Kinetic Interactions between Cyclolinopeptides and Immobilized

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Kinetic Interactions Between Cyclolinopeptides and Immobilized Human Serum Albumin by Surface Plasmon Resonance Youn Young Shim, and Martin J.T. Reaney J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504811x • Publication Date (Web): 01 Jan 2015 Downloaded from http://pubs.acs.org on January 7, 2015

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Journal of Agricultural and Food Chemistry

1

Kinetic

Interactions

Between

Cyclolinopeptides

and

2

Immobilized Human Serum Albumin by Surface Plasmon

3

Resonance

4 5

Youn Young Shim* and Martin J.T. Reaney*

6 7

Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada

8

and Prairie Tide Chemicals Inc. 102 Melville Street, Saskatoon, Saskatchewan S7J 0R1, Canada

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

TITLE RUNNING HEAD: Binding interaction of cyclolinopeptides and human serum albumin.

24

AUTHOR INFORMATION

25

Email address: [email protected] (YYS); [email protected] (MJTR).

26

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ABSTRACT

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Cyclolinopeptides (CLs) are octa-, nona-, and decapeptides present in flaxseed (Linum usitatissimum L.)

29

that may have immunosuppressive and antitumor activities, but little is known of their pharmacokinetics.

30

Human serum albumin (HSA), the most abundant blood protein, is an important mediator of organic

31

solute flux and hence where compounds bind this protein it potentially affects both their availability and

32

efficacy. Quantitative thermodynamic analysis of the interaction of compounds with HSA is important

33

in the development of biomedical applications. A surface plasmon resonance (SPR) biosensor was

34

utilized to reliably determine binding constants for several CLs with HSA. The maximum binding

35

response of [1–9-NαC]-CLA/HSA was almost 20-fold higher than [1–8-NαC],[1-MetO]-CLE/HSA.

36

Through analysis of an array of peptides, it was possible to correlate the impact of structural changes on

37

CL binding. The oxidation of sulfur in methionine (Met) residues formed methionine S-oxide (MetO)

38

and reduced binding significantly. Most strikingly, the further oxidation of MetO to S,S-dioxide (MetO2)

39

produced CLs with strong binding. The large impact on binding by relatively small modifications of

40

methionine containing CLs suggested that hydrophobic interaction was the predominant intermolecular

41

force stabilizing the complex between CLs and HSA. The SPR binding studies may aid in understanding

42

the fate of CLs after consumption of flaxseed or flaxseed products or the development of CLs as drugs

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or drug carriers.

44 45

KEYWORDS: Linum usitatissimum L.; cyclolinopeptide; orbitide; cyclic peptide; surface plasmon

46

resonance; human serum albumin; interaction kinetics.

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INTRODUCTION

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Cyclolinopeptides (CLs) are plant cyclic peptides or orbitides with potential therapeutic properties1.

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CLs are extracted from flaxseed (Linum usitatissimum L.), which is triglycerides source of essential

51

polyunsaturated fatty acids.2–4 They are composed of eight to nine, mostly hydrophobic natural amino

52

acid residues joined into a ring via a N- to C-terminal peptide bond (Table 1, Figure 1). A number of

53

oxidized CLs have been identified in flaxseed oil.5,6 Orbitides are distinct from cyclotides, which are

54

larger peptides with N- to C-terminal peptide bonds and cysteine bonds1.

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There are many pharmaceutical products that are proteins or peptides or peptide derivatives.7 Several

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CLs, including [1–9-NαC]-CLA (1),8 [1–9-NαC]-CLB (2),9 and [1–8-NαC],[1-MetO]-CLE (8)10 are

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bioactive. CL 1 possesses immunosuppressive activities, through the inhibition of calcium dependent T

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cell activation. The proposed mechanism of inhibition is similar to that of cyclosporine A (CsA).8 Both

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CL 1 and CsA bind to cyclophilin to form a complex. The reaction with cyclophilin inhibits phosphatase

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activity of calcineurin. In turn, the absence of an active calcineurin prevents the interleukin-1 (IL-1) and

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IL-2 dependent pathway from activating T cells.3,11,12 The binding also inhibits the peptidyl proyl cis-

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trans isomerase of cyclophilin.13 Unlike CsA, CL 1 demonstrates a low level of toxicity at therapeutic

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concentrations.14 Therefore, CL 1 might have applications in treating immunological diseases, and

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prolonging graft survival.15 In a related finding, the cyclotide kalata B1 inhibited IL-2 production.11 CL

