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

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from Lentinula edodes

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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

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⊥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

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

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§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],

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

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

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

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

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

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

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

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

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

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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

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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.

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KEYWORDS:

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identification;

Lentinula

edodes;

cadmium-binding

protein;

purification;

characterisation

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Journal of Agricultural and Food Chemistry

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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

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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

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

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

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

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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,

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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

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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

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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

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

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

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

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

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

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metalloproteins can also provide valuable insights to the mechanisms of microbial

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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

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

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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,

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

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

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

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

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

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

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

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

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a fine powder.

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Extraction of cadmium-binding protein.

Mycelia powder was homogenized

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

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

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

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

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cadmium-binding protein extract for subsequent purification.

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

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

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

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The cadmium concentration was determined by a Graphite Furnace Atomic

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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

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

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

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

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

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

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

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

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

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

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

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

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Purification of cadmium-binding protein. A combination of ammonium

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

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

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

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

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The crude cadmium-binding protein extract was saturated by ammonium

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

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

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

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content in each supernatant fraction was determined by BCA assay and

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

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precipitation gradient.

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The protein precipitated by ammonium sulphate saturation was dissolved in

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

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

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

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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.

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

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

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

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

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freeze-dried for further study.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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,

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Germany).25

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Identification of amino acid sequences of cadmium-binding protein by

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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The collected mass spectrometric data were queried against the

most recent

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

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

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

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Inc. (Norristown, PA).

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

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

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using

Taq

polymerase

(Takara)

and

the

following

primers:

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

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

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

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

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

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

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

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

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spectrum background.

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Intrinsic

fluorescence

emission

spectra.

The

intrinsic

fluorescence

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

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

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

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

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

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

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

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

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

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

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without the background solution interference.

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

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

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

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

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(P < 0.05).

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RESULTS AND DISCUSSION

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Purification and determination of cadmium-binding protein. The potential

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

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

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

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

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

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redundant

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

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

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

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cadmium-binding proteins.

proteins

(none

cadmium-binding)

was

removed.

In

addition,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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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