Structural Design of Oligopeptides for Intestinal Transport Model

Article pubs.acs.org/JAFC

Structural Design of Oligopeptides for Intestinal Transport Model Seong-Min Hong,† Mitsuru Tanaka,† Riho Koyanagi, Weilin Shen, and Toshiro Matsui* Division of Bioresources and Bioenvironmental Sciences, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan S Supporting Information *

ABSTRACT: Glycyl-sarcosine (Gly-Sar) is a well-known model substrate for the intestinal uptake of dipeptides through peptide transporter 1 (PepT1). However, there are no other model peptides larger than tripeptides to evaluate their intestinal transport ability. In this study, we designed new oligopeptides based on the Gly-Sar structure in terms of protease resistance. Gly-Sar-Sar was found to be an appropriate transport model for tripeptides because it does not degrade during the transport across the rat intestinal membrane, while Gly-Gly-Sar was degraded to Gly-Sar during the 60 min transport. Caco-2 cell transport experiments revealed that the designed oligopeptides based on Gly-Sar-Sar showed a significantly (p < 0.05) lower transport ability by factors of 1/10-, 1/25-, and 1/40-fold for Gly-Sar-Sar, Gly-Sar-Sar-Sar, and Gly-Sar-Sar-Sar-Sar, respectively, compared to Gly-Sar (apparent permeability coefficient: 38.6 ± 11.4 cm/s). Cell experiments also showed that the designed tripeptide and Gly-Sar were transported across Caco-2 cell via PepT1, whereas the tetra- and pentapeptides were transported through the paracellular tight-junction pathway. KEYWORDS: oligopeptide, intestinal absorption, peptide transporter 1, tight junction, paracellular transport

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INTRODUCTION Bioactive peptides with various sequences have been shown to possess physiological benefits such as antihypertensive, antidiabetic, and antiatherosclerotic effects.1−3 For example, peptides showing angiotensin I-converting enzyme inhibitory activity lowered promoting blood pressure both in spontaneously hypertensive rats and in human subjects.1,4 However, as described by Foltz,5 their bioavailability still remains unclear. For the intestinal transport of peptides, there are two bottlenecks: enzyme hydrolysis by intestinal proteases and the affinity to transporters such as H+-coupled peptide transporter 1 (PepT1), which facilitates in vivo absorption of di- and tripeptides.6 In previous reports, we demonstrated intact absorption of Val-Tyr, which had antihypertensive effect,7 and Trp-His, which had antiatherosclerotic effects,3 via PepT1, together with their simultaneous protease hydrolysis at microvilli using our visualization technique involving matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS).8,9 Ito et al.10 reported that Ptr2p expressed in yeast, which shares a common substrate recognition mechanism with PepT1, preferably reognized dipeptides containing hydrophobic and basic amino acids. In contrast to such extensive studies on di- and tripeptide absorption, few studies have examined the absorption of peptides longer than tetrapeptides. Recent studies of oligopeptide absorption have shown that longer peptides, such as Leu-Lys-Pro-Asn-Met and Ile-Val-Gly-Arg-Pro-Arg, lowered the promoting blood pressure in spontaneously hypertensive rats.11 In Caco-2 cell monolayer experiments, Gln-Ile-Gly-Leu-Phe,12 Lys-Val-Leu-Pro-Val-Pro,13 and GlyAla-Hyp-Gly-Leu-Hyp-Gly-Pro14 were reported to be penetrants, likely via paracellular transport pathways. However, comparative studies on the absorption potential of each absorbable oligopeptide have been limited because of the lack © XXXX American Chemical Society

of standards or transport models corresponding to each oligopeptide length. Thus, the aim of the present study was to develop appropriate transport models for oligopeptides (tri- to pentapeptides) to evaluate intestinal transport ability. In order to exclude factors affecting the intestinal transport of peptides from their characteristics such as hydrophobicity15 and stereoisomeric status16 in amino acid residues, or to include the transport factor for peptide length, oligopeptides with protease resistance were designed based on Gly-Sar that could preferentially be transported through PepT1.17 Since the affinity of Sar-Gly with PepT1 was reported to be lower than for Gly-Gly,18 oligopeptides with Gly at the N-terminus were synthesized. In addition, taking into consideration that no intact transport of His-Trp, a Caco-2 cell monolayer permeable dipeptide, was observed in rat intestinal membrane due to its hydrolysis by brush border proteases,9 transport experiments of designed oligopeptides in this study were conducted using Caco-2 cell monolayers and rat intestinal membrane.

