Purification and Characterization of a Cadmium-Binding Protein from

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Food Safety and Toxicology

Purification and characterization of a cadmiumbinding protein from Lentinula edodes Xiaobo Dong, Ying Liu, Xi Feng, Defang Shi, Yinbing Bian, Salam A. Ibrahim, and Wen Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05924 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Journal of Agricultural and Food Chemistry

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Purification and characterization of a cadmium-binding protein

2

from Lentinula edodes

3

Xiao-bo Dong, † Ying Liu,*,† Xi Feng, ⊥ De-fang Shi, †,‡ Yin-bing Bian, § Salam A.

4

Ibrahim,❈ Wen Huang*,†

5

†College

6

Hubei 430070, China

7

⊥Department

8

San Jose, CA 95192, United States

9

‡Research

of Food Science & Technology, Huazhong Agricultural University, Wuhan,

of Nutrition, Food Science and Packaging, California State University,

Institute of Agricultural Products Processing and Nuclear-Agricultural

10

Technology, Hubei Academy of Agricultural Sciences, Wuhan, Hubei 430064, China

11

§Institute

12

430070, China

13

❈Department

14

University, 171 Carver Hall, Greensboro, NC 27411, United States

of Applied Mycology, Huazhong Agricultural University, Wuhan, Hubei

of Family and Consumer Sciences, North Carolina A&T State

15 16

*Corresponding

17

Tel.: 086-027-87282426

18

E-mail

author: Ying Liu, Wen Huang

address:

[email protected],

ACS Paragon Plus Environment

[email protected]


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ABSTRACT: Many organisms possess the ability to produce metal-binding proteins

20

to absorb cadmium. Metallothioneins, an important family of cysteine-rich

21

metal-binding proteins, have been isolated and well characterized. However,

22

Lentinula edodes may have a different type of cadmium-binding protein that contains

23

fewer cysteine residues. In the present study, we purified a cadmium-binding protein

24

from L. edodes (LECBP) by gel filtration and anion exchange chromatography and

25

then identified LECBP by LC-MS/MS. We found LECBP to be a novel

26

cadmium-binding protein which contained 220 amino acid residues but no cysteine

27

residue. LECBP had a high binding affinity for Cd (Ⅱ) with a Kd value of 97.3 μM.

28

The percentages of α-helix, β-sheet, β-turn, and random coil in LECBP were 15.7%,

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39.4%, 8.0%, and 37.1%, respectively. In addition, high temperatures and an acidic

30

environment influenced the conformation of LECBP. Our results will thus provide a

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new perspective to understand the mechanism of cadmium accumulation in L. edodes.

32

KEYWORDS:

33

identification;

Lentinula

edodes;

cadmium-binding

protein;

purification;

characterisation


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INTRODUCTION

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Lentinula edodes, also known as the shiitake mushroom, is the second most cultivated

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edible mushroom in the world, accounting for about 25% of worldwide production.1

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The shiitake mushroom has a relatively high nutritional value and contains various

38

bioactive compounds such as lentinan, ergosterol, and triterpene compounds. This

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mushroom is very popular with consumers, particularly those in China and Japan.2 In

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fact, China is the world’s largest producer and exporter of shiitake mushrooms, with

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an annual production output of around 5.02 million tons which accounts for about

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70% of total worldwide production of L. edodes.3

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Cadmium, is a highly toxic heavy metal, and exposure to even low levels of

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cadmium can lead to a variety of diseases such as bone fragility, kidney disease, and

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several cancers.4 Recently, there is an increasing focus on the excessive level of

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cadmium in L. edodes. For example, He et al.5 reported that the cadmium content in

47

several species of L. edodes from different regions exceeded the legal limit. Similar

48

results were obtained in our previous studies of the cadmium content in shiitake

49

mushrooms from several primary cultivation regions in China. It has been reported

50

that heavy metals can be efficiently accumulated in various edible mushrooms such as

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Coprinus comatus, Agaricus bisporus, and Russula alutacea, which have shown

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considerably higher concentrations of heavy metals than those in vegetables, fruits,

53

and agricultural crop plants.6-8 Consequently, in addition to potential environmental

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pollutants from cultivation substrates, higher levels of cadmium in L. edodes can be

55

attributed to this mushroom’s ability to accumulate cadmium.



