A Ferritin from Dendrorhynchus zhejiangensis with Heavy Metals Detoxification Activity

Ferritin, an iron homeostasis protein, has important functions in transition and storage of toxic metal ions. In this study, the full-length cDNA of ferritin was isolated from Dendrorhynchus zhejiangensis by cDNA library and RACE approaches. The higher similarity and conserved motifs for ferritin were also identified in worm counterparts, indicating that it belonged to a new member of ferritin family. The temporal expression of worm ferritin in haemocytes was analyzed by RT-PCR, and revealed the ferritin could be induced by Cd2+, Pb2+ and Fe2+. The heavy metal binding activity of recombinant ferritin was further elucidated by atomic force microscopy (AFM). It was observed that the ferritin protein could form a chain of beads with different size against three metals exposure, and the largest one with 35∼40 nm in height was identified in the Cd2+ challenge group. Our results indicated that worm ferritin was a promising candidate for heavy metals detoxification.


Introduction
As one of a major member of iron homeostasis proteins, ferritin plays an important role in storage and detoxification of excess iron in living cells. Structure analysis indicates the protein complex usually composed of 24 subunits, which surrounds an inorganic microcrystalline hollow capable of accommodating up to 4500 Fe 3+ [1,2]. In vertebrates, two types of subunits called heavy (H) and light (L) chains are identified and demonstrated to be encoded by separate genes. The H subunit has been studied in a variety of species including vertebrate and invertebrate animals, plants and bacteria [3,4]. The L subunit, however, has been only found in vertebrates [5,6]. The H subunits from different species contain seven conserved residues that confer ferroxidase activity for converting Fe 2+ to Fe 3+ allowing rapid detoxification of iron cations. The L subunit does not have ferroxidase activity, but serves as a salt bridge that stabilizes the ferritin structure, thus playing a role in iron nucleation and long-term storage [7].
As environmental issue attracted much attention in recent times, development and implication pollutant binding or degradation related genes were considered to be promising way, especially for heavy metals. Accumulative results showed that natural ferritin could store multiple toxic metal ions (Zn 2+ , Pb 2+ , Ni 2+ ) by its great storage capacity, resulting to detoxification of heavy metals in vivo [8]. Additionally, ferritin had also potential application for chemotherapy and cancer treatment [9].
Dendrorhynchus zhejiangensis are one of marine animals belonged to Neatinea, Heteronemertea, Lineidae, Dendrorhynchus. The worm lives in the bottom of shrimp ponds, in which the concentration of heavy metal usually higher than that in seawater. Therefore, the worm was considered to be a promising material to study heavy metal detoxification. In the present study, we firstly isolated and characterized a new source of ferritin from D. zhejiangensis. Then, its enrichment capacity for different metals in vitro was further addressed by atomic force microscopy to fully elucidate the roles of worm ferritin.

cDNA library annotation
The QC procedure was performed to evaluate the quality of the cDNA library, and the titer of the cDNA library was 1.7610 6 cfu/ mL. Colony PCR found that the recombinant rate was 87.5% with average size of 800 bp, indicating that the inserted fragment length was ideal and the library quality was good.
Random sequencing of 458 clones using T3 primer yielded 428 effective sequences. After removing low quality sequences, adaptor and vector sequences, the remaining were subjected for BLASTx analysis with 349 sequences successfully matched. These ESTs were classified into seven categories, including 152 unknown genes, 49 genes related to protein expression, 50 mitochondrial genes, 35 ribosomal structural genes, 19 metabolism-related genes, 11 immune-related genes, and 33 genes related to translation, intercellular material transport, and endocrine.

Transcriptional level expression of ferritin under heavy metals exposure
Temporal effect of heavy metals exposure on the transcriptional activities of worm ferritin were investigated by qPCR (Fig. 3). In Fe 2+ groups, an increase expression profile was detected at the first 36 h, and the peak expression was reached at 36 h with 18.5-fold increase compared to the control group (P,0.05). In Pb 2+ group, the ferritin mRNA level increased and reached maximal level at 12 h with 7-fold increase compared to the control group (P,0.05). In Cd 2+ group, the ferritin mRNA expression was sharply induced during the first 6 h, then slowly decreased with time going on (Fig. 3).

