Interaction of Soybean 7S Globulin Peptide with Cell Membrane

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Food and Beverage Chemistry/Biochemistry

Interaction of Soybean 7S Globulin Peptide with Cell Membrane Model via ITC, QCM-D and Langmuir Monolayer Study Yuan Zou, Run-Ting Pan, Qi-Jun Ruan, Zhili Wan, Jian Guo, and Xiao-Quan Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00414 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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Journal of Agricultural and Food Chemistry

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Interaction of Soybean 7S Globulin Peptide with Cell Membrane Model via ITC, QCM-D

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and Langmuir Monolayer Study

3 Yuan Zou†, Runting Pan†, Qijun Ruan†, Zhili Wan†, Jian Guo† and Xiaoquan Yang*†

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†

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Processing of Natural Products and Product Safety, South China University of Technology,

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Guangzhou 510640, P. R China

Food Protein Research and Development Center, Guangdong Province Key Laboratory for Green

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Corresponding to：Xiao-Quan Yang

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Tel: +86 20 87114262； Fax: +86 20 87114263

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E-mail address: [email protected], [email protected]

13 14 15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment


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ABSTRACT: To understand an underlying molecular mechanism on the cholesterol-lowering

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effect of soybean 7S globulins, the interactions of their pepsin-released peptides (7S-peptides)

25

with

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dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL) were systematically studied. The

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results showed that 7S-peptides were bound to DPPC/DOPC/CHOL liposomes mainly through

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Van der Waals forces and hydrogen bonds, and the presence of higher CHOL concentrations

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enhanced the binding affinity (e.g. DPPC/DOPC/CHOL = 1:1:0, binding ratio = 0.114;

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DPPC/DOPC/CHOL = 1:1:1, binding ratio = 2.02). Compression isotherms indicated that the

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incorporation of 7S-peptides increased the DPPC/DOPC/CHOL monolayer fluidity and the lipid

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raft size. The presence of CHOL accelerated the 7S-peptide accumulation on lipid rafts, which

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could serve as platforms for peptides to develop into β-sheet rich structures. These results allow us

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to hypothesize that 7S-peptides may indirectly influence membrane protein functions via altering

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the membrane organization in enterocyte.

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KEYWORDS: 7S-peptide, cell membrane model, liposome, Langmuir monolayer, interaction

cell

membrane

models

consisting

of

dipalmitoylphosphatidylcholine

37 38 39 40 41 42 43 44

2


(DPPC),

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INTRODUCTION

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The beneficial effects of dietary soybean proteins in the control of lipidemic levels of

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hypercholesterolemic patients have been extensively studied.1-3 Several meta-analysis of

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randomized controlled trials have underlined the cholesterol-lowering properties of soybean

49

proteins,4-7 especially the soybean 7S globulin,8 which has received increasing attention in food

50

industry. Soybean 7S globulin has a trimeric structure and is composed of three subunits including

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α, α′ and β. The α and α′ subunits contain an N-terminal extension and a common core region,

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while the β subunit consists of only the core domain.9 It has been demonstrated that the α′ subunit

53

is the main component responsible for the up-regulation of liver low-density lipoprotein

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(LDL)-receptors as to the dramatic reductions in plasma cholesterol and triglycerides.10

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Particularly, the N-terminal extension region of α′ subunit (142 amino acid residues) can

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significantly increase the uptake and degradation of LDL in Hep G2 cell, which may be a

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molecular determinant for cholesterol homeostasis.11

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The underlying cholesterol-lowering mechanisms proposed for the soybean 7S globulin seem

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to be numerous but have not been fully clarified. It is generally agreed that the effects on lipid

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metabolism may be due to the so-called 7S-peptides arising from the gastrointestinal digestion of

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7S globulin.12, 13 It has been reported that the soybean 7S-peptides exert their functions via the

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up-regulation of LDL receptors, the apolipoprotein B receptor activity, and the fecal excretion of

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bile salts, as well as the down-regulation of the hepatic transcription factor in the sterol regulatory

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element binding protein expression pathways, the 3-hydroxy-3-methylglutaryl CoA reductase

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(HMG-CoA-R) activity, and the fatty acid synthase activity and gene expression.13-16 For in vivo

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functions of 7S-peptides, they may adsorb onto and interact with the enterocyte membranes. 3



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However, the information on the effects of 7S peptides on the cell membranes is still lacking. It is

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well known that the soybean 7S globulin, especially α and α′ subunits, are recognized as major

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food and feed allergens in soybean-allergic humans and animals.17 Soybean 7S-peptides could

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increase the permeability and expression of tight junction protein in piglet intestinal epithelial

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cells, leading to the intestinal damage.18 They also showed a strong ability to bind to the

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components of the rat intestinal brush border membranes and to stimulate cholecystokinin release

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and appetite suppression.19 These results imply that the soybean 7S-peptides have biomembrane

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reactive activity.

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Biomembranes are fundamental components of all living cells, and their biophysical

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properties are critical for the numerous functions of mammalian cells including anchorage of

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protein receptors and signal transduction.20 Lipid rafts are highly dynamic, submicroscopic

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assemblies of lipids that float freely within the liquid disordered bilayer in cell membranes,

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forming platforms that function in the membrane signaling and trafficking.21 Recent studies have

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reported that the bioactive peptides can alter the organization and physicochemical properties of

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biomembranes, including fluidity, microviscosity, order, elasticity, and permeability, which may

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further lead to the modifications of membrane protein functions.22,

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focused on the interactions between soybean 7S-peptides with cell membrane models including

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liposomes and Langmuir monolayers, and pursued to find the influence of 7S-peptides on the

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structural organization of lipid domains in biological membranes, which might provide a more

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comprehensive picture for the cholesterol-lowering mechanisms of 7S-peptides at the molecular

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level.

