Cloning, Expression and Characterization of Rat

UDP-glucuronosyltransferase 1A8 (UGT1A8) and its induction

by Licorice Extract and 18/3- Glycyrrhetinic Acid

LEE Kai Woo

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Philosophy in Biochemistry

© The Chinese University of Hong Kong August 2006

The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School |i 2 OCT 1? i Acknowledgements

I extend my appreciation to Professor John Wing Shing Ho who has been a good mentor throughout my study. I am thankful for his comments during the preparation of this work.

I would like to thank Professor Lai Kwok Leung for his support in my difficult times.

His sagacious guidance and support have been valuable.

I must offer my heartfelt thanks to my family. Their supports have always been the

greatest encouragement to me.

Last but not least, I would like to thank all members of MMW 612A for their helps

during my years of work with them.

ii Thesis Committee

Professor Leung Lai Kwok (Chair)

Professor Ho Wing Shing, John (Thesis Supervisor)

Professor Wang Jun (Committee Member)

Professor Chan Pui Kwong (External Examiner)

iii I declare that the thesis here submitted is original except for source material explicitly acknowledged. I also acknowledge that I am aware of University policy and regulations on honesty in academic work, and of the disciplinary guidelines and procedures applicable to breaches of such policy and regulations, as contained in the website http://www.cuhk.edu.hk/policv/academichonestv/

T 3�,Wrt

Signature Date

Name Student ID

iv Abstract

UDP-glucuronosyltransferases (UGTs), which are one of the important detoxifying enzymes, catalyze the glucuronidation of a wide range of endobiotic and xenobiotic compounds. The glucuronidation can lead to the clearance of drugs and xenobiotics.

The induction of UGTs is believed to enhance elimination of metabolites. This study was to investigate the induction of UGT1A8 in the rat liver, and to study the substrate

specificity of this newly identified UGT1A8. Study of the biochemical properties and the substrate specificity of UGT1A8 would allow us to understand the functions of the

enzyme.

The expression and the properties of UGT1A8 were studied. Rat liver cells (clone 9

and H4IIE) and rats (Wistar and j/j rats) were treated with licorice and 18 ^

glycyrrhetinic acid to study the expression of the liver UGT1A8. It was found that the

gene could be induced by both treatments. The liver UGT1A8 cDNA was obtained

from the rat liver and the gene was cloned and expressed in E. coli system. The gene

encoded for a protein with 531 a.a. with a molecular weight of 55kDa. The purified

protein was assayed with different substrates: bilirubin, couramin, 1-naphthol,

4-nitrophenol and phenol red. The results showed that UGT1A8 can be expressed in E.

coli JM109 and the protein can be purified with centrifligation and Ni+-column. The

enzyme showed activity towards couramin and l-naphthol. The present findings

V indicated that UGT1A8 has overlapping substrate specificity with UGT1A7. UGT1A8 can be induced by 18 GA and licorice. The study provides useful information on the functional roles of UGTl A8 and the substrate specificity of the protein.

vi 論文槪要

尿苷二磷酸葡糖苷酸基轉移酶(UGTs)是眾多重要解毒酶的其中一員，該酶催化多

種內源化合物與外源化合物的醣化作用。以清除藥物和外源化合物。尿苷二磷酸

葡糖苷酸基轉移酶類的誘導被認爲有助於有害化學物體內之清除。本研究調查了

大鼠肝臟尿苷二磷酸葡糖苷酸基轉移酶1A8信使核酸的誘導和表達及該酶底物

之特異性。硏究該酶的上述特性，可使我們增加對該酶的認識。

爲硏究該酶於大鼠肝臟中的表達與該酶的生物化學特性，甘草及甘草次酸

分別施用於大鼠肝臟細胞(clone9與H4IIE)和大鼠(wistar鼠與j/j鼠），然後分析

甘草及18 3甘草次酸對肝臟尿苷二磷酸葡糖苷酸基轉移酶1A8表達的影響。本

硏究發現甘草及18/3甘草次酸均可誘導尿苷二磷酸葡糖苷酸基轉移酶1A8之表

達。同時，我們從大鼠肝臟提取分離了該酶的基因序列，並於大腸桿菌內重新表

達了該酶。根據基因序列，該酶由531個氨基酸組成，分子量約爲55kDa�該酶

在提純後用於舗選不同的底物：膽紅素、香豆素�1-奈8分�4-硝基苯酣及酚紅。

數據顯示尿苷二磷酸葡糖苷酸基轉移酶1A8可在大腸桿菌JMIOQ內表達，並以

離心法與鎳離子螯合層析法提純。該酶對香豆素及1-奈酷有活性。本硏究顯示出

尿苷二磷酸葡糖苷酸基轉移酶1A8之底物特異性與尿苷二磷酸葡糖苷酸基轉移

酶1A7有相類近之處。尿苷二磷酸葡糖苷酸基轉移酶1A8可被18/5甘草次酸及

甘草誘導。本硏究提供了更多有關尿苷二磷酸葡糖苷酸基轉移酶1A8之功能與

底物特異性之資料。

vii List of figures

Figure 1. Reaction catalyzed by the enzyme UDP-glucuronosyltransferase 3 Figure 2. Chemical structures of some phytoalexins and antioxidants 5 Figure 3. Organization of the human and rat UDP-glucuronosyltransferase-1 10 gene locus Figure 4. Phylogenetic tree of the 18 human UDP-glucuronosyltransferases 12 Figure 5. Genomic organization of UGTl gene clusters 15 Figure 6. Phylogenetic tree of human, mouse, and rat UGTl gene clusters 19 Figure 7. Tissue distribution of rat UGT1A8 23 Figure 8. Structure of 18 /3 lycyrrhetinic acid 25 Figure 9. Time dependent cellular response of H4IIE cell (licorice) 49 Figure 10. Time dependent cellular response of clone9 cells (licorice) 50 Figure 11. Time dependent cellular response of H4IIE cells (18(3GA) 51 Figure 12. Time dependent cellular response of clone9 cells (18|3GA) 52 Figure 13. Dose dependent cellular response of H4IIE (licorice) 53 Figure 14. Dose dependent cellular response of clone9 (licorice) 54 Figure 15. Dose dependent cellular response of H4IIE (18|3GA) 55 Figure 16. Dose dependent cellular response of clone9 (18pGA) 56 Figure 17. RT PGR result showed the P-actin level in the cells 59 Figure 18. RT PGR result showed the UGTlAl level in the cells 60 Figure 19. RT PGR showed the gene expression level of UGTl A7 61 Figure 20. RT-PCR showed the gene expression level of UGTl A8 62 Figure 21. RNA extraction from the rat liver 65 Figure 22. The results of RT-PCR after treatment showed the level of P-actin 66 Figure 23. RT-PCR result of UGTl A8 level after treatmen 67 Figure 24. Northern blot analysis of UGTl A8 with RNA^ samples after rat 68 treatment Figure 25. UGTl A1 gene expression level after treatment of rats 69 Figure 26. Amplification of the UGT1A8 72 Figure 27. Restriction digestion of the constructed plasmid 73 Figure 28. Blast search of the sequenced target insert 74 Figure 29. SDS-PAGE showed the expression of the target protein after 75 IPTG induction Figure 30. SDS-PAGE showing the stepwise purification of the target protein 76 Figure 31. Different compounds for the activity test of the purified protein 77 Figure 32. Chemicals that UGT1A8 would show glucuronidation 85

viii List of Abbreviations

18 j3 GA 18|3-glycyrrhetinic acid B[a]P Benzo[a]pyrene cDNA complementary DNA DEPC Diethylpyrocarbonate DMEs Drug metabolizing enzymes IMAC Immobilized metal affinity chromatography IPTG Isopropyl-B-D-thiogalactopyranoside PGR Polymerase Chain Reaction PMSF Phenylmethylsulfonyl fluoride SDS-PAGE Sodium Dedocyl Sulfate Polyacrylamide Gel Electrophoresis SULT Sulfotransferases UDPGA Uridine Diphosphate Glucuronic Acid UGTs UDP-glucuronosyltransferases

ix Table of contents

Title Acknowledgements ii Thesis Committee iii Abstracts v 論文槪要 vii List of figures viii List of abbreviations ix

Chapter one Introduction 1 1.1 Drug metabolism and UGTs 1 1.2 Natural substrates of UGTs 4 1.3 Functions of UGT isoforms: roles of UGT polymorphisms 6 1A Evolution of the UGTl gene locus in vertebrates 8 1.5 Multiple Variable First Exons: A Mechanism for Cell- and 13 Tissue-Specific Gene regulation 1.6 Evolutionary Origin of the Variable and Constant Genomic 14 Organization 1.7 Variable and Constant Genomic Organizations Exist in 20 Mammalian UGTs 1.8 The history of recombinant UGT expression 20 1.9 UGTl A8 21 1.10 Licorice and its active component 24 1.11 Enzyme induction in the liver 25 1 12 Objectives 28

Chapter two Methods and Materials 29 2.1 UGT1A8 induction studies 30 2.1.1 Drug preparation 30 2.1.2 Cell viability study with Neutral Red Assay Rat treatment 30 2.1.3 Cell treatment 31 2.1.4 Rat treatment 31 2.1.5 RNA extraction from rat liver and cell culture 31 2.1.6 Quantization of RNA 32 2.1.7 Denaturing gel electrophoresis for RNA 33 2.1.8 Northern hybridization 33 2.1.9 Probe for Northern Blotting 34

xi 2.1.10 Agarose Gel analysis and Northern Blot analysis 34

2.2 Recombinant expression of UGT1A8 in E.coli JM109 35 2.2.1 cDNA synthesis 35 2.2.2 Polymerase chain reaction 35 2.2.3 Agarose gel electrophoresis for DNA 35 2.2.4 Amplification of target gene, UGT1A8 36 2.2.5 Restriction enzyme digestion of plasmid and insert 36 2.2.6 Ligation of plasmid and insert DNA 37 2.2.7 Amplification of target plasmid 37 2.2.8 Screening of target plasmid 37 2.2.9 DNA sequencing 38 2.2.10 Transformation of protein expression host 38 2.2.11 Confirmation of transformation of protein expression host 38 2.2.12 Protein expression 39 2.2.13 Protein purification 39 2.2.14 Sodium dodecyl sulfate polyacrylamide gel electrophoresis 40 2.2.15 Confirmation of the protein 40

2.3 Characterization of recombinant UGT1A8 41 2.3.1 UGT assay 41

2.4 Routine experiment methods 41 2.4.1 Determination of protein 41 2.4.2 Nucleic acid purification 42 2.4.3 Preparation of chemically competent bacterial cells 42 2.4.4 Colony PGR 43 2.4.5 Plasmid rescue by alkaline lysis 44 2.4.6 Charging of His-tagged column 44 2.4.7 Washing of His-tagged column 45

Chapter three Results 46 3.1 UGT1A8 Expression in clone9 and H4IIE after treatment with 46 licorice and 18 ^ glycyrrhentinic acid 3.2 UGT1A8 induction in wistar and j/j rats after treatment 63 3.3 Construction of pRset-UGTlA8 Vector 70 3.4 Purification of recombinant UGT1A8 75 3.5 Screening of substrate of the purified enzyme 77

xi Chapter four Discussion 78 4.1 Effects of licorice and 1 SPglycyrrhetinic acid in the induction 78 of UGT1A8 in different cell lines 4.2 Comparison of wistar and j/j rats in the induction of UGT1A8 79 4.3 Comparison of licorice and 18(3 glycyrrhetinic acid in the 81 induction of UGT1A8 in rats 4.4 Comparison of in vzvo and in vitro of drug treatment 81 4.5 Expression of UGT1A7 after drug treatment in vitro 82 4.6 Protein expression and purification 83

4.7 Substrates of UGTl A8 83

Chapter Five Conclusions 86

References 90

Appendix 105

xii Chapter One. Introduction

1.1 Drug metabolism and UGTs

Drug metabolism is one of the most important processes in the liver. The drug

metabolizing process can be divided into two steps: phase I reaction and phase

II reaction. When different drugs and chemicals enter cells by passive diffusion

or with the actions of uptake transporters, phase I enzymes, mainly cytochromes

P-450 (CYPs), would convert them into nucleophilic phenols, polyphenols or

electrophiles, such as epoxides and quinones, which would then be conjugated

by phase II enzymes, such as UDP glucuronosytransferase (UGT) and

sulfotransferase (SULT) or glutathione S-transferase (GST). The resulting

hydrophilic organic anions are removed from the cell by export transporters.

UGTs represent major phase II drug metabolizing enzymes (DMEs),

conjugating a wide variety of drugs, such as external chemicals like dietary

phytoalexins and endobiotics like bilirubin and steroid hormones. Glucuronic

acid conjugation dictates the elimination of those chemicals, and the action is

determined by the UGT contents of different cells and tissues. There are

obvious inter-individual differences in the amount and expression level of UGTs

in liver and other organs. The drug metabolizing enzymes convert lipid-soluble

compounds into water-soluble and excretable forms. A group of drug

1 metabolizing enzymes (DMEs) rapidly interconvert nucleophiles and electrophiles, for example, NQOl and peroxidases. The balance between phase

I and II enzymes often determines the accumulation of reactive intermediates which may cause oxidative/electrophile stress and toxicity. The latter often initiates the covalent binding of metabolites to DNA and protein. Hence, when dealing with UGT functions, glucuronide transporters and compensatory enzymes, for examples, sulfotransferases (SULTs), have to consider the balance and control of these types of reactions, which are very important in the homeostasis of the body.

2 ：丫。H�

"h)^ + R—OH HO \ OHO-UDP

UGT

〇 OH \ Y o + UDP + water HO \ OHO-R

Figure 1. Reaction catalyzed by the enzyme UDP-glucurono sytransferase. The substrate, after the conjugated with UDPGA, would become more water soluble. The increase in the solubility of substrate can enhance the excretion of it through urine.

