Transport Study of Egg-Derived Antihypertensive Peptides (LKP and

Aug 7, 2017 - Del Mar Contreras , M.; Sancho , A. I.; Recio , I.; Mills , C. Absorption of casein antihypertensive peptides through an in vitro model ...
2 downloads 0 Views 800KB Size
Subscriber access provided by WILFRID LAURIER UNIV

Article

Transport Study of Egg Derived Antihypertensive Peptides (LKP and IQW) Using Caco-2 and HT29 Co-culture Monolayers Qingbiao Xu, Hongbing Fan, Wenlin Yu, Hui Hong, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02176 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

Journal of Agricultural and Food Chemistry

1

Transport Study of Egg Derived Antihypertensive Peptides (LKP and IQW)

2

Using Caco-2 and HT29 Co-culture Monolayers

3 4

Qingbiao Xu†‡, Hongbing Fan‡, Wenlin Yu‡, Hui Hong‡, Jianping Wu*,‡

5 6



7

Wuhan 430070, China

8



9

Edmonton, Alberta, Canada T6G 2P5

College of Animal Sciences and Technology; Huazhong Agricultural University,

Department of Agricultural, Food and Nutritional Science, University of Alberta,

10 11

Short title: Transport of LKP and IQW across Caco-2/HT29 monolayers.

12

*Corresponding author: Jianping Wu, Department of Agricultural, Food and

13

Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5.

14

Phone: 780-492-6885. Fax: 780-492-4265. E-mail: [email protected].

15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

16

ABSTRACT

17

The objective of this study was to investigate the mechanisms of the transport of

18

antihypertensive tripeptides LKP (Leu-Lys-Pro) and IQW (Ile-Gln-Trp) derived from

19

egg white using a co-culture system of Caco-2 and HT29 cell monolayers. The results

20

revealed that LKP and IQW have no cytotoxicity to the cell viability after 2

21

h-incubation, and could be transported intact across co-culture monolayers (apparent

22

permeability coefficient: (18.11 ± 1.57) × 10-8 and (13.21 ± 1.12) × 10-8 cm/s,

23

respectively), and were resistant to peptidase secreted by enterocytes. In addition, the

24

transports were significantly inhibited by dipeptide Gly-Pro (P < 0.05), a competitive

25

substance of peptide transporter 1 (PepT1). The transports from apical to basolateral

26

side were significantly higher than that of the reverse direction (P < 0.05). These

27

results suggest that PepT1 is involved in LKP and IQW transports. The transports

28

were also significantly decreased by theaflavin-3′-O-gallate (P < 0.05), an enhancer of

29

tight junction (TJ), and increased by cytochalasin D (P < 0.05), a disruptor of TJ, but

30

no influenced by wortamanin, a transcytosis inhibitor, suggesting that passive

31

paracellular route via TJs is also involved in LKP and IQW transports, but not

32

transcytosis. In addition, siRNA was also used to knockdown the expression of PepT1,

33

and significantly inhibited the transport (P < 0.05), confirming that PepT1 is involved

34

in transport process. In conclusion, both passive paracellular route via TJ and active

35

route via PepT1 coexist in the transport of antihypertensive LKP and IQW across

36

Caco-2/HT29 co-culture monolayers.

37

KEYWORDS: antihypertensive peptide, LKP, IQW, Caco-2, transport

ACS Paragon Plus Environment

Page 2 of 34

Page 3 of 34

Journal of Agricultural and Food Chemistry

38

INTRODUCTION

39

Hypertension, affecting nearly one third of adult population, is a serious threat to

40

human health. Antihypertensive peptides derived from food proteins are attracting

41

more and more interests as alternatives to manage hypertension.1 Angiotensin

42

converting enzyme (ACE) inhibitory tripeptides IRW, LKP, and IQW were identified

43

previously from egg white protein ovotransferrin.2 All three peptides exerted blood

44

pressure lowering activities in SHR at various degrees.3,4 LKP was also characterized

45

from chicken and bonito protein, and had been developed for uses of mild- or

46

pre-hypertensive subjects.5,6 To exert action in the target organ, bioactive peptides

47

need to reach the intestine or to be transported intact across the intestinal epithelium

48

into the circulatory system in an active form.7,8

49

There are three possible pathways to transport peptides across enterocyte

50

monolayers: transcytosis route via endocytosis, passive paracellular diffusion via tight

51

junction (TJ), and active route via transporters.8,9 Transcytosis prefers to transport

52

large peptides or certain peptides via apical transcytotic vesicles or basolateral

53

secretion,10 such as BCM-5 (YPFPG),11 bovine β-casein (193-209) derived

54

17-residues peptide,10 bradykinin,12 and fluorescence-derivatized cationic peptide

55

001-C8-NBD.13 However, paracellular route via TJ exists more extensively in the

56

transport process of a large number of bioactive peptides, such as VGPV, GPRGF,14

57

RVPSL,15 QIGLF,16 RWQ, WQ,17 GAXGLXGP18, KVLPVP,19 VLPVP,20 HLPLP,21

58

and VPP.22 The peptide transporter 1 (PepT1) is an H+-coupled carrier present mainly

59

in the membrane of gastrointestinal tract and first cloned in rabbit,23 and plays a vital

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 34

60

role in the small peptide transport.9,24 It was reported that PepT1 was also mediated to

61

transport bioactive peptide PH and YPI.25,26 Another antihypertensive tripeptide

62

derived from egg white, IRW, is transported across Caco-2 monolayers via both

63

passive (paracellular diffusion) and active (PepT1) routes.27 A famous ACE-inhibitory

64

tripeptide VPP, derived from milk, was reported to be transported via paracellular

65

route, but not PepT1, due to its quick hydrolysis by intracellular peptidases.22 Hence,

66

different peptides may have different transport mechanisms. However, the

67

permeability of antihypertensive tripeptides LKP and IQW have not been studied. It is

68

of much significance to understand the mechanism of the transport of

69

antihypertensive peptides for their pharmacological application and bioavailability in

70

the future. Therefore, the aim of this study is to investigate the transport mechanism

71

of LKP and IQW across Caco-2/HT29 co-culture monolayers.

