1 Characterization of Angiotensin-Converting Enzyme Inhibitory

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Bioactive Constituents, Metabolites, and Functions

Characterization of Angiotensin-Converting Enzyme Inhibitory Activity of X-Hyp-Gly-Type Tripeptides: Importance of Collagen-Specific Prolyl Hydroxylation Yuki Taga, Osamu Hayashida, Ahmed Ashour, Yhiya Amen, Masashi Kusubata, Kiyoko Ogawa-Goto, kuniyoshi shimizu, and Shunji Hattori J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03648 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 4, 2018

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Journal of Agricultural and Food Chemistry

Characterization of Angiotensin-Converting Enzyme Inhibitory Activity of X-Hyp-GlyType Tripeptides: Importance of Collagen-Specific Prolyl Hydroxylation

Yuki Taga,*,† Osamu Hayashida,† Ahmed Ashour,‡,§ Yhiya Amen,‡,§ Masashi Kusubata,† Kiyoko Ogawa-Goto,† Kuniyoshi Shimizu,‡ and Shunji Hattori†

†

Nippi Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan

‡

Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581,

Japan §

Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516,

Egypt

To whom correspondence should be addressed: Yuki Taga Nippi Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan Tel: +81-297-71-3046; Fax: +81-297-71-3041 E-mail: [email protected]

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ABSTRACT

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Hydroxyproline (Hyp) is a collagen-specific amino acid formed by post-translational

3

hydroxylation of Pro residues. Various Hyp-containing oligopeptides are transported into the

4

blood at high concentrations after oral ingestion of collagen hydrolysate. Here we investigated

5

the angiotensin-converting enzyme (ACE) inhibitory activity of X-Hyp-Gly-type tripeptides. In

6

an in vitro assay, ginger-degraded collagen hydrolysate enriched with X-Hyp-Gly-type

7

tripeptides dose-dependently inhibited ACE, and various synthetic X-Hyp-Gly-type tripeptides

8

showed ACE-inhibitory activity. In particular, strong inhibition was observed for Leu-Hyp-Gly,

9

Ile-Hyp-Gly, and Val-Hyp-Gly with IC50 values of 5.5, 9.4, and 12.8 µM, respectively.

10

Surprisingly, substitution of Hyp with Pro dramatically decreased inhibitory activity of X-Hyp-

11

Gly, indicating that Hyp is important for ACE inhibition. This finding was supported by

12

molecular docking experiments using Leu-Hyp-Gly/Leu-Pro-Gly. We further demonstrated that

13

prolyl hydroxylation significantly enhanced resistance to enzymatic degradation by incubation

14

with mouse plasma. The strong ACE-inhibitory activity and high stability of X-Hyp-Gly-type

15

tripeptides highlight their potential for hypertension control.

16 17

Keywords: collagen hydrolysate, hydroxyproline, X-Hyp-Gly, ginger, angiotensin-converting

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enzyme

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INTRODUCTION

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Collagen is the most common protein in the body and is particularly abundant in connective

21

tissues, such as skin, bone, and tendon. Features of the amino acid sequence of collagen are

22

repeating Gly-Xaa-Yaa triplets and numerous post-translational modifications catalyzed by

23

specific enzymes before triple helix formation in the endoplasmic reticulum.1 Most Pro residues

24

lying at the Yaa position are hydroxylated to 4-hydroxyproline (4-Hyp) (~100 residues/1000

25

amino acid residues in type I collagen), contributing to the stabilization of the collagen triple

26

helix.2 Other collagen-specific post-translational modifications, including prolyl hydroxylation at

27

the Xaa position forming 3-Hyp and lysyl hydroxylation/glycosylation at the Yaa position, are

28

rare compared to 4-Hyp (hereinafter simply referred to as Hyp).3

29

Oral ingestion of collagen hydrolysates prepared by enzymatic hydrolysis of gelatin has shown

