锦鲤Hepcidin基因的克隆及其组织特异性表达分析

【目的】克隆锦鲤hepcidin全长cDNA序列(k-hepc),并获得此基因在鱼体内的表达模式。【方法】利用RT-PCR和RACE PCR的方法,从锦鲤肝脏中克隆锦鲤hepcidin的全长cDNA,进行序列测定和分析;锦鲤经肌肉注射维氏气单胞菌0、4、8、12、24和48 h后,分别取其肝、脾、肾、肠、脑、心、肌肉和鳃组织,采用实时荧光定量PCR的方法,以β-actin为内参基因,检测k-hepc基因的表达量。【结果】锦鲤抗菌肽(GenBank登录号KC795559)全长755 bp,编码序列276 bp,编码91个氨基酸,包括信号肽、原肽和成熟肽,成熟肽C端含有8个半胱氨酸,可形成4个分子内二硫键。与已报道的普通鲤鱼hepcidin氨基酸序列的一致性为93%,与其他鱼类hepcidin氨基酸序列的一致性为29%?93%。在本研究所检测的正常锦鲤的组织中,k-hepc均有表达,其中在肝组织中表达量最高,鳃组织中表达量最低。经维氏气单胞菌感染后,k-hepc在肝和心组织中的表达量明显增加,在其余组织中变化不显著。【结论】k-hepc编码的蛋白是Hepcidin家族的成员之一。锦鲤Hepcidin的表达主要受内在调节因素影响。     

Adaptive evolution of hepcidin genes in antarctic notothenioid fishes

Hepcidin is a small bioactive peptide with dual roles as an antimicrobial peptide and as the principal hormonal regulator of iron homeostasis in human and mouse. Hepcidin homologs of very similar structures are found in lower vertebrates, all comprise approximately 20-25 amino acids with 8 highly conserved cysteines forming 4 intramolecular disulfide bonds, giving hepcidin a hairpin structure. Hepcidins are particularly diverse in teleost fishes, which may be related to the diversity of aquatic environments with varying degree of pathogen challenge, oxygenation, and iron concentration, factors known to alter hepcidin expression in mammals. We characterized the diversity of hepcidin genes of the Antarctic notothenioid fishes that are endemic to the world's coldest and most oxygen-rich marine water. Notothenioid fishes have at least 4 hepcidin variants, in 2 distinctive structural types. Type I hepcidins comprise 3 distinct variants that are homologs of the widespread 8-cysteine hepcidins. Type II is a novel 4-cysteine variant and therefore only 2 possible disulfide bonds, highly expressed in hematopoietic tissues. Analyses of d(N)/d(S) substitution rate ratios and likelihood ratio test under site-specific models detected significant signal of positive Darwinian selection on the mature hepcidin-coding sequence, suggesting adaptive evolution of notothenioid hepcidins. Genomic polymerase chain reaction and Southern hybridization showed that the novel type II hepcidin occurs exclusively in lineages of the Antarctic notothenioid radiation but not in the basal non-Antarctic taxa, and lineage-specific positive selection was detected on the branch leading to the type II hepcidin clade under branch-site models, suggesting adaptive evolution of the reduced cysteine variant in response to the polar environment. We also isolated a structurally distinct 4-cysteine (4cys) hepcidin from an Antarctic eelpout that is unrelated to the notothenioids but inhabits the same freezing water. Neighbor-Joining (NJ) analyses of teleost hepcidins showed that the eelpout 4cys variant arose independently from the notothenioid version, which lends support to adaptive evolution of reduced cysteine hepcidin variants on cold selection. The NJ tree also showed taxonomic-specific expansions of hepcidin variants, indicating that duplication and diversification of hepcidin genes play important roles in evolutionary response to diverse ecological conditions.     

Two hepcidin-like antimicrobial peptides in Barramundi Lates calcarifer exhibit differing tissue tropism and are induced in response to lipopolysaccharide

Genes encoding two hepcidin-like antimicrobial peptides were discovered in Barramundi, Lates calcarifer (barramundi, Giant sea perch). Analysis of the coding regions indicated that genes for each hepcidin comprised 3 exons and 2 introns. The deduced amino acid sequences for each molecule resulted in a protein comprising a signal sequence of 24 aa in each case, coupled to a prepropeptide of 75 aa for hepcidin 1 and 78 aa for hepcidin 2. A cleavage site was identified in each prepropetide at amino acid 64 with the cleavage motif--QKR/QS--resulting in mature peptides of 25 and 28 amino acids respectively. Each mature peptide contained 8 conserved cysteine residues and 3 dimensional modeling predicted a β-hairpin and β-sheet structure characteristic of human Liver Expressed Antimicrobial Peptide (LEAP). Analysis of the deduced amino acid sequences by BLAST with phylogenetic supported indicated that hepcidin 1 was a HAMP1-type peptide closely related to hepcidins identified in other Perciformes (Micropterus and Pseudosciaena), whilst hepcidin 2 was a HAMP2-type peptide most similar to a hepcidin previously identified in black rock fish (Sebastes schlegeli). Both hepcidin genes were inducible in barramundi following intraperitoneal injection with lipopolysaccharide, with elevated expression detected in liver and head kidney 3 h post IP injection for hepcidin 1 and in liver only for hepcidin 2. The elevated expression was transient with return to normal levels within 24-48 h. No significant expression of either peptide was detected in spleen, skin or gill following IP injection with LPS.     

Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone

Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of the present study was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin fusion proteins. The corresponding peptides, purified after cleavage from thioredoxin, were properly folded and contained the expected four-disulfide bridges without the need of any renaturation or oxidation steps. Human and mouse hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation.     

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.     