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Generally, the accumulation of heavy metals in edible mushrooms can be

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summarized as the following three processes: the heavy metals are intercepted by cell

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wall,9 and then they are transported via carriers locating in the plasma membrane,10,11

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and finally they are chelated by bioactive compounds in plasma12. It has been reported

60

that metalloproteins play a crucial role in the accumulation of heavy metals involved

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in the uptake, binding, release, and transport of metals in vivo.13 Metallothioneins

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(MTs) and phytochelatins (PCs), cysteinyl-rich polypeptides with an affinity for

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binding heavy metals, are reported to regulate metal homeostasis and facilitate

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cellular detoxification in some edible mushrooms such as Paxillus involutus, Agaricus

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bisporus, and Boletus edulis.14-16 However, various non-MTs are also involved in the

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metabolism of heavy metals in many organisms.13 For example, Chen et al.17 reported

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that flagellin in Serratia Se1998, with no cysteinyl, exhibited a high Pb-binding

68

capacity. Varieties of non-MT cadmium-binding proteins have been identified in the

69

Protein Data Bank (PDB). However, none of them was found in mushrooms.

70

Thus, the aim of this study was to purify, identify and characterize the potential

71

cadmium-binding protein from L. edodes in order to have a better understanding of

72

the mechanism of cadmium accumulation in L. edodes. The identification of novel

73

metalloproteins can also provide valuable insights to the mechanisms of microbial

74

resistance to toxic metals.

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MATERIALS AND METHODS

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Materials. L. edodes strain W1 (ACCC50926) was provided by the Institute of

77

Applied Mycology, Huazhong Agricultural University. DEAE-Sepharose Fast Flow,

78

Sephadex G-50 and Sephadex G-25 were purchased from GE Healthcare (Pittsburgh,


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PA, U.S.). Protein marker samples were from Thermo Scientific Inc. (Waltham, MA,

80

U.S.). Phenylmethane-sulfonyl fluoride (PMSF), Tris, tricine, SDS, Acrylamide (Acr),

81

N,N'-methylene diacrylamide (Bis), Ammonium persulfate (Ap), TEMED, bovine

82

serum albumin (BSA) and trypsin were purchased from Sigma Chemical Co. (St.

83

Louis, MO, U.S.). All other chemicals and solvents were of analytical grade.

84

Cultivation of L. edodes mycelium. L. edodes mycelium was cultivated in an

85

MYG media (2% malt extract, 2% glucose, 0.1% yeast extract, 0.1% peptone, and 2%

86

agar) containing 2 mg/L Cd (Ⅱ) at 25 ℃. Mycelia were harvested after 15 days and

87

freeze-dried (Beta 2-8 LD plus, Christ, Osterode am Harz, Germany) and ground into

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a fine powder.

89

Extraction of cadmium-binding protein.

Mycelia powder was homogenized

90

at 4 ℃ in a 50 mM Tris-HCl buffer (pH 8.6) containing 1 mM PMSF by a Midea®

91

disintegrator (Foshan, Guangdong, China). The homogenate was then centrifuged at

92

12,000 × g for 25 min at 4 ℃ by an Avanti® J-E refrigerated centrifuge (Beckman

93

Coulter Inc., Fullerton, CA, USA).18 The supernatant was then used as a crude

94

cadmium-binding protein extract for subsequent purification.

95

Determination of protein, cadmium and cadmium-protein contents. The

96

protein concentration was determined by a BCA Assay Kit (Dingguo Biotechnology

97

Ltd., Beijing, China) using BSA as a standard.19

98

The cadmium concentration was determined by a Graphite Furnace Atomic

99

Absorption Spectrophotometer (GFAAS, Agilent 240Z, Agilent Technologies Co.,

100

Ltd., Palo Alto, CA, U.S.) using cadmium chloride as a standard.20 A cadmium hollow

101

cathode lamp was used with a lamp current of 4.0 mA, wavelength of 228.8 nm, and

102

slit width of 0.5 nm. 10-μL of sample and 5-μL of matrix modifier (NH4H2PO4, 0.5%,

103

w/v; Mg(NO3)2, 0.03%, w/v) were injected into the furnace tube by an automatic



104

sampler. The optimized temperature program is shown in Table S1.