Recombiant expression of worm ferritin
The positive transformants were analyzed by SDS-PAGE to characterize the recombinant product of worm ferritin. After IPTG induction, an obvious protein band with molecular weight between 20.1 kDa and 29 kDa was detected in the positive transformants ( Fig. 4), which could be purified to homogeneity by HiTrap Chelating Columns. The molecular mass of the purified product was in good agreement with the Predicted MW of worm ferritin. Moreover, the intensity of the recombinant protein band was increased with time went on. The peak expression level of recombinant protein was observed at 6 h after IPTG was introduced into the culture.

Wstern blot analysis of worm ferritin expression
With the purified recombinant worm ferritin, polyclonal antibodies were generated for Western blot (Fig. 5). The results showed that antiserum could be specifically identified by not only the recombinant protein, but also the native protein from haemocytes. No signals were detected in other control samples. The size difference between the pET-FER and native ferritin was contributed to the extra fusion expression tag in the pET28 vector.

Characterization of recombiant worm ferritin binding by atomic force microscopy
The majority of ferritin molecules were well dispersed and had height of 6,12 nm in cross-section analysis before refolding (Fig 6aa''). In addition to a small amount of protein aggregates, most of the ferritin molecules formed a chain of beads with height of 5,10 nm and diameter of about 90,100 nm ( Fig. 6bb''). After adding Fe 2+ , Pb 2+ , and Cd 2+ , respectively, ferritin molecules

Discussion
Atomic force microscopy (AFM) has become an important tool for studying the interaction between biological molecules based on its higher resolution and capability. The technique had been suceessfully utilized in antigen and antibody, DNA and protein complex, and DNA conformation [10]. In this study, a full-length cDNA of worm ferritin was firstly cloned by cDNA library and RACE, then, its expression profiles were characterized at mRNA level under heavy metal exposure. Its binding activity to heavy metals was further investigated by atomic force microscopy (AFM).
In order to better understand the role of the worm ferritin in response to heavy metals challenge, temporal expression levels of worm ferritin were analyzed by real-time PCR. We showed that the expression of the ferritin in all treated groups was significantly increased after 6 h of exposure to three heavy metals, indicating that worm ferritin could be induced by Cd 2+ , Pb 2+ and Fe 2+ . The ferritin expression level of Cd 2+ at same dose was about three times higher than that of Pb 2+ , suggesting that worm ferritin may be the least sensitive to Pb 2+ , compared with Cd 2+ and Fe 2+ . The similar patterns of ferritin toward heavy metals exposure was also reported in giant prawn [11], clam [12], fruit fly [13] and amphioxus [14]. Given these facts, our results suggested that the enhanced expression of worm ferritin by heavy metals treatments was probably a protective mechanism of the cell to environmental stress [15].
In order to utilize the gene in practice, knowledge on how worm ferritin binding with heavy metals should be further investigated. The dynamic process between worm ferritin and three heavy metals was observed with AFM. The size of recombinant ferritin was very similar to that of the native ferritin protein after refolding [16], with small amount of irregularly aggregated molecules and fragments. However, when divalent ions was introduced in the culture, the subunit number of ferritin shell gradually increases, and ferritin shell becomes thicker, resulting in the size of ferritin structure was greater than that of native ferritin [3]. The interactions between metal and its binding ligands, such as hydrophobic bonds, hydrogen bonds, electrostatic interaction and van der Waals force are changed, which significantly impacts protein folding. Harrison et al [17] classified ferritin's iron storage procedure into three stages, namely, the oxidation of Fe 2+ to Fe 3+ , movement of Fe 3+ and the nucleation of iron core. Once the small organic molecules occupied part of the iron core, unstable iron ions in ferritin were released, resulting in decrease of electron density within the iron core [18]. As a continuation of storage reaction, the amount of released iron increased. The three-phase material exchange tunnel was narrowed by ferrtin shell through a flexible regulation. Thus, the width of ferritin was decreased. Our result was in agreement with the theory that free or denatured ferritin subunits could quickly wrap bare iron core to form native structure.
Regarding to Cd 2+ challenge, the dramatic conformation changes of ferritin was also observed through forming a large hollow, which was speculated to be a cadmium core formed by a lot of Cd 2+ gathering and surrounded by many ferritin molecules. Previous reports had shown Cd 2+ was capable of binding to both the inside and outside of horse spleen ferritin [19]. All these evidences indicated worm ferritin might be promising candidates for Fe 2+ and Cd 2+ detoxification.
The introduce of heavy metal into recombinant ferritin was also changed its size. The diameter of ferritin increased from 35,40 nm to 400,1000 nm when added with Pb 2+ . It was hypothesized that the hydration of metal ions destroyed the hydration layer surrounding ferritin molecules, making it easier to form folding intermediates. The stronger the metal ion hydration was, the greater role it played in promoting ferritin aggregation, leading to the formation of large ferritin aggregates. Therefore, metals with large electron could be more likely to promote ferritin aggregation because they had higher hydration free energy [20]. In addition to metal hydration, the electrostatic interaction among metal ions shielded electrostatic repulsion among protein molecules, the so-called Debye screening. In alkaline solution, ferritin molecules had negative charges, thus the electrostatic repulsion among them made aggregation difficult. These phnomena was also expained by the fact that metal ions could accelerate protein nucleation. Metal ions with high positive charges exerted much stronger Debye screening effect. Therefore, they were more likely to enhance nucleation than metal ions with lower positive charges. Nucleation rate could also affect the protein shape, thus exposure to different metal ions had the potential to form different protein shapes [21].
In summary, we cloned the full-length cDNA of D. zhejiangensis ferritin and characterized its expression patterns during exposure to iron ion. Subsequently, the recombinant ferritin was produced to investigate its binding activity to Fe 2+ , Pb 2+ , and Cd 2+ by AFM.  All our results supported that worm ferritin was a promising candidate for heavy metals detoxification. In order to utilize the protein in practice, its binding capcity to different heavy metals should be elucidated in our further work.

