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cDNA cloning and expression analysis of a hepcidin gene from yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae)
Accepted Manuscript cDNA cloning and expression analysis of a hepcidin gene from yellow catfish Pelteobagrus fulvidraco (siluriformes: Bagridae) Qiu-Ning Liu, Zhao-Zhe Xin, Dai-Zhen Zhang, Sen-Hao Jiang, Xin-Yue Chai, ZhengFei Wang, Chao-Feng Li, Chun-Lin Zhou, Bo-Ping Tang PII:
S1050-4648(16)30692-1
DOI:
10.1016/j.fsi.2016.10.049
Reference:
YFSIM 4277
To appear in:
Fish and Shellfish Immunology
Received Date: 18 July 2016 Revised Date:
15 October 2016
Accepted Date: 30 October 2016
Please cite this article as: Liu Q-N, Xin Z-Z, Zhang D-Z, Jiang S-H, Chai X-Y, Wang Z-F, Li C-F, Zhou CL, Tang B-P, cDNA cloning and expression analysis of a hepcidin gene from yellow catfish Pelteobagrus fulvidraco (siluriformes: Bagridae), Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.10.049. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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cDNA cloning and expression analysis of a hepcidin gene from yellow
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catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae)
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Qiu-Ning Liu, Zhao-Zhe Xin, Dai-Zhen Zhang, Sen-Hao Jiang, Xin-Yue Chai,
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Zheng-Fei Wang, Chao-Feng Li, Chun-Lin Zhou, Bo-Ping Tang*
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Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Synthetic
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Innovation Center for Coastal Bio-agriculture, Jiangsu Provincial Key Laboratory of
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Coastal Wetland Bioresources and Environmental Protection, School of Ocean and
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Biological Engineering, Yancheng Teachers University, Yancheng 224051, Jiangsu
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Province, People's Republic of China.
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* Corresponding author: Bo-Ping Tang
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E-mail: [email protected] (Bo-Ping Tang)
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Address: Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Provincial
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Key
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Protection, Jiangsu Synthetic Innovation Center for Coastal Bio-agriculture, School of
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Ocean and Biological Engineering, Yancheng Teachers University, Yancheng 224001,
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Jiangsu Province, People's Republic of China.
Coastal
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ABSTRACT
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Hepcidin is a small, cysteine-rich antimicrobial peptide with a highly conserved
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β-sheet structure that plays a vital role in innate host immunity against pathogenic
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organisms. In this study, a hepcidin gene was identified in Pelteobagrus fulvidraco, an
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economically important freshwater fish in China. The gene is named PfHep. The
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complete PfHep cDNA was 723 bp, including a 5'-untranslated region (UTR) of 102 1
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of 93 amino acids, which includes a predicted signal peptide and the Hepcidin domain.
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The predicted mature, cationic PfHep protein has a typical hepcidin RX(K/R)R motif
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and eight conserved cysteine residues. The deduced PfHep protein sequence has 70%,
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54% and 39% percent identity with hepcidins from Ictalurus punctatus, Danio rerio,
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and Homo sapiens, respectively. The predicted tertiary structure of PfHep is very
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similar to that of hepcidin in other animals. Phylogenetic analysis revealed that PfHep
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is closely related to the hepcidins of I. punctatus and I. furcatus. Real-time
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quantitative reverse transcription-PCR showed that the PfHep gene was expressed
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most in liver of healthy P. fulvidraco, and expressed to some extent in all the tissues
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tested. After challenge with lipopolysaccharide and polyriboinosinic:polyribocytidylic
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acid (poly I:C), respectively, the expression levels of PfHep were markedly
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upregulated in liver, spleen, head kidney and blood at different time points. Together
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these results imply that PfHep may be an important component of the innate immune
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system and be involved in immune defense against invading pathogens.
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Keywords: Hepcidin; Pelteobagrus fulvidraco; Expression analysis; Immune response
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1. Introduction
Antimicrobial
peptides
(AMPs),
important
antibacterial
proteins
with
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evolutionarily conserved peptides, are widely-distributed in vertebrates, invertebrates,
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and plants, and play important roles in innate immunity against various pathogens
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including fungi, Gram-positive and Gram-negative bacteria, viruses, and protozoa,
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and even various types of cancer cells [1,2,3,4]. In recent years, a large number of
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AMPs have been isolated and characterized in fish; they are classified into four major
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groups based on their structures: I. α-helical linear AMPs (chrysophsin [5], misgurnin
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[6], moronecidin [7], pardaxin [8], piscidin [9], pleurocidin [10], etc.); II. cyclic or
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open-ended cyclic cysteine-containing AMPs (cathelicidin [11], defensin [12], 2
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hepcidin [13,14], etc.); III. specific amino-acid-rich AMPs (parasin I [15], hipposin
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[16], oncorhyncin III [17], oncorhyncin II [18], bactericidal permeability-increasing
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protein [19], etc.); and IV. larger and globular AMPs (NK-lysin [20], etc.). Hepcidin is a small, cysteine-rich AMP that is involved in innate immune defense
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against pathogenic organisms [21]. Hepcidin proteins in different species share highly
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conserved motifs, such as an RX(K/R)R motif. They also contain six to eight cysteine
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residues at conserved positions, suggesting that the disulfide bridges of hepcidin are
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essential for the antimicrobial activity of the peptide [22]. Hepcidin was first isolated
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from human plasma ultrafiltrate and urine [23]. Fish hepcidin was initially identified
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and isolated from the gill of hybrid striped bass, white bass (Morone chrysops) ×
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striped bass (M. saxatilis) [13], and since then hepcidins have been identified in many
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fish species [24]. The teleost hepcidin genes contain three exons separated by two
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introns that encode a prepropeptide which consists of a highly conserved signal
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peptide (24 residues), a prodomain (approximately 38–40 residues), and a mature
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peptide (19–27 residues) [25]. The expression of fish hepcidins has been found to be
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most abundant in liver, but the protein is transcribed in a variety of other tissues
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[26,27,28]. Similar to mammalian hepcidins, the expression levels of fish hepcidins in
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different tissues can be upregulated conspicuously by various pathogens and
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simulated
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polyinosinic:polycytidylic acid (poly I:C), vaccination, and so on [29,30,31,32,33].
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However, little information is available on the regulation and genomic structure of
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hepcidin in the yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae).
