Accepted Manuscript
Identification and involvement of ferritin in the response to pathogen challenge in the abalone, Haliotis diversicolor
Jian He, Jingzhe Jiang, Lu Gu, Manman Zhao, Ruixuan Wang, Lingtong Ye, Tuo Yao, Dr. Jiangyong Wang
PII: S0145-305X(16)30022-2 DOI: 10.1016/j.dci.2016.01.022 Reference: DCI 2544
To appear in: Developmental and Comparative Immunology
Received Date: 2 December 2015 Revised Date: 27 January 2016 Accepted Date: 28 January 2016
Please cite this article as: He, J., Jiang, J., Gu, L., Zhao, M., Wang, R., Ye, L., Yao, T., Wang, J., Identification and involvement of ferritin in the response to pathogen challenge in the abalone, Haliotis diversicolor, Developmental and Comparative Immunology (2016), doi: 10.1016/j.dci.2016.01.022.
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1 Identification and involvement of ferritin in the response to pathogen challenge in the
2 abalone, Haliotis diversicolor
3 Jian He a, Jingzhe Jiang a, Lu Gu a,b , Manman Zhao a,b , Ruixuan Wang a, Lingtong Ye a, Tuo Yao a,
4 Jiangyong Wang a*
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6 a Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of
7 Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,
8 Guangzhou 510300, China
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10 b College of Fisheries and Life, Shanghai Ocean University, Shanghai 201306, China
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12 * Correspondence : Dr. Jiangyong Wang
13 Division of Fishery Organism Disease Control
14 South China Sea Fisheries Research Institute
15 Chinese Academy of Fishery Sciences, 231 Xingang West Road, 16 Guangzhou 510300, China MANUSCRIPT 17 Tel: +86-20-89108321
18 E-mail: [email protected] (J. Wang)
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31 Abstract
32 Accumulating data has demonstrated that ferritin plays an important role in host defense
33 responses against infection by pathogens in many organisms. In this study, ultracentrifugation was
34 used to isolate ferritin from abalone, Haliotis diversicolor , and sodium dodecyl
35 sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that this ferritin
36 consisted of two subunits (designated as HdFer1 and HdFer2). There are no disulfide bonds
37 between the HdFer1 and HdFer2 subunits; however, these subunits co-assemble to form
38 heteropolymers. A novel ferritin subunit (HdFer2) was cloned from H. diversicolor by 5ʹ and 3 ʹ
39 RACE (rapid amplification of cDNA ends) approach. The full-length HdFer2 cDNA sequence
40 consists of 878 bp with an open reading frame of 513 bp that encodes a protein that is 170 amino
41 acids in length. Quantitative real-time PCR analysis revealed that HdFer1 and HdFer2 were
42 transcribed in various tissues, such as the mantle, gill and hepatopancreas, with the highest levels
43 of expression in the hepatopancreas. Following a challenge with the pathogen, Vibrio harveyi , the
44 expression of HdFer1 and HdFer2 were markedly induced at different times. This study has
45 identified a novel ferritin subunit in H. diversicolor which will contribute to further exploration of 46 the role of ferritin in mollusk innate immune defense MANUSCRIPTagainst invading pathogens. 47
48 Key words: Haliotis diversicolor ; Ultracentrifugation; Ferritin; Immune defense
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61 1. Introduction
62 Ferritin, a protein macromolecule first identified by Laufberger in 1937 (Arosio et al., 2009),
63 possesses a wide spectrum of biological functions, including iron storage and release (Anderson
64 and Frazer, 2005), developmental regulation (Levenson and Fitch, 2000), and inflammation (Torti
65 et al., 1988). Ferritin is widely distributed in almost all organisms, including bacteria, fungi, plants,
66 and animals (Shi et al., 2008). Ferritins consist of 24 subunits arranged to form a hollow shell,
67 designed to accommodate up to 4500 Fe 3+ atoms as a biomineral. The molecular weight of the
68 ferritin complex reaches up to 450 kDa (Arosio et al., 2009). Ferritins are composed of two major
69 subunits in higher vertebrates, namely the heavy (H) subunit and the light (L) subunit (Harrison
70 and Arosio, 1996). There is a ferroxidase activity center in the H subunit, whereas the L subunit
71 does not contain this activity center. However, the L subunit can accelerate the transfer of iron
72 from the ferroxidase center to the iron core and improve the overall iron sequestering process
73 (Harrison and Arosio, 1996; Rucker et al., 1996; Theil, 1990). In various animal tissues, and at
74 different developmental stages, the ferritin H and L subunits assemble in a different H/L ratio 75 (Harrison and Arosio, 1996). Furthermore, numerousMANUSCRIPT studies have revealed the existence of 76 another subunit, the M (middle) subunit, in additio n to the H and L subunits in lower vertebrates 77 (Andersen et al., 1995; Andersen et al., 1998; Dickey et al., 1987; Giorgi et al., 2008). The M
78 subunit not only contains the ferroxidase center of the H subunit, but also possesses the iron
79 nucleation site of the L subunit characterized in mammals (Dickey et al., 1987; Giorgi et al., 2008).
