Molecular Cloning, Expression, Purification and Characterization of the Zebrafish Catechol-O-Methyltransferases

Home , Colterol

A Thesis

entitled

Molecular Cloning, Expression, purification and Characterization of the Zebrafish

Catechol-O-methyltransferases

Adnan Alazizi

Submitted to the Graduate Faculty as a partial fulfillment of the requirement for the

Master of Science in Pharmacology and Toxicology

______Dr. Ming-Cheh Liu, Committee Chair

______Dr. Frederick Williams, Committee Member

______Dr. Zahoor Shah, Committee Member

______Dr. Patricia Komuniecki, Dean College of Graduate Studies

The University of Toledo May 2011

An Abstract of

Molecular Cloning, Expression, purification and Characterization of the Zebrafish Catechol-O-methyltransferases

Adnan Alazizi

Submitted to the Graduate Faculty as a partial fulfillment of the requirement for the Master Degree in Pharmacology and Toxicology

The University of Toledo

May 2011

The zebrafish is emerging as an important animal model for biomedical research. This study represents part of an effort to establish the zebrafish as a model for investigating the methylation pathway of drug metabolism. Two zebrafish catechol O-methyltransferases (COMTs), designated COMT-1 and COMT-2, were cloned, expressed, purified, and characterized. Substrate specificity of these two zebrafish COMTs were analyzed using a panel of catechol drugs including dobutamine, methyldopa, and isoproterenol, as well as endogenous catecholestrogens, catecholamines, and dopa. The expression of COMT-1 was examined from embryogenesis to maturity by employing both the conventional RT-PCR and RT- quantitative PCR. Data on the COMT-1 expression provided insights into the developmental expression of the COMT enzyme, which may serve as a basis for further investigation into its possible protective role against the adverse effects of catechol drugs and other xenobiotic catechols during the developmental process.

iii

Acknowledgments

Without the help and support of many people this thesis would never have been completed. Many thanks and gratitude goes to Dr. Ming-Cheh Liu for giving me the chance to participate in this project, also for his advice, support and patience at every step of this project. I also would like to appreciate the help and assistance of the committee members, Dr. Frederick Williams and Dr. Zahoor Shah.

Plenty of help and support to thank for goes to my lab mates Zheng Xu, Yasser

Mohammed, and Amani Alshaban.

Last, but not least, my greatest thanks and gratitude goes to my family for their support, love and inspiration though this journey.

Contents

Abstract iii

Acknowledgments iv

Contents v

List of Tables vii

List of Figures viii

Introduction 1

1.1 Drug metabolism 1

1.2 Conjugation reactions 2

1.3 Methylation 4

1.4 Catechol-O-methyltransferase (COMT) 6

1.5 O-Methylation in vivo 8

1.6 The zebrafish (Danio rerio) as a model for biomedical research 14

1.7 Objectives and goals 17

Material and methods 18

2.1 Materials 18

2.2 Methods 19

2.2.1 Cloning, bacterial expression, and purification of recombinant

zebrafish COMTs 19

2.2.2 Analysis of the development stage-dependent expression of the

zebrafish COMTs 21 v

2.2.3 Quantitative Analysis of the development stage-dependent

expression of the zebrafish COMT-1 23

2.2.4 Enzymatic assay 23

2.2.5 Miscellaneous methods 24

Results 25

3.1 Molecular cloning of the zebrafish COMTs 25

3.2 Expression and purification of recombinant zebrafish COMTs 27

3.3 Characterization of recombinant zebrafish COMT-1 and COMT-2 28

3.4 pH-dependence study 30

3.5 The developmental stage-dependent expression of the zebrafish

COMT-1 32

Discussions 36

References 40

List of Tables

Table 1.1. List of conjugation reactions...... …...... ……..…………...... ….….3

Table 1.2. Lists of methyltransferases...... 4

Table 1.3. Periods of Early Embryonic Development of the Zebrafish…….……...... 15

Table 2.1. Oligonucleotide primers used for the cDNA cloning of zebrafish COMTs and for the RT-PCR analysis of the developmental stage-dependent expression of the zebrafish COMT-1…………………..……...…...... …....……....………...... …..22

Table 3.1. Specific activities of the zebrafish COMT-1 and COMT-2 toward catecholic compounds………...... ………………………...... …...... …29

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List of Figures

Figure 1-1. O-methylation of catechol substrates to form two mono-methyl ether

products...... 6

Figure 1-2. Structure of human COMT gene …………………....…………...... 7

Figure 3-1. Alignment of the amino acid sequences of the zebrafish, mouse, rat, and human COMTs……………………………………………………...... 26

Figure 3-2. SDS gel electrophoretic pattern of purified recombinant zebrafish

COMTs..…...... 28

Figure 3-3. pH-dependency of the methylating activity of the zebrafish COMT-1 and

COMT-2 with 2-OH-E1 (A), (C) and dobutamine (B), (D) as substrate………...... 30

Figure 3-4. RT-PCR Developmental stage-dependent expression of the zebrafish

COMT-1...... 34

Figure 3-5. Quantitative PCR Developmental stage-dependent expression of the zebrafish COMT-1…………………………………...... …..……………………...... 35

viii

Introduction

1.1 Drug metabolism

Drug metabolism, as well as the biotransformation of other xenobiotics, may

proceed through three phases. Phase I consists of the functionalization reactions. This is

where a functional group is introduced on the parent compound through the processes of

oxidation, reduction, hydrolysis, cyclization, or decyclization. These are the main

reactions groups that may subsequently be used in Phase II conjugations (Gonzalez and

Tukey, 2006). Phase II reactions involve the conjugation of the metabolites from Phase I

reactions or other chemicals that already carry conjugatable functional groups. Phase II

conjugation reactions include methylation, sulfonation, acetylation, glucuronidation and

glutathione conjugation. They are considered the true ‘detoxification’ pathways (Mulder,

1990). Phase III involves the excretion of the Phase II derivatives via a system of efflux

pumps (Coleman, 2005).

The metabolism and clearance of drugs and other xenobiotics may involve the

same enzymatic pathways and transport systems that are used for the metabolism of

dietary constituents (Mulder, 1990). The metabolizing enzymes work by converting

hydrophobic drugs and xenobiotics to hydrophilic derivatives. This is done by adding a hydrophilic moiety, thereby facilitating its elimination via excretion into the aqueous

compartments of the tissues (Brunton, 2006; Mulder, 1990). While the metabolizing

enzymes are responsible for facilitating the elimination of chemicals from the body, they

may in some cases convert certain chemicals to highly reactive and carcinogenic metabolites (Brunton, 2006).

1.2 Conjugation reactions

Most of the conjugation reactions were discovered in the 19th century when

various compounds were fed to animals or human volunteers, and their urinary samples

were analyzed (Mulder, 1990). In 1930, Williams and his collaborators initiated a more systemic research by investigating the biotransformation of a related series of compounds

(Williams, 1959). Dose dependence of metabolism, biotransformation in various species,

metabolite patterns (at times for very complex structures) and excretory pathways were

explored. Many of the ‘rules’ were discovered during this time. Today, it has become

possible to predict the metabolism of a new compound based on its chemical structure,

the test species, the dose level and the route of administration (Mulder, 1990).