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.     

A novel hepcidin-like in turbot (Scophthalmus maximus L.) highly expressed after pathogen challenge but not after iron overload

Hepcidins are antimicrobial peptides with an important role in the host innate immunity. Moreover, it has been reported that mammalian hepcidins present a dual-function being a key regulator in the iron homeostasis. Here, we describe the coding sequence of a novel hepcidin-like peptide in turbot, Scophthalmus maximus. This molecule presents several differences with regard to the previously characterized hepcidin in this flatfish species and it has not the hypothetical iron regulatory sequence Q-S/I-H-L/I-S/A-L in the N-terminal region. Therefore we propose the existence of at least two types of hepcidin in turbot. Moreover, results revealed a higher variability in the mRNA sequences of the novel hepcidin compared with the other form. Constitutive expression of turbot hepcidins (Hepcidin-1 and Hepcidin-2) was analyzed in several tissues and as expected, both molecules were highly represented in liver. On the other hand, the effect of three different stimuli (bacterial or viral infection and iron overloading) in the level of hepcidin mRNA was also examined and a differential response to pathogens and iron was observed. Whereas both hepcidins were affected by pathogen challenge, only Hepcidin-1 was up-regulated after iron overloading. Therefore, this and other evidences suggest that these peptides could be involved in different functions covering the dual role of mammalian hepcidins.     

Identification of centrarchid hepcidins and evidence that 17β-estradiol disrupts constitutive expression of hepcidin-1 and inducible expression of hepcidin-2 in largemouth bass (Micropterus salmoides)

Hepcidin is a highly conserved antimicrobial peptide and iron-regulatory hormone. Here, we identify two hepcidin genes (hep-1 and hep-2) in largemouth bass (Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Hepcidin-1 contains a putative ATCUN metal-binding site in the amino-terminus that is missing in hepcidin-2, suggesting that hepcidin-1 may function as an iron-regulatory hormone. Both hepcidins are predominately expressed in the liver of largemouth bass, similar to other fish and mammals. Experimental exposure of pond-raised largemouth bass to 17β-estradiol and/or the bacteria Edwardsiella ictaluri led to distinct changes in expression of hep-1 and hep-2. Estradiol reduced the constitutive expression of hep-1 in the liver. Bacterial exposure induced expression of hep-2, suggesting that hepcidin-2 may have an antimicrobial function, and this induction was abolished by estradiol. To our knowledge, this is the first report of the regulation of hepcidin expression by estradiol in either fish or mammals.     

Iron regulatory and bactericidal properties of human recombinant hepcidin expressed in Pichia pastoris

Hepcidin is a circulating cysteine-rich peptide with antimicrobial properties. It functions as a hormonal regulator of iron homeostasis by controlling iron efflux from target cells via ferroportin (FPN1), which is internalized and degraded upon hepcidin binding. Because of its profound biomedical significance, hepcidin has become the target of intense biochemical studies. The aim of this study was to produce functional recombinant hepcidin in sufficient quantities for advanced research or potential clinical use, as the native hepcidin can be isolated from urine in very low yield. We report the expression, purification and functional characterization of hepcidin variants in yeast P. pastoris. The yield of untagged hepcidin 20- and 25-mer peptides was too low for complete functional characterization. By contrast, Hep20 and Hep25 tagged with either single 6xHis or double Myc-6xHis epitopes were expressed at high quantities (5-7mg/l of culture), yet mostly in oligomeric forms. Purification of monomeric tagged hepcidins was achieved by size exclusion chromatography, with a yield of 0.5-1mg/l of culture. All recombinant hepcidins exhibited bacteriostatic activity and the ability to control cellular iron homeostasis, with Hep25-His being the most potent. Thus, Hep25-His promoted an increase in the levels of the labile iron pool (LIP) in macrophages and consistently bound to ferroportin (FPN1) causing its internalization and the subsequent downregulation of transferrin receptor 1 (TfR1) expression. Analysis by mass-spectrometry suggested that all eight cysteines participated in disulfide bond formation. Our results suggest that only the recombinant Hep25-His monomer was a fully active peptide. As Hep25-His faithfully recapitulates the functional properties of native Hep25, it represents a powerful tool for biochemical studies and potential diagnostic and therapeutic applications.     

Calcitonin increases hepatic hepcidin expression through the BMP6 of kidney in mice

BackgroundOsteoporosis is frequently accompanied by iron disorders. Calcitonin (CT) was approved as a clinical drug to treat osteoporosis. Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin deficiency leads to iron overload diseases. This study was aimed at investigating the effect of CT on hepatic hepcidin and the mechanism by which CT modulates hepatic hepcidin pathways and iron metabolism.MethodRT-PCR, Western blot, ELISA and siRNA were used to detect the effect of CT on iron metabolism in vivo and in vitro. In addition, the regulatory signal molecules of hepcidin were measured to explore the molecular mechanism of its regulation.ResultsThe results showed that CT strongly increased hepcidin expression and altered iron homeostasis, after mice were intraperitoneal injection of CT. In response to CT administration, BMP6 level in kidney and the serum BMP6 was increased significantly. The phosphorylation of Smad1/5/8 proteins in liver was increased at 3 h and 6 h. Moreover, the Bmp inhibitor LDN-193,189 pretreatment significantly attenuated the CT-mediated increases in phosphorylated Smad1/5/8 and Hamp1 mRNA levels. Calcitonin receptor (CTR) siRNA transfection significant suppressed the role of CT on BMP6 expression in Caki-1 cells.ConclusionOur results suggest that CT strongly induces hepcidin expression and affected iron metabolism. It will provide a new strategy for the treatment of calcium iron related diseases.