Inhibition of Methylglyoxal-Induced Histone H1 NÎµ ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

Bioactive Constituents, Metabolites, and Functions

The Inhibition of Methylglyoxal-induced Histone H1 N#-Carboxymethyllysine Formation by (+)-Catechin Lijun Yang, Xinping Li, Zhaozhen Wu, Cuixia Feng, Tianyu Zhang, Shaohua Dai, and Qiang Dong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00826 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

The Inhibition of Methylglyoxal-induced Histone H1

2

Nε-Carboxymethyllysine Formation by (+)-Catechin

4

Lijun Yang #, Xinping Li #, Zhaozhen Wu, Cuixia Feng, Tianyu Zhang, Shaohua Dai, Qiang Dong *

5

College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of

6

China

7

*Corresponding author: Tel: +86 029-87092429; fax: +86 029-87091032

3

8 9

E-mail: [email protected] #

These authors contributed equally to this study and share first authorship.

10

ACS Paragon Plus Environment


11

Abstract

12

Reactive dicarbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal

13

(GO) are common intermediates in protein damage, leading to the formation of

14

advanced glycation end products (AGEs) through nonenzymatic glycation.

15

(+)-Catechin, a natural plant extract from tea, has been evaluated for its ability in

16

trapping GO and MGO. However, (+)-catechin is also reported to have both

17

anti-oxidant ability and pro-oxidant properties. Till now, whether (+)-catechin can

18

inhibit the formation and its mechanism in nucleoprotein nonenzymatic glycation is

19

still unclear. In the present study, histone H1 and MGO were used to establish in vitro

20

(100 mM phosphate buffer solution (PBS), pH 7.4, 37℃) protein glycation model to

21

study the trapping ability of (+)-catechin. Our data show that MGO caused

22

dose-dependent protein damage and the content of MGO-induced Schiff base

23

formation was inhibited by (+)-catechin when the molecular ratio of catechin : MGO

24

is 1:6. The formation of Nε-carboxymethyllysine (CML) was reduced significantly

25

when the ratio of (+)-catechin and MGO was 1:1, which was similar to the inhibition

26

effect of aminoguanidine (AG). The formation of CML under in vitro conditions can

27

be inhibited by low concentration (12.5-100 µM) of (+)-catechin but not with high

28

concentration (200-800 µM) of (+)-catechin. The reason is that the high concentration

29

of (+)-catechin did not inhibit CML formations due to H2O2 produced by (+)-catechin.

30

In the presence of catalase, catechin can inhibit MGO-induced CML formation.

31

In conclusion, the trapping ability of (+)-catechin may be more effective at the

32

early stage of non-enzymatic glycation. However, high concentration (200-800 µM)


Page 2 of 38

Page 3 of 38


33

of (+)-catechin may not inhibit the formation of CML because it induced the increase

34

of H2O2 formation.

35

Key words: Histone H1; methylglyoxal; Nε-carboxymethyllysine; Schiff base

36

formation; (+)-catechin; advanced glycation end products

37 38



39

1. Introduction

40

The reactive dicarbonyl species (RCS) such as methylglyoxal (MGO) (Figure 1)

41

and glyoxal (GO) has been of great interest lately because of increasing recognition of

42

its link to many health complications through inducing protein damage [1]. In human,

43

plasma MGO levels have been measured about 0.15 µM [2] and found to be elevated

44

several fold in diabetics failure [3], while other researchers have demonstrated that

45

plasma MGO concentration in poorly controlled human diabetic patients is about 400

46

µM [4]. Our previous study has already shown that MGO is a highly reactive carbonyl

47

compound known to induce cytotoxicity and dicarbonyl-induced protein damage

48

contribute to cytotoxicity [5]. Protein damage by RCS through glycation is involved

49

in a number of pathophysiological conditions, including hyperglycemia, diabetes

50

related complications, atherosclerosis, Alzheimer’s disease and inflammation [3].

51

Glycation, a nonenzymatic reaction between reducing sugars and lysine residues, is an

52

important source of RCS that leads to the formation of protein advanced glycation end

53

products (AGEs) such as Nɛ-carboxymethyllysine (CML), Nɛ-carboxyethyllysine

54

(CEL), pentosidine, argpyrimidine, and methylglyoxal lysine dimers [6]. CML is

55

formed by small reactive carbonyl and dicarbonyl compounds that are either

56

metabolites or autoxidation products of glucose, ascorbate, Schiff bases, Amadori

57

intermediates, or polyunsaturated lipids in the presence of protein [3]. GO and MGO

58

are implicated as sources of dicarbonyl for non-oxidative protein modification,

59

because they are much more efficient precursors for the formation of non-fluorescent