72 73 74

MATERIALS AND METHODS Chemicals. Dulbecco’s modified Eagle’s medium (DMEM), hanks balanced salt

75

solution

(HBSS,

with

calcium

76

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), fetal bovine serum

77

(FBS), nonessential amino acids (NAA), and antibiotics were all obtained from Gibco

78

(Burlington, ON, Canada). Triflouroacetic acid (TFA) and acetonitrile (ACN) were

79

purchased from Acros Organics (Morris Plains, NJ). Wortamanin, cytochalasin D, and

80

theaflavin-3′-O-gallate (TF3′G) were obtained from Sigma (Oakville, ON, Canada).

81

Peptides LKP, IQW, and GP were synthesized in Genscript Corp (purity: > 97%;

ACS Paragon Plus Environment

and

magnesium),

Page 5 of 34

Journal of Agricultural and Food Chemistry

82

Piscataway, NJ). Lipofectamine 3000, siRNA (small interfering RNA), Opti-MEM

83

reduced serum medium, and antibody (rabbit origin) for gene PepT1 (SLC15A1) were

84

purchased from Invitrogen (Burlington, ON, Canada).

85

Co-culture of Caco-2 and HT29 Cells. Caco-2 and HT29 cells at passage 22 to 35

86

were seeded onto a 12-well-transwell polyester permeable membrane support (0.4 µm

87

pore size, 12 mm diameter, 1.12 cm2 grown surface, Costar, Corning, NY) at a ratio of

88

3 : 1 at a density of 1.0 × 105 cells/cm2. The cells were grown at 5% CO2 and 37 °C in

89

a humidified atmosphere, in DMEM medium (high glucose) supplied with 10% FBS,

90

1% NAA, and 1% antibiotics. The culture medium was replaced every other day and

91

the cells were allowed to differentiate for at least 21 days. Caco-2 monolayers with

92

transepithelial electrical resistance (TEER, World Precision Instruments, Sarasota, FL)

93

values higher than 400 Ω/cm2 were used for the transport studies, showing that the

94

monolayers had reached confluency and polarized.28 On day 21, cell monolayers were

95

pre-incubated in incubation solution (HBSS with 10 mM D-glucose) with apical pH

96

6.0 (adjusted by 25 mM MES and Tris) and basolateral pH 7.4 (adjusted by 25 mM

97

HEPES and Tris).

98

Cytotoxicity Assay. The cytotoxicity of LKP and IQW on Caco-2 cell viability was

99

measured by Alamar Blue dye (Thermo Fisher Scientific Inc., USA). The cells were

100

seeded onto 96-well plates at a density of 1.0 × 104 cells/well. After incubation with

101

peptides (1, 5, and 10 mM) for 2 or 24 h, the medium was removed and incubated

102

with 10% Alamar Blue dye in the medium at 37 °C for 4 h. Then, the fluorescence

103

was measured at 590 nm (excited at 560 nm) using a plate reader (Molecular Devices,

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

104

Spectra max, Sunnyvale, CA).

105

Stability of LKP and IQW in the Co-culture System. LKP and IQW were

106

prepared at a concentration of 5 mM with incubation solution. Before incubation, the

107

monolayers were gently washed 3 times with pre-warmed HBSS (37 °C). Then

108

solution containing peptide was added into the apical compartments. After incubation

109

for 60 min at 37 °C, the samples from both apical and basolateral chambers were

110

collected and characterized using liquid chromatography-mass spectrometry/mass

111

spectrometry (LC-MS/MS).

112

Transport Assay. Transcytosis inhibitor (wortmannin, 1 µM) and TJ disruptor

113

(cytochalasin D, 1 µg/mL) were dissolved in DMSO and diluted in HBSS (pH 6.0,

114

final concentration: 0.05% DMSO). The TJ enhancer TF3′G (20 µM) was prepared

115

with DMEM containing 0.05% DMSO and 10% FBS.29 Before transport assay, cell

116

monolayers were pre-incubated with wortmannin, or cytochalasin D for 30 min, or

117

TF3′G for 24 h. The HBSS solution containing 0.05% DMSO was used as a control.

118

After pre-incubation, the monolayers were washed three times and incubated with 0.5

119

mL HBSS containing 5 mM tripeptide (pH 6.0) in the apical side and 1.5 mL HBSS

120

(pH 7.4) in the basolateral side of the monolayers for 60 min. After transport assay,

121

the samples were collected from the basolateral chambers and the TEER of the

122

monolayer was measured to ensure its value higher than 400 Ω/cm2. The samples

123

from apical or basolateral sides were collected and characterized using ultrahigh

124

performance liquid chromatography (UPLC).

125

Dipeptide GP is a well known substrate of PepT1 and used widely for competitive

ACS Paragon Plus Environment

Page 6 of 34

Page 7 of 34

Journal of Agricultural and Food Chemistry

126

inhibition study. The transport of tripeptides was measured at the presence of 25 mM

127

GP in HBSS incubation solution. The transport of tripeptides from apical to

128

basolateral side (AP-BL) was compared with that of the reverse direction (BL-AP).

