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. 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. 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ACCEPTED MANUSCRIPT 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* 5 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 9 10 b College of Fisheries and Life, Shanghai Ocean University, Shanghai 201306, China 11 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) 19 20 21 22 23 24 25 26 ACCEPTED 27 28 29 30 ACCEPTED MANUSCRIPT 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 49 50 51 52 53 54 55 56 ACCEPTED 57 58 59 60 ACCEPTED MANUSCRIPT 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, numerous studies have revealed the existence of 76 another subunit, the M (middle) subunit, in additionMANUSCRIPT 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 aquaculture industry, as well as exploring the 105 immune system of the hosts. The purpose of the presMANUSCRIPTent 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. 113 114 115 ACCEPTED 116 117 118 ACCEPTED MANUSCRIPT 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 µl (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.