3 1.2 Natural substrates of UGTs

Phytoalexins are antimicrobial compounds produced in plant cells exposed to

bacteria and fungi. They are important in protecting plants against insects and

other animal predators. Many of them act as polyphenolic pro/antioxidants,

such as quercetin, present, for example, anthocyanidine, the red color of roses

Ford et al 1998], in onions and in honey [Galijatovic et al, 2000], the

phytoestrogen resveratrol in grapes and wine [Gehm et al, 1997], and

zeaxanthin in bacteria [Hundle et al, 1992]. They are stored in plant cells and

bacteria as glucosides which are readily hydrolyzed. Vertebrates take in these

plants and so the UGT in the liver and in the intestinal tract would provide a

protective role to neutralize these different phytoalexins. In the gastrointestinal

(GI) tract and the liver, the protective role of UGTs is indicated by their large

numbers and high expression levels, including the specific expression of some

isoforms, such as UGT1A7 in the human upper GI tract (orolaryngeal mucosa,

esophagus, and stomach) and UGTIAIO in the small intestinal and the colon

mucosa [Tukey et al, 2001]. This protective function may be most relevant at

earlier stages of evolution when fish and terrestrial animals tried to live on a

plant diet, and plants developed phytoalexins for survival, a process termed

'animal-plant warfare' [Nebert et al, 1990].

4 OH

^ Chrysin HO"^^^^ Resveratrol OH O

r^oH + a�H

V^OH y^OH I OH OH C Anthocyanidin Quercetin OH

三 63X3 门 thin

Figure 2. Chemical structures of some phytoalexins and antioxidants. They are some common substrates of the enzyme UGTs.

5 1.3 Functions of UGT isoforms: roles of UGT polymorphisms

UGTs work at several different levels in the organism: in presystemic or

systemic drug metabolism and locally within various types of cells. It is known

that a number of factors control glucuronide formation and disposition in cells

and in the whole organism. Nevertheless, when using isoform-specific probe

substrates, in vitro microsomal UGT activity correlates well with in vivo

glucuronide formation. For example, the interindividual hepatic bilirubin UGT

activity correlated with the blood level of bilirubin in Gilbert individuals

[Raijmakers ct al, 2000] and the morphine UGT activity with presystemic

glucuronidation of oral morphine [Save et al, 1985]. Locally, UGT activity has

been shown to be important, for example, in controlling steroid hormone levels

[Sun et al, 1998]. Therefore, the in vitro analysis of UGT activity is very

important for prediction of in vivo metabolic clearance [Mistry et al 1987 and

Soars etam^lX

Certain polymorphisms have been discovered in UGT families 1 [Mackenzie et

al 1997]. In the UGTl locus a TATA box mutation of bilirubin-metabolizing

UGTlAl, leading to a reduced enzyme activity, has received a lot of interest

since it is frequently related to Gilbert's syndrome in Caucasians [Bosma et al

6 1995 and Monogan et al 1996]. In addition to be exposed to mild hyperbilirubinemia, Gilbert individuals have shown unwanted side effects of the chemotherapeutic agent irinotecan since its effective major metabolite SN38 is mostly inactivated by UGTlAl, thus resulting in intoxication [Ando et al 1998,

Ando et al 2000]. Repeated sequences, such as A(TA)nTAA, in the promoter of

UGTlAl are unstable and tend to lengthen or shorten as a result of unequal crossing over in meiosis. It has been shown that increasing TA repeats of the

UGTlAl TATA box alters the transcription, enzyme level, and activity [Beutler et al 1998]. The allelic variants A(TA)6TAA (UGTlAl*：!) and A(TA)7TAA

(UGTlAl 28) are frequent in different populations. Bilirubin may be either harmful or beneficial; severe hyperbilirubinemia in neonates is known to cause kernictems and brain damage. However, low bilirubin level together with high biliverdin reductase activity appears to be a powerful antioxidant [Baranano et al

2002]. In fact, Gilbert individuals seem to be protected from coronary heart

disease [Vitek et al 2002]. The levels of UGTs and the body condition have

complicated relations. There are still major differences in allele frequencies

among ethnic groups. For example, the frequency of UGTlAl'28 was found to

be ca. 0.29 in Caucasians and 0.26 in Egyptians [Kohle et al 2003] and only ca.

7 0.09 in Japanese [Hall et al 1999]. Hence, the disease conditions of different

races would vary.

1.4 Evolution of the UGTl gene locus in vertebrates

The organizations of human and rat UGTl gene locus show considerable

similarities to each other. (FigureS). It is believed that the gene was present

before the radiation of rodents and man, before the beginning of the age of

Paleocene, i.e. 65 million years ago [Strickberger M.W., 2000]. This gene locus

was also found in many other mammalian species, for example, rabbit, monkey,

dog, mouse and sheep. Herbivorous lagomorphs have evolved duplicated

UGT1A6 genes in their UGTl locus [Li et al, 2000]. The detoxification of

dietary plant polyphenolic phytoalexins, which have been discussed before, and

the degradation of phytoestrogens (which share similar structure with steroid

hormones) are believed to be the evolutionary driving force for the generation

of the UGTl locus. [Nebert et al 1990, Gehm et al 1997]. Certainly, sex

hormone-like phytoestrogens would greatly affect the reproduction of

organisms. Prey-predator relation represents a major process in evolution. That

is, herbivorous predators evolved phytoalexin detoxifying enzymes and these

adaptively generated phenol UGTs were no longer necessary in carnivorous

8 species because their prey (cattle, sheep, etc.) had already done the job of

detoxifying phytoalexins. For example, in carnivorous cats and leopards

UGT1A6 was found to be present as a pseudogene [Court et al, 2000:.

9 Human ,, ^ AhR 池R 她R ：I

nil 1:2P11P S 10 13P 9 7 6 5 4 3 2P 1 2 3 4 5

Exon f s Comrnort ex_s

Rat AhR A:hR T1 T2 I广―、 • I��率 I 产I举I 广1令 • 广I• I广棚 I III! ID 9P a 7 6i6 5 4P 3 2 1^ 2 3 4 S E游m fs C_m« exons

Figure 3. Organization of the human and rat UDP-glucuronosyltransferase-1 gene locus; P, pseudogenes; Tl, transcription unit 1; T2, transcription unit 2 (Adapted from Bock, 2003). The expression of different isozymes is highly controlled. AhR is one of the regulatory components of UGTs.

10 Conjugation of compounds, including steroids, by glucuronidation has been demonstrated in all vertebrates studied to date. This reaction, which corresponds to the transfer of the glucuronosyl group from UDP-glucuronic acid to small hydrophobic molecules, is catalyzed by enzymes of the UGT superfamily. The resulting glucuronide products are more polar, generally water soluble, less toxic and more easily excreted from the body than is the parent compound.

More than 45 different UGT cDNA clones have been isolated from seven mammalian species, including 18 human UGT clones [Court et al 2000 and

Clarke et al 1992]. Based upon homology of primary structures, the UGT

proteins have been categorized into two families, UGTl and UGT2, with UGT2

being subdivided into two subfamilies, UGT2A and UGT2B (Figure 4).

Members of the UGTl family share >50% identity with each other, but <50%

identity with members of the UGT2 family.

11 r UGTl AS |L UGTl A3 i I ‘ ^ UGT1A4 UGT1A1

ri t "^ 1 f- UGT1A7 Ii I! JH UGT1A8 j I I I

I �\.��.��.�-��:>^s ����“ -���:��I森 :.vx.�"i 、��‘-\�� .��1 1i ^― LIGItAS ___J IUGT^BIO j fi 厂 UGT2828 j I L IIGT2B11 j yQT287 i i ! 、；、 I——IIGT2B4 ‘\£ / S、，.， r UGT2815 ! L. UGT2B1? I ^^ (UGT2A2 Viy UGT2A1

Figure 4. Phylogenetic tree of the 18 human UDP-glucuronosyltransferases (UGTs). The UGT proteins have been categorized into two families, UGTl and UGT2, with UGT2 being subdivided into two subfamilies, UGT2A and UGT2B. Amino acid sequences of human UGTl A and UGT2B enzymes were from the GenBank database. UGT1A2, UGTl All, UGT1A12 and UGT1A13 are pseudogenes. The human UGT2B enzymes isolated to date are named according to their chronological order of isolation. Five human UGT2B pseudogenes have also been isolated, whereas 16 UGT2B enzymes have been characterized in rats, monkeys and rabbits.

12 1.5 Multiple Variable First Exons: A Mechanism for Cell- and

Tissue-Specific Gene regulation.

The organization of UGTl was studied for the multiple variable first exon. This

genomic organization provides genetic multiplicity for generating diversity. In

the cases of UGTl genes, the variable exons are very similar to each other and

encode diverse polypeptides.

A tandem array of UGTl variable exons, encoding highly similar but distinct

polypeptides, provides a means for generating such diversity. UGTl proteins

are typical type I transmembrane proteins, that is, they have a single

transmembrane segment located close to the C terminus. The oligomer structure

of UGTl proteins may provide diverse substrate specificity (lyanagi et al. 1998).

The UGTl genes, each variable exon only encodes the N-terminal half of the

protein, without a transmembrane segment. In contrast, the constant region

encodes the C-terminal half of the protein, including the transmembrane

segment (lyanagi et al. 1998). Thus, a single variable exon itself does not appear

to encode a functional UGTl protein. In addition, by using the BLAST program

we searched the human, mouse, and rat EST and cDNA databases with intron

sequences immediately downstream from each variable exon, but could not find

13 any transcripts that contain a variable exon and its immediately downstream

intron sequences. A similar search for the Pcdh clusters reveals many such

transcripts. Therefore, unlike Pcdh clusters, the UGTl cluster does not have

variable-only forms of diversity.

1.6 Evolutionary Origin of the Variable and Constant Genomic

Organization

The variable exons aligned raise important implications regarding the

evolutionary flow. For the UGTl cluster, the variable exons of the bilirubin and

phenol groups appear to be duplicated separately from two congenital variable

exons (Figure 5), which are themselves repeated from a single congenital

variable exon. Some variable exons, for example, mouse UGT1A12 and A13,

are believed to result from species-specific amplification of the same variable

exon. Different from the case of UGTl gene locus, the duplication of the IGnT

variable exons occurred before the disrehtion of the human, mouse, and rat,

because the three variable exons reveal totally orthologous relationships.

14 Variable Region , n ConstanRegion t f 1 i \

I -190 kb 1 Variablw , u^ie Regio. n ConstanRegiont I 1 \ 1

3 , •i~~I ~i—~k- L I �110kb 1 Go 门 stsnt Variable Region Region [ \ \ 1

^ 冷9 令 ^ ^^^ ^ ^ P 一 P 2 3 4 3 -WI • W—f~藥爾丨”^誦 [I 180 kb 1 Figure 5. Similar genomic organization of UGTl gene clusters. Shown are comparisons of the genomic organization of mouse, rat, and human UGTl clusters. Each cluster has multiple, highly similar, tandem variable exons followed by one set of constant exons. They are indicated by vertical colored bars: (green) phenol-type UGTl variable exons; (orange) bilirubin-type UGTl variable exons; (blue) pseudogenes or relics (present in both Pcdh and UGTl clusters); (turquoise) non-UGTl genes in the UGTl cluster; (pink) constant exons (present in both Pcdh and UGTl clusters). The approximate length of each cluster is shown below the corresponding panels. The pseudogenes are represented by a letter "p" following the gene symbol, whereas the relic sequences are represented by a letter "r." (Pcdh) Protocadherin; (UGT) UDP glucuronosyltransferase; (Hsp40L) heat-shock protein 40kd like gene.

15 For variable exons that reveal high similarities，the unit of repeat appears to include both the coding sequence and its upstream regulatory region. For UGTl, within a certain cluster, the coding regions of multiple variable exons not only

share exact sequence, but also have a similar number of amino acids residues.

In fact, the conserved sequence motifs are upstream from the variable exon of

each clusters (Wu et al. 2001). However, a large part of the regulatory

sequences have been diversified. For example, the flanking sequences of the

highly conserved motifs are different among distinct variable exons. This

heterogeneity shows tissue specific or cell specific type of gene expression in

order to interact with development process and the change in the environment.

In some cases, however, for example, Pcdh 计6 and yb7, the promoter

sequences show high homogeneity. This exception was found to be the

consequence of selective pressure during evolution or simply not enough time

for the diversification after the repeat of the cluster; however, this case rarely

shows in the UGTl gene cluster.

In the cases the transcriptional direction for variable exons is the same, that is,

towards the C terminal constant region. Even in the cases of pseudogenes or

relics, the remnant codons are always on the same DNA strand. This shows an

exon repeating process that can exclusively result in a parallel contingent

16 arrangement. In addition, this may also demostrate that the parallel arrangement of variable exons and their promoters ensures that the functional mRNAs can be formed by the cz^-splicing mechanisms.

A number of models have been proposed to explain the conservation of duplicated genes. The classical model for the evolution of repeated genes

hypothesized that paralogous genes arise by gaining new adaptive functions

(Ohno 1970). Recently, the duplication-degeneration-complementation (DDC)

model hypothesized that paralogous genes achieve interconnected subfunctions

of the familial gene (Force et al. 1999). The organization of UGTl is unusual in

that only the first exon was duplicated. Anyway, this organization supports both

different aspects of the two models. The vast expansion of variable exons by

organized arrangements in the UGTl clusters may reflect the narrow-down

selection for the numbers of possibilities required for detoxification. On the

other hand, the prototypical UGTl genes may have been expressed ubiquitously

and had much broader functions in the past.

Compared with variable exons that do not show sequence similarity, such as in

the plectin, NOSl, and GR genes, selective splicing that joins a cap-proximal 5'

splice site to the 3' splice site of the first constant exon removes all the introns.

