Importance of Terminal Amino Acid Residues to the Transport of

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Importance of Terminal Amino Acid Residues to the Transport of Oligopeptides across Caco-2 Cell Monolayer Long Ding, Liying Wang, Zhipeng Yu, Sitong Ma, Zhiyang Du, Ting Zhang, and Jingbo Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03450 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Journal of Agricultural and Food Chemistry

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Importance of Terminal Amino Acid Residues to the Transport of Oligopeptides

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across Caco-2 Cell Monolayer

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Long Ding†, Liying Wang†, Zhipeng Yu‡, Sitong Ma†, Zhiyang Du†, Ting Zhang†,

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Jingbo Liu†*

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†

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China

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‡

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China

College of Food Science and Engineering, Jilin University, Changchun 130062, P.R.

College of Food Science and Engineering, Bohai University, Jinzhou 121013, P.R.

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Running title: Terminal Amino Acid Residues and Transport of Oligopeptides

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*Corresponding author: Tel.: +86 431 87836351. Fax: +86 431 87836391. E-mail:

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[email protected].

1

ACS Paragon Plus Environment


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ABSTRACT: The objective of this paper was to investigate the effects of terminal

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amino acids on the transport of oligopeptides across Caco-2 cell monolayer. Ala-based

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tetra- and penta-peptides were designed and the N-terminal or C-terminal amino acid

17

residues were replaced by different amino acids. The results showed that the

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oligopeptides had a wide range of transport permeability across Caco-2 cell

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monolayer and could be divided into 4 categories: non/poor permeability, low

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permeability, intermediate permeability, and good permeability. Tetrapeptides with

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N-terminal Leu, Pro, Ile, Cys, Met, Val or C-terminal Val showed the highest

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permeability with apparent permeability coefficient (Papp) values over 10×10-6 cm/s (P

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< 0.05), suggesting that non-polar hydrophobic aliphatic amino acids or polar

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sulfur-containing amino acids were the best for the transport of tetrapeptides.

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Pentapeptides with an N-terminal or C-terminal Tyr also showed high permeability

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levels with Papp values about 10×10-6 cm/s. The amino acids Glu, Asn, Thr at the

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N-terminus or Lys, Asp, Arg at the C-terminus were also beneficial for the transport of

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tetra- and penta-peptides with Papp values ranging from 1×10-6 cm/s to 10×10-6 cm/s.

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In addition, peptides with amino acids replaced at the N-terminus generally showed

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higher permeability than those with amino acids replaced at the C-terminus (P < 0.05),

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suggesting that N-terminal amino acids were more important for the transport of

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oligopeptides across Caco-2 cell monolayer.

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KEYWORDS: oligopeptide, amino acid, transport permeability, Caco-2 cell

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monolayer, brush border membrane peptidase

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INTRODUCTION

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Bioactive peptides derived from enzymatic hydrolysis of food proteins have

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generated much attention for their potential human health-promoting functions and

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limited adverse effects. In the last few decades, a large number of bioactive peptides

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have been identified from food proteins, including animal and plant proteins, as well

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as marine proteins. These bioactive peptides have been shown to have different

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biological activities such as antihypertensive, antioxidant, anti-inflammatory,

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immunomodulatory, anticancer, antimicrobial, and calcium-binding activities.1

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However, it has been found that the in vitro bioactivities of peptides do not always

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correlate with in vivo activity.2 This is mainly because, most food-derived bioactive

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peptides have to overcome two important physiological barriers, extensive enzymatic

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degradation in the gastrointestinal tract, poor permeability through the intestinal

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epithelium, and be absorbed in an intact form into the circulation to exert their

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bioactive functions in vivo after oral administration.3 This implies the importance of

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resistance to various peptidases, including brush border membrane peptidases, and

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absorption through the intestine for the bioavailability of food-derived bioactive

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peptides.

