Expression Of Human Recombinant UGT1A6 And Study Of Glucuronidation To Compounds

Uridine 5'diphosphate glucuronosyltransferases(UGTs) are the phase Ⅱ metabolising emzymes, which can catalyze the uridine 5'diphosphate glucuronic acid(UDPGA) to conjugate with the suitable compounds. This process is one of the important excretion routes in body. It used to evaluate the glucuronidation of the compounds with tissue microsomes that contain various enzymes. The UGTs are a superfamily of enzymes, and all UGT isoforms are characterized by broad and overlapping substrate specificities. Further more, it's lack of selective inhibitor of each UGT isoform, so it is difficult to identify which one catalyze the compound's glucuronidation. Fortunately, cloning and expression single drug metabolizing enzyme in vitro has been widely used, and most of UGTs have been expressed in vitro. For example, UGT1A3, 1A9 have also been expressed in our laboratory with mammalian cells such as CHO, CHL or V79. Nowadays, expressing heterologous proteins with baculovirus in insect cells are widely used, and this project is utilizing this method to express a member of UGT1 subfamily UGT1A6, then to investigate the metabolism with the recombinant protein to drugs or compounds. A single UGT isoform will be obtained if the gene components have been identified. On this condition, it is possible for us to research the metabolism function of some UGTisoform, learn about the glucuronidation of the compounds, and predict the degree of the glucuronidation and interaction of the compounds.1 Cloning of the gene UGT1A6 and construction of the recombinant virusesThe cDNA of the UGT1A6 was transcripted from mRNA in a Chinese human liver by reverse transcriptase polymerase chain reaction (RT-PCR). The PCR products were isolated and ligated with pGEM-T vector by T4 DNA ligase. The recombinant pGEM-UGTlA6 was amplified by tansforming into E.coli DH5 a .The harvested recombinants were cleaved with endonucleases Hindlllf Xhol, and the segments UGT1A6 were subcloned into the vector pcDNA3.1(+).The harvested recombinants pcDNA3.1(+)-￡/G7V.46 were cleaved by the same way with endonucleases BSP TVXhoI, and the segments UGT1A6 were subcloned into the vector pFastBac. The harvested recombinants pFastBac-UGT1A6 were then transformed into E.coli DHlOBac in which recombinant Bacmid (Bacmid-UGT1A6 ) would generate by transposition. After Bacmid-UGT1A6 transfected the Sf9 cell, the recombinant virus would generate.2 Expression of the recombinant UGT1A6The recombinant viruses were amplified with MOI 0.05~0.1until the viral titer reached 108pfu/mL or higher, then to infect the Sf9 cells with MOI 20 to express the recombinant UGT1A6. The infected Sf9 cells were harvested and socinated to obtain the cell homogenate containing the recombinant UGT1A6. The activity would be identified after the protein concentration was assayed by the method of BCA.3 Validation of the recombinant UGT1A6 transcriptionThe total cDNA was transcripted from mRNA in infected Sf9 cell by reverse transcriptase polymerase chain reaction (RT-PCR), with the parent Sf9 cell as control. The UGTJA6 specific primers were used to identify whehter the UGT1A6 gene was transcripted or not. As a result, the UGT1A6 was transcripted in infected Sf9 cell, not in parent Sf9 cell.4 Activity identification of the recombinant UGT1A6 and kinetic parameters assay of 4-nitrophenolThe activity of the recombinant UGT1A6 was assayed by 4-nitrophenol assubstrate with HPLC, and found it catalyze the 4- nitrophenol to be glucuronidatedsignificantly and the parent Sf9 cell as control did not. The values of Km and Fmax ofthe recombinant UGT1A6 to 4-nitrophenol were 358.0±44.9umol/L and13.1±1.0nmol/min- mg protein respectively assayed in a series of substrateconcentrations.5 Glucuronidation of some compounds with the recombinant UGT1A6UGT1A6 exhibits strict substrate specificity toward short and planar phenols and some carboxyl compounds, so some compounds containing aryl hydroxy or carboxy groups were tested with the recombinant UGT1A6 in our laboratory.5.1 a -naphthol: The glucuronidation reaction was carried out in an incubation mixture of lOOuL total volume in which a -naphthol was added. After pre-incubation, the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of