60

AGEs such as CML and CEL and fluorescent AGEs such as argpyrimidine [7]. CML


Page 4 of 38

Page 5 of 38


61

is one of the major non-fluorescent AGEs in vivo [8] and it was originally believed

62

that CML is chemically modified by GO [9] more than 30 years ago. However, in past

63

ten years, some researchers believed that MGO can react with protein to form CML

64

[10]. The fact that MGO can induce CML was confirmed by fluorescence

65

spectroscopy method with enzyme-linked immunosorbent assay (ELISA), mass

66

spectrometry and E1 conformation [11]. Furthermore, more and more researches have

67

used CML as a target of MGO-derived AGEs [12, 13]. As to the intracellular protein

68

glycation, study has also proved that pentoses are efficient precursors for the

69

formation of different fluorescent AGEs [14]. One of important and abundant

70

intracellular pentoses is ADP-ribose (ADPR) and it is the most potent non-oxidative

71

inducer of AGEs such as CML and protein fluorescent adducts when histone H1 is the

72

target protein [15]. Furthermore, cells produce large amounts of MGO with a speed

73

that 0.4 mM methylglyoxal is formed per cell per day [16] and intracellular levels are

74

probably much higher than plasma levels [17]. Besides, MGO is reported to penetrate

75

the cell membrane through anion channels [18]. Therefore, MGO contributes to the

76

generation of most intracellular AGEs [19] and histone H1 in nucleosome can be a

77

target of intracellular MGO. To study the cytotoxicity or glycation ability of this

78

endogenous metabolite in cell free condition or in vitro, high concentration from 0.33

79

mM to 25 mM MGO [20, 21] and from 0.05 mM to 5 mM MGO was used,

80

respectively [22, 23] although concentration of MGO in healthy human plasma is 0.15

81

µM [2] and in healthy human urine is 1.5 µM [3]. In present cell free research,

82

considering the healthy concentration, pathological concentration and high



Page 6 of 38

83

instantaneous concentration happen in vivo, from 25 µM to 6.4 mM MGO under a

84

2-fold gradient will be used to intermolecular interactions between MGO and histone

85

H1 during the MGO-induced glycation. Natural plant extracts and purified constituents have been evaluated for their role

86 87

in

preventing

AGEs

formation

88

(−)-epigallocatechin-3-gallate

89

(−)-epigallocatechin (EGC), (−)-epicatechin (EC) and (+)-catechin (Figure 1) [25], are

90

a group of polyphenolic flavonoids which are essentially nontoxic. They have

91

demonstrated protective abilities against oxidative stress and the ability to bind to and

92

trap dicarbonyls because they have the same A ring structure like genistein, phloridzin,

93

and phloretin [21]. Catechins are reported to have stronger affinity for lipid bilayer

94

and can easily enter the cell membrane [26] although some researchers think their

95

stereochemical structures will influence the affinity for lipid bilayers and the

96

altercation of membrane structures [27]. Previously we showed for the first time that

97

walnut extracts, almond skin extracts or (+)-catechin, the major polyphenolic

98

compound of nuts, inhibited GO-induced or MGO-induced hepatocyte cytotoxicity

99

and hepatocyte protein carbonylation [28, 29]. In addition, it is showed in vitro

100

experiments that (+)-catechin effectively captures the carbonyls of MGO in

101

incubation conditions, thus inhibiting the carbonylation reaction and the production of

102

AGEs [30]. Moreover, our recent study also shows that (+)-catechin can prevent

103

MGO-induced cytotoxicity in EA.Hy926 cells through inhibiting apoptosis and

104

mitochondrial damage [31]. However, our previous study and other study also find

(EGCG),

[24].

Catechins,

(−)-epicatechin


gallate

including (ECG),

Page 7 of 38


105

that (+)-catechin may enhance the generation of H2O2, resulting in an oxidative

106

environment that can induce the increase of reactive oxygen species (ROS)

107

production and even increase the formation of CML [31, 32]. It seems that

108

(+)-catechin has a paradoxical effect between its antioxidant ability and pro-oxidant

109

effect. Therefore, whether (+)-catechin can inhibit the AGEs formation and if there is

110

a balance between anti-oxidant ability and pro-oxidant effect should be further studied

111

in cell free model.

112

The objective of our research is to determine if (+)-catechin can inhibit

113

nonenzymatic glycation of nucleoprotein through its trapping ability and anti-oxidant

114

ability. Histone H1 and MGO is used to establish an in vitro glycation model to

115

determine Schiff base formation, AGEs formation, and CML formation.