129

Knockdown of PepT1 in Caco-2 Cells. siRNA targeting PepT1 gene was

130

purchased from Invitrogen (Burlington, ON, Canada) and used to interfere the

131

expression of PepT1 gene in cell monolayers. The sequence of siRNA targeting

132

PepT1

133

5′-AAAUGCCUUACUCCGAUGCCT-3′ (antisense). On the day before transport

134

assay, the cell monolayers were washed twice with OPTI-MEM medium and added

135

500 µL OPTI-MEM containing 1 µL Lipofectamine 3000 and 80 nM siRNA into the

136

apical chamber. After 6 h transfection, the transfection medium was replaced with the

137

growth media, and incubated cells for another 24 h. Then the monolayers were rinsed

138

three times and pre-incubated with HBSS for 30 min. Subsequently, the transports

139

were started with incubation with 5 mM tripeptide and the samples in the basolateral

140

sides were collected after 60 min incubation. The group treated with siRNA with

141

disordered

142

5'-GCGCGCUUUGUAGGAUUCGDTDT-3'

143

3'-DTDTCGCGCGAAACAUCCUAAGC-5' (antisense) was used as a control. The

144

knockdown efficiency of PepT1 was analyzed by western blot.

was

5′-GCAUCGGAGUAAGGCAUUUTT-3′

sequence

(scrambled

(sense)

and

nucleotides), (sense)

and

145

Western Blot. After treatment with siRNA, the cells were lysed with hot

146

Laemmli’s buffer containing 2% DTT as described previously.30 Then, the cell protein

147

was run in 9% using sodium dodecyl sulfate polyacrylamide gel electrophoresis and

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

148

transferred to the membranes. Subsequently, the target proteins were incubated with

149

anti-rabbit antibody for PepT1 (Invitrogen, Burlington, ON, Canada) and tubulin

150

(Abcam, Toronto, ON, Canada) overnight at 4°C. Then the members were incubated

151

with second antibody (Abcam, Toronto, ON, Canada) for 60 min at room temperature.

152

The bands were detected using a Licor Odyssey BioImager (Odyssey, Licor

153

Biosciences) and analyzed by Image Studio Lite 5.2 (Licor Biosciences). The protein

154

bands of PepT1 were normalized using the tubulin bands.

155

UPLC. The quantity of peptides in the samples collected after incubation was

156

measured using UPLC (Waters, Miliford, MA, USA) with an Acquity UPLC BEH C18

157

column (1.7 µm, 2.1 × 100 mm). The injection volume was 20 µL. Samples were

158

eluted with 100% solvent A (0.1% TFA in water) within 5 min, and increased to 50%

159

solvent B (0.1% TFA in ACN) in 25 min at a flow rate of 0.3 mL/min. The

160

absorbance was monitored at 220 nm.

161

LC-MS/MS. The peptides transported across monolayers were identified by

162

LC-MS/MS as described.31 The eluents were used as follows: (A) water containing

163

0.1% formic acid, and (B) ACN with 0.1% formic acid. A volume of 5 µL desalting

164

sample dissolved in solvent A was injected into 5 µm trapping column (180 µm × 20

165

mm, Symmetry C18 nanoAcquity column, Waters), and trapped at a flow rate of 10

166

µL/min for 2 min using a gradient as follows: 1-5% B (0-2 min), 5-20% B (2-25 min),

167

20-40% B (25-40 min), 40-65% B (40-45 min), and 65% B in 5 min. Ionisation was

168

conducted using an electrospray ionisation technique with a positive capillary voltage

169

of 3.6 kV and an ion transferred tube temperature of 100 °C. Spectra were recorded

ACS Paragon Plus Environment

Page 8 of 34

Page 9 of 34

Journal of Agricultural and Food Chemistry

170

with a m/z ranges of 100-575 in MS mode and 50-1000 in MS/MS mode, and peptide

171

mass was detected using a Q-TOF analyzer (Waters). The peptides sequences were

172

characterized using a Peaks Viewer 4.5 (Bioinformatics Solutions Inc., Waterloo, ON,

173

Canada).

174

Statistical Analysis. Statistical analysis between two groups was performed by

175

unpaired Student's t-test, and analysis between multiple groups was performed by a

176

one-way analysis of variance (ANOVA) followed by Tukey test for post hoc analysis

177

using the SPSS software (version 22.0, SPSS Inc., IL, USA). The data were presented

178

as the means ± standard error of the mean (SEM). The criterion for significance was

179

established at P < 0.05.

180 181 182

Apparent permeability coefficient (Papp, cm/s) was calculated according to previous report as follows:20 Papp = (dQ/dt) / (A × C0)

183

where dQ/dt is the permeability rate (µmol/s) in the acceptor chamber; A is the

184

monolayer surface area (cm2); C0 is the initial concentration in the donor chamber

185

(µM).

186 187

RESULTS

188

The Cytotoxicity of LKP and IQW. As shown in Figure 1, after treatment with 1

189

mM LKP and IQW for 24 h, there was no significantly difference on the cell viability.

190

However, a concentration of 5 or 10 mM of tripeptides significantly decreased the

191

viabilities of Caco-2 cells (P < 0.05). However, there was no significantly difference

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

192

of cell viability after treatment with 5 mM LKP or IQW for 2 h (Figure 1C). As the

193

transport time used was 60 min, much shorter than 24 h for cytotoxicity, there will be

194

no harm for the cells used in the transport study.

