Tea catechins as potent non-competitive inhibitorsof angiotensin converting enzyme

September, 2016

Zhe Liu

Graduate School of Environmental and Life Science(Doctor’s Course)
OKAYAMA UNIVERSITY, JAPANCONTENTS

List of Figures4

Abbreviations

5

Chapter 1

General Introduction

7

1.1

Cardiovascular disease (CVD)7

1.2

Tea and tea catechins7

1.3

Cardiovascular-protective role of tea and tea catechins9

1.4

Angiotensin converting enzyme (ACE)

10

1.5

Purpose of this study

12

Galloylated catechins as potent inhibitors of angiotensin converting enzyme activity

13

2.1.

Abstract

13

2.2.

Introduction

13

2.3.

Materials and methods

15

Chapter 2

2.3.1.

Chemicals

15

2.3.2.

ACE activity assay

15

2.3.3.

SDS-PAGE and redox-cycling staining

15

2.3.4.

Statistical analysis

16

2.4.

Results

16

2.4.1.

Inhibitory effect of tea catechins on ACE activity

16

2.4.2.

Counteracting effect of borate on the ACE inhibition by tea catechins17

2.4.3.

Covalent modification of ACE by tea catechins18

2.4.4.

Determination of enzyme kinetic parameters of EGC and ECg by Lineweaver-Burk plots 19

2.5.
Chapter 3

Discussion

20

EGCg inhibits the activity of recombinant human angiotensin-converting enzyme through an

autoxidation-dependent mechanism
3.1

23

Abstract

23
1

3.2

Introduction

23

3.3

Materials and Methods

25

3.3.1

Chemicals

25

3.3.2

Cell culture

25

3.3.3

ACE activity assay

26

3.3.4

Influence of external factors on the ACE inhibitory activity of EGCg

26

3.3.5

Determination of hydrogen peroxide by the FOX assay

26

3.3.6

SDS-PAGE and redox-cycling staining

27

3.4

Results27

3.4.1

Inhibitory effects of EGCg on the ACE activity in vitro and cellular systems

27

3.4.2

The Zn2+ chelation mechanism should be ruled out in the ACE inhibition by EGCg

30

3.4.3

Generated hydrogen peroxide might not be involved in the ACE inhibition by EGCg

34

3.4.4

EGCg inhibited the ACE activity through an autoxidation-dependent manner

37

3.4.5

ACE inhibitory activity of EGCg was suppressed by the formation of complex with borate 39

3.4.6

A Lineweaver-Burk plot

42

3.5

Discussion

43

Detection of ACE by a novel tag-free probe

46

4.1

Abstract

46

4.2

Introduction

46

4.3

Materials and methods

47

4.3.1

Chemicals

47

4.3.2

ACE activity assay

48

4.3.3

Effects of Zn2+ on the ACE inhibitory activity of DOPAC

48

4.3.4

SDS-PAGE and redox-cycling staining

49

4.3.5

Synthesis of DPE via Fisher esterification

49

4.3.6

Click chemistry (CuAAC reaction)

49

4.3.7

Statistical analysis

50

4.4

Results50

4.4.1

Inhibitory effect of DOPAC on ACE activity.

50

Chapter 42

4.4.2

Effect of Zn2+ on the ACE inhibitory activity of DOPAC

52

4.4.3

Detection of DOPAC-bound ACE by SDS-PAGE/blotting with redox-cycling staining

53

4.4.4

Detection of ACE by click chemistry

54

4.5

Discussion

55

Summary

57

Acknowledgements

60

References

61

3

LIST OF FIGURES
Figure 1.1 Chemical structures of four major tea catechins ..................................................................... 8
Figure 1.2 Model of human somatic ACE ............................................................................................. 11
Figure 2.1 Inhibitory Effect of tea catechins on ACE activity. .............................................................. 17
Figure 2.2 Counteracting effect of borate on the ACE inhibition by tea catechins. ............................... 18
Figure 2.3 Covalent modification of ACE by tea catechins. .................................................................. 19
Figure 2.4 Lineweaver-Burk plots of ACE inhibition by EGC and ECg. .............................................. 20
Figure 3.1 Inhibitory activity EGCg on the enzymatic activity of ACE. ............................................... 29
Figure 3.2 The involvement of Zn2+-chelation in the ACE inhibition induced by EDTA or EGCg ....... 33
Figure 3.3 The involvement of the hydrogen peroxide formation in the ACE inhibition by EGCg ...... 36
Figure 3.4 Involvement of autoxidation in ACE inhibition by EGCg ................................................... 38
Figure 3.5 Effect of borate on the ACE inhibitory activity of EGCg. .................................................... 41
Figure 3.6 A Lineweaver-Burk plot of ACE inhibition by EGCg. ......................................................... 42
Figure 4.1 Inhibitory effect of DOPAC on the enzymatic activity of ACE ........................................... 51
Figure 4.2 Effect of Zn2+ on the ACE inhibitory activity of DOPAC .................................................... 52
Figure 4.3 Covalent modification of ACE by DOPAC .......................................................................... 53
Figure 4.4 Detection of ACE by click chemistry. .................................................................................. 54

4

ABBREVIATIONS
CVD, cardiovascular disease
EC, (-)-epicatechin
EGC, (-)-epigallocatechinECg, (-)-epicatechin gallate
EGCg, (-)-epigallocatechin gallate
LDL, low-density lipoprotein
ACE, Angiotensin converting enzymesACE, somatic ACE
tACE, testis ACE
RAS, renin-angiotensin system
Ang I, angiotensin I
Ang II, angiotensin II
HHL, hippuryl-histidyl-leucine
OPA, o-phthaldialdehyde
NBT, nitro blue tetrazolium
CBB, coomassie Brilliant Blue G-250
GAPDH, glyceraldehyde 3-phosphate dehydrogenaserh-ACE, recombinant human ACE
EDTA, ethylenediaminetetraacetic acid
ROS, reactive oxygen species
FBS, fetal bovine serum
FOX, ferrous ion oxidation-xylenol orange
DOPAC, 3,4-Dihydoxyphenylaceetic acid
Q4'G, quercetin 4'-glucoside
TBTMA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
BNPP, bis(4-nitrophenyl) phosphate
DPE, 3,4-dihydroxyphenylacetic acid propargyl ester56CHAPTER 1
General Introduction
1.1 Cardiovascular disease (CVD)

Cardiovascular disease (CVD) is a group of disorders that involve the heart and blood vessels.
Cardiovascular disease includes hypertension, coronary heart disease, cerebrovascular disease,
peripheral arterial disease, rheumatic heart disease, as well as congenital heart disease. CVDs are
the leading cause of death worldwide. According to the World Health Organization, an estimated
17.5 million people died from CVDs in 2005, which accounted for 30% of all global deaths. It has
been estimated that, by 2030, almost 23.6 million people will die from CVDs, mainly from heart
attack and stroke. However, most of the premature heart attacks and strokes are preventable bychecking and controlling the risk factors, such as high blood pressure, high cholesterol, and diabetes.

1.2 Tea and tea catechinsTea is considered to be one of the most popular beverages in the world. The consumption of tea
has a long history in Asia, which was literally recorded nearly 5,000 of years ago. Nowadays, tea isbecoming very popular in North America and Europe. It is cultivated in more than 30 countries
around the world. Although there are many types and grades of tea available in the market, basedon the manufacturing process, three main types of tea can be classified: green tea which does notundergo fermentation, oolong tea which is subject to partial fermentation, and black tea which ismade by full fermentation of plant.

Tea leaves are produced from the Camillia Sinensis plant. Fresh tea leaves contain aconsiderable amount of polyphenols (about 30% of the dry leaf weight), which include flavanols,
flavadiols, flavonols, and phenolic acids (Hertog et al., 1993). Flavanols are the major componentof tea, which are mainly composed of catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC),(-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg). Total polyphenol content is7

similar in different types of tea, but the composition varies based on the degree of polyphenol
oxidation during the manufacturing process. Unlike oolong tea and black tea, the green tea leaves
undergo a quick steam or heat treatment after being picked, in order to previously inactivate theoxidative enzymes and retain the polyphenol contents during the following manufacturing processes.
Green tea contains mainly catechins, predominantly EGCg which may account for more than 50%of the total catechins (Khan et al., 2006). In the manufacture of black tea, the monomeric flavanols
are susceptible to oxidative polymerization resulting in the formation of bisflavanols, theaflavins,
thearubigins, and other oligomers during fermentation (Menet et al., 2004; Obanda et al., 2001).
Nearly 10-20% of the dry weight of black tea is due to thearubigins, which are more extensively
oxidized and polymerized (Lin et al., 1999). Oolong tea contains monomeric catechins, theaflavins,
and thearubigins (Dou et al., 2007).

(-)-Epicatechin (EC)(-)-Epigallocatechin (EGC)(-)-Epicatechin gallate (ECg)(-)-Epigallocatechin gallate (EGCg)

Figure 1.1 Chemical structures of four major tea catechins8

1.3 Cardiovascular-protective role of tea and tea catechins

In recent years, tea has attracted significant attention due to its numerous health benefits, suchas antioxidant, anti-cancer, anti-inflammatory, and neuroprotective effects (Azam et al., 2004;
Higdon and Frei, 2003; Kakuda, 2002; Tipoe et al., 2007). The cardiovascular-protective effects oftea consumption have also been intensively studied. Accumulating epidemiological and clinicalstudies indicated that consumption of green tea is inversely associated with the risk of developing
cardiovascular diseases. According to the Ohsaki National Health Insurance Cohort Study, which
followed 40,530 Japanese adults aged 40 to 79 years, green tea consumption was associated with
reduced mortality due to cardiovascular disease. This inverse association was strongest in womenwith a relative risk of 0.69 (95% CI, 0.53-0.90) for women who consumed five or more cups perday (Kuriyama et al., 2006). Arab and colleagues found an inverse dose-response association withtea consumption on both incidence and mortality of stroke (Arab et al., 2013). A case-control study,
which was conducted in southern China, also demonstrated that long-term tea drinking significantly
decreased the risk of ischemic stroke (Liang et al., 2009). Increasing evidence shows that tea
consumption has a direct effect on cardiovascular disease risk factors. A meta-analysis of 13
randomized controlled trials examined the effects of green tea consumption on blood pressure. The
overall outcome of this meta-analysis suggested that green tea consumption significantly decreases
both systolic and diastolic blood pressure level (Peng et al., 2014). Furthermore, inclusion of black
tea in a controlled weight-maintaining diet significantly reduces total and LDL cholesterol in mildly
hypercholesterolemic adults (Davies et al., 2003).