lOOuL methanol and internal standard (P -naphthol in methanol). The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microliters of the supernatant was applied to a reversed phase column Diamonsil? C18 (4.6mm ID X 200mm, 5^m particle size). The mobile phase was made up with H2O (containing 0.01 mol/L KH2PO4, pH3.5)-methanol (38:62). The flow rate was l.OmL/min. Wavelength of UV detector was 280nm. The Km and Fmax value of the recombinant UGT1A6 to a -naphthol were 3.8 ± 1.6 umol/L and 6.5 ±1.0 nmol/min-mg protein respectively.5.2 P -naphthol: The glucuronidation reaction was carried out in an incubation mixture of lOOuL total volume in which P -naphthol was added. After pre-incubation, the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of lOOuL methanol and internal standard (a -naphthol in methanol). The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microliters of the supernatant was applied to a reversed phase column Diamonsil? C18C4.6 mm ID X 200 mm, 5 (am particle size). The mobile phase was made up with H2O (containing 0.01 mol/L KH2PO4, pH3.5)-methanol (38:62). The flow rate was l.OmL/min. Wavelength of UV detectorwas 280 nm. The values of Km and Fmax of the recombinant UGT1A6 to (3 -naphthol were 19.0 ± 7.8 umol/L and 20.4 ± 6.6 nmol/min-mg protein respectively.5.3 Catechol: The glucuronidation reaction was carried out in an incubation mixture of 100|iL total volume in which catechol was added. After pre-incubation, the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of IOOjjL methanol and internal standard (p-aminosalicylic acid in methanol). The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microliters of the supernatant was applied to a reversed phase column Diamonsil? C18 (4.6mm ID X 200mm, 5|am particle size) .The mobile phase was made up with H2O (containing 0.01 mol/L KH2PO4, pH3.5)-acetonitrile (92:8). The flow rate was l.OmL/min. Wavelength of UV detector was 276nm. The values of Km and Fmax of the recombinant UGT1A6 to catechol were 445.8 + 54.7u.mol/L and 7.1 ± 0.9nmol/min-mg protein respectively.5.4 Acetaminophen: The glucuronidation reaction was carried out in an incubation mixture of lOOuL total volume in which acetaminophen was added. After pre-incubation, the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of lOOuL methanol. The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microliters of the supernatant was applied to a reversed phase column Diamonsil? C18 (4.6mm ID X 200mm, 5 um particle size) . The mobile phase was made up with H2O (containing 0.01 mol/L KH2PO4, pH4.5)-methanol (95:5). The flow rate was l.OmL/min. Wavelength of UV detector was 280nm. The enzyme activity to acetominophen couldn't be detected after incubation for 120 min.5.5 Flavonoids (quercetin, kaempferol, isorhamnetin): The glucuronidation reaction was carried out in an incubation mixture of lOOuL total volume in which quercetin, kaempferol and isorhamnetin were added respectively. After pre-incubation, the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of lOOuL methanol. The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microlitersof the supernatant was applied to a reversed phase column Diamonsil? C18 (4.6mm IDX200mm, 5um particle size) . The mobile phase was made up with H2O (containing 0.01 mol/L KH2PO4, pH2.5)-methanol (44:56). The flow rate was l.OmL/min. Wavelength of UV detector was 370nm. The enzyme activity to these three compounds couldn't be detected after incubation for 120 min.5.6 Tolterodine: The glucuronidation reaction was carried out in an incubation mixture of lOOuL total volume in which tolterodine was added. After pre-incubation, the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of lOOuL methanol. The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microliters of the supernatant was applied to a reversed phase column Diamonsil? C18 (4.6mm IDX200mm, 5um particle size) . The mobile phase was made up with H2O (containing 0.01 mol/L KH2PO4, pH3.5)-acetonitrile (70:30). The flow rate was l.OmL/min. Wavelength of UV detector was 280nm. The enzyme activity to tolterodine couldn't be detected after incubation for 90 min.5.7 Salicylic acid and p-aminosalicylic acid: The glucuronidation reaction was carried out in an incubation