30

various beneficial effects, such as increasing bone density,4, 5 modulating lipid metabolism,6, 7

31

lowering blood sugar levels,8,

32

oligopeptides were identified in blood at markedly high concentrations after oral ingestion of

33

collagen hydrolysate.14 The total concentration of Hyp-containing oligopeptides reaches 100 µM

34

in the blood after the ingestion,15,

35

conferred by the presence of Hyp within the peptide sequence.14, 17-19 Various Hyp-containing

36

peptides appear in the blood, including Pro-Hyp, Hyp-Gly, and X-Hyp-Gly-type tripeptides.14, 16-

37

18, 20

38

oligopeptides, such as growth stimulation of skin fibroblasts,17,

39

differentiation,23, 24 and improvement of skin barrier dysfunction.25

9

and lowering blood pressure.10-13 In 2005, Hyp-containing

16

probably due to their high peptidase/protease resistance

Recent studies have demonstrated physiological activities of these collagen-specific 21, 22

promotion of osteoblast

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Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that converts an

41

inactive form of decapeptide, angiotensin I, into a potent vasoconstrictor, angiotensin II.26 This 3



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enzyme also catalyzes inactivation of a vasodilator, bradykinin. Therefore, inhibition of ACE is a

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major target for the prevention and treatment of hypertension. A wide variety of food proteins

44

have been reported as a source of ACE-inhibitory peptides.26, 27 For example, well-established

45

ACE-inhibitory peptides, Val-Pro-Pro and Ile-Pro-Pro, were isolated from sour milk and

46

demonstrated to decrease blood pressure after oral administration.28, 29 To date, various types of

47

enzymatic hydrolysates of collagen have been discovered to possess ACE-inhibitory activity and

48

in vivo antihypertensive effects.10-13,

49

peptides was determined in some studies, such as Gly-Pro-Leu,30, 31 Gly-Pro-Met,30 Gly-Pro-

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Val,31 Met-Gly-Pro,13 and Val-Gly-Pro-Val.32 In addition, Hyp-containing peptides possessing

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high ACE-inhibitory activity were identified in chicken breast muscle hydrolysate (Gly-Phe-

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Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe) and chicken leg collagen hydrolysate (Gly-Ala-Hyp-

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Gly-Leu-Hyp-Gly-Pro).11, 33

30, 31

The structure of collagen-derived ACE-inhibitory

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We recently developed a novel type of collagen hydrolysate using ginger protease.18 The

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unique substrate specificity of ginger protease recognizing Pro and Hyp at the P2 position enables

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efficient generation of X-Hyp-Gly-type tripeptides, which are almost undetectable in

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commercially available collagen hydrolysates. Although X-Hyp-Gly-type tripeptides appear in

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blood at high concentrations after oral ingestion regardless of the types of collagen hydrolysates,

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the ginger-degraded collagen hydrolysate (GDCH) shows more higher absorption of this type of

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tripeptide.18 Iwai et al. investigated the ACE-inhibitory activity of Hyp-containing peptides

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detected in human blood after oral intake of chicken collagen hydrolysate, such as Ala-Hyp (IC50

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= 0.177 mM) and Pro-Hyp (IC50 = 18.118 mM).34 The IC50 value against ACE was also

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measured for some X-Hyp-Gly-type tripeptides (0.601 mM for Ser-Hyp-Gly, 0.711 mM for Ala-

64

Hyp-Gly, 1.129 mM for Pro-Hyp-Gly, and 82.330 mM for Glu-Hyp-Gly).34 The inhibitory 4


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activity of X-Hyp-Gly-type tripeptides was moderate in their study. However, in the current

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study, we found that X-Hyp-Gly-type tripeptides where X is branched-chain aliphatic amino

67

acids strongly inhibit ACE. We also showed that Hyp is a contributing factor to the ACE-

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inhibitory activity of X-Hyp-Gly.

