Identification and characterization of a clam ferritin from Sinonovacula constricta

Fish & Shellfish Immunology 30 (2011) 1147e1151

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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Identification and characterization of a clam ferritin from Sinonovacula constricta Chenghua Li a, Hui Li a, Xiurong Su a, Taiwu Li a, b, * a b

Faculty of Life Science and Biotechnology, Ningbo University, 818 Fenghua Road, Ningbo, Zhejiang Province 315211, PR China Ningbo City College of Vocational Technology, Ningbo 315100, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 November 2010 Received in revised form 16 February 2011 Accepted 20 February 2011 Available online 6 March 2011

Ferritin, a major iron storage protein of most living organisms, plays a crucial role in iron metabolism. Here we reported the isolation and characterization of a cDNA of ferritin gene from Sinonovacula constricta (denoted as ScFER). The full-length cDNA of ScFER was of 996 bp, consisting of a 50 -UTR of 120 bp, a 30 -UTR of 360 bp, and a complete open reading frame of 516 bp encoding a polypeptide with 171 amino acid residues. The predicted molecular mass of deduced amnio acid of ScFER was 19.76 kDa and the theoretical pI was 5.07. Quantitative real-time PCR was employed to analyze the expression profiles of ScFER mRNA in muscle, mantle and visceral mass after iron exposure. The peak expression level of ScFER in the three tissues was 1.79-fold, 1.31-fold and 3.51-fold increases in muscle, mantle and visceral mass, respectively. The ployclonal antibodies generated from the recombinant product of ScFER could be specifically identified not only the recombinant product, but also the native protein from muscle. All these results strongly suggested that ScFER was involved in the iron metabolism regulation in S. constricta. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Sinonovacula constricta Ferritin Quantitative real-time PCR Western blot

1. Introduction As an essential nutrient, iron functions as a cofactor in various biological processes, such as oxygen transportation, electron transfer, DNA replication and photosynthesis in living organisms [1,2]. However, uncontrolled high level of free iron in living cells can also seriously affect on organism’s viability and increase the risk of cell damage. Therefore, organisms have evolved to use some regulated proteins to tightly control free iron under optimal concentration, such as ferritin and transferrin [3]. Ferritin, a conserved iron homeostasis protein, had been identified in a wide range of organisms, such as bacteria, fungi, plants, invertebrates and vertebrates. Except to uptake and detoxification of iron, ferritin had been demonstrated to be involved in innate immunity [4,5], shell formation [6], TNFa-induced apoptosis [7], and coupling of nitric oxide [8]. Ferritin complex is composed of 24 subunits, which surrounds an inorganic microcrystalline hollow capable of accommodating up to 4500 Fe3þ [9]. In vertebrates, two types of ferritin subunits (H and L) with different rates of iron uptake and mineralization have been identified [10,11]. The H

* Corresponding author. Ningbo University, 818 Fenghua Road, Ningbo, Zhejiang Province 315211, PR China. Tel.: þ86 574 87415009; fax: þ86 574 88122209. E-mail addresses: [email protected] (C. Li), [email protected] (T. Li). 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.02.017

subunit with a larger molecular mass contains a ferroxidase center for oxidation of Fe2þ, whereas the L subunit is responsible for the formation of the iron core for nucleation of Fe3þ. In lower vertebrates, a third subunit type named M subunit has been reported to possess both the ferroxidase center of H subunits and the iron nucleation site of L subunits [12,13]. Most identified invertebrates ferritins shared higher identities with the H-type subunit found in vertebrates [14]. The expression profile of ferritin was regulated by a number of simulators, including pathogen [13,15], iron conditions [2,16], pH stress [17], oxidative stress [13], temperature [18], and poly(I:C) [13]. Concerning the tissues expression, ferritin was particularly evident in tissues with iron storage function, such as liver and spleen, or in tissues with high iron load [19]. Sinonovacula constricta, one of the four major economic cultivated shellfish in China, has been cultured for more than 500 years in Fujian and Zhejiang Provinces. However, with the development of intensive culture and environmental deterioration, various diseases caused by bacteria, protozoa occurred in cultured juvenile, resulted in enormous losses to its aquaculture. Therefore, better understanding of the immune defense mechanisms of S. constricta might be contributed to the development of management strategies for disease control and long-term sustainability of clam farming. To date, only a few mollusk ferritins have been reported at sequence level and fewer have been investigated at functional level.