Worms
Dendrorhynchus zhejiangensis was collected from shrimp ponds along the coast of Hudoudu (29.6u N, 121.6u E), Fenghua City, Zhejiang Province, China in May 2011. The animals were acclimated in our laboratory for two days prior to the experiment. No specific permits were required for the described field studied, and no specific permissions were required for these locations.

cDNA library construction and EST analysis
Eight D. zhejiangensis were randomly collected and homogenized with liquid nitrogen. The lysate was used for cDNA library construction. The cDNA library was constructed using the ZAP-cDNA synthesis kit and ZAP-cDNA GigapackIII Gold cloning kit (Stratagene). Random sequencing of the library using T3 primer yielded 458 successful sequencing reactions. BLAST analyses of all the 458 EST sequences revealed that one EST of 387 bp was highly similar to the previously identified ferritin. Therefore, the EST sequence was selected for the full-length cDNA cloning.
Cloning the full-length cDNA of worm ferritin Two specific primers, Fer-R: 59-GTGGAAGCCATCAGG-GAA-39 and Fer-F: 59-AAGGGCATGAAGGTCCAAGAGGG-39, were designed based on the EST to amplify the 39 and 59 ends of worm ferritin combined with vector prime T7 or T3, respectively. PCR was performed at 94uC for 5 min followed by 35 cycles of 94uC for 30 s, 58uC for 30 s, and 72uC for 1 min and final extension at 72uC for 10 min. The obtained PCR amplicons were gel-purified and cloned into pMD18-T (Takara, China). After transforming into competent E. coli, the positive colonies were identified by colony PCR. Three positive clones were sequenced at Invitrogen (Shanghai, China) and were analyzed using BLAST (http://www.ncbi.nlm.nih.Gov/BLAST/).