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1 also inhibits hepatocytes from absorbing bile salt, phallotoxin,16 ethanol, and cysteamine.3 This

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activity is similar to that of antamanide and somatostatin, two other bioactive peptides.17 The Pro-Phe-

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Phe tri-peptide region, which is conserved between CL 1 and antamanide, appears to be critical for this

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activity.7 CL 1 is represented by the strong interaction of the cyclopeptides with components of the

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cholate transport system on hepatocyte membranes.18 This property can help to prevent cell poisoning.19

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Moreover, CL 1 was also found to have anti-malarial activities.20 In addition, CLs 2 and 8 also possess



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immunosuppressive properties, where they inhibit the mitogen-induced (concanavalin A) proliferation

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response of peripheral blood lymphocytes.21

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Human serum albumin (HSA) is the most abundant protein present in the human blood stream.22

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HSA constitutes over half of the total plasma proteins, at a concentration of 35–50 g/L, in healthy

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individuals.23 It is a globular protein consisting of a single peptide chain of 585 amino acid residues. As

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the major soluble protein constituent of the circulatory system, it has many physiological roles and

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pharmacological effects. One of its main functions is to regulate osmotic potential between the blood

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and tissues. It also plays a role in the maintenance of blood pH. Another very important role of albumin

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is its function as a transport molecule. This role is based on albumin’s unique ability to bind a variety of

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exogenous and endogenous compounds, such as metal cations, fatty acids, amino acids and diverse

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drugs.24–26 The distribution, free concentration and metabolism of various drugs can be significantly

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altered as a result of binding to HSA.23 HSA has multiple binding sites that bind a wide range of

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different ligands with high affinity.22 There are two Sudlow’s sites, site I and site II, which have been

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shown to bind drug molecules, endogenous hormones, and ions.27 Site I is composed of a pocket with

85

hydrophobic walls and an opening lined with positive charged residues, allowing it to bind a variety of

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different ligands.27 It binds dicarboxylic acid or bulky heterocyclic molecules with a negative charge in

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the middle.27 Site II is smaller and less flexible than Site I. It binds aromatic carboxylic acids with

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negatively charged acidic groups such as those present on non-steroidal anti-inflammatory drugs.27

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Since the amino acid residues on CLs are primarily hydrophobic, it is probable that CL interacts with

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HSA when introduced to the human body.

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The interaction of HSA with drugs has been investigated using fluorescence,28,29 Fourier transform

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infrared spectroscopy,30–32 and circular dichroism spectroscopy,33 but the binding of CLs other than CL

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1 to HSA has yet to be investigated. Investigation of the kinetics and equilibrium of CL/HSA binding

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and release is necessary as cyclic peptides are being considered as drug-delivery molecules.34 For

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instance, binding information is essential to understand how cyclic peptides can be delivered to a target

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environment or tissue. The information provided by surface plasmon resonance (SPR) is ideal for 4 ACS Paragon Plus Environment

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studying the association/dissociation kinetics of CL/HSA binding. One advantage of SPR is that the

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different binding kinetics of a number of different analytes to the immobilized ligand can be studied.

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Comparisons between the association/disassociation kinetics between different analytes can be made

100

easily. The equilibrium constants of association (KA) and dissociation (KD) are determined from the

101

association and dissociation rate constants.

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MATERIALS AND METHODS

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General. HSA (99% purity, fatty acid and globulin free) was purchased from Sigma-Aldrich

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Canada, Ltd. (Oakville, ON) and used without further purification. CLs were extracted and purified

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from flaxseed using procedures previously reported.5,6,35,36 An amine coupling kit [0.4 M 1-ethyl-3-(3-

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dimethylaminopropyl)carbodiimide hydrochloride (EDC), 0.1 M N-hydroxysuccinimide (NHS), and 1.0

107

M

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hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 150 mM NaCl, 0.005% (v/v) surfactant

109

polysorbate 20, and 3.4 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4], and a research-grade

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CM5 sensor chip were obtained from Biacore (GE Healthcare, Montreal, QC). The sensor chip CM5 has

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a 100 nm thick carboxylated dextran layer on a 50 nm thick gold layer surface. All other reagents were

112

of analytical grade and Milli-Q water (Millipore, Bedford, MA, USA) was used throughout all the

113

ethanolamine

experiments.