Conjugation (Phase II) reactions such as methylation, sulfonation, and

glucuronidation (Table 1.1) have been reported to play an important role in the

metabolism of key endogenous compounds such as catecholamines and steroid/thyroid

hormones. Conjugation reactions have always been thought to deactivate drugs and

xenobiotics, and to concomitantly increase their water-solubility. In contrast however, it has been reported that some drugs may actually be activated through conjugations reactions. And, if we consider conjugation reactions like acetylation and methylation, water-solubility may actually decrease (Lemke, 2003).

Table 1.1: List of conjugation reactions (Gordon, 2001)

Reaction Enzyme Functional group

–OH

–COOH Glucuronidation UDP–Glucuronosyltransferase –NH2

–SH

–OH

Glycosidation UDP–Glycosyltransferase –COOH

–SH

–NH2

Sulfation Sulfotransferase –SO2NH2

–OH

–OH Methylation Methyltransferase –NH2

–NH2

Acetylation Acetyltransferase –SO2NH2

–OH

–COOH Amino acid conjugation

Epoxide Glutathione-S-transferase Glutathione conjugation Organic halide

–OH Fatty acylation conjugation Condensation Various

1.3 Methylation

Methylation was first discovered by Axelrod et al. (1958), and is known as the transfer of a methyl group from a methyl donor to a substrate, which is one of the most widespread conjugation reactions in nature (Mulder, 1990; Axelrod et al., 1958; Axelrod and Tomchick, 1958). It has also been recognized as an important element in the metabolism of drugs. Different types of methylations that have been discovered include

O-methylation, N-methylation, and S-methylation. Although the methylation reactions are mainly involved in the metabolism of endogenous compounds, some drugs may also be methylated (Mulder, 1990; Gordon, 2001).

Table 1.2: Lists of methyltransferases (Gordon, 2001)

Enzyme Substrate Site

Phenylethanolamine N-methyltransferase Noradrenaline Adrenals

Various Non-specific N-methyltransferase Lung (desmethylimipramine) Imidazole N-methyltransferase Histamine Liver Liver Kidney Catechol-O-methyltransferase Catechols Skin Nerve tissue

Hydroxyindole-O-methyltransferase N-Acetylserotonin Pineal gland

Liver S-Methyltransferase Thiols Kidney Lung

Different methylating enzymes, e.g., hydroxyindole-O-methyltransferase and proteincarboxy-O-methyltransferase (Table 1.2), have been reported. Among them, the catechol-O-methyltransferase (COMT) is the most important one for metabolic conjugation through methylation (Mulder, 1990). The COMT is responsible for catalyzing the methylation of the phenolic groups of endogenous catecholamines and,

catecholesterogens, as well as catechol drugs, using S-adenosyl methionine as the methyl

group donor in the presence of Mg+2 (Axelrod, 1966; Lautala et al., 2001; Taskinen et al.,

2003)

S-adenosyl methionine (SAM) and N5-methyltetrahydrofolic acid both are methyl

group donors for the different methylations reactions. Of the two, SAM is the methyl

donor when methylation involves a sulfur, nitrogen, or oxygen nucleophile (Mulder,

1990). The responsible enzyme, ATP: L-methionine S-adenosyl transferase (EC 2.5.1.6),

catalyzes the synthesis of SAM through two steps. The enzyme first cleaves the P-O bond

in the ATP to release di-phosphate and adenosine then transfers adenosine to the sulfur of

the methionine (Cantoni, 1953; Oh et al; 2010). The enzyme requires divalent cations for

its activity and is activated by monovalent cations (Haba, 1959; Conforth, 1977; Mulder,

1990). The SAM diastereomer ((S) configuration at the sulfonium centre) is required for

all the methyltransfer reactions (de la Haba et al., 1959; Zapia et al., 1969, Borchardt et

al., 1976; Mulder, 1990). As SAM contributes as a methyl donor in a methylation reaction, it is converted to S-adenosylhomocystine (SAH), which can be further cleaved to generate homocysteine and adenosine (Figure 1-1) (Miller et al., 1997; Zhu, 2007).

Figure1-1: O-methylation of catechol substrates to form two mono-methyl ether products

(Zhu, 2007).

1.4 Catechol-O-methyltransferase (COMT)

Catechol-O-methyltransferase (COMT) is an important enzyme for the metabolism of not only endogenous compounds but also many catechol drugs such as levodopa, carbidopa, benserazide, apomorphine, dobutamine, isoprenaline

(isoproterenol), rimiterol, inamrinone, and isoetharine (Mannisto, 1999; Zhu, 1993).

Extensive studies have been conducted concerning methylation and the COMT enzymes.

In humans, COMT exists in two isoforms, soluble (S-COMT) and membrane-bound

(MB-COMT) (Salminen et al., 1990; Lundström et al, 1991). Although the two forms are encoded by distinct mRNAs (Figure 1-2), they are derived from the same gene through differential transcription/translation. (Grossman, 1992; Tenhunen, 1994; Lundström,

1991)

Figure 1-2: Structure of human COMT gene. The boxes represent exons, and the thin lines in between the boxes represent introns. The hatched boxes indicate the protein coding regions. The size of each exon and intron is as indicated (Zhu, 2007).

The human MB-COMT contains 271 amino acids and resides mainly in

endoplasmic reticulum with the active site facing the cytoplasm. The S-COMT contains

221 amino acids and is present in the cytosol. The extra amino acids in MB-COMT

resides at the N-terminus provide a hydrophobic membrane anchor (Tenhunen, 1994).

Studies conducted on COMT showed that S-COMT predominates in the peripheral

tissues while approximately 70% of the COMT in the brain is MB-COMT (Mulder, 1990;

Axelrod, 1966). The major physiological function of COMT-mediated methylation is

generally thought to be for the deactivation of biologically-active or chemically-reactive

endogenous as well as xenobiotic catechols. For catechol drugs, COMT plays a dual role.

COMT-mediated methylation may lower the efficacy of catechol drugs, as in the case of

levodopa used in treating Parkinson’s disease and furnish a protective mechanism against

their potential adverse effects (Bonifácio, 2007; Zhu, 2002; Lautala. 2001). A variety of

COMTs from different tissues of rats, humans and pigs appear to have molecular weights

ranging from 23000 to 29000 Daltons (Mulder, 1990). Other forms of the enzyme with

higher molecular weights (45000 to 65000 Daltons) have been reported as well (Huh and

Friedhoff, 1979; Grossman et al., 1985; Veser and Martin, 1986).