195

Stability of LKP and IQW in the System of Co-culture Monolayers. After

196

incubation for 60 min in the apical sides of the transwells, the remaining rate of LKP

197

and IQW were 91.8% and 94.0%, respectively. After incubation for 60 min, the

198

samples from basolateral sides of cell monolayers were collected and measured using

199

LC-MS/MS. As shown in Figure 3, tripeptide LKP and IQW were identified

200

according to the spectrum, suggesting that they can be transported intact across

201

co-culture cell monolayers. The major peak eluted by UPLC from the AP to BL side

202

was LKP or IQW. In addition, dipeptide QW degenerated from IQW was also found

203

in basolateral side (Figure 3B). However, LKP were kept intact and not degenerated

204

in basolateral side (Figure 3A), indicating that LKP may be more stable than IQW in

205

basolateral side of the cell monolayers.

206

Effects of Dipeptide GP, Wortmannin, Cytochalasin D and TF3′G on the

207

Transport of LKP and IQW. Dipeptide GP is a classical substrate of PepT1 and

208

used widely for competitive binding study of transporter.10 The addition of GP

209

significantly decreased the transport of both LKP and IQW (P < 0.05), suggesting

210

PepT1 is involved in the transport process of them. In addition, cytochalasin D is a TJ

211

disruptor.10 As shown in Figure 4, cytochalasin D significantly increased the transport

212

of LKP and IQW (P < 0.05), suggesting that TJ is involved in the transport process.

213

Moreover, the pretreatment of cells with TF3′G significantly decreased the transport

ACS Paragon Plus Environment

Page 10 of 34

Page 11 of 34

Journal of Agricultural and Food Chemistry

214

of LKP and IQW (P < 0.05). Therefore, the passive paracellular transport via TJ is

215

also involved in the transport process of LKP and IQW. However, there is no

216

influence of wortmannin on the tripeptide transport, indicating transcytosis may be

217

not the transport mechanism for LKP and IQW transport.

218

Apical and Basolateral Transport. The transport of LKP and IQW from apical to

219

basolateral side is also compared with that of BL-AP. As shown in Figure 5, the

220

transports of LKP and IQW from AP to BL were significantly higher than those of the

221

inverse direction (P < 0.05), indicating that the peptides could be transported from

222

human intestinal mucosal membrane to the serosal side. In addition, the transport

223

from AP to BL of LKP was significantly higher than that of IQW (P < 0.05), but there

224

was no significant difference from BL to AP.

225

Effect of siRNA for PepT1 on the Transports of LKP and IQW. As shown in

226

the western blot results (Figure 6A, B), siRNA knocked down more than 50% of the

227

expression of PepT1 in Caco-2 cells (P < 0.05). Moreover, the transports of LKP and

228

IQW were significantly decreased by the treatment of siRNA compared with control

229

(P < 0.05, Figure 6C, D), suggesting PepT1 plays a role in LKP and IQW transport. In

230

summary, as shown in Figure 7, there are two pathways involved in the mechanism of

231

the transports of antihypertensive tripeptides LKP and IQW: paracellular route and

232

PepT1 route.

233 234

DISCUSSION

235

In this study, peptide transport was studied using a co-culture of Caco-2 cells with

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

236

goblet and mucus-secreting cells HT29, at a physiological relevant ratio of 3/1,32 to

237

mimic closely to the intestinal tissues.33 The co-culture has lower TEER value than

238

the only Caco-2 culture. Taken together, as shown in Figure 7, there are two pathways

239

of transepithelial transport of antihypertensive peptides LKP and IQW across

240

enterocyte monolayers: passive paracellular route via TJ, and active route via PepT1.

241

This is consistent with a previous review, demonstrating that both passive and

242

carrier-mediated processes coexisted and contributed to drug (peptide analogue)

243

transport across biological membranes.34

244

Antihypertensive tripeptides LKP and IQW were found stable in the co-culture

245

system, indicating that they are resistance to peptidase and could possibly reach to the

246

site of action, which was in agreement with previous in vivo results.35 IRW was

247

previously shown effective in lowering blood pressure in vivo and could be

248

transported Caco-2 cell monolayers intact for function.30,27 Another antihypertensive

249

egg-derived tripeptide YPI can also be transported across Caco-2 monolayer intact

250

with partial degradation.26 Another bioactive tripeptide GPH derived from collagen

251

was also hydrolyzed into free amino acids in brush-border membrane vesicles.25 The

252

stability of peptides may be relevant to the structure of peptides.27 It was also reported

253

that small peptides (di- and tripeptides) were more prone to resistant to enzymes

254

activity compared with large peptides.36 Therefore, tripeptide LKP and IQW are

255

stable to cross epithelial cells and can be used for subsequent transport experiments.

256

Wortmannin, an inhibitor of phosphoinositide 3-kinase, can inhibit the transcytosis

257

route, which was used widely to investigate the role of transcytosis in peptide

ACS Paragon Plus Environment

Page 12 of 34

Page 13 of 34

Journal of Agricultural and Food Chemistry

258

transport.37 In this study, the transport of LKP and IQW was hardly inhibited by

259

wortmannin, indicating the transcytosis was not involved. Transcytosis prefers to

260

transport large peptides,10 such as BCM-5 (YPFPG),11 bradykinins,12 and cationic

261

peptide.13

262

Cytochalasin D could disrupt TJ by altering the cytoskeletal structure and increase

263

the transport of passive paracellular pathway,38 which is energy independent. The

264

increase of LKP and IQW transport caused by cytochalasin D suggests paracellular

265

route mediated by TJs may be a mechanism of tripeptide transport across co-culture

266

monolayers. In addition, TF3′G was used to evaluate the transport pathways, which

267

can enhance the barrier function via increase the expression of TJ-related proteins

268

(claudin-1, occludin, and zonula occluden-1).29,39 In this study, the decrease of

269

tripeptide transport caused by TF3′G confirms TJ is involved in LKP and IQW

270

transport across co-culture monolayers. Paracellular pathway to transport peptides can