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MATERIALS AND METHODS

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Chemicals. ACE from porcine kidney and hippuric acid (Hip) were purchased from Sigma-

72

Aldrich (St. Louis, MO, USA). Hippuryl-histidyl-leucine (HHL) was purchased from Peptide

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Institute (Osaka, Japan), and N-benzoyl-d5-glycine (d5-Hip) was purchased from CDN Isotopes

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(Quebec, Canada). Gly-Ala-Hyp (>99% purity), Ala-Pro-Gly (>99% purity), and Ile-Pro-Pro

75

(>98% purity) were purchased from Bachem (Bubendorf, Switzerland), and other peptides

76

(>95% purity) were custom synthesized by AnyGen (Kwangju, Korea).

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Ethics Statement. All animal studies were approved by the Experimental Ethical Committee of Nippi Research Institute of Biomatrix.

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Preparation of Collagen Hydrolysates. GDCH was prepared from bovine bone gelatin using

80

ginger protease as reported previously,18 with a slight modification for industrial production.

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GDCH was heated at 81 °C and pH 5.5 for 6 h to prepare heat-treated GDCH (H-GDCH). The

82

collagen hydrolysates were dissolved in 0.1% formic acid for liquid chromatography–mass

83

spectrometry (LC–MS) analysis of oligopeptides.

84

ACE Inhibition Assay. ACE-inhibitory activity of collagen hydrolysates and synthetic

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peptides was measured as reported previously with some modifications.35, 36 Peptide samples at

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varied concentrations were pre-incubated with ACE in 75 mM Tris-HCl (pH 8.3) at 37 °C for 10

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min, and the reaction was started by adding HHL as a substrate. The final concentrations of ACE

88

and HHL in the reaction mixture were 1 mU/mL and 0.1 mM, respectively. Aliquots were taken

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at 5, 10, 20, and 30 min during incubation at 37 °C, and an equal volume of 1% formic acid

90

containing d5-Hip was added to stop the reaction. An external calibration curve was prepared

91

using a standard of Hip after mixing with an equal volume of 1% formic acid containing d5-Hip.

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Hip was quantified by LC–MS in multiple reaction monitoring (MRM) mode using a hybrid

93

triple quadrupole/linear ion trap 3200 QTRAP mass spectrometer (AB Sciex, Foster City, CA)

94

coupled to an Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA).

95

Samples were loaded onto an Ascentis Express C18 HPLC column (5 µm particle size, L × I.D.

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150 mm × 2.1 mm; Supelco, Bellefonte, PA) at a flow rate of 500 µL/min and separated by a

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binary gradient as follows: 100% solvent A (0.1% formic acid) for 2 min, linear gradient of 0–

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70% solvent B (100% acetonitrile) for 4 min, 90% solvent B for 2 min, and 100% solvent A for

99

2 min. The MRM transitions of Hip and d5-Hip were m/z 180.2→105.2 and m/z 185.2→110.2,

100

respectively, with collision energy of 17 V. Concentrations of liberated Hip in test samples were

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estimated using the external calibration curve with correction using d5-Hip.

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Enzymatic activity of ACE was calculated from the time course of the liberated Hip

103

concentration (nmol/min), and inhibitory activity was calculated from the ACE activity of the

104

sample and blank control as follows: inhibitory activity (%) = 100 × (blank − sample)/blank. The

105

IC50 value was determined by plotting the logarithm of ACE-inhibitory activity against peptide

106

sample concentration.

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The mode of ACE inhibition and Ki value were determined by Dixon plot37 under the same

108

reaction conditions with two substrate concentrations of 0.1 and 1.0 mM. Three peptide

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concentrations were used for the reaction (0, 1.9, and 4.7 µM for Leu-Hyp-Gly and 0, 50.1, and

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125.2 µM for Leu-Pro-Gly).