80 In addition, ferritins possess different functions depending on their subcellular location and are
81 divided into three subgroups to reflect this: secreted, cytosolic, and mitochondrial (Arosio et al.,
82 2009). Approximately sixty ferritin genes have been identified in mollusks, the overwhelming
83 majority of which belong to the non-secreted ferritins (cytosolic and mitochondrial). A few studies
84 have revealed that mollusk ferritin subunits contain signal peptides, suggesting that secretory 85 ferritins existACCEPTED in this group (De Zoysa and Lee, 2007; Deleury et al., 2012; von Darl et al., 1994). 86 Apart from their function in iron storage and release, an increasing number of studies have
87 focused on the roles of ferritins in oxidative stress and host defense responses. However, the
88 mechanisms of ferritin action in these processes are not completely understood. One mechanism
89 for ferritin involvement in innate immunity is through its iron-withholding ability (Beck et al., ACCEPTED MANUSCRIPT
90 2002). Organisms produce excessive ROS (reaction oxygen species) as a defense mechanism
91 following infection by pathogens, but this generates oxidative stress which is also detrimental to
92 the host (Pipe, 1990, 1992). The production of ROS leads to the generation of reactive hydroxyl
93 radicals which can have damaging effects on DNA, protein, and lipids (Orino et al., 2001). Ferritin
94 is able to inhibit the formation of ROS by restricting excess free iron to inhibit the Fenton reaction,
95 thereby reducing the production of OH (Storey, 1996). This regulation of ferritin gene expression
96 generally occurs at the transcriptional level, but the post-transcriptional regulation of ferritin
97 expression has also been studied in animals. The iron responsive proteins (IRP) inhibit ferritin
98 mRNA translation by binding to the iron regulatory elements (IRE), a regulatory site located in the
99 5ʹ-untranslated region (UTR) of ferritins (Hentze et al., 1989; Theil and Eisenstein, 2000).
100 Abalone, Haliotis diversicolor , is a high-value marine mollusk species that is cultured in
101 southern China. Haliotis diversicolor was the major cultured species off the southern coast of
102 China 20 years ago. However, disease outbreaks have severely limited the development of the
103 abalone aquaculture industry. Discovering and identifying abalone pathogens is necessary for the 104 facilitation of the healthy development of the aquaMANUSCRIPTculture industry, as well as exploring the 105 immune system of the hosts. The purpose of the pres ent study was to extract and isolate ferritin 106 subunits from the tissue of H. diversicolor and to explore their potential roles in host defense
107 response under pathogen V. harveyi challenge. Our results showed two distinct ferritin subunits
108 (HdFer1 and HdFer2) were extracted and isolated directly at the protein level for the first time in
109 mollusk. A full-length cDNA for a novel ferritin subunit (HdFer2) was cloned from abalone (H.
110 diversicolor ), the expression profile of HdFer1 and HdFer2 was examined in adult abalone tissues,
111 and the effect of V. harveyi challenge on ferritin expression was analyzed. The results of this study
112 suggest the involvement of abalone ferritin in innate immune defense against invading pathogens.
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119 2. Materials and methods
120 2.1. Animals, challenge experiments, and sample collection
121 Healthy abalones ( H. diversicolor ) averaging 20 mm in shell length were collected from an
122 aquaculture farm in Dongshan, Fujian province, China. Abalones were stored at –80°C in an
123 ultra-low temperature freezer until use for ferritin extraction and isolation.
124 Healthy adult abalones (40–50 mm shell length) used for the immune challenge experiments
125 were collected from a commercial farm in Huizhou, Guangdong province, China. Animals were
126 acclimatized to the laboratory for seven days before beginning experiments in 60-l tanks filled
127 with recirculating seawater (salinity 30; temperature 24°C) that was continuously aerated.
128 Two-thirds of the water in each group was renewed once daily.
129 For the challenge experiments, abalones were injected with 100 l (1 x 10 7 CFU/ml) of a
130 dilution of V. Harveyi (kept by Division of Fishery Organism Disease Control, South China Sea
131 Fisheries Research Institute, Chinese Academy of Fishery Sciences) into the muscle. Five
132 individuals of challenged abalones were randomly collected at 0, 3, 6, 12, 24, and 48 h after
133 challenge. Their mantle, gill and hepatopancreas were dissected, frozen in liquid nitrogen and 134 stored at –80°C until use. MANUSCRIPT 135
136 2.2. Cesium chloride density gradients and protein extraction
137 A discontinuous density gradient composed of a 2.5-ml solution of CsCl (w/v dissolved in
138 PBS) at different concentrations was prepared in polypropylene centrifuge tubes by fraction
139 under-layering. From the top to the bottom of the tube, the gradient concentration profiles were
140 15%, 25%, 35%, and 45%. The CsCl density gradients were used for protein isolation.