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pathogen
bacteria,
lipopolysaccharide
(LPS),
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P. fulvidraco is a freshwater fish widespread in lakes and rivers in eastern Asia
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that is widely cultured in Asian countries, especially China, because of its delicious
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meat and high market value [34]. Recently, P. fulvidraco has been used as an
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experimental model fish to study breeding technique, development, lipid metabolism,
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and toxicology [35-38]. Many infectious diseases occur in farmed P. fulvidraco
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causing large economic losses; however, little is known about its immunity. 3
ACCEPTED MANUSCRIPT Suppression subtractive hybridization (SSH) libraries have been constructed to
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selectively amplify and identify cDNAs that are differentially expressed in P.
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fulvidraco in response to LPS [39]. An expressed sequence tag (EST) similar to
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hepcidin was identified that showed high similarity with hepcidins of other fish. Here,
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the full-length cDNA of the P. fulvidraco hepcidin gene was obtained by RACE-PCR,
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and expression patterns of the gene were investigated in different tissues and in
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response to immune system challenge. The data provide novel insight into the role of
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hepcidin in P. fulvidraco.
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2. Materials and methods
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2.1. Experimental fish and immune response
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P. fulvidraco with an average weight of 200 ± 10 g were collected from the tiger
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bridge market, Yancheng, Jiangsu Province, China, in April 2015 and were
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acclimated at 24 °C for two weeks before commencing the experiment. Thirteen
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tissues were dissected to assess hepcidin tissue distribution: blood, brain, gill, head
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kidney, heart, intestine, liver, muscle, ovary, skin, spleen, testis, and trunk kidney.
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Sixty individuals were distributed equally and randomly in three PVC tanks at room
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temperature. Twenty fish were treated with 100 µl of phosphate buffered saline (PBS)
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as a control, 100 µL of LPS (5 mg/L; L-2654, Sigma), and 100 µL of polyriboinosinic
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polyribocytidylic acid (poly I:C, 50 mg/L, P9582, Sigma), respectively. After
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treatment, four tissues (blood, head kidney, liver and spleen) were collected at 3, 6, 12,
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24, 36, and 48 h, frozen in liquid nitrogen and then stored at −80 °C.
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2.2. RNA extraction and cDNA synthesis
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Total RNA was extracted using TRIzol reagent (Aidlab, China) according to the
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manufacturer’s instructions. RNase-free DNase I was used to remove contaminating
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genomic DNA (Promega, USA). RNA integrity and DNA contamination were
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assessed on a 1% formaldehyde gel. The concentration of RNA in the samples was
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measured using a NanoDrop 2000c spectrophotometer (NanoDrop, USA). Only 4
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First strand cDNA was generated by using 1 µg of total liver RNA per sample with a
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TruScript cDNA Synthesis Kit (Aidlab, China). For rapid amplification of the cDNA
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ends (RACE), single-stranded cDNAs were synthesized from liver using the
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SMART™ RACE cDNA Amplification Kit (Clontech, USA).
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2.3. Cloning of the PfHep gene
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A suppression subtractive hybridization library of P. fulvidraco head kidney
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cDNA has been constructed in our lab [39]. An EST encoding a hepcidin homolog
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was isolated by random EST sequencing. Oligonucleotide primers (Table 1) were
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designed using Primer Premier 5.0 Software to obtain the full-length cDNA. The
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primers RC3 and RC5 were used for RACE-PCR. The program consisted of 5 min at
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94 °C, followed by five cycles of 94 °C for 1 min and 65 °C for 2 min, and then 35
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cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s. The PCR products were
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analyzed on 1% agarose gels (Aidlab, China), then purified PCR products were
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ligated into the T-vector (Sangon, China) and sequenced (Sunbiotech, China).
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2.4. Sequence analysis of PfHep
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BLAST searches were performed at http://www.ncbi.nlm.nih.gov/blast.cgi.
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Translation of the cDNA was performed using the Expert Protein Analysis System
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(ExPAsy; http://au.expasy.org/). An open reading frame (ORF) and the deduced
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amino acid sequence were identified using DNASTAR software (Madison, USA).
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The isoelectric point (pI) and molecular weight (MW) of the deduced amino acid
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sequences were predicted using the Compute pI/MW Tool at the ExPAsy site
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(http://web.expasy.org/compute_pi/).
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performed using the SignalP 4.0 Server (http://www.cbs.dtu.dk/services/SignalP/). A
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motif scan was performed at http://hits.isb-sib.ch/cgi-bin/motif_scan. Functional
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domains were predicted using SMART software (http://smart.embl-heidelberg.de/).
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The transmembrane protein topological structure was analyzed with the TMHMM
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server on-line tool (http://www.cbs.dtu.dk/services/TMHMM/). The deduced amino
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Putative
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signal
peptide
prediction
was
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acid sequence of PfHep was submitted for automated protein structure homology
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modeling using the SwissModel protein fold server (http://swissmodel.expasy.org).
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2.5. Homologous alignment and phylogenetic analysis Homology searches were performed using BLASTn and BLASTp at the National
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Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). The
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amino acid sequences of hepcidin from different organisms used for phylogenetic
147
analysis were downloaded from the GenBank database. Multiple sequence alignments
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were carried out using Clustal X software [40]. A phylogenetic tree was constructed
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using Molecular Evolutionary Genetics Analysis (MEGA) version 6.0 [41]. The data
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were analyzed using Poisson correction, and gaps were removed by complete deletion.
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The topological stability of the neighbor-joining (NJ) trees was evaluated by 1000
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bootstrap replications.
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2.6. qPCR analysis of expression of PfHep
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Real-time quantitative reverse transcription-PCR (qPCR) was performed to
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determine mRNA expression levels of the PfHep gene in several tissues and following
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simulated pathogen infection. The β-actin gene (GenBank No. EU161065) was used
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as an internal reference. The primers used in qPCR, designed using Primer Premier
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5.0 software, are listed in Table 1. The qPCRs were performed using a Mastercycler
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ep realplex (Eppendorf, Germany) using the 2× SYBR Green qPCR Mix Kit (Aidlab,
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China). Reaction mixtures (20 µL) contained 10 µL 2× SYBR Green qPCR Mix, 1 µL
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forward and reverse primers, 1 µL cDNA, and 7 µL RNase-free H2O. The PCR
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procedure was as follows: 95 °C for 10 s, followed by 40 cycles of 95°C for 15 s,
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58°C for 15 s and 72°C for 30 s. At the end of the reaction, a melting curve was
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produced by monitoring the fluorescence continuously while slowly heating the
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sample from 60 to 95°C. Each independent experiment was conducted in triplicate
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and the relative expression level of the gene of interest was determined using the
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method described in [42].