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23

In this perspective, we

In the present study, we prepared pepsin-released peptides derived from soybean 7S globulin 4


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by using pepsin digestion combined with ultrafiltration fractionation methods.24 The interactions

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of soybean 7S-peptides with liposomes were monitored by using isothermal titration calorimetry

91

(ITC) and quartz crystal microbalance with dissipation (QCM-D). The effects of soybean

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7S-peptides on the compression isotherms for lipid Langmuir monolayers were monitored under

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the physiological pH using Langmuir monolayer-Wilhelmy plate method. In addition, the

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dose-dependent phase behavior of 7S-peptides and domain variation in the model membranes

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were morphologically revealed by using the Langmuir–Blodgett method in combination with

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confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM). It has been

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reported that mammalian membranes contain high proportion of phosphatidylcholines (PC, both

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saturated and unsaturated PC) and cholesterol (CHOL).25 Therefore, we constructed model

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membranes

containing

a

CHOL

and

a

model

phospholipid

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1,2-Dipalmitoyl-sn-glycero-3-phosphocholine,

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1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) in the present study.

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MATERIALS AND METHODS

DPPC;

(saturated: unsaturated:

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Materials. Soybean 7S globulin (β-conglycinin) was isolated according to the method of

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Nagona et al.26 The protein content was 85.80 ± 1.06% (determined by Dumas combustion method,

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N × 5.71, wet basis). Pepsin (P7000) and Thioflavin T (Th T, 75%) were purchased from

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Sigma-Aldrich, Inc. (St. Louis, MO, USA). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),

107

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (CHOL) were purchased from

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Shanghai Advanced Vehicle Technology (AVT) Pharmaceutical Ltd. (Shanghai, China).

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1-palmitoyl-2-6-[(7-nitro-2-1,

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phosphocholine (NBD-PC, >99%) was purchased from Avanti Polar Lipids (Alabaster, AL, USA).

3-benzoxadiazol-4-yl)

amino]

5


hexanoyl

sn-

glycero-3-


111

All other chemicals were of analytical grade.

112

Preparation and characterization of pepsin-released peptides from soybean 7S globulin.

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The pepsin-released peptides derived from soybean 7S globulin (7S-peptides) were prepared

114

according to the method described in our previous study.24 7S-peptides were prepared by using

115

pepsin digestion combined with the ultrafiltration fractionation method. It was found that the

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resulting 7S-peptides mainly consisted of three large fragments with a molecular weight of about 6,

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10, and 11 kDa, respectively, and their amino acid profiles were very similar with the theoretical

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one of the extension regions in α and α' subunits of 7S globulin.24 For further evaluating whether

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the pepsin-released peptides were indeed derived from the extension region of α and α' subunits,

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these peptides were digested with trypsin to prepare smaller peptide fragments and then subjected

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to HPLC-MS/MS for peptide sequence identification. Fig. S1 shows the resulting

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UV-chromatogram, and Table S1 shows the amino acid sequences of the identified small peptides

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derived from the 7S-peptides. It was found that most of the peptide sequences were derived from

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the extension region of α and α' subunits, although some peptide sequences from the core region

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of α, α', and β subunits were also observed. Taken together, these results demonstrate that the

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pepsin-released 7S peptides are indeed mainly derived from the extension regions in α and α'

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subunits.

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Interactions of 7S-peptides with liposomes. The liposomes were prepared through the

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sonication of thin films according to the method of Lόpez-Pinto and coworkers with

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modifications.27 Briefly, 50 mg of ternary mixed phospholipids with the DPPC/DOPC/CHOL

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molar ratios of 1:1:0 and 1:1:1 were dissolved in 5 mL chloroform/methanol (2:1, v/v) mixture.

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After stirring for 10 min, the organic solvent was evaporated at 50 ºC by using an RV 10 digital 6


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rotary evaporator (IKA-Works Inc., Germany) to collect a thin lipid film adhering on the wall of

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the evaporative flask. The lipid film was air-dried in a fume hood at room temperature overnight.

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Then 5 mL pH 7.4 PBS (phosphate buffer saline) was added to the flask to hydrate the dried lipid

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film at 50 ºC, and the mixture was vibrated for 10 s in every 10 min. After hydration for 1 h, the

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mixture was sonicated for 15 min to obtain separated liposomes. The size of the resulting

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liposomes was measured to be about 86.35±0.43 nm by a Nano ZS Zetasizer Instrument (Malvern

139

Instruments Ltd., Worcestershire, UK).

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Isothermal Titration Calorimetry (ITC). The ITC measurements were performed in a

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MicroCal PEAQ-ITC instrument (Malvern Instruments Ltd., Worcestershire, UK) to investigate

142

the interactions between 7S-peptides and liposomes. First, all the samples were prepared with the

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pH 7.4 PBS buffer and degassed abundantly. In a titration, 250 µL liposomes with a concentration

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of 47.2 µM were injected into the sample cell, and 60 µL 7S-peptides with a concentration of 3.75

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mM were filled into the syringe. After reaching equilibrium, one injection of 0.4 µL 7S-peptides

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and then a sequence of 18 injections of 2 µL 7S-peptides were titrated into the sample cell. The

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time delay between successive injections was 150 s. During reaction, the mixture in the sample

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cell was stirred with a speed of 750 r/min at 25 ºC. The control experiments were also measured

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including the titration of 7S-peptides into buffer, buffer into liposomes, and buffer into buffer. The

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corrected data obtained as a plot of heat flow (µcal/s) vs time (min) was then integrated

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peak-by-peak and normalized to obtain a plot of enthalpy change per mole of 7S-peptide (∆H,

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kJ/mol) vs the molar ratio of 7S-peptide to liposome. The experimental data were further analyzed

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by Malvern MicroCal PEAQ-ITC Analysis, providing the thermodynamic parameters including Ka

154

(the binding constant), ∆H (the enthalpy change), ∆S (the entropy change), and N (the number of 7



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7S-peptide bound per mole of liposome). All of the experiments were performed in three

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replicates.

