Interaction between Ester-Type Tea Catechins and Neutrophil

Article Cite This: J. Agric. Food Chem. 2018, 66, 1147−1156

pubs.acs.org/JAFC

Interaction between Ester-Type Tea Catechins and Neutrophil Gelatinase-Associated Lipocalin: Inhibitory Mechanism Wei Zhang,†,§ Xiao Li,†,§ Fang Hua,† Wei Chen,‡ Wei Wang,† Gang-Xiu Chu,*,† and Guan-Hu Bao*,† †

Natural Products Laboratory, International Joint Lab of Tea Chemistry and Health Effects, State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, 230036 People’s Republic of China ‡ Department of Nephrology, Affiliated Anhui Provincial Hospital, University of Science and Technology of China, Hefei, 230026 People’s Republic of China S Supporting Information *

ABSTRACT: Tea is thought to alleviate neurotoxicity due to the antioxidative effect of ester-type tea catechins (ETC). Neutrophil gelatinase-associated lipocalin (NGAL) can sensitize β-amyloid (Aβ) induced neurotoxicity, and inhibitors of NGAL may relieve associated symptoms. As such, the interactions of ETC with NGAL were investigated by fluorescence spectrometry and molecular simulation. NGAL fluorescence is quenched regularly when being added with six processing types of tea infusion (SPTT) and ETC. Thermodynamic analyses suggest that ETC with more catechol moieties has a stronger binding capacity with NGAL especially in the presence of Fe3+. (−)-Epicatechin 3-O-caffeoate (ECC), a natural product isolated from Zijuan green tea, shows the strongest binding ability with NGAL (Kd = 15.21 ± 8.68 nM in the presence of Fe3+). All ETC are effective in protecting nerve cells against H2O2 or Aβ1−42 induced injury. The inhibitory mechanism of ETC against NGAL supports its potential use in attenuation of neurotoxicity. KEYWORDS: tea, ester-type catechins (ETC), fluorescence spectrometry, modeling simulation, nerve cell protection, chelator

■

INTRODUCTION As the most consumed traditional and widespread beverage in the world, tea has attracted continuous academic interest toward its various health benefits, which are based on its rich content of chemical compounds such as polyphenols, alkaloids, saponins, amino acids, and polysaccharides.1 Tea catechins are considered to be one of the most effective dietary constituents, with intensive research conducted on its effects on different diseases such as cancer, cardiovascular, inflammatory, and neurodegenerative diseases.2 The neuroprotective effect of a green tea infusion was attributed to the excellent antioxidative activity and metal chelating effect of tea catechins.3 Green tea shows better neuroprotective effects than other types of teas processed from the leaves of Camellia sinensis, as green tea is a type of unfermented tea that has the highest amount of catechins, especially the ester-type catechins (ETC).4,5 The adjacent phenolic dihydroxyl (catechol) and/or trihydroxyl (pyrogallol) groups in the structure of ETC are excellent electron donors, effective free radical scavengers, and siderophores (metal chelators) in cells, which help protect against metal ions-induced cell oxidative stress, in turn inhibiting free radical-induced neuronal degeneration and apoptosis around the area damaged by brain injury.6 In addition to the antioxidative activity, green tea and ETC have also been reported to reduce β-amyloid (Aβ) induced neurotoxicity.7 Aβ, which is deposited in nerve cells to form related plaques, is the hallmark of Alzheimer’s disease (AD). Recently, researchers have tried to intervene AD at an earlier stage when AD symptoms are mild but already with the presence of Aβ plaques and to discover “primary prevention” agents.8 To accomplish this, many challenges lie ahead. First, the identification and validation of suitable therapeutic targets are needed. Accumulated evidence © 2018 American Chemical Society

suggest that neutrophil gelatinase-associated lipocalin (NGAL) or lipocalin-2 (LCN2) could be an earlier biomarker in multiple aging-related diseases and also a therapeutic target for brain injury.9,10 NGAL is an acute phase protein with a primarily bacteriostatic function. It can finely tune the intracellular iron content through a catechol complex in a nontransferrin iron delivery pathway.11−13 Through tuning the content of iron, NGAL regulates mammalian neurogenesis and controls the maintenance of neural stem cells.14 Aβ toxicity to astrocytes can also be sensitized by NGAL through silencing tumor necrosis factor receptor 2 (TNFR2)-mediated neuroprotection, which suggests that NGAL is a modulator of neuro-inflammation at an earlier stage of AD.15,16 Therefore, NGAL is expected to be a therapeutic target for primary prevention of aging-related diseases, and development of small-molecule inhibitors or neutralizing antibodies to target NGAL could be a related approach for the early intervention of the diseases. The main ligands of NGAL are structurally distinct, with a catechol moiety in their structures.17 Strong binding between ligands and NGAL occurs in the presence of Fe3+. Catechol (CAT) and enterobactin are considered to be two typical ligands of NGAL. (−)-Epigallocatechin gallate (EGCG), the major tea polyphenol, is also a ligand of NGAL.18 Just like EGCG, most ETC have catechol moiety (moieties) in their structures. Therefore, we hypothesize that ETC can act as inhibitors of NGAL and play roles in prevention of aging-related diseases. Received: Revised: Accepted: Published: 1147