271

be affected by many properties, such as molecular size, volume, hydrophilicity, and

272

surface area, and tends to transport water-soluble and low molecular peptides.40

273

Paracellular route is also involved in the transport of many other bioactive peptides,

274

such as antihypertensive peptide GGYR,12 VGPV, GPRGF,14 RVPSL,15 QIGLF,16

275

RWQ, WQ,17 GAXGLXGP,18 KVLPVP,19 VLPVP,20 HLPLP,21 VPP,22 and collagen

276

peptides.41 Studies also demonstrated that a large number of pores existed in TJs of

277

Caco-2 monolayers, which have a radius of 5.8-10.4 Å.12,42 The radiuses of tripeptide

278

LKP and IQW are approximately 5 Å, smaller than those of the pores in TJs, thus,

279

resulting in their possible transport through paracellular pathway.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

280

Dipeptide GP is a classical competitive inhibitor for PepT1 with a low Km

281

value.10,23 The addition of GP significantly inhibited the transport of LKP and IQW,

282

indicating that carrier PepT1 is involved in their transport across the cell monolayers.

283

In addition, the transport of LKP and IQW of AP-BL was much higher than that of

284

BL-AP, suggesting they can be transported from intestinal lumen into plasma. The

285

reason of the unidirection may be the asymmetry of the expression of PepT1 at the

286

apical and basolateral membrane of the gastrointestinal epithelial cell layers resulting

287

in the vectorial peptide transport.43 The transport of LKP was significantly higher than

288

IQW may be due to the lower hydrolytic action of LKP by peptidase in the basolateral

289

sides, compared with IQW. As shown in LC-MS/MS, QW generated from IQW was

290

found in the basolateral sides; however, no decomposed fragments of LKP were

291

determined in the basolateral sides.

292

To investigate the role of PepT1 in the transport of LKP and IQW across co-culture

293

monolayers, siRNA interrupting the expression of PepT1 was used. As far as we know,

294

this is the first report to use siRNA in the study of bioactive peptide transport across

295

monolayers. The siRNA interrupting is a useful and direct way to study the certain

296

gene function, which has been used to study the transepithelial transports of drug

297

simvastatin across Caco-2 cell monolayers.44 In the present study, the knockdown of

298

PepT1 decreased significantly the transport of LKP and IQW, further supporting that

299

PepT1 is involved in the transport process. Transporter PepT1 is reported to carry di-

300

and tripeptides, and of great significance for animal health and nutrition,45 but it’s

301

hard to transport for longer peptides, because they are not the substrates of PepT1.8,24

ACS Paragon Plus Environment

Page 14 of 34

Page 15 of 34

Journal of Agricultural and Food Chemistry

302

Our data are consistent to previous studies, describing that PepT1 can transport many

303

small bioactive peptides, such as IRW,27 YPI,26 and PH,25 but not RVPSL,15 QIGLF,16

304

or GGYR.12

305

The peptide transport is influenced by the size, hydrophobicity, charge, and side

306

chain flexibility of the peptides. For passive paracellular pathway via TJs, the peptides

307

with smaller size are more preferable to be transported.46 For active pathway, PepT1

308

prefers to transport the peptides with shorter chains (di- and tripeptides),

309

hydrophobicity, apolarity, and neutral charge, but hardly to transport peptides with

310

extreme bulk.47 Hydrophobicity of peptides could increase the binding affinity of

311

PepT1.48 Antihypertensive tripeptides IRW, LKP, and IQW are hydrophobic peptides

312

and have small size, therefore they should be the preferable substrates of PepT1 and

313

paracellular pathway.

314

Antihypertensive tripeptides LKP and IQW had a permeability rate of 10-7 cm/s,

315

which was comparable with the reported Papp values ranging from 10−9 to 10−6 cm/s in

316

the previous permeability study of antihypertensive peptides across Caco-2 cell

317

monolayers.15-17,20,21,33,49 Although in low oral permeability (less than 1-2%),

318

bioactive peptides resistant to peptidase could act function at low concentration in the

319

blood stream.50 By the way, Caco-2 cell monolayers have a higher TEER value and

320

tighter TJs than human intestinal wall, therefore, the real peptide permeability in

321

human might be higher than that in Caco-2 monolayers.14-17,22

322

In summary, our results suggest that antihypertensive tripeptides LKP and IQW can

323

be transported intact across co-culture of Caco-2 and HT29 cell monolayers and the

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

324

resistant to peptidase secreted by enterocytes, and the mechanisms for tripeptides LKP

325

and IQW transport are paracellular route and transporter PepT1. In the future, the

326

strategies to enhance the absorption of LKP and IQW need to be explored.

327 328

AUTHOR INFORMATION

329

Corresponding Author

330

*(J.W.) Phone: 780-492-6885. Fax: 780-492-4265. E-mail: [email protected].

331

Funding

332

This research was funded by grants from Alberta Livestock Meat Agency, and Natural

333

Science and Engineering Research Council of Canada to J. Wu.

334

Notes

335

The authors declare no competing financial interest.

336 337

ABBREVIATIONS

338

ACN, acetonitrile; ACE, angiotensin-converting enzyme; AP, apical side; BL,

339

basolateral side; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl

340

sulfoxide; FBS, fetal bovine serum; GP, Gly-Pro; IQW, Ile-Gln-Trp; IRW, Ile-Arg-Trp;

341

HBSS, hanks balanced salt solution; LC-MS/MS, liquid chromatography-mass

342

spectrometry/mass spectrometry; LKP, Leu-Lys-Pro; NAA, nonessential amino acids;

343

Papp, apparent permeability coefficient; PepT1, peptide transporter 1; PH, Phe-Hyp;

344

siRNA, small interfering RNA; TEER, transepithelial electrical resistance; TF3′G,

345

theaflavin-3′-O-gallate; TJ, tight junction.