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Peptide Degradation Using Mouse Plasma. The experiment was performed as reported

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previously.18 In brief, a synthetic tripeptide mixture (Ala-Hyp-Gly, Leu-Hyp-Gly, Ala-Pro-Gly,

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Leu-Pro-Gly, and Ile-Pro-Pro) was incubated with fresh plasma prepared from male ICR mice

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(15 weeks of age; Japan SLC, Shizuoka, Japan) at a concentration of 20 µg/mL each at 37 °C.

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Time-course samples were collected at 0, 15, 30, 60, and 90 min during the incubation. The

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reactants were deproteinized by adding three volumes of ethanol, and the ethanol-soluble

117

fractions were diluted with 0.1% formic acid for LC–MS analysis.

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LC–MS Analysis of Oligopeptides. We quantified oligopeptides in collagen hydrolysates and

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peptide degradation samples by LC–MS in MRM mode. Sample separation was performed using

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an Ascentis Express F5 HPLC column (5 µm particle size, L × I.D. 250 mm × 4.6 mm; Supelco)

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at a flow rate of 400 µL/min with a binary gradient as follows: 100% solvent A (0.1% formic

122

acid) for 7.5 min, linear gradient of 0–90% solvent B (100% acetonitrile) for 12.5 min, 90%

123

solvent B for 5 min, and 100% solvent A for 5 min. The MRM transitions of the oligopeptides

124

are shown in Table S1.

125

Molecular docking. A molecular binding experiment was designed to predict the binding

126

mode of Leu-Hyp-Gly and Leu-Pro-Gly at the active sites of ACE. The crystal structure of ACE

127

was downloaded from the Protein Data Bank (www.rcsb.org; access code 4CA5)38 and then

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imported into the work place of the software AutoDock Vina implemented in PyRx.39 The ligand

129

with the most favorable binding energy (increasingly negative value) was considered a potential

130

inhibitor.

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RESULTS

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ACE-Inhibitory Activity of Collagen Hydrolysates Prepared Using Ginger Protease. We

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used two types of collagen hydrolysates for the ACE inhibition assay. GDCH was prepared by

135

enzymatic hydrolysis of bovine gelatin using ginger protease, and GDCH was further subjected

136

to heat treatment at 81 °C for 6 h to prepare H-GDCH. The contents of X-Hyp-Gly- and X-Pro-

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Gly-type-tripeptides in these collagen hydrolysates are shown in Table 1. Consistent with

138

previous observations,18, 40, 41 we detected substantial amounts of various X-Hyp-Gly in GDCH.

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Pro-Hyp-Gly was only slightly generated because ginger protease cannot cleave the Gly-Pro

140

bond.18 We also detected small amounts of X-Pro-Gly originating from partial hydroxylation of

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Pro residues. In contrast, all X-Hyp-Gly-type tripeptides were significantly diminished in H-

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GDCH due to efficient cyclization into cyclo(X-Hyp) through heating as reported recently.40

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Similar heat-induced decreases were also observed for X-Pro-Gly, probably due to conversion

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into cyclo(X-Pro). Gly-Pro-Y-type tripeptides, which are another major type of oligopeptides in

145

GDCH, were only slightly decreased by the heat treatment (data not shown) as reported

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previously.41

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We used LC–MS to detect Hip liberated from HHL by porcine kidney ACE for measurement of

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ACE-inhibitory activity of the collagen hydrolysates. Figure 1 shows ACE inhibition curves of

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GDCH and H-GDCH. Both collagen hydrolysates dose-dependently inhibited ACE activity, and

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the IC50 value of GDCH (0.210 mg/mL) was markedly lower than that of H-GDCH (0.336

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mg/mL). Since the most striking difference between GDCH and H-GDCH was the X-Hyp-Gly

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content (Table 1), it was suggested that this type of Hyp-containing tripeptide has high ACE-

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inhibitory activity.