November 17, 2017 January 18, 2018 January 22, 2018 January 22, 2018 DOI: 10.1021/acs.jafc.7b05399 J. Agric. Food Chem. 2018, 66, 1147−1156

Article

Journal of Agricultural and Food Chemistry

Tea Material and Extraction for UPLC Analysis. The cultivar Longjingchangye (C. sinensis var. sinensis) was processed to give six major processing types of tea (SPTT) samples (green, yellow, white, oolong, dark, and black teas) with corresponding tea manufacturing methods.5 In May 2015, tea leaves were plucked from the tea base of Anhui Agricultural University (Hefei, Anhui, China). Ground tea powder (0.5 g) was extracted in 20 mL of 70% aqueous methanol by ultrasonic extraction. The tea extract were centrifuged at 10 000 rpm and then run through a 0.22 μm filter to store the supernatanrot. Quantification of Catechins in SPTT by UPLC Analysis. The gradient elution of mobile phase A was 0.17% aqueous acetic acid and mobile phase B was acetonitrile in UPLC (Waters) analysis. Details about the UPLC method can be found in our previous paper.23 The UPLC method was set as follows (A%): 94%, 0−1.08 min; from 94% to 85%, 1.1−3.0 min; from 85% to 80%, 3.0−7.5 min; from 80% to 76%, 7.5−10.0 min; from 76% to 72%, 10.0−11.5 min; form 72% to 40%, 11.5−17.0 min; from 40% to 94%, 17.0−19.0 min; keep 94%, 19.0−22.0 min. The injection volume was 1 μL. The flow rate was 0.22 mL/min, and the wavelength was set at 274 nm. Each sample was repeated thrice. We calculated the regression equation, correlation coefficient, relative standard deviation (RSD, including repeatability and reproducibility), limit of detection (LOD), limit of quantification (LOQ), and the recovery ratio as in the literature.5 SPTT Solutions Preparation for Fluorescence Quenching. To detect NGAL binding ability of a tea infusion with different degrees of fermentation, the SPTT infusions were diluted 100 times by DMSO as tested ligands. A standard TBS (the tris buffered saline, pH = 7.4) buffer was prepared with an experimental volume of 3 mL containing 5% DMSO. The experimental volume was added with 10 μL of ligands. In the fluorescence quenching experiment, the final reaction solution contained 0.0034% SPTT infusion. ETC−Iron Complex (Chelator) Solutions Preparation for Fluorescence Quenching. Previous studies on EGCG suggested that NGAL:EGCG:Fe3+ = 1:3:1 is the best stoichiometric condition for their binding.18 So, chelator solution was prepared as follows: ETC (0.15 mM, 0.5 mL) and FeCl3·6H2O (0.05 mM, 0.5 mL) were mixed, vigorously shaken, and diluted with DMSO (no other metal added for the titrations of ligand). Fluorescence Quenching Experiment of NGAL. To detect NGAL binding ability of different ETC in the absence or presence of Fe3+, the concentration of NGAL was 100 nM except for the blank group. Different concentrations of ligands were added to shake and react at room temperature for 12 h. Fluorescence quenching of NGAL was measured at three temperatures (277, 293, and 310 K) on a Cary Eclipse fluorescence spectrophotometer (Agilent Inc., Santa Clara, CA). A 10 nm slit band-pass was used for excitation and emission with a highvoltage detector. The excitation was set at 281 nm, and the emission spectra at wavelengths of 300−500 nm were collected.24 Type of Fluorescence Quenching of NGAL by ETC. Fluorescence quenching includes dynamic quenching (collision, diffusion-limited) and static quenching (diffusion-independent).25 The quenching can be identified by different trends in the excited state lifetime fluctuations with temperature.26 The Stern−Volmer equation was used to describe the dynamic fluorescence quenching process of small molecules and proteins (Figure 2).27 Binding Constants KA and Dissociation Constant Kd. For a single type of fluorescence quenching experiment, the Lineweaver− Burk formula can be used to calculate the binding constant KA (Figure 3).26 For chelators, the dissociation constant Kd is a necessary parameter

A new type of ETC called hydroxycinnamoylated catechins (HCCs, Figure 1) have been identified from tea recently. These

Figure 1. Structure of hydroxycinnamoylated catechins (HCC) and (−)-epigallocatechin gallate (EGCG).