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Quartz Crystal Microbalance with Dissipation Measurements (QCM-D). The interactions

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between 7S-peptides and liposomes were further investigated using a QCM-D instrument

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(Q-Sense, Biolin Scientific Inc., Vasteras, Sweden). Before measurements, both 7S-peptides and

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liposomes were diluted to a concentration of 0.1% (w/v) with pH 7.4 PBS buffer. The normalized

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frequency changes in the gold coated quartz crystals (∆f) and dissipation changes (∆D) were

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recorded at 25 ºC upon a sequential addition of PBS buffer (baseline, 10 min), liposome

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suspension (60 min), PBS buffer (rinsing, 10 min), 7S-peptide solution (40 min), and PBS buffer

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(rinsing, 10 min). The injection rate was 0.1 mL/min. Before and after each experiment, the sensor

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was sonicated in sulphuric acid-hydrogen peroxide (3:1, v/v) mixed solution for 10 min, washed

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with Milli-Q water, and then dried with nitrogen. The microbalance section was also rinsed with

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Milli-Q water and dried with nitrogen. All of the experiments were performed in three replicates.

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Interactions of 7S-peptides with Lipid Langmuir monolayers. The surface pressure

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(Π)-area (A) isotherms were measured at 25 ºC to study the interactions between 7S-peptides and

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lipid Langmuir monolayers using a platinum Wilhelmy plate (perimeter of 39.44 mm, KSV,

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Finland) calibrated via the weighing method. The Langmuir trough (KSV NIMA, liquid-liquid

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trough, Tietäjäntie 2, FI-02130 Espoo, Finland) used in the present study had a total area of 40176

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mm2 and was equipped with two hydrophilic Delrin® barriers. Before the measurements, 300 mL

174

pH 7.4 PBS buffer was poured into the Langmuir trough as subphase, and the liquid interface was

175

compressed and cleaned by aspiration until the surface pressure was lower than 0.5 mN/m to

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ensure the absence of surfactant contamination. The ternary mixed phospholipids with various 8


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DPPC/DOPC/CHOL molar ratios of 1:1:0, 1:1:0.2, and 1:1:1 were dissolved in the

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chloroform/methanol mixture with a volume ratio of 2:1, and a drop of 40 µL sample (1 mg/mL)

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was slowly dropped on the interface of subphase by using a Hamilton syringe. After 20 min of

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solvent evaporation and lipid spreading, 7S-peptide solutions with various lipid/peptide molar

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ratios (1:0, 1:0.12, 1:0.40, and 1:0.78) were dropped on the subphase. After 30 min of

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equilibration, the two compression barriers started to compress with a rate of 8 mm/min. All of the

183

experiments were performed in three replicates.

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Morphology observation. An automatic dipper was also equipped on the Langmuir trough

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used in the present study for Langmuir-Blodgett (LB) monolayers deposition onto solid substrates.

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When the surface pressure reached desired values, the LB monolayers were transferred on a clean

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glass slide or a freshly cleaved mica plate by a vertical pulling with a constant velocity of 2

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mm/min. After air-drying for 1 h, the resulting films were observed with confocal laser scanning

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microscopy (CLSM) and atomic force microscopy (AFM).

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Confocal Laser Scanning Microscopy (CLSM). The LB monolayers deposited on clean glass

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slides were observed at 25 ºC using a Zeiss 710 Confocal Microscope (Zeiss, Oberkochen,

192

Germany). The phospholipids were dyed with NBD-PC (excite at 488 nm, emit at 526 nm), and

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the 7S-peptides were stained by Th T (excite at 405 nm, emit at 482 nm).

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Atomic Force Microscopy (AFM). AFM images of LB monolayers deposited on mica plates

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were recorded at 25 ºC in tapping mode using a MultiMode SPM microscope equipped with a

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Nanoscope III controller (Digital Instruments, Veeco, Santa Barbara, CA). The drive frequency

197

was around 300 kHz, and the scan rate was around 1.0 Hz. Images were treated with Digital

198

Nanoscope software (version 5.30r3, Digital Instruments, Veeco). 9



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Statistics. Statistical analyses were performed using an analysis of variance (ANOVA)

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procedure of the SPSS 21.0 statistical analysis program, and the differences between means of the

201

trials were detected by a least significant difference (LSD) test (P ≤ 0.05).

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RESULTS AND DISCUSSION

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Interactions of 7S-peptides with liposomes. The interactions between peptides and cell

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membranes mediate a wide variety of biological processes, and characterization of the molecular

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details of these interactions is central to our understanding of cellular events, such as protein

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trafficking, cellular signaling, and the insertion and folding of membrane proteins.28 Herein, the

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mixed phospholipid liposomes with the DPPC/DOPC/CHOL molar ratios of 1:1:0 and 1:1:1 were

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used to mimic the cell membranes. The adsorption behaviors of 7S-peptides to these mixed

209

phospholipid liposomes were determined by using isothermal titration calorimetry measurements

210

(ITC) and quartz crystal microbalance with dissipation measurements (QCM-D).

