UGT1A3 And UGT1A9: Heterogenous Expression, Flavonoids Glucuronidation And Functional Characterization Of UGT1A3 Variants

Drugs are metabolized by bodys as xenobiotics in a defensing mechanism. Glucuronidation is one of the most popular phaseâ…¡metabolisms to facilitate drugs excretion,in which drugs are conjugated with glucuronic acid catalyzed by uridine diphosphate glucuronosyltransferases(UGTs).The development and use of in vitro approaches to predict aspects of human drug metabolism and pharmacokinetics in vivo has attracted intense interest over the past decades.The recombinant enzyme has been developed as an effective model besides traditional microsomes or animal models.In this system,the specific UGT enzyme is obtained by introducing its cDNA sequence into heterogeneous expression systems.It has been proved as an effective way to detect the substrate spectrum of individual UGT and make clear the sophisticated metabolism mechanisms.To date,most human UGTs have been cloned in a number of laboratories,and many are also available from commercial sources,while research using this model is still in the preliminary stage in China.In this project,we obtained the active recombinant UGT1A3 and UGT1A9 in CHL and Bac-to-Bac^?expression systems, and performed the metabolism research using some flavonoids as substrates. â… .Expression of active human UGT1A3 and UGT1A9 in Chinese hamster lung(CHL)cells or Bac-to-Bac^?baculovirus infected insect cells.UGTs superfamily contains four UGT subfamilies,UGT1,UGT2,UGT3 and UGT8.The UGT1 and UGT2 are invaluable in detoxifying chemicals of both endogenous and foreign origins,especially the UGT1.The human UGT1 gene on chromosome 2q37 spans approximately 200 kb and contains 13 individual promoters/first exons and a shared set of exons 2-5.In this project,UGT1A3 gene and UGT1A9 gene,were expressed in CHL cells or Bac-to-Bac^?baculovirus infected insect cells to obtain the active enzymes for the following metabolism research.1.Heterogeneous expression of active UGT1A3 in CHL cells.The full-length UGT1A3*2a gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR)using total RNA from human liver or stomach as templates,which contains three point mutations compared with wild-type UGT1A3*1:Two are sense mutations(T31C,W¹¹R and T140C,V⁴⁷A),and the rest is silent one(G81A,E²⁷E).The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1(+),and recombinant vector was transfected into CHL cells using a calcium phosphate method. Concentration of G418 was kept at 400μg/ml in medium in the first selection passage to eliminate the cells failed to be transfected until untransfected cells in control group were killed completely.After selection,surviving cells were diluted into 96-well plates to obtain resistant monoclones and subjected to RT-PCR analysis for UGT1A3 transcription.Sf9 proteins were prepared and subjected to further glucuronidation assay.In the reaction mixture using quercetin as substrate,the recombinant UGT1A3 catalyzed the production of a glucuronide,which was detected by reverse phase-high performance liquid chromatography(RP-HPLC)coupled with diode array detector (DAD).The quercetin glucuronide was further confirmed by hydrolysis withβ-glucuronidase.No glucuronide was observed in negative control experiments.It suggested UGT1A3 expressed heterogenously in CHL cells was functional.The production of recombinant UGT1A3 was 4.3±1.2 mg/ml.The enzyme lost its activity quickly after 10 min.Considering the poor enzyme activity and stability,the active UGT1A3 expressed in CHL cells was not suitable for the quantitative assay.2.Expression of human UGT1A3 and UGT1A9 in Bac-to-Bac^?baculovirus infected insect cells.Here we tried a Bac-to-Bac^?baculovirus infected Sf9 insect cell expression system to get stable and high-activity recombinant UGT1A3 and UGT1A9.The wild-type UGT1A3*1 sequence was obtained by point mutation in PCR amplification using pcDNA3.1(+)- UGT1A3*2a as template.A His-tag sequence was added at the 3'-side of UGT1A3 sequence.The UGT1A9 flanked by Aftâ…¡and Xhoâ… was obtained by PCR using pcDNA3.1(+)- UGT1A9 as template.Both sequences were ligated into pFastBac^TM1 vector and transformed into MAX Efficiency DH10Bac^TMcompetent Escherichia coli.The PCR was used for identification of successful transposition.The Sf9 cells were transfected with recombinant bacmid-UGTs and produced the recombinant baculovirus.High-titer baculovirus stocks were used to express UGT1A9 and UGT1A3 proteins.RT-PCR proved the UGTs transcription,and the His-tagged recombinant UGT1A3 was detected using anti-His antibody.We tested the recombinant UGTs catalyzing activity to quercetin.Both UGT1A3 and UGT1A9 incubation produced four quercetin glucuronides previously detected in human liver microsome incubates.The activity of recombinant enzymes produced in Bac-to-Bac^?was higher than that of mammalian system and stable during 100 min incubation,which is suitable for the further quantitative analysis.