HCCs, including (−)-epigallocatechin 3-O-ferulate, (−)-epigallocatechin 3-O-p-coumaroate, and (−)-epicatechin 3-Ocaffeoate (ECC), were found to be strong acetylcholinesterase inhibitors.19 As the most active one in this category, ECC is also the major subject studied in this study. Previous studies on ETC suggested that its neuroprotective effect was attributed to its antioxidant property. However, ETC is easily oxidized and cannot be stably delivered. Therefore, how ETC stably travel to the target area and exert neuroprotective effects remain uncertain. On the basis of the studies on NGAL’s roles in aging-related diseases, we hope to explain the neuroprotective mechanism of a green tea infusion through the interaction between ETC and NGAL. As such, fluorescence quenching was used.20 The strength and types of binding were detected in the presence or absence of Fe3+. Binding model was established by the molecular docking technique to elucidate both the binding sites between ETC and NGAL and their binding mechanisms as well.21 Neuroprotective effects of ETC were assayed in an H2O2 or Aβ1−42 injured nerve cells model.22 Besides, measurement of NGAL expression in culture medium and nerve cells explains the mechanism by which ETC may protect nerve cells through the interaction with NGAL.

■

MATERIALS AND METHODS

Chemicals. NGAL was expressed in BL-21 bacteria as reported.11 FeCl3·6H2O was bought from Sinopharm (Shanghai, China, purity >99%), and catechol (CAT) was bought from Shanghai Zhongqin Chemical Reagent Co. Ltd. (Shanghai, China). Phosphate buffered saline (PBS) was purchased from Solar-bio (Beijing, China). Deionized water (Watsons, Guangzhou, China) was used throughout the whole study. Small molecular ligands were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, reagent grade). Methanol (HPLC grade), acetonitrile (HPLC grade), and formic acid (LC-MS grade) were purchased from Duksan (Ansansi, Korea). Thermo-Fisher provided the DMEM/F12 and fetal bovine serum (Grand Island, NY). The SH-SY5Y cells were bought from ATCC (New York, NY). GL Biochem Ltd. (Shanghai, China) provided Aβ1−42 protein. Small molecular ligands used in the experiment were isolated from our laboratory, and the purity of these compounds was ≥98% confirmed by HPLC or UPLC analysis.5,19,23 All materials were stored in the refrigerator before use.

Figure 2. Stern−Volmer equation. F0 and F are the fluorescence intensities of NGAL in the absence and presence of ligands. KSV is the dynamic quiescent constant, and Kq is the diffusion collision quenching rate constant in dynamic fluorescence quenching. The τ0 represents the average lifetime of the fluorescent molecules when the quencher is absent, and the value was taken as 10−8 s. 1148

DOI: 10.1021/acs.jafc.7b05399 J. Agric. Food Chem. 2018, 66, 1147−1156

Article


protein were determined according to the relevant report.11,24 We performed multiple sets of dockings and selected the optimal values. SH-SY5Y Cell Culture. In 5% CO2, at 37 °C, cells were cultured in DMEM/F12 with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL streptomycin. After being cultivated for 48 h, the cells were transferred to 6- or 96-well plates. Neuroprotective Effects Assay of ETC.22 The medium containing 10 μM of ETC was added and cultured for 24 h. The cells were treated with Aβ1−42 (5 μM) or H2O2 (200 μM) for 24 h. The MTT assay was performed to study the survival rate of cells. The medium was replaced, and 20 μL 5 mg/mL MTT was added and kept for 4 h. The cells were washed with PBS and then added DMSO. Absorbance was measured at 490 nm using a microplate reader. The experiment was repeated thrice. Cell viability was expressed as a percentage relative to untreated cells as a control group, and positive control was set.31 NGAL Level Detected by Human NGAL Elisa Kits.32 Cells were cultured in 6-well plates, 10 μM EGCG, ECC, vitamin-E, and huperzineA were added to 2 mL culture medium, respectively, and then Aβ1−42 protein was added for further culturing after 24 h. The control group and model group were set up. After culturing for 24 h, the cell culture fluid was collected in a sterile tube and the supernatant was centrifuged. The trypsinized cells were suspended in PBS at one million/mL. Cells were repeatedly frozen and thawed, and the supernatant was centrifuged. NGAL protein levels were quantified using the Human NGAL Elisa Kits (Senbeijia Nanjing Biotechnology Co., Ltd., Nanjing, China). All experiments were performed according to the steps of the manufacturer’s protocol. Statistical Analysis. All bioassays were repeated thrice, and the values are presented as the mean ± SD, unless otherwise specified. One way ANOVA with Turkey tests was applied to determine significant differences (*P < 0.05, **P < 0.01, ***P < 0.001). GraphPad Prism (version 6.0) software was applied for statistical analysis.

for describing the binding system and providing relative affinity information between ligands and proteins.

Figure 3. Lineweaver−Burk formula. The least-squares curve fitting of these fluorescence data was used to calculate Kd. Analysis process used DynaFit (http://www.biokin.com/dynafit/). KA was calculated by Kd: KA = 1/Kd.. Binding Forces between NGAL and ETC. Proteins and small molecules usually form supramolecular complexes by classical forces: hydrophobic, hydrogen bonds, van der Waals, and other noncovalent bond forces. The thermodynamic parameters of the reaction are important evidence for binding forces. Also the parameters between small molecules and proteins can be calculated by the formula (Figure 4).28

Figure 4. Formula used to calculate the thermodynamic parameters of the reaction between small molecules and proteins. KA is the binding constant for the Lineweaver−Burk formula at different temperatures. R is the gas constant (R = 8.314 J mol−1 K−1).