105

The cadmium-protein content was determined by cadmium-saturation assay.21 A

106

0.2 mL Cd ( Ⅱ ) solution (20 µg/mL) was mixed with 0.2 mL of crude

107

cadmium-binding protein extract and kept at room temperature for 10 min after which

108

0.2 mL of bovine hemoglobin (2%) was added to the mixture. The resultant sample

109

was incubated at 100 ℃ for 1 min, followed by centrifuging at 12, 000 × g for 20

110

min at 4 ℃. The supernatant was collected. Then, the procedures were repeated twice.

111

The cadmium content in the final collected supernatant was determined by GFAAS.

112

Purification of cadmium-binding protein. A combination of ammonium

113

sulphate precipitation, Sephadex G-50 gel filtration chromatography (column

114

dimension 1.6 × 50 cm) and DEAE-Sepharose Fast Flow ion-exchange

115

chromatography (column dimension 1.6 × 20 cm) was employed to separate and

116

purify the cadmium-binding protein from L. edodes mycelia.

117

The crude cadmium-binding protein extract was saturated by ammonium

118

sulphate from 10% (w/v) to 90 % (w/v) with a 10% (w/v) increment, and then mixed

119

gently for 20 min using a magnetic stirrer, and stored at 4 ℃ overnight, followed by

120

centrifuging at 10, 000 × g for 25 min at 4 ℃. The total protein and cadmium-protein

121

content in each supernatant fraction was determined by BCA assay and

122

cadmium-saturation assay in order to acquire the optimal ammonium sulphate

123

precipitation gradient.

124

The protein precipitated by ammonium sulphate saturation was dissolved in

125

Tris-HCl buffer (50 mM, pH 8.6) and dialyzed with the same buffer at 4 ℃ overnight.

126

The resulting crude cadmium-binding protein was loaded onto a gel filtration

127

Sephadex G-50 column with same Tris-HCl buffer at a follow rate of 0.8 mL/min (2

128

mL per tube). The cadmium and protein content of each fraction was measured.


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Fractions contain both cadmium and protein content were collected and lyophilized.

130

The lyophilized sample was dissolved and then eluted from a DEAE-Sepharose Fast

131

Flow by a stepwise elution of 0, 0.1, and 0.2 M NaCl in the same Tris-HCl buffer at a

132

flow rate of 1 mL/min (2 mL per tube). The purified potential cadmium-binding

133

protein fractions were then desalted using a Sephadex G-25 column (1.0 × 20 cm) and

134

freeze-dried for further study.

135

Tricine-SDS-PAGE analysis. Tricine sodium dodecyl sulfate-polyacrylamide

136

gel electrophoresis (Tricine-SDS-PAGE) was conducted according to Schägger.22

137

Concentrations of stacking gel and separating gel contained 4% and 10% acrylamide,

138

respectively. Electrophoresis samples were dissolved in a buffer containing 5%

139

β-mercaptoethanol. Protein brands were visualized by staining with Coomassie

140

Brilliant Blue R250, and gels were scanned using Bio-Rad Gel Doc XR system

141

(Bio-Rad, Hercules, CA, U.S.).

142

Protein bands in the Tricine-SDS-PAGE were cut and ground into powders,

143

dissolved into distilled water with the aid of ultrasonication, and then decomposed.23

144

The cadmium content in the resulting solution was determined by GFAAS.