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The main objectives of the present study were: 1) to clone the fulllength cDNA of ferritin from S. constricta (denoted as ScFER); 2) to investigate the temporal expression profile of ScFER transcript in muscle, visceral mass and mantle after exposure to Fe2SO4$7H2O; 3) to generate the antibody and detect the native protein in muscle. 2. Materials and methods 2.1. Animals and challenge experiment S. constricta were obtained from Changjie, Ningbo city, Zhejiang province, China, and acclimated for a week in 500 L tanks equipped with air-lift circulating water at 26  1  C before commencement of the experiment. In the challenge experiment, around 120 S. constricta were divided in to three tanks and were exposed to Fe2SO4$7H2O with the final concentration of 2.8 mg/ml [5], while other twenty individuals cultured in seawater were selected as control group. The tissues of muscle, mantle and visceral mass were dissected from experimental and negative control samples after 0, 3, 6, 12 and 24 h iron ion exposure. Samples were immediately frozen in liquid nitrogen and stored at 80  C for RNA extraction and cDNA synthesis. 2.2. Cloning of the full-length ferritin cDNA A SMART cDNA library was constructed from the whole bodies of S. constricta, using SMARTÔ cDNA Library Construction Kit (Clontech). Random sequencing of the library using T3 primer yielded 1220 successful sequencing reactions. BLAST analysis of the EST sequences revealed that an EST of 456 bp was highly similar to the known ferritin sequences. This sequence was then selected for further cloning of full-length cDNA of ferritin gene from S. constricta. Two specific primers, sense primers P1: 50 -TCATCAGGACAGC GAAGC-30 and reverse primers P2: 50 -GCTAAGGAGTTTCTGGTCGT-30 , were designed based on the known sequence to clone the full-length cDNA of ScFER. The nested PCR strategy was applied to clone the 30 end of ScFER using sense primer P1 and reverse primer T7, while sense primer T3 and reverse primer P2 were used to get the 50 end of ScFER. The PCR products were cloned into the pMD18-T simple vector (TaKaRa) and sequenced bi-directionally with primers M1347 and RV-M. The sequencing results were verified and subjected to cluster analysis. 2.3. Sequence and phlyogenetic analysis of ScFER The ScFER cDNA sequence was analyzed by the BLAST algorithm at National Centre for Biotechnology Information (http://www.ncbi. nlm.nih.gov/blast) and the deduced amino acid sequence was analyzed with the Expert Protein Analysis System (http://www. expasy.org/). The percentages of similarity and identity of fulllength amino acid sequences between ScFER and ferritin from other organisms were calculated by the Identity and Similarity Analysis program (http://www.biosoft.net/sms/index.html). The SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) was employed to predict the signal sequence of ScFER and potential glycosylation sites were forecasted by NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/ NetNGlyc/). The motif sequences search was performed using InterPro Scan software (http://www.ebi.ac.uk/InterProScan/). The deduced amino acid sequences of ScFER and all the identified mollusk ferritins were used for phylogenetic analysis. A NJ tree was constructed with Mega3.1 software package (http://www. megasoftware.net/) and Clustal X (1.81). To derive the confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times.