17 Hence, specific promoter initiation and cap-proximal cw-splicing may have evolved for this type of multiple regulatory strategies. In this case, the duplication of UGTl variable exons, would come to the same evolution for arrays of multiple first exons. At the same time, the origin of aligned tandem was from varying first exons. It was very likely that this organization provides a genetic mechanism for the diverse gene regulation required for complex mammalian development and adaptation.

18 mlJGTIAlO rUGTIA8 ,,, \ j ni UGTl All 丨 All hUGTlAloX / I / liLiGTIAS \ /rUGTlAy^ hlJGTlA? \ \ \ U X MjGTlA9 1 淑丨'丨丨A丨2 — mUGTIA13

70

mU(.rnA9

hUGTlAl rUGTlA? \ \ < / / r^ 100 100 / _^niUGTlAl hUGTlA6 N；^

100 rUGTlAl

rU GT1A 6 Xj ()() 力J /\ .扑hUGTlA4 mliGTlA'S rliGTlA：/ \ / hUGT!A5 mUGTlA2 rUG_TlA4 Figure 6. Phylogenetic tree of human, mouse, and rat UGTl gene clusters. The tree was reconstructed based on variable region polypeptides by using the neighbor-joining method of the CLUSTAL W package. The tree branches are labeled with the percentage support for that partition based on 1000 bootstrap replicates. The scale bar equals a distance of 0.1. [Adapted from Zhang et al 2003:

19 1.7 Variable and Constant Genomic Organizations Exist in Mammalian

UGTs

High throughput genomic sequencing has enhanced the process of annotating

sequences. To date, DNA sequencing of cDNA clones is still the most reliable

method to identify unknown genes. Although EST sequencing can give

valuable information about the annotation in a given genome, it is usually not

possible to provide reliable information about the 5'-end of genes since most

ESTs are derived from the 3'-end. Recently, the Institute of Physical and

Chemical Research (Japan; RJKEN) has sequenced 60,770 full-length mouse

cDNA clones (http://fantom2.gsc.iiken.go.jp/; Okazaki et al. 2002). This large

amount of data still provides limited information on the study of the UGTl gene

cluster, allowing a further study of the genes.

1.8 The history of recombinant UGT expression

Twenty years ago, it was difficult to study UGTs in the liver. It required a

freshly separated tissue, usually an ox liver. The purification of the protein was

one of the most time consuming step. The purification technique was not

advanced enough to give a homogeneous protein. The study was not significant

20 enough since the data was from a species distant from us. The characterization

of the UGT isoforms was greatly enhanced by the modem technology of

cloning and recombinant protein expression. The study of an enzyme was

greatly enhanced when a large amount of the protein can be expressed and

purified. Recombinant protein expression in E.coli provides a low-cost and

efficient means for protein study. In 1988, Harding D. et al successfully

expression phenol-UGT in mammalian culture for the first time (Harding et al

1987). Since then, the study of the family has been rapidly remedied by the

recombinant expression. The study of substrate specificity of different isoforms

can be achieved. The isoform UGTlAl, UGT1A6, UGT1A7 etc. were also

studied through this way. In this study, bacterial expression system was used.

1.9 UGT1A8

Rat UGT1A8 was first detected in 2002 in our lab [Leung et al, 2002]. It was

found with the induction of 3-methycholanthrene in rat hepatoma cells. In 2003,

the tissue expression of this protein in rat was found [Shelby et al, 2003].

However, the range of inductive agents and the properties of this newly

identified protein remained unknown. A more detailed study of the protein

21 would provide insights into UGT. The rat UGT1A8 shares high homology

(higher than 80%) amino acid sequence homology with human UGT1A8;

however, the major site of human UGT1A8 expression is the intestine.

22 1 ^ 1 UGT1A8 "Male 2 4 - * _ Female 1 3- 1 < o 圓女

§ 终 ^ • pi

H 各• M'^il — Jtti --////// //

、。令// ///

Figure 7. Tissue distribution of rat UGT1A8, liver and kidney were found to be the major site expressing the protein. Female rats showed a higher level of UGT1A8 expression when compared to male rats [Shelby et al, 2003]

23 1.10 Licorice and its active component

The word "licorice" originated from Greek words “glyks，，，meaning sweet and

rhiza meaning root. It is the sweet tasting rhizomes that are used as flavourings.

Licorice is native to south-eastern Europe and the Middle East, where it grows

in the wild. Chinese herbalists have long been using licorice, concentrating the

root's extract and prescribing it for a wide range of conditions. The root

contains vitamin E, B-complex, biotin, niacin, pantothenic acid, lecithin,

manganese and other trace elements. It also has beneficial medicinal qualities.

Licorice Root has been used as a laxative; to adjust blood sugar, reduce pain

from ulcer and arthritis. It has been shown that chewing licorice root has been

very helpful in giving up smoking as it gives the hands something to do and has

the shape/texture of a cigarette.

Many researchers have shown that licorice helps treat and relieve pains that

accompany certain types of ulcers, with more and more researchers gaining

positive results with its use as an ulcer treatment. Licorice is getting more

popular in North America. Licorice is shown to have anti-inflammatory

properties [Kawakami et al, 2003, Amaryan et al, 2003] and may therefore help

relieve the discomforts that accompany arthritic conditions.

18(3 glycyrrhetinic acid, one of the active components of licorice, has known to

24 possess pharmacologic effects. It is most well known for its anti-inflammatory

properties, which can inhibit the prostaglandins metabolizing enzymes. It is also

known as a hepatoprotective agent. However, the mechanism of

hepatoprotection is still unclear but it is believed that the chemical can induce

expression of several genes in the liver. o H3q少、OH ...、.八、

O�〜.、火..、.., HsC I cH.j rCH,

Ljq I H CH3

、二 ��z - � A — H /\ H H3C CH3 Figure 8. Structure of 18(3 glycyrrhetinic acid

1.11 Enzyme induction in the liver

Liver is the primary site for xenobiotics metabolism. Different nuclear receptors

like aryl hydrocarbon receptor (AhR) and orphan nuclear receptors have been

shown to be the key mediators of drug-induced changes in phase I and phase II

metabolizing enzymes. It is well known that the expression of CYPl genes can

be induced by AhR, which can dimerize with the AhR nuclear translocator, due

to the presence of many poly cyclic aromatic hydrocarbons. Similarly, orphan

25 nuclear receptors, the constitutive androstane receptor (CAR) and pregnane X receptor (PXR), both form hetero-dimers with the retinoid X receptor (RXR), and are shown to transcriptionally activate the promoters of CYP2B and

CYP3A gene by xenobiotics such as phenobarbital and dexamethasone and rifampin family agents. The peroxisome proliferator activated receptor (PPAR), which is one of the first characterized members of the nuclear hormone receptor,

also dimerizes with RXR and shown to be activated by cholesterol reducing

agent leading to transcriptional activation of the promoters on CYP4A gene.

CYP7A was discovered as the first target gene of the liver X receptor (LXR), in

which the elimination of cholesterol depends on CYP7A. The physiological and

the pharmacological implications of common partner of RXR for CAR, PXR,

PPAR, LXR and FXR receptors largely remain unknown and are under lots of

studies. For the phase II DMEs, there are plenty of phase II gene inducers, for

example, the phenolic compounds butylated hydroxyanisol (BHA),

tert-butylhydroquinone (tBHQ), green tea polyphenol (GTP),

(-)-epigallocatechin-3 -gallate (EGCG) and the isothiocyanates (PEITC,

sulforaphane) generally appear to be electrophiles. They all possess

electrophilic-mediated stress response, resulting in the initiation of transcription

factors Nrf2 which dimerizes with Mafs and binds to the

26 antioxidant/electrophile response element (ARE/EpRE) promoter, which is located in phase II DMEs as well as many cellular defensive enzymes, with the successive induction of the expression of these genes. Along with phase I and phase II enzyme induction, pretreatment with several kinds of inducers has been shown to alter the excretion of the xenobiotics, which reflects that transporters may also be regulated, at the same time gives an important way to protect the body from chemical insults. The stress would lead to the increase in gene expression, which eventually enhances the elimination of these xenobiotics and reactive oxygen species (ROS). The induction of UGTs belongs to the induction of the phase II enzyme, and the detail of the induction is lacking. A novel gene of UGTl A8 was discovered in the rat hepatoma cell-line [Leung, et al, 2002]. It has been shown that the gene can be induced by some polyaromatic compounds.

However, in vivo study is required for a better understanding of the induction

process in an organism.

27 1.12 Objectives

The biochemical functions of the newly identified UGT1A8 in rat are lacking. The present study was aimed at

1. To quantitatively prepare UGT1A8 in E.coli expression system;

2. To purify the enzyme;

3. To understand the biochemical properties of UGT 1A8

The study would provide useful information on the functional roles and substrate specificity of UGTl A8 in detoxification.

28 Chapter Two. Materials and Methods

General Procedure

The project was divided into two main parts: UGT1A8 induction in in vitro and

in vivo systems and recombinant UGT1A8 expression in E. coli. In the first part

of the project, the inductive effects of licorice and 18(3-glycyiThetinic acid on

UGT1A8 were studied with two types of liver cells: clone 9 (isolated from rat

epithelial cells of normal liver) and H4IIE (isolated from rat liver hepatoma).

The results in in vitro study were confirmed with two types of rats: Wistar (rats

commonly used for the study of liver metabolism) and j/j (rats with UGT

deficiency). Livers of treated rats were isolated and the expression levels of

UGTs were investigated with RT PGR and Northern blot analysis. In the protein

expression experiment, the rat liver UGT1A8 coding fragment was isolated and

cloned into prokaryotic expression system. Recombinant protein expression was

performed with E.coli (strain JM109) system in order to prepare sufficient

amount of protein for further studies. The purified protein was characterized for

its substrate specificity.

29 2.1 UGT1A8 induction studies

2.1.1 Preparation of inducers

The dried herb was purchased from a local store and used for the whole project.

Licorice (50 grams) was soaked in 500 ml of distilled water for one hour and

boiled to a final volume of 100 ml in two hours. The extract was kept in 4�C

before rat feeding. 18(3-glycyrrhetinic acid was dissolved in nano pure water.

The solution was also kept at 4� Cin a fridge before use. All the reagents were

discarded after one month to prevent any auto-degradation. Reagents that used

in cell culture experiments were 0.2jam syringe-filtered just before use.

2.1.2 Cell viability study with Neutral Red Assay

Rat hepatoma cells (H4IIE) and rat liver epithelial cells (clone 9) were

previously purchased from ATCC and used to study UGT1A8 induction. Cells

were seeded with a 96-well format (5000 cells in each well) and after

incubation for twenty four hours, different concentrations of drug were added to

the cells. After the desired incubation period of time, the plates were harvested.

First, wells were washed with 0.2mL of IXPBS, and then 0.15mL of 5mg/mL

neutral red (Sigma) solution was added. The plate was allowed to be further

incubated at 37°C for two hours in incubator. The solution was removed and

30 the plate was allowed to dry overnight in a 55� Coven. The red pigment was resuspended in 1% SDS just before spectrophotometric measurement at 545nm.

2.1.3 Cell treatment

Cells were seeded 24 hours before the addition of drugs with 100mm dish

format (0.5 x 10^ cells). The desired amount of drug was added to the plate and

incubated for different periods of time. RNA was extracted and analyzed for the

expression of the target gene.

2.1.4 Rat treatment

Both wistar rats (weighing around 200gram) and j/j (weighing around 200gram)

rats were divided into three groups, with five rats in each group. The dosage of

the licorice extract was 20mg/kg/day and 160mg/kg/day of 1 Sp-glycyrrhetinic

acid for the treatment group to yield the over-expression of UGTl A8. Rats in

the control group received water instead.

2.1.5 RNA extraction from rat liver and cell culture

Rat liver RNA was extracted with Trizol reagent (Invitrogen) with an

established protocol. Briefly, 100 mg of liver tissue or one 100 mm dish of cells

31 was homogenized with 1 mL Trizol reagent. The sample was subjected to 13000

X g for fifteen minutes at 4°C ； the supernatant was taken and mixed with 0.2mL

of chloroform. The sample was vortexed and allowed to stand at room

temperature for ten minutes. The process was followed by centrifugation

(13000 X g) for fifteen minutes at 4°C. The supernatant was taken and mixed

with ImL of isopropanol. The mixture was allowed to stand for fifteen minutes

at -20°C, followed by centrifugation (13000 x g). The pellet was washed with

75% ethanol and air dried. The pellet was subsequently dissolved in 45 //1 of

DEPC-treated water. The samples were stored at -20for less than three

months.

2.1.6 Quantization of RNA

RNA was diluted to 1000 fold with IXTE buffer, pH8.0 and the optical density

was measured at 260nm with a Beckman DU650 spectrophotometer. It is

known that OD260 times the factor 40 would be equal to the RNA concentration.

Each RNA sample was measured three times and the average reading was taken

for RNA concentration.

32 2.1.7 Denaturing gel electrophoresis for RNA

The gel consisted of 1.5% agarose, 0.04mM MOPS and 6.8% formaldehyde.

RNA was sepearated by electrophoresis at lOOV for thirty minutes. The resultant gel was stained with O.lmg/ml ethidium bromide and observed under

UV. Both 28S and 18S RNA bands were observed as an indicator of RNA

integrity.

2.1.8 Northern hybridization

RNA (30|ig) was separated in a 1.5% denaturing gel. RNA was transferred to

Hybond-N+ (Amersham Bioscience) membrane by capillary force overnight.

RNA was immobilized by a UV cross-linker with maximum power for thirty

seconds. The membrane was hybridized with ^^P-labelled cDNA probe at 65�C

for two hours, followed by thirty minutes of wash with 2XSSC, 0.1% SDS at 65

�C tw, o rounds of twenty minutes of 0.2XSSC with 0.1% SDS wash at 65

and 2XSSC at room temperature for five minutes. The membrane was subjected

to autoradiography at -70� Cfor sixteen hours. The densities of the bands were

analyzed with ImageJ imaging program.