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Neway and Smith first found that some dipeptides, such as glycyl-glycine, could be

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absorbed through the intestine without hydrolysis.4,5 Two important H+-coupled

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transporters for dipeptides, peptide transporter 1 (PepT1) and peptide transporter 2

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(PepT2), were successfully cloned and identified in the 1990s, providing evidence for

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the intact absorption of dipeptides at the molecular level.6,7 Moreover, many 3



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tripeptides or peptidomimetic drugs were also demonstrated to be transported by

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PepT1 and PepT2.8 However, the exact transport pathways for oligopeptides,

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especially peptides containing 4 to 10 amino acid residues, remain unknown.

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Compared with di- or tri-peptides, oligopeptides are generally more easily hydrolyzed

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by brush border membrane peptidases and have a lower permeability through the

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intestine. It is speculated that some oligopeptides might be transported through the

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intestinal epithelium by transcytosis and the tight junction-mediated paracellular

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pathway,9 although convincing empirical evidence is lacking. In addition, Ananth and

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colleagues10,11, and Chothe and colleagues12,13 reported two novel Na+-coupled

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oligopeptide transporters SOPT1 and SOPT2, that can transport peptides consisting of

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five or more amino acids. These two new peptide transporters differ from PepT1 and

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PepT2, as the transport of oligopeptides through SOPT1 and SOPT2 can be

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stimulated or inhibited by some di- or tri-peptides. Nevertheless, the genes encoding

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SOPT1 and SOPT2 and their protein structures have not yet been fully identified.

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Actually, regardless of the oligopeptide transport pathway, it is important to

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understand the relationship between transport and structural features of oligopeptides

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or whether there are structural determinants. In our previous study, the relationship

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between the structure of RVPSL and transport across Caco-2 cell monolayer was

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investigated. A series of pentapeptides derived from RVPSL was obtained by amino

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acid replacement with proline. It was found that N-terminal Pro was more beneficial

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for the transport of pentapeptides across Caco-2 cell monolayer than Arg and Val

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residues, while no similar results were observed at the C-terminus.14 To further verify 4


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the effects of terminal amino acid residues on the transport of oligopeptides, up to 78

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Ala-based tetra- and penta-peptides with different N-terminal or C-terminal amino

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acids were designed and their transport across Caco-2 cell monolayer was

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investigated in the present study. In addition, the enzymatic degradation of peptides

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by brush border membrane peptidases of Caco-2 cells was studied.

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MATERIALS AND METHODS

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Chemicals. Caco-2 cell lines were purchased from American Type Culture

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Collection. All peptides were synthesized by solid-phase procedures using

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Fmoc-protected amino acid synthetic methods with purity over 95% by ChinaPeptide

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Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal

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bovine serum, penicillin-streptomycin, nonessential amino acid solution, and Hanks’

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balanced salt solution (HBSS) were purchased from Gibco BRL Life Technology

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(Carlsbad, CA, USA). Trypsin, L-glutamine, dimethyl sulfoxide (DMSO),

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ethylenediaminetetraacetic acid (EDTA), and collagen type I were all purchased from

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Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN), trifluoroacetic acid (TFA),

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and methanol were purchased from Fisher Scientific (Waltham, MA, USA). All other

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reagents were analytical grade.

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Cell culture. Caco-2 cells were grown in DMEM containing 10% fetal bovine

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serum, 1% nonessential amino acid solution, 100 units/mL penicillin, 100 µg/mL

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streptomycin, and 4 mM L-glutamine at 37 °C in an atmosphere of 5% CO2 and 90%

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relative humidity. Stock cultures were grown in 75 cm2 tissue culture flasks and were

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split at 80% to 90% confluency using 0.25% trypsin and 0.02% EDTA solution. The 5



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cells from passage numbers of 30−40 were used and seeded on collagen type I-coated

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permeable polycarbonate inserts (12 mm diameter, 0.4 µm pore size, 1.12 cm2 grown

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surface area; Costar, Corning, NY, USA) in 12 transwell plates at a density of 1 × 105

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cells/insert. The culture medium (0.5 mL on the apical side (AP) and 1.5 mL on the

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basolateral side (BL)) was replaced every 2 days for the first week and then daily until

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the cells were used for experiments 21 days after seeding. The integrity of the cell

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monolayer was evaluated by measuring transepithelial electrical resistance (TEER)

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values with an EVOM epithelial volt/ohmmeter (Millicell-ERS, Millipore, Billerica,

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MA, USA). Only Caco-2 cell monolayers with TEER values higher than 300 Ω·cm2

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were used for transport studies.