The O-methylation by COMT enzymes takes place through the transfer of the

methyl group from SAM to either the meta- or the para-hydroxyl group of a substituted catechol (Figure 1-1). The methylation appears to occur predominantly at the meta-

phenolic group of catecholamines, and accommodates catechol substrates with positively

charged, negatively charged, or neutral substituents (Creveling et al., 1970; Creveling et

al., 1972).

1.5 O-Methylation in vivo

The in vivo O-methylation mediated by COMT plays an important role in the

inactivation of many endogenous catecholic compounds. It has been demonstrated that

COMT plays an important role during the developmental process from early to adult

stages (Mulder, 1990; Zhu et al., 2010). The formation of O-methylated metabolites in

vivo was first discovered in 1951 (Maclagan and Wilkinson, 1951). In the 1950’s,

Armstrong and McMillan showed that the major metabolic product of norepinephrine

(noradrenaline) in human is 3-methoxy-4-hydroxymandelic acid (Armstrong, 1957).

Axelrod in 1957 investigated the O-methylation of epinephrine and other catechols both

in vitro and in vivo (Axelrod, 1957). By incubating the epinephrine with a soluble

fraction of the rat liver, adenosine-triphospate, and methionine, they noticed the

disappearance of the catecholamines (Axelrod, 1957). A more rapid disappearance was

noticed when the S-adenosylmethionine was used to substitute adenosine-triphosphate

and methionine (Axelrod, 1960, 1962, 1966). These observations showed the presence of

a methylating enzyme that is capable of catalyzing the transfer of a methyl group from

the S-adenosylmethionine to one of the phenolic groups of epinephrine or other

catecholic compounds.

There are many catechol drugs, e.g., levodopa, carbidopa, benserazide (dopa

decarboxylase inhibitors) and apomorphine (dopamine agonist), currently in use for

treating different disorders. There are many more in the process of development as well

(Mendis, 1999; Ghanem, 2003). Understanding the metabolism of these drugs regarding

the mechanisms and pathways involved is vital to ensure the safety and proper use of

these drugs.

Among the catechol drugs currently in use, Tadalafil (Cialis), is a potent,

reversible and competitive inhibitor of phosphodiesterase 5 (PDE5). It is used in the

treatment of erectile dysfunction. Tadalafil was found to be mainly cleared via hepatic

metabolism CYP3A to form a catecholic derivative, which was then subjected to

methylation and glucuronidation to form methylcatechol and methylcatechol glucuronide

conjugate. Methylcatechol detected was less than 10% of the glucuronide metabolite

(Forgue, 2006; Tadalafil 2008). Micafungin is an antifungal agent that has a broad range

of antifungal activity. It works by inhibiting the 1, 3-ß-D-glucan synthase, an enzyme

involved in fungal cell wall synthesis. Micafungin was shown to be metabolized in the

liver by arylsulfatase to the M-1 metabolite (a catechol form), then to a secondary

metabolite by COMT to form the M-2 metabolite (a methoxy form). Approximately 90% of the parent compound or metabolites are eliminated through the biliary system primarily into the feces (Hebert, 2005; Joseph.2007). Levodopa is a prescription drug for the treatment of Parkinson’s disease, which is associated with low levels of dopamine in

the brain. Administration of levodopa, which is a precursor of dopamine, has been the

most effective means of therapy for Parkinson’s disease. Levodopa is predominantly

metabolized to form 3-O-methyldopa by COMT (Manuela, 2010). Inhibition of COMT

has been utilized to reduce the loss of levodopa through methylation and increase

levodopa levels in the brain. The inhibitors of COMT may work both peripherally and

centrally (Männistö et al., 1992; Blessing et al., 2003), thereby increasing the amount of

levodopa entering the brain and enhancing the concentrations of levodopa and dopamine

in the brain (Goetz, 1998; Blessing et al., 2003). Methyldopa is a pro-drug which works

via its active metabolite as an aromatic-amino-acid decarboxylase inhibitor in animals

and in human. It is metabolized mainly by sulfation, O-methylation and decarboxylation,

forming methyldopa sulfate, 3-O-methyl-α- methyldopa, and α-methyldopamine respectively. Previous studies revealed incidence of depression among patients treated with α-methyldopa (Campbell et. al., 1984). Canadian practitioners reported methyldopa

(Aldomet) as one of the most frequently prescribed drugs for the treatment of mild to moderate hypertension in pregnancy (Borghi 2002). In a study performed by Redman et al. (2005), however, 14.5% of the pregnant women assigned to methyldopa had to be transferred to another drug or had to stop treatment completely because of side effects

(Redman, 1977). An important aspect with regard to risk is for the developing fetus during the use of methyldopa. This was indicated by a study by Todoroki et al. (2006) showing that neonatal suppurative parotitis may be associated with congenital cytomegalovirus (CMV) infection and maternal use of methyldopa in some of patients

(Todoroki et al., 2006).

For the management of certain cardiovascular disorders in pediatrics, various

catechol drugs have been used. Some of these drugs include epinephrine, isoproterenol, dopamine, dobutamine, and inamrinone (Chen, 1998; Ravishankar, 2003; Yaffe, 2004).

Dobutamine is a commonly used drug for the treatment of heart failure and shock.

Previous studies illustrated that the major metabolite of dobutamine in humans is 3-O-

methyldobutamine by COMT (Yan et. al., 2002). It has been reported that dobutamine

may affect platelet aggregation function when used to treat myocardial dysfunction in

asphyxiated neonates (Al-Salam, 2008). Isoproterenol (Isoprenaline) is a beta adrenergic

agonist which is used for treating bronchospasm and cardiac arrest. Myocardial ischemia

has been reported as a complication with the administration of IV isoproterenol to

asthmatic children (Maguire, 1991). Isoproterenol is metabolized to a sulfate conjugate if

giving by inhalation. However, it is deactivated and metabolized to 3-O-

methylisoproterenol sulfate by the COMT when administered directly to the bronchial

tree (Pesola, 1993; Szefler, 1979). Bitolterol is a bronchodilator and selective beta-2-

adrenoreceptor agonists. It is considered a pro-drug which is activated in the lung by

esterase hydrolysis. Its active form is the colterol catecholamine N-t-butyl-arterenol. The

pharmacological activity of this active metabolite can be terminated via conjugation or 3-

O-methylation. (Kass, 1980; Alice, 1985) Apomorphine (Spontane, Uprima, Apokyn) is a non-narcotic dopamine derivative and works as a potent dopamine agonist. Apomorphine was the first dopamine agonist given to patients with Parkinson’s disease. It has a very short elimination half life (30 to 90 min) and high clearance rate (3 to 5 L/kg /h), and it is mainly excreted and metabolized by the liver. It has been reported that apomorphine is metabolized by glucuronidation and O-methylation (COMT) which gives apocodeine as a

metabolite in several animal species. (Kaul, 1961; Neef, et. al., 1999; Gancher, et. al.,

2004). Madopar is an effective drug used for the treatment of Parkinson's disease. It is a

combination of levodopa and benserazid (decarboxylase inhibitor). The advantage of combining benserazide with levodopa is the inhibition of the peripheral decarboxylation of levodopa without significantly affecting its metabolism in the brain. It has been reported that benserazide may lower the optimum dose of levodopa by about 70 to 80%.