JO 2.1.9 Probe for Northern Blotting

DNA probe was synthesized with PGR. The sequence of the probe was checked

with DNA sequencing. Radioactive labeling was performed with Rediprime

labeling kit (Amersham Bio Science).

2.1.10 Agarose Gel analysis and Northern Blot analysis

Gel photos and exposed films were scanned with Epson Perfection® 3170

Scanner. Gel analysis was performed by the software Image J (NCBI). Film of

Northern blotting was also scanned and analyzed by the software.

Table 1. Sequences of primers used in RT-PCR

Forward primer Reverse primer

UGT 1A1 5' acaccggaactagaccatcgS’ 5’ tgggtcttggatttgtgtgaS’

UGT 1A7 5' ttcaccatgcagatggttgtS‘ 5, ctgggcagggctagtcagta3，

UGT1A8 5' atggctccttcaggctgtcS' 5 ‘ actttcccatgtgccaactc3 ‘

(3actin 5 ’ agccatgtacgtagccatccB ’ 5 ’ ctctcagctgtggtggtgaaS,

34 2.2 Recombinant expression of UGTl A8 in E.coli JM109

2.2.1 cDNA synthesis

RNA (3jig) was mixed with 0.5mM dNTP mix, IX reaction buffer and 200

units MMuLV reverse transcriptase (Amersham) and incubated at 37°C for 45

minutes. Autoclaved water (20|i,L) was added to dilute the mixture. Synthesized

cDNA samples were stored at -20°C until use. Samples were not stored for

more than half a year.

2.2.2 Polymerase chain reaction

Polymerase chain reaction was performed with 1 |iL of cDNA, 1X PCR buffer,

0.75mM MgCli, 0.6|iM sequence specific primer, lU of Taq DNA polymerase,

followed by different PCR conditions with ABI GeneAmp ® 9700 thermocycler.

2.2.3 Agarose gel electrophoresis for DNA

Agarose (Amersham Biosciences) was added to 0.5X TBE to a final

concentration of 1.5%, Ethidium bromide (USB) was added to a final

concentration of 0.05jig/mL. DNA was separated at a constant voltage of 120 V

for twenty minutes. The gel was visualized with a gel documentation system

(UVItec).

35 2.2.4 Amplification of target gene, UGT1A8

UGT1A8 insert was amplified from liver cDNA with UGT1A8 specific primers

(forward: 5’ ctcgagatggctccttcaggct 3'; reverse: 5’ aagctttcagtgggttcttg 3') under the following conditions: 94� Cfor five minutes; 94� Cfor forty five seconds,

55� Cfor forty five minutes, 72°C for one minute and thirty seconds for 28

cycles; 72� Cfor ten minutes with Pfu polymerase (Promega). The amplified

DNA fragment was purified with Nucleospin DNA purification kit (BD Falcon)

and used as template for another round of PGR with the same conditions. The

DNA was quantified; part of the sample was taken for restriction enzyme

digestion.

2.2.5 Restriction enzyme digestion of plasmid and insert

pRset (Invitrogen) plasmid (l|ag) or PGR product was mixed with Img/mL BSA

and IX buffer and incubated with lU of Hindlll and Xhol (New England

Biolab) in a 37°C water bath for one hour. Sample was analyzed by an agarose

gel to confirm complete digestion. The resultant DNA fragment was purified by

Mini-M gel purification system (Viogene).

36 2.2.6 Ligation of plasmid and insert DNA

The digested plasmid and DNA fragment was incubated at 4°C with lOU of T4

DNA ligase (New England Biolabs) with IX T4 ligase reaction buffer in a 20[iL reaction for eighteen hours.

2.2.7 Amplification of target plasmid

The total 20 fiL ligation reaction was used in the transformation of DH5a

competent cells (Invitrogen). The DNA was first incubated with the cells for

twenty minutes on ice. Immediately, the whole mixture was placed in a water

bath at 42°C and subjected to heat shock for exactly 45 seconds. The bacteria

were allowed to recover on ice for 5 minutes. Then LB medium (500|jL) was

added to the mixture and the whole reaction was incubated at 37� Cfor one hour.

Bacteria (50jiL) were spread on a LB plate containing 1 OOjLig/mL ampicillin.

The plate was incubated for 16-18 hours at 37�.C

2.2.8 Screening of target plasmid

Twenty colonies of bacteria were randomly picked and grown in 5 mL LB

medium with lOOp^g/ml ampillcin for ten hours. The bacterial cells were taken

for plasmid extraction with plasmid Mini-M purification kit (Viogene). The

37 DNA was taken for restriction digestion. Plasmid that can be digested into two

and with the expected size was taken for DNA sequencing for further

confirmation of the insert.

2.2.9 DNA sequencing

DNA sequencing was performed by Tech-Dragon Company which provided the

service. The result was checked by NCBI (BLAST) for the identity.

2.2.10 Transformation of protein expression host

The confirmed plasmid was used to transform protein expression host JM109.

The procedure was similar to the one described previously.

2.2.11 Confirmation of transformation of protein expression host

Transformed colonies were randomly selected and picked out for colony PCR

with the following conditions: single colony was picked and mixed with IxPCR

buffer, 1 ！1M gene specific primer, ImM magnesium chloride, 0.2mM dNTP

mix, lU of Taq DNA polymerase. Clones that showed the expected band were

inoculated into LB medium.

38 2.2.12 Protein expression

Bacteria were cultured to mid-log phase before IPTG induction. When the

OD600 of the culture was between 0.4 and 0.6, a final concentration of 0.5mM of

IPTG was added to the culture and six hours later the cells were harvested by centrifugation. Successful protein expression was confirmed by SDS-PAGE

showing protein bands with expected size (55kDa).

2.2.13 Protein purification

Bacterial cells were harvested by centrifugation at 5000xg for 20 minutes. Cell

pellet was resuspended in binding buffer (O.IM Tris-HCl buffer, pH8.0). The

cells were lysed by sonication on ice. The mixture was centrifuged at 5000 x g

to remove unbroken cells and large debris; then the sample was further

centrifuged at 12000 x g to remove any remaining debris. Protein purification

was performed with a Ni+ column. The sample was allowed to bind with the

matrix at 4� Cfor one hour. Then the matrix was washed with 10 volumes of

binding buffer under gravity to remove any unbound protein. After this, the

matrix was washed with five volumes of washing buffer (with lOOmM

imidazole). The protein of interest was eluted with three volumes of elution

buffer (with SOOmM imidazole). The protein was collected in fractions of

39 0.5mL •

2.2.14 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE containing 0.1 % SDS was performed in 12 % slab gel according to the method of Laemmi (Laemmi, 1970). BioRad Mini-PROTEAN system was employed for electrophoresis. Precipitated samples were incubated in

SDS-PAGE sample buffer (125 mM Tris-HCl, pH6.8, 1 %w/v SDS, 5 %v/v glycerol, 10 mM dithiotheritol) at boiling temperature for 5 minutes. Protein marker (BENCHMARK™ Jnvitrogen) was used. Protein bands were stained with Coomassie brilliant blue R-250 (Boehringer Mannheim) in fixing solution of 30%v/v ethanol (Merck) and 10 %v/v acetic acid (Merck) for 30 minutes.

Stained gel was destained in destaining solution (30 %v/v ethanol, 10 %v/v acetic acid) till protein bands were visible.

2.2.15 Confirmation of the protein

Different fractions of the chromatography were analysed by SDS-PAGE to check for the target protein. A protein band at 55kDa was believed to be the target protein.

40 2.3 Characterization of recombinant UGT1A8

2.3.1 UGT assay

UGT assay was performed under the following condition: O.IM Tris HCl,

pH7.4, 3mM UDPGA, 5mM MgCb and 50^M of the substrate. The mixture

was pre-incubated at 37°C for 5 minutes followed by the addition of the purified

target protein. After 30 minutes of incubation, the reaction was stopped by

NaOH. The concentration of the substrate was measured. The substrate

concentration was determined using a standard curve.

2.4 Routine experiment methods

2.4.1 Determination of protein

A commercial kit, purchased from BioRad, was employed for the determination

of protein. This assay kit was based on the method described by Bradford

(Bradford, 1976). In each determination, protein samples were diluted with

water, to make up a volume of 800 \xL A volume of 200 jil of the assay dye was

added. Color development was allowed for 5 minute. Absorbance was measured

at 595 nm.

41 2.4.2 Nucleic acid purification

PCR products were purified by using Nucleospin gel purification kit (BD

Biosciences) according to procedures supplied by the kit manufacturer. For products excised from agarose gel, agarose gel was dissolved in NTl buffer supplied at 55°C. For any PCR products in solution form, the volume of solution was first added to 100 |il by TE buffer (0.01 M Tris, 1 mM EDTA, pH

7.4) before mixing with NT2 buffer. Samples were loaded to a spin column, which was washed twice with NTS buffer. Nucleic acid was eluted from column by NE buffer (5 mM Tris-HCl, pH 8.5) which had previously warmed (55°C).

2.4.3 Preparation of chemically competent bacterial cells

Competent cells were prepared by rubidium chloride method. One ml of culture of competent cells grown overnight was inoculated into 100 ml Psi broth (5 g/L bacto yeast extract, 20 g/L bacto tryptone, 5 g/L magnesium sulfate, pH 7.6).

The culture was grown at 37°C with aeration until OD550 reached 0.4. The

culture was cooled for 15 minutes. Cells were collected by centrifugation at

5,000 g for 8 minutes. The pellet obtained was resupended in Tfbl buffer (30 mM potassium acetate, 100 mM rubidium chloride, 10 mM calcium chloride,

50 mM manganese chloride, 15 %v/v glycerol, pH 5.8) and iced for 15 minutes.

42 After incubation, centrifugation was carried out at 5,000 g for 8 minutes. The pellet was respended in Tfbll buffer (lOmM MOPS, 75 mM calcium chloride,

10 mM rubidium chloride, 15 %v/v glycerol, pH 6.5). Resupended cells were quickly frozen in liquid nitrogen, and stored at -80°C. These cells were now ready for transformation by heat shock.

2.4.4 Colony PGR

Bacterial colonies were picked and lysed in lysis buffer (TE buffer, pH 8.0, 0.1

% Tween 20) at boiling temperature for 10 minutes. Plasmid DNA released was

used as the template for the subsequent PGR. A primer pair flanking the

multiple cloning site of the plasmid was used. Each reaction contained 1 mM

dNTP mixture, 0.25 jiM primers and 0.2 |il Taq polymerase (Sangon). The

reaction was performed in reaction buffer supplied by manufacturer. The PGR

mixture was incubated at 94°C for 3 minutes followed by 30 cycles of

amplification. Each cycle consisted of 45 seconds of denaturation at 94°C, 1

minute of annealing at 55°C and 1 minute of extension at 72� CA. final

extension step of 72°C for 10 minutes was performed after thermo-cycling had

been completed.

43 2.4.5 Plasmid rescue by alkaline lysis

Plasmids were purified from overnight-cultured competent bacterial cells by alkaline lysis method. The Rapid Plasmid Minipreps system (Marligen) was employed in this work. Cells were collected by centrifugation at 10,000 g for 2 minutes. Any pellets obtained were suspended in cell suspension buffer (50 mM

Tris-HCl, pH 8.0, 10 mM EDTA, 20 mg/ml RNase A) supplied. Cells were

subsequently disrupted by incubating in cell lysis buffer (200 mM NaOH, 1 %

SDS w/v) for 5 minutes. After neutralization of cell lysate, lysate was loaded

into purification column. The column was washed by two successive washings.

Plasmids were finally eluted by NE buffer (5 mM Tris-HCl, pH 8.5) previously

wanned to 60�C.

2.4.6 Charging of His-tagged column

The His-tagged column was first washed with ten volumes of nanopure water.

Three volumes of 50mM nickel sulphate solution were added, then followed by

5 volumes of nanopure water and five volumes of equilibration buffer.

44 2.4.7 Washing of His-tagged column

The His-tagged column was washed with five bed volumes of IM imidazole solution, followed by ten bed volumes of nanopure water. The column was stored in 20% EtOH at 4°C.

45 Chapter Three. Results 3.1 UGT1A8 expression in clone 9 and H4IIE after treatment with licorice and ISp glycyrrhetinic acid

Previous studies have shown that licorice was used in formulations to treat and

relieve pains associated with ulcers and liver diseases. Licorice was shown to

have therapeutic benefits including anti-inflammatory properties and may

therefore help relieve the discomforts with arthritic conditions [Kawakami et al,

2003, Amaryan et al, 2003].

ISP glycyrrhetinic acid, one of the active components of licorice, is known for

its anti-inflammatory properties, which can inhibit the prostaglandins

metabolizing enzyme [Tsukahara et al, 2005]. It is also known to have

hepatoprotective properties. However, the mechanism of hepatoprotection is

still unclear. Therefore, to study the biologic activity of licorice and 18(3 GA in

the liver would allow us to get a better understanding of the pharmacological

effects of licorice and its components on the liver.

In order to study the effect of licorice extract and 18(3 GA on the liver, clone 9

and H4IIE cell lines were employed to study the cellular responses. The cell

viability in the presence of licorice was measured to evaluate its potential

toxicity. Figure 9 shows that the viability of H4IIE decreased with the

46 incubation time. Licorice extract seemed to induce considerable toxic effect after twenty hours incubation. The cellular response of clone 9 showed similar trend (Figure 10). Both cellular responses appeared to decrease in a time-dependent manner. The period of incubation is important in affecting cell viability.