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MATERIALS AND METHODS

Chemicals. Gly-Sar (Gly-N-methyl-Gly) and 2′,4′,6′-trihydroxyacetophenone (THAP) were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). Gly-Sar-Sar and Gly-Gly-Sar were obtained from Bachem (Bubendorf, Switzerland). Gly-Sar-Sar-Sar and Gly-SarSar-Sar-Sar were obtained from Biomatik Co. (Cambridge, ON, Canada). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Phytic acid was obtained from Tokyo Chemical Ind. Co. (Tokyo, Japan). 2,4,6-Trinitrobenzenesulfonate Received: January 18, 2016 Revised: February 25, 2016 Accepted: February 29, 2016

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DOI: 10.1021/acs.jafc.6b00279 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

the apical or basolateral side was derivatized with 50 μL of 150 mM TNBS solution (pH 8.5) at 30 °C for 30 min. The reaction was stopped with 50 μL of 0.2% formic acid (FA) solution, and the mixture (20 μL) was injected into an LC-TOF-MS. LC separation was performed using an Agilent 1200 series (Agilent, Waldbronn, Germany) on a Cosmosil 5C18-AR-II column (Φ 2.0 mm × 150 mm, Nacalai Tesque) at 40 °C with linear gradient elution with 0.1% FA (solvent A) and acetonitrile (ACN) with 0.1% FA (solvent B) for 20 min at a flow rate of 0.2 mL/min. The target trinitrophenyl (TNP) peptides (TNP-Gly-Sar, m/z 358.0630; TNP-Gly-Gly-Sar, m/z 415.0844; TNP-Gly-Sar-Sar, m/z 429.1001; TNP-Gly-Sar-Sar-Sar, m/z 500.1372; TNP-Gly-Sar-Sar-Sar-Sar, m/z 571.1743) were analyzed using a microTOF II (Bruker Daltonics, Bremen, Germany) in an electrospray ionization positive mode. The MS conditions were as follows: drying gas, N2; flow rate, 8.0 L/min; drying gas temperature, 200 °C; drying gas pressure, 1.6 bar; HV capillary voltage, −4500 V; capillary exit, 70.0 V; skimmer 1, 50.0 V; hexapole 1, 23.0 V; hexapole RF, 100.0 Vpp; skimmer 2, 23.0 V; lens 1 transfer, 52.0 μs, and mass range m/z 100−1000. The calibration solution containing 10 mM sodium formate in 50% ACN was injected at the beginning of each run. The data were analyzed using Bruker Data Analysis version 3.2 software. Calibration curves for quantification were obtained using standards (0.1−10 μM). Analysis of Metabolites and Tissue Distribution of Oligopeptides in Rat Intestinal Membrane. The metabolites of oligopeptides after the 60 min transport experiment across the SD rat intestinal membrane were analyzed in a solution on the basolateral side by LC-TOF-MS. The tissue distribution was also analyzed using our proposed MALDI-MS imaging (MSI) technique.8 A frozen intestinal segment obtained from the transport experiments was sliced into 12 μm thick sections at the cross-sectional face using a CM1100 Leica Cryomicrotome (Leica, Wetzler, Germany). Each intestinal section was thaw-mounted on an indium−tin oxide (ITO) coated conductive glass slide (Bruker Daltonics) and dried under the flow of N2 gas. For MALDI-MS analysis, the THAP matrix (5 mg/mL) was dissolved in ACN/water (3:2, v/v) containing 5 mM phytic acid and 0.1% trifluoroacetic acid (TFA).8 The matrix was sprayed with an ImagePrep automatic matrix sprayer (Bruker Daltonics) to spray uniformly over the ITO glass slide. MALDI-MSI was performed using an autoflex III mass spectrometer equipped with a smartbeam III (Bruker Daltonics) in the positive ion linear mode. MS data were acquired in the range m/z 100−1000 by averaging the signals of 100 consecutive laser pulses. The MS parameters were as follows: ion source 1, 20.00 kV; ion source 2, 18.80 kV; lens voltage, 7.50 kV; gain, 5.0; laser frequency, 200 Hz; and laser power, 100%. MALDI-MSI analysis was performed with a spatial resolution of 20 μm, and the acquired MS spectra were analyzed using Bruker Flex Analysis software (ver. 3.3). The image data were reconstructed for visualization with a mass filter of m/z ± 0.2 by Bruker Flex Imaging software (ver. 2.1). Statistical Analysis. All data were expressed as the mean ± standard error of the mean (SEM). The statistical significance between two groups was evaluated by unpaired Student’s t-test. Analysis between multiple groups was conducted using one-way analysis of variance followed by the Tukey-Kramer’s t-test for post hoc analysis. A p value 300 Ω·cm2 were used for the experiments. Caco-2 cells treated with cytochalasin D (0.5 μg/mL, 30 min) or TF3′G (20 μM, 24 h) before peptide transport experiments were used to evaluate the transport pathways of oligopeptides. The apparent permeability coefficient (Papp, cm/s) was calculated using eq 1:

Papp (cm/s) =

V dC × AC0 dt

(1)

where dC/dt is the change in concentration on the basolateral side over time (mM/s), V is the volume of solution in the basolateral compartment (1.5 mL), A is the surface area of the membrane (0.9 cm2), and C0 is the initial concentration on the apical side (mM). Transport Experiments of Oligopeptides across Intestinal Membrane of Sprague−Dawley (SD) Rats. Rat intestinal membrane for transport experiment of oligopeptides were prepared from SD rats (SPF/VAF Crj:SD; Charles River Japan, Kanagawa, Japan) as described previously.9 A segment of the jejunum (15−20 cm below the duodenum) was excised from the small intestine immediately after the sacrifice of 16 h fasted SD rats. The isolated jejunum was washed with Krebs−bicarbonate Ringer’s solution (2.5 mM CaCl2·2H2O, 4.8 mM KCl, 1.3 mM KH2PO4, 1.2 mM MgSO4· 7H2O, 118.1 mM NaCl, 10 mM D-glucose, and 25 mM NaHCO3), and then cut along the mesenteric border to expose the mucosal side. The prepared intestinal segments were mounted in an Ussing chamber (dual channel Ussing chamber model U-2500, Warner Instrument LLC, Hamden, CT, USA), followed by a 60 min transport experiment. The transport experiments of oligopeptides were the same as described for the Caco-2 cell monolayers. The segments used for the transport experiments were immediately frozen in powdered dry ice and stored at −40 °C until use. All animal experiments were carried out under the Guidance for Animal Experiments in the Faculty of Agriculture and in the Graduate Course of Kyushu University and the Law (No. 105, 1973) and Notification (No. 6, 1980) of the Prime Minister’s Office of the Japanese Government. All experiments were reviewed and approved by the Animal Care and Use Committee of Kyushu University (Permit Number: A26-075). Determination of Transport Amounts of Oligopeptides. The amounts of transport oligopeptides were determined using TNBS derivatization liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS).20,21 An aliquot (50 μL) of sample solution from either

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RESULTS AND DISCUSSION In this study, oligopeptides (tri-, tetra-, and pentapeptides) were newly designed to obtain insight into their intestinal absorption characteristics. Gly-Sar was used as a template to develop an appropriate transport model, as the dipeptide was well-designed for PepT1-mediated transport with no protease hydrolysis or requirements for specific amino acid residues.17 In addition, we synthesized oligopeptides with Gly at the Nterminus, as Sar-Gly shows lower permeability than Gly-Gly and Gly-Sar in Caco-2 cell monolayers.18 B