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Transport experiments. The transepithelial transport of peptides across Caco-2

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cell monolayer was conducted according to the method described by Hubatsch.15 The

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Caco-2 cell monolayers were rinsed with HBSS twice and then incubated with HBSS

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for 30 min at 37 ℃ in 5% CO2 prior to transport experiments. The HBSS was

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removed and replaced with 0.5 mL of 3.0 mM peptides (dissolved in HBSS, pH 7.4)

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on the AP and 1.5 mL fresh HBSS on the BL (pH 7.4). All plates were incubated at

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37 ℃ in 5% CO2 for 2 h. Peptide concentrations on both the AP and BL were

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determined by RP-HPLC analysis.

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The apparent permeability coefficient (Papp, cm/s) was calculated as follows: =

1 1 × × ×

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where dC/dt was the change of peptide concentration on the BL (mM/s); A was the

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area of the membrane (cm2); C0 was the initial peptide concentration on the AP (mM); 6


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V was the volume of the BL (mL).

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During the process of absorption, peptides were partly degraded by various brush

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border membrane enzymes into smaller peptides and amino acids and this might have

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decreased the concentrations of peptides on the AP and affected the permeability of

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peptides. For many peptides, the more they were degraded by brush border membrane

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enzymes, the less well absorbed they were. Thus, we introduced a new permeability

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coefficient (Papp’) here to account for the final peptide concentrations on the AP in the

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present study.

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The Papp’ (cm/s) was calculated as follows: ′ =

1 1 × × ×

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where dC/dt was the change of peptide concentration on the BL (mM/s); A was the

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area of the membrane (cm2); Ct was the peptide concentration on the AP after 2 h

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(mM); V was the volume of the BL (mL).

134 135

Degradation of peptides by various brush border membrane enzymes (cytosolic peptidases were not included) was calculated as follows: Degradation % = 1 −

× + × " × 100% ×

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where C0 and Ct were the initial and final peptide concentrations on the AP (mM),

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respectively, and C was the peptide concentration on the BL (mM); V0 and V were the

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volumes of the AP and BL (mL), respectively.

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RP-HPLC. Peptides were quantified using a Shimadzu HPLC system equipped

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with LC solution software, an LC-20AD binary gradient pump, an SIL-20AC

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autosampler, a CTO-20AC column oven and SPD-20AV UV detector (Shimadzu, 7



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Kyoto, Japan). The analytical column was a C18 reverse-phase column (Symmetry C18

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column, 250×4.6 mm, particle size 5 mm, Waters, Milford, MA, USA). The mobile

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phases were 0.1% TFA in water (solvent A) and 0.1% TFA in ACN (solvent B).

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Samples were eluted with a gradient of solvent B from 15% to 55% over 15 min and

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held at 55% for 2 min at a flow rate of 0.5 mL/min. The injection volume was 20 µL,

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and the absorbance detector wavelength was set at 220 nm. Peptide concentrations

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were calculated by a linear regression analysis of known concentrations ranging from

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1 µM to 3000 µM of peptide standard and corresponding peak areas. The calibration

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curves of peak area vs concentration and the coefficients were then analyzed.

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Statistical analysis. All data were expressed as the mean ± SD (n = 3). Differences

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between two means were detected by unpaired Student’s t-tests. When there were

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more than two means, the differences were analyzed by one-way analysis of variance

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(ANOVA) followed by the least significant difference (LSD) test with the significance

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level at P < 0.05 using SPSS 19.0 software.

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RESULTS

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Transport of peptides. An Ala-based tetrapeptide (Ala-Ala-Ala-Ala) and

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pentapeptide (Ala-Ala-Ala-Ala-Ala) were firstly designed and their N-terminal or

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C-terminal amino acid residues were then replaced with other amino acids. In total, 78

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tetra- and penta-peptides with different N-terminal or C-terminal amino acid residues

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were obtained and synthesized. The permeability coefficients of Papp and Papp’ of these

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oligopeptides were determined in Caco-2 cell monolayer.