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ITC. Figs. 1A and B show the corrected heat rate-time plots obtained by titrating 19 aliquot

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drops of 7S-peptides into the mixed phospholipid liposomes. The corrected heat trace peaks

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demonstrated the exothermic enthalpy released during the titration process, reflecting the

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adsorption behaviors of 7S-peptides to the mixed phospholipid liposomes.29 With an increase in

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the addition of 7S-peptides, a decrease in the exothermic peaks was seen, indicating the number of

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available binding sites on the mixed phospholipid liposomes decreased. Finally the exothermic

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peaks reached to a plateau, suggesting the mixed phospholipid liposomes were totally saturated by

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7S-peptides. It was noted that the corrected heat rate-time plots of the mixed phospholipid

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liposomes with and without CHOL showed remarkable difference. For the mixed phospholipid

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liposomes with a DPPC/DOPC/CHOL molar ratio of 1:1:0 (Fig. 1A), the exothermic peaks 10


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quickly decreased to a level of baseline after only 3 injections of 7S-peptides, indicating the small

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number of available binding sites on such liposomes. However, for the mixed phospholipid

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liposomes with a DPPC/DOPC/CHOL molar ratio of 1:1:1 (Fig. 1B), the exothermic peaks

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showed a slow decrease and reached to a plateau after 19 injections of 7S-peptides, suggesting

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more exothermic enthalpy releasing and more available binding sites on such liposomes. These

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results indicated that the presence of CHOL increased the binding affinity of 7S-peptides to the

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mixed phospholipid liposomes. The thermodynamic parameters, which were important evidences

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for confirming the binding force, were also determined from the titration curves in Figs. 1A, and B,

229

as shown in Figs. 1A' and B', respectively. The binding ratio, N, for the interaction between

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DPPC/DOPC/CHOL liposome and 7S-peptide was calculated to be 2.02, suggesting that

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liposomes became saturated when 2.02 molecules of monomer 7S-peptide were bound to per

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molecule of liposomes. However, the binding ratio was calculated to be 0.114 for the interaction

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between DPPC/DOPC liposome and 7S-peptide. These results suggest that the interaction between

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DPPC/DOPC/CHOL liposome and 7S-peptide becomes stronger as compared to that between

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DPPC/DOPC liposome and 7S-peptide. The affinity represents the binding ability between two

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substances, which can be determined by a KD value. A low KD value (＜10-9 M) means high

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affinity, while a high KD value (＞10-3 M) indicates low affinity.30 Herein, a KD value of 3.08×10-4

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M was observed in the interactions between 7S-peptides and mixed phospholipid liposomes with a

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DPPC/DOPC/CHOL molar ratio of 1:1:1, indicating their weak affinity. In addition, the Gibbs free

240

energy change (∆G), entropy of binding (∆S = -T∆S/T, T = 298.15 K), enthalpy of binding (∆H)

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were calculated to be -20 kJ/mol, -0.362 kJ/mol, and -128 kJ/mol, respectively. The negative ∆G

242

indicates the binding process is initiative. Both ∆H and ∆S were negative, reflecting the formation 11



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of Van der Waals force and hydrogen bond.31 It has been reported that OH groups of CHOL at the

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membrane-water interfaces are able to form hydrogen bonds with the C=O and OH groups of

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peptides, and CHOL can form van der Waals interactions with the amino acids (e.g. Ile, Val, and

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Leu) in the peptides.32, 33 The corrected heat rate-time plots for mixed phospholipid liposomes

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without CHOL could not be analyzed satisfactorily in the analysis software, and hence no

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thermodynamic parameters were present.

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QCM-D. For further understanding the interactions between mixed phospholipid liposomes

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and 7S-peptides, QCM-D responses including the shift in resonance frequency of the piezoelectric

251

sensor (∆f) and the shift in energy dissipation (∆D) were also measured. ∆f is proportional to the

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mass change of thin film on the gold-coated quartz crystal, and ∆D is related to the property of

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thin film adsorbed on the quartz crystal, such as thick, soft, and loose.34 Fig. 2 shows the QCM-D

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responses of the binding of 7S-peptides to the mixed phospholipid liposomes in the absence (Fig.

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2A) and presence (Fig. 2B) of CHOL. For the mixed phospholipid liposomes without CHOL (Fig.

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2A), at the point (i), the addition of liposome suspensions caused a quick decrease in ∆f and a

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rapid increase in ∆D, and only small changes in both ∆f and ∆D were seen after rinsing with PBS

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buffer at the point (ii). This result indicates that the most of liposomes can strongly adsorb on the

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crystal surface and remain intact.35 At the point (iii), the introduction of 7S-peptides into the

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QCM-D chamber led to a small decrease in ∆f and a small increase in ∆D, indicating the liposome

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film on the quartz crystal became thicker due to the increase of mass. This one-step mass increase

262

on the quartz crystal indicates that 7S-peptides do not rupture the mixed phospholipid liposomes.

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After rinsing with PBS buffer again at the point (iv), ∆f showed a small increase but ∆D slightly

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decreased, suggesting some 7S-peptides were washed away and only a small number of 12


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7S-peptides were left on the quartz crystal. For the mixed phospholipid liposomes with CHOL

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(Fig. 2B), much significant decrease in ∆f and more obvious increase in ∆D were observed after

267

injection of 7S-peptides at the point (iii). After rinsing with PBS buffer at the point (iv), only a

268

little number of 7S-peptides were removed, and thus the most of 7S-peptides remained on the

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crystal interface. These results suggest that the presence of CHOL significantly improves the

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binding affinity of 7S-peptides to mixed phospholipid liposomes, leading to a large change in the

271

liposome structure. Taken together, the results of ITC and QCM-D suggest that 7S-peptides

272

interact with mixed phospholipid liposomes without any rupture, and the presence of CHOL

273

improves the binding affinity of 7S-peptides to liposomes through hydrogen bonds and van der

274

Waals interactions between CHOL and 7S-peptides.