ACS Paragon Plus Environment

Page 16 of 34

Page 17 of 34

Journal of Agricultural and Food Chemistry

346

REFERENCES:

347

(1)

Hernández-Ledesma, B.; del Mar Contreras, M.; Recio, I. Antihypertensive

348

peptides: production, bioavailability and incorporation into foods. Adv. Colloid

349

Interface Sci. 2011, 165 (1), 23–35.

350

(2)

351 352

Wu, J.; Acero-Lopez, A. Ovotransferrin: Structure, bioactivities, and preparation. Food Res. Int. 2012, 46 (2), 480–487.

(3)

Majumder, K.; Chakrabarti, S.; Morton, J. S.; Panahi, S.; Kaufman, S.; Davidge,

353

S. T.; Wu, J. Egg-derived tri-peptide IRW exerts antihypertensive effects in

354

spontaneously hypertensive rats. PLoS One 2013, 8 (11), 1–14.

355

(4)

Majumder, K.; Chakrabarti, S.; Morton, J. S.; Panahi, S.; Kaufman, S.; Davidge,

356

S. T.; Wu, J. Egg-derived ACE-inhibitory peptides IQW and LKP reduce blood

357

pressure in spontaneously hypertensive rats. J. Funct. Foods 2015, 13, 50–60.

358

(5)

Iroyukifujita, H.; Eiichiyokoyama, K.; Yoshikawa, M. Classification and

359

antihypertensive activity of angiotensin I-converting enzyme inhibitory

360

peptides derived from food proteins. J. Food Sci. 2000, 65 (4), 564–569.

361

(6)

Gleeson, J. P.; Heade, J.; Ryan, S. M.; Brayden, D. J. Stability, toxicity and

362

intestinal permeation enhancement of two food-derived antihypertensive

363

tripeptides, Ile-Pro-Pro and Leu-Lys-Pro. Peptides 2015, 71, 1–7.

364

(7)

Horner, K.; Drummond, E.; Brennan, L. Bioavailability of milk protein-derived

365

bioactive peptides: a glycaemic management perspective. Nutr. Res. Rev. 2016,

366

29 (1), 91–101.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

367

(8)

Page 18 of 34

Miner-Williams, W. M.; Stevens, B. R.; Moughan, P. J. Are intact peptides

368

absorbed from the healthy gut in the adult human? Nutr. Res. Rev. 2014, 27 (2),

369

308–329.

370

(9)

Gilbert, E. R.; Wong, E. A.; Webb, K. E. Board-invited review: Peptide

371

absorption and utilization: Implications for animal nutrition and health. J. Anim.

372

Sci. 2008, 86 (9), 2135–2155.

373

(10)

Regazzo, D.; Mollé, D.; Gabai, G.; Tomé, D.; Dupont, D.; Leonil, J.; Boutrou,

374

R. The (193-209) 17-residues peptide of bovine β-casein is transported through

375

Caco-2 monolayer. Mol. Nutr. Food Res. 2010, 54 (10), 1428–1435.

376

(11)

377 378

derived bioactive peptide VLPVPQK. Food Chem. 2016, 190, 681–688. (12)

379 380

Vij, R.; Reddi, S.; Kapila, S.; Kapila, R. Transepithelial transport of milk

Shimizu, M.; Tsunogai, M.; Arai, S. Transepithelial transport of oligopeptides in the human intestinal cell, Caco-2. Peptides 1997, 18 (5), 681–687.

(13)

Sai, Y.; Kajita, M.; Tamai, I.; Kamata, M.; Wakama, J.; Wakamiya, T.; Tsuji,

381

A.

Intestinal

absorption

of

fluorescence-derivatized

cationic

peptide

382

001-C8-NBD via adsorptive-mediated transcytosis. Bioorg. Med. Chem. 1998,

383

6 (6), 841–848.

384

(14) Fu, Y.; Young, J. F.; Rasmussen, M. K.; Dalsgaard, T. K.; Lametsch, R.; Aluko,

385

R. E.; Therkildsen, M. Angiotensin I-converting enzyme-inhibitory peptides

386

from bovine collagen: insights into inhibitory mechanism and transepithelial

387

transport. Food Res. Int. 2016, 89, 373–381.

ACS Paragon Plus Environment

Page 19 of 34

Journal of Agricultural and Food Chemistry

388

(15)

Ding, L.; Wang, L.; Zhang, Y.; Liu, J. Transport of antihypertensive peptide

389

RVPSL, ovotransferrin 328-332, in human intestinal Caco-2 cell monolayers. J.

390

Agric. Food Chem. 2015, 63 (37), 8143–8150.

391

(16)

Ding, L.; Zhang, Y.; Jiang, Y.; Wang, L.; Liu, B.; Liu, J. Transport of egg

392

white ACE-inhibitory peptide, Gln-Ile-Gly-Leu-Phe, in human intestinal

393

Caco-2 cell monolayers with cytoprotective effect. J. Agric. Food Chem. 2014,

394

62 (14), 3177–3182.

395

(17)

Fernández-Musoles, R.; Salom, J. B.; Castelló-Ruiz, M.; Contreras, M. del M.;

396

Recio, I.; Manzanares, P. Bioavailability of antihypertensive lactoferricin

397

B-derived peptides: Transepithelial transport and resistance to intestinal and

398

plasma peptidases. Int. Dairy J. 2013, 32 (2), 69–174.