hydrochloride-NaOH

(EA),

pH

8.5],

HBS-EP

buffer

[10

mM

4-(2-

-1 (M-1 s ) constant tion rate the associa

ka

-1 (M-1 s )

constant tion rate A+B AB te constant (M s ) MWA the associa ra n tio cia Ri - R114 ) x S x so blank Theoretical Aspects. The the as kinetic model for a 1:1 interaction is normally represented by eqs 1 and 2: MWL ka A+B AB nt (s ) ka MW A te consta ra n io iat (1) ssoc RA+B Rblank) x S x theidi115 AB max =(R MW A MWL (R i - Rblank) x S xMWA Rmax = Rim x S x MWL kd (s ) constant MWL (2) iation rate AB A+B constant (s ) the dissoc te ra n iatio oc ss MW di A e th 116 Where,MW ka and k represent R the = Rim x S xand dissociation rates,kdrespectively. The affinity constant KA maxassociation A d MWL Rmax = Rim x S x AB A+B kd MWL AB A+B 37 ka 117 is the ratio of ka and kd, shown in eq 3. =1/KD kd ka KA[CLs] (3) KA = =1/KD ka max k d KA[CLs]+1 =1/K D kd KA[CLs] a 118KA[CLs] The ligands are=immobilized on204(HSA the SPR sensing surface by spotting serial dilutions in a microarray Req== Rkmax 1/K D KA Rmax kd KA[CLs]+1 KA[CLs]+1 204(HSA ka 119 format (Figure 2A). The immobilized ligands have differentKdensities 204(HSA 1/KD = A = k as well as different orientations on ka d 1/KD =KA = k 5 d -1 -1

-1

-1

-1



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the sensor surface. The interactions are measured in real time at several regions of interest on various

121

spots simultaneously (Figure 2B). -1 s-1 )

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(M constantsites are equally available The affinity of interaction is estimated by first assuming that HSA binding tion rate the associa

123

a and second that only high affinity sites are occupied working concentration range.38 Plots of the A+B AB MWA in the

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measured response versus different CL concentrations [CLs] are shown inte coFigure ) 3 and Figure S1 (A– nstant (s

k

Rmax =(Ri - Rblank) x S x

MWL

-1

MWA MWL

iation ra

the dissoc

125

Rmax = respectively. Rim x S x KA and the response H) of the Supporting Information, kd values at the binding site saturation

126

(Rmax) were obtained by fitting the measured response values (Req) obtained with different CLs

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concentrations to eq 4.

AB

A+B

ka =1/KD kd KA[CLs] Req = Rmax KA[CLs]+1 KA =

(4)

ka

204(HSA

128

A= Analyte and Buffer Preparation. Nine CLs 1/K ranging length D =Kin kd 8–9 amino acids (915.17–1090.38

129

Da; Table 1 and Figure 1) were used. The CLs were purified to >98% homogeneity using reversed

130

phase high-pressure liquid chromatography (HPLC). Analyte solutions were prepared by diluting CL

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stock solutions twenty-fold into phosphate-buffered saline (PBS; 10 mM phosphate buffer, 2.07 mM

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KCl, 137 mM NaCl, pH 7.4), giving a final PBS buffer containing 5% (v/v) dimethylsulfoxide

133

(DMSO). The CL concentrations in the analyte solutions injected on the biosensor ranged from 40 to

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150 µM. Analyte solutions were prepared preferably just before use, but in some cases were stored at

135

4 °C. Running buffer for all experiments was PBS (pH 7.4) containing 5% (v/v) DMSO. Running

136

buffers were filtered and degassed daily before use. A 10 mM sodium acetate buffer (pH 5.3) is

137

generally adequate for immobilizing amine ligands onto a CM5 sensor chip and was used in all

138

experiments. All buffer solutions were freshly prepared, degassed, and filtered with a 0.22-µm filter.

139

Surface Plasmon Resonance Analysis. SPR measurements were performed using a Biacore

140

XTM instrument (Biacore AB, Uppsala, Sweden) equipped with an internal injection system (500 µL

141

Hamilton syringe). This instrument has a flow-injection system to enable real time measurement of

142

molecular binding. The SPR angle shift is used as a response unit (RU) to quantify the binding of

143

biomolecules to the sensing surface (Figure 2B). The fluidic unit of the instrument was cleaned prior to ACS Paragon Plus Environment

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experiments by injecting 0.5% sodium dodecyl sulfate, followed by 50 mM glycine (pH 9.5). In order to

145

reuse a chip for SPR analysis bound analyte must be regenerated by stripping prior test compounds from

146

the surface after each injection, usually with mild detergents. However, standard regeneration treatments

147

did not effectively remove CLs from the HSA-immobilized chip. The only reagent that reproducibly did

148

so was 6 M guanidine hydrochloride (GdnHCl) applied as a brief pulse (10 µL at 10 µL/min flow rate),

149

followed by continued HBS-EP buffer flow (>5 min at 25 µL/min) to stabilize HSA prior to the next

150

injection.