Tea catechins, the most important bioactive components of tea, are considered to be responsible
for a broad variety of health-promoting effects, particularly cardiovascular-protective effects. Basedon the data from the Zutphen elderly study, Arts et al., found that catechin intake was inversely
associated with ischemic heart disease mortality (Arts et al., 2001). Moreover, in a placebo
controlled double-blind study, a beverage containing tea catechins (197.4 mg) was given twice a
day to the adult males and females with mild and borderline hypercholesterolemia. A significant
decrease on the serum total cholesterol level was observed at 8th week (228 ±23 mg/dl to 220 ±21
mg/dl, p < 0.01) and maintained until 12 weeks (222 ± 20 mg/dl, p < 0.05). The results suggested
9the benefits of tea catechins in individuals with mild or borderline hypercholesterolemia (Kajimoto
et al., 2003). Similarly, a meta-analysis of randomized controlled trials was conducted and foundthat green tea and tea catechins significantly reduced the total cholesterol and low-density
lipoprotein cholesterol. It also suggests that green tea and its tea catechins might be associated withimproved blood pressure, especially in subjects with systolic blood pressure ≥ 130 mmHg (Khalesi
et al., 2014).

As mentioned above, increasing observational studies suggest a possible association between
the intake of tea or tea catechins and the beneficial effects on cardiovascular disease. Several
mechanistic studies have also shown tea and tea catechins display promoting effects on systemicrisk factors and direct benefits on the vasculature and platelets which might be responsible for lower
cardiovascular risk. Taken together, tea and its catechins can be considered as potential candidates
for treatment or adjunctive treatment of cardiovascular disease.

1.4 Angiotensin converting enzyme (ACE)

Angiotensin converting enzyme (ACE) is known as a transmembrane dipeptidase. There are
two distinct isoforms of ACE in humans, the somatic and testicular isoforms (Sibony et al., 1993).
The somatic ACE is a 170-kDa glycoprotein, which is abundantly expressed in many tissues, suchas lung, kidney, liver, and brain (van Sande et al., 1985). The somatic ACE is composed of two
independent catalytic domains, termed the N- and C-domains. The two domains are about 60%
homologous in both DNA and amino acid sequences. In the regions containing residues involved in
catalysis, homology reaches 89% (Araujo et al., 2000; Wei et al., 1991). Both of the two domains
contain the zinc coordinating amino acid sequence HEXXH, which is crucial for enzymatic activity.
The testis ACE (tACE), a smaller molecular form (90-110 kDa), contains only the C-domain, which
is attached to cell membranes. The tACE is exclusively expressed in male germinal cells, and isrelated with generation (Ehlers et al., 1989; Hagaman et al., 1998).

10Figure 1.2 Model of human somatic ACE (Bernstein et al., 2013).
ACE was first identified as a key component of the renin-angiotensin system. It plays a crucialrole in the regulation of blood pressure and electrolyte homeostasis, by catalyzing angiotensin I toangiotensin II. The binding between angiotensin II and the AT1 receptor leads to vasoconstrictive
effects, stimulation of aldosterone secretion from the adrenal gland, retention of salt and water, as
well as growth stimulation. It has been already documented that angiotensin II is associated withcardiovascular disease, including hypertension, cardiac failure, coronary heart disease, and postmyocardial infarction. ACE inhibitors, such as captopril, lisinopril, and enalapril, which are capableof lowering the angiotensin II level, are initially introduced as antihypertensive agents. Their effects
are greater than expected by their ability to moderate blood pressure alone. It has been reported that
ACE inhibitors display clinical efficacy in the treatment of a wide range of cardiovascular diseases,
such as heart failure, peripheral artery disease, and acute myocardial infarction (Coppola et al., 2008;
Mujib et al., 2013; Shi et al., 2010). Thus, ACE inhibitors are generally used as the first-line drugfor the treatment of cardiovascular disease.

11

1.5 Purpose of this study

Angiotensin converting enzyme is an important member of renin-angiotensin system, whose
main function is to regulate the blood pressure, fluid and electrolyte balance. Inhibition of ACE is
one of the most effective strategies for the treatment of hypertension. Synthetic ACE inhibitors,
such as captopril and enalapril, are also widely used for the therapy of cardiovascular disease.
However, certain adverse effects, such as cough and skin rashes, are associated with the clinical useof these artificial ACE inhibitors. Therefore, the investigation of safe and efficient ACE inhibitors
could greatly benefit CVD patients. Tea catechins, the major biologically active components of tea,
have been reported to inhibit the enzymatic activity of ACE. However, the precise mechanism
involved in the inhibition remains to be clarified. The objective of the present study is to investigate
the molecular mechanism involved in the ACE inhibition by tea catechins.

12CHAPTER 2
Galloylated catechins as potent inhibitors of angiotensin converting enzyme
activity
2.1.

Abstract

In the present study, I investigated the inhibitory effects of four tea catechins, including (-)epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg) and (-)-epigallocatechingallate (EGCg), on the activity of purified ACE from rabbit lung in vitro. Each catechin treatment
significantly reduced the ACE activity with the order of potency being EGCg > ECg > EGC = EC.
The addition of 1 mM borate significantly recovered the reduced ACE activities by tea catechins,suggesting that hydroxyl groups at B-ring or at a galloyl moiety play an important role in the ACEinhibitory mechanism. The covalent modification of ACE by tea catechins was also observed by aredox-cycling staining experiment. A Lineweaver-Burk plot indicated that EGC and ECg were noncompetitive inhibitors. The galloylated catechins might more potently inhibit ACE activity in anallosteric manner through the interaction of the galloyl moiety with the non-catalytic site of ACE.

2.2.

Introduction

Accumulating epidemiological and intervention studies indicated that green tea consumption isconversely associated with the risk of hypertension, which might be largely ascribed to the presenceof polyphenols (Arab et al., 2013; Kuriyama, 2008; Nakachi et al., 2000). Tea catechins, including(-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg) and (-)epigallocatechin gallate (EGCg), are the primary class of polyphenols in green tea. The protective
effects of the catechins on cardiovascular diseases might be partially associated with its antioxidant
and free radical scavenging properties (Dragsted, 2003). In addition to the antioxidant action, somestudies have demonstrated that tea catechins have the ability to inhibit the activity of angiotensin
converting enzyme (ACE) (Actis-Goretta et al., 2006a; Persson et al., 2006). ACE, a dipeptidylcarboxypeptidase involved in renin-angiotensin system regulating blood pressure and electrolyte
balance, contributes to conversion of angiotensin I (Ang I) into angiotensin II (Ang II), a strong13vasoconstrictor, as well as metabolism of biologically active peptides such as bradykinin.

Like well-known artificial ACE inhibitors, tea catechins including EGCg have the capacity tochelate metal ions (Hayakawa et al., 1999; Sun et al., 2008). Alternatively, autoxidation ofpolyphenols with a catechol structure leads to the formation of polyphenol quinones and ROS suchas hydrogen peroxide, both of which can modify the protein function through the covalent adductformation and oxidation, respectively (Nakamura and Miyoshi, 2010). Our group previously
demonstrated that catalase significantly abolished the cytotoxicity induced by the pyrogallol-type
catechins (EGC or EGCg), whereas it did not influence the EC- or ECg-induced effect on human T
lymphocytic leukemia Jurkat cells (Tang et al., 2014). These findings suggested that tea catechinsare able to induce cytotoxicity in hydrogen peroxide-dependent and -independent manners. It has
also been reported that tea catechins could bind and inhibit several cellular enzymes, such as fattyacid synthase, glyceradehyde-3-phosphate dehydrogenase, and catalase (Mori et al., 2010; Pal et al.,2014; Wang et al., 2003). However, the precise mechanism underlying the ACE inhibition by tea
catechins remains to be clarified.

Tea catechins have a flavan-3-ol structure composed of A-, B-, and C-rings with or without agalloyl groups. The number and arrangement of phenolic hydroxyl groups in catechins are regarded
to be responsible for their differences in biological activities. Because all tea catechins share several
chemical properties, I hypothesized that they would inhibit ACE activity through a common
mechanism to the different extent. To test this hypothesis, I compared the inhibitory effect of eachcatechin on ACE activity in vitro. The effect of borate on the inhibition of ACE activity by catechins
was also examined, because it is a potent inhibitor of autoxidation of catechins by the formation ofcomplex with coordinate linkage (Mochizuki et al., 2002). The covalent modification of ACE bytea catechins was determined by a redox-cycling staining to confirm its involvement in ACE
inhibition. Finally, I employed a Lineweaver-Burk plot to confirm tea catechins as non-competitive
inhibitors.

14

2.3.

Materials and methods

2.3.1.

Chemicals

ACE from rabbit lung, hippuryl-histidyl-leucine (HHL) and o-phthaldialdehyde (OPA) werepurchased from Sigma (St. Louis, MO, USA). EC, EGC, ECg, and EGCg were obtained from Wako
Pure Chemical Industries (Osaka, Japan). Nitro blue tetrazolium (NBT) was from Tokyo Chemical
Industry (Tokyo, Japan). All other chemicals were obtained from Nacalai Tesque (Kyoto, Japan).

2.3.2.

ACE activity assay

The enzymatic activity of ACE was determined by a fluorimetric assay as previously reported(Santos et al., 1985) with some modifications. Briefly, 1 mU ACE was incubated with 1 mM HipHis-Leu in 0.1 M potassium phosphate buffer (pH 8.3) with 0.3 M NaCl in a total volume of 500 μl
at 37ºC for 30 min. In an inhibitory assay with tea catechins, ACE was pre-incubated with thechemicals at 4ºC for 15 min before the addition of substrate. Enzymatic reaction was terminated by
the addition of 1.2 ml of 0.34 M NaOH, followed by a 10-min incubation at room temperature with
100 μl of OPA (20 mg/ml). The solution was acidified with 200 μl of 3 N HCl and centrifuged at
3,000 × g at 4ºC for 10 min. The fluorescence intensity of the OPA-His-Leu adduct was measured
by a Hitachi F-2500 fluorescence spectrophotometer (Tokyo, Japan) at 360 nm excitation and 490
nm emission wavelengths. As for the effect of borate on the ACE inhibitory activity of tea catechins,
tea catechins were pre-incubated with or without borate at 4ºC for 15 min, followed by the additionof enzyme and substrate.

2.3.3.