399

(18)

Shimizu, K.; Sato, M.; Zhang, Y.; Kouguchi, T.; Takahata, Y.; Morimatsu, F.;

400

Shimizu, M. The bioavailable octapeptide Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro

401

stimulates nitric oxide synthesis in vascular endothelial cells. J. Agric. Food

402

Chem. 2010, 58 (11), 6960–6965.

403

(19)

Sun, H.; Liu, D.; Li, S.; Qin, Z. Transepithelial transport characteristics of the

404

antihypertensive peptide, Lys-Val-Leu-Pro-Val-Pro, in human intestinal

405

Caco-2 cell monolayers. Biosci. Biotechnol. Biochem. 2009, 73 (2), 293–298.

406

(20)

Lei, L.; Sun, H.; Liu, D.; Liu, L.; Li, S. Transport of Val-Leu-Pro-Val-Pro in

407

human intestinal epithelial (Caco-2) cell monolayers. J. Agric. Food Chem.

408

2008, 56 (10), 3582–3586.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

409

(21)

Quirós, A.; Dávalos, A.; Lasunción, M. A.; Ramos, M.; Recio, I.

410

Bioavailability of the antihypertensive peptide LHLPLP: Transepithelial flux of

411

HLPLP. Int. Dairy J. 2008, 18 (3), 279–286.

412

(22)

Satake, M.; Enjoh, M.; Nakamura, Y.; Takano, T.; Kawamura, Y.; Arai, S.;

413

Shimizu, M. Transepithelial transport of the bioactive tripeptide, Val-Pro-Pro,

414

in human intestinal Caco-2 cell monolayers. Biosci. Biotechnol. Biochem. 2002,

415

66 (2), 378–384.

416

(23)

Fei, Y.-J.; Kanai, Y.; Nussberger, S.; Ganapathy, V.; Leibach, F. H.; Romero,

417

M. F.; Singh, S. K.; Boron, W. F.; Hediger, M. A. Expression cloning of a

418

mammalian proton-coupled oligopeptide transporter. Nature 1994, 368 (6471),

419

563–566.

420

(24)

421 422

Daniel, H. Molecular and integrative physiology of intestinal peptide transport. Annu. Rev. Physiol. 2004, 66 (1), 361–384.

(25)

Aito-Inoue, M.; Lackeyram, D.; Fan, M. Z.; Sato, K.; Mine, Y. Transport of a

423

tripeptide, Gly-Pro-Hyp, across the porcine intestinal brush-border membrane.

424

J. Pept. Sci. 2007, 13 (7), 468–474.

425

(26)

Miguel, M.; Dávalos, A.; Manso, M. A.; De La Peña, G.; Lasunción, M. A.;

426

López-Fandiño, R. Transepithelial transport across Caco-2 cell monolayers of

427

antihypertensive egg-derived peptides. PepT1-mediated flux of Tyr-Pro-Ile.

428

Mol. Nutr. Food Res. 2008, 52 (12), 1507–1513.

ACS Paragon Plus Environment

Page 20 of 34

Page 21 of 34

Journal of Agricultural and Food Chemistry

429

(27)

Bejjani,

S.;

Wu,

J.

Transport

of

IRW,

an

ovotransferrin-derived

430

antihypertensive peptide, in human intestinal epithelial caco-2 cells. J. Agric.

431

Food Chem. 2013, 61 (7), 1487–1492.

432

(28)

Fialka, I.; Pasquali, C.; Lottspeich, F.; Ahorn, H.; Huber, L. Subcellular

433

fractionation

434

organelle-specific

435

Electrophoresis 1997, 18 (14), 2582–2590.

436

(29)

of

polarized proteins

epithelial by

cells

and

two-dimensional

gel

identification

of

electrophoresis.

Park, H.-Y.; Kunitake, Y.; Hirasaki, N.; Tanaka, M.; Matsui, T. Theaflavins

437

enhance intestinal barrier of Caco-2 Cell monolayers through the expression of

438

AMP-activated protein kinase-mediated Occludin, Claudin-1, and ZO-1. Biosci.

439

Biotechnol. Biochem. 2015, 79 (1), 130–137.

440

(30)

Majumder, K.; Chakrabarti, S.; Davidge, S. T.; Wu, J. Structure and activity

441

study of egg protein ovotransferrin derived peptides (IRW and IQW) on

442

endothelial inflammatory response and oxidative stress. J. Agric. Food Chem.

443

2013, 61 (9), 2120–2129.

444

(31)

Majumder, K.; Wu, J. Angiotensin I converting enzyme inhibitory peptides

445

from simulated in vitro gastrointestinal digestion of cooked eggs. J. Agric.

446

Food Chem. 2009, 57 (2), 471–477.

447

(32)

Mahler, G. J.; Shuler, M. L.; Glahn, R. P. Characterization of Caco-2 and

448

HT29-MTX cocultures in an in vitro digestion/cell culture model used to

449

predict iron bioavailability. J. Nutr. Biochem. 2009, 20 (7), 494–502.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

450

(33)

Del Mar Contreras, M.; Sancho, A. I.; Recio, I.; Mills, C. Absorption of casein

451

antihypertensive peptides through an in vitro model of intestinal epithelium.

452

Food Dig. 2012, 3 (1–3), 16–24.

453

(34) Sugano, K.; Kansy, M.; Artursson, P.; Avdeef, A.; Bendels, S.; Di, L.; Ecker, G.

454

F.; Faller, B.; Fischer, H.; Gerebtzoff, G.; et al. Coexistence of passive and

455

carrier-mediated processes in drug transport. Nat. Rev. Drug Discov. 2010, 9

456

(8), 597–614.