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2.7. Data analysis Data are presented as the mean ± standard error of the mean, the SPSS 16.0
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program (SPSS, USA) was used to Statistical analysis. The data were subjected to a
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one-way analysis of variance (ANOVA), followed by Duncan’s Multiple Range test,
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and differences were considered statistically significant when the P-value was < 0.05
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and highly significant for P < 0.01.
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3. Results and discussion
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3.1. Sequence analysis of the PfHep gene
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The PfHep gene was isolated from P. fulvidraco liver. The nucleotide and
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deduced amino acid sequences of PfHep are shown in Fig. 1. The full-length cDNA
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fragment of PfHep was obtained by using RACE-PCR, and contains a 102-bp
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5′-untranslated sequence, a putative ORF of 282 bp encoding a polypeptide of 93
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amino acids which contains the Hepcidin domain, and a 339-bp 3′-untranslated region
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with an 18-bp poly(A) tail. However, no putative polyadenylation signal was observed
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in the P. fulvidraco gene, similar to observations for Cyprinus carpio and Epinephelus
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coioides [43,44]. This cDNA sequence has been deposited in NCBI under GenBank
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accession number KX505868.
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Based on the entire amino acid sequence, the predicted molecular weight and the
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isoelectric point of PfHep were 10.36 kDa and 7.76, respectively. A signal peptide
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was predicted by the SignalP 4.1 Server: the signal peptide cleavage sites of PfHep
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were between Ala23 and Val24, suggesting that it is a secretory protein.
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Transmembrane topological structure was found in PfHep protein (at position 4–26) .
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Motif-scan results indicated that the PfHep protein contains two casein kinase II
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phosphorylation sites (at positions 28–31 and 53–56), a N-myristoylation site (at
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position 86–91), a protein kinase C phosphorylation site (at position 66–68), a cAMP-
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and cGMP-dependent protein kinase phosphorylation site (at position 67–70), and a
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microbody C-terminal targeting signal (at position 91–93) (Fig. 1). As shown in Fig.
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ACCEPTED MANUSCRIPT S1, the tertiary structure of the PfHep protein predicted by the SwissModel protein
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fold server contains two distorted β-sheets with an unusual vicinal disulfide bridge
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found at the turn of the hairpin, which is probably of functional significance. Such a
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disulfide would be similar to that in other animals and suggest that Hep proteins have
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similar functions [45]. By using SMART software, conserved domains were predicted:
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the PfHep protein contains the Hepcidin domain, which is involved in innate immune
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defense against invading pathogens and also acts as a signaling molecule in iron
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metabolism [46].
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3.2. Homologous alignment and phylogenetic analysis
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As Fig. 2 shows, the deduced amino acid sequence of PfHep was aligned with
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several other known and predicted hepcidin peptides using Clustal X software. The
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PfHep protein sequence has 70%, 54% and 39% percent identity with those from
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Ictalurus punctatus, Danio rerio, and Homo sapiens, respectively. In addition,
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sequence alignment and prediction of functional domains revealed that the amino acid
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sequences of the conserved features of PfHep were very similar or identical to those
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of known hepcidins; for example, the signal peptide sequence of hepcidin is highly
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conserved among all the tested animals; an RX(K/R)R motif for recognition and
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cleavage by furin propeptide convertases was observed at position 65–68 [47]; and a
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key feature of hepcidins, a stretch of eight cysteines in the C-terminal region, was
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conserved compared with the hepcidins of other animals. This is an unusually high
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amount of Cys compared with the composition of other “cysteine-rich” antimicrobial
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peptides, and all of the Cys residues are bridged, which makes this peptide highly
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constrained [48]. Further studies need to be conducted in fish to identify the
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relationship between the structural features and the immune activities of hepcidin.
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To classify and analyze the molecular evolution of hepcidins, 42 representative
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hepcidin sequences were used to reconstruct their phylogenetic relationships based on
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amino acid sequences. The evolutionary history was inferred using the NJ method,
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with complete deletion of gaps and 1000 bootstrap replications. As shown in Fig. 3, 8
ACCEPTED MANUSCRIPT the sequences used could be classified into three main clades. Fish hepcidins clustered
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in two main groups; PfHep was closely related to the hepcidins of I. punctatus and I.
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furcatus, and then clustered with D. rerio, and then fell into a clade with H. sapiens.
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Overall, the phylogenetic tree analysis was consistent with the phylogenetic
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relationships of the organisms concerned, confirmed the identification of the PfHep
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gene, and showed the strong orthology of PfHep to its counterparts identified in other
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species. Together, these results suggest that hepcidin proteins have remained highly
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conserved throughout the evolution of animals [49].
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3.3. Expression of the hepcidin gene in healthy P. fulvidraco
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qPCR was used to establish the expression pattern of PfHep in different tissues.
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The expression level in each of the tissues examined was normalized to that of β-actin.
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Relative expression fold-differences were calculated by comparison with the
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expression in muscle. The PfHep gene was expressed in all tested tissues. The highest
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expression level was observed in liver, which is thought to be an important
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metabolizing organ, followed by spleen and intestine. Relatively low expression
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levels were observed in brain, head kidney, blood, skin, gill and trunk kidney (Fig. 4).
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This pattern suggests that PfHep plays an important role throughout the entire life
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cycle of the fish. Similar results were found in other fish: the expression of hepcidin
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in other fish species was detected in a wide range of tissues; it was highly expressed
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in the liver of all the reported fish, and expressed in the spleen of most of these
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species [26,29,31,51,52]; however, high expression has been detected in the head
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kidney of some species [28,33]. Hepcidin in human and mouse is mainly produced by
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hepatocytes [53]. In addition, it has been shown that hepcidin isoforms have various
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expression patterns in different development stages of different organisms; the
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expression of hepcidin can be detected as early as the fertilized egg of Megalobrama
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amblycephala and channel catfish [50,54]. Taken together, we conclude that hepcidin
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is primarily located in the immune organs, including spleen, head kidney and liver.