141 Approximately 10 g of abalone visceral mass (gill, mantle and hepatopancreas) was weighted
142 and immediately frozen in liquid nitrogen. The frozen tissue was ground into a powder using a
143 laboratory mortar mill (Pulverisette 2, Fritsch, Sweden), suspended in sterile PBS (1:10 w/v), and 144 centrifugedACCEPTED three times at 12000 rpm at 4°C for 20 min. Supernatants were collected and 145 ultra-centrifuged at 70000 rpm for 1 h at 4°C in a Hitachi CP100WX ultracentrifuge with a P70AT
146 rotor (Hitachi, Tokyo, Japan). The pellet was dissolved in 600 l of ice cold PBS and carefully
147 pipetted onto the top of the CsCl gradient. This preparation was centrifuged at 30000 rpm for 20 h
148 at 4°C in a Beckman Optima TM LE-80K ultracentrifuge equipped a SW41Ti rotor (Beckman ACCEPTED MANUSCRIPT
149 Coulter, Brea, CA, USA). Subsequently, the supernatant was discarded and the orange protein
150 pellet was suspended in ice cold PBS.
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152 2.3. SDS-PAGE, native-PAGE, mass spectrometric analysis and database search
153 For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing
154 or non-reducing conditions, the protein samples were denatured in 2 × SDS loading buffer in the
155 presence or absence of 5% β-mercaptoethanol, respectively. The samples were then boiled at
156 100 °C for 5 min and separated on a 12% SDS-PAGE gel by electrophoresis at 120 V until the dye
157 front reached the end of the gel. For native-PAGE, samples were incubated in 2 × loading buffer
158 without β-mercaptoethanol or SDS at 4°C for 15 min. The native gel did not contain SDS and
159 electrophoresis was performed on ice at 120 V until the dye front reached the end of the gel.
160 Subsequently, the bands containing the proteins of interest were excised from the native-PAGE gel
161 and incubated in 2 × SDS loading buffer for 10 min at room temperature. The proteins extracted
162 from the gel were then separated by two-dimensional 12% SDS-PAGE after boiling at 100 °C for 5
163 min. The gels were stained with Coomassie brilliant blue R-250 and scanned using an 164 Imagescanner (GE, Fairfield, CT, USA). The protein MANUSCRIPTbands were excised from the SDS-PAGE gel, 165 destained in 25 mM NH 4HCO 3 / 50% acetonitrile, and incubated with 20 ng/ µl trypsin (Promega,
166 Fitchburg, WI, USA) at 37 °C overnight. The peptides were extracted using 2.5% trifluoroacetic
167 (TFA)/90% acetonitrile (ACN) at room temperature for 30 min, dried in a vacuum concentrator for
168 3 h, and subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass
169 spectrometry (Ultraflex III MALDI-TOF/TOF MS; Bruker, Billerica, MA, USA). Mascot software
170 (Matrix science, London, UK) was used to search the NCBI databases
171 (http://www.ncbi.nlm.nih.gov/).
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173 2.4. RNA isolation and cDNA synthesis 174 Total ACCEPTED RNA was extracted from the mantle, gill and hepatopancreas using TRAzol reagent 175 (Dongsheng, China) according to the manufacturer’s instructions. RNA concentration and purity
176 were determined by NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
177 The OD 260 /OD 280 ratio of all RNA templates was between 1.8 and 2.0. RNA integrity was
178 determined by agarose gel electrophoresis. For cloning the full length ferritin cDNA, the total ACCEPTED MANUSCRIPT
179 RNA was immediately transcribed into 5 ʹ and 3 ʹ end RACE cDNA template using a SMART
180 cDNA kit (Clontech, Mountain View, CA, USA). For RT-qPCR analysis, total RNA was reverse
181 transcribed into cDNA using a PrimeScript 1st strand cDNA synthesis kit (Takara Bio, China).
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183 2.5. Cloning the full-length HdFer2 cDNA
184 The whole tissue H. diversicolor EST library (unpublished) was searched for cDNAs
185 encoding ferritin and the selected partial sequences were identified using BLAST
186 (www.ncbi.nlm.nih.gov/blast). Four gene-specific primers were designed for cloning the
187 full-length HdFer2 cDNA based on the EST sequence and amino acid sequences of peptides from
188 mass spectrographic analysis. P1 (outer) and P2 (inner) nested primers were designed for 5 ʹ RACE,
189 and P3 (outer) and P4 (inner) nested primers were designed for 3 ʹ RACE (Table 1). For all RACE
190 experiments, a SMART cDNA kit (Clontech, Mountain View, CA, USA) was used according to
191 the manufacturer’s instructions.
192 The PCR products were purified using a 1% agarose gel and a gel extraction kit and
193 subsequently cloned into the PMD18-T vector (Takara Bio, China). Plasmids were transferred into 194 Escherichia coli DH5 α and the recombinant bacteria MANUSCRIPT were identified by PCR. The clones were 195 then sequenced by Invitrogen (Guangzhou, China).