275

Compression isotherms of DPPC/DOPC/CHOL monolayers and 7S-peptides. The lipid

276

Langmuir monolayers formed at the air-water interfaces represent half of a bilayer, and this

277

technique has been widely used to study the effect of molecules on biophysical properties of lipid

278

monolayers, such as the surface pressure, the phase separation, and the membrane

279

organization.36-38 The surface pressure (Π) - area (A) isotherms of ternary DPPC/DOPC/CHOL

280

monolayers incorporated with different concentrations of 7S-peptides (lipid/peptide molar ratio =

281

1:0.0, 1:0.12, 1:0.40, and 1:0.78) were measured at 25 ºC, as shown in Fig. 3. The ternary

282

DPPC/DOPC/CHOL monolayers were formed with three different DPPC/DOPC/CHOL molar

283

ratios of 1:1:0, 1:1:0.2, and 1:1:1. Fig. 3A shows the Π–A isotherms for DPPC/DOPC/CHOL

284

monolayers with a molar ratio of 1:1:0 obtained in the absence or presence of different

285

concentrations of 7S-peptides. For DPPC/DOPC monolayer, it presented a typical curve with a

286

lipid expanded (LE) phase at lower surface pressures, a lipid condense (LC) phase at higher 13



287

surface pressures, and a collapse pressure (Πc, where monolayers began to transform into bilayer

288

or multilayer structures) of 45.02 mN/m. In the presence of 7S-peptides, the surface pressure of

289

DPPC/DOPC monolayers gradually increased with an increase in the peptide concentration during

290

the whole compression process. Also, an obvious decrease in the collapse pressure Πc was

291

observed in the presence of 7S-peptides. Similar phenomena were also observed in the ternary

292

DPPC/DOPC/CHOL monolayers with molar ratios of 1:1:0.2 (Fig. 3B) and 1:1:1 (Fig. 3C). These

293

results indicate that 7S-peptides can penetrate into the DPPC/DOPC/CHOL monolayers with

294

various concentrations of CHOL and lower the stability of these monolayers, and these behaviors

295

are dependent on the peptide concentration.38,

296

concentration, the surface pressure during the whole compression process increased with an

297

increase of CHOL concentration in lipid monolayers, especially at the high 7S-peptide

298

concentration with a lipid/peptide molar ratio of 1:0.78, suggesting the presence of CHOL can

299

attract more 7S-peptides to adsorb on lipid monolayers. This is in accordance with the ITC (Fig. 1)

300

and QCM-D (Fig. 2) results mentioned above.

39

It was noted that at the same 7S-peptide

301

To further investigate the influence of 7S-peptides on properties of cell membrane models,

302

the static elasticity was calculated on the basis of Π-A isotherms according to the equation:40

303

‫ܧ‬଴ = ሺ−‫ܣ‬ሻ ቀ ቁ , where A was the molecular area at a given surface pressure, Π. The static

304

elasticity can provide information related to the packed state of lipid monolayer upon compression,

305

which has important biological implications including cell signaling, surface enzymatic reaction,

306

and trans-membrane transport.41 The surface pressure (Π) - static elasticity (E0) curves of ternary

307

DPPC/DOPC/CHOL monolayers with three different molar ratios of 1:1:0, 1:1:0.2, and 1:1:1

308

obtained in the presence of various 7S-peptide concentrations are shown in Figs. 3A', B' and C',

பగ

ப஺ ୘

14


Page 14 of 37

Page 15 of 37


309

respectively. In all cases, the static elasticity gradually increased as the surface tension increased

310

to a maximum, after which it showed a sharp decrease, indicating the monolayers became more

311

compact, and were transformed into bilayer and multilayer structures at the end. The presence of

312

7S-peptides significantly decreased the static elasticity of DPPC/DOPC monolayers (Fig. 3A').

313

Similar phenomena were also seen in the ternary DPPC/DOPC/CHOL monolayers with molar

314

ratios of 1:1:0.2 (Fig. 3B') and 1:1:1 (Fig. 3C'). These results indicate that the presence of

315

7S-peptides can induce the rearrangement of the micro-domains in lipid monolayers to increase

316

the fluidity of ternary DPPC/DOPC/CHOL monolayers with different CHOL concentrations. It

317

was noted that the static elasticity of ternary DPPC/DOPC/CHOL monolayers increased with an

318

increase in the concentration of CHOL, indicating the formation of more compact monolayers.

319

This may be related with the fact that CHOL tends to interact with the long saturated acyl chain in

320

DPPC to form a liquid-condensed phase.38,

321

elasticity (e.g. the maximum E0 value) of DPPC/DOPC/CHOL monolayers in the presence and

322

absence of 7S-peptides became larger when the CHOL concentration in monolayers increased,

323

which may be caused by interactions between CHOL and 7S-peptides.

42

In addition, the difference between the static

324

Morphological observations of DPPC/DOPC/CHOL monolayers with or without

325

7S-peptides. The morphological changes of the ternary DPPC/DOPC/CHOL monolayers induced

326

by their interactions with 7S-peptides were investigated with CLSM and AFM. Fig. 4 shows

327

CLSM images of equimolar ternary DPPC/DOPC/CHOL (1:1:1) monolayers with various

328

7S-peptide concentrations (lipid/peptide molar ratios = 1:0, 1:0.4, and 1:0.78) at three different

329

surface pressure levels (10, 20, and 30 mN/m). The fluorescent probe, NBD-PC, can dissolve

330

selectively into the LE phase in the monolayer state.43 Therefore, in CLSM images, the bright 15



331

domains refer to the LE phases (dis-ordered), while the dark domains refer to the LC phases

332

(ordered, lipid rafts). The long saturated acyl chains in DPPC can strongly interact with CHOL to

333

form a liquid condensed phase, while unsaturated phospholipids DOPC are loosely packed to form

334

a liquid expanded phase. These different properties in packing states induce phase separation to

335

organize microdomains so-called lipid rafts, which float freely within the liquid disordered bilayer.

336

Such lipid rafts provide dynamic scaffolding for various different cellular processes, such as signal

337

transduction, membrane trafficking, transportation of CHOL.44, 45 In the absence of 7S-peptides,

338

the ternary DPPC/DOPC/CHOL monolayer was almost bright at a low surface pressure of 10

339

mN/m. With an increase in the surface pressure, the bright domains gradually decreased and the

340

dark domains gradually grown up. Similar observations were also seen in the ternary

341

DPPC/DOPC/CHOL monolayers in the presence of two different 7S-peptide concentrations.