■

Molecular Modeling Interaction between ETC and NGAL. The crystal structure of NGAL protein was obtained from the PDB database (PDB ID Code: 1L6M).11 The structure of protein was optimized using the molecular docking software AutoDock 4.2 (http://autodock. scripps.edu/).29 ETC chelating ferric iron are considered to be the ETC−iron complex formed by the coordination bond. The ETC−iron complex was constructed and minimally optimized using the software GROMACS 3.3.1 (www.gromacs.org).30 Ligand and NGAL interaction were investigated by AutoDock 4.2 software.29 The default is a semiflexible docking to maintain the rigid structure of the protein and the flexible structure of small molecules during the molecular docking process. The target binding sites of

RESULTS AND DISCUSSION Determination of Catechins Content in SPTT. As the main functional components of tea, catechins including (−)-epigallocatechin gallate (EGCG), epicatechin-3-gallate (ECG), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), gallocatechin (GC), (−)-gallocatechin-3-gallate (GCG) in SPTT were analyzed by UPLC (Table S1). The content of catechins, especially ETC, decreases with the degree of fermentation of teas, with the sequence as green, yellow, white,

Figure 5. UPLC analysis of (−)-epigallocatechin gallate (EGCG), epicatechin-3-gallate (ECG), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), gallocatechin (GC), (−)-gallocatechin-3-gallate (GCG) in six processing types of tea samples (green, yellow, white, oolong, black, and dark tea, SPTT) and the increase in the relative NGAL fluorescence intensity at 340 nm (Fl 340 nm) with the decrease in amount of ester-type catechins (ETC, mainly EGCG and ECG) in SPTT. Fluorescence test solution contains 100 nM NGAL and 0.0034% tea infusion (T = 293 K, pH = 7.40, λex = 281 nm and λem = 340 nm). 1149


Article


Figure 6. Relative fluorescence intensity of NGAL at 340 nm quenched by ETC and ETC−iron complex (T = 293 K, pH = 7.40). (A) Determination of the affinity of different catechins (EGCG, ECG, GCG, EGC, EC, and GC) in a complex with NGAL. (B) Determination of the affinity of chelators in a complex with NGAL. Chelators are different ligands complexed with Fe3+ including EGCG, ECG, GCG, (−)-epicatechin 3-O-caffeoate (ECC), (−)-epigallocatechin 3-O-ferulate, (−)-epigallocatechin 3-O-p-coumaroate, caffeic acid, and the positive control catechol (CAT).

than NETC (P = 2.80 × 10−8) due to the galloyl group in the structures of ETC. ECC Is the Strongest Ligand of NGAL Among Different ETC. NGAL was found to bind the siderophore−iron complex (chelator) by interaction with the three positively charged residues (R81, K125, and K134).11 In the structure of siderophore, one or more poly dentate ligands can provide multiple pairs of electrons to form a coordination bond with the central body. The ferric iron−ETC complex was used in this experiment, unless otherwise stated. Six ETC including EGCG, ECG, GCG, ECC, (−)-epigallocatechin 3-O-ferulate, and (−)-epigallocatechin 3-O-p-coumaroate together with the reference compound caffeic acid and the positive ligand CAT were tested. Addition of chelators resulted in a regular decrease of NGAL fluorescence intensity (Figure 6B). Table 1 shows the Kd values of ETC binding with NGAL. ECC (Kd = 15.21 ± 8.68 nM), GCG (Kd = 32.06 ± 9.79 nM), EGCG

