Both PepT1 and GLUT Intestinal Transporters Are Utilized by

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Both PepT1 and GLUTs Intestinal Transporters Are Utilized by a Novel Glycopeptide Pro-Hyp-CONH-GlcN Mengmeng Feng, and Mirko Betti J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00815 • Publication Date (Web): 08 Apr 2017 Downloaded from http://pubs.acs.org on April 9, 2017

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Journal of Agricultural and Food Chemistry

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Both PepT1 and GLUTs Intestinal Transporters Are Utilized by a Novel Glycopeptide Pro-

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Hyp-CONH-GlcN

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Mengmeng Fenga, and Mirko Bettia*

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Affiliations:

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a

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410 Agriculture/Forestry Centre

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Edmonton, AB T6G 2P5 Canada

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

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*Corresponding Author: Dr. Mirko Betti;

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E-mail: [email protected]

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Tel: (780) 248-1598;

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Fax: (780) 492-4265.

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Abstract

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Pro-Hyp (PO) accounts for many beneficial biological effects of collagen hydrolysates for skin

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and bone health. The objective of this study was to conjugate PO with glucosamine (GlcN) to

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create a novel glycopeptide Pro-Hyp-CONH-GlcN (POGlcN) and then to investigate the

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potential involvement of multiple transepithelial transport pathways for this glycopeptide. NMR

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results revealed the amide nature of this glycopeptide with α and β configuration derived from

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GlcN. This glycopeptide was very resistant to simulated gastrointestinal digestion. Also, it

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showed a superior transepithelial transport rate (permeability coefficient (Papp) of (2.82 ±0.15)

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×10-6 cm/s) across the Caco-2 cell monolayer compared to either the parental dipeptide PO or

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GlcN (Papp of (1.45 ± 0.17) ×10-6 cm/s and (1.87 ± 0.15) ×10-6, respectively). A transport

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mechanism experiment indicated that the improved transport efficiency of POGlcN is attributed

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to the introduction of glucose transporters GLUTs.

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Keywords: Glucosamine, Pro-Hyp, Glycopeptide, Pro-Hyp-CONH-GlcN, GLUT transporters

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Introduction

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When collagen is chemically or enzymatically hydrolyzed numerous collagen-derived

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peptides are released to impart beneficial bioactivities, such as anti-aging effects on skin

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conditions and growth-promoting effects on joints and bones. Some clinical trials have revealed

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the efficacy of collagen hydrolysates (CHs) to improve skin hydration, elasticity, and thereby

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reduce wrinkles.1 Others have proposed an important role of CHs to improve bone mineral

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density and bone collagen metabolism.2,3 In addition, CHs may stimulate the synthesis of Type II

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collagen and inhibit the loss of cartilage mass thus maintaining the structure and function of joint

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cartilage.4 Some bioactive collagen peptides have also been identified. Shigemura et al.5,6

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reported that Pro-Hyp (PO) and Hyp-Gly were able to stimulate the growth of fibroblasts.

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Recently, PO and Hyp-Gly were demonstrated to alleviate a skin barrier dysfunction as well as

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modulate gene expression in skin.7 Moreover, the dipeptide PO was able to promote the activity

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of osteoblastic cells and the differentiation of chondrocytes.8,9

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Unlike native collagen which is resistant to in vivo digestion, CHs are relatively easily

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digested and absorbed upon oral administration. Iwai et al.10 identified food-derived collagen

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peptides in human blood such as PO, Ala-Hyp, Pro-Hyp-Gly, and others, among which the

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dipeptide PO was the most prevalent peptide accounting for up to 95% of CHs in the blood after

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the ingestion of porcine type I collagen peptides. These findings were confirmed by Ohara et

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al.11 who also reported that CHs were absorbed mainly in Hyp-peptide form rather than free Hyp

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form due to the great enzymatic stability of Hyp-containing peptides. Although the dipeptide PO

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is more stable and bioavailable than other collagen peptides, its bioavailability is still limited due

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to its low transepithelial permeability.12 Therefore, in the present study, a representative

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collagen-derived bioactive dipeptide PO was selected to study the possibility of improving its

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transepithelial permeability.