Likewise, 18(3 GA was used to treat H4IIE (Figure 11) and clone 9 (Figure 12) to study its effects on the cell lines. Both cell lines responded in a similar manner with time. The cell viability decreased in a time-dependent manner.

To determine the potential toxicity of the licorice extract and 18(3 GA, different concentrations of the licorice extract and 18(3 GA were incubated with H4IIE and clone 9 cells. Figure 13 showed the viability of H4IIE significantly decreased and become level off when the concentration of the licorice extract exceeded 0.1 mg/ml.

Table 2. Summary of IC50 in cell lines

Licorice extract (mg/ml) 18(3 GA (fiM)

H4IIE 0.076 50

Clone 9 0.058 52

47 The IC50 of licorice and 18(3 GA in H4IIE and clone 9 were summarized in table

2. Figure 14 indicated toxic effect of licorice extract on clone 9 was observed.

Also, Figure 15 showed the toxic effect of ISp GA on H4IIE after 48 hours of incubation while Figure 16 showed the same in clone 9. The results showed that

18PGA had toxic effects on H4IIE and clone 9. The viability of both cell lines

decreased significantly when the concentration of 18(3 GA exceeded 5jiM and

become level off when concentration was greater than 125jiM. As a result, the

concentration of licorice extract (20mg/kg/day) and ISp GA (160mg/kg/day,

approximately 60}iM of 18(3 GA based on its molecular weight) were used to

study their effects on UGT expression. UGTs are one of the important

detoxifying enzymes in the liver. The activity of UGTl A1 could affect

detoxification of foreign substances.

48 120�

J ~•~ treated cells 100 -. • - - control T .T _

一 80 - T

I T 、、；… a 60 - p--—

> 40 - ^^^^^^ …

20 -

Q 1 L_ I I I I L_ 1 1 1_ 1 1

0 10 20 30 40 50 60 70 80 90 100 110 120

Time (hours)

Figure 9. Time dependent cellular response analyzed with neutral red assay of H4IIE cells. H4IIE cells were treated with licorice extract for different periods of time up to 120 hours. The data represent the average of five independent experiments. (n=3, p<0.05)

49 120�

100 p f--. _ —^treated cells •、- -- -. , T •• - - - --令--control i i 丄 T

尝

罢 60 - I ^^

40 - ^^^^^

20 -

Q I I \ I I I I I I ] I I

0 10 20 30 40 50 60 70 80 90 100 110 120

Time (hours)

Figure 10. Time dependent cellular response analyzed with neutral red assay of clone 9 cells. Clone 9 cells were treated with licorice extract for different periods of time up to 120 hours. The data represent the average of five independent experiments. (n=3, p<0.05)

50 100 「 了-.... •treated cells 90 _ 丄 --•…control _、--.•. 80 - T 丁

一 7。_ .、、•••“、..•〜....： I 60 - i 50 _ I 4�L

30 -

20 -

10 -

Q I i I I 1 i 1 1 1 1 1 1

0 10 20 30 40 50 60 70 80 90 100 110 120

Time (hours)

Figure 11. Time dependent cellular response analyzed with neutral red assay of H4IIE cells. H4IIE cells were treated with 18(3GA for different periods of time up to 120 hours. The data represent the average of five independent experiments. (rr=3, p<0.05)

51 100「

I� .� T 90 I " • • - . T i " ... 80 , " 丁 -^treatedcells 丄T •-�---. LJ • - • control -7�[ T

I 60 一 、\ • I T ：； ^ 50 L I 丢 I ^ 40 L

20 - 1

10 - 0 ’——^ —^ ^ 1 1 1 I 0 1 0 20 30 4 0 50 60 70 80 90 100 110 120 Time (hours)

Figure 12. Time dependent cellular response analyzed with neutral red assay of clone 9 cells. Clone 9 cells were treated with 18[3GAfor different periods of time up to 120 hours. The data represent the average of five independent experiments. (n=3, p<0.05)

52 100 -r 9。\

80 - \

70 - }\

I 60 - \ !5。_ \

I 40 - \

3� _ T 2。 ^^^^^^^ T

10 - 0 ‘ ‘ ^ ‘ ^ J 0 0.1 0.2 0.3 0.4 0.5 0.6 Licorice concentration mg/ml

Figure 13. Cellular response of H4IIE cells after 48 hours of licorice treatment. The data represent the average of five runs. The viability (%) shows the change relative to the control. (n=3, p<0.05)

53 100「

\ I 50 - \ 1 40 I \J

0 ‘ ‘ ‘ ‘ ‘ ‘ 0 0.1 0.2 0.3 0.4 0.5 0.6 Licorice concentration mg/ml

Figure 14. Cellular response of clone 9 cells after 48 hours of licorice treatment. The data represent the average of five runs. The viability (%) shows the change relative to the control. (n=3, p<0.05)

54 100「 ！

90 I

80 p :。l 書 6。[k

I 50 [

：2 40 L \ ^ I 丄\

30 一 X

20 - T

10 - ~ 0 ^ ^ ‘ ‘ ‘ 0 50 100 150 200 250 300 Concentration ofl8/S G A( M)

Figure 15. Cellular response of H4IIE cells after 48 hours of ISp GA treatment. The data represent the average of five runs. The viability (%) shows the change relative to the control. (n=3, p<0.05)

55 100 r

90

80 一

70 I 60 - M, I 5。 ^^

I 40 -

30 -

20 - 、 _

10 -

Q ‘ i ： 1 I 1 1

0 50 100 150 200 250 300 Concentration ofl8/SGA M)

Figure 16. Cellular response of clone 9 cells after 48 hours of 18(3 GA treatment. The data represent the average of five runs. The viability (%) shows the change relative to the control. (n=3, p<0.05)

56 In order to study the effect of treatment process on the expression level of UGT,

RT-PCR of P-actin as the standard in H4IIE and clone 9 was carried out. Cells were treated with licorice extract and ISp GA for different periods of time

(Figure 17). Essentially, there was no difference on the expression level of

P-actin among cell lines compared with the control (n=3, p<0.05). In addition, longer incubation period did not seem to affect the expression level of P-actin.

Likewise, the expression level of UGTl A1 in H4IIE and clone 9 was measured

(Figure 18). There was a slight increase of UGTl Al after treatment of cells for

48 hours of incubation, while the expression level slightly decreased after 96 hours of incubation with licorice extract. On the other hand, treatment of cells with ISp GA showed a noticeable increase in the expression level after 96 hours

incubation. By the same approach, the expression of UGTl A7 which showed a

higher homology with UGT1A8 (Figure 19) and UGT1A8 (Figure 20) were

measured in H4IIE and clone 9. Figure 19 showed that the expression level of

UGT1A7 considerably increased after treatment (n二3, p<0.05). 18(3 GA was

more effective on the induction of UGT1A7 in H4IIE and clone 9. The

expression level of UGT1A7 after 48 hours was slightly higher than that at 96

hours.

57 Furthermore, the expression level of UGTl A8 was measured in cells treated with licorice extract and ISp GA for 48 hours and 96 hours. Similar trends of expression to UGTl A7 were observed. Effects of licorice extract and 18(3 GA on UGTl A8 expression was about the same after 48 hours incubation while the expression level decreased after 96 hours incubation.

58 1.2 -

品 0.8 - •S a 0.6 - cd S

P^ 0.4 -

0.2 -

0 ~——~‘——•~——‘~——‘―^~——~‘ ‘ ‘ ‘ control H4IIE licorice clone9 licorice H4IIE 18 j3 clone9 18 /3 H4IIE licorice clone9 licorice H4IIE 18 曰 clone9 18 冷 48 hours 48 hours GA 48 hours GA 48 hours 96 hours 96 hours GA 96 hours GA 96 hours Figure 17. RT PGR results of P-actin level in cells after treatment with licorice extract and 18(3 GA. RNA was extracted from treated cells and cDNA was synthesized with the RNA. (3-actin specific primers were used to amplify the specific gene from the cDNA. PGR products were visualized with agarose gel with ethidium bromide, (n二3, p<0.05)

59 1.6 -

1.2 - 丄 T rt"

QJ 丁 03 1 T

8 內 0.8 -

1。.6 -

0.4 -

0.2 -

0 ~——丨~——~^——^~——~‘“——~‘“——~‘~——‘——• control H4IIE licorice clone9 licorice H4IIE 18/3 clone9 18^ H4IIE licorice clone9 liconce H4IIE 18/3 c]one9 18 j3 48 hours 48 hours GA 48 hours GA 48 hours 96 hours 96 hours GA 96 hours GA 96 hours

Figure 18. RT PCR results of UGTlAl level in cells after treatment with licorice extract and 18(3 GA. RNA was extracted from treated cells and cDNA was synthesized with the RNA. UGTlAl specific primers were used to amplify the specific gene from the cDNA. PCR products were visualized with agarose gel with ethidium bromide. (n=3, p<0.05)

60 2[ r^内 内 ^ n h M h n r^ h 1.4 - 丄

！卜r^ I 0.8 -

0.6 -

0.4 -

0.2 -

0 ——‘ 丨——~‘——~‘——~‘~——‘~——~‘——~^~——~‘ control H4IIE licorice clone9 liconce H4IIE18/3 clone9 18/S H4IIE licorice cloneQ licorice H4nE18/3 clone9 18/3 48 hours 48 hours GA 48 hours GA 48 hours 96 hours 96 hours GA 96 hours GA 96 hours

Figure 19. RT PGR results of UGTl A7 level in cells after treatment with licorice extract and 18p GA. RNA was extracted from treated cells and cDNA was synthesized with the RNA. UGT1A7 specific primers were used to amplify the specific gene from the cDNA. PCR products were visualized with agarose gel with ethidium bromide. (n=3, p<0.05)

61 2 -

1.8 - 1.6 - pi T

1.4 - I 1.2 - T r^ T 区 1 - m n

CO 丄 ""i 0.8 -

0.6 -

0.4 -

0.2 • 0 ~——‘~——~^~——~‘ ‘ ‘ ‘ ‘ ‘—”‘ control H4IIE licorice clone9 licorice H4IIE18yS clone9 18/3 H4IIE licorice clone9 licorice H4I正 18 召 clone9 18/3 48 hours 48 hours GA 48 hours GA 48 hours 96 hours 96 hours GA 96 hours GA 96 hours

Figure 20. RT PGR results of UGTl A8 level in cells after treatment with licorice extract and 18(3 GA. RNA was extracted from treated cells and cDNA was synthesized with the RNA. UGT1A8 specfic primers were used to amplify the specific gene from the cDNA. PGR products were visualized with agarose gel with ethidium bromide. (rr=3, p<0.05)

62 3.2 UGT1A8 induction in wistar and j/j rats after treatment

(

The in vitro study showed positive effects on the expression of UGTs. Thus,

effects of licorice extract and 18(3 GA on wistar and j/j rats were studied. Wistar

serves as a wild type while j/j is UGTl A1 deficient. After the treatment, rat

livers were isolated and RNA was extracted in rat livers (Figure 21) and the

expressions of genes of interest were evaluated. (3 actin serves as a standard for

the expression level. Figure 22 shows the expression of p actin in the control

and rats treated with licorice extract and ISp GA for different periods of time.

The results indicate that there is no difference in (3 actin expression among the

control and the treated group [Lee et al, 2004].

Figure 23 shows that the level of UGTl A8 was increased after treatment of rats

with licorice extract and 18(3 GA for different periods of time. The level of

increment in wistar group was found to be higher than that of the j/j group. The

induction with ISP GA was stronger than that of licorice. The induction

processes in the treated rats appeared to increase in a time-dependent manner.

The results in Figure 24 shows that both licorice and 18[3 GA treatment could

induce UGT1A8 to a different extent. The level of induction of UGTl A8 after

treatment with 18|3 GA were higher than that with licorice treatment. The levels

of induction of UGTl A8 in wistar were higher than that in j/j group [Lee et al,

63 2004]. The results confirmed the expression of UGT1A8 in rats in RT-PCR

experiment. In relation with the UGT1A8 expression, the expression level of

UGTlAl in rats was also analyzed. The results (Figure 25) show that there is

no significant difference in the expression level among the treated group (n=3,

p<0.05). The treatment process did not seem to affect UGTl A1 induction in

wistar and j/j rats.

64 I 2 3 4 5 6 SS SS SS^ IB! •,

__Piiti m^mmmm^mmmm^^^m^^^^mmmmrn^^^*__觀_圖___圓__画議_____ 厂：“'…K 、/ , 二’ /a “。 V 、-':',/ ''' ‘ ‘ 7, ‘； ；

…、：‘冗。雄r;:力、

':、'‘“‘；〜义乂 ‘广“义"、•么'/,/'"‘'' ‘ ''' ：‘ ‘ ‘‘ '二

讓?______

“‘;,:';〜“；；‘' 7, r'r?, ‘ '/>f。'l二 驗塞織ill 戀乡iliiliiii 凝iii ii^iijiili凝縫毅凝ii 毅錢i Hiijii^il絲縫敏法麵越逾®磁资翁鍵效叙被級

Lane 1 and 2: rats in control group Lane 3 and 4: rats treated with licorice extract Lane 5 and 6: rats treated with ISp GA

Figure 21. RNA extraction from the Wistar rat livers RNA samples were separated under denatured condition with formaldehyde with 1% agarose gel under constant voltage (lOOV) for 35 minutes. The gel was stained with ethidium bromide. Both 28S and 18S were analyzed in order to check for the integrity of the RNA samples. The samples were taken as starting materials for RT-PCR and Northern Blot. The results showed good quality of samples prior to RT-PCR and Northern Blot analysis.

