was detected and quantified using the Typhoon 9400 Variable Mode Imager and ImageQuant analysis Amersham Biosciences Inc. To confirm that the samples were evenly loaded, the blot was subsequently stripped in a stripping buffer (2% SDS, 100 mM 2-mercaptoethanol and 62.5 mM Tris-HCl, pH 6.7) at 50°C for 30 min, and re-probed with a polyclonal anti-calnexin antibody (diluted at 1:10,000; StressGen Biotechnologies, San Diego, CA).

Enzyme Assays.

A 0.2-ml reaction mixture containing 0.1 mg/ml of the membrane fractions, 1 mM uridine diphosphoglucuronic acid (UDPGA), 10 mM magnesium chloride, 0.025 mg/ml alamethicin, and HMTFC (1.3–50 μM) in 50 mM Tris, pH 7.5, was incubated at 37°C for 5 or 10 min. After incubation, the reaction was stopped by the addition of glacial acetic acid/acetonitrile (6:94, 100 μl) and centrifuged at 10,000g for 3 min at 4°C. The HPLC system consisted of Shimadzu LC-10AD pumps, an SIL-10A auto injector, a CTO-10A column oven, an SPD-10AV UV-VIS detector and RF-10A spectrofluorometric detector. An aliquot (50 μl) of the supernatant was injected into a 4.6 × 150 mm C₁₈ HPLC column (5 μm) (Inertsil ODS-80Al; GL Sciences, Tokyo, Japan) and separated at 30°C using a linear gradient. Initial HPLC conditions were 80% of 10% methanol solution (mobile phase A), 10% of methanol (mobile phase B), and 10% of aqueous solution containing 30% acetonitrile and 1 mM perchloric acid (mobile phase C). Ratio of the mobile phase B was increased to 90% over 15 min, whereas the ratio of mobile phase C remained constant during the course of the HPLC run. The HPLC flow rate was 1 ml per minute. The glucuronide was detected by its absorbance at 325 nm and quantified using HTFMC glucuronide as a standard. The limit of quantification for HTFMC glucuronide was 0.15 nmol/ml, which corresponded to the enzyme activity of 0.2 nmol/min/mg of protein. The HTFMC glucuronide production was linear for at least 60 min using an enzyme concentration up to 0.5 mg/ml.

E2 glucuronidation activities were assayed as described by Alkharfy and Frye (2002) except that product formation was quantified using the external standard method. The incubation mixture (0.25 ml) consisted of 30 μg of the membrane fraction, 15 μg/ml alamethicin, 1 mM magnesium chloride, 5 mM UDPGA, E2 (0.63–50 μM), and 0.1 M potassium phosphate, pH 7.1. The reaction was initiated by adding UDPGA, and the mixture was incubated at 37°C for 5 or 10 min. The reaction was terminated by the addition of 6% perchloric acid (25 μl) and then clarified by centrifugation at 10,000g for 5 min at 4°C. An aliquot (50 μl) of the resulting supernatant was injected into a 4.6 × 150 mm phenyl HPLC column (5 μm) (Inertsil Ph; GL Sciences). Isocratic elution with acetonitrile and 50 mM ammonium phosphate buffer, pH 3.0 (32:68, v/v), was used at the flow rate of 1 ml/min. The E2 glucuronides were detected by fluorescence (210 nm excitation, 300 nm emission) and quantified using E2 3-O-glucuronide and E2 17-O-glucuronide as standards. The limit of quantification for each E2 glucuronide was 0.1 nmol/ml, which corresponded to an enzyme activity of 0.1 nmol/min/mg of protein. The E2 3-O-glucuronide production was linear for at least for 60 min using an enzyme concentration up to 0.5 mg/ml.

Data Analysis.

Results were analyzed using one-way ANOVA followed by Dunnett's test as a post hoc test. Kinetic parameters were calculated with Prism 3.0 (GraphPad Software, Inc., San Diego, CA) using nonlinear regression of Michaelis-Menten equation, and the results are shown as mean ± S.D. from four-independent preparations of UGT1A10 protein.

Results

Expression of Wild-Type and Variant UGT1A10s in COS-1 Cells.

