Egg white-derived tripeptide IRW (Ile-Arg-Trp) is an activator of

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Bioactive Constituents, Metabolites, and Functions

Egg white-derived tripeptide IRW (Ile-Arg-Trp) is an activator of angiotensin converting enzyme 2 Wang Liao, Khushwant S. Bhullar, Subhadeep Chakrabarti, Sandra T. Davidge, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03501 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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Journal of Agricultural and Food Chemistry

Egg white-derived tripeptide IRW (Ile-Arg-Trp) is an activator of angiotensin converting enzyme 2

Wang Liao1,4, Khushwant S. Bhullar1,2, Subhadeep Chakrabarti1,3,4, Sandra T. Davidge3,4,5,6 and Jianping Wu1,4* 1. Department of Agricultural, Food & Nutritional Science, 2. Department of Pharmacology, 3. Women and Children’s Health Research Institute, 4. Cardiovascular Research Centre, 5. Department of Obstetrics & Gynecology, 6. Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.

Correspondence: Dr. Jianping Wu Tel: 1-780- 492-6885; Fax: 1-780- 492-4265; E-mail: [email protected]

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Abstract

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Angiotensin converting enzyme 2 (ACE2) plays beneficial roles in the renin angiotensin

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aldosterone system. Our previous studies indicated that egg white-derived antihypertensive

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peptide IRW could upregulate ACE2 mRNA level in mesenteric artery of spontaneously

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hypertensive rats (SHRs), suggesting the potential of IRW as an in vivo ACE2 activator. In this

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study, the effects of IRW on activity and protein expression of ACE2 were investigated. Results

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indicated that IRW activated human recombinant ACE2 with an EC50 value of 7.24×10-5 M. In

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rat aortic vascular smooth muscle cell line A7r5 cells, IRW treatment (50 μM) significantly

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increased the expression and activity of ACE2. Oral administration of IRW to SHRs upregulated

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ACE2 protein levels in kidney and aorta. Molecular docking study suggested that IRW might

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activate ACE2 through interaction with the subdomain I near the active site through hydrogen

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bonds. Overall, this study established IRW as the first food derived ACE2 activating peptide.

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Keywords: antihypertensive peptides, IRW, ACE2, ACE2 activating peptide, A7r5 cells,

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spontaneously hypertensive rats

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Introduction

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Hypertension is a key risk factor for cardiovascular, neural and renal diseases, and is present in

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approximately about a-quarter of the adult population.1 In 2017, hypertension has been redefined

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to be above 130/80 mmHg rather than 140/90 mmHg for systolic/diastolic blood pressure.2

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Hyperactive renin angiotensin system (RAS), a classic endocrine system, is a common attribute

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of hypertension and related pathologies.3 While angiotensin converting enzyme (ACE) is

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responsible for the generation of angiotensin II (Ang II), one of the most potent vasoconstrictors

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in the body, another RAS enzyme, ACE2 (angiotensin converting enzyme 2) is involved in its

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conversion into angiotensin (1-7) [Ang (1-7)]. Ang (1-7) is a smaller peptide with pronounced

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anti-inflammatory and anti-hypertensive properties.4 As such, ACE2 is considered a negative

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regulator of RAS pathology in cardiovascular diseases.5 Therefore, increased expression and

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activity of ACE2 has been correlated with improvements in cardiovascular health while

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downregulation of ACE2 is associated with adverse outcomes in multiple experimental models.6-

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Reducing Ang II formation through ACE inhibition or blockage of angiotensin type I receptor

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(AT1), have been the major therapeutic approaches to control RAS overactivity and

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consequently hypertension; indeed, a range of ACE inhibitors and AT1 receptor antagonists have

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been developed and widely prescribed in clinical settings over the past two decades.11,12

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However, these anti-hypertensive agents are generally associated with significant side-effects

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over prolonged use, while many cases of essential hypertension only show modest benefits upon