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Two major challenges in developing an effective peptide-delivery system are

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gastrointestinal instability and low intestinal permeability. The intestinal transport of peptide can

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be improved para- and transcellularly, by co-administrating with permeation enhancers and by

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using nano-carriers, respectively. However, the former strategy may introduce significant

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toxicity resulting from the perturbation of intestinal barrier, while the latter strategy may require

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large dose usage and be lack of control on peptide release.13, 14 Another promising approach is to

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conjugate with a specific molecule, such as glucose or chimeric vectors like insulin fragments,

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since these would be able to naturally penetrate the cell membrane via specific pathways.15

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Studies here offer encouraging results of enhanced permeability, stability and lower chances of

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toxicity.

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Glucosamine (GlcN) is widely used as a natural dietary supplement to mitigate symptoms

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of osteoarthritis and improve joint function.16 Previous research has demonstrated that the

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chemical conjugation of GlcN by amidation with etodolac can reduce the toxicity of etodolac.17

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Alternatively, esterification with Gly-Val can improve the intestinal permeation of GlcN itself.18

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Therefore, in the present study, the amino sugar GlcN, which is naturally transported by GLUT1,

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2, and 4,19 was used to conjugate the target dipeptide PO. PO is primarily absorbed by the brush-

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border membrane through the di- and tripeptide transporter PepT1.12 We hypothesized that this

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conjugation may introduce multiple mechanisms of absorption to the synthesized glycopeptide,

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including the ones devoted to transport GlcN (i.e. GLUTs) and the one specific for PO (i.e.

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PepT1). Our objective was to create an enzymatically stable PO glycopeptide with improved

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intestinal permeability, and to study its transepithelial transport mechanism using a Caco-2 cell

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model.

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Materials and Methods

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Chemicals

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D-(+)-Glucosamine hydrochloride, dimethylformamide (DMF), triethylamine (TEA), N-

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methylmorpholine (NMM), Fmoc-Cl, dichloromethane (DCM), piperidine, amantadine

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hydrochloride (ADAM), acetonitrile (ACN), porcine pepsin (3,200-4,500 units/mg protein),

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porcine pancreatin (8×USP specifications), monobasic potassium phosphate, trichloroacetic acid

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(TCA), and ammonium formate were purchased from Sigma-Aldrich (St. Louis, MO, USA).

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Benzotriazol-l-yl-N-oxy-tris-(dimethylamino) phosphonium hexafluorophosphate (BOP) was

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from Novabiochem® products (EMD Millipore Corporation, Etobicoke, ON, CA). 1-

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Hydroxybenzotriazole (HOBt) was provided by ApexBio Technology (Boston, MA, USA). The

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protected dipeptide Fmoc-Pro-Hyp (>98%) was purchased from Biomatik Corporation

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(Cambridge, ON, CA), dipeptide Pro-Hyp (>99%) from Bachem Americas, Inc. (Torrance,

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USA), and Pro-Hyp-Gly (>98%) from GenScript (Piscataway, NJ, USA).

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Cell culture supplies such as Caco-2 cells (HTB-37) and methylthiazolyldiphenyl-

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tetrazolium bromide (MTT) Cell Proliferation Assay Kit were purchased from American Type

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Culture Collection (ATCC, Manassas, VA, USA). Morpholineethanesulfonic acid (MES),

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Lucifer yellow carbohydrazide lithium salt, cytochalasin D, wortmannin, Gly-Sar, phloretin,

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phloridzin dihydrate, quercetin, and dimethyl sulfoxide (DMSO) were the product from Sigma-

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Aldrich (St. Louis, MO, USA). Other cell supplies were obtained from Gibco (Life Technologies

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Inc., ON, CA), including Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum

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(FBS), Hank’s balanced salt solution (HBSS), penicillin, and N-2-hydroxyethylpiperazine-N-2-

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ethane sulfonic acid (HEPES).

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Synthesis and Purification of Collagen-derived Glycopeptide Pro-Hyp-CONH-GlcN

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(POGlcN)

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The collagen-derived glycopeptide POGlcN was synthesized by a stepwise solution phase

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synthesis according to Kohan et al.18 with some modifications. Firstly, GlcN (1eq) was dissolved

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in the mixture of DMF/TEA (5/1, v/v), and Fmoc-Pro-Hyp-OH (1eq) was dissolved in DMF.

