Transport Study of Egg-Derived Antihypertensive Peptides (LKP and

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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

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Journal of Agricultural and Food Chemistry

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

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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,

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

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

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

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

15

ACS Paragon Plus Environment


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ABSTRACT

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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

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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

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

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Caco-2/HT29 co-culture monolayers.

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


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INTRODUCTION

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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

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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

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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

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

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

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

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

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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



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

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

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

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

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

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

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different peptides may have different transport mechanisms. However, the

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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

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antihypertensive peptides for their pharmacological application and bioavailability in

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

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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

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

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

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

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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).

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


and

magnesium),

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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).

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

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

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

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

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

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

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

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

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

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

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

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

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HEPES and Tris).

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

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

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

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

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

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



104

Spectra max, Sunnyvale, CA).

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

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

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

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

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

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collected and characterized using liquid chromatography-mass spectrometry/mass

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spectrometry (LC-MS/MS).

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

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

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

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

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

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

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

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

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

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

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

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from apical or basolateral sides were collected and characterized using ultrahigh

124

performance liquid chromatography (UPLC).

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


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

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

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

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

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

131

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

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PepT1

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

134

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

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

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

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

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

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

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

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disordered

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5'-GCGCGCUUUGUAGGAUUCGDTDT-3'

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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



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.

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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

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bands of PepT1 were normalized using the tubulin bands.

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

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

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

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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.

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

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

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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),

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

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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


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

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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

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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



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

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basolateral side of the cell monolayers.

206

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

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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

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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


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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



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


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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.



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


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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



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

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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;

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

345

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


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Figures captions

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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

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

Transport Study of Egg-Derived Antihypertensive Peptides (LKP and

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