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ACE-Inhibitory Activity of X-Hyp-Gly-Type Tripeptides. We investigated ACE-inhibitory

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activity of X-Hyp-Gly using a series of synthetic peptides (Table 1). The IC50 value of Ile-Pro-

156

Pro used as a positive control was 0.9 µM. This was lower than a previously reported value (5

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µM) estimated by a conventional method with UV detection of Hip,28 probably due to the low

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substrate concentration achieved by sensitive LC–MS detection. Among the X-Hyp-Gly-type

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tripeptides, Leu-Hyp-Gly most strongly inhibited ACE (IC50 = 5.5 µM). This ACE-inhibitory

160

activity was lower than, but not far from, that of the known ACE-inhibitory peptide, Ile-Pro-Pro.

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In addition, higher ACE-inhibitory effects were also detected for Ile-Hyp-Gly and Val-Hyp-Gly

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with IC50 values of 9.4 µM and 12.8 µM, respectively. Other X-Hyp-Gly-type tripeptides,

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including Ala-Hyp-Gly, Glu-Hyp-Gly, Phe-Hyp-Gly, Pro-Hyp-Gly, and Ser-Hyp-Gly, displayed

164

moderate inhibition (IC50 = 130.6–404.5 µM). ACE-inhibitory activity of Leu-Pro-Gly was

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extremely low (IC50 = 373.8 µM) compared to that of Leu-Hyp-Gly. Other X-Pro-Gly-type

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tripeptides (X = Ala, Pro, Ser, and Val) also showed 5- to 10-fold decreases in ACE-inhibitory

167

activity compared to corresponding X-Hyp-Gly-type tripeptides. In contrast, ACE-inhibitory

168

activity of another type of Hyp-containing tripeptide, Gly-X-Hyp, was lower than that of

169

corresponding Gly-X-Pro-type tripeptides, showing 5- and 16-fold increases in IC50 values for

170

Gly-Ala-Hyp and Gly-Leu-Hyp, respectively (Table S2). A pronounced change was observed by

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removing the C-terminal Gly residue from Leu-Hyp-Gly (IC50 = 3462.1 µM for Leu-Hyp).

172

We estimated the mode of ACE inhibition and Ki value for Leu-Hyp-Gly and Leu-Pro-Gly by

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Dixon plot (Fig. 2). Both peptides were determined to be competitive inhibitors, and the Ki value

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significantly differed between the peptides (5.5 µM for Leu-Hyp-Gly and 270.6 µM for Leu-Pro-

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Gly). These values were close to their IC50 values against ACE.

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Molecular docking 9



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Molecular docking is a computational procedure that is useful for predicting the optimal pose

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and binding affinity of a ligand (tripeptide) when it interacts with a target protein (ACE) to form

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a stable complex. Guided by the docking scores, it is noted that Leu-Hyp-Gly showed excellent

180

binding activity with a binding energy value of −7.7 kcal/mol compared to that of Leu-Pro-Gly

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(−7.1 kcal/mol). Careful investigation of the interactions of Leu-Hyp-Gly with the target enzyme

182

indicated that the hydroxyl group showed strong binding interactions with the Tyr-523 residue of

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the amino acid sequence of the binding site of ACE (Fig. S1), while the Tyr residue did not

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interact with Leu-Pro-Gly (Fig. S2). These results could at least in part explain the superior

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ACE-inhibitory activity of Leu-Hyp-Gly over that of Leu-Pro-Gly.

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Stability of X-Hyp-Gly-Type Tripeptides in Mouse Plasma. We previously showed that Ala-

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Hyp-Gly was highly stable as well as Pro-Hyp in mouse plasma, while Gly-Pro-Ala and Gly-

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Ala-Hyp were rapidly degraded.18 The data suggested that X-Hyp-Gly is resistant to enzymatic

189

digestion due to the presence of Hyp located in the middle position. In the present study, we

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similarly studied the stability of Ala-Hyp-Gly and Leu-Hyp-Gly by incubation with mouse

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plasma using comparisons among Ala-Pro-Gly, Leu-Pro-Gly, and Ile-Pro-Pro. As shown in Fig.

