Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops x Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel beta-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, alpha-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4-5 orders of magnitude above base-line 24-48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.     

哺乳动物Hepcidin表达调控的研究进展

Hepcidin是由生物体肝脏表达的一种具有抵抗外界微生物侵害的小分子阳离子多肽,与干扰素、补体等组成了宿主的免疫防御系统.哺乳动物的Hepcidin表达调控受多种因素的影响.本文主要介绍了机体内铁水平、感染和炎症、贫血和缺氧以及运动对Hepcidin表达调控的影响.铁超负荷、脂多糖和病原体可诱导Hepcidin的表达,而贫血、缺氧和运动可下调其表达.本文还对Hepcidin的临床应用研究中存在的问题进行了初步讨论.     

Hepcidin研究进展

Hepcidin是肝脏特异性表达的一种小分子抗菌肽,是铁代谢的负调节激素。与炎症性贫血、遗传性血色沉着病等疾病的发病机制密切相关。证据显示,Hepcidin直接抑制肠上皮细胞铁吸收和诱导单核巨噬细胞铁滞留。同时,Hepcidin还具有广谱抗菌活性,与固有免疫密切相关。铁超载、感染、炎症及细胞因子可诱导Hepcidin表达,而贫血和缺氧则抑制其表达。Hepcidin的发现及其相关的铁离子运输机制的研究,将为铁离子吸收及分配的铁稳态调节和炎症性贫血、遗传性血色沉着病中的铁代谢障碍的分子机制探索开辟新的途径。本文就Hepcidin的分子特征、表达调控及生物学功能等方面研究进展进行综述。     

Molecular characterization of hepcidin AS-hepc2 and AS-hepc6 in black porgy (Acanthopagrus schlegelii): expression pattern responded to bacterial challenge and in vitro antimicrobial activity

There are more diversified isoforms of the hepcidin gene that exist in fishes than in mammals, and elucidating the differences between these isoforms should provide insight into the functioning of hepcidin in fishes. In our study, AS-hepc2 and AS-hepc6 hepcidin isoforms from black porgy were characterized for their in vivo expression patterns following bacterial challenge, and their in vitro antimicrobial activities against Gram-positive and Gram-negative bacteria as well as fungi. As a result, two isoforms were observed to be widely distributed in all the tissues tested. AS-hepc2 was a liver-expressed hepcidin peptide which was always highly more expressed in the liver than in the other tissues tested no matter whether this was before or after bacterial challenge. AS-hepc6 was detected mainly in the head kidney and trunk kidney of normal fish, but, in the challenged fish, its expression involved more tissues than just the kidneys. The mature peptides of AS-hepc2 and AS-hepc6 were modeled for 3D structure and then synthesized for antimicrobial assay. AS-hepc6 had a wider antimicrobial spectrum than AS-hepc2 and, in particular, had more potent antifungal activity. Our study indicated that the two hepcidin isoforms had different characteristics in terms of their expression patterns and antimicrobial activity, and they were assumed to play an overlapping role in the innate immune system of black porgy against invading pathogens.     

Hepcidin的生物学特性及其研究进展

Hepcidin是一种由肝脏合成的富含半胱氨酸的小分子肽。近几年的研究证实hepcidin对于调节机体铁离子的代谢平衡发挥着重要的作用,其可抑制肠道铁吸收和单核巨噬细胞系统铁释放。此外,除了机体铁状况,感染、炎症、贫血和缺氧等原因也会改变hepcidin的表达水平。通过对hepcidin的分子生物学特点、表达调控及生物活性、医学及药用价值等方面研究进展的概述,对采用基因工程的方法生产hepcidin进行了评述及展望。     

Hepcidin在哺乳类及鱼类中的表达和作用

Hepcidin也称为铁调素,是肝脏特异性表达的一种阳离子小分子抗菌肽,具有抑制多种细菌、真菌、病毒和原生动物生长繁殖的作用,是机体天然免疫的一种效应分子;同时也是一种信号分子,参与机体铁代谢,通过直接抑制肠上皮细胞铁吸收和单核巨噬细胞铁释放调节机体铁平衡,与炎症性贫血、遗传性血色素沉着病等铁代谢紊乱性疾病的发病机制密切相关。脂多糖（LPS）、铁超载和病原体可诱导hepcidin表达,而贫血和缺氧可下调其表达。目前,鱼类hepcidin的研究也成为热点,但主要集中在hepcidin的抗菌活性方面,有关其在鱼类铁代谢方面的功能仍需要进一步研究。     