oolong, dark, and black teas, among which green tea has the highest content of ETC (Figure 5). NGAL Binding Ability of SPTT Decreases with the Degree of Fermentation of Teas. The amino acid residue Trp79 is the host fluorescence origin of NGAL. Fluorescence intensity of protein decreases when the protein is bound by ligands, which is known as the fluorescence quenching effect. Changes of intrinsic fluorescence of a protein reflect the conformational changes of the structure when it is bound by a biologically active compound. Thus, fluorescence measurements can help to analyze the intrinsic binding mechanism between the protein and its ligands.33 The NGAL solution was added with quantitative SPTT, which resulted in the fluorescence changes. As shown in Figure 5, the value of the fluorescence intensity was converted to the relative fluorescence intensity by setting the fluorescence intensity of empty NGAL (Apo-NGAL) as 1, in which SPTT was not added.11 SPTT can quench NGAL fluorescence as the following sequence: green (0.60 ± 0.0021), yellow (0.63 ± 0.0045), white (0.69 ± 0.0022), oolong (0.71 ± 0.0056), dark (0.79 ± 0.0033), and black (0.85 ± 0.0068) teas. The NGAL fluorescence quenching effect of SPTT was positively correlated with the content of catechins, especially the content of ETC (ECG and EGCG). In addition, the types of catechins also have affected the fluorescence quenching of NGAL. SPSS Statistic 17.0 was used to analyze the relationship between the content of different catechins in SPTT and the fluorescence intensity of NGAL (Table S4). The content of EGCG (τ = −0.996) and ECG (τ = −0.992) in SPTT were negatively correlated with the fluorescence intensity of NGAL when NGAL was interacted with SPTT. Therefore, we can infer that the presence of ETC, especially EGCG and ECG in tea infusions being the main contributor to quench NGAL. Green tea infusion, which has the highest content of ETC, shows the strongest binding activity with NGAL among SPTT. NGAL Binding Ability of ETC Is Stronger than Those of Non-Ester Types of Catechins (NETC). To further study the inhibitory effect of catechins against NGAL, we used EGCG, ECG, GCG, EGC, EC, and GC to quench NGAL. As shown in Figure 6A, quenching of NGAL with ETC (EGCG, ECG, GCG) are much more stronger than those of NETC (EGC, EC, GC). The results indicated that ETC bound NGAL more efficiently

Table 1. Calculated Dissociation Constant (Kd), Binding Constants (KA), and Free-Energy Change (ΔG) of Chelators in Complex with NGAL (293 K) ligands epigallocatechin3-O-ferulate epigallocatechin3-O-pcoumaroate caffeic acid catechol (CAT) (−)-epicatechin 3-O-caffeoate (ECC) (−)-epigallocatechin gallate (EGCG) epicatechin-3-gallate (ECG) (−)-gallocatechin-3-gallate (GCG)

10−7 KA (L mol−1)

ΔG (kJ mol−1)

−b −b

−b −b

>300a 1.91 ± 0.95 15.21 ± 8.68

−b 52.62 6.67

−b −48.92 −43.89

38.12 ± 10.85

2.63

−41.62

41.96 ± 17.49 32.06 ± 9.79

2.38 3.13

−41.38 −42.04

Kd (nM) >300a >300a

a

The minimum quenching value is not available at a concentration of 0−400 nM. b−, no value.

(Kd = 38.12 ± 10.85 nM), and ECG (Kd = 41.96 ± 17.49 nM) are all remarkable inhibitors. Especially, ECC is the strongest one among all tested catechins. However, epigallocatechin 3-Oferulate and epigallocatechin 3-O-p-coumaroate bind NGAL poorly as they lack the second catechol moiety. As the representative ETC, EGCG and ECC were added in NGAL to 1150


Article


Figure 7. Fluorescence quenching of NGAL by ester-type catechins (ETC) and ETC−iron chelator (EIC) at λex = 281 nm (T = 293 K, pH = 7.40). (A) Effect of EGCG on fluorescence spectra of NGAL. C (EGCG)/(nM): 0, 100, 200, 300, 400, 500, 600, 700 and 800, respectively (from spectra 1 to 9). (B) Effect of EGCG−iron chelator on fluorescence spectra of NGAL. C (EGCG−iron chelator)/(nM): 0, 50, 100, 150, 200, 250, 300, 350 and 400, respectively (from spectra 1 to 9). (C) Effect of ECC on fluorescence spectra of NGAL. C (ECC)/(nM): 0, 100, 200, 300, 400, 500, 600, 700 and 800, respectively (from spectra 1 to 9). (D) Effect of ECC−iron chelator on fluorescence spectra of NGAL. C (ECC−iron chelator)/(nM): 0, 50, 100, 150, 200, 250, 300, 350 and 400, respectively (from spectra 1 to 9).

Figure 8. Stern−Volmer plots for the quenching of NGAL by ETC and EIC at different temperature (λex = 281 nm and λem = 340 nm): (A) EGCG, (B) EGCG−iron chelator, (C) ECC, and (D) ECC−iron chelator.

ETC Binds NGAL in a Static Type. In general, F0/F will be linear if the fluorescence quenching type is a single dynamic or

further explore the inhibitory mechanism of ETC on NGAL (Figure 7). 1151


Article


Table 2. Calculated Quenching Constants (Ksv), Diffusion Collision Quenching Rate Constant (Kq), Binding Constants (KA), and Thermodynamic Parameters of Interacting ETC with NGAL T (K) EGCG

ECC

a

277 293 310 277 293 310

Ra2

equation

10−6Ksv (L mol−1)

10−14Kq (L mol−1)

0.9908 0.9926 0.9928 0.9945 0.9911 0.9933

F0/F = 0.7482 + 4.1 × 106[Q] F0/F = 0.6785 + 3.7 × 106[Q] F0/F = 0.7172 + 3.2 × 106[Q] F0/F = 0.99 + 3.9 × 106[Q] F0/F = 0.9 + 3.5 × 106[Q] F0/F = 0.98 + 2.9 × 106[Q]