342

These results indicate that the size of lipid rafts becomes larger. It was noted that at each value of

343

surface tension, the area of dark domains and the size of lipid rafts gradually increased with an

344

increase in the 7S-peptide concentration. This indicates that the presence of 7S-peptides can

345

induce the phase transformation in the ternary DPPC/DOPC/CHOL monolayer, and this behavior

346

is dependent on the 7S-peptide concentration.

347

For more insight into the effect of CHOL on the morphological changes of lipid monolayers

348

in the presence of 7S-peptides, ternary DPPC/DOPC/CHOL monolayers with various molar ratios

349

of 1:1:0, 1:1:0.2, and 1:1:1 at a surface pressure of 30 mN/m were dyed with NBD-PC, and

350

observed by using CLSM, as shown in Fig. 5. For the monolayers dyed with only NBD-PC,

351

obvious dark domains (lipid rafts) were seen after the addition of a small amount of CHOL, and

352

further addition of CHOL promoted their formation. These results were in accordance with 16


Page 16 of 37

Page 17 of 37


353

previous studies of ternary monolayers (PSM/DOPC/CHOL and egg SM/DOPC/CHOL).46, 47 To

354

investigate the structural changes of 7S-peptides on lipid monolayers, the ternary

355

DPPC/DOPC/CHOL monolayers with various molar ratios of 1:1:0, 1:1:0.2, and 1:1:1 at a surface

356

pressure of 30 mN/m were also dyed with Th T. Th T is widely used for the identification of

357

β-sheet structure in vitro. It fluoresces strongly when it is added to samples containing β-sheet-rich

358

deposits, while its free style shows only weak fluorescence.48 For the DPPC/DOPC monolayer, no

359

Th T fluorescence (in red) was observed. Interestingly, fluorescence was clearly observed after the

360

addition of a small amount of CHOL, and further addition of CHOL enhanced the fluorescence.

361

These results suggest that the presence of CHOL induces the β-sheet structure formation of

362

7S-peptides, and this behavior is dependent on the CHOL concentration. To further investigate the

363

distribution of such β-sheet structures, the lipid monolayers were dyed with NBD-PC and Th T at

364

the same time. It was found that the β-sheet structures mainly located in lipid rafts of ternary

365

DPPC/DOPC/CHOL monolayers with molar ratios of 1:1:0.2 and 1:1:1. Since the lipid rafts are

366

enriched with CHOL, it is therefore hypothesized that the interactions between CHOL and

367

7S-peptides induce the β-sheet structure formation of 7S-peptides on lipid rafts.

368

Smaller-scale images observed by AFM were further used to study the effect of the CHOL

369

concentration on morphological changes of 7S-peptides in the ternary DPPC/DOPC/CHOL

370

monolayers at a surface tension of 30 mN/m, as shown in Fig. 6. For the lipid monolayer without

371

CHOL, there were several large LC domains with a height of about 0.2-1.0 nm and a diameter of

372

around 5 µm, as well as many brighter dots with about 2-6 nm in height in LE domains or on the

373

edge of LC domains. These brighter dots were obviously higher than LC domains, suggesting that

374

they were peptide aggregates. In the presence of a small amount of CHOL, a large twisted coil 17



375

structure with a contour length of 10 µm and a height of 10-23 nm as well as few dots were

376

observed in the LC domain with a height of about 1-2.5 nm. This twisted coil structure showed a

377

similar size with that of β-sheet structures dyed with Th T (Fig. 5, in red), suggesting that the

378

twisted coil structure might be β-sheet structure of 7S-peptides on the lipid rafts. Upon further

379

increase of the CHOL concentration in the ternary DPPC/DOPC/CHOL monolayer, a large deposit

380

with an island shape (long axis was up to 250-550 nm) was observed in the LC domain with a

381

height of about 1-15 nm. These results indicate that the size of the β-sheet structure of 7S-peptide

382

is sensitive to the CHOL concentration in the lipid monolayer.

383

General discussion. The cholesterol-lowering property of the dietary soybean 7S globulin has

384

been confirmed in animal models 49-51 and human subjects.8 Recent reports further acknowledged

385

the important contributions played by the N-terminal extension region of its α' subunit.11 It has

386

been reported that compared to the soybean protein isolate, daily intake of the soybean 7S globulin

387

in rats can specifically down-regulate the mRNA expression of ATP-binding cassette transporters

388

sub-family G member 5 (ABCG5, as a reversal sterol transporter to shuttle the excessive CHOL

389

from enterocytes to the lumen of the intestine for elimination), down-regulate both mRNA and

390

protein mass of HMG-CoA-R (a rate-limiting enzyme of the CHOL metabolism in the liver), and

391

up-regulate both mRNA and protein mass of hepatic cholesterol-7α-hydroxylase (CYP7A1, a

392

regulating enzyme responsible of bile acid synthesis in the liver), leading to an increase in the

393

excretion of CHOL and an inhibition of CHOL synthesis in the liver.51 Considering the important

394

role of membranes on protein trafficking and cellular signaling, for example, the adsorption of

395

proteins at membranes can initiate a cascade of signaling activities,52 we studied the interactions

396

of 7S-peptides with membranes and the structural changes of 7S-peptides post-membrane 18


Page 18 of 37

Page 19 of 37


397

localization to further understand the possible cholesterol-lowering mechanism for soybean 7S

398

globulin.

399

We prepared the pepsin-released peptides from the extension region of soybean 7S globulin

400

(7S-peptides), and found that these peptides had an ability to associate with liposomes and lipid

401

monolayers. These binding behaviors could be controlled by the presence of CHOL, and those

402

7S-peptides adsorbed on the raft-like membranes could further develop into β-sheet rich structures.

