School of Life Sciences and Food Engineering, Nanchang University, No. 999 Xuefu Road, Hong Gu Tan New District, Nanchang, Jiangxi 330031, China.
Fish Shellfish Immunol. 2013 Mar;34(3):865-74. doi: 10.1016/j.fsi.2012.12.021. Epub 2013 Jan 20.
Ferritin is a conserved iron-binding protein involved in cellular iron metabolism and host defense. In the present study, two distinct cDNAs for ferritins in the freshwater pearl mussel Hyriopsis schlegelii were identified (designated as HsFer-1 and HsFer-2) by SMART RACE approach and expressed sequence tag (EST) analysis. The full-length cDNAs of HsFer-1 and HsFer-2 were of 760 and 877 bp, respectively. Both of the two cDNAs contained an open reading frame (ORF) of 522 bp encoding for 174 amino acid residues. Sequence characterization and homology alignment indicated that HsFer-1 and HsFer-2 had higher similarity to H-type subunit of vertebrate ferritins than L-type subunit. Analysis of the HsFer-1 and HsFer-2 untranslated regions (UTR) showed that both of them had an iron response element (IRE) in the 5'-UTR, which was considered to be the binding site for iron regulatory protein (IRP). Quantitative real-time PCR (qPCR) assays were employed to examine the mRNA expression profiles. Under normal physiological conditions, the expression level of both HsFer-1 and HsFer-2 mRNA were the highest in hepatopancreas, moderate in gonad, axe foot, intestine, kidney, heart, gill, adductor muscle and mantle, the lowest in hemocytes. After stimulation with bacteria Aeromonas hydrophila, HsFer-1 mRNA experienced a different degree of increase in the tissues of hepatopancreas, gonad and hemocytes, the peak level was 2.47-fold, 9.59-fold and 1.37-fold, respectively. Comparatively, HsFer-2 showed up-regulation in gonad but down-regulation in hepatopancreas and hemocytes. Varying expression patterns indicate that two types of ferritins in H. schlegelii might play different roles in response to bacterial challenge. Further bacteriostatic analysis showed that both the purified recombinant ferritins inhibited the growth of A. hydrophila to a certain degree. Collectively, our results suggest that HsFer-1 and HsFer-2 are likely to be functional proteins involved in immune defense against bacterial infection.
铁蛋白是一种参与细胞铁代谢和宿主防御的保守铁结合蛋白。本研究采用 SMART RACE 方法和表达序列标签（EST）分析，从淡水珍珠贝（Hyriopsis schlegelii）中鉴定出两种不同的铁蛋白 cDNA（命名为 HsFer-1 和 HsFer-2）。HsFer-1 和 HsFer-2 的全长 cDNA 分别为 760 和 877 bp，均包含 522 bp 的开放阅读框（ORF），编码 174 个氨基酸残基。序列特征和同源性比对表明，HsFer-1 和 HsFer-2 与脊椎动物铁蛋白的 H 亚基比 L 亚基具有更高的相似性。对 HsFer-1 和 HsFer-2 的非翻译区（UTR）进行分析表明，它们的 5'-UTR 中均存在一个铁反应元件（IRE），被认为是铁调节蛋白（IRP）的结合位点。采用定量实时 PCR（qPCR）检测方法分析 mRNA 表达谱。在正常生理条件下，HsFer-1 和 HsFer-2 的 mRNA 在肝胰腺中表达水平最高，在性腺、斧足、肠、肾、心、鳃、后闭壳肌和套膜中表达水平中等，在血细胞中表达水平最低。用嗜水气单胞菌刺激后，HsFer-1 在肝胰腺、性腺和血细胞中的组织中经历了不同程度的增加，峰值水平分别为 2.47 倍、9.59 倍和 1.37 倍。相比之下，HsFer-2 在性腺中表现出上调，而在肝胰腺和血细胞中则下调。不同的表达模式表明，珍珠贝中的两种铁蛋白可能在应对细菌挑战时发挥不同的作用。进一步的抑菌分析表明，两种纯化的重组铁蛋白均在一定程度上抑制了嗜水气单胞菌的生长。综上所述，我们的结果表明 HsFer-1 和 HsFer-2 可能是参与免疫防御细菌感染的功能蛋白。