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After Fmoc-Pro-Hyp-OH was pre-activated in DMF by BOP (1eq) in the presence of HOBt (1 eq)

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and NMM (1eq), the GlcN mixture was added to react overnight with continuous shaking (200

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rpm). In this step, the coupling reaction occurred between the activated carboxylate group of the

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dipeptide and the primary amine group of GlcN to form an amide derivative Fmoc-Pro-Hyp-

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CONH-GlcN. The temporary protective N-Fmoc group was then removed by adding piperidine

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to a final concentration of 20% (v/v), performed for 45 min to achieve a complete removal. The

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reaction mixture was then precipitated with 15-fold volume of cold diethyl ether for at least 4

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hours at 4°C. The precipitate was rinsed two more times with diethyl ether and air-dried

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overnight to obtain the crude glycopeptide.

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The crude product was then redissolved in distilled deionized water and purified

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according to its LC-MS profile. The samples were firstly separated by C18 column (Ascentis®

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Express, 2.7 µm, 15 cm × 4.6 mm) using an HPLC unit (Agilent 1100 series) with a binary pump.

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The samples were eluted at flow rate of 0.6 mL/min with 0.1% acetic acid (eluent A) and 0.1%

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acetic acid in ACN (eluent B) by a linear gradient from 0-99% of eluent B. The signal was

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monitored at 214 nm by UV detector. Followed, the samples were subjected to an analysis using

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a triple quadrupole mass spectrometer, 4000 Q Trap LC/MS/MS System (Applied 6


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Biosystems/MDS Sciex) acquiring spectra in a positive ion mode. The parameters applied to the

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MS were: ion source temperature 500°C, capillary voltage 4500 V and the curtain gas 20 psi.

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The corresponding peak with m/z 390 was collected as eluted out from LC. The collected

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solution was pooled and lyophilized to obtain the final POGlcN acetate. This lyophilisation

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process was repeated two more times to maximize the removal of excess acetic acid. The final

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product was stored at -20˚C for later experiments.

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Structure Characterization of the Collagen-derived Glycopeptide POGlcN by Tandem

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Mass Spectrometry (MS/MSn) and Nuclear Magnetic Resonance Spectroscopy (NMR)

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The identification of POGlcN was firstly confirmed by MS/MSn using a LTQ Orbitrap

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XL hybrid ion trap-orbitrap mass spectrometer (Thermo Scientific) calibrated with standard ion

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calibration solutions (Pierce LTQ, Thermo Scientific). Direct infusion of sample was performed

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at a flow rate of 5 uL/min using the on-board syringe pump. The mass spectrometer was operated

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using an electrospray ionization source in a positive mode with a source voltage of 4.0 kV and

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sheath gas flow rate of 10.0. The analyses were conducted at a resolving power of 60.000 full-

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width/half-maximum peak height and spectra were acquired over the range of m/z 100 - 500. The

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peaks at 390.19 and 391.19 m/z were isolated and fragmented at a normalized collision energy

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(NCE) of 27. Then the product ion with m/z at 372.18 was further isolated and a MS/MS/MS

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was performed at an NCE of 25. The data was acquired and reported by Tune Plus 2.7 software

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(Thermo Scientific).

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To verify the structure, purity and the composition of the synthesized glycopeptide, the

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final product was dissolved in deuterium oxide and subjected to 1H and 13C NMR spectroscopy.

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The data were acquired on a four channel 700 MHz Agilent VNMRS spectrometer using a HCN

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z-gradient pulsed field gradient cold probe. The data were processed with VNMRJ 4.2A software

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(Agilent Technologies).

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Simulated Gastrointestinal (GI) Digestion of the Collagen-derived Glycopeptide POGlcN

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To study the degradation in GI tract prior to the intestinal absorption, the glycopeptide

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POGlcN was subjected to a simulated GI digestion model. The simulated gastric and intestinal

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fluids were prepared as described in the United States Pharmacopeial Convention (Test Solutions,

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United States Pharmacopeia 35, NF 30, 2012). Both simulated fluids were freshly prepared

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immediately before use, with pH of 1.2 and 6.8 for gastric fluid (0.32% w/v pepsin) and

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intestinal fluid (0.68% w/v pancreatin), respectively.