mixture of lOOuL total volume in which salicylic acid and p-aminosalicylic acid were added respectively. After pre-incubation, the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of lOOuL methanol. The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microliters of the supernatant was applied to a reversed phase column Diamonsil? C18 (4.6mm IDX200mm, 5um particle size) . The mobile phase was made up with H2O (containing 0.01 mol/L KH2PC>4, pH3.5)-methanol (60:40) for salicylic acid and (85:15) for p-aminosalicylic acid. The flow rate was l.OmL/min. Wavelength of UV detector was 280nm. The enzyme activity to these two compounds couldn't be detected after incubation for 90 min.5.8 Mandelic acid: The glucuronidation reaction was carried out in an incubation mixture of lOOuL total volume in which mandelic acid was added. After pre-incubation, the glucuronidation in the mixtures were started by the addition ofUDPGA and stopped by the addition of lOOpl methanol. The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microliters of the supernatant was applied to a reversed phase column Diamonsil? C18 (4.6mm ID X 200mm, 5um particle size) . The mobile phase was made up with H2O (containing 0.01 mol/L KH2PO4, pH2.5)-methanol (70:30). The flow rate was l.OmL/min. Wavelength of UV detector was 266nm. The enzyme activity to mandelic acid couldn't be detected after incubation for 90 min.5.9 Ibuprofen: The glucuronidation reaction was carried out in an incubation mixture of lOOuL total volume in which ibuprofen was added. After pre-incubation, the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of lOOuL methanol. The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microliters of the supernatant was applied to a reversed phase column Diamonsil? C18 (4.6mm IDX200mm, 5um particle size) . The mobile phase was made up with H2O (containing O.Olmol/L KH2PO4, pH2.5)-acetonitrile (45:55). The flow rate was l.OmL/min. Wavelength of UV detector was 271nm. The enzyme activity to ibuprofen couldn't be detected after incubation for 90 min.5.10 Naproxen: The glucuronidation reaction was carried out in an incubation mixture of lOOuL total volume in which naproxen was added. After pre-incubation, the glucuronidation in the mixtures were started by the addition of UDPGA and stopped by the addition of lOOuL methanol. The mixtures were stirred thoroughly and centrifuged at 4°C with 16000g for 5 min to remove the proteins. Twenty microliters of the supernatant was applied to a reversed phase column Diamonsil? C18 (4.6mm ID X 200mm, 5um particle size) .The mobile phase was made up with H2O (containing 0.01 mol/L KH2PO4, pH2.5)-acetonitrile (55:45). The flow rate was 1.0 mL/min. Wavelength of UV detector was 271nm. The enzyme activity to naproxen couldn't be detected after incubation for 90 min.From the results of the glucuronidation of these compounds with the recombinant UGTl A6, we found that the compounds a -naphthol, P -naphthol and catechol can be markedly glucuronidated by the recombinant UGTl A6, but the othercompounds above did not. Different position, size and electronegativity of the substituents will influence the glucuronidation. For example, 4-nitrophenol and acetaminophen are accepted as the substrates of UGT1A6, but in this experiment we didn't detect the glucuronide of the acetaminophen, perhaps the substituent of the acetaminophen is the group of electron giving, which resulted in difficult inozation of the phenol hydroxy. As a result, we couldn't detect the activity of the recombinant UGT1A6 for poor glucuronidation.In a word, we successfully expressed the recombinant UGT1A6 with catalytic activity by the baculovirus expression system, and the values of Km and Fmax of the recombinant UGT1A6 toward 4-nitrophenol were 358.0±44.9umol/L and 13.1±1.0 nmol/min- mg protein. Using the recombinant UGT1A6 to react with some compounds, we found it catalyze a -naphthol, ￡ -naphthol and catechol obviously, but not catalyze the compounds tolterodine, acetaminophen, quercetin, kaempferol, isorhamnetin, salicylic acid, p-aminosalicylic acid, mandelic acid, ibuprofen, naproxen. The enzyme UGT1A6 tends to catalyze the simple planar phenols as references reported, and the results in our experiment were in accordance with it on the whole.