Benserazide itself is hydroxylated to trihydroxybenzylhydrazine. This metabolite acts as a potent inhibitor of the aromatic amino acid decarboxylase, thus reducing the peripheral

decarboxylation of methyadopa and increasing the plasma levels of levodopa and 3-O- methyldopa. (Pinder et.al., 1976; Da Prada et. al., 1987; Shen et. al., 2003). Isoetharine

(Bronkosol) is β 2-adrenergic receptor agonist. It was the drug of choice for relieving airway spasm until when it was substituted by albuterol. Metabolites of isoetharine found in urine from man and dog were O-methylisoetharine, isoetharine sulphate, isoetharine glucuronide. Upon intravenous or intrabronchial administration, soetharine first became

O-methylated then conjugated (Williams, 1974). Parkinson’s disease and pregnancy is a relatively rare situation and the treatment could be dangerous for the fetus. There is only a limited literature on anti-Parkinson drugs for pregnant animals where malformations of the skeletal and circulatory systems were reported (Scott, 2005). Another drug,

Diclofenac (Voltaren), is processed by the Phase I cytochrome P450 enzymes and subsequently becomes a substrate of COMT. Diclofenac has been reported to cause severe pulmonary hypertension and transient right-sided hypertrophic cardiomyopathy in neonates, if used by the mother (Zenker, 1998; Bort, 1999).

In the brain COMT plays an important role in the metabolism of catecholamine neurotransmitters including dopamine, epinephrine, and norepinephrine. Recently the

COMT enzyme started to gain an interest with respect to emotional and cognitive brain functions (Herrmann, 2009; Barnett, 2008). Significantly, Val158Met, the substitution of

Val-to-Met at amino acid position 158 in COMT, is a common single nucleotide polymorphism in the COMT gene (Barnett, 2008). This mutation causes lower enzymatic activities which in turn results in an increase in synaptic dopamine and strengthen dopaminergic tone (Herrmann, 2009). A number of studies also linked the COMT

Val158Met polymorphism to early onset of major depressive disorders and suicide,

(Massat, 2005; Jia, 2005, Abdolmaleky et al., 2006). A study by Zhu et al. (2010) showed that COMT is involved in the development of placenta and embryo, likely via the formation of 2-methoxyestradiol (2-MeO-E2) (Zhu et al., 2000, 2010). The concentration of 2-MeO-E2 was shown to increase considerably during pregnancy, but decrease significantly in women with pre-eclampsia (Barnea et al., 1988; Rosing and Carlstrom,

1984). This reflects a protective role of the COMT in vivo. In another study, the COMT-/- mice were used to show that COMT is critical to the normal development of the placenta and embryo (Kanasaki et al., 2008). The study demonstrated that the administration of 2-

MeO-E2 could effectively rescue the pregnant COMT-/- phenotype in these mice. The study also showed that COMT directly, or via its products, plays a role in regulating the utero-placental vascular homeostasis, blood pressure, kidney glomerular structure, and hypoxia response during pregnancy (Kanasaki et al., 2008; Zhu et al., 2010). Studies by

Zhu et al. (1994) also showed a protective role of COMT against quercetin and other mutagenic catechol-containing flavonoids in vivo. They showed that catechol-containing

flavonoids quercetin and fisetin are rapidly O-methylated by COMT both in vitro and in

vivo. They suggested that the COMT-mediated methylation is the major reason for the lack of carcinogenicity of the mutagenic flavonoids in vivo. It therefore seems that

COMT may play an important and protective role against the adverse effects of catecolic compounds throughout the early developmental stages until adulthood (Zhu 1993, 1994).

1.6 The zebrafish (Danio rerio) as a model for biomedical

research

Mice and rats have been the most common animal models for biomedical research. During the past three decades the zebrafish (Danio rerio) has became a valuable and powerful alternative as an experimental organism in developmental biology and genetics (Eisen, 1996).

Zebrafish are vertebrate creatures and they have several features that make them a valuable model for biomedical research. The zebrafish is small in size (adult ~ 1- 1.5 inch

long), which allows for the maintenance of a large number of fish. A female fish can lay

a relatively large number of eggs. Under ideal conditions, a single zebrafish female can

lay 200 eggs once every 2–3 days in ideal conditions. The virtually transparent embryos

undergo rapid external development and the generation time is short (Westerfield, 1993).

The embryos are optically clear. Thus, it is easy to recognize and allows for easy

detection of morphological changes and the manipulation of the transparent embryos

(Eisen, 1996).

It is somewhat cumbersome to stage the zebrafish development since different embryos tend to develop at different rates. Kimmel, et al. (1995) staged the development of the zebrafish based on their morphological criteria and thus managed to resolve this problem.

Table 1.3: Periods of Early Embryonic Development of the Zebrafish (Kimmel, et al.,

1995)

Period Hour Description

The newly fertilized eggs through the completion of the first Zygote 0 zygotic cell cycle

Cleavage ¾ Cell cycles 2 through 7 occur rapidly and synchronously

Rapid, metasynchronous cell cycles (8,9) give way to 2¼ Blastula lengthened, saynchronous ones at the midbalstula transition;

epiboly then begins.

Morphogenetic movements of involution, convergence, and

Gastrula 5¼ extension form the epiblast, hypoblast, and embryonic axis;

through the end of epiboly.

Somites, pharyngeal arch primorida and neuromeres

Segmentation 10 develop; primary organogenesis; early movements; the tail

appears.

Phylotypic-stage embryo; body axis straightens from its

Pharyngula 24 early curvature about the yolk sac; circulation,

pigmentation, and fins begin development.

Completion of rapid morphogenesis of primary organ

Hatching 48 systems; cartilage development in head and pectoral fin;

hatching occurs asynchronously.

Swim bladder inflates; food-seeking and active avoidance Early larva 72 behaviors

As the zebrafish became a valuable model organism and for biomedical research, many questions have been raised as to how to optimize the use of zebrafish in the examination of the efficacy and toxicity of drugs and other xenobiotics. Researchers became more interested in knowing whether there are any instances of zebrafish mutations with phenotypes that can be reasonably viewed and examined as resembling human diseases and disorders. It also posed the question as to whether the COMT- knockdown zebrafish embryos/larvae, compared with their normal counterparts, may be

(more) susceptible to the adverse effects of (lower concentrations of) the tested drugs.

From the view point of analyzing the adverse effects of certain obstetric or pediatric drugs, researchers are more interested in determining the occurrence and functional relevance of COMT-mediated methylation in developing fetus/infant/child. Fortunately, the zebrafish has all the tissues/organs (except lungs) found in humans, and it has been reported that many phenotypes of zebrafish mutations resemble human diseases, thus making the zebrafish a suitable model (Shin, 1999).

