Preliminary Study Of Phosphorylation Effect On UGT1A3 Metabolism Activity Towards Drugs

Protein phosphorylation is the most common and important post-translational modification, which plays vital roles in completion and change of protein function. Phosphorylation is catalysed by a class of enzymes referred to as protein kinases, while removal of phosphate is controlled by enzymes called phosphatases. Protein kinase C (PKC) is a family of calcium-and/or phospholipid-dependent serine/threonine protein kinases that play a central role in signal transduction pathways, especially transmembrane signal regulations. To date, phosphorylation has been investigated to be involved in regulations of UGT activities and their substrate specificity. In addition, phosphorylation mostly catalyzed by PKC is showed to be required for UGT1A activities.This study focused on the relationship between phosphorylation and UGT1A3 activity as phosphorylation of UGT1A3 is unclear. Firstly, we tried a Bac-to-Bac baculovirus infected Sf9 insect cell expression system to get stable and high-activity recombinant UGT1A3. Secondly, the correlation between treatment of general kinase inhibitor curcumin or calphostin-C, a selective pan-PKC inhibitor, on Sf9 cells respectively and UGT1A3 activity towards quercetin was investigated.1 Expression, activity analysis and purification of UGT1A3 expressed in Sf9 cellsObjectives To express UGT1A3 protein in Sf9 insect cell and analyze its activity to quercetin substrate. To establish a reliable reversed-phase high-performance liquid chromatographic method for the determination of quercetin in cell lysate. Explore the purification condition of 6His-tagged UGT1A3 via Ni-NTA agarose.Methods Spodoptera frugiperda (Sf9) insect cells were infected with Bacmid-UGT1A3 to generate recombinant baculoviruses carrying human UGT1A3 gene. High-titer baculovirus stocks were used to express UGT1A3 protein. The His-tagged recombinant UGT1A3 was detected by western blot using anti-His antibody. The catalytic activity of the recombinant enzyme toward quercetin was determined by using an HPLC method developed and validated. In addition, the relative metabolic kinetic parameters were measured. Eventually, the purification condition for 6His-tagged was determined.Results After Sf9 cells were infected with high-titer baculovirus stock (34.4μg·mL-1), western blot was employed to confirm the expression of the protein of interest. Active recombinant UGT1A3 was confirmed by using quercetin as substrate, and four new peaks on HPLC analysis were found, which disappeared after incubation withβ-glucuronidase. The established HPLC method had a linear concentration range of 5-200μmol·L-1 (r=0.9999) for quercetin. The limit of detection was 1.25μ.mol·L-1 (S/N>3), and the limit of quantification was 5μmol·L-1 (S/N>10, RSD=6.99%, n=5). The method afforded recoveries of 99.1%～103.5%, and precisions for inter-and intra-assay were<2.5%and<8%(n=5), respectively. In addition, kinetic analysis indicated that the Km, Vmax and CLint (Vmax/Km) values for quercetin glucuronide were (62.95±13.16)μmol·L-1, (284.5±24.35) nmol-miV-1·g-1 and 4.52ml-min-1·g-1, respectively. Finally, the target single protein band of UGT1A3 was observed on SDS-PAGE after purification and ultrafiltration.Conclusion The recombinant UGT1A3 protein expressed in Sf9 cells has respectable activity on quercetin. And the HPLC method established is specific, robust, sensitive and accurate, which could be used for the assay and quantification of quercetin from cell lysate. In addition, the purified UGT1A3 could be used as sample analyzed by mass spectrometry.2 Effects of kinase inhibitors on UGT1A3 activityObjective To investigate effects of general kinase inhibitor curcumin and protein kinase C-specific inhibitor calphostin-C on UGT1A3 activity expressed in Sf9 cells.Methods The toxicity of curcumin or calphostin-C to Sf9 cells was evaluated via MTT method. Insofar as non-toxic to cells, curcumin or calphostin-C with variable concentrations was incubated with cell lysate containing UGT1A3, respectively. The cells expressing target protein were exposed to the two inhibitors with concentrations of 1-25μmol·L-1 and 1-1000nmol·L-1, respectively. Western blot and incubation using quercetin as substrate were investigated to evaluate the expression level and enzyme activity variations of UGT1A3. Besides, effects of both inhibitors on UGT1A3 activity towards quercetin with changing concentrations were also investigated.Result The results from MTT tests showed that inhibitors curcumin and calphostin-C had no apparent toxicities towards Sf9 cells within 50μmol·L-1 and 1000nmol·L-1, respectively. Addition of either curcumin or calphostin-C directly to incubation samples did not affect the enzyme activity of UGT1A3 towards quercetin, which remained over 95%or 93%, respectively. The result indicated that both inhibitors had no immediate influence on UGT1A3 protein. Cell lysates treated with curcumin (1,5,15,25μmol·L-1) or calphostin-C (1,10,50,125,250,500, 1000nmol·L-1) were analyzed via western blot and incubation tests. It showed that the targeted protein had no obvious changes on expression level, while curcumin or calphostin-C-dependent inhibition of UGT1A3 activity on quercetin was observed. At the concentration of 25μmol·L-1 curcumin or 500 nmol·L-1 calphostin-C, UGT1A3 activity towards quercetin was reduced to 41.6%and 21.9%, respectively. In addition, when incubated with quercetin of varying concentrations, UGT1A3 from cells treated with either inhibitor was always less active than corresponding positive controls. Besides, UGT1A3 had better sensitivity to PKC specific inhibitor calphostin-C except for the incubation with quercetin of 10μmol·L-1.Conclusion As curcumin is a recognized general kinase inhibitor, its concentration-dependent inhibition effect on UGT1A3 activity toward quercetin suggested that recombinant UGT1A3 expressed in Sf9 cells requires phoshporylation to maintain its enzymatic activity. Meanwhile, the specific protein kinase C (PKC) inhibitor calphostin-C also showed concentration-dependent inhibition towards UGT1A3, which strongly indicated the involvement of PKC in UGTlA3 phosphorylation.