The wild-type human UGT1A10 cDNA was successfully cloned from stomach cDNA, according to the information on the gastrointestinal expression of UGT1A10 (Strassburg et al., 1997). The DNA sequence of our wild-type UGT1A10 cDNA was identical to that reported by Mojarrabi and Mackenzie (1997) and the current version of GenBank U39550.2 submitted by I. S. Owens, J. W. Cho, N. Gholami, and C. Potter (unpublished data). The sequencing results of the UGT1A10 gene from Japanese subjects (Saeki et al., 2002) were also consistent with the sequence of the wild-type UGT1A10 cDNA cloned here. However, our UGT1A10 cDNA was different from the published UGT1A10 cDNA sequence (GenBank accession number U89508) in several positions resulting in the following amino acid substitutions; the putative signal peptide “RAGWTSPVPLCVCLLLTCGFA” in our deduced amino acid sequence (residues 3–23) was replaced by “PRRVDQPRSFMCVSTADLWLC” in U89508.U89508 contained additional three amino acid substitutions, T40A, H175R, F224L. At present, we have no information as to how these alterations influence the UGT1A10 activities. Thus the enzyme characteristics shown below might be different from those reported byStrassburg et al. (1998).

T202I and M59I substitutions were introduced into the wild-type UGT1A10 cDNA by PCR-based site-directed mutagenesis. The wild-type and variant UGT1A10s were transiently expressed in COS-1 cells. Figure1A shows the representative Western blot of pooled samples from four-independent transfections. For each sample preparation, relative UGT1A10 levels were determined using one of the wild-type samples as a standard. As shown in Fig. 1B, the relative expression level of T202I was slightly lower (0.73 ± 0.10) than that of the wild-type, whereas that of M59I was marginally elevated (1.15 ± 0.21). These alterations in the expression levels, however, were not statistically significant by one-way ANOVA and Dunnett's test (p > 0.05). These relative expression levels were used for the normalization of glucuronidation activities of UGT1A10s described below.

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Figure 1

Expression of WT and variant (T202I and M59I) human UGT1A10s in COS-1 cells.

A, aliquots (20 μg) of the pooled membrane fraction from four-independent preparations were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to a polyvinylidene difluoride membrane, and detected immunochemically with rabbit anti-human UGT1A antiserum (diluted at 1:5000) as described underMaterials and Methods. To confirm that the samples were evenly loaded, the blot was subsequently stripped and re-probed with a polyclonal anti-calnexin antibody (diluted at 1:10,000). B, the stained UGT1A10 bands of each Western blot of four-independent preparations were quantified using the ImageQuant analysis software, and the expression levels of UGT1A10 proteins were normalized to the average of WT. The results are expressed as the mean ± S.D. from four-independent preparations.

Glucuronidation Activities of Wild-Type and Variant UGT1A10s.

Functional characterization of the expressed UGT1A10s was first carried out using HTFMC as a substrate. Table 1summarizes the apparent kinetic parameters of the wild-type and variant UGT1A10s for HTFMC glucuronidation. Wild-type UGT1A10 catalyzed HTFMC glucuronidation with a V_max value of 4.03 nmol/min/mg membrane protein, whereas those of T202I and M59I were 1.52 (38% of WT) and 4.28 (106%) nmol/min/mg of membrane protein, respectively. Their K_m values were 4.9, 5.4, and 4.2 μM for wild-type, T202I, and M59I, respectively. With respect to the normalized efficiencies (V_max/K_m), where the V_max values were normalized by the expressed UGT1A10 protein levels, the efficiency of T202I was just half those of wild-type and M59I. These results indicate that the T202I variant resulted in a lower HTFMC glucuronidation activity, whereas the M59I alteration had no significant influence on the activity.

The wild-type and variant UGT1A10s were further characterized using an endogenous substrate, E2. E2 is known to be glucuronidated at two different positions to form 3-O- and 17-O-glucuronides (as reviewed by Ritter, 2000). Figure2 shows the representative HPLC chromatograms of E2 glucuronide standards (A) and a 10-min incubation sample with E2 (50 μM) and wild-type UGT1A10 (0.5 mg/ml). As depicted in Fig. 2B, wild-type UGT1A10 exclusively catalyzed E2 3-O-glucuronidation. The same specificity was also found for the T202I and M59I variants (data not shown). Table 2 summarizes the apparent kinetic parameters of wild-type and variant UGT1A10s toward E2 3-O-glucuronidation. TheV_max and normalizedV_max/K_mvalues of T202I were 36 and 50% of that for the wild-type enzyme, respectively, and no statistically significant difference was observed in the K_m values (2.3, 2.3, and 1.9 μM for wild-type, T202I and M59I, respectively).

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Figure 2

Representative HPLC chromatograms of E2 glucuronides obtained after incubation of E2 with wild-type UGT1A10.

A, incubation buffer spiked with 250 pmol of E2 and its 3-O- and 17-O-glucuronides. B, a 10-min incubation mixture of E2 (50 μM) and COS-1-expressed wild-type UGT1A10 (0.5 mg/ml).

Therefor the kinetic analysis using both HTFMC and E2 revealed that the M59I variant was functionally identical to wild-type UGT1A10, whereas the catalytic activity of the T202I variant was almost half that of the wild-type enzyme without influencing the apparentK_m values.