1.7 Objectives and goals

This study represents part of an effort to establish the zebrafish as a model for the investigation of the methylation pathway in drug metabolism. The goal of this thesis was to identify, clone, purify, and characterize zebrafish COMT enzymes. In this research, two zebrafish COMTs (designated COMT-1and COMT-2) were identified, cloned, purified, and characterized. The developmental expression of the COMT was examined in order to gain insights into its possible protective role against the adverse effects of catechol drugs and other xenobiotic catechols during the developmental process.

Materials and methods

2.1 Materials.

Dopamine, epinephrine, L-3,4-dihydroxyphenylalanine (L-dopa), methyl-dopa,

Trizma base, S-adenosyl-l-methionine (AdoMet), sodium dodecyl sulfate (SDS), sodium acetate, 2-morpholinoethane sulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid

(MOPS), N-2-hydroxylpiperazine-N-2-ethanesulfonic acid (HEPES), 3-[N-tris

(hydroxymethyl) methylamino]-propanesulfonic acid (TAPS), 2-(cyclohexylamino) ethanesulfonic acid (CHES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), dithiothreitol (DTT), carbidopa, apomorphine, isoproterenol, dobutamine, and isopropyl-

β-D-thiogalactopyranoside (IPTG) were products of Sigma Chemical Company. 1, 3, 5

(10)-estratrien-2, 3-diol-17-one (2-OH-E1), 1, 3, 5 (10)-estratrien-3, 4-diol-17-one (4-

OH-E1), 1,3,5(10)-Estratriene-2,3-17β-triol (2-OH-E2), 1, 3, 5 (10)-estratrien-3, 4, 17β-

triol (4-OH-E2) were products of Steraloids, Inc. TRI Reagent was from Molecular

Research Center, Inc. Total RNA from a 3-month-old female zebraﬁsh was prepared using the TRI Reagent based on the procedure described previously (Sugahara et al.,

2003a). Taq DNA polymerase was a product of Promega Corporation. Takara Ex Taq

DNA polymerase was purchased from Fisher Scientiﬁc. T4 DNA ligase and Bam HI restriction endonuclease were from New England Biolabs. Oligonucleotide primers were synthesized by MWG Biotech. pSTBlue-1 AccepTor Vector Kit and BL21 (DE3) competent cells were from Novagen. Protein molecular weight standards were from

Fermentas Life Sciences. pGEX-2T glutathione S-transferase (GST) gene fusion vector,

GEX-5` and GEX-3` sequencing primers, and glutathione-Sepharose 4B were products of

GE Healthcare. DyNAmo HS SYBR Green qPCR Kit was from New England Biolabs.

Cellulose thin-layer chromatography (TLC) plates were products of EM Science. [14C]-

labeled AdoMet was from American Radio labeled Chemicals, and Ecolume scintillation

cocktail was from MP Biomedicals. All other reagents were of the highest grades

commercially available.

2.2 Methods

2.2.1 Cloning, bacterial expression, and purification of recombinant zebrafish COMTs

By searching the GenBank database, two sequences encoding COMT-like

proteins were identified (GenBank Accession # CR457452 and GeneID # 565370;

tentatively designated COMT-1 and COMT-2). To generate the cDNA for subcloning

into the pSTBlue-1 vector, sense and antisense oligonucleotide primers designed based

on 5`- and 3`-regions of the coding sequence were synthesized (Table 2.1). Using these

primer sets, PCR was carried out under the action of EX Taq DNA polymerase, with the

first-strand cDNA reverse-transcribed from the total RNA isolated from a 3-month-old

adult female zebrafish as the template. Amplification conditions were 2 min at 94°C and

25 cycles of 94°C for 30 sec, 60°C for 35 sec, and 72°C for 45 sec, followed by a 5-min incubation at 72°C. The final reaction mixture was applied onto a 1% agarose gel, separated by electrophoresis, and visualized by ethidium bromide staining. The PCR product band detected was excised from the gel, and the DNA therein was isolated by

spin filtration. Purified PCR product was cloned into the pSTBlue-1 vector and verified

for authenticity by nucleotide sequencing. To amplify a truncated cDNA encoding the

“soluble-form” of the two zebrafish COMTs, different sets of sense and antisense primers

(Table 2.1) were used in a PCR reaction with pSTBlue-1 harboring the full-length zebrafish COMT-1 or COMT-2 cDNA as the template. Amplification conditions were the same as described above. At the end of the PCR reaction, the PCR product was purified, subjected to Bam HI restriction, and subcloned into Bam HI-restricted pGEX-2TK (for

COMT-1) or pMAL-c5x (for COMT-2) vector. To express the recombinant zebrafish

COMT, competent Escherichia coli BL21 (DE3) cells transformed with pGEX-2T or pMAL-c5x harboring the COMT cDNA were grown in 1 L LB medium supplemented with 60μg/ml ampicillin. After the cell density reached 0.6 OD600 nm, IPTG (0.1 mM final

concentration) was added to induce the production of recombinant zebrafish COMT.

After an overnight induction at room temperature, the cells were collected by

centrifugation and homogenized in 25 ml ice-cold lysis buffer (20 mM Tris-HCl, pH 8.0,

150 mM NaCl, and 1 mM EDTA) using an Aminco French Press. Twenty μl of 10 mg/ml

aprotinin (a protease inhibitor) was added to the crude homogenate. The crude

homogenate was subjected to centrifugation at 10,000 x g for 15 min at 4°C. The

supernatant collected was fractionated using 2.5 ml of glutathione-Sepharose (for

COMT-1) or amylose resin (for COMT-2), and the bound GST- or MBP-fusion protein

was eluted by an elution buffer (50 mM Tris-HCl, pH 8.0, plus 10 mM reduced

glutathione or maltose) at 4ºC or treated with 3 ml of a thrombin digestion buffer (50 mM

Tris-HCl, pH 8.0, 150 mM NaCl, and 2.5 mM CaCl2) containing 5 unit/ml bovine

thrombin at room temperature. Following 15-min incubation with constant agitation, the

preparation was subjected to centrifugation. The recombinant zebrafish COMT was

analyzed for purity by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and

subjected to enzymatic characterization.

2.2.2 Analysis of the development stage-dependent expression of the zebrafish COMTs

RT-PCR was employed to investigate the developmental stage-dependent

expression of the zebrafish COMTs .Total RNAs from zebrafish embryos, larvae, and

adult (3-months-old male or female) fish at different developmental stages were isolated

using TRI Reagent, based on manufacturer’s instructions. Aliquots containing 5 μg each

of the total RNA preparations were used for the synthesis of the first-strand cDNA using

the First-Strand cDNA Synthesis Kit (Amersham Biosciences). One microliter aliquots of

the 33 μl first-strand cDNA solutions prepared were used as the template for the

subsequent PCR amplification. PCRs were carried out in 25 μl reaction mixtures using

EX Taq DNA polymerase, in conjunction with gene-specific sense and antisense

oligonucleotide primers (Table 2.1). Amplification conditions were 2 min at 94°C

followed by 35 cycles of 30 sec at 94°C, 35 sec at 60°C, and 45 sec at 72°C. The final

reaction mixtures were applied onto a 1% agarose gel, separated by electrophoresis, and visualized by ethidium bromide staining. As a control, PCR amplification of the sequence encoding zebrafish β-actin was concomitantly performed using the above-mentioned first-strand cDNAs as templates, in conjunction with gene-specific sense and antisense oligonucleotide primers (Table 2.1) designed based on reported zebrafish β-actin nucleotide sequence (GenBank Accession No. AF057040).