145

Microscale Thermophoresis (MST) analysis. MST was performed using the

146

Monolith NT.115 (NanoTemper Technologies GmbH, Munich, Germany) according

147

to Wienken et al.24 The Cd (Ⅱ) stock solution (4 mM) was serially diluted by PBS

148

buffer (50 mM, pH8.6). Next, the purified protein (137.46 nM) in PBS buffer was

149

added to the tubes. Samples were loaded into NT.115 premium capillaries

150

(NanoTemper Technology), and measurements were performed at 25 ℃, 100% LED

151

power and 20% MST power. The dissociation constant (Kd) was calculated by

152

measuring the change in normalized fluorescence (Fnorm), and Kd was estimated

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using the NT analysis software offered by NanoTemper Technology GmbH (Munich,



154

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Germany).25

155

Identification of amino acid sequences of cadmium-binding protein by

156

LC-MS/MS. The cadmium-binding protein band from Tricine-SDS-PAGE was cut

157

and treated with dithiothreitol and iodoacetamide. The resulting sample was digested

158

with sequencing-grade trypsin and subjected to peptide sequencing by a nano-LC-MS

159

system with a Nano electrospray ionization (ESI) source (Thermo Scientific,Waltham,

160

MA) coupled with a high-performance liquid chromatography (HPLC) system

161

(Agilent).26 The digested protein was then loaded onto a reverse phase C18 capillary

162

column (75 μm × 80 mm, 3 μm ), followed by a 60 min elution at a flow rate of 800

163

nL/min consisting of mobile phase A ( 2% acetonitrile and 0.5% formic acid in water)

164

and B (9.5% water, 90% acetonitrile, and 0.5% formic acid), where B was increased

165

from 2 to 90%. The nanoLC eluate was directly electrosprayed into the mass

166

spectrometer in positive-ion mode. The ionization voltage was in a range of 1.5 to 1.8

167

KV, and the capillary temperature was 100 ℃. The MS/MS data were acquired via a

168

low energy collision induced dissociation (CID) process with a collision energy of

169

33% and charge state of 3. The MS microscan was conducted in a mass range of 350

170

to 1650 amu following a MS/MS microscan of the most intense MS ions. MS/MS

171

spectra were acquired in the ion trap with an exclusion width of 1.6 Da.

172

The collected mass spectrometric data were queried against the

most recent

173

non-redundant protein database (NR database) from the National Center for

174

Biotechnology Information (NCBI). The L. edodes protein database was predicted

175

using the L. edodes genome27 and the ProtQuest software suite provided by ProtTech

176

Inc. (Norristown, PA).

177

Gene clone. Total RNA extracted from L. edodes was used as a template for

178

cDNA synthesis using random hexamers. The cDNA (663 bp) was PCR amplified


Page 9 of 33


179

using

Taq

polymerase

(Takara)

and

the

following

primers:

180

5`-ATGTCTGCTGAACAACCC-3` and 5`-CTAGCCACTCAATTGCTG-3`.

181

Circular dichroism (CD) measurements. The far-ultraviolet (UV) CD spectra

182

of the purified protein were determined using a CD spectropolarimeter (JASCO

183

J-1500, JASCO Corp., Tokyo, Japan ). The purified protein sample was dissolved in

184

distilled water and adjusted the concentration to 0.1 mg/mL. Quartz Cells with 0.1 cm

185

path length and photo multiplier high voltage less than 700 V in the recorded spectral

186

region (190-250 nm ) were used. Each spectrum was calculated by the average of

187

three time measurements and baseline was corrected by subtracting the distilled water

188

spectrum background.

189

Intrinsic

fluorescence

emission

spectra.

The

intrinsic

fluorescence

190

measurements of the purified protein were performed on a Hitachi F-4600

191

fluorometer (Hitachi, Tokyo, Japan) according to Viseu et al.28 with some

192

modifications. The excitation wavelength was 290 nm, and the emission spectra were

193

recorded from 300 to 400 nm. Both the excitation and emission slit widths were set at

194

5 nm. In order to evaluate the effects of temperature on the protein intrinsic

195

fluorescence, the purified protein was dissolved in distilled water (150 μg/mL) and

196

incubated at temperatures (20 ℃, 40 ℃, 60 ℃, 80 ℃, 100 ℃, respectively) for 10 min.

197

Then, the samples were immediately cooled in ice water for subsequent measurement.

198

For the pH effects, the purified protein was dissolved in buffers (150 μg/mL) in the

199

pH range of 3-11, respectively. Then, the samples were analyzed by fluorometer

200

without the background solution interference.