2.4. Quantitative PCR analysis Total RNAs from different tissues were extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNAs were synthesized with reverse transcription system (Promega). Quantitative PCR was performed in Rotor-GeneÔ 6000 real-time PCR detection system. Two ScFER specific primers (P3: 50 GTGATGAGTGGGGAAGTGGC-30 and P4: 50 -TA GTGATGCGGTCGGA CAGG-30 ) were used to amplify an 192 bp fragment. b-actin gene was chosen as the reference for internal standardization. Two b-actin specific primers (P5:50 -AGTTG CCGCTCTTGTCGTGG-30 and P6:50 -TGCTCTGGGCTTCATCTCCG-30 ) were used to amplify an 170 bp fragment. The real-time PCR amplifications were carried out in triplicate in a total volume of 20 mL containing 10 mL Mix (Takara), 5 mL of the 1:10 diluted cDNA, 1 mL primers (20 mM) and 8 mL PCRgrade water. The real-time PCR parameter was denaturation at 95  C for 10 s, followed by 35 cycles of 95  C for 5 s, 60  C for 15 s, 72  C for 20 s. Melting analysis of the amplified products was performed at the end of each PCR to confirm that a single PCR product was produced and detected. The internal reference gene (b-actin) of the same sample was amplified in the same PCR plate. Standard curves for the target and the internal control were constructed using 10-fold serial dilutions of the corresponding purified plasmid. The amount of target and internal reference genes in each sample was determined using the appropriate standard curve. The 2DDCT method was used to analyze the expression level of ScFER. All data were given in terms of relative mRNA expressed as mean  S.D. The data were then subjected to analysis by one-way analysis of variance (ANOVA). Differences were considered significant at P < 0.05. 2.5. Expression and purification of the recombinant protein PCR fragment encoding the mature peptide of ScFER was amplified with gene-specific primers P7 50 -CGGGATCCATGGCTGA GACAATG-30 and P8 50 -CCCAAGCTTCTAGCTAAGGAGTTTCTGGTC-30 with BamH I and Hind III sites at their 50 end, respectively. The PCR product was cloned into pMD18-T simple vector (Takara), digested completely by restriction enzymes BamH I and Hind III (NEB), and then subcloned into the BamH I/Hind III sites of expression vector pET-28a(þ) (Novagen). The recombinant plasmid (pET-28a-ScFER) was transformed into Escherichia coli BL21 (Novagen) and subjected to DNA sequencing. After sequencing to ensure in-frame insertion, positive clones were incubated in SOB medium (containing 25 mg/L Kanamycin) at 37  C with shaking at 220 rpm. When the culture reached OD600 of 0.6, IPTG with final concentration of 1 mmol/L was added to the culture, and incubated for additional 1 h, 2 h, 3 h and 4 h under the same conditions. Cells were harvested by centrifugation at 10,000g for 2 min, and suspended in 50 mM Tris containing 5 mM EDTA, 50 mM NaCl, and 5% Glycerol (pH7.9). After being sonicated at 4  C for 60 min, the rScFER was purified by HiTrap Chelating Columns (Amersham Biosciences) according to the manufacturer’s instruction. The purified protein was subjected to 15% SDSePAGE according to the method of Laemmli [20]. After washing the PAGE in 3 M KCl solution for 3e5 min, the target protein band was excised from the gel, grinded into small pieces and dissolved in PBS for antibody preparation. 2.6. Western blot analysis Polyclonal antibodies against the purified recombinant ScFER protein were generated according to our previously work [21]. The total protein extraction from the muscle of S. constricta, IPTGinduced E. coli BL21 cell lysate and negative control strain were boiled for 10 min and separated by 12% SDSePAGE. The gel was

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firstly blotted onto a sheet of nitrocellulose transfer membrane by electrophoretically at 20 V for 14 h. Then, the membrane was blocked TBST (10 mM TriseHCl, pH 7.5, 100 mM NaCl, 0.05% (w/v) Tween 20) containing 5% skimmed milk for 4 h at room temperature. Subsequently, the membrane was incubated with mouse antiferritin polyclonal antibody with 1:5000 dilution in the block buffer for 2 h. The membrane was washed extensively before secondary antibody (1:10,000) was added and incubated for 1 h at 37  C. After three times of washing, signal was then visualized with BCIP/NBT in the dark condition. The reaction was quenched with distilled water after bands were sufficiently displayed.

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H-specific ferroxidase center in mammalian ferritins, are completely conserved in the deduced amino acid sequences of ScFER. Moreover, a potential N-glycosylation site (Asn-Gln-Ser) present in most ferritins is also identified in ScFER located from 109aa to 111aa. No signal peptide was found in the N-terminus of ScFER by the SignalP program. Blastp analysis indicated that the deduced amino acid of ScFER shared 80% identity with Meretrix meretrix (AAZ20754), 78% with Crassostrea gigas (AAP83794) and 75% with Haliotis discus hannai (ABH10672). All these results indicated ScFER was a novel member of ferritin family. 3.2. Phylogenetic analysis

3. Results 3.1. Cloning and analysis the full-length cDNA of ScFER One EST from the cDNA library of S. constricta was homologous to the previously known ferritin. Based on this EST, two fragments around 850 bp and a 650 bp were amplified by 30 -RACE and 50 RACE technique, respectively. A 996 bp nucleotide sequence representing the complete cDNA sequence of ScFER was obtained by overlapping two fragments with this EST. The full-length cDNA of ScFER was deposited in GenBank under accession no. GQ906972. The full-length cDNA sequence and the deduced amino acid sequence were showed in Fig. 1. The full-length cDNA of ScFER consisted of a 50 -terminal untranslated region (50 UTR) of 120 bp, a 30 UTR of 360 bp with a poly (A) tail, and an open reading frame (ORF) of 516 bp. The ORF encoded a polypeptide of 171 amino acids with a predicted molecular mass of 19.76 kDa and pI of 5.07 (Fig. 1). The putative iron responsive element (IRE) sequence, including a conserved loop, 50 CAGUGN-30 , and a bulged C located six nucleotides upstream of the loop and interrupts the stem was also conserved in the 50 -UTR of ScFER. Seven amino acid residues, known as metal ligands at the