Table 2.1: Oligonucleotide primers used for the cDNA cloning of zebrafish COMTs and for the RT-PCR analysis of the developmental stage-dependent expression of the zebrafish COMT-1

Target Sense and antisense oligonucleotide primers used sequence

I. For cDNA cloning*:

Sense: 5’-CGCGGATCCATGCTGTGGGTTGTGTTGGCAGTGGTGGTG-3’ COMT-1 (Full-length) Antisense: 5’-CGCGGATCCTCATCCTAAGAAGACCGATTTCTCCAGGCC-3’

Sense: 5’-CGCGGATCCCACGATGTGGTCCATCAGCGGCTCCTGAAC-3’ COMT-1 (Truncated) Antisense: 5’-CGCGGATCCTCATCCTAAGAAGACCGATTTCTCCAGGCC-3’

Sense: 5’-CGCGAATTCCATGATGTCATCGTGGAGCGAGCTTTGGATTCC COMT-2 CTGA-3’

Antisense: 5’-CGCGAATTCCTAGCCCAAAAACACAGACTTCTCCAAGCCAT CCTCTGC-3’ II. For RT-PCR analysis**:

Sense: 5’-CGCGGATCCATGCTGTGGGTTGTGTTGGCAGTGGTGGTG-3’ COMT-1 Antisense: 5’-CGCGGATCCTCATCCTAAGAAGACCGATTTCTCCAGGCC-3’

β-Actin Sense: 5’-ATGGATGAGGAAATCGCTGCCCTGGTC-3’

Antisense: 5’-TTAGAAGCACTTCCTGTGAACGATGGA-3’

III. For quantitative -PCR analysis***:

Sense: 5’-TCACGACCACAGCGCATCT-3’ COMT-1 Antisense: 5’-CCCACATTCATGGCCCATT-3’

Sense: 5’-CGAGCTGTCTTCCCATCCA-3’ β-Actin Antisense: 5’-TCACCAACGTAGCTGTCTTTCTG-3’

* Recognition sites of BamHI restriction endonuclease in the oligonucleotides are underlined. Initiation and termination codons for translation are in bold type. ** The sense and antisense oligonucleotide primer sets listed were verified by BLAST Search to be specific for the zebrafish COMTs or β-actin nucleotide sequence. *** The sense and antisense oligonucleotide primer sets for the quantitative-PCR analysis.

2.2.3 Quantitative Analysis of the development stage-dependent

expression of the zebrafish COMT-1.

Quantitative real-time PCR was employed to investigate the developmental stage-

dependent expression of the zebrafish COMT-1. For use as templates in real-time PCR,

first-strand cDNAs were reverse-transcribed from total RNA samples isolated from zebrafish embryos and larvae at different developmental stages, and adult (3- months-old male or female) fish. Oligonucleotide primers (Table 2.1) for quantitative real-time PCR were designed using Primer Express software (Applied Biosystems, Foster City, CA) based on the following parameters: primer size between 18 and 22 base pairs; primer Tm

range between 58°C and 62°C; and GC content between 50% and 60%. The nucleotide

sequences for respective target PCR products were blasted to confirm their specificity.

PCR amplification was performed using the Eppendorf mastercycler® ep realplex system. Reactions were carried out in triplicate. The reaction mixtures (25 μl final volume) contained 12.5μl of SYBR® 2X Master mix, 500 nM each of sense and antisense oligonucleotide primers, and 1μl (1.25 ng) of the DNA template. Reaction conditions were as follows: 15 min at 95◦C for initial denaturation followed by 94°C for

denaturation, 25 sec 56°C for annealing and 30 sec 72°C for extension. The expression

values obtained were normalized against those from the control zebrafish β-actin.

2.2.4 Enzymatic assay.

The methylating activity of purified recombinant zebrafish COMTs was assayed

using radioactive [14C]-labeled AdoMet as the methyl group donor. The standard assay

mixture, with a final volume of 20 μl, contained 50 mM Tris-HCl buffer at pH 7.5, 0.1

14 mM [ C]-labeled S-adenosyl-L-methionine, 5 mM DTT, 1.5 mM MgCl2, and 1 mM substrate. Controls with DMSO or water, in place of substrate, were also prepared. The reaction was started by the addition of the enzyme, allowed to proceed for 60 min at

28°C, and terminated by the addition of 10 μl of 1 N HCl. The precipitates formed were cleared by centrifugation, and the supernatant was subjected to the analysis of [14C] methylated product using a previously developed TLC procedure (Liu and Lipmann,

1984) with n-butanol/isopropanol/88% formic acid/water (3:1:1:1; by volume) as the solvent system. The TLC plate was then dried and autoradiographed. The radioactive spot on the TLC plate due to the [14C] methylated product was cut out and eluted by shaking in 0.5 ml H2O in a glass vial, four milliliter of scintillation fluid was then added and thoroughly mixed, radioactivity counted using a liquid scintillation counter. To examine the pH-dependence of the methylation of 2-OH-E1 or dobutamine, different buffers (50 mM Mes at pH 5.5 or 6.5; Hepes at pH 7.5; Taps at pH 8.5; Ches at pH 9.5; Caps at pH

10.5 or 11.5), were used in the reactions with 1 mM of each substrate.

2.2.5 Miscellaneous methods

SDS-PAGE was performed on 12% polyacrylamide gels using the method of

Laemmli (Laemmli, 1970). Protein determination was based on the method of Bradford

(Bradford, 1976) with bovine serum albumin as the standard.

Results

3.1 Molecular cloning of the zebrafish COMTs

By searching the zebrafish GenBank database at NCBI, we identified two

sequences encoding COMT-like proteins (GenBank Accession # CR457452 and GeneID

# 565370; tentatively designated COMT-1 and COMT-2). Based on the sequence

information, we designed and synthesized oligonucleotide primers corresponding to 5’-

or 3’-coding regions of each of these two sequences. The first-strand cDNA was reverse- transcribed from the total RNA isolated from a 3-month-old adult female zebrafish.

Using the first-strand cDNA as a template, cDNA encoding the zebrafish COMT-1 was

PCR-amplified. The resulting PCR product was cloned into the pSTBlue-1 vector and sequenced to confirm its authenticity. The nucleotide sequences obtained were submitted to the GenBank database under the Accession No. HM997189. To amplify cDNA encoding COMT-2, a cDNA (GenBank Accession # BC134931; obtained from Open

Biosystems) was used as a template. The resulting PCR product was cloned into the pSTBlue-1 vector and sequenced to confirm its authenticity. Figure 3-1 shows the alignment of the deduced amino acid sequences of the zebrafish COMT-1 and COMT-2, together with mouse, rat, and human COMTs. As indicated in the Figure, conserved regions are found among the five COMT enzymes.