403

Similar results were also found in Aβ-peptide (1-42 amino acids).53 Upon binding to raft-like

404

membranes containing ganglioside clusters, Aβ-peptide underwent a conformational transition

405

from an α-helix-rich structure to a β-sheet-rich one with increasing peptide density on the

406

membrane. The presence of CHOL enhanced the interaction between Aβ-peptide and membrane,

407

and promoted the formation of β-sheet-rich structure. The β-sheet served as a seed for the

408

formation of amyloid fibrils, which could exert toxicity against neuronal cells and result in the

409

Alzheimer’s disease.53

410

We also proposed a possible schematic illustration to explain the interaction process between

411

7S-peptide and cell membrane, as shown in Fig. 7. In the lipid monolayers, the long saturated acyl

412

chains in DPPC strongly interact with CHOL to form a liquid condensed phase (LC, ordered),

413

so-called CHOL-rich lipid rafts, while unsaturated phospholipids are loosely packed to form a

414

liquid expended phase (LE, disordered).45 The polar environment accommodates the OH group of

415

CHOL to locate at the membrane-water interface.32 7S-peptides has been characterized with a

416

large number (35%) of carboxyl amino acid (Glu and Asp) residues,24 facilitating high binding

417

affinity to OH groups of CHOL through Van der Waals forces and hydrogen bonds (Fig. 1). This

418

binding behavior is an initiative process (Fig. 1), and does not result in the destruction of 19



419

liposomes (Fig. 2). Our previous study also found that the carboxyl groups of Glu and Asp

420

residues in the 7S-peptides exhibited a high binding capacity to hydroxyl groups and Ca2+ of CaP

421

particles, and the interactions guided the assembly of these particles to balance between

422

electrodynamics properties and wettability.24 Therefore, 7S-peptides prone to adsorb and

423

accumulate on the CHOL-rich lipid rafts (Fig. 5). An increase in the CHOL concentration also can

424

improve the absorption of 7S-peptides at the lipid monolayers, especially on the lipid rafts (Figs. 1,

425

3, and 5). It is therefore proposed that lipid rafts in cell membranes are specific targets for

426

7S-peptides. The incorporation of 7S-peptides increases the fluidity of lipid monolayers (Fig. 3)

427

and promotes the formation of lipid rafts (Fig. 5), and these effects depend on the peptide

428

concentration. Finally, 7S-peptides adsorbed on the lipid rafts are induced to develop into a

429

β-sheet-rich structure (Fig. 5). The formation of β-sheet structures in the lipid rafts and the

430

accompanying alteration of membrane organization may affect the functions of enterocyte

431

membrane proteins (e.g. ABCG 5) and cellular signaling (the CHOL metabolism), leading to an

432

increase in the excretion of CHOL and an inhibition of CHOL synthesis in the liver.51

433

In conclusion, 7S-peptides can strongly bind to the ternary DPPC/DOPC/CHOL membrane

434

models mainly through hydrogen bonds and Van der Waals forces, and an increase in the CHOL

435

content can enhance the binding affinity of 7S-peptides to cell membranes. The incorporation of

436

7S-peptides results in a decrease in the static elasticity of ternary DPPC/DOPC/CHOL monolayers

437

and an increase in the size of lipid rafts. The presence of CHOL also accelerates the accumulation

438

of 7S-peptides on the lipid rafts, where peptides can be induced to develop into β-sheet rich

439

structures. These results obtained in this work provide valuable information on the interactions

440

between 7S-peptides and cell membranes during the adsorption of peptides to the enterocytes, 20


Page 20 of 37

Page 21 of 37


441

which may be associated with the cholesterol-lowering effect of soybean 7S globulin at the

442

molecular level.

443

ABBREVIATIONS USED DPPC,

444

dipalmitoylphosphatidylcholine;

DOPC,

dioleoylphosphatidylcholine;

CHOL,

445

cholesterol; ITC, isothermal titration calorimetry; QCM-D, quartz crystal microbalance with

446

dissipation; 7S-peptides, pepsin released 7S-globulin peptides; LDL, low-density lipoprotein;

447

HMG-CoA-R, the inhibition of 3-hydroxy-3-methylglutaryl CoA reductase; CLSM, confocal laser

448

scanning microscopy; AFM, atomic force microscopy; Th T, Thioflavin T; NBD-PC,

449

1-palmitoyl-2-6-[(7-nitro-2-1,

450

phosphocholine; ∆f, frequency change; ∆D, dissipation change; Π, surface pressure; A, area; LB,

451

Langmuir-Blodgett; Ka, the binding constant; ∆H, the enthalpy change; ∆S, the entropy change; N,

452

the number of 7S-peptide bound per mole of liposome; LE, lipid expanded phase; LC, lipid

453

condense phase; Πc, collapse pressure; E0, static elasticity; ABCG5, ATP-binding cassette

454

transporters sub-family G member 5; CYP7A1, hepatic cholesterol-7α-hydroxylase.

455

ACKNOWLEDGMENTS

3-benzoxadiazol-4-yl)

amino]

hexanoyl

sn-

glycero-3-

This work is supported by grants from the Chinese National Natural Science Foundation

456 457

(Serial numbers 31771923).

458

SUPPORTING INFORMATION The sequence of pepsin-released peptides derived from soybean 7S globulin was analyzed by

459 460

UPLC-MS/MS after digestion with trypsin.

461

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462

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592 593 594

27



595

FIGURE CAPTIONS

596

Fig. 1 ITC determination of the binding of 7S-peptides to the mixed phospholipid liposomes with

597

various DPPC/DOPC/CHOL molar ratios of 1:1:0 (A and A') and 1:1:1 (B and B'). A and B show

598

the raw data for the titration of liposomes with 7S-peptides. A' and B' show the integrated heats of

599

binding obtained from the raw data, after subtracting the heat of dilution. The solid line is the

600

theoretical fit.

601 602

Fig. 2 ∆f-t and ∆D-t plots obtained for the interactions between 7S-peptides and mixed

603

phospholipid liposomes with various DPPC/DOPC/CHOL molar ratios of 1:1:0 (A) and 1:1:1 (B).

604

The specific time points (i), (ii), (iii) and (iv) correspond to liposome addition, PBS buffer rinse,

605

7S-peptide addition, and PBS buffer rinse, respectively. All the presented data are from the 3rd

606

overtone (n=3).