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Figure 1. LC-TOF-MS stacked chromatograms of Gly-Sar, Gly-Sar-Sar, and Gly-Gly-Sar for 60 min Caco-2 cell transport. Transport experiments of 1 mM Gly-Sar (A), Gly-Sar-Sar (B), and Gly-Gly-Sar (C) were performed using Caco-2 cell monolayers for 60 min. Solutions taken from the basolateral side at 15, 30, 45, and 60 min were subjected to TNBS derivatization-LC-TOF-MS analysis in [M + H]+ at m/z 358.0630, m/z 415.0844, and m/z 429.1001 for TNP-Gly-Sar, TNP-Gly-Gly-Sar, and TNP-Gly-Sar-Sar, respectively. Lowerside stacked chromatograms in B and C indicate the MS detection of Gly-Sar as a possible degraded peptide during the 60 min transport experiments of Gly-Gly-Sar and Gly-Sar-Sar in Caco-2 cell monolayers. Asterisk (*) indicates endogenous Gln (or TNP-Gln) from Caco-2 cells with same m/z as TNP-Gly-Sar. EIC, extracted ion chromatogram.

Transports of Designed Tripeptides in Caco-2 Cell Monolayers and SD Rat Small Intestinal Membrane. Figure 1 shows the waterfall plots of transported tripeptides, Gly-Gly-Sar and Gly-Sar-Sar, as well as a template dipeptide, Gly-Sar, on the basolateral side during Caco-2 cell transport experiments for up to 60 min. Each designed peptide, together with their expected metabolite, Gly-Sar, was detected by TNBS derivatization, followed by LC-TOF-MS. Enhanced MS detection of small and polar amines using the TNBS derivatization technique20,21 was also achieved for the designed peptides in this study. For Gly-Sar detection under the present MS conditions, TNBS derivatization (as TNP-Gly-Sar, retention time (RT), 22.9 min) showed 173-fold higher detection compared to intact Gly-Sar (RT, 1.9 min) (Figure S1). The detection limits of Gly-Sar, Gly-Gly-Sar, and Gly-SarSar by TNBS-LC-TOF-MS were 15.6, 10.3, and 21.1 nM, respectively (data not shown). In addition, we could exclude the TNBS derivatization efficiency on target peptides from their transport characteristics, as the evaluation of peptide transport as Papp or the time-dependent increase in analyte (slope, eq 1) was not influenced when the efficiency was constant for each target peptide. As shown in Figure 1, time-dependent increases in MS intensity for Gly-Gly-Sar (RT, 22.1 min) and Gly-Sar-Sar (RT, 22.4 min) were observed, together with Gly-Sar transport,

indicating that both designed tripeptides can be used as Caco-2 transport models. As shown in the MS chromatogram for GlySar in each tripeptide transport (Figure 1B, C), no degradation of the tripeptides by peptidases occurred during the 60 min Caco-2 transport experiments, because metabolic Gly-Sar from Gly-Gly-Sar and Gly-Sar-Sar was not detected. Additionally, other degradation of Gly-Sar-Sar such as to Sar-Sar by peptidases in Caco-2 cells did not appear to occur. Although endogenous Gln as TNP-Gln (denoted with an asterisk in Figure 1) from the cells was also detected because it had the same m/z 358.0630 as TNP-Gly-Sar, they were separately detected in the present TNBS-LC conditions. In order to confirm the intestinal protease resistance of the designed tripeptides, transport experiments using intestinal membranes from SD rats were performed. As shown in Figure 2A, intact transport of Gly-Sar-Sar in the basolateral solution was successfully detected after the 60 min experiments, along with no MS detection of degraded Gly-Sar, which was similar to the Caco-2 experiments (Figure 1). In contrast, after the 60 min transport experiments of Gly-Gly-Sar across the rat intestinal membrane, no MS detection of Gly-Gly-Sar on the basolateral side and significant MS detection of its degraded Gly-Sar on both the apical and basolateral sides were observed (Figure 2B). The reports that peptides with N-methylated C


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Figure 2. LC-TOF-MS chromatograms of Gly-Sar-Sar and Gly-Gly-Sar for 60 min rat intestinal membrane transport. Transport experiments of GlySar-Sar (A) and Gly-Gly-Sar (B) were performed using SD rat intestinal membrane for 60 min. After 60 min transport of 1 mM Gly-Sar-Sar or GlyGly-Sar, solutions taken from both apical and basolateral sides were subjected to TNBS-LC-TOF-MS analysis. Target peptides by MS analysis were Gly-Sar-Sar, Gly-Gly-Sar, and their possible degraded peptide, Gly-Sar. Asterisk (*) indicates endogenous Gln (or TNP-Gln) from SD rat membrane.