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The digestion of POGlcN was investigated according to the method from Sontakke et

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al.20 with some modifications. Briefly, 40 µM of POGlcN was incubated in the simulated gastric

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or intestinal fluid at 37°C for 2 h in a shaking incubator (200 rpm). The digestion was terminated

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and precipitated at 0, 15, 30, 60, 120 min with TCA containing Pro-Hyp-Gly as internal standard

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to make the final TCA concentration of 20% (w/v). Meanwhile, matrix blank without POGlcN

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was prepared. To facilitate the precipitation, the sample was then placed in an ice bath for 10 min

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before centrifugation at 10,000 rpm for another 10 min at 4°C. Aliquot of the supernatant was

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collected, neutralized, and immediately analyzed to determine the remaining amount of intact

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POGlcN using UHPLC according to the method described below. Simultaneously, the chemical

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stability of the glycopeptide POGlcN was assessed by incubating with the enzyme-free

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gastric/intestinal fluid and determined the same way as described above for the experimental

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fluids. The results were calculated as follows:

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Remaining percentage % =

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Caco-2 Cell Culture

! " # $$ % ! "

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× 100%

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The Caco-2 cells were grown in DMEM supplemented with 10% FBS, 25 mM HEPES,

184

and 1% penicillin in a 37°C incubator which maintains a 90% humidified atmosphere and 5%

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carbon dioxide. To ensure the cell monolayer integrity and differentiation, cells at a density of 2

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× 105/cm2 were seeded on polycarbonate membrane transwell inserts (12 well, 12 mm diameter,

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0.4 µm pore size, Corning, NY, USA); and then gently shook to obtain uniform cell distribution.

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During the incubation, cell growth media were carefully replaced every two days without

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damaging the monolayer. After 21-23 days’ growth and differentiation, the integrity of the cell

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monolayers was assessed by measuring the transepithelial electrical resistance (TEER) and the

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permeability of Lucifer yellow. Only monolayers with TEER higher than 450 Ω•cm2 were used

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for transport experiments. The cytotoxicity assay was performed using MTT assay as described

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in the instructions from ATCC (ATCC, MTT cell proliferation assay). The cells used were

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between passages 18 and 24.

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Permeability Assay of Glycopeptide POGlcN, PO, and GlcN in the Caco-2 Cell Model

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To verify the hypothesis that conjugation of the collagen-derived dipeptide PO with GlcN

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promotes its transport efficiency, the permeability of the glycopeptide POGlcN, dipeptide PO,

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and GlcN were compared in the human intestinal epithelial Caco-2 cell model. According to

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Feng & Betti

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maintain the performance of the cell monolayer. To mimic the in vivo intestinal acid

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microclimate22, the transport experiments were conducted with a pH gradient, i.e. HBSS/MES

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buffer (pH 6.5, 10 mM MES in HBSS) in the apical chamber, and HBSS/HEPES buffer (pH 7.4,

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10 mM HEPES in HBSS) in the basolateral chamber. On the day of transport, the cell

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monolayers were firstly rinsed twice with pre-warmed HBSS to wash out the remaining DMEM,

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and then equilibrated with 0.5 mL HBSS/MES (apical) and 1.5 mL HBSS/HEPES (basolateral)

21

, the growth media were changed 12 to 24 h before the transport experiment to

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for 30 min. For the apical to basolateral transport (AB transport) experiment, 0.5 mL of the

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POGlcN, PO, and GlcN at a non-toxic concentration of 1 mM in HBSS/MES were applied to the

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apical side, and 1.5 mL HBSS/HEPES without test compounds were filled in the basolateral side.

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To measure the transport efficiency of each compound, 300 µL of each sample was withdrawn

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from the basolateral chamber at pre-determined time points of 0, 15, 30, 45, and 60 min.

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Meanwhile an equal volume (300 µL) of test compound-free and pre-warmed HBSS/HEPES

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media were supplemented. In addition, the bi-direction transport of POGlcN was carried out to

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better understand its transport efficiency. For the basolateral to apical transport (BA transport),

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similarly, the 1.5 mL of 1 mM POGlcN sample in HBSS/HEPES was loaded at basolateral side,

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while 0.5 mL compound-free HBSS/MES at apical side. Fifty microliters were withdrawn from

216

the apical side and replaced with equal volume of HBSS/MES at the same time points mentioned

217

above. Collected samples were subjected to analysis using UHPLC as described below.

