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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
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
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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
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%,
29
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.
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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
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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
97
Ltd., Beijing, China) using BSA as a standard.19
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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
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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
195
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
203
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
209
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
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
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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
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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
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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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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
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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
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.
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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
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