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198 2.6. Sequence analysis
199 The cDNA and deduced amino acid sequence of HdFer2 were analyzed using DNAstar and
200 BLAST (http://blast.ncbi.nlm.nih.gov/). The SignalP4.0 Server
201 (http://www.cbs.dtu.dk/services/SignalP/) was adopted for predicting the presence and location of
202 a signal peptide. The sub-cellular location of HdFer2 was predicted by CELLO (Yu et al., 2006).
203 Protein sequences from different animals were aligned using the ClustalW program. The 204 phylogeneticACCEPTED tree was constructed with the MEGA 6.0 program using the neighbor-joining (NJ) 205 algorithm and the reliability was tested using bootstrap resampling (with 1000 pseudo-replicates).
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207 2.7. Quantitative real-time PCR analysis of ferritin expression
208 A quantitative real-time PCR was performed to measure the HdFer expression levels in ACCEPTED MANUSCRIPT
209 different tissues and in response to V. harveyi challenge. All of the primers used in this study were
210 designed by Primer Premier 5.0 and the primer sequences are given in Table 1. Each assay was
211 performed with HdL5 as the internal control. The real-time PCR was performed on a Mastercycler
212 ep realplex (Eppendorf, Hamburg, Germany) using the THUNDERBIRD SYBR ®qPCR Mix
213 (Toyobo, Osaka, Japan) with reaction conditions as recommended by the kit manufacturer. The
214 qPCR for each sample was performed twice in triplicate using a 20- l reaction volume containing
215 10 l 2× qPCRmix, 1 l of each of sense and anti-sense primer (10 µM), 1 l of a 1:10 dilution of
216 sample cDNA, and 7 l of PCR grade water. Forty PCR cycles were followed by melting curve
∆∆ 217 analysis. The 2 - CT method was used to analyze the relative expression of the two ferritin subunits,
218 HdFer1 and HdFer2. Data are expressed as mean ± SD (n = 5). Statistical analysis was performed
219 using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA,
220 followed by Newman-Keuls’s multiple comparison test, was used to analyze the statistical
221 significance among multiple groups. Differences were considered statistically significant when P
222 values < 0.05 were obtained.
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239 3. Results
240 3.1. Ferritin extraction and identification
241 Density gradient ultracentrifugation was adopted to isolate ferritin from abalone. To obtain
242 ferritin as many as possible, the visceral mass (mantle, gill and hepatopancreas) of healthy abalone
243 was chosen as experiment material. An abundance of macromolecular proteins was obtained at the
244 bottom of the centrifuge tubes containing samples from abalone. The SDS-PAGE results indicated
245 that two different proteins existed in the protein precipitate and that their molecular weights were
246 between 15 and 25 kDa (Fig. 1). Subsequently, the proteins were extracted from the SDS-PAGE
247 gel and subjected to mass spectrometry analysis. The data collected from the MALDI-TOF-TOF
248 was compared to the NCBI databases using the MASCOT online search tool. The protein score
249 represents the similarity between the peptide data obtained from the mass spectrometric analysis
250 and the theoretical peptide data in the NCBI database. The protein scores of heavy and light
251 protein brands were 219 and 146, respectively. The highest score obtained by the target protein
252 was considered the most credible. The two protein bands were identified as abalone ferritin (Table
253 2). As the availability of protein information in mollusks is limited, the database search only 254 identified one protein band as a ferritin subunit inMANUSCRIPT H. diversicolor successfully. Moreover, the 255 full-length cDNA encoding this light ferritin band had been previously cloned in H. diversicolor
256 (Genbank# EU244336.1, hereafter referred as HdFer1 ). The heavy protein band was identified
257 highly similar as ferritin from H. discus hannai , which has not been previously described in H.
258 diversicolor .
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260 3.2. Biochemical analysis of ferritin from H. diversicolor
261 In order to detect whether the two ferritin subunits from H. diversicolor are linked by a
262 disulfide bond, SDS-PAGE under reducing and non-reducing conditions was performed. As a
263 strong reducing agent, β-mercaptoethanol can dissociate disulfide bonds between or among 264 different subunitsACCEPTED so that they may be separated by SDS-PAGE. As shown in Fig. 2, two protein 265 bands (HdFer1 and HdFer2) were observed in the gel in both the presence and absence of 5%
266 β-mercaptoethanol. This result suggested that there is no disulfide bond linking the HdFer1 and
267 HdFer2 subunits.
268 Subsequently, native-PAGE followed by a second dimension SDS-PAGE was adopted to ACCEPTED MANUSCRIPT
269 analyze the assembly of the two ferritin subunits. Only one protein band was observed in the
270 native-PAGE gel (Fig. 3A). As expected, two protein bands were detected in the second dimension
271 SDS-PAGE gel (Fig 3B). Collectively, these data suggested that HdFer1 and HdFer2 co-assemble
272 to form heteropolymers.