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Moreover, Lucifer yellow (1 mM) was used as paracellular marker during the experiment. The

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concentration of Lucifer yellow was determined using UHPLC with a fluorescent detector

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(excitation 428 nm, emission 536 nm). The transport efficiency of each compound was evaluated

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by apparent permeability coefficient (Papp) which was calculated with the following equation:

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)$$ cm/s =

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amount change of the intact test compound over time (pmol/s) at the receiver side, A is the

224

surface area of the transwell insert (cm2), C0 is the initial concentration of the test compound at

225

the donor side (pmol/cm3).

226

Transport Experiment of POGlcN, PO and GlcN in the Presence of Various Transport

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Inhibitors/Interrupters

,,.

× 1⁄ / × 01 , where t is the incubation time (s), dQ/dt is the cumulative

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To investigate the transport mechanism of POGlcN across the Caco-2 cell monolayer, the

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effects of different transport inhibitors/interrupters on POGlcN were studied in comparison to

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that on GlcN and dipeptide PO. The experiment was performed according to Miguel et al.23 and

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Alzaid24 et al. with some modifications. Similar to the permeability assay, the cell monolayers

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were firstly gently rinsed with HBSS, and pre-incubated for 30 min with 0.5 ug/mL cytochalasin

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D (a tight junction interrupter), 500 nM wortmannin (a transcytosis inhibitor), 10 mM Gly-Sar (a

234

competitive PepT1 substrate), 0.1 mM quercetin (a GLUT1 and 2 inhibitor), 0.1 mM phloretin (a

235

GLUTs inhibitor), and 0.1 mM phloridzin (a SGLTs inhibitor), respectively. One millimolar of

236

glycopeptide POGlcN, dipeptide PO, and GlcN were then applied apically on the monolayer in

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the presence of inhibitors/interrupters for 60 min, respectively. Gly-Sar was dissolved in

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HBSS/MES, while all other inhibitors were dissolved in DMSO and diluted with HBSS/MES

239

before adding on the monolayers (the final DMSO concentration was 0.044%). The control

240

experiments were conducted by pre-incubating with 0.044% DMSO and HBSS/MES. Samples

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were then collected from the basolateral compartment and detected by UHPLC analysis as

242

described below.

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Analysis by UHPLC with Fluorescence Detection

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Fmoc-Cl has been used as an established derivatization reagent for the determination of

245

amino acids, and was also extended to be used for the analysis of GlcN due to the amino group

246

presence. In the present study, the determination of POGlcN, PO, and GlcN were performed with

247

Fmoc-Cl pre-column derivatization using a UHPLC unit (Shimadzu, Columbia, MD, USA)

248

according to Ibrahim and Jamali 25 with some modifications. In brief, 100 µL of the sample was

249

firstly mixed with 40 µL of 0.2 M borate buffer (pH 8.0) before being derivatized with 60 µL of

250

0.16 mM Fmoc-Cl (dissolved in ACN) for 30 min at 30°C. Fifty microliters of 6 mM ADAM 11



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(dissolved in 80% ACN (v/v)) was then added to consume the excess Fmoc-Cl and terminate the

252

derivatization. Thirty microliters of the final derivatized sample was then injected into the

253

UHPLC and separated by a Ascentis® Express HILIC column (10 cm × 4.6 mm I.D., 2.7 µm

254

particles). The sample was eluted at 0.5 mL/min by mobile phase A: 13 mM ammonium formate

255

in water and mobile phase B: 13 mM ammonium formate in 90% ACN with a 40 min gradient:

256

0-8 min with 100% B, 8-23 min from 100% to 55% B, then gradually increase to 100% B within

257

2 min, and finally maintain 100% B for 15 min. The column oven temperature was controlled at

258

35°C during the elution and the sample was finally detected by a fluorescent detector (excitation

259

at 263 nm, emission at 315 nm). Standard curves for GlcN and POGlcN were linear quantifiable

260

over the concentration range of 0.1 to 4 µg/mL (R2≥ 0.99), and for PO 0.05 to 4 µg/mL (R2≥

261

0.99). All the analyses were repeated at least two times.