Quantitative expression analysis of ferritin
For heavy metals challenge experiment, worms were divided in to four tanks and were exposed to three metals of Fe 2+ , Pb 2+ and Cd 2+ with the final concentration of 10 mM. The fourth tank was served as control group. The seawater was changed daily and metal stock solution was added to the seawater everyday. After 6 h, 12 h, 24 h and 48 h exposure, the haemocytes from each group were collected from the control and the treated groups for RNA extraction and cDNA synthesis.
For expression analysis, the ferritin specific primers, FER-F (59-GAGGAATGCGAGGCTGGT-39) and FER-R (59-CACG CTGATTCTGGAACTTG-39), were designed to amplify a product of 205 bp. For normalizing the ferritin transcripts, two b-actin specific primers actin-F (59-GGACCTCTACGCCAA-CACTG-39) and actin-R (59-ATGCAAGGATGGAGCCAC-39) were used to amplify an 170 bp fragment. The fluorescent realtime PCR assay was carried out performed in Rotor-Gene 6000real-time PCR detection system and the condition was according to our previous work [12]. The 2 2DDCT method was used to analyze the expression level of worm ferritin. All data were given in terms of relative mRNA expressed as mean 6 S.D. The data were then subjected to analysis by one-way analysis of variance (ANOVA). Differences were considered significant at P,0.05.

Expression and purification of worm ferritin
PCR product corresponding to ferritin ORF was amplified by two gene specific primers with BamH I and Hind III sites at 59ends. The PCR amplicons were digested with BamH I and Hind III, and then inserted into digested pET-28a(+) vector. After transforming into competent E. coli BL21, positive clones were identified by kanamycin screening and sequencing. After sequencing to ensure in-frame insertion, positive clones were incubated at 37uC at 220 rpm. When the culture reached OD 600 nm = 0.5 to 0.7, 1 mM isopropyl-b-D-thiogalacto-pyranoside (IPTG) were added to induce the ferritin expression for additional 6 h. Cells were harvested at 1 h, 2 h, 3 h, 4 h, 5 h and 6 h by centrifugation at 4000 r/min for 15 min and resuspended in PBS for sonication. The expressed ferritin was purified using Ni-NTA affinity column (GE healthcare) according to the manufacturer's instruction.The expression and purified products were separated by SDS-PAGE and stained by Coomassie brilliant G250.

Western blot analysis
Protein samples from positive bacteria and D. zhejiangensis tissue were separated by SDS-PAGE. Proteins were electronically transferred onto nitrocellulose membrane, and blocked with 5% non-fat milk in TBST. Then the membrane was incubated with anti-ferritin serum for 1 h and washed three times with TBST. The membrane was then incubated with 1:10000 diluted rabbit anti-mouse secondary antibody for 1 h and washed three times with TBST. Signals were visualized using NBT/BCIP method and the reaction was terminated with distilled water. The anti-ferritin serum was prepared according to our previous work [4].

Functional characterization of worm ferritin
To achieve recombinant ferritin with biological activity, the purified protein was refolded against stepwise decrease of urea concentration in GSH/GSSG buffer (50 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, 10% glycerol, 1% glycine, 2 Mm reduced glutathione, 0.2 Mm oxide glutathione, pH 8.0) overnight at 4uC. The concentration of purified protein was quantified by BCA method.
To determine heavy metal enrichment capacity, 1 mL purified ferritin (20 mg/mL) was incubated with 20 mL of 2 mM FeCl 2 , PbCl 2 and CdCl 2 at 22uC for 10 min, respectively. Then, 5 mL of each mixture was evenly distributed on the surface of pretreated mica sheet and dried for 3 h at room temperature at environmental humidity of 5%. The samples were observed with a Nanoscope IIIa multimode scanning probe microscopy (Veeco Instruments, USA) using Tapping mode. The NSC11 triangular cantilever probe (MikroMasch, USA) was used for scanning at a resonance frequency of 330 kHz, scan rate of 1-1.5 Hz under spring constant of 50 N/m.