3.95 ± 0.11 3.51 ± 0.15 3.03 ± 0.13 3.93 ± 0.01 3.41 ± 0.05 2.87 ± 0.02

3.95 ± 0.11 3.51 ± 0.15 3.03 ± 0.13 3.93 ± 0.01 3.41 ± 0.05 2.87 ± 0.02

a

Rb2

10−6KA (L mol−1)

ΔH (kJ mol−1)

ΔG (kJ mol−1)

ΔS (J (mol.K)−1)

0.9915 0.9932 0.9919 0.9973 0.9953 0.998

2.64 1.98 1.51 3.81 2.89 2.2

−12.12

−34.05 −35.32 −36.67 −34.92 −36.25 −37.68

79.13 79.13 79.15 83.04 83.96 83.53

b

−11.78

Ra2 is the coefficient of decision in the Stern−Volmer equation fitting. bRb2 is the coefficient of decision in the Lineweaver−Burk formula.

Figure 9. Lineweaver−Burk curves for quenching of NGAL with ETC at different temperatures (λex = 281 nm and λem = 340 nm): (A) EGCG and (B) ECC.

static quiescent within defined concentration range.34 Figure 8 presents the Stern−Volmer plots of the quenching of NGAL fluorescence by ETC and ETC−iron chelator (EIC). For ETC, the curve showed a good linearity in the concentration range from 100 nM to 800 nM at different temperatures (Figure 8A,C). Linear Stern−Volmer plots indicated the existence of a single type of quenching. Typically, there are two criteria for judging a static quench or dynamic quench: compare diffusion collision quenching rate constant Kq with the maximum diffusion quenching constant or compare the size of KSV at different temperatures.35 The values of KSV were obtained from the slopes of curves listed in Table 2. If the quenching is dynamically quenched, the rising of temperature will increase the diffusion coefficient and result in increasing KSV. However, the results showed that the quenching constants to the temperature were inversely related. In addition, the Kq of ETC is larger than the maximum diffusion quenching constant for various macromolecules (2.0 × 1010 L mol−1 s−1). So, fluorescence quenching of NGAL by ETC is a typical static quenching. These results suggested that ETC interacted with NGAL as a stable complex rather than collided with each other to achieve fluorescence quenching. EIC Binds NGAL in a Combined Type. The fluorescence quenching of NGAL by EIC was considered to be a combined quenching (including static and dynamic) since the Stern− Volmer curves were upward (Figure 8B,D).33,35 The change of temperature affected the generation of EIC, thus explaining that the quenching type is a combined one36 Binding Constants of NGAL with ETC and EIC. The values of 1/ (F0 − F) were the Y-axis and 1/[Q] were the X-axis in Figure 9. The KA decreased with increasing temperature, and the complex had lower stability in higher temperature (Table 2). On the basis of the thermodynamic analysis, we believe that temperature increase makes the molecular move intensively, affecting the stability of the complex. For a combination

quenching, KA was calculated by Kd (Table 1). By comparing different binding constants KA, the binding ability of ETC and NGAL was enhanced in the presence of iron. Regularly, iron is deemed to play an important role in fluorescence quenching of NGAL. For example, although CAT bound NGAL with poor affinity (Kd = 200 nM), hundreds of fold increase was detected in the presence of iron.11 The stability of protein−siderophore−iron complexes was affected by the catechol or pyrogallol moiety of ETC. The complex will decompose when catchol group was replaced by other functional group. For examples, epigallocatechin3-O-ferulate and epigallocatechin 3-O-p-coumaroate have poor ability to bind NGAL. Furthermore, the number of catechol moieties in catechin determines the stability of catechin-iron complex. Formation of a catechin−iron complex was the first step in the binding process, and later the NGA−catechin−iron complex was formed. Different Binding Forces of ETC and EIC with NGAL. The major forces between small molecules and proteins depend on the species of small molecules. Fluorescence experiments confirmed that ETC can form stable complex with NGAL without the influence of temperature in the absence of iron. The enthalpy change (ΔH) is regarded as a constant when temperature varies in a small range. The values of ΔH, entropy change (ΔS), and free-energy change (ΔG) for ETC binding to NGAL are presented in Table 2. For EIC, we can obtain ΔG by the binding constants KA (Table 1). In fluorescence experiment, ETC has a stronger ability to bind NGAL in the presence of iron. For instance, the EGCG−iron−protein complex (ΔG = −41.62 kJ mol−1) (Table 1) is more stable than the EGCG−protein complex (ΔG = −35.32 kJ mol−1) (Table 2), and ECC also follows the rule. According to the thermodynamic parameters, Ross summarized the thermodynamic laws that determine the binding properties of biomacromolecules and small molecules.37 For the interaction in aqueous solution, ΔS > 0 can be considered to 1152


Article


Figure 10. Ligands formed hydrogen bonds with NGAL protein: (A) comparison of CAT and EGCG and (B) comparison of CAT and ECC.