116 117

2. Materials and methods

118

2.1 Materials

119

Histone H1 protein was extracted in our lab. Bovine serum albumin (BSA),

120

aminoguanidine (AG), Tris, β-Mercaptoethanol, glycine, sodium dodecyl sulfate,

121

N,N,N′,N′-tetramethylethylenediamine,

122

(+)-catechin, 2,4-dinitrophenylhydrazine (DNPH), guanidine HCl, aminoguanidine

123

(AG),

124

(±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and all other

125

chemicals were purchased from Sigma (Shanghai, China). The mouse monoclonal

126

antibody against histone H1 (AE-4) was purchased from Santa Cruz Biotechnology

N-Acetyl-L-cysteine

acrylamide/bis-acrylamide,

(NAC),

Chelex


，

MGO,

catalase,


127

Inc. (Santa Cruz, California, USA). The mouse monoclonal antibody was supplied by

128

R&D Systems (Minneapolis, MN, USA). The secondary antibody, goat anti-mouse

129

IgG conjugated with horseradish peroxidase was from Jackson ImmunoResearch

130

Laboratories (West Grove, PA, USA).

131 132

2.2 Histone H1 extractions from calf thymus and purification

133

Histone H1 protein extraction and purification were done using a modification of

134

the previous method [33]. The entire preparation was carried out at 4 ℃ unless

135

otherwise stated. Calf thymus was obtained directly after scarification and transported

136

directly to the laboratory on an ice pack. To minimize proteolysis of thymus histone

137

H1, membranes and connective tissue were quickly removed and 200 g of thymus was

138

minced with scissors into 2 cm chunks. Fifty-gram batches were homogenized for 2

139

min with 700 mL of 0.14 M NaCl, and adjusted to pH 5 with HCl, while keeping the

140

solution on ice. The pH of the homogenate was readjusted to pH 5 with 1 M HCl and

141

then centrifuged for 30 min at 1500 × g. The supernatant fluid was discarded and 350

142

mL 0.14 M NaCl was added to each tube. Tubes were shaken to dislodge the pellet,

143

and the suspension was transferred to a blender and homogenized for 1 min and the

144

homogenate was then centrifuged at 1500 × g for 20 min. This process was repeated

145

three to five times until the supernatant fluid was very clear. H1 was extracted from

146

the final supernatant by suspending the chromatin pellet in 800 mL of 5% perchloric

147

acid (PCA; final concentration) and blending for approximately 2 min, followed by

148

centrifugation at 1500 × g for 30 min. The process was repeated using 400 mL of PCA


Page 8 of 38

Page 9 of 38


149

and the combined supernatants were clarified by filtration through a fine sintered

150

glass funnel. Trichloroacetic acid (TCA; 100%; 100 g in 100 mL) was added to the

151

clarified solution dropwise with continuous stirring to give a final TCA concentration

152

of 18%. The precipitate was collected by centrifugation at 10000 × g for 30 min in

153

glass tubes, resuspended in water, and clarified by centrifugation if some insoluble

154

material was present. After dialysis (molecular-mass cut-off, 8±14 KD) against water

155

for 48 h, the sample was freeze-dried and stored at 4 ℃ [34]. The protein

156

concentration of Histone H1 was assayed using the method of Bradford [35].

157 158

2.3 MGO-induced glycation model

159

The glycation model is the modification on the basis of the predecessors [33, 34].

160

Histone H1 (1 mg/mL) was incubated in 100 mM PBS (pH 7.4) with 200 µM MGO in

161

the presence of 0.03% sodium azide. The buffers and water, used to prepare solutions,

162

were pretreated with Chelex (10 g/100 mL) using 24 h incubations. Samples were

163

incubated for 24 h. Dissolved oxygen was purged from the buffer with argon gas and

164

the samples were incubated for 24 h at 37℃ under dark conditions. Similar

165

conditions were used to study the effects of the (+)-catechin, at concentrations of

166

50-400 µM. Negative controls included the (+)-catechin and histone H1 in 100 mM

167

PBS without MGO.

168 169 170

2.4 Determination of reactive dicarbonyls by Girard’s reagent The concentration of MGO was determined using Girard’s reagent [36]. Briefly,



171

an aliquot (5 µL of 30 mM solution of MGO) was mixed in a 1.5 mL Eppendorf tube

172

with 995 µL of 120 mM fresh sodium borate buffer, pH 9.3. An aliquot of this mixture

173

(200 µL) was then added to 800 µL of 100 mM Girard’s reagent T dissolved in 120

174

mM fresh sodium borate buffer. After the reaction had reached equilibrium (10 min at

175

room temperature), the amount of reacted dicarbonyl groups was determined by

176

measuring absorbance at 295 nm. The concentration of MGO in the reaction mixtures

177

was calculated using a standard curve of MGO (1-40 mM) and the % remaining of

178

reactive MGO was calculated.