Figure 3. LC-TOF-MS stacked chromatograms of Gly-Sar-Sar-Sar and Gly-Sar-Sar-Sar-Sar for 60 min Caco-2 cell transport. Transport experiments of 1 mM oligopeptides were performed using Caco-2 cell monolayers for 60 min. Solutions taken from the basolateral side at 15, 30, 45, and 60 min were subjected to TNBS derivatization-LC-TOF-MS analysis in [M + H]+ at m/z 500.1372 and m/z 571.1743 for TNP-Gly-Sar-Sar-Sar and TNPGly-Sar-Sar-Sar-Sar, respectively.

peptide bond22 and/or N-terminal Gly23 had high protease resistance may support the intact transport of Gly-Sar-Sar, but not Gly-Gly-Sar. In turn, Gly-Sar-Sar with intestinal protease resistance at the microvilli and/or intracellular region is a template that can be used for further oligopeptide design. More susceptive hydrolysis of the Gly-Gly sequence by proteases was also reported by Adibi, who demonstrated that intravenous injection of Gly-Gly into rats caused a marked increase of Gly in the plasma.24 Transports of Designed Tetra- and Pentapeptides in Caco-2 Cell Monolayers and SD Rat Small Intestinal Membrane. Based on the findings described above, we designed tetra- and pentapeptides using Gly-Sar-Sar as a

template. Apparent MS detections of the designed Gly-Sar-SarSar and Gly-Sar-Sar-Sar-Sar across Caco-2 cell monolayers were successfully observed over a transport time of up to 60 min (Figure 3), which was similar to the designed tripeptides (Figure 1). Their intact transport or absence of protease degradation across the SD rat intestinal membrane was investigated using 60 min transported intestinal membrane segments. Phytic acid aided MALDI-MS and imaging analyses8 were used in this study, as some expected metabolites with no amino groups (i.e., Sar-Sar, Sar-Sar-Sar, and Sar-Sar-Sar-Sar) from corresponding oligopeptides do not undergo the TNBS derivatization reaction for LC-TOF-MS detection. As shown in Figure 4, the MALDI-MSI clearly revealed the overall D


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Figure 4. MALDI-MS detection of Gly-Sar-Sar-Sar, Gly-Sar-Sar-Sar-Sar, and their metabolites in SD rat intestinal membrane. Intestinal membrane segments from SD rats were subjected to MALDI-MS and imaging analyses. After 60 min transport of 5 mM Gly-Sar-Sar-Sar (A) or Gly-Sar-Sar-SarSar (B), segments from the SD rat intestinal membrane were subjected to MALDI-MS analysis. Red arrows indicate target peptides in [M + H]+ of Gly-Sar (m/z 147.1), Sar-Sar (m/z 161.1), Gly-Sar-Sar (m/z 218.1), Sar-Sar-Sar (m/z 232.1), Gly-Sar-Sar-Sar (m/z 289.2), Sar-Sar-Sar-Sar (m/z 303.2), and Gly-Sar-Sar-Sar-Sar (m/z 360.2). Visualization of ions corresponding to Gly-Sar-Sar-Sar and Gly-Sar-Sar-Sar-Sar on the segment was performed by MALDI-MSI at a spatial resolution of 20 μm, at which the section was sprayed with 5 mM phytic acid−THAP solution (5 mg/mL) for 120 cycles. Scale bar: 1 mm.