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274 3.3. HdFer2 cDNA cloning and sequence analysis
275 To clone the full-length cDNA of HdFer2, four-specific primers were designed based on the
276 the EST library and amino acid sequence of HdFer2 from mass spectrometry data. The full-length
277 HdFer2 cDNA sequence (Genebank# KU555407) was 878 bp in length, with an open reading
278 frame of 513 bp, and encoded a 170 amino acid protein (Fig. 4). The deduced molecular weight of
279 HdFer2 was 19828.39 Da with a theoretical pI of 5.14. The HdFer2 5 ʹ untranslated region (UTR)
280 was 72 bp long, and the 3 ʹ UTR was 293 bp long and contained a polyadenylation signal
281 (AATAAA). No signal peptide or transmembrane domain was found in the HdFer2 amino acid
282 sequence and the CELLO program predicted HdFer2 to be a kind of cytoplasmic protein. The
283 analysis of the deduced amino acid sequence revealed that HdFer2 contained only two conserved 284 residues in the ferroxidase activity center, namely Y 31MANUSCRIPT and E 104 . Only one conserved residue of the 61 115 128 285 ferrihydrite nucleation center was observed (E ). All three iron ion channel residues (H , D ,
286 and E 131 ) were present in HdFer2.
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288 3.4. Homologous and Phylogenetic analysis of HdFer1 and HdFer2
289 Analysis of the deduced amino acid sequences with the BLASTP program indicated that
290 HdFer1 and HdFer2 shared 63% and 55% identity with the human ( Homo sapiens ) ferritin heavy
291 (Genbank# NP_002023.2) subunit, respectively. However, HdFer1 and HdFer2 shared lower
292 identity (52% and 51%) with the human ferritin light subunit (Genbank# NP_000137.2). In other
293 words, both HdFer1 and HdFer2 were closer matched to the ferritin heavy subunit than to the 294 ferritin lightACCEPTED subunit. The deduced amino acid sequences of HdFer1 and HdFer2 shared the 295 highest identity (96% and 92%) with those of H. diversicolor supertexta (Genbank# ACU09496.1)
296 and H. discus hannai (Genbank# ADK60915.1), respectively. However, HdFer1 only shared 64%
297 identity with HdFer2.
298 Multiple sequence alignment of HdFer1 and HdFer2 with other ferritins from vertebrates and ACCEPTED MANUSCRIPT
299 invertebrates indicated that they were highly conserved (Fig. 5). In the unrooted phylogenetic tree,
300 two distinct branches, consisting of cytoplasmic and secreted ferritins, were separated in the tree.
301 Both HdFer1 and HdFer2 were placed within the cytoplasmic ferritins group (Fig. 6).
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303 3.5. Tissue distribution of ferritin gene expression
304 Quantitative PCR analysis was performed to compare the relative mRNA expression of the
305 two HdFer subunits in abalone tissues, including the hepatopancreas, gill, and mantle (Fig. 7).
306 Expression of both HdFer subunits was detected in all tissues examined and the highest level of
307 expression was observed in the hepatopancreas. There were no significant differences in the level
308 of expression of HdFer1 between different tissues (P > 0.05). However, the expression of HdFer2
309 was markedly higher in the hepatopancreas when compared to the mantle (P < 0.01).
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311 3.6. Ferritin gene expression in response to pathogen challenge
312 The expression of both HdFer1 and HdFer2 were obviously increased at different times in the
313 examined tissues following pathogenic challenge with V. harveyi (Fig. 8). HdFer1 was 314 significantly up-regulated at 6 h post-challenge in theMANUSCRIPT hepatopancreas (2.8-fold, P < 0.01) and at 315 48 h post-challenge in the mantle (1.9-fold, P < 0.01). The expression of HdFer2 was markedly
316 induced after a 6 h and 24 h V. harveyi challenge in the hepatopancreas and gill, in which levels
317 were 3.9-fold and 2.4-fold, respectively (P < 0.01). The highest transcript abundance for HdFer2
318 was observed at 48 h after bacterial challenge in the mantle (1.4-fold, P < 0.05).
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329 4. Discussion
330 Density gradient centrifugation is a widely used method for isolating and purifying viruses
331 and macromolecular proteins (Dantas-Lima et al., 2013). Numerous ferritin genes have previously
332 been identified and cloned from mollusks, but the current study represents the first successful
333 isolation and purification of ferritin from mollusks at the protein level. In a previous study of
334 human ferritins, the heart was found to contain a higher proportion of the H subunit, whereas the
335 liver contained a higher proportion of the L subunit (Harrison and Arosio, 1996). The molecular
336 weight of the H subunit is higher than that of the L subunit. However, it has been reported that the
337 molecular weight of the ferritin L subunit is higher than the H subunit in insects (Hamburger et al.,
338 2005). In fact, the categorization of ferritin subunits is based on their activity centers rather than
339 their molecular weight. The H and L subunits often co-assemble to form heteropolymers in higher
340 eukaryotes (Arosio et al., 2009). In this study, HdFer1 and HdFer2 were demonstrated to assemble
341 to form a ferritin composed of both subunits based on the results of the two-dimensional
342 native-PAGE/SDS-PAGE. Additionally, two protein bands were visualized following separation
343 by SDS-PAGE under non-reducing conditions, suggesting that there is no disulfide bond linking 344 HdFer1 and HdFer2 subunits. To our knowledge, this MANUSCRIPT is the first report regarding the linkage and 345 assembly of ferritin subunits in mollusks.