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N-terminal amino acids had a large influence on the transport of tetrapeptides 8


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(Figure 1A and B). It was found that tetrapeptides with N-terminal amino acid

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residues of Leu, Ile, Pro, Cys, Met or Val had the highest transport permeability with

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Papp and Papp’ values over 20×10-6 cm/s and 30×10-6 cm/s, respectively. Tetrapeptides

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with N-terminal Tyr, Glu, Asn, Thr, Asp, Gly or His all had relatively low

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permeability with Papp and Papp’ values ranging from 1×10-6 cm/s to 10×10-6 cm/s,

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respectively. Tetrapeptides with N-terminal Phe, Gln, Ser, Lys, Ala, Arg and Trp were

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either poorly transported or not transported at all. Compared with the N-terminal

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amino acids, tetrapeptides with altered C-terminal amino acid residues generally

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showed lower permeability (Figure 1C and D). Only the tetrapeptide with the Val at

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C-terminus had a Papp value greater than 10×10-6 cm/s, followed by that with Cys, Glu,

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Lys, Asp, Thr and Arg, ranging from 1×10-6 cm/s to 10×10-6 cm/s. However, the two

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tetrapeptides with C-terminal Val and Cys residues both exhibited Papp’ values greater

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than 40×10-6 cm/s. The amino acids Tyr, Trp, Ala, Ile, Asn, Gln, Ser, His, and Gly at

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the C-terminus all caused tetrapeptides to be either poorly transported or not

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transported at all across Caco-2 cell monolayer.

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Pentapeptides with the N-terminal amino acids Asp, Gly and Tyr had Papp values

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greater than 10×10-6 cm/s, followed by Asn, Arg, Glu, Gln, Thr, Lys, His and Cys that

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ranged from 1×10-6 cm/s to 10×10-6 cm/s (Figure 2A). Pentapeptides with N-terminal

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Ser, Ala, Pro, Leu, Phe, Trp, and Val residues had poor permeability or were not

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transported at all. However, what should be noted was that the pentapeptide with an

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N-terminal Tyr residue had the highest Papp’ value of approximately 70×10-6 cm/s and

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it was significantly higher than pentapeptides with N-terminal Asp, Gly and Asn 9



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residues (Figure 2B). Interestingly, the pentapeptide with C-terminal Tyr also showed

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the highest Papp value compared with other C-terminal amino acids, although the Papp

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value was only approximately 7×10-6 cm/s. Pentapeptides with the C-terminal amino

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acids Pro, Lys, Asp, Cys, Gln, Arg and Val had lower Papp values ranging from 1×10-6

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cm/s to 5×10-6 cm/s. Pentapeptides with Ala, Phe, Met, Ile, Trp, Leu, Glu, and Thr at

191

the C-terminus were non-permeable or had poor transport permeability (Figure 2C).

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Notably, pentapeptides with different C-terminal amino acids showed lower Papp’

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values compared with N-terminal replaced pentapeptides. Only the pentapeptides with

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C-terminal Cys and Tyr had Papp’ values greater than 10×10-6 cm/s. However, it was

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found that the Papp’ value of the pentapeptide with C-terminal Cys was higher than

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that with Tyr. Pentapeptides with C-terminal Asp, Lys, Pro, Val, Gln, Arg, Gly, Thr,

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Asn and Leu also showed intermediate permeability with Papp’ values ranging from

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1×10-6 cm/s to 10×10-6 cm/s (Figure 2D).