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Immobilization of HSA. HSA was covalently immobilized onto a research-grade CM5 sensor

152

surface at 25 °C using standard amine-coupling chemistry.37 Briefly, CM5 sensor chips were

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preconditioned prior to HSA immobilization by priming the instrument 3 times with HBS-EP buffer,

154

followed by 3 successive 1 min injections of 50 mM NaOH at 50 µL/min. Flow cell 1 (Fc1) and flow

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cell 2 (Fc2) were activated by a 7 min injection of a 1:1 mixture of 0.1 M NHS and 0.4 M EDC using a

156

flow rate of 5 µL/min. HSA has the isoelectric point (pI) of 5.67 and is positively charged below this pH.

157

Immediately after activation, HSA (30 µg/mL) was diluted in 10 mM acetate buffer (pH 5.3) to impart a

158

positive charge on the HSA (Figure 4). Thereafter, the HSA is attracted to the negatively charged matrix

159

of the sensor chip and immobilized onto Fc2 (Figure 5). A solution of 10 mM acetate buffer without

160

HSA was passed over Fc1, to act as the reference cell. A 7 min injection of 1.0 M EA (pH 8.5) using a

161

flow rate of 5 µL/min was passed over Fc1 and Fc2 to deactivate unbound areas of the sensor surfaces.

162

The thickness of the bilayer was calculated by assuming that 1 kRU corresponded to a 1 nm thick

163

layer.39 This bilayer was subsequently used as a model membrane surface to study the CL/HSA

164

interactions. When performing kinetic analyses, the ligand density should be as low as possible,

165

provided signal-to-noise ratios are adequate. Direct detection of CL (ca. 1.0 kDa) binding to the

166

immobilized HSA ligand (ca. 66 kDa) on a Biacore X generally requires immobilization responses.

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CL Binding to HSA. For all CLs, 40 µL of analyte solution (40–150 µM) were injected, in

168

duplicate, and flowed over reference and HSA surfaces for 2 min at a flow rate of 20 µL/min, followed 7 ACS Paragon Plus Environment


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by a dissociation phase of 2 min for all experimental conditions. At the end of the CL injection, the flow

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solution was switched to the running buffer and a decrease in the response towards the baseline was

171

observed within minutes. This decrease in response indicated that CLs were dissociating from the HSA

172

immobilized to the sensor surface. All experiments were performed at 25 °C. In instances where the

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baseline RU was not readily achieved, a short pulse of 50% (v/v) DMSO solution was injected at a high

174

flow rate to remove any remaining CLs. Repeated experiments indicated that the regeneration procedure

175

did not change the binding characteristics of the immobilized HSA (data not shown).

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The sensorgrams were analyzed by the global fitting procedure using a 1:1 (Langmuir type) drifting

177

baseline model-derived equation26 available in the BIA evaluation software (version 4.2). RU curves

178

taken from the sensorgrams 10–30 s after association and the dissociation were used in the model. The

179

association rate constant (ka), the dissociation rate constant (kd), and the dissociation equilibrium

180

constants (KD) were determined from the model at the minimal chi-squared (χ2) value.

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RESULTS AND DISCUSSION

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Pre-concentration Assays. Pre-concentration assays were performed in order to determine the

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best immobilization buffer. Different pre-concentration pHs were used to determine optimal binding of

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HSA to the sensor chip surface (Figure 4). Using ca. 12 kRU as a reasonable immobilization level for

185

the direct kinetic assay of CL binding to a HSA surface, pH 5.5 produces inefficient pre-concentration,

186

resulting in low levels of the ligand binding to the carboxymethyl-dextran matrix of the sensor chip. At

187

pH 5.3, efficient HSA pre-concentration occurred, producing a satisfactory final response level (Figure

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4B). In contrast, HSA uptake by the surface is too rapid at pH 5.0, resulting in an excessive final

189

response (Figure 4C). Thus, for immobilization of HSA via the amine coupling reaction, a pre-

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concentration pH of 5.3 was chosen.