be hydrophobic and ΔH < 0 can be considered to be the electrostatic interaction between ions. The binding forces between protein and small molecules are not a single force but the role of several forces together according to the complex structure. The binding process of a ligand and NGAL is a spontaneous one with an increase in entropy and a decrease in free energy. Thus, from the thermodynamic theory, we deduced that the interaction between NGAL and ETC is mainly composed of hydrophobic and electrostatic interactions. In addition, the process also includes hydrophilic interaction and dipole−dipole interactions since ligands have polyphenol structure. Binding Sites Decided by Molecular Modeling of EIC− NGAL Complex. The binding of ligand and NGAL is a dynamic process. Binding sites of NGAL were determined from the amino acid residues. At the binding sites, the conformation with the lowest energy of ligand was determined to bind NGAL. We deduced the binding sites of other ligands at NGAL based on those of CAT.11 The hydrogen bond was the most important force and the existing distance was less than 0.35 nm. CAT formed hydrogen bonds with Arg81, Tyr106, Lys125, and Lys134 of NGAL protein. EGCG formed hydrogen bonds with Asn39, Glu44, Gln49, Try52, Arg72, Ser99, Lys125, Phe133, and Lys134. ECC formed hydrogen bonds with Lys50, Pro101, Tyr106, Phe123, and Phe133 (Figure 10). Besides hydrogen bonds, hydrophobic contact was also important (Figure 11). We listed the best docking results about the NGAL−EIC complex comparable to those of CAT (Table 3). Lys125 and Lys134 are the key binding sites for NGAL protein to sequester the iron−siderophore complex.11 As discussed above, CAT and EGCG formed hydrogen bonds with Lys125 and Lys134 residues. The structural characteristic of ECC makes it impossible to form hydrogen bonds with these two residues, but it can interact with them through hydrophobic forces. Usually, in search for small molecules with bioactivity, we tend to focus on the molecules with smaller molecular weight, which can effectively reduce the steric hindrance to bind target protein. Catechins do not conform to this practice. Compared with CAT, the molecular weight of EIC was huge, which resulted in a greater steric hindrance during the binding process (torsional free

energy = 8.05 kcal/mol). However, it still bound NGAL strongly because more hydrophilic and hydrophobic interactions were obtained during its binding process with NGAL (Figure 11). Energy Effect of the Interaction between EIC and NGAL. To study the intermolecular interactions, Gibbs free energy is a very important parameter. Generally, selection of binding mode with the lowest energy is carried out for molecular docking.38 In the fluorescence experiment, although the lowest combination of energy is the most stable one, each combination to achieve the lowest energy is impossible. Usually, the complexity of binding processes leads to better results from molecular docking than those of the fluorescence experiments as docking results are received from an ideal condition. However, the free energy obtained by molecular docking experiments (Table 3) was higher than the free energy obtained by fluorescence quenching experiments (Table 1) in this study. We speculate that the flexibility of small molecules is weakened, and the steric hindrance is increased due to the artificial construction of EIC in the process of molecular docking. In addition, coordinate covalent bonds are also important factors which affect the docking results. Nerve Cells Protection by ETC. In vitro neuroprotective assays showed that all ETC (10 μM) exhibited significantly protective effects on SH-SY5Y cells against H2O2 or Aβ1−42 injury (Table 4). As EGCG was reported to have neuroprotective activity against Aβ1−42-induced cell death,39 the neuroprotective effect of ETC on human neuroblastoma SH-SY5Y cells was evaluated here using MTT method. As shown in Table 4, ETC have significant neuroprotective effect against H2O2- or Aβ1−42induced cytotoxicity in SH-SY5Y cells. Among them, ECC showed more neuroprotective effects than epigallocatechin 3-Oferulate and epigallocatechin 3-O-p-coumaroate. The bioassay data confirmed that ETC possessed potential neuroprotective effects. ETC Can Inhibit NGAL Level in Nerve Cells. Data in Figure 12 illustrate the influence of Aβ1−42 without or with addition of EGCG or ECC on NGAL expression in nerve cells and cell culture medium. The increase of NGAL level in culture medium was observed with Aβ1−42 treatment. In the presence of Aβ1−42, ETC decreased the level of NGAL expression in cells 1153