346 The ferritin subunits are currently divided into three types based on their activity centers: the
347 H subunits have a ferroxidase center, the L subunits possess a ferrihydrite nucleation center
348 (Santambrogio et al., 1996), and the M subunits contain both types of activity centers. In this study,
349 two of the conserved residues in the ferroxidase activity center and only one conserved residue in
350 the ferrihydrite nucleation center were identified in HdFer2. This lack of conservation
351 demonstrates that the abalone HdFer2 does not contain two intact functional activity centers. This
352 special type of ferritin subunit had also been discovered in Patinopecten yessoensis (Sun et al.,
353 2014) . These data imply that HdFer2 is a novel ferritin subunit that has not been previously
354 recognizedACCEPTED in abalone and that a potentially novel iron regulation mechanism in mollusks needs to
355 be further explored. In addition, HdFer2 appears to resemble to a cytoplasmic type ferritin more
356 than a secreted type ferritin based on the bioinformatic prediction of the signal peptide, the
357 sub-cellular localization prediction, and the phylogenetic analysis. Only one secretory ferritin ACCEPTED MANUSCRIPT
358 subunit has been identified in abalone H. discus discus (De Zoysa and Lee, 2007). To date,
359 cytoplasmic type and secreted type ferritins in mollusks have been identified through
360 bioinformatic analysis alone. However, preliminary bioinformatic analysis of ferritin genes may
361 not be sufficient to determine ferritin types. For instance, human serum ferritin and tissue ferritin
362 L are encoded by the same gene, which lacks a signal peptide sequence (Santambrogio et al.,
363 1987). The detailed serum ferritin secretory pathway remains largely unknown. Therefore, it is
364 necessary to perform further protein experiments to determine ferritin subunit types (cytoplasmic
365 or secreted) in mollusks. As there is only limited genetic information available for abalones, it is
366 difficult to confirm whether cytoplasmic type and secreted type ferritin subunits coexist in any
367 species. Further research is required to identify and explore additional ferritin subunit genes in
368 mollusks.
369 Consistent with previous studies of the tissue distribution of ferritin in mollusks, the highest
370 levels of HdFer1 and HdFer2 expression among the examined tissues in this study were observed
371 in the hepatopancreas. Transcripts for ferritin subunits are widely distributed in most organisms. In 372 vertebrates, the liver is a major organ involved in ironMANUSCRIPT metabolism and storage (Xie et al., 2012). 373 The hepatopancreas in invertebrates is generally co nsidered to be the functional equivalent of the 374 vertebrate liver. The invertebrate hepatopancreas produces many molecules involved in innate
375 immunity, such as hemocyanin, lectins, ferritin, and antibacterial peptides (Roszer, 2012). In
376 addition, the hepatopancreas plays important roles in pathogen clearance and antigen processing
377 (Alday-Sanz et al., 2002). Low levels of HdFer1 and HdFer2 expression were observed in the
378 mantle in the current study, which is inconsistent with a study in which ferritin was found to be
379 highly expressed in the mantle where it participated in shell formation in pearl oysters (Fang et al.,
380 2011; Zhang et al., 2003). One possible explanation for this discrepancy is that ferritin has roles in
381 numerous physiological functions in mollusks. 382 V. harveyiACCEPTED is a kind of Gram-negative bacteria that has been recognized as an opportunistic 383 pathogen in marine organisms (Austin and Zhang, 2006). Accumulating evidence indicates that V.
384 harveyi has been responsible for serious losses of farmed abalone H. diversicolor in China (Jiang
385 et al., 2013). In the current study, the expression of HdFer1 and HdFer2 were increased
386 significantly at the transcriptional level at different times following V. harveyi challenge. Mollusks ACCEPTED MANUSCRIPT
387 rely on their strong innate immunity to resist invasion and infection by environmental pathogens
388 since they lack an adaptive immune response. Ferritin is an important macromolecular protein that
389 is involved in innate immunity in animals. It was reported that ferritin transcripts were
390 significantly up-regulated in another species of abalone ( H. tuberculata ) after a 24 h exposure to V.
391 harveyi (Cardinaud et al., 2015). Another study involving H. tuberculata also demonstrated a
392 marked up-regulation of ferritin mRNA in survivors of successive V. harveyi infections. (Travers
393 et al. 2010). In addition, the expression of ferritins was increased in bay scallop, red drum, and
394 Chinese mitten crab after challenge with Listonella anguillarum (Hu et al., 2010; Kong et al.,
395 2010; Li et al., 2012). In turbot ( Scophthalmus maximus ), expression of the ferritin M subunit was
396 markedly up-regulated in a time-dependent manner after infection with a Gram-negative fish
397 pathogen Listonella anguillarum , a Gram-positive fish pathogen Streptococcus iniae , and after
398 poly (I:C) artificial challenge (Zheng et al., 2010). Ferritin injection enhanced resistance to WSSV
- 399 (white spot syndrome virus) infection through increased total hemocyte count, O 2 levels,
400 superoxide dismutase activity, and phenoloxidase activity in shrimp Litopenaeus vannamei (Ruan
401 et al.). These previous studies reveal an important role for ferritin in host defense against infection 402 by pathogens. MANUSCRIPT 403 Ferritin gene expression is regulated at both the transcriptional and post-transcriptional levels.