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HSA Covalent Immobilization. Typical SPR response sensorgram of HSA immobilization are

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shown in Figure 5. In a first stage (A), the EDC/NHS activating mixture is injected with the consequent

193

increase in the SPR signal due to a change in the bulk refractive index. The HSA solution is then ACS Paragon Plus Environment

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injected and the binding event (B) can be followed in real time. Once the adequate binding level is

195

reached, the remaining active carboxyl-NHS esters are blocked with ethanolamine hydrochloride (C),

196

causing a significant change in the bulk refractive index. The biospecific HSA surface is then ready to

197

be used (D). The final immobilized HSA response is 12–13 kRU at the sensorgram.

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Binding of the Bioactive Compound CLs to HSA. Interactions between 1 and immobilized

199

HSA were measured (Figure 3). As 1 concentration increased from 40 to 150 µM, the binding signal

200

steadily increased in RU. This result was similar to that reported by Rempel et al. though the response is

201

significantly stronger in this study.40 Interestingly, when the chip surface was saturated with analyte, the

202

response normalized by the concentration-dependent analysis should have yielded equal maximum

203

responses. A CL: HSA (1:1) binding stoichiometry was observed, using a drifting baseline model (Table

204

2 and Figure S1 of the Supporting Information). Residual values from Figure 6 also support the

205

closeness of this 1:1 ratio between the experimental and theoretical curves. All plots illustrated the

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experimental curve-fitting methodology for a simple binding model (1:1 Langmuir).

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Data Processing and Evaluation. The concentration dependency of different CLs binding to

208

immobilized HSA for concentrations ranging between 40 µM and 150 µM were determined (Figure S1

209

of the Supporting Information). Sensorgrams corresponding to a nonspecific CL 8 injected on a HSA

210

surface are shown in Figure 6 and Figure S1G. The sensorgrams are square-wave shaped due to a

211

refractive index jump, which is confirmed by the fact that no CL 8 is bound to the HSA at the beginning

212

of the dissociation phase: 1) CL 8 did not appreciably bind HSA; 2) the increase in RU is caused by an

213

increase in refractive index is largely the result of buffer injection, and 3) all the other CLs examined

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formed HSA complexes as the association curve is relatively higher than observed in CL 8.

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In contrast, other sensorgrams correspond to a specific interaction between an injected CL and the

216

immobilized HSA. Compared to the other CLs tested, CL 1 has slow association kinetics with HSA

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(Figure 6). At higher concentrations, the steady-state plateau was not reached after 120 s. The gradual

218

increase of response to a steady state during CL 1 injection indicates slow recognition kinetics of CL 1 9 ACS Paragon Plus Environment


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with the HSA binding site compared to other CLs tested (Figure 6). Quantitative kinetic data were

220

initially extracted using the kinetics profiling within the Biacore Evaluation software. The slow

221

recognition may be due to CL becoming available for interaction with HSA. It is likely that CL 1 has a

222

large hydration sphere, which must dissociate before interaction with HSA is possible.

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HSA has seven known fatty-acid binding sites, each of which is known to bind other small,

224

hydrophobic molecules. Two of the binding sites for small molecule drugs are known as Sudlow site I

225

(warfarin binding site), and Sudlow site II (indole-benzodiazepine binding site).41 Site I binds drug

226

molecules that are structurally bulky, heterocyclic compounds with centrally localized negative

227

polar/charges and hydrophobic substitutions. It has been suggested that binding is through hydrophobic

228

interactions between the analyte and a hydrophobic crevice within the binding cavity of site I.42 The

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nature of CL binding was not proven in the present study.

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The equilibrium parameters for several CL binding reactions with immobilized HSA were

231

determined (Table 2). The KA value indicated that the affinity tends to increase as the CL becomes more

232

hydrophobic though there are significant exceptions. The KD showed more rapid dissociation of CLs: 4,

233

5, and 8 than CL 1 (Table 2 and Figure 6). This suggests that there is more binding of 1 at the highly

234

hydrophobic crevice. All CLs tested were able to bind, showing more than an order of magnitude

235

increase in the 8/HSA binding (RU 71.0) and 1/HSA, which was almost 20-fold higher (RU 1370.6)

236

than 8/HSA (Figure 6). Also the oxidation state of the sulfur-containing amino acid residue in position 1

237

of CLs (2–9) was a critical component to binding. To examine the importance of this sulfur-containing

238

residue (S), we synthesized CLs with 2-aminobutanoic acid (Abu) instead of methionine residues at this

239

position (Table 1 and Figure 1). These modified compounds are named [1–9-NαC],[1-Abu]-CLB (3)35