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3.4. Expression of PfHep gene in response to simulated pathogen infection
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levels of PfHep were identified using qPCR in immune tissues in response to LPS and
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poly I:C challenge, respectively. Four tissues which are involved in immune response
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or pathogen attachment were selected — blood, head kidney, liver and spleen — and
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analyzed at early time points after challenge. As Fig. 5 shows, following poly I:C
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challenge, PfHep showed different expression patterns in the four tissues. The
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expression of PfHep was significantly upregulated in liver and head kidney at was at
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its highest observed level at 3 h. In spleen, PfHep was highly expressed and reached
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its peak at 12 h, and the peak in blood was detected at 24 h. After LPS challenge, the
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expression of PfHep reached a peak at 12 h in spleen, at 24 h in liver and head kidney,
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and at 36 h in blood (Fig. 6). Expression of hepcidin has been shown to be induced in
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fish after pathogen challenge. The mRNA transcript levels of hepcidin from Alphestes
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immaculatus were upregulated in the head kidney and spleen after injection with LPS
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[32]. Similar results were also found in Pseudosciana crocea, where the expression of
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hepcidin was induced in stomach, spleen and head kidney after stimulation by LPS
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[55]. The mRNA levels of Scophthalmus maximus hepcidin were significantly
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increased in liver and spleen upon injection of Listonella anguillarum (a
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Gram-negative γ-proteobacterium) [56]. The expression levels of Oncorhynchus
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mykiss and Gadus morhua hepcidin were upregulated in response to poly I:C
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challenge [28,57]. Two hepcidins from Scatophagus argus synthesized in vitro
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possessed antibacterial activity and antiviral activity against Siniperca chuatsi
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rhabdovirus (an enveloped single-stranded RNA virus) and Micropterus salmoides
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reovirus (a non-enveloped double-stranded RNA virus) [58]. These results imply that
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hepcidin plays a role in the innate immune system of fish.
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4. Conclusions
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Hepcidin, a cysteine-rich AMP, plays a key role in host innate immune systems
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and displays other functions such as iron regulation. The gene PfHep was isolated and
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cloned for the first time from Pelteobagrus fulvidraco, and we analyzed its 10
ACCEPTED MANUSCRIPT phylogenetic relationships and expression pattern following different simulated
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pathogen challenges. The results suggest that PfHep might play an important role in
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mediating an innate immune response to pathogen infection. Our data on the
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expression pattern of this gene should provide valuable information that may yield
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insights into the molecular mechanisms of immune responses. However, further
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functional studies should be carried out to characterize the precise roles of hepcidin in
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the disease resistance of fish.
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Acknowledgements
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The authors declare no competing interests. This work was supported by the
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Natural Science Foundation of Jiangsu Province (BK20160444), the National Natural
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Science Foundation of China (31640074, 31600740, and 31672267), the Natural
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Science Research General Program of Jiangsu Provincial Higher Education
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Institutions (15KJB240002 and 12KJA180009), the Special Guide Fund Project of
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Agricultural Science and Technology Innovation of Yancheng city (YKN2014022),
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the Open Project of Jiangsu Provincial Key Laboratory of Coastal Wetland
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Bioresources and Environmental Protection (JLCBE14006), the Jiangsu Provincial
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Key
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JKLBS2015004).
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for
Bioresources
of
Saline
Soils
(JKLBS2014013
and
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Fig. 1. Complete nucleotide and deduced amino acid sequence of the Pelteobagrus
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fulvidraco hepcidin (PfHep) gene. The amino acid residues are represented by
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one-letter symbols. The open reading frame (ORF), from the initiation codon ATG to
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the termination codon TAA is to be uppercase. Functional features predicted using
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Motif-scan and SMART analysis are indicated.
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Fig. 2. Sequence alignment of the PfHep protein with homologs from other organisms.
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The Hepcidin proteins from species: I. furcatus AAX39714, I. punctatus ABA43709,
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G. rarus AKL71657, D. rerio AAR18594, A. mississippiensis KYO28626, C.
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siamensis ADA68357, B. mutus ELR56119, and H. sapiens NP_066998 were
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included. Identical amino acids are highlighted in black, similar amino acids are
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highlighted in gray. Key conserved domains, motifs and residues are indicated.
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Fig. 3. A phylogenetic tree of hepcidin protein sequences constructed using MEGA
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software version 6.06, the neighbor-joining method and 1000 bootstrap replicates.
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Accession numbers of the sequences are shown.
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Fig. 4. Expression analysis of the PfHep gene in various tissues of healthy P.
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fulvidraco. Relative expression levels were analyzed by qPCR and the β-actin gene
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was used as an internal standard. Gene expression level in the control group was set to
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1.0. The data were expressed as the mean fold change (means ± SE, n = 3).
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Fig. 5. Relative mRNA expression levels of PfHep in different tissues in response to
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polyriboinosinic:polyribocytidylic acid (poly I:C) challenge, determined by qPCR.
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The β-actin gene was used as an internal standard. Gene expression level in the
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control group was set to 1.0. The data were expressed as the mean fold change (means
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± SE, n = 3) relative to the untreated group. The values were significantly different to
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the control at the same time point when marked with asterisks (*P < 0.05, **P < 0.01,
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***P < 0.001).
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Fig. 6. Relative mRNA expression levels of PfHep in different tissues in response to
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lipopolysaccharide challenge, determined by qPCR. The β-actin gene was used as an
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internal standard. Gene expression level in the control group was set to 1.0. The data
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group. The values were significantly different to the control at the same time point
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when marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).
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Fig. S1. Tertiary structure of PfHep protein predicted by the SwissModel protein fold
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server.