404 It has been demonstrated that pathogen infection induced oxidative stress in various
405 aquatic animals, such as catfish, oyster, rock bream, clam and others (Wang, et al., 2010;
406 Elvitigala, et al., 2013; Wang, et al., 2013; Adeyemi, et al., 2014). Infection by an invading
407 pathogen may be prevented by the phagocytes of the innate immune system through phagocytosis.
408 During this process, oxygen within cells is transformed into ROS, which is an important
409 component for eliminating invaders. However, excess accumulated ROS can result in damage to
410 the host. There is an antioxidant responsive element (ARE) region upstream of the ferritin gene.
411 Several proteins are involved in the transcriptional activation of ferritin via the ARE following
412 oxidative stressACCEPTED (Tsuji, 2005). Ferritin can inhibit Fenton’s reaction, which prevents the formation
413 of OH in order to protect the host. On the other hand, ferritin gene expression is also regulated at
414 the post-transcriptional level through an interaction between IRP and IRE that occurs upstream in
415 the 5 ʹ -UTR. When cells need iron, IRP is separated from IRE, increasing the synthesis of ferritin ACCEPTED MANUSCRIPT
416 to ensure iron storage. In the current study, transcripts for HdFer1 increased significantly in the
417 hepatopancreas at 6 h post-challenge, whereas this marked rise occurred at 48 h post-injection in
418 the mantle. In comparison, the expression of HdFer2 was increased significantly at 6 h
419 post-challenge in all sampled organs, including the gill, mantle, and hepatopancreas. An
420 alleviation of the damage induced by oxidative stress in host abalones would result as a
421 consequence of these increases in HdFer1 and HdFer2. Additionally, abalone ferritin may function
422 to compete with invading pathogens for iron, resulting in suppression of microorganism
423 proliferation and growth. This study illustrates a relationship between abalone ferritin and the host
424 defense response, but the mechanisms of action of ferritins need to be further explored and
425 elucidated.
426 In summary, the current study has demonstrated the existence of two ferritin subunits at the
427 protein level and provided a full-length cDNA sequence for one ferritin subunit in the abalone (H.
428 diversicolor ) for the first time. Analysis of the deduced amino acid sequence of HdFer2 indicated
429 that it was a novel ferritin that has not been previously described in abalone. There is no disulfide 430 bond linking the HdFer1 and HdFer2 subunits, andMANUSCRIPT these two subunits co-assemble to form 431 heteropolymers in H. diversicolor . Both HdFer1 and HdFer2 were widely distributed in the gill, 432 hepatopancreas, and mantle, but the highest level of expression for both subunits was observed in
433 the hepatopancreas. Moreover, transcripts for HdFer1 and HdFer2 were significantly increased
434 following V. harveyi challenge, suggesting that ferritins are involved in the abalone immune
435 response. Further investigation is needed to elucidate the underlying mechanisms of action of
436 mollusk ferritins in the host defense response.
437
438
439
440
441 ACCEPTED
442
443
444 ACCEPTED MANUSCRIPT
445
446 Acknowledgements
447 We are grateful to Prof. Dr. Xianhui He and Ouyang Dongyun (Jinan University, Guangzhou,
448 China) for their assistance in experiment technical of protein biochemical analysis. This work was
449 supported by grants from the Earmarked Fund for Modern Agro-industry Technology Research
450 System (No. CARS-48),the Special Scientific Research Funds for Central Non-profit Institutes,
451 South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (No.
452 2014TS16), the grants from the National Natural Science Foundation of China (No. 31172428 and
453 No. 41206118).
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572 (Crassostrea virginica ) reveals a novel combination of antimicrobial and oxidative stress host 573 responses after dermo ( Perkinsus marinus ) challenge. Fish Shellfish Immunol., 29, 921-929. 574 Xie, J.S., Cao, X.H., Wu, L.J., Luo, M., Zhu, Z.W., Huang, Y.Q. and Wu, X.Z., 2012. Molecular and 575 functional characterization of ferritin in abalone Haliotis diversicolor supertexta . Acta Oceanol. 576 Sin.31, 87-97. 577 Yu C.S., Chen Y.C., Lu C.H. and Hwang J.K., 2006. Prediction of protein subcellular localization. 578 Proteins. 64, 643-651. 579 Zhang, Y., Meng, Q., Jiang, T., Wang, H., Xie, L. and Zhang, R., 2003. A novel ferritin subunit 580 involved in shell formation from the pearl oyster (Pinctada fucata ). Comp. Biochem. Physiol. B 581 Biochem. Mol. Biol. 135, 43-54. 582 Zheng, W.J., Hu, Y.H., Xiao, Z.Z. and Sun, L., 2010. Cloning and analysis of a ferritin subunit from 583 turbot ( Scophthalmus maximus ). Fish Shellfish Immunol. 28, 829-836. 584 585 586 587 588 589 590 591 592 593 594 595 MANUSCRIPT 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 ACCEPTED 611 612 613 614 615 ACCEPTED MANUSCRIPT
616 617 Figure legends 618 619 Fig. 1. SDS-PAGE analysis of the protein pellet obtained by ultracentrifugation of protein
620 extracted from the visceral mass of abalone ( Haliotis diversicolor ). The gel was stained with