607 608

Fig. 3 Surface pressure (Π) - area (A) isotherms of monolayers with various DPPC/DOPC/CHOL

609

molar ratios of 1:1:0 (A), 1:1:0.2 (B), and 1:1:1 (C), obtained in the presence of 7S-peptides with

610

various lipid/7S-peptide molar ratios of 1:0.00, 1:0.12, 1:0.40, and 1:0.78. Surface pressure (Π) -

611

static elasticity (E0) isotherms of monolayers with various DPPC/DOPC/CHOL molar ratios of

612

1:1:0 (A'), 1:1:0.2 (B'), and 1:1:1 (C'), obtained in the presence of 7S-peptides with various

613

lipid/peptide molar ratios of 1:0.00, 1:0.12, 1:0.40, and 1:0.78.

614

28


Page 28 of 37

Page 29 of 37


615

Fig. 4 CLSM images of the ternary equimolar DPPC/DOPC/CHOL (1:1:1) monolayers with

616

various lipid/7S-peptide molar ratios of 1:0.00, 1:0.40, and 1:0.78 at various surface pressures (10,

617

20, and 30 mN/m). The monolayers contain 1 mol % NBD-PC.

618 619

Fig. 5 CLSM images of the lipid monolayers with various DPPC/DOPC/CHOL molar ratios of

620

1:1:0, 1:1:0.2, and 1:1:1 in the presence of 7S-peptide with a lipid/peptide molar ratio of 1:0.78 at

621

a surface pressure of 30 mN/m. The monolayers contain 1 mol % NBD-PC or 1 w/w % Th T.

622 623

Fig. 6 AFM images of the lipid monolayers with various DPPC/DOPC/CHOL molar ratios of

624

1:1:0, 1:1:0.2, and 1:1:1 in the presence of 7S-peptide with a lipid/peptide molar ratio of 1:0.78 at

625

a surface pressure of 30 mN/m.

626 627

Fig. 7 Schematic mechanism for the interaction between 7S-peptide and membrane model and the

628

structural change of 7S-peptide on the lipid raft.

29



Page 30 of 37

0.4

0.0

B 0.0

Corrected heat rate (µcal/s)

A Corrected heat rate (µcal/s)

Fig. 1

-0.4

-0.8

-1.2 0

10

20

30

40

-0.7 -1.4 -2.1 -2.8 0

50

10

A'

5

B' 0

30

40

50

0 -5

-5

∆H (kJ/mol)

∆H (kJ/mol)

20

Time (min)

Time (min)

Thermodynamic parameters

-10

N

0.114 ± 0.027

-10

Thermodynamic parameters -15

-15

-20

-20

-25

0

3

6

9

12

15

N -6 KD (10 M) ∆H (kJ/mol) -T∆S (kJ/mol) ∆G (kJ/mol) 0

Molar ratio

3

6

9

Molar ratio

30


2.02 ± 0.452 308 ± 44.9 -128 ± 38.1 108 ± 24.5 -20.1 ± 3.67 12

15

Page 31 of 37


Fig. 2 ∆f ∆D

30

-50

∆f (Hz)

20 -100 10

-6

0

∆D (× 10 )

A

-150

(ii) (iii)

(i) 0

20

40

60

80

100

0

(iv) 120

140

Time (min) 0 30

∆f (Hz)

20 -100 10

∆f ∆D

-150

(ii) (iii)

(i) 0

20

40

60

80

0

(iv) 100

120

Time (min)

31


140

-6

-50

∆D (× 10 )

B


Page 32 of 37

Fig. 3 1:0.00

1:0.12

1:0.40

A'

1:0.78

150

Static elasticity (mN/m)

Surface pressure (mN/m)

A 50 40 30 20 10

120 90 60 30 0

0 48

60

72

84

96

108

120

0

132

10

Area/molecule (Å2)

40 30 20 10

50

120 90 60 30 0

48

60

72

84

96

108

120

132

0

10

Area/molecule (Å2)

20

30

40

50


C'

50

150



40

150

0

C

30

B'

50



B

20


40 30 20 10 0

120 90 60 30 0

48

60

72

84

96

108

120

132

0

Area/molecule (Å2)

10

20

30

40


32


50

Page 33 of 37


Fig. 4

33



Fig. 5

34


Page 34 of 37

Page 35 of 37


Fig. 6

35



Page 36 of 37

Fig. 7

A

OH

OH

OH

B

OH

OH

OH

OH OH

OH OH OH

OH

C

OH OH

OH OH

OH

OH

OH OH

CHOL-rich LC phase 7S-peptides prefer to bind to (lipid raft) the lipid raft

OH

OH

CHOL

Unordered 7S-peptide

36


OH

7S-peptides form a β-sheet rich structure on the lipid raft

OH

Phospholipid

OH OH

OH

OH

OH

OH

OH

OH OH

OH

OH

OH

OH OH

OH

OH

OH

OH

LE phase

OH

OH OH

OH

7S-peptide β-sheet

Page 37 of 37


TOC GRAPHIC

A

OH

B

OH

OH OH

OH

OH OH OH

OH

OH

OH

OH OH

OH

OH OH

OH

OH

OH

C

OH

OH

OH

OH

OH

LE phase CHOL-rich LC phase (lipid raft)

OH

OH

OH

OH

OH OH

OH OH

OH OH

OH OH

OH

OH OH

OH

OH

OH

OH OH

OH

OH OH

OH

OH

7S-peptides prefer to bind to the lipid raft

OH

OH

7S-peptides form a β-sheet rich structure on the lipid raft

OH

Phospholipid

CHOL

Unordered 7S-peptide

7S-peptide β-sheet

Schematic mechanism for the interaction between 7S-peptide and model membrane and the structural changes of 7S-peptide on the lipid raft.

37


Interaction of Soybean 7S Globulin Peptide with Cell Membrane

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