absorption studies and that these peptides did not undergo protease hydrolysis. Characteristics of Designed Oligopeptide Transport across Caco-2 Cell Monolayers. Table 1 summarizes the Papp values of the designed oligopeptides across Caco-2 cell monolayers. During the transport experiments of each oligopeptide, no significant differences in TEER values of Caco-2 cell monolayers with >400 Ω·cm2 were observed (Figure S2), indicating that designed oligopeptides did not affect the membrane barrier properties. For transport directed

distribution of each designed oligopeptide in the 60 min transported intestinal membrane segments, while other m/z signals corresponding to their possible metabolites were not observed within the present MS detection limits of 400 pmol/ spot;8 there were no MS signals for Gly-Sar (m/z 147.1), SarSar (m/z 161.1), Gly-Sar-Sar (m/z 218.1), Sar-Sar-Sar (m/z 232.1), Gly-Sar-Sar-Sar (m/z 289.2), and Sar-Sar-Sar-Sar (m/z 303.2). This strongly indicated that the newly designed tetraand pentapeptides were appropriate penetrants for intestinal E


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AP, apical side; BL, basolateral side. Values are expressed as the mean ± SEM, n = 3.

tentative criteria proposed by Farrell et al.,28 at ratio of 0.5 passive transport precedes. Together with the ratio of each oligopeptide (Table 1) and the above criteria, we performed competitive transport experiments of each oligopeptide with Gly-Sar as a positive PepT1 substrate.17 As shown in Figure 5A, the 60 min uptake of 1 mM Gly-Sar-Sar across Caco-2 cell monolayers was significantly (p < 0.01) inhibited by 10 mM Gly-Sar. No significant reduction of designed tetra- and pentapeptide uptake by Gly-Sar was observed (Figures 5B

from the apical (AP) to the basolateral side (BL) Gly-Sar showed the highest Papp of 38.6 ± 11.4 × 10−6 cm/s among the designed peptides in this study, in descending order of Gly-Sar > Gly-Sar-Sar (1/10-fold lower than Gly-Sar) > Gly-Sar-Sar-Sar (1/25 lower) > Gly-Sar-Sar-Sar-Sar (1/40 lower). It was also likely that the transport ability of oligopeptides across Caco-2 cell monolayers occurred in a peptide length dependent manner. These results are consistent with those of Terada et al.,18 who found that Gly-Gly uptake was higher than those of Gly-Gly-Gly and Gly-Gly-Gly-Gly in PepT1-transfected pig kidney epithelial cells. Papp values of the designed oligopeptides (Table 1) may be useful for comparative evaluation of the reported transport abilities of oligopeptides. The tripeptides Val-Pro-Pro (Papp: 0.5 × 10−8 cm/s) and Ile-Pro-Pro (0.9 × 10−8 cm/s) showed approximately 1/200-fold lower transport ability25 compared to our designed tripeptide, Gly-Sar-Sar, indicating that the amino acid residues in the tripeptide sequence greatly affect PepT1mediated transport in Caco-2 cells. Gao et al.26 reported that a methylated tripeptide, Ala-N-methyl-Phe-Ala, with protease resistance showed a preferable PepT1-mediated transport with Papp value of 2.5 × 10−6 cm/s, which was comparable to the Papp value of designed Gly-Sar-Sar. Within the limited information on tripeptide transport so far reported, Papp values of tripeptides showing 0.5. According to the

Figure 5. Effect of inhibitors on the transport of oligopeptides across Caco-2 cell monolayers. Transport experiments of 1 mM Gly-Sar-Sar (A), Gly-Sar-Sar-Sar (B), and Gly-Sar-Sar-Sar-Sar (C) in the presence or absence of 10 mM Gly-Sar. For Gly-Sar-Sar-Sar and Gly-Sar-SarSar-Sar transport experiments, Caco-2 cells treated with or without cytochalasin D (0.5 μg/mL, 30 min) or TF3′G (20 μM, 24 h) were used. After 60 min transport, the uptake of each oligopeptide in basolateral solution was measured by TNBS-LC-TOF-MS. Values are expressed as the mean ± SEM, n = 3. Statistical differences between two groups were analyzed by Student’s t-test. N.S., not significant.