621 Coomassie brilliant blue R250.
622
623 Fig. 2. Reducing and non-reducing SDS-PAGE analysis of ferritin from abalone ( Haliotis
624 diversicolor ). The protein samples were denatured in SDS loading buffer in the presence or
625 absence of β-mercaptoethanol (5%, v/v). The gel was visualized by Coomassie brilliant blue R250
626 staining.
627
628
629 Fig. 3. Detection of the assembly of HdFer1 and HdFer2 by (A) native-PAGE followed by (B) a
630 second dimension SDS-PAGE. The protein sample was first separated by native-PAGE in one
631 dimension, followed by gel extraction, denaturation with SDS, and two-dimensional SDS-PAGE. 632 The gels were stained with Coomassie brilliant blue R250.MANUSCRIPT 633
634 Fig. 4. Nucleotide and deduced amino acid sequence of HdFer2 from H. diversicolor . Initial
635 codon (ATG) and terminal codon (TAA) was in bold. The letters of polyadenylation signal
636 sequence were in box. The two amino acid residues of ferroxidase activity center were in blue, one
637 conserved residue of ferrihydrite nucleation center was in green, and three iron ion channel
638 residues were in pink. 639 640 Fig. 5. Multiple alignment by the ClustalW method of HdFer1 and HdFer2 with ferritins from
641 other species. Amino acid residues that are conserved in at least 50% sequence are shaded in dark, 642 and similarACCEPTED amino acids shaded in grey. The protein sequences used for alignment analysis 643 including: Homo sapiens (NP_002023.2), Danio rerio (NP_001002378.1), Branchiostoma
644 belcheri tsingtauense (AAQ21039.1), Ostrea edulis (AFK73708.1), H. rufescens (ACZ73270.1),
645 H. discus hannai (ADK60915.1 and ABH10672.1), H. discus discus (ABG88846.1 and
646 ABG88845.1) and H. diversicolor (ABY87353.1). ACCEPTED MANUSCRIPT
647 648 Fig. 6. An neighbor-joining tree based on the amino acids sequences of different ferritins. The
649 phylogenetic tree was constructed based on ClustalW generated multiple sequence alignment of
650 amino acid sequences using MEGA 6.0. Bootstrap values (%) from 1000 replicates are indicated
651 at the tree nodes. The protein sequences used for phylogenetic analysis including: H. rufescens
652 (ACZ73270.1), H. diversicolor supertexta (ACU09496.1), H. discus hannai (ABH10672.1), H.
653 discus discus (ABG88846.1), H. diversicolor (ABY87353.1), Tegillarca granosa (ADC34696.1),
654 Crassostrea ariakensis (ACU25551.1), O. edulis (AFK73708.1), C. gigas (EKC30759.1),
655 Scrobicularia plana (AFV81451.1), Ruditapes philippinarum (ADX31290.1), Mercenaria
656 mercenaria (AFH73817.1), Meretrix meretrix (AAZ20754.1), B. belcheri tsingtauense
657 (AAQ21039.1), H. discus hannai (ADK60915.1), H. diversicolor (HdFer1, ABY87353.1), H.
658 sapiens (NP_000137.2), D. rerio (NP_001002378.1), H. sapiens (NP_002023.2), Mus musculus
659 (AAA37613.1), C. gigas (EKC42967.1), H. discus discus (ABG88845.1), C. gigas (EKC42968.1),
660 Carcinoscorpius rotundicauda (AAW22505.1 and AAW22506.1), Drosophila melanogaster
661 (NP_733358.1), Tribolium castaneum (XP _967058.1) and Eriocheir sinensis (ADF87491.1 and 662 ADF87490.1) MANUSCRIPT 663 664 Fig. 7. The relative expression levels of HdFer1 and HdFer2 in different tissues. Data are shown
665 as mean ± SD (n=5). **, P < 0.01 vs mantle. 666 667 Fig. 8. The relative expression levels of HdFer1 and HdFer2 in different tissues after pathogen V.
668 harveyi challenge . Data are shown as mean ± SD (n=5). *, P < 0.05, **, P < 0.01 vs 0 h. 669 670
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>We extracted and purified ferritin from abalone Haliotis diversicolor . >A novel ferritin gene HdFer2 was cloned and characterized from H. diversicolor . >HdFer transcript levels were altered in abalones upon Vibrio harveyi challenge. >HdFer may play an important role in abalone innate immune defense against invading pathogens.
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