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3, Ala-Pro-Gly and Leu-Pro-Gly were completely degraded after incubation for 60 min, but

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stability significantly increased for corresponding X-Hyp-Gly. Ala-Hyp-Gly remained

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unchanged during the 90 min incubation, consistent with the previous observation.18 In addition,

195

Leu-Hyp-Gly exhibited only modest reduction comparable to Ile-Pro-Pro, which was reported to

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have high resistance to enzymatic degradation.42 The high stability of X-Hyp-Gly-type

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tripeptides would help exert their high ACE-inhibitory activity in vivo.

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DISCUSSION 10


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In the present study, we demonstrated that X-Hyp-Gly-type tripeptides, especially of which X

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is branched-chain aliphatic amino acids, have high ACE-inhibitory activity. It was clearly shown

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that prolyl hydroxylation is critical for ACE inhibition by X-Hyp-Gly. Saiga et al. previously

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identified Hyp-containing ACE-inhibitory peptides, including Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-

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Leu-Hyp-Gly-Phe (IC50 = 42.4 µM) and Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro (IC50 = 29.4 µM),

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derived from chicken collagen.11,

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antihypertensive effects against spontaneously hypertensive rats.43,

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evidence that these peptides are transported into the blood in an intact form. We speculate that X-

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Hyp-Gly-type tripeptides are generated by partial hydrolysis in the gastrointestinal tract and

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blood and are main contributors to the antihypertensive effects. In fact, Leu-Hyp-Gly showing

210

the highest ACE-inhibitory activity is present both within the previously established Hyp-

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containing antihypertensive peptides.

33

The two kinds of Hyp-containing peptides showed 44

However, there is no

212

Surprisingly, substitution of Hyp with Pro dramatically decreased the ACE-inhibitory activity

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of X-Hyp-Gly regardless of the amino acid residue at the N-terminal. Alemán et al. investigated

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the effect of Hyp on ACE-inhibitory activity using Gly-Pro-X-Gly-X-X-Gly-Phe-X-Gly-Pro-X-

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Gly-X-Ser where X was either Leu or Hyp.45 While higher inhibition of ACE was observed when

216

all X were Leu, a corresponding peptide with X being Hyp did not show any inhibitory activity.

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In contrast, Saiga et al. suggested the importance of Hyp for ACE inhibition by replacing all Hyp

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within Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe with Pro, which decreased the

219

inhibitory activity 10-fold.33 However, in a subsequent paper, they concluded that Phe at the C-

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terminal most critically affects the ACE-inhibitory activity of the peptide.43 These previous data

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were obtained for the relatively long peptides. In contrast, our findings are based on observations

222

of tripeptides, which would more directly reflect the effect of Hyp on ACE-inhibitory activity. In 11



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addition to X-Hyp-Gly-type tripeptides, we analyzed Gly-Ala-Hyp and Gly-Leu-Hyp with

224

comparison to Gly-Ala-Pro and Gly-Leu-Pro, which were previously reported to strongly inhibit

225

ACE.46, 47 The ACE-inhibitory activity was dramatically decreased by prolyl hydroxylation in the

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case of this type of Hyp-containing tripeptide, indicating that the position of Hyp is also

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important for the enhancement of ACE inhibition. Molecular docking experiments comparing

228

Leu-Hyp-Gly with Leu-Pro-Gly suggested that the hydroxyl group of Hyp contributes to the

229

ACE-inhibitory activity of X-Hyp-Gly through its interaction with the enzyme. However, further

230

studies using various types and lengths of Hyp-containing peptides are needed to clarify the

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essential requirements for enhancement of ACE inhibition by prolyl hydroxylation.