179 180

2.5 Determination of protein carbonyl content of Histone H1

181

In a Histone H1 glycation assay, Histone H1 (1 mg/mL) was incubated with

182

MGO and the protein carbonyl content (Schiff base formation) was determined by

183

derivatizing the protein carbonyl adducts with DNPH. Histone H1 (0.5 mL) was

184

incubated for 1 hour at room temperature with 0.5 mL of DNPH (0.1% w/v) in 2 M

185

HCl. 1 mL of TCA (20% w/v) was added to the suspension to stop the reaction. The

186

sample was centrifuged at 9000 × g to obtain the sediment pellet, and the supernatant

187

was removed by extracting the pellet three times using 0.5 mL of ethyl acetate:ethanol

188

(1:1) solution. After the extraction, the pellet was dried under a gentle stream of

189

nitrogen and dissolved in 1 mL of Tris-buffered 8 M guanidine-HCl (pH 7.2). The

190

solubilized hydrazones were measured spectrophotometrically using an extinction

191

coefficient of 22000 M−1 cm−1 at 374 nm [37, 38].

192


Page 10 of 38

Page 11 of 38


193 194

2.6 The fluorescence detection of Histone H1 glycation adducts Histone H1 (1 mg/mL) was incubated in PBS (pH 7.4) with 200 µM and 0.03%

195

sodium azide as previously described [34]. Samples were incubated for 5 days at 37 ℃

196

under dark conditions. Similar conditions were used to study the effects of the

197

(+)-catechin at concentrations of 50-400 µM. Negative controls were employed

198

without MGO to evaluate baseline fluorescence changes in histone H1 throughout the

199

incubation. The fluorescence intensity units was measured with excitation/emission at

200

360/420 nm using a SpectraMax i3x Multi-Mode microplate reader (Molecular

201

Devices, Sunnyvale, CA, USA). Percentage of the AGE inhibition was calculated by

202

the following equation:

203 204

Percentage inhibition =（1−Fluorescent intensity units with inhibitor/Fluorescent intensity units without inhibitor) × 100%

205 206

2.7 Western blotting for immunodetection of histone H1

207

Five micrograms of histone H1 with or without MGO incubation was subjected

208

to 12% (w/v) SDS-PAGE and histone H1 bands were stained with Coomassie

209

Brilliant Blue to confirm the protein glycation. After the confirmation, the histone H1

210

sample was transferred to polyvinylidene difluoride (PVDF) membranes. Membranes

211

were blocked with 5% nonfat dried milk in a Tris-buffered saline containing 0.1%

212

Tween-20 (TBS-T solution), pH 7.4, at room temperature for 1 h. A mouse

213

monoclonal antibody against histone H1 (AE-4, Santa Cruz) was used at 1:400

214

dilution. The secondary antibody is goat anti-mouse IgG conjugated with horseradish



215

peroxidase and was used at 1:2000 dilution, followed by three washes with TBS-T.

216

Immunoblots were visualized using ECL detection reagents.

217

2.8 Western blotting for immunodetection of CML

218

Sixteen micrograms of histone H1 from the corresponding glycating incubation

219

reaction mixtures were subjected to 12% (w/v) SDS-PAGE and transferred to PVDF

220

membranes. Membranes were blocked with 5% nonfat dried milk in a TBS-T solution,

221

pH 7.4, at room temperature for 1 h. The mouse monoclonal anti-CML antibody was

222

used at a 1:500 dilution and incubated for 1 day at 4 ℃. The secondary antibody was

223

a goat anti-mouse antibody conjugated with peroxidase and used at a 1:2000 dilution

224

for 2 h at 37 ℃. This was followed by three washes with TBST. Immunoblots were

225

visualized using ECL detection reagents. NAC, another powerful antioxidant and free

226

radical scavenger, Trolox and catalase were used as antioxidants.

227 228

2.9 Measurement of H2O2 formations

229

H2O2 in glycation system was measured by the FOX 1 reagent (ferrous oxidation

230

of xylenol orange) method as described previously [39]. The 50 µL of the samples

231

was added to 950 µL of FOX1 reagent, mixed, and incubated at room temperature for

232

a minimum of 30 min to allow complete color development. The absorbance was

233

measured at 560 nm by a microplate reader (EPOCH Bio Tek, America). The molar

234

concentration of H2O2 was calculated according to the established standard curve.

235 236

2.10 Statistical analyses


Page 12 of 38

Page 13 of 38


237

The statistical significance was determined by performing a one-way analysis of

238

variance (ANOVA), with post hoc Tukey’s analysis to determine differences between

239

treatments. Values were considered statistically significant when p

Inhibition of Methylglyoxal-Induced Histone H1 NÎµ ... - ACS Publications

Recommend Documents