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Statistical Analysis

263

All the data was analyzed using one-way analysis of variance by SAS 9.4 (Cary, NC,

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USA). Multiple comparisons of means were done by Tukey’s test where p ≤ 0.05 was considered

265

significantly different.

266

Results and Discussion

267

Synthesis and Structure Characterization

268

It is conventional to synthesize small peptides in the solution phase. Typically, reagents

269

like carbodiimides and phosphonium salts are used to accelerate the coupling between the amino

270

and carboxyl groups of two amino acids, and protection groups such as Boc and Fmoc are used

271

for the alpha-amino protection to avoid side reactions.26 We gained insight from this solution-

272

phase synthesis method and utilized this strategy in the present study. Here, an amide derivative

273

was rapidly synthesized by conjugating the carboxyl group of Fmoc-protected PO with the C212


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amino group of GlcN under the activation of phosphonium salts BOP (schematic synthesis is

275

summarized in Figure 1). After purification and lyophilisation, the final product in acetate form

276

(white powder) yielded 37.0 ± 1.5% with no detectable contamination signal using HPLC UV

277

detector at 214 nm (Figure S1), and the identity of the glycopeptide was confirmed by MS/MSn

278

(Figure 2). In Figure 2A, the protonated glycopeptide (M+H)+ showed a major peak at m/z of

279

390.19 and a small shoulder peak on its right side with m/z of 391.19 while the latter from the

280

13

281

peaks. The MS/MS fragmentation pattern of these glycopeptide peaks are shown in Figure 2B. A

282

fragment of Hyp-GlcN with a monoisotopic molecular mass of 292.13 Da was found as a

283

pseudomolecular ion (M+H)+ at m/z 293.13 and 294.13 from the precursor ions at m/z 390.19 and

284

391.19, respectively. The major fragment peak at m/z 372.18 was the dehydrated form of ion

285

390.19. The peaks at m/z 229.12 and 211.11 corresponded to Pro-Hyp fragment and its

286

dehydrated form, respectively. GlcN fragment and its dehydrated form were found at m/z 180.09

287

and 162.08, respectively. The major peak at m/z 372.18 was further fragmented to obtain a

288

pattern shown in Figure 2C. Interestingly, this pattern almost mirrors the one for intact

289

glycopeptide presented in Figure 2B. No peak at m/z 293.13 was detected, however the ion at m/z

290

275.12 was probably its dehydrated form. These two similar fragment patterns confirmed the

291

Pro-Hyp-GlcN composition of the glycopeptide.

292

C isotope form. Except for the major glycopeptide peaks, there were minimal contamination

To further confirm the chemical bond between Hyp and GlcN, and purity of the

293

glycopeptide, the 1H and

13

294

the glycopeptide was in the acetate form, a peak occurred with the chemical shift of 1.88 ppm,

295

which indicates the presence of acetic acid was shown in Figure S2 (1H NMR). Only minor

296

peaks from impurities present in the spectra indicated that the final glycopeptide had a purity

C NMR spectroscopy was used to generate spectra (Figure S2). As

13



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around 96%. GlcN usually exists in equilibrium with two anomers when dissolved in water, the α

298

anomer and β anomer, at a ratio of 63:37.27 In this study, the two peaks with chemical shifts of

299

5.20 and 4.77 ppm represent the H1 of α and β anomers of GlcN residue, respectively (Table 1);

300

they gave a ratio of 64:36 upon the integration of their signal intensities. This shows that the

301

GlcN anomers ratio was maintained in the glycopeptide form. Along with the 1H spectra, the 13C

302

NMR spectra confirmed the amide bonds between Hyp and GlcN, and between Pro and Hyp,

303

representing the carbonyl chemical shifts at 176.14 and 171.10 ppm, respectively. Other proton

304

and carbon arrangements were summarized in Table 1 which are consistent with 1H TOCSY, 1H

305

COSY, 1H-13C HSQC and 1H-13C HMBC 2-D NMR spectra (data not shown). The label and

306

position of each hydrogen and carbon is indicated in the POGlcN structure in Figure 1. Taken all

307

together, the glycopeptide was verified as an amide derivative Pro-Hyp-CONH-GlcN (POGlcN)