SDS-PAGE and redox-cycling staining

ACE (15 mU) was incubated with 2 mM of tea catechins (EC, EGC, ECg, and EGCg) in 50
mM potassium phosphate buffer (pH 7.4) at 37ºC for 60 min. The reaction between tea catechinsand ACE was terminated by the addition of DTT (50 mM) as previously reported (Ishii et al., 2008).
The reaction mixtures were separated by SDS-PAGE using 10% gels. For a redox-cycling staining
experiment, the gel bands were transferred onto a PVDF membrane (0.45 μm, Millipores), and then15

EGCg-modified proteins were detected by staining with NBT (0.2 mg/ml in 2 M potassium
glycinate, pH 10). The membrane was immersed in the glycinate/NBT solution for approximately
30 min in dark, resulting in a blue-purple stain of quinoproteins bands and no staining of otherproteins. The membrane was also stained with Coomassie Brilliant Blue G-250 (CBB) for checking
the protein loading.

2.3.4.

Statistical analysis

All results are expressed as means ±SD of more than three independent experiments. Statisticalsignificance is denoted as *P < 0.05.

2.4.

Results

2.4.1.

Inhibitory effect of tea catechins on ACE activity

It has been reported that tea and tea catechins possess ACE inhibitory activity (Actis-Goretta etal., 2006a; Persson et al., 2006). To evaluate the ACE inhibitory activity of four major tea catechins,a fluorimetric assay reported by Santos et al. was used. The maximum absorption and theelimination metabolism of tea catechins occur very fast (Scalbert and Williamson, 2000). Therefore,
to compare the inhibitory effect of tea catechins, ACE was incubated with each catechin at theindicated concentrations for 15 min and then the enzymatic activity of each group was determined.
Each catechin treatment significantly reduced the ACE activity with the order of potency being
EGCg > ECg > EGC = EC (Figure 2.1); EGCg at the concentration of 50 μM completely inhibited
the ACE activity, whereas ECg and EGC or EC at the same concentration reduced it by 55% and15%, respectively. The present result suggested that the presence of the galloyl ester at 3-position
might play a more important role in ACE inhibitory activity than the pyrogallol group at B ring.

16

Relative ACE activity (%)

120
100

EC

80
EGC

60
ECg

40

EGCg

20
0
0

50

100

150

200

Concentration (μM)

Figure 2.1 Inhibitory Effect of tea catechins on ACE activity. The enzymatic activity of purified
ACE (from rabbit lung) was measured in the presence of tea catechins (EC, EGC, ECg, and EGCg)
at the indicated concentrations. The relative values represent means ± SD of three independent
experiments.

2.4.2.

Counteracting effect of borate on the ACE inhibition by tea catechins

To investigate the involvement of ortho-dihydroxyl (catechol, pyrogallol or galloyl) groups intea catechins on their ACE inhibitory activity, borate was used to form the stable complex with thesegroups at a basic condition (Mochizuki et al., 2002). First, tea catechins were treated with or without
1 mM borate at 4ºC for 15 min. After the addition of purified ACE and HHL, the ACE activity ofeach group was measured. As shown in Figure 2.2, the presence of 1 mM borate partly but
significantly recovered the ACE activities reduced by 100 μM catechins. It should be noted that the
influence on the ACE inhibitory activity of the four catechins brought by borate-treatment differ indegree (EGCg > ECg > EGC > EC).

17

Relative ACE activity (%)

120
100

*

*

80

*

60

*

40
20
0

Borate (1 mM)− + − + − + − + − +−EGC (100 μM) −ECg (100 μM)−
EGCg (100 μM) −EC (100 μM)

−
−
−
−

+
−
−
−

+
−
−
−

−
+
−
−

−
+
−
−

−
−
+
−

−
−
+
−

−
−
−
+

−
−
−
+

Figure 2.2 Counteracting effect of borate on the ACE inhibition by tea catechins. Tea catechinswere pre-incubated with or without 1 mM borate at 4ºC for 15 min. Then the ACE activity of eachgroup was measured. The relative values represent means ± SD of three independent experiments.
*P < 0.05 vs. borate (-) group.

2.4.3.

Covalent modification of ACE by tea catechins

The covalent modification of ACE by tea catechins was examined by SDS-PAGE/blotting with
redox-cycling staining. As shown in Figure 2.3, positive bands at about 50 kDa were observed in
the tea catechin-treated ACE groups, suggesting that all the four tea catechins can covalently bindto ACE (N- or C-domain). The order of potency for covalent modification of ACE was EGCg >EGC > ECg > EC, which is consistent with the previous report showing the modification of GAPDH
by tea catechins (Mori et al., 2010).

18

CTL

EC

EGC

ECg EGCg

50 kDa50 kDaFigure 2.3 Covalent modification of ACE by tea catechins. ACE (15 mU) was treated with tea
catechins (2 mM) at 37ºC for 60 min. The catechin-modified ACE was detected by a redox-cycling
staining (above) and CBB staining (below), respectively.

2.4.4.

Determination of enzyme kinetic parameters of EGC and ECg by Lineweaver-Burkplots

To gain further information about inhibitory mechanism, Lineweaver-Burk plots were
employed for the ACE inhibition by EGC and ECg. EGC (100 μM) or ECG (100 μM) was coincubated with various substrate concentrations (0.1, 0.25, 0.5, 1, and 2 mM) and 1 mU ACE, andthe plots are shown in Figure 2.4. In both cases, the two straight lines intersected at one point on the
1/[S] axis, indicating that both EGC and ECg were non-competitive inhibitors. These results
suggested that tea catechins could bind to a non-catalytic but specific site of ACE molecule andproduce a dead-end complex, regardless of whether or not a substrate is bound.

195

1/V (μM/min)−1

4
CTL

3

EGC

2
ECg

1
0
-2

0

2

4

6

8

10

12

1/[S] (HHL, mM−1)

Figure 2.4 Lineweaver-Burk plots of ACE inhibition by EGC and ECg. The ACE activity wasmeasured in the absence (closed circle) or presence of 100 μM EGC (closed triangle) or 100 μM
ECg (closed square). 1/V and 1/[S] represent the reciprocal of velocity and substrate concentration,
respectively.

2.5.

Discussion

Several studies suggested that tea catechins are potential ACE inhibitors. In the present study, I
confirmed the ACE inhibitory activity of the four major tea catechins (Figure 2.1). The inhibitory
tendency is partly consistent with the previous study using human umbilical vein endothelial cells(Persson et al., 2006), but different from the tendency for GAPDH inhibition by tea catechins (Ishii
et al., 2008) and for hydrogen peroxide production (Tang et al., 2014). The ACE inhibitory activityof each tea catechin suggested that the presence of the galloyl ester at 3-position might play a more
important role in ACE inhibitory activity than the pyrogallol group at B ring.

To investigate the involvement of ortho-dihydroxyl (catechol, pyrogallol or galloyl) groups intea catechins on their ACE inhibitory activity, I used borate to form the stable complex with thesegroups at a basic condition (Mochizuki et al., 2002). The results (Figure 2.2) suggested that hydroxyl
groups at B-ring or at a galloyl moiety play an important role in the ACE-inhibitory mechanism.
Although pyrogallol-type catechins are reported to be more susceptible to autoxidation andsubsequent electrophilic reaction than catechol-type catechins (Mori et al., 2010), the borate20

treatment had a stronger influence on the ACE inhibition of ECg than EGC (Figure 2.2). This result
also suggested that the galloyl ester might play a significant role in the ACE inhibitory activity
independently of autoxidation-related phenomena, such as the formation of polyphenol quinones
and hydrogen peroxide. The inhibitory effects of flavonoids on ACE activity have largely been
ascribed to the generation of chelate complexes with Zn2+ ion at the active center of ACE (Loizzo
et al., 2007). The structure-activity relationship and protein-ligand docking studies also supported
this idea (Guerrero et al., 2012). However, I preliminarily observed that the addition of exogenous
Zn2+ ion did not influence the inhibitory effect of EGCg on the ACE activity, suggesting little
contribution of its hydroxyl groups to the Zn2+ chelation.

It has been widely recognized that tea catechins are able to bind several proteins which isclaimed to be responsible for their biological activities (Chen et al., 2011; Ishii et al., 2008; Pal etal., 2014). At alkaline pH, catechins are readily oxidized into their corresponding quinones which
are able to react with thiol groups of cysteine residues in proteins or peptides (Bae et al., 2009; Ishii
et al., 2009). The autoxidation of catechins is initiated by the one-electron oxidation of the B-ring,which is the crucial step in the autoxidation of catechins (Mochizuki et al., 2002). The SDSPAGE/blotting with redox-cycling experiments revealed that tea catechins were covalently modified
the N- or C-domain of ACE (Figure 2.3). The order of potency for covalent modification of ACE
was EGCg > EGC > ECg > EC, which is consistent with the previous report showing themodification of GAPDH by tea catechins (Mori et al., 2010). However, there is an inconsistency
between the activity inhibition (Figure 2.1) and covalent modification of ACE (Figure 2.4),
supporting the idea that the galloyl ester might contribute to the ACE inhibitory activity in an
autoxidation-independent manner. In addition, the stronger activity of EGCg than that of ECg might
be attributed by the difference in their covalent modification abilities.

Furthermore, Lineweaver-Burk plots suggested that both EGC and ECg were non-competitive
inhibitors of ACE. Non-competitive ACE inhibitors are usually to be purified from some foodsources (Duan et al., 2014; Ni et al., 2012). However, the inhibition mechanism and the binding site
on ACE of these non-competitive inhibitors have not fully been understood. Molecular docking has
been applied to study the structure-activity relationship between bioactive peptides and ACE
21

(Gentilucci et al., 2012). The binding of inhibitors with ACE is strongly influenced by the three Cterminal amino acids of the peptide, that interact with the subsites S1, S1’, and S2’ at the ACE active
site; hydrophobic amino acids at the C-terminus such as Leu, Pro, Phe, Trp, and Tyr, would
significantly increase ACE-binding affinity to occupy the active site. Additionally, a high content ofamino acids Leu, Tyr, and Val at the N-terminus helps to enhance the ACE inhibitory activity of thepeptide, which might contribute to the non-competitive inhibition of ACE. In the present study, wedemonstrated that the galloylated catechins such as ECg and EGCg exhibited the more potent
inhibitory activity of ACE than EC and EGC with no galloyl group. This tendency is completely
matched with that for the binding affinity of tea catechins with human serum albumin (Bae et al.,2009), suggesting that the galloyl moiety is present for the specific binding with target proteinsthrough the hydrogen-bonding forces and electrostatic forces. In addition, a comparative study on
distribution coefficient of tea catechins suggested that the galloyl moiety would increase thehydrophobicity of the catechin molecule (Hashimoto et al., 1999). Taken together, I speculated thatthe galloylated catechins inhibit ACE activity in a non-compatitive manner through the interactionof the galloyl moiety with the non-catalytic site of ACE.