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Table 1. Primers used in this study
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Sequence (5’-3’)
Purpose
TCGACACAGAGACAGGTGACT
RACE-PCR
RC3
ACATTAACTTATTTATCCTGCTG
RACE-PCR
F1
CTGGAGAAGCCTGTGGAAAC
R1
CCCATCTTGAGGGATGAAAC
Actin-F
GCACAGTAAAGGCGTTGTGA
Actin-R
ACATCTGCTGGAAGGTGGAC
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RC5
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qPCR
qPCR qPCR
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ACCEPTED MANUSCRIPT Highlights
A hepcidin cDNA sequence was cloned from yellow catfish Pelteobagrus fulvidraco
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Hepcidin mRNA levels were detected in a wide range of P. fulvidraco tissues
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Hepcidin mRNA expression was upregulated on challenge with Poly I:C or LPS
S1050-4648(16)30692-1
DOI:
10.1016/j.fsi.2016.10.049
Reference:
YFSIM 4277
To appear in:
Fish and Shellfish Immunology
Received Date: 18 July 2016 Revised Date:
15 October 2016
Accepted Date: 30 October 2016
Please cite this article as: Liu Q-N, Xin Z-Z, Zhang D-Z, Jiang S-H, Chai X-Y, Wang Z-F, Li C-F, Zhou CL, Tang B-P, cDNA cloning and expression analysis of a hepcidin gene from yellow catfish Pelteobagrus fulvidraco (siluriformes: Bagridae), Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.10.049. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
cDNA cloning and expression analysis of a hepcidin gene from yellow
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catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae)
3
Qiu-Ning Liu, Zhao-Zhe Xin, Dai-Zhen Zhang, Sen-Hao Jiang, Xin-Yue Chai,
4
Zheng-Fei Wang, Chao-Feng Li, Chun-Lin Zhou, Bo-Ping Tang*
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Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Synthetic
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Innovation Center for Coastal Bio-agriculture, Jiangsu Provincial Key Laboratory of
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Coastal Wetland Bioresources and Environmental Protection, School of Ocean and
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Biological Engineering, Yancheng Teachers University, Yancheng 224051, Jiangsu
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Province, People's Republic of China.
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* Corresponding author: Bo-Ping Tang
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E-mail: [email protected] (Bo-Ping Tang)
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Address: Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Provincial
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Key
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Protection, Jiangsu Synthetic Innovation Center for Coastal Bio-agriculture, School of
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Ocean and Biological Engineering, Yancheng Teachers University, Yancheng 224001,
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Jiangsu Province, People's Republic of China.
Coastal
Wetland
Bioresources
and
Environmental
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Laboratory
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ABSTRACT
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Hepcidin is a small, cysteine-rich antimicrobial peptide with a highly conserved
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β-sheet structure that plays a vital role in innate host immunity against pathogenic
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organisms. In this study, a hepcidin gene was identified in Pelteobagrus fulvidraco, an
29
economically important freshwater fish in China. The gene is named PfHep. The
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complete PfHep cDNA was 723 bp, including a 5'-untranslated region (UTR) of 102 1
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32
of 93 amino acids, which includes a predicted signal peptide and the Hepcidin domain.
33
The predicted mature, cationic PfHep protein has a typical hepcidin RX(K/R)R motif
34
and eight conserved cysteine residues. The deduced PfHep protein sequence has 70%,
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54% and 39% percent identity with hepcidins from Ictalurus punctatus, Danio rerio,
36
and Homo sapiens, respectively. The predicted tertiary structure of PfHep is very
37
similar to that of hepcidin in other animals. Phylogenetic analysis revealed that PfHep
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is closely related to the hepcidins of I. punctatus and I. furcatus. Real-time
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quantitative reverse transcription-PCR showed that the PfHep gene was expressed
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most in liver of healthy P. fulvidraco, and expressed to some extent in all the tissues
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tested. After challenge with lipopolysaccharide and polyriboinosinic:polyribocytidylic
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acid (poly I:C), respectively, the expression levels of PfHep were markedly
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upregulated in liver, spleen, head kidney and blood at different time points. Together
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these results imply that PfHep may be an important component of the innate immune
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system and be involved in immune defense against invading pathogens.
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Keywords: Hepcidin; Pelteobagrus fulvidraco; Expression analysis; Immune response
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1. Introduction
Antimicrobial
peptides
(AMPs),
important
antibacterial
proteins
with
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evolutionarily conserved peptides, are widely-distributed in vertebrates, invertebrates,
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and plants, and play important roles in innate immunity against various pathogens
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including fungi, Gram-positive and Gram-negative bacteria, viruses, and protozoa,
53
and even various types of cancer cells [1,2,3,4]. In recent years, a large number of
54
AMPs have been isolated and characterized in fish; they are classified into four major
55
groups based on their structures: I. α-helical linear AMPs (chrysophsin [5], misgurnin
56
[6], moronecidin [7], pardaxin [8], piscidin [9], pleurocidin [10], etc.); II. cyclic or
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open-ended cyclic cysteine-containing AMPs (cathelicidin [11], defensin [12], 2
ACCEPTED MANUSCRIPT 58
hepcidin [13,14], etc.); III. specific amino-acid-rich AMPs (parasin I [15], hipposin
59
[16], oncorhyncin III [17], oncorhyncin II [18], bactericidal permeability-increasing
60
protein [19], etc.); and IV. larger and globular AMPs (NK-lysin [20], etc.). Hepcidin is a small, cysteine-rich AMP that is involved in innate immune defense
62
against pathogenic organisms [21]. Hepcidin proteins in different species share highly
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conserved motifs, such as an RX(K/R)R motif. They also contain six to eight cysteine
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residues at conserved positions, suggesting that the disulfide bridges of hepcidin are
65
essential for the antimicrobial activity of the peptide [22]. Hepcidin was first isolated
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from human plasma ultrafiltrate and urine [23]. Fish hepcidin was initially identified
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and isolated from the gill of hybrid striped bass, white bass (Morone chrysops) ×
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striped bass (M. saxatilis) [13], and since then hepcidins have been identified in many
69
fish species [24]. The teleost hepcidin genes contain three exons separated by two
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introns that encode a prepropeptide which consists of a highly conserved signal
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peptide (24 residues), a prodomain (approximately 38–40 residues), and a mature
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peptide (19–27 residues) [25]. The expression of fish hepcidins has been found to be
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most abundant in liver, but the protein is transcribed in a variety of other tissues
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[26,27,28]. Similar to mammalian hepcidins, the expression levels of fish hepcidins in
75
different tissues can be upregulated conspicuously by various pathogens and
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simulated
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polyinosinic:polycytidylic acid (poly I:C), vaccination, and so on [29,30,31,32,33].
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However, little information is available on the regulation and genomic structure of
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hepcidin in the yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae).
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pathogen
bacteria,
lipopolysaccharide
(LPS),
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P. fulvidraco is a freshwater fish widespread in lakes and rivers in eastern Asia
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that is widely cultured in Asian countries, especially China, because of its delicious
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meat and high market value [34]. Recently, P. fulvidraco has been used as an
83
experimental model fish to study breeding technique, development, lipid metabolism,
84
and toxicology [35-38]. Many infectious diseases occur in farmed P. fulvidraco
85
causing large economic losses; however, little is known about its immunity. 3
ACCEPTED MANUSCRIPT Suppression subtractive hybridization (SSH) libraries have been constructed to
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selectively amplify and identify cDNAs that are differentially expressed in P.