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The effects of the replacement of different amino acids at the N-terminus and

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C-terminus on the transport permeability of Ala-based tetra- and penta-peptides across

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the Caco-2 cell monolayer were compared. As shown in Figure 3, the replacement of

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N-terminal amino acids generally improved the transport permeability of Papp (P
10

> 20

Leu, Ile, Pro, Cys, Met and Val at N-terminus; Val and Cys at C-terminus

Category

Tetrapeptides

Pentapeptides

Lys, Ser, Gln and Phe at N-terminus; Trp and Tyr at C-terminus His and Trp at N-terminus; Pro, His, Gly, Ser and Gln at C-terminus

Ser at N-terminus; Trp, Met, Ile, Phe and Ala at C-terminus

24


Met, Val, Trp, Phe and Ile at N-terminus; Gly, His, Asn, Ser, Thr, Glu and Leu at C-terminus Asn, Arg, Glu, Gln, Thr, Lys and Cys at N-terminus; Tyr, Pro, Lys, Asp, Cys, Gln, Arg and Val at C-terminus Tyr, Asp and Gly at N-terminus

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Figure legends Figure 1. Effects of N-terminal (A, B) and C-terminal (C, D) amino acid residues on the permeability coefficients Papp (A, C) and Papp’ (B, D) for tetrapeptides across Caco-2 cell monolayer. All values are means ± SD (n = 3). Figure 2. Effects of N-terminal (A, B) and C-terminal (C, D) amino acid residues on the permeability coefficients Papp (A, C) and Papp’ (B, D) for pentapeptides across Caco-2 cell monolayer. All values are means ± SD (n = 3). Figure 3. Effects of different amino acids at the N-terminus and C-terminus on the transport permeability of Papp (A) and Papp’ (B) for Ala-based tetra- and penta-peptides across Caco-2 cell monolayer. All values are means ± SD (n = 3). Figure 4. Correlation between the transport permeability of Papp and Papp’ of a number of Ala-based tetra- and penta-peptides with different N-terminal or C-terminal amino acids across Caco-2 cell monolayer. The solid line represents the fitted equation for all plots: y=2.5460x+0.6116; R2=0.8420, n=78; The dotted line represents the fitted equation that did not include the plots of AAAC, AAAV and YAAAA: y=2.3538x-0.0817; R2=0.8626, n=75. Figure 5. Effects of N-terminal amino acid residues on the degradation of tetrapeptides (A) and pentapeptides (B) by brush border membrane enzymes of Caco-2 cells. All values are means ± SD (n = 3). Figure 6. Effects of C-terminal amino acid residues on the degradation of tetrapeptides (A) and pentapeptides (B) by brush border membrane enzymes of Caco-2 cells. All values are means ± SD (n = 3). 25


-6

Papp (1×10 , cm/s) 8 -6

15

10

Papp' (1×10 , cm/s)

-6

-6

Papp (1×10 , cm/s) 20

10

6

4

FA A Q A AA SA A A KA A A RA A A AA A W AA AA HA A A DA A A G A AA NA A A EA A A TA A A YA A A VA A A PA A A M A AA A IA A CA A A LA A AA

16

Papp' (1×10 , cm/s)

FA A Q A AA SA A A KA A A AA A A RA A W AA AA HA A A G A AA DA A A TA A A NA A A EA A A YA A A VA A A M A AA CA A A IA A A PA A A LA A AA

35

AA AA AY A AA W A AA N A AA A A AA Q A AA S AH AA AA AI A AA G A AA P A AA M A AA F A AA L A AA D A AA T A AA R A AA E A AA K A AA C AV

AA AA AY A AA W A AA A AA AI A AA N A AA Q A AA S A AA F A AA G A AA M A AA H A AA L A AA P A AA R A AA T AD AA A AA K A AA E A AA C AV


(A)

Papp 90

30

5

0

(C) 80

2 10

0 0

(B)

Papp'

80

25 70

60

50

40

30

20

10 0

14

(D)

12 70

60

50

40

30

20

Figure 1

26


Page 26 of 32

-6

Papp (1×10 , cm/s) 4 -6

5

3

2

Papp' (1×10 , cm/s)

-6

12 60

10 50

6

4

Papp' (1×10 , cm/s)

-6

Papp (1×10 , cm/s) 8

8 20

SA A AA AA A LA AA A IA AA W AAA AA FA AA A VA AA A M AA AA HA AA A PA AA A KA AA A CA AA A TA AA A Q AA AA EA AA A RA AA A NA AA A G AA AA DA AA A YA AA AA A