308

which maintained the structure of both components. The synthesis and purification processes

309

were effective to produce this very pure glycopeptide POGlcN with a molecular weight of

310

389.19 Da.

311

Stability of the POGlcN

312

Similar to a tripeptide, the amide bond between Hyp and GlcN can be digested either by

313

the GI proteases or by the harsh acidic environment of the stomach. Therefore, the stability of

314

this newly synthesized glycopeptide POGlcN was assessed under simulated gastric and intestinal

315

fluid conditions. As Figure 3 shows, around 5% of POGlcN was digested in simulated gastric

316

fluid while around 10% of POGlcN was digested in simulated intestine fluid. The newly

317

synthetized glycopeptide is relatively stable in the simulated GI digestion, unlike the findings of

318

Pandey et al.17 where etodolac-CONH-GlcN, a nonsteroidal anti-inflammatory prodrug against

319

arthritis, was easily digested in simulated intestinal fluid. The good stability of this amide 14


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POGlcN implies the potential protective effect of the dipeptide PO as compared to the etodolic

321

acid in etodolac-CONH-glucosamine. In the testing of control systems, the degradation of the

322

POGlcN was negligible in pancreatin-free intestinal juice and pepsin-free acidic gastric juice,

323

indicating its good chemical stability. In summary, the results suggest POGlcN exerted

324

satisfactory GI enzymatic and chemical stability for at least 2 h. This is consistent with the

325

findings of Sontakke et al.20 about the good stability of tripeptide Gly-Pro-Hyp which shares a

326

similar structure with the glycopeptide POGlcN.

327

Permeability of the Glycopeptide POGlcN, PO, and GlcN Across the Caco-2 Cell

328

Monolayer

329

Caco-2 cells are grown on a semi-permeable membrane which allows the nutrients, ions,

330

and water to diffuse to both sides, and the cells are able to well differentiate into polarized

331

epithelial monolayer to exhibit the typical morphological and functional characteristics of human

332

intestine mucosa.28 This well characterized system has been a widely used in vitro model for

333

studying the transport and metabolism of drugs and chemicals in the last three decades. As a

334

small hydrophilic compound, Lucifer yellow normally crosses the Caco-2 cell monolayer

335

through the tight junctions among the cells.29 In the present study, Lucifer yellow was used as a

336

reference compound to validate the suitability of the Caco-2 cell monolayer. We found that

337

Lucifer yellow had a Papp < 3.05×10-7 cm/s with a permeability lower than 0.24% after 60 min,

338

when TEER of the monolayers were above 450 Ω•cm2. This performance of Lucifer yellow is

339

comparable with previously reported screening standards, for the Lucifer yellow transport < 0.25%

340

and Papp < 3×10-6 cm/s.28,30 The Caco-2 cell model in this study was therefore reliable to predict

341

the transepithelial transport of test compounds. Moreover, a concentration of one millimolar of

342

POGlcN was justified as non-saturable condition in our model (Figure S3). 15



Page 16 of 35

343

As shown in Figure S4, the good chromatographic separation of each compound indicates

344

the effectiveness this method to determine POGlcN, PO, and GlcN concentrations. The

345

permeability of POGlcN, PO, and GlcN were compared in Figure 4A, which shows that the

346

transported amount of all three compounds from apical to basolateral showed good linearity (R2

347

≥ 0.96) over time in the range from 15 to 60 min. The incubation time axis intercept of each

348

linear line indicates the lag time for each compound, i.e. 9.5 min for POGlcN, 16.3 min for GlcN

349

and 12.9 min for PO. The lag time is defined as the time needed for one compound to reach

350

steady-state flux from which the Papp is calculated. It takes some time for the test compounds to

351

partition into the absorption surface and well equilibrate, so these lag times are expected and

352

typical.31 During the permeability assay, POGlcN exerted significantly greater Papp ((2.82 ±0.15)

353

×10-6 cm/s) than that of PO ((1.45 ± 0.17) ×10-6 cm/s) and GlcN ((1.87 ± 0.15) ×10-6 cm/s),

354

respectively. These values are similar to those reported for bioactive oligopeptides such as

355

QIGLF and RVPSL in other studies.32, 33 The Papp of PO is much greater than that of PO (0.13 ×

356

10-6 cm/s) as reported by Sontakke et al.,20 while the Papp of POGlcN is comparable to that of

357

Gly-Pro-Hyp (1.09 × 10-6 cm/s) in the same study. The difference is likely due to the different

358

transport mechanism involved, variable culturing conditions and protocols, and different cell

359

origins. The greater permeability of POGlcN implies the positive effect of conjugation on

360

transepithelial transport, and this additive effect was derived from the sugar moiety, GlcN.