65 1.4 - 1.2' - rt fi rnp-jp-r, T fflT H "

0.8 - ：；：；：：：：

C .：.；.；.； ；.：.；.； ：•：•：•：• •：•：•：• I ^ 1：!：；：；： ：!：!：!： • wistar i …“� 丨i"� loj/j ^ 0.6 - ……:i ：;;；：：； 0.4 - ：；；：：；：； 丨 0.02 - ：；：；：；；； ；：；；；：： ——^^^^——^^^——‘——^——‘ control licorice 1 week licorice 4 weeks ISySGA 1 weeks 18 GA 4 weeks Figure 22. The results of RT-PCR after treatment showed the level of P-actin. The level of P-actin was not affected by the treatment, (n = 3, p < 0.05).

66 2 ‘

1.8 _

1.6 - J

_ r^ Mr 士 二 1.2 - Mi 3 盡 _ "H •：：i • ： • ：；：；：I ； ： 1 S J ^ ：；:；：；： ：；；；：；： ：；：；:；:； • wistar

I 1 ‘ 丄：丨:⑴：丨 ；;；;；;；: ……：： loj/j Tj :•:•:•:• •：•：•：• ...... ：•：•：• •:•:•:•: DS ：：：：：：：： ：：：：：：;： ::::::: ::::::::

0.8 - :::::::: ：：：：：：； ：丨:::::： ；：：：；：：：

0.6 - ：；：：：；：： ：：；：；：；； ：；；：；：； ；：；：；：；；

0.4 - ：;：；：；：： ;：;：;：; i::丨;：;：;：;：

0.2 - ：;：:：;：; ;：;；;：: i::::::: i；;：;；;： 0 ‘ ‘”” 丨::::::“~‘ ^^^^^ ‘ ‘ control liconce 1 week licorice 4 weeks 18/3GA 1 weeks 18/5GA 4 weeks

Figure 23. RT-PCR result of UGTlA8 level after reatment. The relative unit is defined as the ratio of the intensity of the UGT1A8 band to the intensity of the housing keeping gene ((3-actin) (n=3, p<0.05).

67 2厂 1.8 - r-pTT 1.6 L r^ ⑴：⑴丨

1.4� . T mm丨 ：丨:丨;…

r^ ：：⑴：丨：

.1.2 - _ 11 __

S T T ；:；：；：；： ：：：：：：：： ::::::: :::::::: 口 wistar

I 1 卜 丄 ::::::: I；；;：;：; gyj T3 ； •:•;•: ^ :•:•；•:• :::•： •:•:•:• •:•:•:•; Di ：：：：：：：： ；；：：：：：： • ：：：：：：：：

0.8 [- :::::::: ：：：；：；：； ；：；：；：；： ::::::: i：：：；：；：

0.6 - ；；；；；；；； ：；：；：；：； ；；；：；：；： ：；；；!；： ；:；：；：；：

0.4 - :::::::: ：：：；：；：： ；；；：:：；； ::::::: ；：；：；：：： 0.2 - ：；：；：：：； ：；：；；；；； ⑴::::： ：出：：： ；：；；；：；；

control licorice 1 week licorice 4 weeks IS^SGA 1 weeks 18冷GA 4 weeks

Figure 24. Northern blot analysis of UGT1A8 mRNA samples after treatment of rats for 1 week and 4 weeks. (n=3, p<0.05)

68 1.4� I 1.2 - y

I 丄丁 丄 T^TfT 1 - I—~^T^ ::::::丨 丄丨:::丨;：;：;：; T r^n^ I : j: j: I: I i: i: i 二 i i:!二！二 i 二 二！二 i 二 i 二

0.8 - ：;；：：;：; ;：;；;；; ;；;；;；;； ；：；：；：； ；：；：；；；；

0 ；：：：：：：： ：：：：：：： :::::::: ::::::: :::::::: 口 wistar 1 ::::::: ayj

0.4 - ：；：：；；：； 丨:::::： ：::::::： ；；；：；：； ；：；：；：；： i :::::::: ::::::: :::::::: :::::::

0.2 L ：；：：：；：； 丨：丨:"： :；;：;：;； ::::::: ：：；：；：：：

0 丨:::::::丨 丨：：丨：丨"丨 丨：：：：：：：丨 1::::::;丨 I control liconce 1 week licorice 4 weeks 18 /S GA 1 weeks 18/SGA 4 weeks

Figure 25. Northern blot analysis of UGTl Al after treatment of rats for 1 week and 4 weeks. The levels of UGTl A1 in the livers were not significantly affected (n 二 3, p<0.05).

69 3.3 Construction of pRset-UGTlA8 Vector

In order to study the biochemical properties of UGT1A8, a sufficient quantity

of the enzyme needed to be prepared. However, the expression level of the

enzyme in cell lines is far from sufficiency. Therefore, the enzyme was

expressed in E.coli expression system. UGT1A8 mRNA was isolated from the

rat liver and the UGT1A8 coding fragment was amplified and cloned into

expression vector. E. coli cells were transformed with the expression vector

followed by IPTG induction to express the protein. Subsequently cells were

lysed and purified to prepare the target protein. Figure 26 showed the

amplification of the target gene UGT1A8 in the rat liver. The results indicated

that a successful amplification of the target gene. Figure 27 showed that the

presence of the desired construct in the positive clones. The E. coli expression

system can produce a sufficient amount of protein for the present study.

Figure 28 indicates the BLAST search result of DNA sequence of the plasmid.

The results showed there was no mutations in the insert and provided

confirmative evidence for the plasmid insert sequence. Figure 29 showed

SDS-PAGE of the expression of the target protein after IPTG induction. Lanes 2

and 3 indicated the target protein was successfully expressed in 0.5 // M IPTG

at 37°C for 5 hours. Subsequently, the protein was purified from the cell lysate

70 by centrifugation, followed by chromatography through Ni-column. Figure 30 showed the protein content of fractions isolated from Ni-column and pellets obtained from centrifugation, while Figure 31 showed the activities of purified protein towards different substrates were shown. The protein showed activities towards 1 -naphthol and coumarin. The activity with 1 -naphthol was higher than that of coumarin. On the other hand, phenol red, bilirubin and 4-nitrophenol

showed no detectable activity with the protein.

71 :::：虚 ；：S ：；：：：：, ：：；；：：；：：. ,. •；•：：.. ：：；•；•.：. ：.< •：；% . ：：•••.；：；：：：, ：； ；/：.： ••：：. ,；; ••：；；. ••：：..；分::”.:....:..:;％: . ：；；；：：；；. ：；' ^ '•••> ：；---：：^-'^'；'；-；'-^

I 幽_„ Zkfe

'HF'“‘,

、- ‘ ‘？ “ ‘ W ‘ •‘‘“‘ ‘ ‘, ‘ ‘. ‘ 々f"''^tf^^, ‘‘‘ , ‘ ‘ /:,。；

:::::::叙為.；：..-..、;::..:.:..；•.;：：；：；. 兮::::/.)、；：：>•：...• •；：•".；：•'：•场.:r •；：器；灣錄.:.C 發;|:::::::::::_资:::-:丨:::::窃錄；冬:念./免:.::终::'宏|::.;:::::::_；;；^.》簽麥::(:、.;_:::；;;：黎釋辨;:二:.:::::::'!^ 蔡\ ::〔:(.丨赞妨么...

攀-::.:::::::.:〔:; "-•：•：：丨丨••••f：；；�.:: �:: .¥�-：-:: >::: i---： ‘ 丫鲜，： ： 镇:j：::簿• 。::.: ；；丨••••y ::: -；--；：：-： ：

；；：；：；：1 ^^^^ ：/：;<|.;；；：.：lif•：；；；；；；^：|；：;：；：；^：• 丨;.:__||_:|」;丨::_^

；：‘；^'：繁；|-；^ ...：：‘丨衫；^-i;"：!''：臂:._实:'《:签:•‘‘

Lanel: No template control Lane2: rat liver sample 1 from control group LaneS: rat liver sample 2 from control group

Figure 26, Amplification of the target gene in the rat liver cDNA pool, the expected product size is 1500bp. The DNA sample was cut out and purified for ligation with pRset vetor. The bands with the expected size were isolated and purified for the cloning expreiment.

72 ^ 、： ...... ：:、::..

.:.:」，、. ‘,。广‘，，‘‘y - ： ,二；辟

• • . -i--^ ： ••-.••••••.. -..:-_.,.. .:::.•:• . .. ；;: ..；^ •； • ； ：•>：：« • ？:：:：^ 觀驟s;::::驚飄资 •• . .:: . :•.::::..^^ ：

_____奢；...... 驟^

圖 1_::::”.:： ：琴 r/.v ； ‘ ‘ -jr-^o； i 仏“‘.，样—‘'“‘二‘…,‘修寧�Z ：‘；

liiilg 麵 _圓^^ 醫：：個

_疆:__藝 J丨 ’ ： ”‘"楼 颜

： ..:..》.义津 - ‘： 、.. ：： -遍 :1:循

丨I蔡丨 lii；；?；:^；；?:」...... 錦•；纖

.丨丨...• 丨：..'••...... ,:::.；<；：；;：：丨丨j;::於丨丨邀丨：絮口涛彭缀

1|；：|||||« . .,::.警_f ：：：；：s ：：：^ ；；；：S ？ ？ •., •• ：： + +- + ,,.:.:::裕鶴毅 :•:•:<：:：>.•；.：：•.•；.V；"：-.i ;' ：•：'：:••• . -;：...... ••••：；. • ‘ • •••：：：：:. •.•；.••：：^^ ^ ：：- ：: ^-：： •‘'：；•••：•'；• ••• ； , 梦W;丨取感彳;•资：窓彳:S弹•辨?

Figure 27. Restriction digestion of the constructed plasmid. Successful digestion with expected product size indicated the presence of the desired construct. These positive clones were proceeded to DNA sequencing.

73 > •giI 2已461126 I I：印NM 175846.11 19 Rattua norvegicus UDP glycosyltransferase 1 丸曰（Ugtla8), mRNA Length=2241

Score = 162 6 bits (巳20) , Expect = 0.• Identities = 820/已20 (100%), Gaps = 0/820 (•%) Strand=Plus/Plu3

Query 1 ACCATGGCTCCTTCAGGCTGTCCACCCTCCCTTCCTCTGTGTGTTTGTCTGTTCCTGGCC 6Q 11丨111丨丨m丨I m I丨11丨丨1111丨丨丨m 111 M m丨丨丨丨M I丨I M丨丨丨丨I M丨丨

5t)jct 1 ACCATGGCTCCTTCAGGCTGTCCACCCTCCCTTCCTCTGTGTGTTTGTCTGTTCCTGGCC 60

Query 61 TC TGGC TTTGC C C AGGC AGGC AGGC TGC TGGTAGTGC C C ATGGAC GGGAGC C AC TGGTTC 12D

I丨I丨丨丨I M I III I m丨I丨I m 11111 m m M丨m M丨丨111丨丨M M 111 m

Stajct 61 TCTGGCTTTGCCCAGGCAGGCAGGCTGCTGGTAGTGCCCATGGACGGGAGCCACTGGTTC 12•

Query 121 ACCATGCAGATiSATTGTGGiLGAAACTGAGTCACAGJlGGGC ATGAGGTGGTGGTAGTCATC IBD I M丨1111丨11丨丨11 m m III I丨I丨m丨丨I丨I丨丨丨丨I丨丨I丨11 M M I丨M丨M 11

Sbjct 121 AC C ATGC AGATGATTGTGGAGAAAC TGAGTC ACAGAGGGC ATGAGGTGGTGGTAGTC ATC IBO

Query 181 CCAGAGGTGAGTTGGCACATGGGAJUIGTCGCTGAATTTTACCGTGAAAACTTACTCAGTT 240

丨m I丨m m 11 m I丨M m I m 11丨M丨丨M m丨I M I M丨丨III m I丨M I Sbjct 161 CCAGAGGTGAGTTGGCACATGGGAAAGTCGCTGAATTTTACCGTGAAAACTTACTCAGTT 240 Query 241 TC TTAC AC TC TGGAAGATTTAAAC TAC C AC TTC AAGTTTTTTGC TC AC AAC C AATGGAAA 3DD I I I 丨 I I M I I I M I M I I II I m M I I 丨 m I I 丨 I I I I M M 丨 M I I I I 丨丨 I I M I M I

Sbjct 241 TCTTACACTCTGGAAGATTTAAACTACCACTTCAAGTTTTTTGCTCACAACCAATGGAAA 300

Query 301 AC TC AAGAAGTAGGGATGTTTTC TC TAC TGAAGC ATTC AGGC Jl AAGGTTTC TTC GAATTA 3 60

I 丨 I I I I 丨丨 I I I 丨 I I III M I I 丨 M I I M I M I I II I I 丨 I I I m 丨 M 丨 I I M 丨 I I I 丨 I I I

Stojct 301 ACTCAAGAAGTAGGGATGTTTTCTCTACTGAAGCATTCAGGCAAAGGTTTCTTCGAATTA 360

Query 3 61 C TGTTTTC AC AC TGTAGAAGTTTGTTTAAGGAC AAGAAGTTAGTGGAGTAC TTGAAGC AG 420

I m I m I M m I丨I M丨I丨I M 11 M I丨丨M丨丨丨丨I M I丨丨11111111 m M I丨丨

Sbjct 3 61 CTGTTTTCACACTGTAGAAGTTTGTTTAAGGACAAGAAGTTAGTGGAGTACTTGAAGCAG 420

Query 421 AGTTCGTTTGATGCTGTGTTTCTGGATCCTTTTGATGTGTGTGGCTTAATTCTTGCCAAG 480 M 11111 m 111 m I Ml I m M III11 M I m 丨 I 丨 I m 1111 丨 I 丨 m m m