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their use.13,14 Therapeutic options, particularly using food-derived products or nutraceuticals,

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targeting ACE2 are rare. Moreover, rationally designed ACE2 activators are still emerging on a

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languid pace. Some established anti-hypertensive drugs such as losartan and telmisartan have 3 ACS Paragon Plus Environment


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shown concomitant upregulation of ACE2 expression,15-17 while synthetic compounds like

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diminazine

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dihydrochloride)

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methylphenyl) sulfonyloxy]-9H-xanthene-9-one) have been claimed as specific ACE2

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activators.18,19 Indeed, diminazine administration appears to reduce the severity of cardiovascular

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diseases in animal models suggesting the therapeutic potential of ACE2 activator.20-22 However,

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these promising approaches have been insufficiently investigated for their safe therapeutic

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applications.

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Food proteins are a rich source of bioactive peptides that may beneficially affect our health

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through modulation of various physiological systems including the RAS pathway.23 Indeed,

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previous work from our lab has identified several egg-derived ACE inhibitory peptides.24 One of

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these peptides, IRW (Ile-Arg-Trp), exhibits additional anti-inflammatory and anti-oxidant effects

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in both cells and SHRs.25,26 Its antihypertensive activity is associated with reduced Ang II and

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inflammation, as well as enhanced NO-mediated vasorelaxation.25 It was found by a

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transcriptomics study that IRW could significantly up-regulate the mRNA level of ACE2 in

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mesenteric artery, thus supporting its candidacy as an ACE 2 activator.27 However, effect(s) of

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IRW on the ACE2 protein expression and enzyme activity remain elusive. Therefore, to bridge

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this research gap, this study was conducted to understand if IRW could affect the protein

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expression and activity of ACE2. Experimentally, the potential of IRW as an ACE2 activator

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was first studied using purified human recombinant ACE2 (in situ cell free system) and

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molecular docking study, followed by rat aortic vascular smooth muscle cells (VSMCs), and in

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tissues of SHRs.

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Material and Methods

(4-[2-(4-carbamimidoylphenyl) and

XNT

iminohydrazinyl]benzenecarboximidamide

(1-[(2-dimethylamino)

ethylamino]-4-(hydroxymethyl)-7-[(4-


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Reagents

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Dulbecco’s phosphate buffered saline (PBS) and dithiothreitol (DTT) were purchased from

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Sigma Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine

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serum (FBS), and antibiotics (Penicillin-Streptomycin and Gentamicin) were purchased from

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Thermo Fisher Scientific (Waltham, MA, USA). Triton-X-100 was from VWR International

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(West Chester, PA, USA). IRW was synthesized by Genscript (Piscataway, NJ, USA) with a

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minimum purity of 99%.

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In vitro (cell-free) ACE2 activity assay

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In vitro ACE2 activity under cell-free system was calculated as described by Hernández Prada et

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al.18 Human recombinant ACE2 (hrACE2; R&D systems, Minneapolis, MN, USA) was

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incubated with different concentrations of IRW for 5 min before 50 µM of the ACE2 fluorogenic

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substrate Mca-Dnp-OH (R&D systems) was added. The reaction was performed in a total

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volume of 100 µL containing 10 nM hrACE2, 1 M NaCl, 75 mM Tris-HCl and 0.5 µM ZnCl2, at

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the physiological pH (7.4). All assays were performed in triplicates and read at the interval of 30

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sec for 30 min at 37℃ using SpectraMax M3 spectrophotometer (Molecular devices, Sunnyvale,

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CA, USA). Background of each measurement was subtracted from the observed readings.

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Relative fluorescence unit (RFU) of each time point was plotted to generate the kinetic curves.

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Maximum response under each IRW concentration was expressed as maximum RFU in

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percentage, with respect to the corresponding control and plotted to fit into sigmoidal

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concentration response curve, followed by calculation of EC50 value (the concentration eliciting

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50% of maximal response) using PRISM 5 statistical software (Graph Pad Software, San Diego,

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CA, USA).