The occurrence of having duplicated or multiple copies of certain genes in

zebrafish is well established and is believed to be a consequence of the whole genome

duplication event taking place in the teleost ancestor (Woods, 2011). This event was

followed by incomplete loss of duplicated genes in derivative species, leaving a subset of

genes present in two or more copies (Amores et al. 1998; Gates et al. 1999; Woods,

2011)

Figure 3-1: Alignment of the amino acid sequences of the zebrafish, mouse, rat, and human COMTs. Different colors shows percentage of conservation among the five sequences. Amino acid residues previously shown to be important for AdoMet-binding 26

are indicated by solid triangles ; and those important for catechol-binding are indicated

by solid dots . The transmembrane regions of the four enzymes are underlined.

3.2 Expression and purification of recombinant zebrafish

COMTs

The zebrafish COMTs were expressed in soluble form in order to avoid problems that may be encountered in the expression and purification of membrane proteins. Figure

3-2 shows the SDS gel electrophoretic pattern of purified recombinant zebrafish COMT-

1 and COMT-2. Samples analyzed in lanes 1, 2 and 3 were GST-COMT-1 and MBP-

COMT-2 fusion protein and protein molecular weight markers respectively. The GST- and MBP-fusion protein form of the recombinant zebrafish COMT-1 and COMT-2 migrated respectively at ~ 48 kDa and 65 kDa positions upon SDS-polyacrylamide gel electrophoresis. It is noted that the zebrafish COMT-2 still had some impurities which, however, did not affect the methylatying activity of the enzyme.

Figure 3-2: SDS gel electrophoretic pattern of purified recombinant zebrafish COMTs.

SDS-PAGE was performed on a 10% gel, followed by Coomassie blue staining. Samples analyzed in lanes 1, 2 and 3 correspond to the protein molecular weight markers, MBP-

COMT-2, and GST-COMT-1, respectively.

3.3 Characterization of recombinant zebrafish COMT-1 and

COMT-2

A panel of endogenous and xenobiotic catecholic compounds were selected and

tested as substrates for purified zebrafish COMT-1 and COMT-2 under standard assay

conditions as described in the Methods section. The activity data obtained are compiled in Table 3-1. Both zebrafish COMT-1 and COMT-2 displayed strong methylating

activities towards the catechol estrogens (2-OH- E1, 4-OH-E1, 2-OH-E2, and 4-OH-E2)

and dobutamine. Lower, but significant activities were also detected towards other

endogenous (L-Dopa, dopamine and epinephrine) and xenobiotic (methyl-Dopa, carbidopa, isoproterenol, and apomorphine) catecholic compounds tested as substrates. It was noted that, in general, zebrafish COMT-1 displayed higher methylating activities than COMT-2.

Table 3.1: Specific activities of the zebrafish COMT-1 and COMT-2 toward catecholic compounds

COMT-1 COMT-2 Substrate Specific Activity Specific Activity (nmol/min/mg)* (nmol/min/mg)* L-DOPA 5.44 ± 0.51 0.01 ± 0.00

Methyl-DOPA 0.20 ± 0.05 0.02 ± 0.01

Carbidopa 2.19 ± 0.22 1.17 ± 0.7

Dopamine 37.43 ± 1.07 3.07 ± 0.52

Epinephrine 18.94 ± 0.96 3.22 ± 0.30

Isoproterenol 41.04 ± 1.24 1.15 ± 0.40

Apomorphine 0.529 ± 0.18 0.02 ± 0.00

2-OH-E1 157.33 ± 6.67 18.65 ± 0.61

4-OH-E1 46.31 ± 3.46 2.94 ± 0.59

2-OH-E2 144.57 ± 4.29 19.30 ± 0.70

4-OH-E2 85.80 ± 1.12 7.24 ± 0.47

Dobutamine 128.54 ± 5.66 16.30 ± 0.45

*Data represents mean ± S.D. derived from four experiments

3.4 pH-dependence study:

The pH-dependence study was subsequently carried out under standard assay

conditions. The pH-dependence of the zebrafish COMT-1 and COMT-2 were examined

using 2-OH-E1and dobutamine as substrates. Results showed that the methylating activity of the zebrafish COMT-1 and COMT-2 exhibited a broad pH optimum from pH 6.5 to

10.5 using 2-OH-E1 as a substrate (Figure 3-4 A and C). Using dobutamine as a substrate,

both enzymes also exhibited a broad pH optimum from pH 7.5 to 10.5 (Figure 3-4 B and

(A) COMT-1 With 2-OH-E1 as a substrate

(B) COMT-1 With dobutamine as a substrate

(D) COMT-2 With dobutamine as a substrate

Figure 3-3: pH-dependency of the methylating activity of the zebrafish COMT-1 and

COMT-2 with 2-OH-E1 (A), (C) and dobutamine (B), (D) as substrate. The enzymatic

assays were carried out under standard assay conditions as described in section 2, using

different buffer systems as indicated.

3.5 Developmental stage-dependent expression of the zebrafish

COMT-1

The expression of the mRNA encoding COMT throughout different

developmental stages was examined in order to better understand the physiological

involvement of the zebrafish COMT-1. The expression was examined from embryogenesis to maturity by employing the conventional RT-PCR (Figure 3-4) and RT- quantitative PCR (Figure 3- 5). Results from the conventional RT-PCR showed a significant level of the COMT-1 mRNA in unfertilized eggs (lane1), indicating clearly its

presence as a maternal transcript. Following fertilization, the level of the COMT-1 mRNA of decreased considerably and remained low throughout the early embryonic development until the pharyngula period (24-hpf; lane7). Upon hatching (48-and72- hpf; lanes 8 and 9), the COMT-1 mRNA showed a dramatic increase, which lasted into the late (4-week) larval stage (lane13). In adult zebrafish, there appears to be a lower level of

COMT-1 mRNA. Interestingly, the level of the COMT-1 mRNA was considerably lower in male fish (lane14) than in female fish (lane15). In contrast to the developmental stage- dependent expression of the zebrafish COMT-1, the expression of β-actin, a housekeeping protein, was found to be consistent throughout the entire developmental process (Figure 3-4B).

Quantitative PCR was preformed to verify the expression of the zebrafish COMT-

1. The expression was analyzed throughout the same developmental stages as mentioned above in conventional RT-PCR (Figure 3-5). The results showed a similar pattern of expression of the COMT-1 mRNA as that found with the analysis using conventional

RT-PCR, except that for adult fish the level of COMT-1 mRNA in female fish was considerably higher than that in male fish.