A NJ phylogenetic tree was reconstructed based on mollusk ferritin protein sequence and showed in Fig. 2. Different types of ferritin from the same species were almost clustered together firstly, then with ferritin from other mollusk. ScFER was identified from clam ferritin subgroup, supporting that it was a new member of mollusk ferritin family. 3.3. Temporal expression profile of ScFER exposure to iron in different tissues Temporal effect of iron exposure on the transcriptional activities of ScFER gene was investigated over a 24 h period in different tissues of muscle, mantle and visceral mass by quantitative real-time PCR

100 Bay-scallop-1 99

Bay-scallop-2

52

Zhikong-Scallop-2

32

Zhikong-Scallop-1 Mussel

23

Abalone-1 Abalone-4

99

25

Abalone-2

72 97 20

Abalone-3

Sea-hare Pearl-shell

99

Pearl-oyster Oyster-1

33 40

Oyster-4 41

Oyster-3 100

Oyster-2 72

Oyster-5

25

Blood-clam 97

Hard-clam

46

Razor-clam

76

Snail-1 Pearl-mussel Chiton Snail-2

0.1

Fig. 1. Complete cDNA sequence of ferritin from Sinonovacula constricta and its deduced amino acid sequence. Nucleotides were numbered from the first base at the 50 end. The start condon was boxed. The asterisk indicated the stop codon. Amino acid residues involved in metal binding was shadowed and underlined.

Fig. 2. Consensus neighbor-joining tree based on the sequences of mollusk ferritins. The numbers at the forks indicated the bootstrap. The detail information for the used sequences were as follows: Abalone-1 (ABY87353), Abalone-2 (ACZ732700), Abalone-3 (ABH10672), Abalone-4 (ABG88846), Pearl-shell (ACS72281), Pearl-oyster (AAQ12076), Oyster-1 (AAP83794), Oyster-2 (CAD92096), Oyster-3 (CAD91440), Oyster-4 (ABE99842), Oyster-5 (AAP83793), Hardclam (AAZ20754), Pearl-mussel (ADK25061), Sea-hare (ABF21074), Bloodclam (ADC34696), Mussel (ACM86786), Snail (AAB24081), Snail-2 (AAL55398), Chiton (BAA21810), Bay-scallop-1 (ADR71732), Bay-scallop-2 (ADR71731), Zhikong-scallop-1 (AAV66904), Zhikong-scallop-2 (AAV66905).

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(Fig. 3). In the tissues of muscle, ScFER mRNA transcript increased gradually in the first 6 h and reached to 1.79-fold and 1.74-fold compared to the control group at 6 h (P < 0.05) and 12 h (P < 0.05). After that, the expression level was gradually drop back to the original level. Concerning the tissue of visceral mass, the expression level of ScFER was obviously decreased in the first 3 h (P < 0.01), then sharply increased to reach the peak at 6 h with 3.5-fold increase compared to the control group (P < 0.05). The expression level of ScFER mRNA in mantle was increased sharply and kept stable during the first 12 h, then drop back to the original level at 24 h post-iron challenge. One-way analysis of variance with control and challenged groups showed statistically significant difference in ScFER gene expression at 3 h for tissues of mantle and visceral mass, at 6 h and 24 h for muscle and visceral mass, and 12 h for mantle and muscle. However, no significant difference was observed in other time points of the challenge group.

3.4. Characteristics of rScFER and specificity of the antibody against ScFER The recombinant plasmid pET-28a-ScFER was transformed and expressed in E. coli BL21. After IPTG induction for 1 h, 2 h, 3 h and 4 h, the whole cell lysate analyzed by SDSePAGE revealed a distinct band with a molecular weight of 24.0 kDa (Fig. 4), which was further purified to homogeneity by HiTrap Chelating Columns (Fig. 4). The intensity of the recombinant protein band is increased with time increased. The peak expression level of recombinant protein was observed at 3 h after IPTG was introduced into the culture (Fig. 4). Western blot analysis showed that the mouse antisera could be specifically identified not only the recombinant protein, but also the native protein from muscle (Fig. 5).