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fulvidraco in response to LPS [39]. An expressed sequence tag (EST) similar to
89
hepcidin was identified that showed high similarity with hepcidins of other fish. Here,
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the full-length cDNA of the P. fulvidraco hepcidin gene was obtained by RACE-PCR,
91
and expression patterns of the gene were investigated in different tissues and in
92
response to immune system challenge. The data provide novel insight into the role of
93
hepcidin in P. fulvidraco.
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2. Materials and methods
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2.1. Experimental fish and immune response
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P. fulvidraco with an average weight of 200 ± 10 g were collected from the tiger
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bridge market, Yancheng, Jiangsu Province, China, in April 2015 and were
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acclimated at 24 °C for two weeks before commencing the experiment. Thirteen
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tissues were dissected to assess hepcidin tissue distribution: blood, brain, gill, head
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kidney, heart, intestine, liver, muscle, ovary, skin, spleen, testis, and trunk kidney.
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Sixty individuals were distributed equally and randomly in three PVC tanks at room
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temperature. Twenty fish were treated with 100 µl of phosphate buffered saline (PBS)
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as a control, 100 µL of LPS (5 mg/L; L-2654, Sigma), and 100 µL of polyriboinosinic
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polyribocytidylic acid (poly I:C, 50 mg/L, P9582, Sigma), respectively. After
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treatment, four tissues (blood, head kidney, liver and spleen) were collected at 3, 6, 12,
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24, 36, and 48 h, frozen in liquid nitrogen and then stored at −80 °C.
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2.2. RNA extraction and cDNA synthesis
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Total RNA was extracted using TRIzol reagent (Aidlab, China) according to the
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manufacturer’s instructions. RNase-free DNase I was used to remove contaminating
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genomic DNA (Promega, USA). RNA integrity and DNA contamination were
111
assessed on a 1% formaldehyde gel. The concentration of RNA in the samples was
112
measured using a NanoDrop 2000c spectrophotometer (NanoDrop, USA). Only 4
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First strand cDNA was generated by using 1 µg of total liver RNA per sample with a
115
TruScript cDNA Synthesis Kit (Aidlab, China). For rapid amplification of the cDNA
116
ends (RACE), single-stranded cDNAs were synthesized from liver using the
117
SMART™ RACE cDNA Amplification Kit (Clontech, USA).
118
2.3. Cloning of the PfHep gene
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A suppression subtractive hybridization library of P. fulvidraco head kidney
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cDNA has been constructed in our lab [39]. An EST encoding a hepcidin homolog
121
was isolated by random EST sequencing. Oligonucleotide primers (Table 1) were
122
designed using Primer Premier 5.0 Software to obtain the full-length cDNA. The
123
primers RC3 and RC5 were used for RACE-PCR. The program consisted of 5 min at
124
94 °C, followed by five cycles of 94 °C for 1 min and 65 °C for 2 min, and then 35
125
cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s. The PCR products were
126
analyzed on 1% agarose gels (Aidlab, China), then purified PCR products were
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ligated into the T-vector (Sangon, China) and sequenced (Sunbiotech, China).
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2.4. Sequence analysis of PfHep
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BLAST searches were performed at http://www.ncbi.nlm.nih.gov/blast.cgi.
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Translation of the cDNA was performed using the Expert Protein Analysis System
131
(ExPAsy; http://au.expasy.org/). An open reading frame (ORF) and the deduced
132
amino acid sequence were identified using DNASTAR software (Madison, USA).
133
The isoelectric point (pI) and molecular weight (MW) of the deduced amino acid
134
sequences were predicted using the Compute pI/MW Tool at the ExPAsy site
135
(http://web.expasy.org/compute_pi/).
136
performed using the SignalP 4.0 Server (http://www.cbs.dtu.dk/services/SignalP/). A
137
motif scan was performed at http://hits.isb-sib.ch/cgi-bin/motif_scan. Functional
138
domains were predicted using SMART software (http://smart.embl-heidelberg.de/).
139
The transmembrane protein topological structure was analyzed with the TMHMM
140
server on-line tool (http://www.cbs.dtu.dk/services/TMHMM/). The deduced amino
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Putative
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ACCEPTED MANUSCRIPT 141
acid sequence of PfHep was submitted for automated protein structure homology
142
modeling using the SwissModel protein fold server (http://swissmodel.expasy.org).
143
2.5. Homologous alignment and phylogenetic analysis Homology searches were performed using BLASTn and BLASTp at the National
145
Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). The
146
amino acid sequences of hepcidin from different organisms used for phylogenetic
147
analysis were downloaded from the GenBank database. Multiple sequence alignments
148
were carried out using Clustal X software [40]. A phylogenetic tree was constructed
149
using Molecular Evolutionary Genetics Analysis (MEGA) version 6.0 [41]. The data
150
were analyzed using Poisson correction, and gaps were removed by complete deletion.
151
The topological stability of the neighbor-joining (NJ) trees was evaluated by 1000
152
bootstrap replications.
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2.6. qPCR analysis of expression of PfHep
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Real-time quantitative reverse transcription-PCR (qPCR) was performed to
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determine mRNA expression levels of the PfHep gene in several tissues and following
156
simulated pathogen infection. The β-actin gene (GenBank No. EU161065) was used
157
as an internal reference. The primers used in qPCR, designed using Primer Premier
158
5.0 software, are listed in Table 1. The qPCRs were performed using a Mastercycler
159
ep realplex (Eppendorf, Germany) using the 2× SYBR Green qPCR Mix Kit (Aidlab,
160
China). Reaction mixtures (20 µL) contained 10 µL 2× SYBR Green qPCR Mix, 1 µL
161
forward and reverse primers, 1 µL cDNA, and 7 µL RNase-free H2O. The PCR
162
procedure was as follows: 95 °C for 10 s, followed by 40 cycles of 95°C for 15 s,
163
58°C for 15 s and 72°C for 30 s. At the end of the reaction, a melting curve was
164
produced by monitoring the fluorescence continuously while slowly heating the
165
sample from 60 to 95°C. Each independent experiment was conducted in triplicate
166
and the relative expression level of the gene of interest was determined using the
167
method described in [42].