232

Although a relationship between the peptide primary structure and ACE-inhibitory activity has

233

not been completely elucidated, common features can be found among ACE-inhibitory peptides;

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for example, peptides having hydrophobic amino acids at terminal positions tend to show high

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ACE inhibition.26, 48, 49 Among X-Hyp-Gly-type tripeptides, Leu-Hyp-Gly, Ile-Hyp-Gly, and Val-

236

Hyp-Gly exhibited especially strong ACE-inhibitory activity in this study. All of these peptides

237

have branched-chain aliphatic amino acids at the Xaa position. This is consistent with previous

238

observations that branched-chain aliphatic amino acids at the N-terminal are suitable for binding

239

to ACE.47-49 A dipeptide, Leu-Hyp, displayed extremely weak ACE-inhibitory activity that was

240

600-fold lower than that of Leu-Hyp-Gly, indicating that C-terminal Gly is also important for

241

ACE inhibition by X-Hyp-Gly-type tripeptides. Taken together, all constituents of X-Hyp-Gly

242

are indispensable to exert high ACE-inhibitory activity.

243

One of the advantages of collagen-derived Hyp-containing peptides is their high

244

bioavailability. While other food-derived ACE-inhibitory peptides, including Ile-Pro-Pro from

245

milk casein and Val-Tyr from sardine muscle, are detected in blood at nM levels after oral 12


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ingestion,50, 51 X-Hyp-Gly-type tripeptides show remarkably higher blood concentrations at µM

247

levels.15, 16 We consider that two factors contribute to the high bioavailability of X-Hyp-Gly. One

248

is that -X-Pro-Gly- sequences are frequently observed in collagen (Fig. S3). Since most Pro

249

residues at the Yaa position are hydroxylated, large amounts of X-Hyp-Gly are potentially

250

generated during production of collagen hydrolysate and in the gastrointestinal tract after oral

251

ingestion. In contrast, only one -Ile-Pro-Pro- sequence is present in bovine β- and κ-casein,

252

respectively, and bovine αs1- and αs2-casein do not have the sequence. Although Ile-Hyp-Gly and

253

Val-Hyp-Gly displayed higher ACE-inhibitory activity next to Leu-Hyp-Gly, their contents in

254

GDCH were small due to the low occurrence of -Ile-Pro-Gly- and -Val-Pro-Gly- sequences in

255

bovine type I collagen (Fig. S3). The use of other animal sources of collagen may increase the

256

yield of these peptides.

257

Another factor responsible for the high bioavailability of X-Hyp-Gly-type tripeptides is their

258

high resistance to peptidase/protease. A previous study reported that Val-Pro-Pro and Ile-Pro-Pro

259

were not very susceptible to digestive enzymes and Caco-2 cells expressing various brush-border

260

peptidases, suggesting that orally administered these antihypertensive tripeptides are absorbed

261

intact into the blood.42 We showed that Ile-Pro-Pro was stable but slightly decreased (~30%)

262

during incubation for 90 min with mouse plasma. Although Ala-Pro-Gly and Leu-Pro-Gly were

263

rapidly degraded, the stability of corresponding X-Hyp-Gly-type tripeptides was higher (Ala-

264

Hyp-Gly) or comparable (Leu-Hyp-Gly) to that of Ile-Pro-Pro. The high bioavailability of the

265

collagen-derived Hyp-containing peptides possessing high ACE-inhibitory activity highlights

266

their potential for hypertension control. Further in vivo experiments are warranted to evaluate the

267

antihypertensive effects of X-Hyp-Gly-type tripeptides and GDCH enriched with the peptides.

268

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ABBREVIATIONS USED

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Hyp, hydroxyproline; ACE, angiotensin-converting enzyme; GDCH, ginger-degraded collagen

271

hydrolysate; Hip, hippuric acid; HHL, hippuryl-histidyl-leucine; H-GDCH, heat-treated ginger-

272

degraded collagen hydrolysate; LC–MS, liquid chromatography–mass spectrometry; MRM,

273

multiple reaction monitoring

274 275

Supporting Information. Figure S1: Molecular docking between Leu-Hyp-Gly and ACE.