Table 1. Apparent Permeability Coefficient (Papp) and the Ratio of Papp(BL→AP)/Papp(AP→BL) of Designed Oligopeptides across Caco-2 Cell Monolayersa Papp (×10−6, cm/s) peptide Gly-Sar Gly-Sar-Sar Gly-Sar-Sar-Sar Gly-Sar-Sar-Sar-Sar

AP → BL 38.6 3.5 1.6 0.9

± ± ± ±

11.4 0.3 0.2 0.1

BL → AP

Papp(BL→AP)/Papp(AP→BL)

± ± ± ±

0.08 0.47 0.67 1.05

2.9 1.6 1.0 0.9

1.4 0.2 0.2 0.2

a

F



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ABBREVIATIONS USED ACN, acetonitrile; AP, apical side; BL, basolateral side; DMEM, Dulbecco’s modified Eagle’s medium; FA, formic acid; FBS, fetal bovine serum; HBSS, Hanks balanced salt solution; LCTOF-MS, liquid chromatography time-of-flight mass spectrometry; MALDI-MSI, matrix-assisted laser desorption/ionization mass spectrometry imaging; Papp, apparent permeability; PepT1/2, H+-coupled peptide transporter 1/2; SD, Sprague− Dawley; TEER, transepithelial electrical resistance; TF3′G, theaflavin-3′-O-gallate; THAP, 2′,4′,6′-trihydroxyacetophenone; TJ, tight junction; TNBS, 2,4,6-trinitrobenzenesulfonate

and 5C). These results clearly demonstrate that the designed tripeptide, Gly-Sar-Sar, may be transported through PepT1, similarly to Ile-Arg-Trp.29 In addition, PepT1-mediated transport of tripeptides showed good agreement with the report that human intestinal PepT1 can primarily recognize peptides within two peptide bonds or di- and tripeptides.18 Passive transport of the designed tetra- and pentapeptides across Caco-2 cell monolayers (Table 1) was confirmed in Caco-2 cells treated with cytochalasin D (as tight-junction (TJ) opener12) or TF3′G (as TJ closer30). As shown in Figure 5B, the uptake of the designed tetrapeptide was significantly (p < 0.05) promoted in cytochalasin D-treated cells (TEER: control, 370 ± 36 Ω·cm2; cytochalasin D, 187 ± 18 Ω·cm2) and was significantly (p < 0.01) reduced in TF3′G-treated cells (TEER: 502 ± 40 Ω·cm2), similarly to the results for the designed pentapeptide (Figure 5C). In turn, both designed oligopeptides were transported through the paracellular TJ route. Paracellular transport of the designed tetra- and pentapeptides via TJ showed good agreement with previous reports of TJ-mediated transport of tetrapeptide (Gly-Gly-Tyr-Arg31), pentapeptides (Gln-Ile-Gly-Leu-Phe,12 Val-Leu-Pro-Val-Pro,27 and His-LeuPro-Leu-Pro32), hexapeptide (Lys-Val-Leu-Pro-Val-Pro13), and octapeptide (Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro14) across Caco-2 cell monolayers. In conclusion, we successfully designed oligopeptides (tri- to pentapeptides) using Gly-Sar as a template. The designed GlySar-Sar (via PepT1), Gly-Sar-Sar-Sar (via TJ route), and GlySar-Sar-Sar-Sar (via TJ route) showed intestinal protease resistance and transport ability across Caco-2 cell monolayers and the SD rat intestinal membrane in a peptide lengthdependent manner. The designed models may be useful as standards for evaluating the transport (or absorption) of oligopeptides.

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ASSOCIATED CONTENT

* Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00279. Figures described in text (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel and fax: +81-92-642-3011. E-mail: [email protected]. ac.jp. Author Contributions †

S.-M.H. and M.T. contributed equally to the experimental work. Funding

This study was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (No. 22248014) to T.M. The cost of publication was supported in part by the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University. Notes

The authors declare no competing financial interest.

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REFERENCES

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ACKNOWLEDGMENTS

The authors thank Kaori Miyazaki for her technical assistance. G


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Structural Design of Oligopeptides for Intestinal Transport Model

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