201

Statistical analysis. Three packages of samples (replications) were used for each

202

analysis. Data were analyzed by the GLM procedure of Origin (Origin 8.0) for

203

different treatments. The differences in the mean values were compared by Duncan's



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204

multiple comparison method, and mean values and standard deviation were reported

205

(P < 0.05).

206

RESULTS AND DISCUSSION

207

Purification and determination of cadmium-binding protein. The potential

208

cadmium-binding proteins were purified from crude protein extract by ammonium

209

sulphate precipitation, gel filtration and anion-exchange chromatography. As shown

210

in Fig. 1a, the cadmium-protein content remained a similar level at the ammonium

211

sulphate saturation of 10% to 40% and the total protein content significantly

212

decreased at the ammonium sulphate saturation of 40%, indicating that partial

213

redundant

214

cadmium-protein content tended to have a remarkable decline at the ammonium

215

sulphate saturation of 40% (14.8 μg/g) to 70% (3.8 μg/g). Therefore, saturation of

216

ammonium sulphate in the range of 40% to 70% was used to precipitate

217

cadmium-binding proteins.

proteins

(none

cadmium-binding)

was

removed.

In

addition,

218

The elution profile of cadmium-binding proteins by the Sephadex G-50 column

219

is shown Fig. 1b. Two peaks (namely peak A and peak B) in the seven eluted protein

220

peaks had high contents of cadmium. In addition, both protein and cadmium contents

221

in the peak A were higher than those of peak B. Further purification was performed

222

by anion exchange chromatography. As shown in Fig. 1c, the first protein peak (peak

223

A-1) eluted by a 0 M NaCl solution showed a high cadmium content (1449 μg/L),

224

whereas the second protein peak eluted by a 0.2 M NaCl solution had no cadmium

225

content. A similar result for peak B is shown in Fig. 1d where the second protein peak

226

with no cadmium content was eluted by a 0.1 M NaCl solution, and the first protein

227

peak (peak B-1) that exhibited a high cadmium content (62.1 μg/L) was collected. The

228

purified peak A-1 and peak B-1 were then freeze-dried for further analysis.


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229

The Tricine-SDS-PAGE profiles of the purified peak A-1 and peak B-1 are

230

shown in Fig. 2a. The results showed that both peak A-1 and peak B-1 had one band

231

of apparent molecular mass of 15 kDa and 20 kDa, respectively.

232

In order to investigate the cadmium-binding abilities of the purified peak A-1

233

and peak B-1, 10 μg of peak A-1 and peak B-1 were used for electrophoresis,

234

respectively. The cadmium content in each of the two protein bands separated by

235

Tricine-SDS-PAGE was determined. As shown in Fig. 2b, the cadmium content in the

236

PA-1 band was 0.05 ng, whereas the content of cadmium in the PB-1 band was 2.05

237

ng. The results demonstrated that the level of cadmium in the PB-1 band was 40 times

238

higher than that of the PA-1 band. However, the amount of cadmium in the purified

239

protein PA-1 (22.8 ng) was about 6 times higher than that of PB-1 (3.8 ng) before the

240

electrophoresis. The results indicated cadmium only loosely bound on the surface of

241

protein PA-1.29 Sodium dodecyl sulfate and thermal treatment might break the binding

242

stability between the purified protein PA-1 and cadmium.30 It also demonstrated that

243

the purified protein PB-1 had a better cadmium-binding stability than that of PA-1.

244

In order to study the cadmium-binding stability of the purified protein PB-1, the

245

cadmium distribution profile in the gel lanes of PB-1 was analyzed. As shown in Fig.

246

2c, 3, 5, and 8 μg of the purified PB-1 was loaded into lanes 1, 2, and 3, respectively.

247

The protein bands located in the A, B, C, D, and E positions of each lane were cut for

248

cadmium content assay. The cadmium content of each band in the gel lanes is shown

249

in Fig. 2d. The results displayed that the cadmium content in the band C (containing

250

PB-1) of each lane was significantly higher than those of other bands (A, B, D, and E)

251

(P

Purification and Characterization of a Cadmium-Binding Protein from

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