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Cell culture

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Cell culture was performed according to our previous studies.28-31 A VSMC cell-line from rat

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aorta, A7r5, was purchased from ATCC (cat# CRL-1444, Manassas, VA, USA). The cells

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between passage 3 and 10 were used for the experiments. Cells were grown in DMEM

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supplemented with 10% FBS and 1% antibiotics until they reached ~70% confluence. The

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confluent cells were then placed in a quiescing DMEM medium supplemented with 1% FBS and

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1% antibiotics. The cell model system was treated with different concentrations of IRW (10 µM

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or 50 µM) for 24 hours.

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Cellular ACE2 activity assay

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ACE2 activity of A7r5 cells was measured by SensoLyte®390 ACE2 activity assay kit (Anaspec,

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Fremont, CA, USA) according to the manufacturer’s instructions with slight modifications.

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Briefly, the cultured cells were detached and lysed using assay buffer containing 0.1% Triton-

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X100. Supernatant from the cell lysates was collected for analysis. ACE2 activity was measured

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in a reaction system with 50 µL of sample and 50 µL of ACE2 substrate solution. All assays

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were read every 5 min for 30 min at 37°C using SpectraMax M3 spectrophotometer (Molecular

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devices, San Jose, CA, USA). Auto-fluorescence of each assay was subtracted from the

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measured values to generate final results. RFU of each sample was normalized to the

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corresponding total protein concentration, which was measured by PierceTM BCA protein assay

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kit (Thermo Fisher Scientific).

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Quantitative reverse transcription PCR (qRT-PCR)

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TRIzol Reagent (Ambion, Carlsbad, CA, USA) was used for total RNA extraction from A7r5

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cells in vitro. RNA from cell lysates was separated and precipitated by chloroform and 6 ACS Paragon Plus Environment

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isopropanol, respectively. Finally, RNA pellets were washed with 80% ethanol and solubilized in

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50 µL of nuclease-free water. Quantity and quality of RNA were evaluated by Qubit® 3.0

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Fluorometer (Invitrogen) and Agilent 2200 TapeStation (Agilent technologies, Santa Clara, CA),

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respectively. Only RNA samples with integrity number higher than 7.0 were eligible for the

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subsequent applications. SuperScript® II Reverse transcriptase (Invitrogen) was used for reverse

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transcription to synthesize complementary DNA. qPCR was performed by StepOnePlusTM Real-

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Time PCR System (Applied Biosystems, Foster City, CA, USA) using TaqMan® gene

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expression assay (Applied Biosystems). Primers and probes (Table 1) were designed by Primer

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Express 3.0 (Applied Biosystems). Thermal conditions were as follow: 95°C for 20 sec; 40

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cycles at 95°C for 1 sec, followed by annealing/extension for 20 sec at 60°C. Fold change of

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ACE2 expression was calculated by using Glyceraldehyde-3-phosphate dehydrogenase

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(GAPDH) as the reference gene based on cycle of threshold (CT) as follows: ΔCT= [CT (ACE2)

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- CT (GAPDH)], Δ Δ CT= [Δ CT (IRW) - Δ CT (Untreated)], fold change= 2- Δ Δ CT.

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Western blotting

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Western blotting was performed the same as our previous study.28 Cell lysates were prepared by

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lysing the cells in boiling hot Laemmle’s buffer containing 50 μM DTT and 0.2% Triton-X-100.

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These cell lysates were then run using 9% SDS-PAGE gel electrophoresis, transferred to

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nitrocellulose membranes and immunoblotted with antibodies against ACE2 (Abcam,

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Cambridge, MA, USA) and α-tubulin (Abcam). Goat anti-rabbit IRDye 680RD or Donkey anti-

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rabbit IRDye 800CW (Licor Biosciences, Lincoln, NE, USA) were used as the secondary

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antibodies. The protein bands were detected by Licor Odyssey BioImager (Licor Biosciences)

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and quantified through densitometry using the corresponding Odyssey v3.0 software (Licor

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Biosciences). Each ACE2 band was normalized to α-tubulin. Bands from untreated cells were 7 ACS Paragon Plus Environment


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used for comparative analysis. Data were expressed as the percentage change with respect to

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untreated control.