â…¡.Quantitative regioselectivity of glucuronidation of quercetin by recombinant UDP-glucuronosyltransferases 1A9 and 1A3 using enzymatic kinetic parametersQuercetin has been suggested to exert its pharmacological effects,at least in part, via its metabolites,such as glucuronides.Glucuronidation is a major conjugation reaction for quercetin,and glucuronidation in different positions leads to dramatic difference in the pharmacological activity.Quantitative regioselectivity analyses are important to understand the contribution of individual UGT to pharmacological activity of quercetin.In this work,we obtained active UGT1A9 and UGT1A3 enzyme with a Bac-to-Bac^?expression system,and quercetin was metabolized to produce four monoglucuronides(7-,3-,4′- and 3′-glucuronide).Quercetin glucuronides were isolated on a HPLC system and identified by sodium acetate and sodium methoxide UV spectra.Enzymatic kinetic parameters of each glucuronide were calculated to quantitatively elucidate UGT1A9's and UGT1A3's regioselectivities for quercetin. UGT1A3's highest glucuronidation efficiency was observed for the 3′-glucuronide, then the 3-,4′- and 7-glucuronide.The catalytic efficiency order for UGT1A9 was:3-＞7-＞3′-＞4′-glucuronide.Considering the low antioxidant activity of quercetin-3′-glucuronide and quercetin-4′-glucuronide,UGT1A3 may play a limited role in the pharmacological activity of quercetin.While the high antioxidant activity of quercetin-7-glucuronides suggests that UGT1A9 has an important contribution to the pharmacological activity of quercetin.The regioselectivities of both enzymes will not change with time.â…¢.Glucuronidations of flavonoids by UGT1A3 and UGT1A9.Flavonoids are widely distributed in the plant kingdom and highlighted for their potential role in the prevention of various diseases associated with oxidative stresses, such as cancer,cardiovascular and neurodegenerative diseases.In order to ascertain the role health-improve benefits of flavonoids in vivo and their possible applications to the functional industry,it is necessary to understand their bioavailability,uptake, metabolism and excretion.Glucuronidation is one of three most important conjugations after flavonoids absorption.Here,the glucuronidation of nineteen flavonoids by UGT1A3 and UGT1A9 was tested.Among these flavonoids,eleven compounds could be catalyzed by both enzymes.Isorhamnetin,morin,silybin,kaempferol,daidzein, quercetin-3',4'-OCHO-,quercetin xylopyranoside and avicularin were reported as substrates of UGT1A3 for the first time.Apigenin,morin,daidzein, quercetin-3',4'-OCHO-,quercetin xylopyranoside and avicularin were the first reported substrates of UGT1A9.Each catalyzed flavonoid produced more than one uneven glucuronides,indicating a regioselectivity of UGT to different hydroxyl group in flavonoid.Both enzymes catalyzed the form the monoglucuronides with no diglucuronides detected in LC-MS.Enzymatic kinetic analysis indicated human UGT1A3 and UGT1A9 preferred flavonoids aglycone to flavonoid glycoside.They showed modest to high catalyzing activity to most flavonoid aglycones,while the catalyzing efficiency changes with structure.In general,the catalyzing efficiency (V_max/K_m)of UGT1A9 was much higher than that of UGT1A3,suggesting its important role in flavonoids glucuronidation.â…£.Functional characterization and frequency distribution of genetic variants of human UGT1A3 in a Chinese Han PopulationUGTs are highly polymorphism.Like CYP450s,the genetic variation of UGTs can lead to the change on xenobiotics toxicity and activity.Some polymorphisms in UGTs indicating the clinic and disease relevance were reported.Recently,a total of six single nucleotide polymorphisms(SNPs)have been identified in the human UGT1A3 gene.Among them,four SNPs(A17G,Q⁶R;T31C,W¹¹R;C133T,R⁴⁵W and T140C, V⁴⁷A)cause amino acid substitutions.Variants caused by these SNPs showed an activity change in estrone metabolism,while their activities towards other substrates were not examined.In the present study,six common flavonoids,quercetin,luteolin,kaempferol, silybin,morin and isorhamnetin,were used as substrates for glucuronidations by wild-type and variant UGT1A3s expressed in Bac-to-Bac^?insect expression system. Our results demonstrated that the activities of two variants,UGT1A3.2 and UGT1A3.3,remarkably decreased to 20%of that of UGT1A3.1.In contrast, UGT1A3.4 exhibited an approximately 3.6～7.8 times increase in the glucuronidation efficiency to all tested flavonoids and a clear preference to produce the high-activity quercetin 7- and 3- glucuronides. The frequency distributions of UGT1A3 alleles and SNPs in UGT1A3 in a Chinese Han population were first reported in this project.The distribution of UGT1A3.4,with a high glucuronidation efficiency and a preference to produce high-activity quercetin glucuronides,was statistically different from the reported value in German-Caucasian or Japanese population.UGT1A3 variants have altered glucuronidation activities towards all tested flavonoids,and may alter human susceptibility to flavonoids exposure.