240

and [1–8-NαC],[1-Abu]-CLE (7)36 according to established conventions. Each of the S free [1-Abu]-

241

CLs had similar binding strength to their 2-Met-containing counterparts (Figure 6). Most strikingly, the

242

conversion of methionine sulfoxide to the sulfone peptide [1–8-NαC],[1-MetO2]-CLE (9) had the

243

greatest effect on binding (Figure 6 and Figure S1 of the Supporting Information). It can be speculated


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that this was an effect of structural differences between CLs 6 and 9 and CLs 8 and 9 induced by the

245

oxygen sulfur bonds or the dipole moment of the S-oxide group. A similar but less pronounced effect

246

was observed with CLs 2–5. The conservative introduction of an oxygen group to 2 leads to a partial

247

disruption of the HSA interaction and weak binding of compound 4, while the addition of two oxygen

248

groups results in 5 with four-fold stronger binding than 4 (the most polar CL tested).

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CLs are produced in plants via expression of genes that code for multiple CLs.1 The peptides

250

produced in the same reading frame are usually produced in constant ratios in the plant determined by

251

transcription and post-translational modification of the peptide gene. In an attempt to observe the

252

sensorgram of peptides encoded by the same gene after different post-translational modification we

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compared three mixtures of three peptides each mixture with a similar modification of methionine. The

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slow release of the stronger binding groups (CL mixtures 1_2_6; 1_3_7; and 1_5_9) suggested that

255

parameters would also be important in understanding their HSA binding (Figure 7). The fast

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dissociation of CL mixture 1_4_8 showed weak binding with HSA. This is due to contribution from

257

breaking of the solution complex and hydrophobic binding. It has been observed that CL mixture 1, 2,

258

and 6 is observed in flaxseed meal products even with prolonged storage. This mixture is the major

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product of a single gene located on Scaffold 1198 coded on the 3’ to 5’ strand bases 162067 to 161526

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available via Phytozome portal (version 9.1). Flaxseed oil CLs 2, and 6 become oxidized to 4, and 8

261

respectively. Finally excessive oxidation will produce mixture 5 and 9.43

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In conclusion, the KD of CL/HSA solution is correlated with both the methionine oxidation state and

263

polarity. The binding of methionine containing CLs with HSA was similar to peptides that had Abu in

264

place of methionine but greater than that of peptides with methionine S-oxide. The dipole moment of the

265

S-oxide likely contributes to decreased affinity. Conversely, increased oxidation of the methionine S-

266

oxide to form the S,S-dioxide produces a molecule with restored HSA binding. These findings show that

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cyclic peptides and particularly CLs can be used to study the discrete effects of changing a single amino

268

acid on receptor binding. Other studies on more complex molecules do not allow the examination of a 11 ACS Paragon Plus Environment


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single substitution. For example, oxidation of methionine residues in human IgG2 Fc decreased SPR

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binding affinities to protein A.44 Comparison of the equilibrium dissociation constants to other literature

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compounds indicates that CLs binding to HSA is not comparatively strong when compared with other

272

drugs, except that the values of KD for 5 and 8 were similar to that reported for pyrimethamine and

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rifampicin bindings, respectively.45 In this study the impact of sulfur oxidation and elimination was

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observed for orbitides containing methionine and its modified analogs.

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ASSOCIATED CONTENT

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Supporting Information

277

Supplemental figure: concentration-dependent analysis of different CLs (2–9) binding to immobilized

278

HSA (Figure S1, A–H). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATIONDID

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Corresponding Authors

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*Tel: +1 306 9665050 (YYS); +1 306 9665027 (MJTR). Fax: +1 306 9665015. E-mail:

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[email protected] (YYS); [email protected] (MJTR).

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS

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This work was funded by the Saskatchewan Agricultural Development Fund (Saskatchewan Ministry of

287

Agriculture, Project 20080205) and Natural Sciences and Engineering Research Council (NSERC). We

288

thank Dr. D. J. Craik (Institute for Molecular Bioscience, The University of Queensland) for helpful

289

comments, Dr. R. Sammynaiken of Saskatchewan Structural Sciences Centre (SSSC), for his advise and

290

help with our Biacore X experience, and Mr. J. Maley of SSSC for resurrecting our Biacore X by

291

performing initial trials with ligand-analyte interactions.


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REFERENCES

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(1) Arnison, P. G.; Bibb, M. J.; Bierbaum, G.; Bowers, A. A.; Bugni, T. S.; Bulaj, G.; Camarero, J.