半滑舌鳎AKT-ineracting protein基因的克隆及免疫应答表达分析

研究克隆了半滑舌鳎Cynoglossus semilaevis膜蛋白AKT-interacting protein (AKTIP)基因, 研究了其在健康组织中的表达模式、鳗弧菌(Vibrio anguillarum)感染后免疫组织和不同病原物刺激外周血淋巴细胞中的表达特征。通过常规克隆和RACE技术, 获得的半滑舌鳎AKTIP基因全长cDNA序列为1224 bp, 其中包括5'-UTR为116 bp, 3'-UTR为117 bp和完整的ORF序列891 bp, 编码296个氨基酸, 预测蛋白质的等电点(PI)为9.12,分子量是33.74 kD; 同源比对发现半滑舌鳎AKTIP的氨基酸序列在不同物种之间具有较高的保守性; 荧光实时定量PCR (qRT-PCR)检测到半滑舌鳎各个组织中均有AKTIP基因的表达, 在卵巢中表达量最高; 鳗弧菌感染半滑舌鳎后, AKTIP基因在肝、鳃、血液、肠、头肾和脾中均上调表达; 病原模拟物PGN、LPS、poly I:C和WGP刺激半滑舌鳎外周血淋巴细胞均诱导AKTIP基因下调表达。研究表明半滑舌鳎AKTIP基因参与了机体免疫反应为给半滑舌鳎免疫防御技术的研究提供理论依据。     

Purification and partial characterisation of recombinant human hepcidin

Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays.     

Bacteriostatic activity of the proregion of human hepcidin

Hepcidin was first identified as an antimicrobial peptide expressed in the liver. It was later demonstrated that hepcidin is in fact the long sought hormone that regulates iron homeostasis in mammals. Hepcidin is encoded as an 84 amino acid prepropeptide that is successively cleaved to yield prohepcidin and the mature 25 amino acid hepcidin. Both the bioactive 25-aa hepcidin and the 35-aa proregion are secreted by liver hepatocytes. The aim of the present study was to assess the antibacterial activity of the proregion peptide from human hepcidin. Using a chemically synthesized peptide corresponding to the proregion, we show that it is bactericidal against Bacillus megaterium (25μM), and inhibits Bacillus subtilis growth at high concentration (200μM). No synergistic interaction of proregion and Hepc25 against Bacillus megaterium was seen. In a further step, the mode of action of proregion on Bacillus megaterium was studied. It caused a slow accumulation of the vital stain SYTOX in the bacteria, indicating that it did not destroy the microbial membranes through a detergent-like mechanism, even at concentrations (80μM) higher than those required to kill the bacteria. This result suggests that the target of proregion might be an intracellular component. Finally, gel retardation assay showed that the DNA binding ability of the hepcidin proregion was equivalent to that observed for magainin 2, an antimicrobial peptide which exerts its antimicrobial effect by interfering with intracellular nucleic acids. In conclusion, we speculate that the proregion of hepcidin may have bacteriostatic effects, and as such may contribute to the innate immune response.     

Hepcidin and Host Defense against Infectious Diseases

Hepcidin is the master regulator of iron homeostasis in vertebrates. The synthesis of hepcidin is induced by systemic iron levels and by inflammatory stimuli. While the role of hepcidin in iron regulation is well established, its contribution to host defense is emerging as complex and multifaceted. In this review, we summarize the literature on the role of hepcidin as a mediator of antimicrobial immunity. Hepcidin induction during infection causes depletion of extracellular iron, which is thought to be a general defense mechanism against many infections by withholding iron from invading pathogens. Conversely, by promoting iron sequestration in macrophages, hepcidin may be detrimental to cellular defense against certain intracellular infections, although critical in vivo studies are needed to confirm this concept. It is not yet clear whether hepcidin exerts any iron-independent effects on host defenses.     