361

Interestingly, when only POGlcN was applied apically, except for POGlcN, an increasing

362

amount of dipeptide PO was also found in the basolateral side as the incubation time increased

363

(Figure 4B). After 60 min, the accumulated PO was around 0.28% of the originally applied

364

POGlcN and 12.5% of total transported peptides. The minor presence of PO in the receptor side

365

can be explained by the action of peptidases from the brush border membrane and endocellular 16


Page 17 of 35


366

enzymes. However, no free Pro was detected, which further confirms that PO and POGlcN are

367

resistant or at least not significantly hydrolyzed when subjected to transepithelial transport.

368

To have a more comprehensive understanding of the permeability of this novel

369

glycopeptide, the POGlcN transport was done bi-directionally, i.e. apical to basolateral (AB

370

transport) and vice versa (BA transport). As shown in Figure 4A, the AB transport of POGlcN

371

was much faster than BA transport, with an uptake ratio (Papp,AB/Papp,BA) of 3.24. This

372

asymmetrical permeation is a first indicator of active transport routes’ involvement. Normally no

373

clear difference of permeability between two directions appears if a compound is only passively

374

transported.22 The di- and tripeptide transporter PepT1 on the epithelial cells may actively shuttle

375

the uptake of POGlcN because of its tripeptide-like structure. The lower uptake ratio of POGlcN

376

than that of Gly-Sar (uptake ratio at 5),22 which is the substrate of PepT 1, suggests that the

377

POGlcN may exert lower affinity to PepT1 transporter. However, the POGlcN uptake may also

378

be the combined effect of passive and active pathways.

379

The Effects of Different Inhibitors/interrupters on the Transepithelial Transport of

380

POGlcN

381

In general, di- and tripeptides uptake in intestinal epithelial cells largely depend on the

382

proton-coupled transporter PepT1. In addition, peptides may also be absorbed through the

383

passive diffusion across the lipid bilayer, endocytosis, and through paracellular channels of the

384

tight junction.29 The four hydroxyl groups and an imino group of POGlcN impart a significant

385

hydrophilic nature, and thus is unlikely to passively merge into the cell membrane and be

386

transported transcellularly.34 Instead, it is possible that POGlcN uses paracellular channels to

387

access the serosal side. POGlcN could also be mediated via the previously mentioned carrier

388

PepT1 due to its PO component which can be a substrate for PepT112. On the other hand, as the 17



Page 18 of 35

389

NMR results indicated, POGlcN retained the structure of the sugar moiety GlcN; its exposed part

390

shares a similar structure with glucose. In this respect, the chemical synthesis used in this study

391

introduced the potential for this glycopeptide to be transported through a shuttle mechanism used

392

by hexose, such as facilitated diffusion by GLUTs and active sodium-dependent transport by

393

SGLTs35. Hence, inhibitors or interrupters for each potential transport pathway were used

394

strategically to better understand the transport mechanism of POGlcN through the Caco-2 cell

395

monolayers.

396

As shown in Figure 5A, after a 60 min incubation, there was no significant effect of

397

cytochalasin D and wortmannin on the accumulated amount of POGlcN and PO in the

398

basalateral chamber compared to POGlcN and PO controls. Cytochalasin D is normally used to

399

induce the structure change of the cytoskeleton and occluding junctions of epithelial cells, thus

400

facilitating the movement of compounds across the tight junctions.36 Cytochalasin D was able to

401

slightly enhance the permeability of PO; however, this was not statistically significant, so this

402

paracellular pathway is not mainly involved. Despite some studies that emphasize the importance

403

of paracellular route for the uptake of high molecular weight peptides (e.g. HLPLP) and peptide

404

drugs (e.g. octreotide)37, 38, the cut off is usually

Both PepT1 and GLUT Intestinal Transporters Are Utilized by

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