276

Figure S2: Molecular docking between Leu-Pro-Gly and ACE. Figure S3: Amino acid

277

sequences of the triple helical region of bovine type I collagen. Table S1: MRM transitions of

278

dipeptides and tripeptides. Table S2: Effect of prolyl hydroxylation on ACE-inhibitory activity

279

of Gly-X-Pro-type tripeptides. This material is available free of charge via the Internet at

280

http://pubs.acs.org.

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REFERENCES

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1.

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a004978.

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2.

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collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen.

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Biochem. Biophys. Res. Commun. 1973, 52, 115-20.

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435

Figure captions

436

Figure 1. ACE inhibition rate of (A) GDCH and (B) H-GDCH. The data represent the mean ±

437

SD of three separate measurements.

438 439

Figure 2. Dixon plot of (A) Leu-Hyp-Gly and (B) Leu-Pro-Gly. The kinetic assays were

440

performed at substrate concentrations of (▲) 0.1 and (■) 1.0 mM. The data represent the mean ±

441

SD of three separate measurements.

442 443

Figure 3. Residual ratio of tripeptides after incubation with mouse plasma. Ala-Hyp-Gly, Leu-

444

Hyp-Gly, Ala-Pro-Gly, Leu-Pro-Gly, and Ile-Pro-Pro were incubated with freshly prepared

445

mouse plasma at 37 °C for 0, 15, 30, 60, and 90 min. The residual ratio was calculated using the

446

peak area of the respective peptides in LC–MS analysis. The data represent the mean ± SD (n =

447

3).

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Tables Table 1. ACE-inhibitory Activity of Collagen-derived Oligopeptides and Their Contents in GDCHs

a

Ala-Hyp-Gly Ala-Pro-Gly

IC50 (µM)a,c 151.3 ± 6.8 1293.3 ± 286.6

Content in GDCH (mg/g)b,c 2.205 ± 0.087 0.204 ± 0.008

Content in H-GDCH (mg/g)b,c 0.197 ± 0.017 0.056 ± 0.004

Glu-Hyp-Gly

404.5 ± 51.4

1.204 ± 0.057

0.310 ± 0.009

Ile-Hyp-Gly

9.4 ± 2.2

0.076 ± 0.002

0.018 ± 0.002

Leu-Hyp-Gly Leu-Pro-Gly Leu-Hyp

5.5 ± 1.5 373.8 ± 77.2 3462.1 ± 1012.2

1.857 ± 0.254 0.008 ± 0.001 ND

0.131 ± 0.018 0.002 ± 0.001 ND

Phe-Hyp-Gly

210.9 ± 37.1

0.380 ± 0.022

0.013 ± 0.001

Pro-Hyp-Gly Pro-Pro-Gly

257.8 ± 41.9 1372.6 ± 297.8

0.017 ± 0.001 ND

0.010 ± 0.004 ND

Ser-Hyp-Gly Ser-Pro-Gly

130.6 ± 4.5 1029.5 ± 163.0

0.183 ± 0.021 0.071 ± 0.003

ND ND

Val-Hyp-Gly Val-Pro-Gly

12.8 ± 0.7 125.9 ± 4.5

0.018 ± 0.001 0.093 ± 0.008

0.007 ± 0.001 0.037 ± 0.001

Ile-Pro-Pro 0.9 ± 0.2 The data were obtained by ACE inhibition assay using synthetic peptides. bThe data were

obtained by LC–MS analysis of the collagen hydrolysates. ND, not detected. cThe data represent the mean ± SD of three separate measurements.

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Figure Graphics Figure 1

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Figure 2

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Figure 3

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GRAPHICS FOR TABLE OF CONTENTS

27


1 Characterization of Angiotensin-Converting Enzyme Inhibitory

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