香鱼hepcidin基因的克隆、序列分析及组织表达特征

Hepcidin是一类富含半胱氨酸的抗菌肽,在鱼类非特异性免疫中起重要作用,具有调节铁代谢的功能。该研究克隆了香鱼hepcidin基因cDNA序列,全长763个核苷酸,包含一个完整的开放阅读框,推测编码一个由85个氨基酸组成的相对分子质量为9.7k的多肽,N端24个氨基酸是信号肽序列。香鱼hepcidin的成熟肽序列由25个氨基酸组成,含有8个半胱氨酸,可形成4个分子内二硫键结构。系统进化树分析表明,hepcidin的物种进化关系与目前接受的物种分类关系基本一致,香鱼hepcidin位于鱼类hepcidin簇中,与大西洋鲑hepcidin的亲缘关系最近,达60%。香鱼hepcidin在肝脏中表达量最大,在脾、肾、心脏和肌肉中也有表达。实时荧光定量PCR(qRT-PCR)结果表明,鳗利斯顿氏菌(Listonella anguillarum)感染以后香鱼肝组织中hepcidin基因mRNA表达量显著增加,在12h增加了8.26倍。hepcidin基因可能在香鱼抗外界病原物感染过程中起重要作用。     

Animal models with enhanced erythropoiesis and iron absorption

The regulation of iron absorption is of considerable interest in mammals since excretion is minimal. Recent advances in iron metabolism have expounded the molecular mechanisms by which iron absorption is attuned to the physiological demands of the body. The pinnacle was the discovery and identification of hepcidin, a hepatic antimicrobial peptide that regulates absorption to maintain iron homeostasis. While the intricacies of its expression and regulation by HFE, transferrin receptor 2 and hemojuvelin are still speculative, hepcidin responsiveness has correlated negatively with iron absorption in different models and disorders of iron metabolism. Consequently, hepcidin expression is repressed to enhance iron absorption during stimulated erythropoiesis even in situations of elevated iron stores. Animal models have been crucial to the advances in understanding iron metabolism and the present review focuses on phenylhydrazine treated and hypotransferrinaemic rodents. These, respectively, experimental and genetic models of enhanced erythropoiesis highlight the shifting focus of iron absorption regulation from the marrow to the liver.     

Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus

The cysteine-rich peptide hepcidin is known to be an antimicrobial peptide and iron transport regulator that has been found in both fish and mammals. Recently, we found two different types (designated Hep-JF1 and Hep-JF2) of hepcidin cDNA in the Japanese flounder, Paralichthys olivaceus, by expressed sequence tag analysis. The identity of amino acid sequences between Hep-JF1 and Hep-JF2 was 51%. The Hep-JF1 and Hep-JF2 genes both consist of three exons and two introns, and both exist as single copies in the genome. The predicted mature regions of Hep-JF1 and Hep-JF2 have six and eight Cys residues, respectively. The first Cys residue of Hep-JF1 was deleted and the second was replaced with Gly. The number and positions of Cys residues in Hep-JF2 are the same as they are in human Hep. Hep-JF1 is specifically expressed in liver while the expression of Hep-JF2 was detected from gill, liver, heart, kidney, peripheral blood leucocytes, spleen and stomach. Gene expression of Hep-JF1 in liver decreased during experimental iron (iron-dextran) overload. Expression of Hep-JF1 in liver was decreased by injecting fish with iron-dextran and increased by injecting lipopolysaccharide. Iron overload did not significantly affect expression of Hep-JF2 in liver but it did increase expression of Hep-JF2 in kidney. Lipopolysaccharide injection increased expression of Hep-JF2 in both liver and kidney. In liver, some cells expressed both Hep-JF1 and Hep-JF2 while some other cells expressed just one of them. Synthesized Hep-JF2 peptide showed antimicrobial activity, while synthesized Hep-JF1 peptide did not against several bacteria including fish-pathogenic bacteria used in this study.