Article


methods and the degree of fermentation.1,5 In this paper, we processed SPTT from the same tea leaves, quantified major catechins (EGCG, ECG, GCG, EGC, EC, GC), and first tested their NGAL binding activity by the fluorescence quenching method. The results showed that NGAL binding activity of SPTT was positively correlated with the content of ETC (especially EGCG and ECG) (Figure 5). Subsequent experiment showed that ETC can bind NGAL more tightly than NETC (P = 2.8 × 10−8) (Figure 6A). Then, the fluorescence quenching of NGAL by different ETC found ECC was the strongest one (Kd = 15.21 ± 8.68 nM, Table 1). ECC is better than ECG (Kd = 41.96 ± 17.49, Table 1), which means a caffeoyl substitute is better than a galloyl one. Interestingly, caffeic acid itself had no NGAL binding activity (Kd > 300 nM, Table 1), and the skeleton compound EC was even worse than caffeic acid, which further confirmed the importance of the caffeoyl substitute in a catechin skeleton. Further study showed that ETC bound NGAL in a static type while EIC in a combined one. The in vitro assay confirmed that ETC can prevent nerve cell injury and ECC was the best one (P < 0.0027) (Table 4). The inhibition of EGCG or ECC against NGAL level in the nerve cells and the culture medium suggested that ETC may protect the nerve cells from injury through the interaction with NGAL. Huperzine A and vitamin E cannot inhibit NGAL level, suggesting that they play protective roles in different mechanisms. Besides able to bind NGAL tightly, ECC is also an excellent siderophore, which is able to chelate iron and may act in iron homeostasis in nerve cells. Additionally, ECC was found to be an effective acetylcolinesterase inhibitor too.19 Above evidence suggest that ECC could be a multitarget agent against aging-related diseases and hopefully be developed as a primary prevention agent for intervention of related diseases at an earlier stage. However, ECC is a minor component in tea. For its quantification and further in vitro or in vivo study, synthesis of ECC is highly needed. The neuroprevention effect of tea consumption may be related to catechins association with NGAL and iron with the following reasons. (1) Tea catechins especially ETC are effective antioxidants, and antioxidants prevention is an effective neuroprevention approach.4 (2) EGCG can be detected in its free form in vivo while EC and EGC were conjugated, methylated, or even decomposed, and all the catechins were cleared in 12 h.40 Free catechins and iron are reactive and hard for delivery. On the basis of related behavior from the NGAL−catechol−iron complex in vivo, tea catechins and iron could also be stably delivered to the target area through the relative stable NGAL−catechins−iron complex, since they all have and use the catechol moiety (moieties) to catch NGAL protein, and tea catechins can inhibit the reactive iron through the complex.11,18 However, further in vivo experiments are needed to confirm the NGAL−catechins− iron behavior. (3) Iron homeostasis is also an important factor to keep nerve cells from iron caused toxicity. As natural iron chelators and effective antioxidants, tea catechins can help to keep iron homeostasis in vivo, which is helpful in the treatment of neurodegenerative disorders.3,18 Green tea drinking can provide ETC and may exert neuroprotection through inhibiting NGAL ability to silence a TNFR2 mediated neuroprotective pathway and thus to sensitize Aβ toxicity to nerve cells, which is also a valuable topic for further study.15,16 Conclusively, green tea infusion is the most active one to bind the proinflammatory protein NGAL among all the six processing types of teas (P < 0.0017), and the binding activity is positively correlated with the content of ETC. NGAL can bind ETC tightly in a static way while it can bind ETC−iron complex in a

Figure 11. Ligands formed hydrophobic contact with NGAL protein: (A) CAT, (B) EGCG, and (C) ECC. The red half circle represents the amino acid residue that interacts with the ligands. The green short line represents the hydrogen bond, and the number indicates the length of the hydrogen bond.

significantly (P < 0.05) while the effect of huperzine-A and vitamin-E were not obvious. Tea can be classified into six major types (green, yellow, white, oolong, dark, and black) based on the different processing 1154


Article

Journal of Agricultural and Food Chemistry Table 3. Best Docking Results About the Chelator Combine with NGAL (298 K)a

CAT EGCG ECC a

estimated free energy of binding (kcal/mol)

estimated inhibition constant Ki (nM)

final intermolecular energy (kcal/mol)

final total internal energy (kcal/mol)

torsional free energy (kcal/mol)

ΔG (kJ mol−1)

−10.76 −9.13 −9.94

12.99 202.69 51.35

−10.76 −17.18 −18.00

0.00 −12.81 −6.25

0.00 8.05 8.05

−45.14 −38.18 −41.74

Cluster analysis of 100 docking runs and select the optimal value.

Table 4. Neuroprotective Effects of ETC Against Hydrogen Peroxide (H2O2 200 μM) and Amyloid-β (Aβ1−42 5 μM) Induced Human Neuroblastoma SH-SY5Y Cells Cytotoxicitya H2O2 (200 μM) group

cell survival rate (% of control)

control model vitamin-Eb huperzine-Ab (−)-epicatechin 3-O-caffeoate (ECC) (−)-epigallocatechin gallate (EGCG) epigallocatechin3-O-ferulate epigallocatechin3-O-p-coumaroate

100 ± 1.14 70.65 ± 2.64c 84.15 ± 2.68e 97.04 ± 1.37f 90.52 ± 1.57f 87.82 ± 2.63f 86.32 ± 2.85e 85.63 ± 3.42e

Aβ1−42 (5 μM) P value

cell survival rate (% of control)

P value

Interaction between Ester-Type Tea Catechins and Neutrophil

Recommend Documents