In conclusion, this study provides basic information concerning the inhibition of ACE
enzymatic activity by tea catechins, and enables us to propose an involvement of the galloyl moiety
in the catechin-protein interaction. The present findings encourage further study using not only theplausible structural model based on the docking simulation but also proteomic and reverse genetic
approaches to identify amino acid residues of ACE involved in the catechin-protein interaction.

22

CHAPTER 3
EGCg inhibits the activity of recombinant human angiotensin-converting
enzyme through an autoxidation-dependent mechanism
3.1 Abstract

Although dietary flavonoids, such as (-)-epigallocatechin-3-gallate (EGCg) and luteolin, havebeen shown to inhibit angiotensin converting enzyme (ACE) activity, the precise mechanisms still
remain to be clarified. In this study, I investigated the molecular mechanisms involved in the ACE
inhibition by EGCg, a major tea catechin. I first confirmed that the activity of HCT-116 cell lysate
and recombinant human ACE (rh-ACE) were inhibited by EGCg in a dose-dependent manner. Coincubation with Zn2+ showed no influence on the ACE inhibition by EGCg, whereas it completelycounteracted the inhibitory effect of EDTA, a representative ACE inhibitor chelating Zn2+ at theactive site of ACE. Although a considerable amount of hydrogen peroxide was produced during the
incubation of EGCg, the treatment of ACE with hydrogen peroxide had little effect on the enzymatic
activity. On the other hand, the co-incubation of EGCg with inhibitors of catechol oxidation, suchas borate or ascorbic acid, significantly diminished the EGCg inhibition. A redox-cycling staining
experiment revealed that rh-ACE was covalently modified by EGCg. Furthermore, LineweaverBurk plot analysis indicated that EGCg inhibited ACE activity in a non-competitive manner. These
results strongly suggested that EGCg might allosterically inhibit the ACE activity through theoxidative conversion into an electrophilic quinone and subsequent binding to the ACE.

3.2 Introduction

Cardiovascular disease (CVD) is the leading cause of mortality worldwide. The majority of
CVD is considered preventable by controlling or modifying its risk factors (Bernstein et al., 2013).
One of the most powerful risk factors for CVD is high blood pressure (Kannel, 1996), which
accounts for an estimated 54% of all strokes and 47% of all ischemic heart disease events (Lawes
et al., 2008). Angiotensin converting enzyme (ACE), a dipeptidyl-carboxypeptidase, plays a crucialrole in renin-angiotensin system (RAS), which is one of the major control systems for blood pressure,23

fluid and electrolyte balance. Within the RAS, ACE catalyzes the conversion of angiotensin I (AngI) into a potent vasoconstrictor angiotensin II (Ang II), and the metabolism of other peptides suchas the vasodilator bradykinin. Therefore, the inhibition of ACE is one of the most promisingstrategies for prevention of hypertension. Synthetic drugs having ACE inhibitory activity, such as
captopril and enalapril, are usually used for the treatment of CVD and for the treatment of heart
failure. However, these artificial ACE inhibitors have side effects such as cough, angioneurotic
edema and deleterious effects in pregnancy (Hanssens et al., 1991; Israili and Hall, 1992b; Shotan
et al., 1994; Simon et al., 1992; Vleeming et al., 1998). Thus, it is desirable for hypertensive patientsto investigate and develop a new type of ACE inhibitors without side effects.

Green tea is regarded as one of the most widely consumed beverages worldwide. Accumulating
epidemiological and intervention studies indicate that green tea or tea extracts consumption isassociated with the beneficial effects on decrease of blood pressure, which might be largely ascribedto the presence of polyphenols, such as flavanols, flavonols, anthocyanidins and phenolic acids(Negishi et al., 2004; Peng et al., 2014; Zaveri, 2006). Among them, flavanols, the collective term
for the catechins, is the primary class of polyphenols in green tea; (-)-epicatechin (EC), (-)epigallocatechin (EGC), (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg). It
has been suggested that the cardioprotective effects of tea catechins are partially associated with its
antioxidant and free radical scavenging properties (Babu and Liu, 2008). In addition to the
antioxidant action, some studies have demonstrated that tea catechins significantly inhibited ACE
activity (Actis-Goretta et al., 2006b; Persson et al., 2006). The well-known artificial ACE inhibitors,
such as captopril, enalaprilat and lisinopril, can inhibit ACE activity by directly interacting with
Zn2+ ions at the active sites of ACE (Natesh et al., 2004; Natesh et al., 2003). Tea catechins are
known to have the capacity to chelate metal ions like Zn2+, Fe2+ and Cu2+ (Esparza et al., 2005;
Khokhar and Owusu Apenten, 2003; Mira et al., 2002). The Zn2+-chelating property of tea catechinsseems to be one of the possible mechanisms for tea catechins to inhibit ACE activity. Alternatively,
autoxidation of polyphenols with a catechol structure leads to the formation of polyphenol quinones
and ROS, both of which can modify the protein function through the covalent adduct formation andoxidation, respectively (Ishii et al., 2008; Raza and John, 2007). Although covalent or non-covalentmodification with ACE might play a role in the ACE inhibition by tea catechins, the precise24mechanism remains to be clarified.

EGCg, the most abundant catechin in green tea, has been reported to possess higher ACE
inhibitory activity than other catechins (Chapter 2). In the present study, the underlying mechanism
involved in ACE inhibition by EGCg was investigated. The ACE inhibitory activity of EGCg wasattenuated by the combinatory incubation with borate or hydrogen peroxide. Incubation with zinc
sulfonate showed no change in the EGCg’s effect, whereas it significantly reduced the ACE
inhibition by a representative chelating agent, EDTA. These results indicate that Zn chelation andhydrogen peroxide production might be ruled out in the mechanism for ACE inhibition by EGCg.
This study provides the evidence showing that EGCg inhibits ACE activity, possibly through thecovalent interaction.

3.3 Materials and Methods
3.3.1

Chemicals

Recombinant human ACE/CD143 somatic form was purchased from R&D Systems(Minneapolis,

MN,

USA).

Hippuryl-histidyl-leucine(HHL),

histidyl-leucine

and

o-

phthaldialdehyde (OPA) were obtained from Sigma (St. Louis, MO, USA). EGCg and ZnSO4 werepurchased from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals including
EDTA were obtained from Nacalai Tesque (Kyoto, Japan).

3.3.2

Cell culture

Human colon tumor cell lines HCT-116 (carcinoma) was obtained from American Type Culture
Collection (Rockville, MD). HCT-116 cells were grown in McCoy’s 5a medium supplemented with
10% v/v fetal bovine serum (FBS), 50 U/mL penicillin and 50 μg/mL streptomycin (Gibco BRL).
Cells were maintained at 37ºC in an incubator under 5% CO2 and 95% air at constant humidity.
After a 3-d preculture, cells were harvested and lysed in 50 mM Tris (pH 7.4) containing 0.5%
Nonidet P-40. After centrifugation at 3,000 × g for 10 min at 4ºC, the supernatant was used for
determination. The measurement of ACE activity was described below.

25

3.3.3

ACE activity assay

The proteolytic activity of ACE was determined by a fluorimetric method, which waspreviously described by Santos et al. with some modifications (Santos et al., 1985). Briefly, a certain
amount of recombinant human somatic ACE (rh-ACE) or HCT-116 cell lysate was incubated with
1 mM Hip-His-Leu in 0.1 M potassium phosphate buffer (pH 8.3) with 0.3 M NaCl in a total volumeof 500 μl at 37ºC for 30 min. In an inhibitory assay with EGCg or EDTA, rh-ACE or cell lysate was
pre-incubated with the chemicals at 4ºC for 15 min before the addition of substrate. Enzymatic
reaction was stopped by the addition of 1.2 ml of 0.34 M NaOH, followed by a 10-min incubation
at room temperature with 100 μl of OPA (20 mg/ml). The solution was acidified with 200 μl of 3 N
HCl and centrifuged at 3,000 × g at 4ºC for 10 min. The fluorescence of the OPA-His-Leu adductwas detected by a Hitachi F-2500 fluorescence spectrophotometer (360 nm excitation, 490 nm
emission wavelength). All assays were carried out in triplicate.

3.3.4

Influence of external factors on the ACE inhibitory activity of EGCg

The effect of Zn2+ ions on the ACE inhibitory activity of EGCg was investigated by measuring
the enzymatic activity in the presence of EGCg supplemented with different concentrations (0, 5,
10, and 20 μM) of ZnSO4. ACE activity was also measured in the presence of Zn-EGCg complex
which was prepared according to method proposed by Sun et al. (Sun et al., 2008). EDTA was used
as a reference zinc chelator, and its ACE inhibitory activity was measured as mentioned above withthe addition of varied concentrations (0, 2, 2.5, 3, and 5 μM) of ZnSO4. Borate, H2O2, or ascorbic
acid was used to interfere the ACE inhibition by EGCg. EGCg was pre-incubated with borate (0,0.25, 0.5, and 1 mM), or hydrogen peroxide (0, 20, 50, and 100 μM) at 4ºC for 15 min, whereas
ascorbic acid (0, 50, 100, and 200 μM) was pre-incubated with EGCg at 37ºC for 15 min. Then the
ACE activity assay was initiated by the addition of enzyme and substrate.

3.3.5

Determination of hydrogen peroxide by the FOX assay

Ferrous ion oxidation-xylenol orange method (Qi et al., 2011) was used to determine the changeof hydrogen peroxide levels during the ACE activity assay. Different concentrations (0, 5, 10, 50,
26

and 100 μM) of EGCg was incubated with recombinant human ACE at 37ºC for 30 min. 45 μl of
resulted solution was collected and mixed with 5 μl of methanol, and incubated at room temperature.
After an incubation period of 30 min, FOX reagent composed of 125 μM xylenol orange, 250 μM
ammonium ferrous sulphate and 100 mM sorbitol in 25 mM H2SO4 was added and incubated foranother 30 min. The solution was then centrifuged at 15,000 rpm for 10 min at room temperatureand the absorbance was measured at 560 nm using Bio-Rad Benchmark Plus Multiplate
Spectrophotometer (Bio-Rad, Hercules, CA). The concentration of hydrogen peroxide wascalculated from standard curve. Stock solution of FOX reagent was previously prepared and wasused in the experiments at 10 times dilution.

3.3.6

SDS-PAGE and redox-cycling staining

To detect the covalent modification by EGCg, 2.5 ng of rh-ACE was incubated with different
concentrations of EGCg (0, 0.5 mM, and 1 mM) in 50 mM potassium phosphate buffer (pH 7.0) at
37ºC for 60 min. The reaction between EGCg and ACE was terminated by the addition of DTT (50mM final), following the method of Ishii (Ishii et al., 2008). The reaction mixtures were separated
by SDS-PAGE using 10% gels. For redox cycling staining, the gel bands were transferred onto a
PVDF membrane (0.45 μm, Millipores), and then EGCg-modified proteins were detected bystaining with NBT (0.2 mg/ml in 2 M potassium glycinate, pH 10). The blotting membrane was
immersed in the glycinate/NBT solution for approximately 3 h in the dark, resulting in a blue-purple
stain of quinoproteins bands and no staining of other proteins. After redox-cycling staining, theproteins were stained with Coomassie Brilliant Blue G-250 (CBB).