4. Discussion In this study, we identified and analyzed a full-length cDNA of ferritin gene from S. constricta (ScFER). Some necessary elements for ferritin, including a putative IRE in the 50 UTR and seven amino acids involved in metal ligand, were totally conserved in the ScFER. Blastp analysis indicated ScFER is remarkably similar to H-type ferritins and ferritin from mollusk, such as 80% identity to M. meretrix (AAZ20754), 78% to C. gigas (AAP83794) and 75% to H. discus hannai (ABH10672). Phylogenetic analysis further revealed that ScFER was close matched to ferritin from other clams (the bootstrap was 76% to hard clam and 50% to blood clam). All these results indicated ScFER is a novel member of H-type ferritin family.

Fig. 4. SDSePAGE analysis of recombinant ScFER. After electrophoresis, the gel was visualized by Coomassie brilliant blue R250 staining. Lane 1: protein molecular standard; lane 2, 3, 4: negative control for rScFER (without induction); lane 5: induced expression for 1 h of rScFER; lane 6: induced expression for 2 h of rScFER; lane 7: induced expression for 3 h of rScFER; lane 8: induced expression for 4 h of rScFER; lane 9: purified rScFER.

Ferritin plays a vital role in iron metabolism like concentration regulation and detoxification of iron. In order to better understand the role of the ferritin in S. constricta response to iron challenge, the temporal expression levels of ScFER were analyzed by real-time PCR. We showed that the expression of the ferritin in the muscle was significantly increased (1.79-fold) after 6 h of exposure to iron. Expression of the ferritin in the mantle was also significantly increased and the peak expression level was detected at 3 h after exposure to iron. The most dramatic increase of ferritin mRNA after iron treatment was found in the visceral mass, in which 3.51-fold of mRNA was detected compared to untreated samples. Then, the ferritin expression in all three tissues was gradually recovered to the original level at 24 h, demonstrating that ScFER expression can be induced by iron treatment and should be invovled in the iron storage in S. constricta. The similar results have also been investigated in several animals. In human hepatocytes, translation of ferritin mRNA was increased up to a 50-fold in the iron-rich medium [22]. Iron treatment also could induce ferritin expression in giant prawn [23], mosquito [24], fruit fly [25] and amphioxus [5]. Given these facts, our results suggest that the enhanced expression of ScFER by iron treatments is probably a protective mechanism of the cell to cope with the stresses induced by iron challenge [13]. Surprisingly, the mRNA level of ferritin in viscera mass dropped after 3 h of iron exposure, given the fact that those in muscle and

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Fig. 3. Time-course expression level of ScFER transcript in muscle, mantle and visceral mass after iron exposure. Each symbol and vertical bar represented the mean  S.D (n ¼ 3). Significant differences between challenged group and control group were indicated by an asterisk (P < 0.05).

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Fig. 5. Specificity of ferritin polyclonal antibody was determined by western blot. Lane 1: protein molecular standard; lane 2, 3: BL21 lysate without rScFER; lane 4: Bl21 lysate including rScFER; lane 5: total protein extraction from muscle of S. consticta.

mantle both went up. The different expression patterns might be attributed to its multiple roles related to the presence of iron in various tissues. Similar result was also reported by Ma et al. [26]. The expression of shrimp alpha-2macroglobulin was significantly up-regulated in the heamocytes after Vibrio challenge and the peak expression level was detected at 37 h post-infection. While in the tissue of lymphoid, the lowest expression level was also found at 37 h post-infection. Western blot analysis is a useful tool to identify the native protein in tissues. In order to further detect and validate the existence of deduced amino acid of ScFER, the polyclonal antibodies were generated with the purified recombinant protein. Western blot results indicated that a unique protein band could be specifically identified from the S. constricta muscle protein extraction. This will be of great help in understanding the biological functions of ScFER. Acknowledgements This work was financially supported by the National High Technology Research and Development Program of China (Grant No. 2006AA10A410), by the Cheung Kong Scholars Programme and the Creative Research Groups of China, by Ningbo Committee of Science and Technology, China (Grant No. 2006C100041), by K.C. Wong Magna Fund of Ningbo University. References [1] De Zoysa M, Lee J. Two ferritin subunits from disk abalone (Haliotis discus discus): cloning, characterization and expression analysis. Fish Shellfish Immunol 2007;23:624e35. [2] Wang D, Bo YK, Kwang SL. Molecular characterization of iron binding proteins, transferrin and ferritin heavy chain subunit, from the bumblebee Bombus ignitus. Comp Biochem Physiol B 2009;152:20e7.

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