(A) Zebrafish COMT-1

(B) Zebrafish β-Actin

Figure 3-4: RT-PCR Developmental stage-dependent expression of the zebrafish COMT-

1. The expression of the COMT-1 mRNA at different stages during embryogenesis and

larval development until maturity was analyzed using RT-PCR. Samples analyzed were

unfertilized zebrafish eggs, zebrafish embryos during the zygote period (0-hour post-

fertilization (hpf)), cleavage period (1-hpf), blastula period (3-hpf), gastrula period (6-

hpf), neurula/segmentation period (12-hpf), pharyngula period (24-hpf), and hatching

period (48- and 72-hpf), 1, 2, 3, 4-week-old zebrafish larvae, and 3-month-old male or

female zebrafish. (A)The PCR products corresponding to the zebrafish COMT-1 cDNA,

were visualized by ethidium bromide staining. (B) RT-PCR analysis of the expression of the zebrafish β-actin at the same developmental stages as those described above.

Figure 3-5: Quantitative PCR Developmental stage-dependent expression of the zebrafish

COMT-1. The expression of the COMT-1 mRNA at different stages during embryogenesis and larval development until maturity was analyzed using quantitative real-time PCR. Samples analyzed were unfertilized zebrafish eggs, zebrafish embryos during the zygote period (0-hour post-fertilization (pf)), cleavage period (1-hpf), blastula period (3-hpf), gastrula period (6-hpf), neurula/segmentation period (12-hpf), pharyngula period (24-hpf), and hatching period (48- and 72-hpf), 1, 2, 3, 4-week-old zebrafish larvae, and 3-month-old male or female zebrafish. The zebrafish COMT-1 mRNA levels were quantified in arbitrary units, normalized against β-actin signal, as described in the section 2.

Discussion

The zebrafish has been gaining popularity as a model organism for different areas

of research due to its advantages over other vertebrate model organisms. (Alestrom et al.,

2006). The small size, high yield, external development, and the ease of phenotypic

analysis of the embryos/larvae are the characteristics which make the zebrafish a

convenient platform for pharmacological assessment and large-scale drug screening

(Milan, et al., 2003). A major focus of our laboratory is the development of the zebrafish as a model for studying the Phase II detoxifying/drug-metabolizing enzymes. A prerequisite is the identification of the different Phase II enzymes present in the zebrafish.

In the present study, two distinct COMT enzymes have identified, cloned, and expressed in E. coli. The expressed proteins were purified and characterized. Sequence similarity of the two zebrafish COMT enzymes compared with COMTs from rat, mouse, and human is illustrated in figure 3-1, which shows some highly conserved domains. The alignment of the zebrafish COMTs revealed a transmembrane domain indicating that they are likely membrane proteins in nature. Sequence analysis based on the BLAST pair wise search showed that the deduced amino acid sequence of the zebrafish COMT-1 displays 55%,

57%, and 58% identity to human, rat, and mouse COMT, respectively. The zebrafish

COMT-2 displays 57%, 56%, and 58% identity to human, rat, and mouse COMT, respectively (Figure 3-1). Both the two zebrafish COMTs were found to contain a number of conserved amino acid residues previously shown to be important for AdoMet-

binding (E140, D191, K194 fot the human COMT) and catechol-binding (W88, P224,

E249, and Y250 for the human COMT) (Vidgren et al., 1994; Rutherford et al., 2008;

Tsuji et al., 2009). These results demonstrated a considerable conservation with regards to the critical amino acid residues between the zebrafish and mammalian COMTs.

The zebrafish COMTs were expressed in soluble form in order to avoid problems that may be encountered in the expression and purification of membrane proteins. The

GST-COMT-1 and MBP-COMT-2 fusion proteins were used for the enzymatic characterization, because the free form of both enzymes, following protease treatment, was found to be unstable. The specific activities determined in the present study were corrected for the molecular mass of the GST moiety (23.9 kDa) or MBP (42kDa) in the fusion protein form of the two COMT enzymes. The purified enzymes showed differential activities toward different substrates as shown in Table 3.1. The differential activities toward different substrates could be due to the differences in chemical structures of the compounds tested as substrates. Both zebrafish COMT-1 and COMT-2 showed a wide pH optimum with regard to their specific activity (Figure 3-3).

The expression of the zebrafish COMT-1 was examined from embryogenesis to maturity by conventional RT-PCR (Figure 3-4) and RT-quantitative PCR (Figure 3- 5). It is generally known that the real-time PCR is more quantitative and sensitive in detecting even small amounts of specific nucleic acid sequences. This latter method was employed to further verify the expression of the COMT-1 mRNA. Comparing the results from both the conventional RT-PCR and RT-quantitative PCR, similar patterns of the expression pattern of COMT-1 mRNA throughout the developmental stages were observed. The only difference is that in the real-time PCR analysis, the expression of COMT-1 mRNA

for the adult stage did not decrease as in the conventional RT-PCR. Interestingly, using

both techniques, the adult female showed more expression of the mRNA than in the male.

This could be explained by the need to metabolize higher levels of the catecholestrogens

in the female. Previous studies have shown that estrogen is an important risk factor in

breast cancer. As estrogens are known to be hydroxylated to form catecholestrogens that

could be converted to quinines or semiquinones with carcinogenic activity (Yager, 1996).

The COMT enzyme can metabolize these caltecholestrogens via methylation generating

less carcinogenic products (Fotsis, 1994; Tenhunen, 1999). Other studies also confirmed

a significant increase in COMT expression in fetal membranes from laboring women,

suggesting the involvement of the COMT in regulating the level of prostaglandin E2

(Harirah, 2009).

Catechol-O-methyltransferase (COMT) is known to methylate endogenous

catecholamines, catecholestrogens, and many catechol drugs such as carbidopa, levodopa, benserazide, apomorphine, isoprenaline (isoproterenol), dobutamine, inamrinone, rimiterol and isoetharine. These drugs, while exerting therapeutic effects, may also have undesirable effects that could be managed through better directional, formulation, or dosage of the drug. With regard to the adverse effects of catecholic drugs, a better understanding about the mechanism of action, distribution, and developmental expression of the COMT enzyme would be valuable for maximizing desirable therapeutic effect and minimizing undesirable adverse effects. The results on zebrafish COMTs obtained in the present study should be valuable toward the understanding about the

Phase II drug metabolism in general and more specifically the O-methylation of catecholic compounds. Future studies including the preparation of specific COMT-

knockdown embryos/larvae are warranted in order to quantitatively analyze the metabolic

fates of different catecholic drugs and xenobiotics.

In summary, two zebrafish COMTs, designated COMT-1 and COMT-2, were

identified, cloned, expressed, purified, and characterized in the present study.

Additionally, the developmental stage-dependent expression of COMT-1 was analyzed in order to gain a better understanding of any possible protective role of COMT against the adverse effects of catechol drugs and xenobiotics during the developmental process. The present study is part of an overall effort to establish the zebrafish as an animal model for investigating the Phase II metabolism pathway of drugs and xenobiotics.

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