3.4 Results
3.4.1

Inhibitory effects of EGCg on the ACE activity in vitro and cellular systems

To confirm the inhibitory effect of EGCg on the enzymatic activity of ACE, a modified
fluorimetric assay was applied to measure the ACE activity of HCT-116 cell lysate in the presenceor absence of EGCg. An aliquot of lysate was pre-incubated with different concentrations of EGCg
(0, 1, 5, and 10 μM) at 4ºC for 15 min, then the enzyme activity assay of each group was initiated27

by the addition of artificial tripeptide substrate, HHL. The cell lysate treated with captopril, acommercial available ACE inhibitor, was used as a positive control. As shown in Figure 3.1A,
captopril (10 μM) treatment decreased the original ACE activity to 10.6%. The ACE inhibitory
activity of 100, 200, and 400 μM of EGCg was 75.6%, 42.9%, and 1.8%, respectively. Although
EGCg was weaker than captopril as ACE inhibitor, the treatment with EGCg could effectively anddose-dependently inhibit the human ACE activity.

To avoid influences coming from the contaminants in the cell lysate, recombinant human ACE(rh-ACE) was used in the following experiments. I first examined the inhibitory effect of EGCg on
the enzymatic activity of rh-ACE. As shown in Figure 3.1B, treatment with EGCg dose-dependently
inhibited the rh-ACE activity. Residual ACE activity after pretreatment of 1, 5 or, 10 μM EGCg was
68.5%, 19.8%, or 5.1%, respectively. These results strongly suggested that EGCg is a potential ACE
inhibitor.

28

(A)

Relative ACE activity (%)

120
100
80
60
40
20
0
CTL

10

100

Captopril (μM)

200

400

EGCg (μM)(B)

Relative ACE activity (%)

120
100
80
60
40
20
0
CTL

0.1

1

Captopril (μM)

510EGCg (μM)

Figure 3.1 Inhibitory activity EGCg on the enzymatic activity of ACE. The enzymatic activityof HCT-116 cell lysate (A) and recombinant human ACE (B) were measured in the presence of
EGCg at the indicated concentrations. The relative values represent means ± SD of three
independent experiments.

29

3.4.2The Zn2+ chelation mechanism should be ruled out in the ACE inhibition by EGCg

ACE has an absolute requirement of Zn2+ for its proteolytic activity. EDTA is a well-known
ACE inhibitor by chelating Zn2+ in the active sites of ACE (Buttery and Stuart, 1993). The rh-ACE
activity was actually suppressed by EDTA in a dose-dependent fashion (Figure 3.2A), which wasconsistent with previous a report (Kasahara and Ashihara, 1981). EDTA (5 μM) decreased the rhACE activity to 3.2%, whereas exogenously added Zn2+ competitively counteracted this inhibition
by EDTA (Figure 3.2B). These results further supported the idea that EDTA act as a Zn2+ chelator
to inhibit the ACE activity.

EGCg, which is capable of chelating metal ions (such as Fe2+, Cu2+, and Zn2+), is believed toinhibit ACE activity by coordinating Zn2+ in the active site (Kumamoto et al., 2001; Chen et al.,2007). To investigate the involvement of Zn chelation in ACE inhibition by EGCg, ACE inhibitory
effect of EGCg was measured in the presence of Zn2+ ions. As shown in Figure 3.2C, the rh-ACE
activity was decreased to 7.4% in the presence of 10 μM of EGCg. However, no significant changes
in the ACE activity had been observed when 5, 10, or 20 μM of Zn2+ was supplemented.

I next prepared an already-formed complex of EGCg with Zn2+ (Zn-EGCg complex) according
to Sun et al (Sun et al., 2008). As shown in Figure 3.2D, the UV-vis absorption spectra of EGCg
with or without Zn2+ were completely matched, whereas that of Zn-EGCg showed a disappearanceof maximum absorption at 322 nm. The UV-vis absorption spectra indicated that Zn-EGCg complex
is less likely to be formed in the potassium phosphate buffer (pH 8.3). Furthermore, Zn-EGCg
complex exhibited much less inhibitory effect on ACE activity than EGCg (Figure 3.2E). These
results suggested that ACE inhibition induced by EGCg might not be attributed to the metal ionschelating property.

30

(A)

Relative ACE activity (%)

120
100
80
60
40
20
0
0

1

1.5

2

5

EDTA (μM)(B)

Relative ACE activity (%)

120
100
80
60
40
20
0

EDTA (μM)

0

5

5

5

5

5

ZnSO4 (μM)

−

−

2

2.5

3

5

31

(C)

Relative ACE activit (%)

120
100
80
60
40
20
0
EGCg (μM)

0

10

10

10

10

ZnSO4 (μM)

0

0

5

10

20

(D)

32

(E)

Relative ACE activity (%)

100
80
60
40
20
0
0

20
Zn-EGCg (μM)

Figure 3.2 The involvement of Zn2+ chelation in the ACE inhibition induced by EDTA or
EGCg. (A) ACE inhibitory activity of EDTA. (B) Effect of Zn2+ on the ACE inhibitory activity of
EDTA. (C) Effect of Zn2+ on the ACE inhibitory activity of EGCg. (D) UV-vis absorption spectraof EGCg alone, zinc-EGCg complex and Zn2+/EGCg mixture. (E) ACE inhibitory activity of ZnEGCg complex.

33

3.4.3Generated hydrogen peroxide might not be involved in the ACE
inhibition by EGCg

EGCg has been reported to easily undergoes autoxidation at alkaline pH, which is accompaniedby the generation of hydrogen peroxide (Mochizuki et al., 2002; Nakayama et al., 2002). The
formation of hydrogen peroxide is considered to contribute to the anti-cancer properties of EGCg
against certain tumor cell lines. The net increase in hydrogen peroxide level triggers several
signaling cascades, which will lead to apoptotic cell death (Nakagawa et al., 2004; Ranzato et al.,2012; Satoh et al., 2013). Thus, the involvement of hydrogen peroxide generation in the ACE
inhibition by EGCg was investigated. After the incubation of EGCg in potassium phosphate buffer(pH 8.3) at 4ºC for 15 min, the FOX assay was used to monitor the changes in hydrogen peroxidelevels. As shown in Figure 3.3A, the hydrogen peroxide amount was elevated as the concentrationof EGCg increased (0, 5, 10, 50, and 100 μM). Further incubation of EGCg with ACE and HHL at
37ºC for 30 min showed no significant change in hydrogen peroxide level (data not shown). EGCg
at 10 μM, which could completely inhibit the enzymatic activity of ACE, produced 16.1 μM ofhydrogen peroxide during the 30-min incubation in assay buffer (Figure 3.3A). In fact, 20 μM of
exogenous hydrogen peroxide had no inhibitory effect on the ACE activity (Figure 3.3B).
Furthermore, co-incubation of hydrogen peroxide with EGCg did not enhance but rather inhibit the
EGCg-dependent effect on the ACE activity. These results indicated that hydrogen peroxide
produced by the autoxidation of EGCg might not be responsible for the ACE inhibition by EGCg.

34

(A)

100

H2O2 (μM)

80
60
40
20
0
0

5

10

50

100

EGCg (μM)(B)

Relative ACE activity (%)

120
100
80
60
40
20
0
0

20

50

H2O2 (μM)

35

100

(C)

Relative ACE activity (%)

100
80
60
40
20
0
0

20

50

100

H2O2 (μM)

Figure 3.3 The involvement of the hydrogen peroxide formation in the ACE inhibition by
EGCg. (A) Hydrogen peroxide formation by EGCg in potassium phosphate buffer (pH 8.3).
Different concentrations (0, 5, 10, 50, and 100 μM) of EGCg were added to potassium phosphatebuffer (pH 8.3), and incubated at 4ºC for 15 min. The hydrogen peroxide concentration wasmeasured by FOX assay. (B) Effect of exogenous hydrogen peroxide on the enzymatic activity of
ACE. Different concentrations (0, 20, 50, and 100 μM) of hydrogen peroxide was mixed with rhACE, and co-incubated at 4ºC for 15 min before the ACE activity assay. (C) Effect of exogenoushydrogen peroxide on the ACE inhibitory activity of EGCg. EGCg (10 μM) was pre-incubated withhydrogen peroxide at the indicated concentrations at 4ºC. After 15 min, ACE activity of each group
was measured.

36

3.4.4EGCg inhibited the ACE activity through an autoxidation-dependent manner

Next, I attempted to investigate whether autoxidation is involved in the ACE inhibition by
EGCg. It has been reported that several reducing agents, such as ascorbic acid, are able to prevent
EGCg oxidation (Chen et al., 1998). To examine the effect of ascorbic acid on the ACE inhibitory
effect of EGCg, EGCg was pre-incubated in potassium phosphate buffer (pH 8.3) in the presence ofdifferent concentrations of ascorbic acid (0, 50, 100, and 200 μM) at 37ºC for 15 min. As shown in
Figure 3.4A, the ACE activity was dose-dependently recovered by the addition of ascorbic acid.
With 200 μM of ascorbic acid, the ACE inhibition by EGCg was completely abolished. These results
suggested that autoxidation of EGCg plays an important role in the inhibitory effect on the ACE
activity.

Numerous evidence reveals that oxidation of EGCg leads to the formation of semiquinone
radicals which electrophilically attacks the thiol groups in proteins to form covalent adducts (Ishii
et al., 2008; Chen et al., 2010). To determine whether EGCg covalently binds to ACE, ACE (2.5 ng)
was co-incubated with varied concentrations of EGCg (0, 0.5, and 1 mM) in potassium phosphatebuffer (pH 7.4) at 37ºC for 60 min, then the reaction mixtures were analyzed by SDS-PAGE/blottingwith redox-cycling staining. As shown in Figure 3.4B, the positive bands around 160 kDa weredetected in the EGCg-treated group, indicating that EGCg covalently binds to ACE.