Sbjct 42 1 AGTTCGTTTGATGCTGTGTTTCTGGATCCTTTTGATGTGTGTGGCTTAATTCTTGCCAAG 480

Query 4已 1 TACTTTTCTCTCCCGTCCGTGGTCTTCAGCGGGGGAJlTATTTTGCCACTJLCCTTGATGAG 540

M M I M M I I I 丨 M I M M M 丨 I I m M I M m I 丨丨 I I m 丨 I I 丨丨 M 丨 M I 丨 M M

Sbjct 4已1 TACTTTTCTCTCCCGTCCGTGGTCTTCAGCGGGGGAATATTTTGCCACTACCTTGATGAG 540

Query 541 GGTGCCCAGTGCCCCAGTCCTCCTTCTTATGTTCCCAGAATTCTCTCAAAATTCACAGAC 6DD

M 丨 I I 丨丨丨丨 I I I I I 丨 I M 丨 I I I 丨丨 I I I I M M I II I M II M M II I I M I M I M I I M Sbjct 541 GGTGCCCAGTGCCCCAGTCCTCCTTCTTATGTTCCCAGAATTCTCTCAAAATTCACAGAC 6DD Query 601 ACCATGACTTTCAAGGAGAGAGTGTGGAACCATCTCTCCTACATGAAGGAGCGTGCATTT 660 I I I I I I I 丨 M I I I I I I I I m I I M I I I M m I I I I I I I 丨 I III I I I M I I I M I I I I I

Stojct 601 ACCATGACTTTCAAGGAGAGAGTGTGGAACCATCTCTCCTACATGAAGGAGCGTGCATTT 65D

Query 661 TGCCCCTACTTTTTCAAAACTGCCGTAGAAATTGCCTCTGAAGTTCTCCAGACCCCGGTG 720

丨丨 I I M I I M I I III I M I I M M I III M I 丨 I m I I 丨 m M I M I I I m I M I I I 丨

Sbjct 651 TGCCCCTACTTTTTCAAAACTGCCGTAGAAATTGCCTCTGAAGTTCTCCAGACCCCGGTG 720

Query 721 ACTATGAGAGACCTCTTCAGCCCAGTGTCCATTTGGATGTTTCGAACTGACTTTGTGCTG 7已0 I m 1111111 丨丨 11 m I M I 丨 M 111 丨 III M I M I M I 丨 11 丨丨 11 丨 I 丨 I m 丨 I m Sbjct 721 ACTATGAGAGACCTCTTCAGCCCAGTGTCCATTTGGATGTTTCGAACTGACTTTGTGCTG 780 Query 7B1 GAGTTCCCCAGACCTGTGATGCCCJUiCATGGTCTACATTG 820 I I I I I III I I I I I III 丨 I 丨丨 III I I M I I 丨丨 M m M I I Sbjct 7曰 1 GAGTTCCCCAGiLCCTGTGATGCCCAACJlTGGTCTACJlTTG 已20 Figure 28. Blast search of the sequenced target insert. The result showed that 74 the submitted sequence was the expected one. Protein expression was performed with the clone. 3.4 Purification of recombinant UGT1A8

：；; ‘i, -爆， iiiiiiMlilB^^^ mm^mm.‘“ ：“ ‘‘‘‘ ””汽私 -Jlr

^Sj-r ,树、。‘_ 、哼'《：“^〉:•一怎it一，，，、,:一…

Lane 1 ： lysate of non-induced cells Lane 2: lysate of 3hrs induced cells (UGTlA8-pRSetB plasmid transformed) Lane 3: lysate of 5hrs induced cells (UGTlAS-pRSetB plasmid transformed) Lane 4: lysate of induced cells (pRSetB plasmid transformed) Lane 5. Benchmark Protein Ladder Figure 29. SDS-PAGE showing the expression of the target protein after IPTG induction. Bands of about 55kD were observed in lane 2 and 3. The target protein in lanes 2 and 3 were successfully expressed under the following conditions: 0.5mM IPTG, 37°C, 5 hours. Cell lysates (lOfil) were mixed with lOul of 2 X SDS-PAGE Loading Buffer. Samples (20^1) were loaded into the well 1 to 4 and the marker (6}il) was loaded into the well 5. The gel was run under 125V for 2 hours. The gel was stained with 0.5 % Coomassie Blue for 1 hours and detained in solution with 10% acetic acid and 30% ethanol.

75 — M 1 — 2一 3 4

-‘ “ ‘‘ ‘, ‘

‘‘_ 繼 。’’“

‘“專^^ "一：’’ ’

60kD —• ‘ “

細—,• ^^、‘‘ ‘ - 、 ‘�-iS'irr

M: Benchmark protein marker 1: Pellet at 20000 xg 2: Supernatant at 20000 x g 3: Fraction 1 4: Fraction 2

Figure 30. SDS-PAGE showing the stepwise purification of the target protein. It was found that a protein of about 55kDa was collected in fraction 1 using Ni-Column eluted with imidazole solution while fraction 2 was found to contain no desired protein.

76 3.5 Screening of substrate of the purified enzyme

coumarin phenol red n ^ CH3

OH I n T I CH2 bilirubin 4-nitrophenol 1-naphthol

1-naphthol 1.21 土 0.14 4-nitrophenol ND coumarin 0.58 士 0.08 bilirubin ND phenol red ND ND: activity not detected

Figure 31. Different compounds were used for the activity test of the purified protein. Values were expressed as nanomoles of substrate converted per milligram per minute. Values represent the average of three runs.

77 Chapter Four. Discussion

4.1 Effects of licorice and 18p glycyrrhetinic acid in the induction of

UGT1A8 in different cell lines

The effects of treatments were studied using clone9 and H4IIE. Clone9 was not

from a cancer origin whereas H4IIE was from a cancerous origin. It is known

that these two types of cell have very different protein expressing profiles,

especially those involved in cell division and cell death [Joyce, 2001; Harrison,

1998]. Although, our target gene did not belong to these two categories, it was

predicted that the response of cell types to the treatment would be different. It

was because some drug metabolizing proteins, for example, glutathione

S-transferase, were found to be up-regulated in tumor cells [Landi, 2000].

However, the basal expression of UGTl A8 in tumor cells was similar to that of

non-cancerous cells. These two types of cells, after the different treatments, also

responded similarly. Both treatments of licorice extract and 18j3 glycyrrhetinic

acid were not toxic to the cell lines at low concentration. The IC50 of ISp

glycyrrhetinic acid in clone 9 and H4IIE were 52|iM and 50jiM, respectively.

The IC50 of licorice extract in clone9 and H4IIE were 0.058mg/mL and

0.076mg/mL, respectively. It was suggested that the cells were killed due to the

change in osmotic pressure. Therefore, the results of cell culture experiment

78 were repeated with a low dose of both extracts and 18(3 glycyrrhetinic acid.

When the incubation time was kept the same, 18(3 glycyrrhetinic acid showed a

higher induction effect on UGT1A8 gene expression in both cell types. The

expression of gene reduced to a lower level after 96 hours of incubation with the

compound.

4.2 Comparison of wistar and j/j rats in the induction of UGT1A8

Two different types of rats were used for the study of the crude extract and one

single isolated component, 18(3-GA. Both wistar rats and j/j rats were found with

no change in the morphology of the liver. No nodules or white spots were

observed. The sizes of the livers were about the same as the control group livers.

In addition, the P-act in levels were about the same as both the control groups

and the treatment groups. The level of this gene was found to be up-regulated

when liver cells were under higher level of division and differentiation after

liver injury [Ramaekers et al, 2004]. The stable level of (3-actin reflected proper

control of the treated and the untreated rats. The levels of UGTlAl expression

were not found to be increased in both types of rats. Both experiments showed

that the treatment did not induce UGTlAl, which is one of the most constantly-

expressed family members in the liver and it metabolizes a large number of

79 endiobiotics such as, bilirubin. The constant expression of this gene suggested that the main drug metabolism pathways remain unchanged [Miners, 2000]. The expression level of UGT1A8 was found to be increased in both types of rats

Lee et al, 2004]. The UGT enzyme of j/j rats with deficiency in UGT genes, was considerably reduced [Komaki, 1991]. This heterozygous mutation in the genome results in the decreased amount of functional UGTl proteins. The induction of UGTl A8 in both types of rat was found to increase by 1.3 to 1.8 folds. These results suggest that the induction of the gene was regulated at the transcriptional level. The treatment of rats can up-regulate the expression level

of the gene. It can be concluded that both types of rats responded very similarly

to the treatment. The induction process did not involve the gene product of the

target gene. It would be helpful to use homozygous Gunn rats to repeat the

experiment since Gunn rat is a homozygous species. In fact, the project did plan

to use Gunn rat, however, only a limited number of Gunn rats (only 3) were

available at the time from the animal centre. The preliminary data showed some

effects with variations in the rats. It would be better to use rats of the same age

and sex in order to eliminate the undesirable factors that could affect the

experiment.

80 4.3 Comparison of licorice and 18(3 glycyrrhetinic acid in the induction

ofUGTlA8 in rats

In the experiment, licorice and 18p glycyrrhetinic acid were administered to rats

for one or four weeks. It was shown that the treatments could up-regulate

UGT1A8 at a different level. With the same period of treatment duration, 18|3

glycyrrhetinic acid induced a higher level of the gene when compared to licorice.

It is suggested that the inductive effect by licorice extract was similar to that of

18(3 glycyrrhetinic acid. With a longer period of treatment, the level of induction

was increased. The up-regulation did not reach the maxima yet at the time point.

It would be worthy to study a longer time course of the treatment. Herbalists

suggest that herbs should be taken at a low dose for a long period of time in

order to achieve better protection effects. A longer time course study was useful

in the study of the long term effects and toxicity of the active components.

4.4 Comparison of in vivo and in vitro study of drug treatment

The in vitro results were different from that in the in vivo study. The induction

level increased to a different extent with time. This can be explained by the

feed-back control mechanism of any enzyme induction process. The enzyme

induction is a well-controlled process in which the gene would go up to a

81 maximum level and then drop back to the basal level. Prolonged induction of a

certain gene would result in loss of equilibrium state of the cell, which may

result in inhibition of normal metabolism. In the in vivo study, the absorption

rate and the metabolism of drugs in the GI tract greatly affect the amount of

drugs reaching the liver. Although the same concentrations of the extract and

ISp GA were administered following the same treatment procedures to rats, the

results were quite different, and the expression of UGTl A1 was different from

UGT1A8 due probably to regulation of different promoters in the expression.

4.5 Expression of UGTl A7 after drug treatment in vitro

Rat UGT1A7 has been shown to be closely related to UGT1A8 both in the

amino acid sequence and the promoter sequence [Gong et al, 2001]. Therefore,

its level of expression after treatment was also studied with the cell model. It

was found that UGT1A7 was also up-regulated. The level of induction of this

gene was even higher than that of UGT1A8. The substrates of UGT1A7 are

mainly polyphenols. Both isozymes could share substrate specificity.

82 4.6 Protein expression and purification

Rat liver UGT1A8 was successfully cloned into pRset vector and protein was

successfully expressed in the host E. coli JM109. The protein was purified with

IMAC with the yield of 8.5mg per litre of E. coli. and the protein was found in

the soluble fraction of the bacterial lysate. It was interesting to see that the

activity of the protein remained about the same when the purification of the

protein was done at room temperature. The His-tag linked protein did not alter

the activity of the protein. The purification could be improved by using a

commercially available chemical-coupling matrix to which the substrate could

be coupled. The column would provide a platform for more efficient

purification of the protein.

4.7 Substrate of UGTl A8

Our results suggest that compounds sharing similar structure with simple

aromatic substituents such as 1 -naphthol or coumarin could be substrates of

UGT1A8. The two compounds differ in their functional groups. The functional

group of 1 -naphthol is a hydroxyl group whereas that of a couramin is a keto

group. It was suggested that the protein interacts more strongly with a hydroxyl

group than a keto one. Bilirubin and phenol red with different structures could

83 not glucuronidated by the enzyme. Phenol red molecule is hydrophilic whereas bilirubin is hydrophobic. Both compounds are non-planar molecules. It can be concluded that UGT1A8 did not glucuronidate molecules with non-planar structure, regardless of its polarity. 4-nitrophenol is basically planar in structure, which is similar to 1 -naphthol and coumarin. However, it interacts poorly with the protein, probably due to steric effects. In a recently published paper [Webb et al, 2005], a number of possible substrates of UGT1A8 were reported (Figure

32). The result was compared with that of UGT1A7. The two proteins exhibited overlapping substrate specificities, but the scope of substrate of UGT1A7 was broader. The present study showed 1 -naphthol-like structure was the target

substrate of the protein.

84 1-Naphtho“I "“I l 1-0HB[a]L PJ OH� OH

5-OH B[a]P OH 11-OH B[a]P rvV^ H〇 f^ IW^ fYi 1

Figure 32. Chemicals that UGT1A8 show high rates of glucuronidation. 1-naphthol is the common basic structure. [Webb et al, 2005]

85 Chapter five. Conclusion

The present study showed that the expression of UGT1A8 could be induced with licorice and ISp GA. The induction was more effective with the active component of licorice, ISP glycyrrhetinic acid. In both in vivo and in vitro studies, the gene was up-regulated after treatment. The treatment did not affect the constantly-expressed member of the family, UGTlAl but specifically induced the target gene UGT1A8.

The response was time and dose dependent. Generally, the responses of wistar rat were slightly greater than that of UGT deficient j/j rats.

In the cell model study, both cancerous cells and non-cancerous cells were used. The cells responded similarly. Clone 9 cells showed a more sensitive response to the treatment. Induction of UGT1A8 in H4IIE cells was not as high as that in clone 9 cells. The low basal level and induction response in H4IIE suggest that UGT1A8 was not an important factor associated with the cellular response that we measured.

Quantitative amount of protein could be expressed in bacterial host JM109 and

purified with IMAC. The protein was found in the soluble fraction with biological

activity. It was the first time that rat UGT1A8 was expressed with the bacterial

expression system. The low cost and efficient way of expressing UGTl A8 enabled

us to prepare a sufficient amount of protein for the characterization study.