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A.; Campopiano, D. J.; Challis, G. L.; Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann, E.;

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Karlsson, R. Biosensor analysis of the interaction between immobilized human serum albumin and drug

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compounds for prediction of human serum albumin binding levels. J. Med. Chem. 2000, 43, 1986–1992.

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Publishing Co.: New York, 2000. (40) Rempel, B.; Gui, B.; Maley, J.; Reaney, M.; Sammynaiken, R. Biomolecular interaction study of cyclolinopeptide a with human serum albumin. J. Biomed. Biotechnol. 2010, 2010, 1–8. (41) Sudlow, G.; Birkett, D. J.; Wade, D. N. The characterization of two specific drug binding sites on human serum albumin. Mol. Pharmacol. 1975, 11, 824–382. (42) Rich, R. L.; Myszka, D. G. Survey of the year 2005 commercial optical biosensor literature. J. Mol. Recognit. 2006, 19, 478–534.

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(46) Shim, Y. Y.; Gui, B.; Arnison, P. G.; Wang, Y.; Reaney, M. J. T. Flaxseed (Linum usitatissimum

414

L.) bioactive compounds and peptide nomenclature: A review. Trends Food Sci. Technol. 2014, 38, 5–

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20.

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417

FIGURE CAPTIONS

418

Figure 1. Structure of CLs used in the HSA binding studies.

419

Figure 2. The principle of SPR phenomenon. (A) Polarized light is applied to the surface of the sensor

420

chip and is reflected. The intensity of the reflected light is reduced at a certain incident angle, the SPR

421

angle. (B) Interacting substances near the surface of the sensor chip increase the refractive index, which

422

alters the SPR angle. The optical detection unit detects position changes in the wedge of the reflected

423

light, corresponding to the SPR angle. The signals produced are measured in RU.

424

Figure 3. Concentration-dependent analysis of CL 1 complexes binding to HSA. All colour plots

425

indicate a spot exposed to 30 µg/mL HSA ligand and with various concentration of CL 1 on the surface

426

respectively with duplicates (omitting the regeneration stage). The response baseline is set to equal the

427

level of HSA while time zero is set at the time of injection of CL 1. One representative experiment is

428

shown. All experiments are performed on the same sensor chip. The asterisk marks the end of the CL 1

429

injection and the beginning of CL 1 desorption.

430

Figure 4. The pH scouting of three successive electrostatic pre-concentration assays for HSA (66 kDa

431

MW and pI 5.67) as ligand. The all HSA was diluted in 10 mM sodium acetate to a final concentration

432

of 30 µg/mL, at three different pH values (A) 5.5, (B) 5.3, and (C) 5.0, respectively.

433

Figure 5. Steps in the immobilization of HSA ligand on a CM5 research-grade sensor chip via the

434

amine coupling reaction (red line: reference surface and green line: HSA surface). The labelled portions

435

of the graph indicate the following: (A) injection of the EDC/NHS mixture (1:1, v/v) onto the surface,

436

activating the carboxymethyl group by forming a highly-reactive succinimide ester, followed by

437

termination of the injection; (B) after surface activation, injection of ligand sample diluted in buffer of

438

the appropriate, pre-determined pH, with continuation of injection resulting in covalent binding of

439

ligand to the reactive surface to yield; (C) blockage of remaining non-reacted-activated carboxymethyl

440

groups by injection of ethanolamine solution, followed by cessation of injection; and (D) point of


18

Page 19 of 29


441

attained immobilization of ligand. Small triangles on time line below designate events such as injection,

442

end of injection, and re-filling.

443

Figure 6. SPR sensorgram responses for 100 µM of different CLs binding to HSA. Colors of

444

sensorgram traces are as the right side legends.

445

Figure 7. SPR sensorgram responses for 100 µM of each of CL mixtures binding to HSA.