37

(A)

Relative ACE activity (%)

120
100
80
60
40
20
0
0

50

100

200

Ascorbic acid (μM)(B)

160 kDa160 kDa
rh-ACE (ng)

2.5

2.5

2.5

EGCg (mM)

0

0.5

1

Figure 3.4 Involvement of autoxidation in ACE inhibition by EGCg. (A) Effect of ascorbic acidon the ACE inhibitory activity of EGCg. EGCg (10 μM) was co-incubated with the indicatedconcentrations of ascorbic acid (0, 50, 100, and 200 μM) in potassium phosphate buffer at 37ºC for
15 min. (B) ACE (2.5 ng) was treated with different concentrations of EGCg (0, 0.5, and 1 mM) at
37ºC for 60 min. The EGCg-bound ACE was detected by redox-cycling staining (above) and CBBstaining (below), respectively.

38

3.4.5

ACE inhibitory activity of EGCg was suppressed by the formation of complex withborateTo further confirm the involvement of o-dihydroxyl groups in the ACE inhibition by EGCg, theeffect of borate on the ACE inhibitory activity of EGCg was investigated. It is well-known that
EGCg is capable of chelating with borate ion, and leads to the formation of EGCg-borate complex.
As shown in Figure 3.5A, the maximum absorption at 322 nm of EGCg was hypsochromicallyshifted to 301 nm in the presence of borate. The concentration-dependent increase of the absorption
at 301 nm clearly supported the formation of the EGCg-borate complex.

Next, the effect of borate on the ACE inhibition by EGCg was examined. The co-incubation of
EGCg and borate at 4ºC for 15 min significantly dose-dependently diminished the ACE inhibitory
activity of EGCg (Figure 3.5B). Furthermore, no EGCg-modified ACE was detected in the presenceof borate, whereas the positive band was observed in the absence of borate. These results suggested
that the generation of EGCg-modified ACE is one of the important mechanism for the ACE
inhibition by EGCg.

39

(A)

(B)

Relative ACE activity (%)

120
100
80
60
40
20
0
EGCg (μM)

0

10

10

10

10

Borate (mM)

0

0

0.25

0.5

1

40

(C)

160 kDa160 kDa

rh-ACE (ng)

2.5

2.5

2.5

2.5

EGCg (mM)

0

1

0

1

Borate (mM)

0

0

50

50

Figure 3.5 Effect of borate on the ACE inhibitory activity of EGCg. (A) UV-vis spectrum changeof 50 μM EGCg in the presence or absence of borate. (B) Effect of borate on the ACE inhibition by
EGCg. EGCg was co-incubated with the indicated concentrations of borate. (C) Effect of borate on
the covalent modification of ACE by EGCg. ACE (2.5 ng) was treated incubated with 1 mM EGCg
in the presence or absence of 50 mM borate at 37ºC for 60 min. Proteins were applied to SDSPAGE/blotting with redox-cycling staining (above) and CBB staining (below).

41

3.4.6A Lineweaver-Burk plot

To determine the inhibition pattern of EGCg on the rh-ACE activity, I employed a LineweaverBurk plot using various concentrations of the substrate and EGCg. As shown in Figure 3.6, twostraight lines intersected at one point on the 1/[S] axis, indicating that the inhibition of ACE by
EGCg is of non-competitive type. This result suggested that EGCg could bind to a non-catalytic but
specific site of ACE molecule and produce a dead-end complex, regardless of whether or not a
substrate is bound.

50

1/V (μM/min)−1

40
30

CTL

20

EGCg

10
0
-2

0

2

4

6

8

1/[S] (HHL, mM-1)

Figure 3.6 A Lineweaver-Burk plot of ACE inhibition by EGCg. The ACE activity wasmeasured in the absence (closed circle) or presence of 2.5 μM EGCg (closed square). 1/V and 1/[S]represent the reciprocal of velocity and substrate concentration, respectively.

42

3.5 Discussion

ACE is a widely distributed dipeptidyl carboxypeptidase in mammalian tissues and can be
expressed in many cell types, such as endothelial cells, and epithelial cells (Bruneval et al., 1986;
Caldwell et al., 1976; Defending et al., 1983; van Sande et al., 1985). In the preliminary study, I
found that the lysate of HCT-116 cells exhibited ACE activity, which could be inhibited by captopril
and EGCg (Figure 3.1A). In addition, the enzymatic activity of rh-ACE was also suppressed by
EGCg (Figure 3.1B). These results clearly supported that EGCg is effective to inhibit ACE activity,
which is consistent with the previous research (Persson et al., 2006).

EGCg is able to chelate metal ions, such as Zn2+, Fe2+ and Cu2+ (Esparza et al., 2005; Khokhar
and Owusu Apenten, 2003; Mira et al., 2002). EGCg coordinates to the Zn2+ complexes in a 1:1
molar ratio (Esparza et al., 2005; Sun et al., 2008). This metal-chelating property has been reported
to be associated with several biological activities, including the inhibition of enzyme activity, tumor
invasion and angiogenesis, as well as the misfolding or aggregation of amyloid β-sheet (Ghosh etal., 2006; Jung and Ellis, 2001; Zhang et al., 2012). ACE is known as a zinc-dependent peptidase
whose main function is to regulate arterial blood pressure as well as electrolyte balance through the
RAS. It is well-known that the pharmacological mechanism of captopril, enalaprilat, as well as
lisinopril, which are applied as the first-line drug therapy for hypertension, is the ability to directlyinteract with the Zn2+ ion at the active site of the enzyme. The ability of EGCg to chelate Zn2+ iswidely regarded as one of the possible explanations for its ACE inhibitory activity (Guerrero et al.,2012; Persson et al., 2006). However, Zn2+ supplementation did not change the ACE inhibitory
activity of EGCg (Figure 3.2C), whereas the ACE inhibition by EDTA was suppressed in thepresence of Zn2+ (Figure 3.2B). Moreover, UV-vis absorption spectra indicated that Zn-EGCg
complex is less likely to be formed in the potassium phosphate buffer (Figure 3.2D). All thesefindings suggest that ACE inhibition induced by EGCg might not be attributed to metal ionschelating property of tea catechins as EDTA did.

Hydrogen peroxide generated during the oxidation of EGCg, is known to be responsible forseveral biological properties, such as bactericidal action and anti-tumor effect (Arakawa et al., 2004;
Ranzato et al., 2012). Hydrogen peroxide has been shown to directly react with thiol groups in43

bioactive peptides or proteins and lead to the transduction of cellular signals (Winterbourn and
Hampton, 2008; Winterbourn and Metodiewa, 1999). Moreover, it has been found that hydrogen
peroxide is able to oxidize the cysteine residues of certain enzymes causing reversible inactivation(Bogumil et al., 2000; Denu and Tanner, 1998). In the present study, I found that exogenoushydrogen peroxide had no effect on the enzymatic activity of rh-ACE (Figure 3.3B). Furthermore,
the addition of hydrogen peroxide showed a slightly counteractive effect on the ACE inhibition by
EGCg (Figure 3.3C). These data suggested that the inhibition of rh-ACE activity by EGCg might
be independent of hydrogen peroxide-related mechanisms.

EGCg is extremely unstable in alkaline solutions and easily undergoes autoxidation. The
generation of hydrogen peroxide provides evidence that EGCg has been autoxidized during the
incubation in potassium phosphate buffer. Ascorbic acid is reported to stabilize EGCg and is able toreduce the oxidized EGCg (Chen et al., 1998; Sang et al., 2007). As shown in Figure 3.4A, ACE
inhibitory effect of EGCg was weakened by the addition of ascorbic acid, suggesting that EGCg
might inhibit the enzymatic activity of ACE through an autoxidation-dependent way. In recent years,
accumulating evidence suggests that catechol type polyphenol has potential to covalently bind tothiol groups of proteins through autoxidation (Chen et al., 2011; Ishii et al., 2009; Ishii et al., 2008).
EGCg has been reported to covalently bind to and inhibit several cellular key enzymes, such as
catalase and fatty-acid synthase (Pal et al., 2014; Wang et al., 2003). Redox-cycling staining, which
can specifically detect the presence of quinoproteins, provides evidence that EGCg covalently binds
to ACE (Figure 3.4B).

To investigate the relation between the ACE inhibition and the covalent modification of ACE
by EGCg, borate was used to form a stable complex with ortho-dihydroxyl groups in EGCg. As
shown in Figure 3.5A, UV-vis absorption spectra support the complex formation between EGCg
and borate. The formation of borate-EGCg complex diminished the ACE inhibitory activity of
EGCg, and prevented EGCg to covalent bind to ACE (Figure 3.5B and 3.5C). These data indicatethat the covalent modification of ACE by EGCG would lead to the enzyme inhibition. Additionally,
the Lineweaver-Burk plot (Figure 3.6) reveals that EGCg is a non-competitive inhibitor of ACE,
which is consistent with previous study. (Liu et al., 2003). Non-competitive inhibition means that44

the ACE inhibitory activity of EGCg is not affected, whether a substrate molecule is bound to theactive site of the enzyme or not. It also indicates that EGCg can bind to a specific site but not to theactive site of ACE.

In conclusion, I provide evidence that the ACE inhibition by EGCg might be independent of Zn
chelation and EGCg-induced hydrogen peroxide generation. Moreover, the study strongly suggested
that EGCg allosterically inhibits the ACE activity through the oxidative conversion into anelectrophilic quinone and subsequent binding to ACE. Further study is encouraged to explore theprecise binding site of ACE by EGCg utilizing proteomic and reverse genetic approaches.

45

CHAPTER 4
Detection of ACE by a novel tag-free probe
4.1 Abstract

3,4-Dihydoxyphenylacetic acid (DOPAC), is one of the colonic microflora-produced
catabolites of quercetin 4ʹ-glucoside (Q4ʹG). It has been reported that the oxidation of DOPAC leads
to the formation of o-quinone and the consequently covalent modification of proteins. In this study,
the effect of DOPAC on ACE activity and the involvement of their interaction were investigated.
Like other polyphenols, DOPAC inhibited the enzymatic activity of ACE in a dose-dependent
manner. The inhibitory activity of DOPAC to inhibit ACE was quite similar to that of a tea catechin,
EC. The covalent binding between DOPAC and ACE was confirmed by both redox-cycling staining
and a tag-free DOPAC probe. These findings suggested that (1) the catechol structure might beassociated with a potential ACE inhibitory activity; (2) ACE might be considered as one cellulartarget of DOPAC.