457

(35)

Majumder, K.; Wu, J. Purification and characterisation of angiotensin I

458

converting enzyme (ACE) inhibitory peptides derived from enzymatic

459

hydrolysate of ovotransferrin. Food Chem. 2011, 126 (4), 1614–1619.

460

(36)

Miguel, M.; Aleixandre, M. A.; Ramos, M.; López-Fandiño, R. Effect of

461

simulated gastrointestinal digestion on the antihypertensive properties of

462

ACE-inhibitory peptides derived from ovalbumin. J. Agric. Food Chem. 2006,

463

54 (3), 726–731.

464

(37)

Hansen, S. H.; Olsson, A.; Casanova, J. E. Wortmannin, an inhibitor of

465

phosphoinositide 3-kinase, inhibits transcytosis in polarized epithelial cells. J.

466

Biol. Chem. 1995, 270 (47), 28425–28432.

467

(38)

Madara, J. L.; Barenberg, D.; Carlson, S. Effects of cytochalasin D on

468

occluding junctions of intestinal absorptive cells: Further evidence that the

469

cytoskeleton may influence paracellular permeability and junctional charge

470

selectivity. J. Cell Biol. 1986, 102 (6), 2125–2136.

ACS Paragon Plus Environment

Page 22 of 34

Page 23 of 34

Journal of Agricultural and Food Chemistry

471

(39)

Hong, S.-M.; Tanaka, M.; Koyanagi, R.; Shen, W.; Matsui, T. Structural design

472

of oligopeptides for intestinal transport model. J. Agric. Food Chem. 2016, 64

473

(10), 2072–2079.

474

(40)

Salamat-Miller, N.; Johnston, T. P. Current strategies used to enhance the

475

paracellular transport of therapeutic polypeptides across the intestinal

476

epithelium. Int. J. Pharm. 2005, 294 (1–2), 201–216.

477

(41)

Shimizu, K.; Sato, M.; Zhang, Y.; Kouguchi, T.; Takahata, Y.; Morimatsu, F.;

478

Shimizu, M. Molecular size of collagen peptide reverses the permeability of

479

Caco-2 cells. Biosci. Biotechnol. Biochem. 2010, 74 (5), 1123–1125.

480

(42) Linnankoski, J.; Makela, J.; Palmgren, J.; Mauriala, T.; Vedin, C.; Ungell, A.-L.;

481

Lazorova, L.; Artursson, P.; Urtti, A.; Yliperttula, M. Paracellular porosity and

482

pore size of the human intestinal epithelium in tissue and cell culture models. J.

483

Pharm. Sci. 2010, 99, 2166−2175.

484

(43)

Ito, K.; Suzuki, H.; Horie, T.; Sugiyama, Y. Apical/basolateral surface

485

expression of drug transporters and its role in vectorial drug transport. Pharm.

486

Res. 2005, 22 (10), 1559–1577.

487

(44)

Qi, R.; Zhang, H.; Xu, L.; Shen, W.; Chen, C.; Wang, C.; Cao, Y.; Wang, Y.;

488

van Dongen, M. A.; He, B.; et al. G5 PAMAM dendrimer versus liposome: A

489

comparison study on the in vitro transepithelial transport and in vivo oral

490

absorption of simvastatin. Nanomed. Nanotechnol. Biol. Med. 2015, 11 (5),

491

1141–1151.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

492

(45)

Xu, Q.; Wu, Y.; Liu, H.; Xie, Y.; Huang, X.; Liu, J. Establishment and

493

characterization of an omasal epithelial cell model derived from dairy calves

494

for the study of small peptide absorption. PLoS One 2014, 9 (3), e88993.

495

(46)

Pauletti, G. M.; Okumu, F. W.; Borchardt, R. T. Effect of size and charge on

496

the, passive diffusion of peptides across caco-2 cell monolayers via the

497

paracellular pathway. Pharm. Res. 1997, 14 (2), 164–168.

498

(47)

Vig, B. S.; Stouch, T. R.; Timoszyk, J. K.; Quan, Y.; Wall, D. A.; Smith, R. L.;

499

Faria, T. N. Human PEPT1 pharmacophore distinguishes between dipeptide

500

transport and binding. J. Med. Chem. 2006, 49 (12), 3636–3644.

501

(48)

Tateoka, R.; Abe, H.; Miyauchi, S.; Shuto, S.; Matsuda, A.; Kobayashi, M.

502

Significance of substrate hydrophobicity for recognition by an oligopeptide

503

transporter (PEPT1). Bioconjugate Chem. 2001, 12 (4), 485–492.

504

(49)

505 506

Chua, H. L.; Jois, S.; Sim, M. K.; Go, M. L. Transport of angiotensin peptides across the Caco-2 monolayer. Peptides 2004, 25 (8), 1327–1338.

(50)

Sánchez-Rivera, L.; Ares, I.; Miralles, B.; Gómez-Ruiz, J. Á.; Recio, I.;

507

Martínez-Larrañaga, M. R.; Anadón, A.; Martínez, M. A. Bioavailability and

508

kinetics of the antihypertensive casein-derived peptide HLPLP in rats. J. Agric.

509

Food Chem. 2014, 62 (49), 11869–11875.

ACS Paragon Plus Environment

Page 24 of 34

Page 25 of 34

Journal of Agricultural and Food Chemistry

510

Figures captions

511

Figure 1. The cytotoxicity of LKP and IQW on the viabilities of Caco-2 cells. The

512

viabilities were significantly decreased after treatment with 5 or 10 mM LKP (A) and

513

IQW (B) for 24 h. However, there are no significantly differences of cell viability

514

after treatment with 5 mM LKP or IQW for 2 h (C). The data are expressed as the

515

means ± SEM (n = 4). Values with different letters are significantly different (P