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Animal model & ethics statement

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Animal experiment was performed in SHR as described previously.25 In brief, male SHRs (16-17

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weeks old) obtained from Charles River (Senneville, QC, Canada) were randomly assigned into

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two groups, untreated (vehicle, n=5) and IRW (15 mg/kg body weight, n=5); after 18 days of

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treatment, the rats were sacrificed, and the tissue/organs were collected for further analysis. The

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experimental procedures were approved by the University of Alberta Animal Welfare Committee

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(Protocol # 611/09/10/D) in accordance with the guidelines issued by the Canada Council on

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Animal Care and also adhered to the Guide for the Care and Use of Laboratory Animals

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published by the United States National Institutes of Health.

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Immunostaining

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Kidney and aorta sections were prepared under -20°C by Leica CM1900 Cryostat (Leica

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Biosystems, Heidelberger, Nussloch, Germany) and mounted on glass slides for analysis. During

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the experiment, tissue sections were fixed by cold acetone and blocked by 1% bovine serum

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albumin (BSA) in PBS for 1 hour. The sections were immunostained with anti-ACE2 antibody

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(rabbit polyclonal antibody from Abcam, cat# ab87436) at a final concentration of 3 μg/mL in

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PBS overnight at 4°C. Upon washing, the sections were incubated with goat anti-rabbit IRDye

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680RD (Licor Biosciences) secondary antibody at room temperature for 30 min. Later, the

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sections were washed and mounted on a coverslip with PBS. The slides were then scanned by

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Odyssey Sa Imaging System (Licor Biosciences) at a resolution of 20 µm. The intensity

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(maximum and minimum) of all the images were adjusted to a similar level by Image Studio lite

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Ver 5.0 software (Licor Biosciences). The data obtained were expressed as mean fluorescence 8 ACS Paragon Plus Environment

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intensity (MFI) as measured by Image J software (http://rsb.info.nih.gov/ij/). For each image,

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MFI of the corresponding secondary antibody control was subtracted to acquire normalized

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results.

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Molecular docking

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The structure of the human ACE2 (PDB: Q9BYF1) was retrieved from the PDB.32 The protein

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was loaded and analyzed using University of California, San Francisco CHIMERA version 1.9

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software (University of California, San Francisco, CA).33 The torsional root and branches of the

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ligands, permitting flexibility for all rotational bonds in the software.33 Docking calculations with

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respect to the active zinc site were performed using the AutoDock Vina software (Molecular

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Graphics Lab at The Scripps Research Institute).34 A grid box of 12Å3 was designed using the

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software draw features and centered at the binding site was used to calculate the atom types

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needed for the calculation. A total of five runs were completed with average exhaustiveness of

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50. The best binding mode of each molecule was selected based on the lowest binding free

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energy and best peptide binding score. Finally, the 3D figures were prepared using PyMOL

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Molecular Graphics System (DeLano Scientific LLC, Palo Alto, CA).

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Statistical analysis

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Data were presented as mean ± SEM (standard error of mean) of 3 to 8 independent experiments.

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For 2 groups, data were analyzed by an unpaired t-test, while for 3 or more groups, a one way

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analysis of variance (ANOVA) with the Turkey’s post-hoc test was used. For the cell-free and

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cellular ACE2 kinetics studies, two way ANOVA was used with Turkey’s post-hoc test. The

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PRISM 5 statistical software (Graph Pad Software, La Jolla, CA, USA) was used for all analyses.

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The P

Egg white-derived tripeptide IRW (Ile-Arg-Trp) is an activator of

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