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2.7. Data analysis Data are presented as the mean ± standard error of the mean, the SPSS 16.0
170
program (SPSS, USA) was used to Statistical analysis. The data were subjected to a
171
one-way analysis of variance (ANOVA), followed by Duncan’s Multiple Range test,
172
and differences were considered statistically significant when the P-value was < 0.05
173
and highly significant for P < 0.01.
174
3. Results and discussion
175
3.1. Sequence analysis of the PfHep gene
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The PfHep gene was isolated from P. fulvidraco liver. The nucleotide and
177
deduced amino acid sequences of PfHep are shown in Fig. 1. The full-length cDNA
178
fragment of PfHep was obtained by using RACE-PCR, and contains a 102-bp
179
5′-untranslated sequence, a putative ORF of 282 bp encoding a polypeptide of 93
180
amino acids which contains the Hepcidin domain, and a 339-bp 3′-untranslated region
181
with an 18-bp poly(A) tail. However, no putative polyadenylation signal was observed
182
in the P. fulvidraco gene, similar to observations for Cyprinus carpio and Epinephelus
183
coioides [43,44]. This cDNA sequence has been deposited in NCBI under GenBank
184
accession number KX505868.
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Based on the entire amino acid sequence, the predicted molecular weight and the
186
isoelectric point of PfHep were 10.36 kDa and 7.76, respectively. A signal peptide
187
was predicted by the SignalP 4.1 Server: the signal peptide cleavage sites of PfHep
188
were between Ala23 and Val24, suggesting that it is a secretory protein.
189
Transmembrane topological structure was found in PfHep protein (at position 4–26) .
190
Motif-scan results indicated that the PfHep protein contains two casein kinase II
191
phosphorylation sites (at positions 28–31 and 53–56), a N-myristoylation site (at
192
position 86–91), a protein kinase C phosphorylation site (at position 66–68), a cAMP-
193
and cGMP-dependent protein kinase phosphorylation site (at position 67–70), and a
194
microbody C-terminal targeting signal (at position 91–93) (Fig. 1). As shown in Fig.
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ACCEPTED MANUSCRIPT S1, the tertiary structure of the PfHep protein predicted by the SwissModel protein
196
fold server contains two distorted β-sheets with an unusual vicinal disulfide bridge
197
found at the turn of the hairpin, which is probably of functional significance. Such a
198
disulfide would be similar to that in other animals and suggest that Hep proteins have
199
similar functions [45]. By using SMART software, conserved domains were predicted:
200
the PfHep protein contains the Hepcidin domain, which is involved in innate immune
201
defense against invading pathogens and also acts as a signaling molecule in iron
202
metabolism [46].
203
3.2. Homologous alignment and phylogenetic analysis
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As Fig. 2 shows, the deduced amino acid sequence of PfHep was aligned with
205
several other known and predicted hepcidin peptides using Clustal X software. The
206
PfHep protein sequence has 70%, 54% and 39% percent identity with those from
207
Ictalurus punctatus, Danio rerio, and Homo sapiens, respectively. In addition,
208
sequence alignment and prediction of functional domains revealed that the amino acid
209
sequences of the conserved features of PfHep were very similar or identical to those
210
of known hepcidins; for example, the signal peptide sequence of hepcidin is highly
211
conserved among all the tested animals; an RX(K/R)R motif for recognition and
212
cleavage by furin propeptide convertases was observed at position 65–68 [47]; and a
213
key feature of hepcidins, a stretch of eight cysteines in the C-terminal region, was
214
conserved compared with the hepcidins of other animals. This is an unusually high
215
amount of Cys compared with the composition of other “cysteine-rich” antimicrobial
216
peptides, and all of the Cys residues are bridged, which makes this peptide highly
217
constrained [48]. Further studies need to be conducted in fish to identify the
218
relationship between the structural features and the immune activities of hepcidin.
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To classify and analyze the molecular evolution of hepcidins, 42 representative
220
hepcidin sequences were used to reconstruct their phylogenetic relationships based on
221
amino acid sequences. The evolutionary history was inferred using the NJ method,
222
with complete deletion of gaps and 1000 bootstrap replications. As shown in Fig. 3, 8
ACCEPTED MANUSCRIPT the sequences used could be classified into three main clades. Fish hepcidins clustered
224
in two main groups; PfHep was closely related to the hepcidins of I. punctatus and I.
225
furcatus, and then clustered with D. rerio, and then fell into a clade with H. sapiens.
226
Overall, the phylogenetic tree analysis was consistent with the phylogenetic
227
relationships of the organisms concerned, confirmed the identification of the PfHep
228
gene, and showed the strong orthology of PfHep to its counterparts identified in other
229
species. Together, these results suggest that hepcidin proteins have remained highly
230
conserved throughout the evolution of animals [49].
231
3.3. Expression of the hepcidin gene in healthy P. fulvidraco
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qPCR was used to establish the expression pattern of PfHep in different tissues.
233
The expression level in each of the tissues examined was normalized to that of β-actin.
234
Relative expression fold-differences were calculated by comparison with the
235
expression in muscle. The PfHep gene was expressed in all tested tissues. The highest
236
expression level was observed in liver, which is thought to be an important
237
metabolizing organ, followed by spleen and intestine. Relatively low expression
238
levels were observed in brain, head kidney, blood, skin, gill and trunk kidney (Fig. 4).
239
This pattern suggests that PfHep plays an important role throughout the entire life
240
cycle of the fish. Similar results were found in other fish: the expression of hepcidin
241
in other fish species was detected in a wide range of tissues; it was highly expressed
242
in the liver of all the reported fish, and expressed in the spleen of most of these
243
species [26,29,31,51,52]; however, high expression has been detected in the head
244
kidney of some species [28,33]. Hepcidin in human and mouse is mainly produced by
245
hepatocytes [53]. In addition, it has been shown that hepcidin isoforms have various
246
expression patterns in different development stages of different organisms; the
247
expression of hepcidin can be detected as early as the fertilized egg of Megalobrama
248
amblycephala and channel catfish [50,54]. Taken together, we conclude that hepcidin
249
is primarily located in the immune organs, including spleen, head kidney and liver.