4.2 Introduction

Renin-angiotensin system (RAS) is one of the major control systems for blood pressure, fluidand electrolyte balance. Angiotensin converting enzyme (ACE), a dipeptidyl-carboxypeptidase,plays a crucial role in RAS by catalyzing the conversion of angiotensin I (Ang I) into
vasoconstrictive angiotensin II (Ang II), and the metabolism of other peptides such as thevasodilator bradykinin. Inhibition of ACE activity is considered to be one of the effective strategiesof the therapy of hypertension. Artificial ACE inhibitors, such as captopril and lisinopril, are thefirst-line drugs for the treatment of hypertension. Additionally, the ACE inhibitors are associatedwith the improvement of the cardiovascular disease, such as heart attack and stroke (Mancini et al.,
1996; McFarlane et al., 2003; Wald and Law, 2003). However, certain side effects, including coughand angioneurotic edema, have been reported due to the administration of these synthetic ACE
inhibitors (Israili and Hall, 1992a; Simon et al., 1992). Thus, there is an urgent requirement for the
exploration and development of a new type of ACE inhibitor without adverse effects.
46

Recent years, it has been widely reported that the oxidation of dietary polyphenols with acatechol structure leads to the formation of a corresponding o-quinone, which electrophilicallyreacts with thiol groups in proteins to form covalent thiol-flavonoid adducts (Ishii et al., 2009; Moriet al., 2010). The covalent protein-binding ability of polyphenols is associated with the inhibitionof several key cellular enzymes including fatty acid synthase, glyceradehyde-3-phosphate
dehydrogenase (GAPDH), and catalase (Mori et al., 2010; Pal et al., 2014; Wang et al., 2003). Tea
catechins, the major polyphenols in tea, are found to be potential ACE inhibitors (Persson et al.,2006). Our previous research indicated that the ACE inhibition by tea catechins might be attributed
to the covalent modification of ACE by tea catechins. Additionally, galloylated catechins were
found to be potent inhibitor of ACE. However, the role of catechol-like structures needs further
investigation.

3,4-Dihydroxyphenylacetic acid (DOPAC), a catechol-like substance, is one of the colonicmicroflora-produced catabolites of quercetin 4ʹ-glucoside (Q4ʹG). It has been reported that thecatechol structure in DOPAC can be oxidized to o-quinone, then electrophilically reacts with thethiol groups in peptides or proteins (Ishii et al., 2009). Although the interaction between DOPAC
and cellular proteins might be contributed to its biological activity, its targeted proteins have notbeen fully identified. A novel tag-free DOPAC probe, designed by Nakashima et al., was confirmed
to be effective to investigate its molecular target for induction of several phase II enzymes(Nakashima et al., 2016).

In the present study, the ACE inhibitory activity of DOPAC was determined. I also confirmedthat Zn chelation was not involved in the ACE inhibition by DOPAC. Redox-cycling staining wasapplied to detect the covalent binding of DOPAC to ACE. Finally, the efficiency of the tag-free
DOPAC probe toward ACE was examined.

4.3 Materials and methods
4.3.1

Chemicals

ACE from rabbit lung, hippuryl-histidyl-leucine (HHL) and o-phthaldialdehyde (OPA),
47

DOPAC, tyrosinase from mushroom, glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTMA), bis(4-nitrophenyl) phosphate (BNPP)
and azide-PEG3-biotin conjugate were purchased from Sigma (St. Louis, MO, USA). Nitro blue
tetrazolium (NBT) and 2-Propyl-1-ol were from Tokyo Chemical Industry (Tokyo, Japan).
Streptavidin, HRP conjugate was purchased from Funakoshi (Tokyo, Japan). Anti-actin antibody
and horseradish peroxidase-linked anti mouse IgG were purchased from Santa Cruz Biotechnology(Santa Cruz, USA). All other chemicals were obtained from Nacalai Tesque (Kyoto, Japan). Allother chemicals such as benzyl azide were purchased from Wako Pure Chemical Industries (Osaka,
Japan).

4.3.2

ACE activity assay

The enzymatic activity of ACE was determined by a fluorimetric assay as previously reported(Santos et al., 1985) with some modifications. Briefly, 1 mU ACE was incubated with 1 mM HipHis-Leu in 0.1 M potassium phosphate buffer (pH 8.3) with 0.3 M NaCl in a total volume of 500 μl
at 37ºC for 30 min. In an inhibitory assay, ACE was pre-incubated with DOPAC at 4ºC for 15 min
before the addition of substrate. Enzymatic reaction was terminated by the addition of 1.2 ml of 0.34
M NaOH, followed by a 10-min incubation at room temperature with 100 μl of OPA (20 mg/ml).
The solution was acidified with 200 μl of 3 N HCl and centrifuged at 3,000 × g at 4ºC for 10 min.
The fluorescence intensity of the OPA-His-Leu adduct was measured by a Hitachi F-2500
fluorescence spectrophotometer (Tokyo, Japan) at 360 nm excitation and 490 nm emission
wavelengths.

4.3.3

Effects of Zn2+ on the ACE inhibitory activity of DOPAC

The effect of Zn2+ on the ACE inhibitory activity of DOPAC was investigated by measuring the
enzymatic activity in the presence of 400 μM DOPAC supplemented with different concentrations(0, 200, 400, and 800 μM) of ZnSO4. After 15 min incubation at 4ºC, the ACE activity assay was
initiated by the addition of ACE and HHL to each group.

48

4.3.4

SDS-PAGE and redox-cycling staining

ACE (15 mU) was incubated with 2 mM (or 4 mM) of DOPAC in 50 mM potassium phosphatebuffer (pH 7.4) at 37ºC for 60 min. The reaction between tea catechins and ACE was terminated by
the addition of DTT (50 mM) as previously reported (Ishii et al., 2008). The reaction mixtures wereseparated by SDS-PAGE using 10% gels. For a redox-cycling staining experiment, the gel bands
were transferred onto a PVDF membrane (0.45 μm, Millipores), and then DOPAC-modified proteins
were detected by staining with NBT (0.2 mg/ml in 2 M potassium glycinate, pH 10). The membrane
was immersed in the glycinate/NBT solution for approximately 30 min in dark, resulting in a bluepurple stain of quinoproteins bands and no staining of other proteins. The membrane was also
stained with Coomassie Brilliant Blue G-250 (CBB) for checking the protein loading.

4.3.5

Synthesis of DPE via Fisher esterification

DPE was synthesized as previously reported (Nakashima et al., 2016). Briefly, DOPAC (30 mg,
0.18 mmol) and PTSA (6 mg, 0.03 mmol) were dissolved in the solution of dehydrated toluene (20ml) and 2-propyl-1-ol (1 ml, 17 mmol), and stirred for 6 h at 40ºC, after cooling to room temperature.
The reaction mixture was washed with 5% NaHCO3 solution and water twice. The aqueous phase
was extracted with 20 ml ethyl acetate and combined with the organic phase. The combined extract
was dried over anhydrous MgSO4, filtered, and evaporated to dryness. The product was purified by
preparative thin-layer chromatography (CHCl3-MeOH (9:1)) to afford 0.34 mg (0.07 mmol) of 3,4dihydroxyphenylacetic acid propargyl ester (DPE). Its structure and purity was confirmed by NMR
and mass spectrometry.

4.3.6

Click chemistry (CuAAC reaction)

CuAAC reaction with DPE and benzyl azide was performed as previously reported (Nakashima
et al., 2016). ACE was incubated with of 100 μM DPE in 50 mM potassium phosphate buffer (pH7.4) at 37ºC for 1 h. Subsequently, the solution was mixed with 1 mM copper (II) sulphate
pentahydrate, 1 mM sodium ascorbate, 0.1 mM TBTMA, and 20 μM azide-PEG3-biotin. Afterincubation in the dark for 1 h at room temperature, the protein samples were used to subject to SDS49

PAGE/blotting followed by the treatment with HRP-conjugated streptavidin for 1 h. The generated
streptavidin-biotin complex was visualized by Chemi-Lumi One Super.

4.3.7

Statistical analysis

All results are expressed as means ±SD of more than three independent experiments.

4.4 Results
4.4.1

Inhibitory effect of DOPAC on ACE activity.

To investigate the ACE inhibitory effect of DOPAC, purified ACE (from rabbit lung) was
incubated with various concentrations of DOPAC (0, 50, 100, 200, and 400 μM) at 4ºC for 15 min
and then the ACE activity of each group was measured. As shown in Figure 4.1C, ACE activity was
dose-dependently inhibited by DOPAC. DOPAC also showed a similar ACE inhibitory activity tothat of a tea catechin, EC.

50

(A)

(B)

(C)

Relative ACE activity (%)

120
100
80

DOPAC

60
EC

40
20
0
0

100

200

300

400

DOPAC (μM)

Figure 4.1 Inhibitory effect of DOPAC on the enzymatic activity of ACE. Chemical structuresof DOPAC (A) and EC (B). (C) The enzymatic activity of purified ACE (from rabbit lung) was
measured in the presence of DOPAC (closed circle) or EC (open circle) at indicated concentrations.
The relative values represent means ±SD of three independent experiments.

51

4.4.2

Effect of Zn2+ on the ACE inhibitory activity of DOPAC

To determine whether ACE inhibition by DOPAC is due to its zinc-chelating effect, ACE
inhibitory activity of DOPAC (400 μM) was measured in the presence of different concentrationsof ZnSO4 (0, 200, 400, and 800 μM). As shown in Figure 4.2, no significant changes in the ACE
inhibitory activity of DOPAC had been observed when 200, 400, and 800 μM of Zn2+ was
supplemented. These results indicated that ACE inhibition by DOPAC might not be attributed to its
zinc-chelating property.

Relative ACE activity (%)

120
100
80
60
40
20
0
DOPAC (μM)

0

400

400

400

400

ZnSO4 (μM)

0

0

200

400

800

Figure 4.2 Effect of Zn2+ on the ACE inhibitory activity of DOPAC. The ACE inhibitory activityof 400 μM DOPAC was measured in the presence or absence of ZnSO4. The relative values representmeans ±SD of three independent experiments.

52

4.4.3Detection of DOPAC-bound ACE by SDS-PAGE/blotting with redox-cycling staining

To examine whether DOPAC could covalently bind to ACE, ACE was co-incubated with 2 mM
(or 4 mM) of DOPAC in potassium phosphate buffer (pH 7.4) at 37ºC for 60 min, then DOPACmodified ACE was detected by SDS-PAGE/blotting with redox-cycling staining. As shown in
Figure 4.3, the DOPAC-treated ACE generated positive bands around 50 kDa, indicating that
DOPAC covalently binds to ACE.

50 kDa50 kDa

DOPAC (mM)

0

2

4

Figure 4.3 Covalent modification of ACE by DOPAC. ACE (15 mU) was treated with different
concentrations of DOPAC (0, 2 or 4 mM) at 37ºC for 60 min. The DOPAC-modified ACE wasdetected by a redox-cycling staining (above) and CBB staining (below), respectively.