In the substrate study，it was found that the protein would convert planar molecules

86 with single hydroxyl group. These findings were consistent with studies on the other isoenzymes. The rate of UGT1A8 activity was within the same order of magnitude with other studies [Webb et al, 2005]. The chemistry of 1-naphthol provides structural information on the substrate specificity of UGT1A8.

The study of the structure-function relationship would be necessary to get a better understanding of this newly identified protein. Site-directed mutagenesis could provide useful information on the functional roles of amino acid residues in the structure-function relationship. The mechanism of induction could provide insights into the properties and modulation of the expression of UGT enzymes.

Further directions of study of rat UGT1A8

At this stage, the substrates of rat UGT1A8 tested were synthetic compounds. There

could be some natural substrates that the protein would glucuronidate, for example

flavones. As the basal expression level of the protein is low, it is believed that the

range of substrates of the protein could be narrow. The natural substrate(s) of the

protein could be found in plants. Phytoalexins are candidates that are worth a trial,

especially those that shares structural similarities with 1-naphthol.

Structure-function relationship is important in the study of enzyme function. As the

87 structure of UGT family members remains unclear, study of the structure of

UGT1A8 could provide useful information on the enzyme. Preliminary study with

circular dichroism, and protein denaturation could be used to study this enzyme.

Site-directed mutagenesis could enable us to identify the structurally important

amino acid residues.

The expression of a mammalian protein in E. coli is a convenient way of preparing

the protein. However, the recombinant expression usually results in a reduction in

the activity and change in properties of the protein. The results should be tested in a

mammalian expression system since mammalian system can provide more valid

results on the functions of the protein. Non-liver tissue such as HeLa cell expression

system is suitable. The functional study of the transformed cell line can provide

information on the function of the enzyme.

The induction mechanism for the enzyme remains unclear. It has been known that

the promoter region shares high homology with that of UGTl A7 [Webb et al, 2005].

However, the spectra of substrates of the two isoforms are different. It would be

interesting to clone the two promoter regions into expression detection systems to

study the range of chemicals that can trigger promoter activities and to differentiate

the two promoter regions. A luciferase promoter detection system can be used for

this study. After cloning of the promoter region at the front of the luciferase gene, the

88 vector could be transfected to cell line. With treatment of different candidates of inducer, the level of gene promoter activity could be determined from the luciferase activity.

Possible improvements of the experiment

In the induction in rats, a homozygous recessive control (Gunn rat) should be used to provide a better control. It would be worthwhile to repeat the treatment with different dosages and time. The number of rats used could also be increased in order to reduce

individual differences. In both in vivo and in vitro experiment, some more parameters

like the inductions of any phase I enzyme could also be investigated. It would

provide a clearer picture of the effect of the treatment process.

In the enzyme assay, the activity would be determined by the specific substrate

concentration. It is inconvenient when a larger number of substrates were required to

be tested. This process could be improved by measuring the common co-factor,

UDPGA with HPLC. The results would be more precise for comparison among

different substrates.

Although it has been suggested that His-tag has minimal level of the effect towards

linked protein, maybe we should delete the His-tagged with EK protease before the

activity measurement.

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104 Appendix Alignment of rat UGT1A8 and human rat UGT1A8

CLUSTAL W (1.82) multiple sequence alignment

ugtlal MSWCRSSCSLLLLPCLLLCVLGPSASHAGKLLVIPIDGSHWLSMLGVIQQLQQKGHEW 60

ugtla8 ---MAPSGCPPSLPLCVCLFLAS-GFAQAGRLLWPMDGSHWFIIQMIVEKLSHRGHEW 56

ugt 1 a6 ----MAaLPAARLPAGFLFLVLWGSVLGDKLL_DGSHWLSMKEIVEHLSERGHDIV 56

ugtlaV ----MACLLPAARLPAGFLFLVLWGSVLGDKLLWPQDGSHWLSMKEIVEHLSERGHDIV 56

* • •氺本本*本：):本氺氺••••* •木氺••：：i

ugtlal VIAPEASIHIKEGSmiRKYPVPFQNENVTAAFVELGRSVro-QDPFLLRVVmNKVK 119

ugt lag WIPEVSWHMGKSLNFTVmSVSYILEDLNYHFKFFAHNQWKTQEVGMFSLLKHSGKG- 115

ugtla6 VLVPEVNLLLGESKYYRRKSFPVPYNLEELRTmSFG_FA-ASSPLMAPLREYRNNM 115

ugtla? VLVPEVNLLLGESKYYRRKSFPVPYNLEELmYRSFGNNHFA-ASSPLMAPLREYRNNM 115

ugtlal RDSSMLLSGCSHLIilNAEFMASLEQSHroALLTOPFLPCGSIVAQYLSLPAVYFLNALPC 179

ugtla8 - FFELLFSHCRSLFKDKKLVEYLKQSSFDAVFLDPFDVCGLILAKYFSLPSWFSGGIFC 174

ugtla6 IVIDMCFFSCQSLLKDSAILSFLRENQFDALFTDPAMPCGVILAEYLKLPSIYLraGFPC 175

UgtlaV IVIDMCFFSCQSLLKDSATLSFLRENQroALFTOPAMPCGVILAEYLKLPSmn^GFPC 175

• . 氺 本•‘. • ；J: ‘ 氺木•• 氺氺 jJcjJ: 氺•氺本氺... .氺

ugtlal SIDLEATQCPAPLSYVPKSLSSNTDRMNFLQRVKNMIIALTENFLCRWYSPYGSLATEI 239

ugtla8 HYLDEGAQCPSPPSYVPRILSKFmiMTFKERWNHLSYMKERAFCPYFFKTAVEIASEV 234

ugtla6 SLEHMLGQSPSPVSYVPRFYTKFSDHMTFPQRLANFIANILENYLYHCLYSKYEILASDL 235

ugtla? SLEHMLGQSPSPVSYVPRFYTKFSDHMTFPQRLANFIANILENYLYHCLYSKYEILASDL 235

^^•氺氺；^：氺本* • •本本本 _本•氺 • •氺 • • •氺•••

ugtlal LQKEVTVKDLLSPASIWL_FVKDYPRPIMPNMVFIGGINCLQKKALSQEFEAYVNAS 299

ugtla8 LQTPVTMRDLFSPVSIWMFRTOFVLEFPRPVMPNMVYIGGINCHQGKPLSKEFEAYVNAS 294

ugtla6 LKRDVSLPAmQN-SLWLLRYDFVFEYPRPWWIFIGGTOCKKKGNLSQEFEAYVNAS 294

UgtlaV LKRDVSLPALHQN-SLWLLRYDFVFEYPRPVMPNMIFIGGTNCKKKGNLSQEFEAYVNAS 294

* . * . . * * . * . . * *** ••木朱朱•：):>!：氺來••：!：：!：氺；!；* • ** .

Ugtlal GEHGIVVFSLGSMVSEIPEKKAMEIAEALGRIPQTVLWRYTGTra>SNLAKNnLVKWLPQ 359

ugtlaS GEHGIWFSLGSMVSEIPEKKAMEIAEALGRIPQTVLWRYTGTRPSNLAKNTILVKWLPQ 354

ugtla6 GEHGIWFSLGSMVSEIPEKKAMEIAEALGRIPQTVLWRYTGTRPSNLAKNTILVKWLPQ 354

UgtlaV GEHGIWFSLGSMVSEIPEKKAMEIAEALGRIPQTVLWRYTGTIRPSNLAKNTILVKWLPQ 354

Ugtlal NDLLGHPKARAFmSGSHGIYEGICNGVPMVMMPLFGDQMDNAKRMETRGAGVTUWLE 419

UgtlaS NDLLGHPKARAFIIBSGSHGIYEGICNGVPMVMMPLFGDQMDNAKRMETRGAGVILNVLE 414

ugtla6 NDLLGHPKARAFIlHSGSHGIYEGICNGVPMVMMPLFGDQMDNAKMETRGAGVnMLE 414

ugtla? NDLLGHPKARAFITHSGSHGIYEGICNGVPMVMMPLFGDQMDNAKRMETRGAGVmWLE 414

Ugtlal _DLENAIJCrVINNKSYKENIMELSSLHKDRPIEPLDLAVFWVEYVMRHKGAPHLRPA 479

105 ugtlaS MTADDLENALKTVINNKSYKENIMRLSSLHKDRPIEPLDLAVFWVEYVMRHKGAPHLRPA 474 ugtla6 MTADDLENAUCIVINNKSYKENIMRLSSLHKDRPIEPLDLAVFWVEYVMRHKGAPHLRPA 474 ugtlaV MTADDLENALmiNNKSYKENIMRLSSLHKDRPIEPLDLAVFWVEYVMRHKGAPHLRPA 474

ugtlal AHDLTWYQYHSLDVIGFLLAIVLTWFIVYKSCAYGCRKCFGGKGRVKKSHKSKIB 535

UgtlaS AHDLTWQYHSLDVIGFLLAIVLTWFIVYKSCAYGCRKCFGGKGRVKKSHKSfCffl 530 ugtla6 AHDLTWYQYHSLDVIGFLLAIVLTWFIVYKSCAYGCRKCFGGKGRVKKSHKSKTH 530

UgtlaV AIDLTWYQYHSUMGFLUIVLTWFIVYKSCAYGCRKCFGGKGRVKKSHKS™ 530

CLUSTAL W (1.82) multiple sequence alignment human.ugtlaS MARTWTSPIPLCVSLLLTGGPAEAGKLLWPMDGaiWFTiqSVVEKLILRGHEVWVMP 60 rat_ugtla8 MAPSGCPPSLPLCVCLFLASGFAQAGRLLWPMDGSHWFUQMIVEKLSHRGHEVWVIP 60

** - * •本氺氺冰 *•氺• 氺•氺本本氺氺氺氺it：；!：氺氺氺氺本:(：.：!：*** 本氺氺氺本：^：•冰 hunian_ugtla8 EVSMXGKSLICIVKTYS 丁 SYTLEDLDEEFMDPAM(^WKA(^VRSIJSLFLSSSNGFFNLP 120 rat_ugtla8 EVSWHMGKSLNFrVKTYSVSYTLEDLN™CFF_WKTQEVGMFSLLKHSGKGFFELL 120

human_ugtla8 FSHCRSLF^^RKLVEYLKESSFDAVFLDPFDACGLIVAKYFSLPSWFARGIACHYLEEG 180 rat_ugtla8 FSHCRSLFKDKKLVEYLKQSSFDAWLDPFDVCGLILAKYFSLPSWFSGGIFCHYLDEG 180

：！：：):；!；本本本本：)：.：(：.本；！：；！：！！：本：！：水.宋块本：)：：!：：!；；!：**氺*木 ** ** 氺;)；.*!(; human_ugtla8 AQCPAPLSYVPRILLGFSDAMTFKERVRNHIMHLEEHLFOQYFSKNALEIASEILQTPVT 240

rat_ugtla8 AQCPSPPSYVPRILSKFmMTFKERWNHLSYMKERAFCPYFFmVEIASEVLQTPVT 240

氺本本氺_氺氺氺氺求;t：水本 •本.氺本氺氺本宋：)；术本• •• •氺• (氺本本 * 本•氺本本氺氺• 氺氺本氺 hunian_ugtla8 AYDLYSHTSIWLLRTOFVLDYPKPVMPNMIFIGGINCHQGKPLPMEFEAYINASGEHGIV 300

rat_ugtla8 MI^LFSPVSIWMFRTOFVLEFPRPWNMVYIGGINCHQGKPLSKEFEAYVNASGEHGIV 300

本华•氺 氺氺氺.•氺氺氺氺本本丨•氺氺本;fc氺氺• • 氺氺本氺伞本氺氺氺;i：* ：！：:}：;^：*;^ _氺氺氺^！：；！；氺氺氺氺 human_ugtla8 VFSLGSMVSEIPEKKAMAIADALGKIPQTVLWRYTGTRPSNLANNTILVKWLPQNDLLGH 360

rat_ugtla8 VFSLGSMVSEIPEKKAMEIAEALGRIPQTVLWRYTGTl^SNLAKNTILVKWLPQNDLLGH 360

氺本:jc氺氺:};氺氺氺氺木氺氺本氺：；；木；}：氺•本宋氺•氺氺氺氺;i：*：!：木氺;1：氺氺;i：氺*本：}：本*；|:氺氺氺氺*氺本氺本;氺氺

human—ugt 1 a8 PMmFimGSHGVYESICNGVPMVMMPLFGDQMDNAKRMETKGAGVILNVLEMTSEDL 420

rat_ugtla8 PKARAFITHSGSHGIYEGICNGVPMVMMPLFGDQMDNAKRMET^GAGVILNVLEMTADDL 420

* •氺本氺•氺氺氺氺•傘氺氺氺:i：；；：伞本本：}：；}：水氺木氺*氺氺氺木:ic本氺:j：氺氺*丨：}：氺氺氺氺氺氺本本氺水* •• ••本氺

human_ugt1a8 ENALKAVINDKSYKENIMRLSSLHKDRPVEPLDLAVFWVEFVMRHKGAPHLRPAAHDLTW 480

rat_ugtla8 ENALKTVINNKSYKENIMRLSSLHKDRPIEPLDLAVFWVEYVMRHKGAPHLRPAAHDLTW 480

；！：木块：}:：!： •:):；!!>(: •本木木*水:)：!|:氺;宋：；！；！：：；：本本•木牟本:！：糸：；：华宋泳本***

human_ugtla8 YQYHSLDVIGFLLAWLTVAFITFKCCAYGYRKCLGKKGRVKKAHKSKIH 530

rat_ugtla8 YQYHSIDVIGFLLAIVLTWFIVYKSCAYGCRKCFGGKGRVKKSHKSm 530

106

CUHK Libraries

004359395