250
3.4. Expression of PfHep gene in response to simulated pathogen infection
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252
levels of PfHep were identified using qPCR in immune tissues in response to LPS and
253
poly I:C challenge, respectively. Four tissues which are involved in immune response
254
or pathogen attachment were selected — blood, head kidney, liver and spleen — and
255
analyzed at early time points after challenge. As Fig. 5 shows, following poly I:C
256
challenge, PfHep showed different expression patterns in the four tissues. The
257
expression of PfHep was significantly upregulated in liver and head kidney at was at
258
its highest observed level at 3 h. In spleen, PfHep was highly expressed and reached
259
its peak at 12 h, and the peak in blood was detected at 24 h. After LPS challenge, the
260
expression of PfHep reached a peak at 12 h in spleen, at 24 h in liver and head kidney,
261
and at 36 h in blood (Fig. 6). Expression of hepcidin has been shown to be induced in
262
fish after pathogen challenge. The mRNA transcript levels of hepcidin from Alphestes
263
immaculatus were upregulated in the head kidney and spleen after injection with LPS
264
[32]. Similar results were also found in Pseudosciana crocea, where the expression of
265
hepcidin was induced in stomach, spleen and head kidney after stimulation by LPS
266
[55]. The mRNA levels of Scophthalmus maximus hepcidin were significantly
267
increased in liver and spleen upon injection of Listonella anguillarum (a
268
Gram-negative γ-proteobacterium) [56]. The expression levels of Oncorhynchus
269
mykiss and Gadus morhua hepcidin were upregulated in response to poly I:C
270
challenge [28,57]. Two hepcidins from Scatophagus argus synthesized in vitro
271
possessed antibacterial activity and antiviral activity against Siniperca chuatsi
272
rhabdovirus (an enveloped single-stranded RNA virus) and Micropterus salmoides
273
reovirus (a non-enveloped double-stranded RNA virus) [58]. These results imply that
274
hepcidin plays a role in the innate immune system of fish.
275
4. Conclusions
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Hepcidin, a cysteine-rich AMP, plays a key role in host innate immune systems
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and displays other functions such as iron regulation. The gene PfHep was isolated and
278
cloned for the first time from Pelteobagrus fulvidraco, and we analyzed its 10
ACCEPTED MANUSCRIPT phylogenetic relationships and expression pattern following different simulated
280
pathogen challenges. The results suggest that PfHep might play an important role in
281
mediating an innate immune response to pathogen infection. Our data on the
282
expression pattern of this gene should provide valuable information that may yield
283
insights into the molecular mechanisms of immune responses. However, further
284
functional studies should be carried out to characterize the precise roles of hepcidin in
285
the disease resistance of fish.
286
Acknowledgements
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The authors declare no competing interests. This work was supported by the
288
Natural Science Foundation of Jiangsu Province (BK20160444), the National Natural
289
Science Foundation of China (31640074, 31600740, and 31672267), the Natural
290
Science Research General Program of Jiangsu Provincial Higher Education
291
Institutions (15KJB240002 and 12KJA180009), the Special Guide Fund Project of
292
Agricultural Science and Technology Innovation of Yancheng city (YKN2014022),
293
the Open Project of Jiangsu Provincial Key Laboratory of Coastal Wetland
294
Bioresources and Environmental Protection (JLCBE14006), the Jiangsu Provincial
295
Key
296
JKLBS2015004).
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for
Bioresources
of
Saline
Soils
(JKLBS2014013
and
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Laboratory
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ACCEPTED MANUSCRIPT Figure and Table captions
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Fig. 1. Complete nucleotide and deduced amino acid sequence of the Pelteobagrus
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fulvidraco hepcidin (PfHep) gene. The amino acid residues are represented by
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one-letter symbols. The open reading frame (ORF), from the initiation codon ATG to
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the termination codon TAA is to be uppercase. Functional features predicted using
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Motif-scan and SMART analysis are indicated.
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Fig. 2. Sequence alignment of the PfHep protein with homologs from other organisms.
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The Hepcidin proteins from species: I. furcatus AAX39714, I. punctatus ABA43709,
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G. rarus AKL71657, D. rerio AAR18594, A. mississippiensis KYO28626, C.
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siamensis ADA68357, B. mutus ELR56119, and H. sapiens NP_066998 were
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included. Identical amino acids are highlighted in black, similar amino acids are
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highlighted in gray. Key conserved domains, motifs and residues are indicated.
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Fig. 3. A phylogenetic tree of hepcidin protein sequences constructed using MEGA
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software version 6.06, the neighbor-joining method and 1000 bootstrap replicates.
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Accession numbers of the sequences are shown.
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Fig. 4. Expression analysis of the PfHep gene in various tissues of healthy P.
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fulvidraco. Relative expression levels were analyzed by qPCR and the β-actin gene
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was used as an internal standard. Gene expression level in the control group was set to
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1.0. The data were expressed as the mean fold change (means ± SE, n = 3).
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Fig. 5. Relative mRNA expression levels of PfHep in different tissues in response to
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polyriboinosinic:polyribocytidylic acid (poly I:C) challenge, determined by qPCR.
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The β-actin gene was used as an internal standard. Gene expression level in the
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control group was set to 1.0. The data were expressed as the mean fold change (means
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± SE, n = 3) relative to the untreated group. The values were significantly different to
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the control at the same time point when marked with asterisks (*P < 0.05, **P < 0.01,
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***P < 0.001).
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Fig. 6. Relative mRNA expression levels of PfHep in different tissues in response to
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lipopolysaccharide challenge, determined by qPCR. The β-actin gene was used as an
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internal standard. Gene expression level in the control group was set to 1.0. The data
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group. The values were significantly different to the control at the same time point
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when marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).
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Fig. S1. Tertiary structure of PfHep protein predicted by the SwissModel protein fold
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server.
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Table 1. Primers used in this study
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Sequence (5’-3’)
Purpose
TCGACACAGAGACAGGTGACT
RACE-PCR
RC3
ACATTAACTTATTTATCCTGCTG
RACE-PCR
F1
CTGGAGAAGCCTGTGGAAAC
R1
CCCATCTTGAGGGATGAAAC
Actin-F
GCACAGTAAAGGCGTTGTGA
Actin-R
ACATCTGCTGGAAGGTGGAC
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RC5
qPCR
qPCR
qPCR qPCR
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A hepcidin cDNA sequence was cloned from yellow catfish Pelteobagrus fulvidraco
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Hepcidin mRNA levels were detected in a wide range of P. fulvidraco tissues
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Hepcidin mRNA expression was upregulated on challenge with Poly I:C or LPS