Discussion

The digestive tract represents the first metabolically active tissue for orally ingested compounds. Recently, there has been a growing concern about the intestinal first pass metabolism, which influences the overall bioavailability of orally administrated drugs (Lin et al., 1999). The limited expression of UGT1A10 in biliary and gastrointestinal tissues suggests that this isoform participates in the removal of such drugs and their metabolite. For instance, Kemp et al. (2002) suggested that intestinal glucuronidation by UGT1A8 and UGT1A10 was a significant contributor to the presystemic clearance of a selective estrogen receptor modulator raloxifene. Thus alterations in function resulting from genetic polymorphisms may influence systemic drug levels and therapeutic outcome.

To date, a number of single nucleotide polymorphisms have been identified in the human UGT1A locus including UGT1A1, UGT1A6, UGT1A7, and UGT1A8 (Burchell et al., 2000; Guillemette et al., 2000; Tukey and Strassburg, 2000; Huang et al., 2002). Recently, we found two novel nonsynonymous single nucleotide polymorphisms inUGT1A10 exon 1 which resulted in amino acid alterations, T202I and M59I (Saeki et al., 2002). So far, no other polymorphisms inUGT1A10 have been reported in the literature. In the present study, these variants and wild-type UGT1A10 were transiently expressed in COS-1 cells, and their glucuronidation activities were characterized using HTFMC and E2 as substrates. Our results demonstrate that the glucuronidation activity (normalizedV_max value) of the T202I variant was nearly half that of the wild-type without influencing the apparentK_m values. The alignment of deduced amino acid sequences shown in Fig. 3indicates that the residue T202 is located within a highly conserved region of UGT1A isoforms. The amino acid sequence “MTF” at the residues 200–202 in UGT1A10 is shared among all the UGT1A isoforms. Interestingly, a substitution at the same residue (T202A) was also found in one of the controversial cDNA sequences of UGT1A8, which led to the generation of a protein with no enzymatic activity (Strassburg et al., 1998). The reduced activity of the UGT1A10 T202I variant toward HTFMC and E2 suggests that the conserved T202 residue is functionally important for catalytic activity.

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Figure 3

Alignment of deduced amino acid sequences of UGT1A isoforms in the region corresponding to the amino acid residues 192–212 of UGT1A10.

The arrow indicates the position corresponding to the residue 202 of UGT1A10. Shaded amino acids show those shared among all the UGT1A isoforms. The GenBank accession number is shown in the parenthesis.

Another intriguing observation of this study is that UGT1A10 catalyzes the 3-O-glucuronidation of E2, but not the 17-O-glucuronidation. Previous studies have shown that the hydroxyl groups at positions 3 and 17 undergo glucuronidation mediated by at least three different UGT isoforms. The glucuronidation at 17β-OH is mediated by human UGT2B7 (Gall et al., 1999), while UGT1A1 and UGT1A3 glucuronidate E2 at the phenolic 3-OH (Senafi et al., 1994;Gall et al., 1999). Although UGT1A4 (Green and Tephly, 1996), UGT1A9 (Albert et al., 1999), and UGT1A10 (Strassburg et al., 1998) have also been shown to possess the E2 glucuronidation activities, theregio-specificity has not been reported for these UGT1A isoforms. Human intestinal microsomes have been shown to glucuronidate E2 exclusively at 3-position with 2 to 15 times higher activities than those seen in human liver microsomes (Czernik et al., 2000). The apparent K_m values were approximately 40 μM with the V_max values of 6.4–7.3 nmol/min/mg. Fisher et al. (2000) have also reported a comparable apparent K_m value of 31 μM for the 3-O-glucuronidation of E2 by human small intestinal microsomes. In the human small intestine, several UGT1A isoform transcripts have been detected, including UGT1A1, UGT1A3, UGT1A4, UGT1A6, and UGT1A10 (Strassburg et al., 2000). As suggested byFisher et al. (2000) and Czernik et al. (2000), UGT1A1 and another UGT1A isoform would catalyze 3-O-glucuronidation in a cooperative manner. Our data suggest that UGT1A10 could be an important candidate for the UGT isoforms involved in the intestinal 3-O-glucuronidatition of E2.

In conclusion, of the two UGT1A10 variants characterized in this study, the T202I variant had a lower glucuronidation activities toward HTFMC and E2, compared with the wild-type enzyme, whereas the M59I variant had no effect on these activities. These results suggest that the T202I substitution can influence the intestinal absorption of orally administrated drugs and ingested environmental pollutants. In addition, enterohepatic circulation of biliary excreted chemicals may also be influenced.