53

4.4.4

Detection of ACE by click chemistry

Redox-cycling staining suggested that DOPAC can covalently bind to ACE. Therefore, to getfurther evidence for covalent binding of DOPAC with ACE, a tag-free DOPAC probe with the azide
labeled biotin and a horseradish peroxidase (HRP)-streptavidin complex was used. DPE wasincubated with ACE in the presence of laccase, followed by the CuAAC reaction with the azidelabelled biotin and identification using the HRP-conjugated streptavidin. Several positive bands
were observed in the DPE-treated groups, whereas no bands had been detected in the absence ofDPE (Figure 4.4). This result further supported the idea that DPE can covalently bind to ACE. It
also suggested that the tag-free probe using click chemistry is extremely effective for the detectionof the covalently modified ACE.

ACE (mU)

20

20

20

ACE (mU)

20

20

20

DPE (μM)

0

50

100

DPE (μM)

0

50

100

140 kDa

80 kDa70 kDa55 kDa40 kDaFigure 4.4 Detection of ACE by click chemistry. Detection of ACE tagged by CuAAC reaction
with DPE and the azide-labelled biotin. Purified ACE (20 mU) was incubated with or without DPE
in the presence of 30 U tyrosinase in 50 mM potassium phosphate buffer (pH 7.4) for 1 h at 37ºC.
DPE-tagged ACE was detected by CBB staining (left panel) and HRP-streptavidin (right panel).

544.5 Discussion

Flavonoids are polyphenolic compounds which are widely distributed in plants, such as fruits,
vegetables, tea, and wine (Harnly et al., 2006). Accumulating evidence suggests that certain
flavonoids are able to inhibit ACE activity, which plays a crucial role in the regulation of bloodpressure (Kameda et al., 1987; Loizzo et al., 2007; Oh et al., 2004). Structure-activity relationshipstudies have found that the catechol group in flavonoids is important for the extent of ACE inhibition(Guerrero et al., 2012). DOPAC (Figure 4.1A), one of the colonic microflora-produced catabolites
of quercetin 4'-glucoside, exhibited inhibitory effect on the ACE activity (Figure 4.1C). Additionally,
the ACE inhibitory potency of DOPAC was extremely similar as that of EC. It indicates that thecatechol group presented in DOPAC or EC might contribute, at least in part, to their ACE inhibitory
activity. Here, I speculated that both DOPAC and EC would inhibit ACE through a similar
mechanism.

ACE is a zinc-dependent metalloproteinase and has an absolute zinc requirement for its
enzymatic activity (Bünning and Riordan, 1981; Reeves and O'Dell, 1985). Numerous studies
suggest that certain health-promoting effects of flavonoids, including anti-oxidation, antiAlzheimer’s activity, might be ascribed to their metal ion chelating properties (Fernandez et al.,
2002; Mira et al., 2002). It has been revealed that the metal chelating ability of flavonoids depends
largely on the number of hydroxyl groups associated to the aromatic ring (Andjelković et al., 2006;
Khokhar and Owusu Apenten, 2003). Thus, zinc-chelating property of catechol or galloyl group
was usually proposed to explain the ACE inhibition by certain flavonoids (Guerrero et al., 2012).
However, ACE inhibition by DOPAC was independent of zinc chelation (Figure 4.2). In addition,
my previous work shows that ACE inhibitory activity of EGCg might not due to zinc chelation.
These findings suggested that although the presence of catechol or galloyl group might be associatedwith the ACE inhibition by certain polyphenols, their metal-chelating properties might be ruled out
in the mechanism for the ACE inhibition.

Because of the presence of catechol structure, DOPAC is oxidized to form o-quinone, which isable to covalently bind to thiol groups in GSH or proteins (Ishii et al., 2009). This covalentmodification of protein is usually associated with the protein function change. To confirm whether
55

ACE inhibition by DOPAC is due to the covalent binding, redox-cycling staining was applied. The
result indicated that DOPAC could covalently bind to ACE (Figure 4.3). These findings suggested
that DOPAC was initially autoxidized into its quinone form, then the corresponding quinone
electrophilically attacked the thiol groups of ACE. The results also indicated that ACE might be apotential intracellular target of DOPAC.

In conclusion, it is interesting to note that both DOPAC and tea catechins could covalently bindto ACE, and inhibit the enzymatic activity. It might suggest that the presence of catechol (orpyrogallol) group could inhibit the ACE activity through oxidative conversion into an electrophilic
quinone and subsequent binding to ACE. Further study is needed to gain structural insight into the
autoxidation-dependent interaction between catechol (or pyrogallol) group and ACE.

56

SUMMARY
Renin-angiotensin system (RAS) is one of the major control systems for blood pressure, fluidand electrolyte balance. Angiotensin converting enzyme (ACE) plays a crucial role in the RAS by
converting angiotensin I to angiotensin II, a potent vasoconstrictor. The inhibition of ACE is one of
the most promising strategies for treatment of hypertension. Synthetic ACE inhibitors, such as
captopril and enalapril, are also used as the first-line drugs for the therapy of cardiovascular diseases.
However, these artificial ACE inhibitors are believed to have certain side effects such as cough, skinrashes, and angioneurotic edema. Thus, there is an urgent requirement for the exploration anddevelopment of a new type of ACE inhibitors without adverse effects. Accumulating
epidemiological and intervention studies indicated that green tea consumption is inversely
associated with the risk of cardiovascular disease, which might be largely attributed to the presenceof polyphenols. Tea catechins, including (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), are the primary class ofpolyphenols in green tea. Although green tea and tea catechins has been shown to be promising as
potential ACE inhibitors, the precise mechanisms underlying ACE inhibition by catechins remain
to be clarified. In this thesis, the molecular mechanisms involved in the ACE inhibition by tea
catechins are investigated.

In Chapter 2, the inhibitory effects of four tea catechins, including (-)-epicatechin (EC), (-)epigallocatechin (EGC), (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), on
the enzymatic activity of ACE (purified from rabbit lung) were compared. Each catechin treatment
significantly reduced the ACE activity with the order of potency being EGCg > ECg > EGC = EC.
The addition of 1 mM borate significantly recovered the reduced ACE activities by tea catechins,suggesting that hydroxyl groups at B-ring or at a galloyl moiety play an important role in the ACEinhibitory mechanism. The covalent modification of ACE by tea catechins was also observed by aredox-cycling staining experiment. A Lineweaver-Burk plot indicated that EGC and ECg were noncompetitive inhibitors. The galloylated catechins might more potently inhibit ACE activity in anallosteric manner through the hydrophobic and hydrogen-bonding interaction of the galloyl moiety
with the non-catalytic site of ACE.
57

In Chapter 3, the molecular mechanisms involved in the ACE inhibition by EGCg, a major tea
catechin, were further investigated. The enzyme activity of recombinant human ACE was inhibitedby EGCg in a dose-dependent manner. Co-incubation with Zn2+ ion showed no influence on the
ACE inhibition by EGCg, whereas it completely counteracted the inhibitory effect of EDTA, arepresentative ACE inhibitor chelating Zn2+ at the active site of ACE. Although a considerable
amount of hydrogen peroxide was produced during the incubation of EGCg, the treatment of ACE
with hydrogen peroxide showed little effect on its enzymatic activity. On the other hand, the coincubation of EGCg with inhibitors of catechol oxidation, such as borate or ascorbic acid,significantly diminished the EGCg inhibition. A redox-cycling staining experiment revealed thatACE was covalently modified by EGCg. Furthermore, a Lineweaver-Burk plot analysis indicatedthat EGCg also inhibited the ACE activity in a non-competitive manner. These results strongly
suggested that EGCg allosterically inhibits the ACE activity through the oxidative conversion into
an electrophilic quinone and subsequent binding to the ACE.

3,4-Dihydoxyphenylacetic acid (DOPAC), having a catechol group, is one of the colonicmicroflora-produced catabolites of quercetin 4´-glucoside. A previous study indicated that DOPAC
is oxidized to form o-quinone, then covalently binds to sulfhydryls in GSH or proteins due to its
catechol structure. In Chapter 4, the modulating effect of DOPAC on ACE activity was also studied.
The co-incubation of DOPAC significantly inhibited the enzymatic activity of ACE in a dosedependent manner. Among the four tea catechins, DOPAC has the similar potency to inhibit ACE
to that of EC. In addition, the covalent binding of DOPAC with ACE was detected by a tag-free
DOPAC probe with the azide labeled biotin and a horseradish peroxidase (HRP)-streptavidin
complex. These findings suggested that (1) the catechol structure and its oxidative conversion into
electrophilic species are involved in the potential ACE inhibitory activity; (2) ACE is one of thepotential cellular targets of DOPAC.

In conclusion, this study indicates that: (1) tea catechins and DOPAC, both of which are derived
from food stuffs, are potential ACE inhibitors; (2) Zn2+ chelation as well as hydrogen peroxidedependent mechanism are ruled out in the mechanisms of the ACE inhibition by tea catechins; (3)the galloylated catechins (ECg and EGCg) allosterically inhibit the ACE activity through the58

oxidative conversion into an electrophilic quinone and subsequent binding to the ACE; (4) the
presence of galloyl moiety might play an important role in the hydrophobic and hydrogen-bonding
interaction with the non-catalytic site of ACE, associated with more potent ACE inhibitory activity.
The present findings encourage further study using not only the plausible structural model based on
the docking simulation but also proteomic and reverse genetic approaches to identify amino acidresidues of ACE involved in the catechin-protein interaction. It is likely that basic knowledge of theACE-binding site of tea catechins will provide a platform for the rational design of new domainselective ACE inhibitors with improved efficacy and pharmacological profiles. Taken together, thisstudy suggests that the galloylated catechins could be potential candidates to develop nutraceuticals
against hypertension and its related disease.

59

ACKNOWLEDGEMENTS
Three years ago, I was extremely grateful and honored that Professor Yoshimasa Nakamura
provided me the opportunity to come to Japan and study as a doctoral student in his lab. I would
like to express the deepest appreciation to him for his professional advice, valuable suggestions andcontinuous encouragement I received throughout the course of this study. Without his guidance andpersistent help, it would not have been possible to finish this dissertation.

I would like to thank Professor Yoshiyuki Murata for his amazing ideas and academic
suggestions. Special thanks to him for all the efforts he did throughout my research.

Thanks go to Shintaro Munemasa and Toshiyuki Nakamura for their valuable suggestions andmeaningful academic discussions.

Special thanks go to Yue Tang, Yinye Li, Wenxiu Ye, Xiaoyang Liu, Yujia Liu, Ying Liang,
Wensi Xu, Qifu Yang, as well as other foreign students in this lab. Their company and supporting
makes me feel less homesick and more concentrated on my research.

Thanks go to all the Japanese students here for their sincere help no matter in my research workor in my daily life. I really feel appreciated!

Finally, I would love to give my special thanks to my wonderful parents for their love andcontinuous support. I hope I can make my parents proud of me.

60

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