SA A AA AA A PA AA A LA AA AA IA A A FA AA W AAA AA VA AA A M AA AA CA AA A HA AA A KA AA A TA AA A Q AA AA EA AA A RA AA A NA AA A YA AA A G AA AA DA AA AA A

14

AA A AA AA A AA AF AA AA M AA AA A I AA AW A AA AE A AA AH A AA AS A AA AL A AA AN A AA AT A AA AG A AA AR A AA AQ A AA AV A AA AP A AA AK A AA AD A AA AY AA C

AA A AA AA A AA AF AA AA M AA AA AA I AA W A AA AL A AA AE A AA AT A AA AS A AA AN A AA AH A AA AG A AA AV A AA AR A AA AQ A AA AC A AA AD A AA AK A AA AP AA Y

Page 27 of 32 Journal of Agricultural and Food Chemistry

(A)

Papp 70

2 10

0 0

(C)

7 18

1

0

(B)

Papp'

40

30

20

(D)

6 16

14

12

10

8

6

4

2

0

Figure 2

27



(A)

35

N-terminus C-terminus

30

-6

Papp (1×10 cm/s)

Page 28 of 32

25 20 15 10 5 0 0 90

5

10

15

20

25

30

35

40

35

40

number of peptides

(B)

N-terminus C-terminus

80

60 50

-6

Papp' (1×10 cm/s)

70

40 30 20 10 0

0

5

10

15

20

25

number of peptides

30

Figure 3

28


Page 29 of 32


y=2.5460x+0.6116, 2 R =0.8420, n=78

80

YAAAA 70

AAAV

-6

Papp' (1×10 cm/s)

60 50

AAAC

y=2.3538x-0.0817, 2 R =0.9626, n=75

40 30 20 10 0 0

5

10

15

20

25

30

-6

Papp (1×10 cm/s) Figure 4

29


35

H A R AA A A G AA A A D AA AA A TA AA EAAA A KAAA A A IA A A Q AA A A W AA A A C AA AA A N A A A VAAA A FAAA A M AA AA A LA A A YAAA A AAAA A PAAA A SAAA AAA A

Degradation (%) PA LAAA VAAA H AA AA IA A C AA A YAAA M AA A D AA A EAAA N AA A SAAA TAAA G AA A W AA AA R A A KAAA Q AA A FAAA AAAA AA

Degradation (%)


90

80

100

90

(A)

70

60

50

40

30

20

10

0

(B)

80

70

60

50

40

30

20

10

0

Figure 5

30


Page 30 of 32

AA AAAAH AAAA Y AAAA P AAAA S AAAA K AA AA E AAAA G AAAA D AA AA R AAAA Q AAAA N AA AA V AAAAC AAAAT AA AA A L AA AM A A AF AA AA A AI AA AW AA A

Degradation (%) AA AA AE AAAD AAAH AA AT AA AS AAAN AA AP AAAQ AA AK AAAG AA AV AAAC AAAR AAAA AAAY A AA L AA AI AA AM A AA W AF

Degradation (%)

Page 31 of 32 Journal of Agricultural and Food Chemistry

100 90

100

90

(A)

80

70

60

50

40

30

20

10

0

(B)

80

70

60

50

40

30

20

10

0

Figure 6

31



XAAA/XAAAA AAAX/AAAAX Caco-2 cell monolayers

Good permeability 35 30

-6

Papp (1×10 , cm/s)

25

Intermediate permeability

20

Non/Poor permeability 15 10

Low permeability

5

AA AA S W SAAA A A AA AA W AW A AAAA AA AS W A AA G AA AA AA A H AA AA S A AA A AA H A G AAAA R TAAA AAR A A AAAA A D AAAD TAAK N AA AAAA A A EAAK EA A A N AA AA A YA A A G AA AA A D A AA A AAAA VAAV M AA A C AA AA IA A PAAA LAAA AA

0

Table of content graphic

32


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Importance of Terminal Amino Acid Residues to the Transport of

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