446



447

Page 20 of 29

Table 1. Primary Structural Formula of CLs. code new namea 1

[1–9-NαC]-CLA

2

[1–9-NαC]-CLB c

old name CLA

amino acid sequence (NαC-)b Ile-Leu-Val-Pro-Pro-Phe-Phe-Leu-Ile

chemical formula C57H85N9O9

CLB

Met-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile

C56H83N9O9S

1058.38

CLB-S

Abu-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile

C55H81N9O9

1012.29

MW (Da) 1040.34

3

[1–9-NαC],[1-Abu]-CLB

4

[1–9-NαC],[1-MetO]-CLB

CLC

MetO-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile C56H83N9O10S 1074.38

5

[1–9-NαC],[1-MetO2]-CLB

CLK

MetO2-Leu-Ile-Pro-Pro-Phe-Phe-Val-Ile C56H83N9O11S 1090.38

6

[1–8-NαC]-CLE

CLE'

Met-Leu-Val-Phe-Pro-Leu-Phe-Ile

c

C51H76N8O8S

961.26

CLE'-S Abu-Leu-Val-Phe-Pro-Leu-Phe-Ile

C50H74N8O8

915.57

7

[1–8-NαC],[1-Abu]-CLE

8

[1–8-NαC],[1-MetO]-CLE

CLE

MetO-Leu-Val-Phe-Pro-Leu-Phe-Ile

C51H76N8O9S

977.26

9

[1–8-NαC],[1-MetO2]-CLE

CLJ

MetO2-Leu-Val-Phe-Pro-Leu-Phe-Ile

C51H76N8O10S

993.26

448

a

46

All new CLs listed are proposed by Shim et al. , except 3 and 7.

449

b

The first positions of amino acid sequences are highlighted in Figure 1. Abbreviations are Met for

450

methionine, Abu for 2-aminobutanoic acid, MetO for methionine S-oxide, and MetO2 for methionine

451

S,S -dioxide.

452

c

This work proposed the use of 3 and 7 for Abu substituted forms 2 and 6, respectively.

453


20

Page 21 of 29


454

Table 2. CL/HSA Equilibrium Binding Constants of Dissociation Determined From SPR

455

Sensorgrams Using A 1:1 Interaction Model with a Drifting Baseline. interaction CLsa

ka (Ms–1)

kd (s–1)

KA (mM–1)

KD (mM)

Rmax (RU)

χ2

1

6.55E–1

2.38E–4

2.75E+0

3.64E–1

6.27E+3

7.04E+2

2

5.88E+0

2.62E–2

2.25E–1

4.45E+0

1.92E+4

1.94E+3

3

8.47E+0

1.21E–1

7.01E–2

1.43E+1

7.86E+4

9.64E+2

4

1.91E+2

5.88E–2

3.25E+0

3.08E–1

1.17E+3

5.75E+2

5

1.94E+2

1.60E–2

1.21E+1

8.27E–2

2.78E+2

2.00E+1

6

2.78E+0

2.05E–2

1.35E–1

7.38E+0

9.05E+4

8.90E+3

7

3.51E+0

2.70E–2

1.30E–1

7.68E+0

1.70E+4

5.14E+2

8

1.96E+2

3.47E–2

5.64E+0

1.77E–1

4.38E+3

2.99E+1

9

7.06E–1

1.20E–3

5.89E–1

1.70E+0

1.01E+4

1.02E+3

456

All experiments were performed on the same HSA-immobilized CM5 chip.

457

Values are means of SPR assays performed in duplicate.

458

a

459

Supporting Information.

The slopes were determined from curve-fitting dose-response curves in Figure 3 and Figure S1 of the



Page 22 of 29

460

461 462

Figure 1.


22

Page 23 of 29


463 464 465 466

Figure 2.

467


Fig5


Page 24 of 29

Relative Response (RU)

2500

*

2000

1500

150 µM

1000

40 µM

500

0 -20 -20

468 469

-500

30 30

80 80

130 130

180 180

230 230

280 280

Time (s)

470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488

Figure 3.


24

Page 25 of 29


489

490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507

Figure 4.

508


es on time line below designate events such as injection, end of injection, and re-filling.

Relative Response (kRU)


COO–

COO–

COO–

COON

EDC/NHS COO– A

COO–

COO–

COON

O

O

H2N-HSA

O

B

COO–

COO–

CONHHSA

CONHHSA H2NCH2CH2OH

COO–

C

D

COO–

CONHCH2CH2OH O

C

B

A

50

O

COON O

60

Page 26 of 29

40

D

30 20 10

0

509 510

500

1000

1500

2000

2500

3000

Time (s)

511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529

Figure 5.


26

Page 27 of 29


530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548

Figure 6.



Page 28 of 29

549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569

Figure 7.


28

Page 29 of 29

570


TABLE OF CONTENTS GRAPHICS

Ligand

(Human serum albumin)

Analyte

(Cyclolinopeptide)

Carrier

571

Gold sensor surface

Sheath


Kinetic Interactions between Cyclolinopeptides and Immobilized

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