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Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates
Developmental and Comparative Immunology 28 (2004) 747–754 www.elsevier.com/locate/devcompimm
Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates Hiroko Shike, Chisato Shimizu, Xavier Lauth, Jane C. Burns* Department of Pediatrics, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, San Diego, CA 92093-0830, USA Received 9 September 2003; revised 20 November 2003; accepted 29 November 2003
Abstract Hepcidin is an antimicrobial peptide and iron-regulatory molecule that is conserved among vertebrates. Mutations or overexpression of the human hepcidin gene have been found in patients with hemochromatosis and refractory anemia. To further understand the function and regulation of hepcidin, animal models are needed. We sequenced cDNA, genes and upstream regions of zebrafish hepcidin and analyzed gene expression by kinetic PCR. Zebrafish hepcidin genes consist of two introns and three exons that encode a prepropeptide (91 amino acids). The amino acid sequences and gene organization were remarkably conserved between zebrafish and other species. Elevated gene expression was observed in abdominal organs, skin, and heart in fish that developed signs of infection following bacterial injection. Zebrafish may be a suitable model organism for further study of hepcidin gene regulation. q 2003 Elsevier Ltd. All rights reserved. Keywords: Fish; Streptococcus iniae; Infection; Hepcidin; Bacterial challenge
1. Introduction Hepcidin is an antimicrobial peptide and ironregulatory molecule primarily expressed in liver, and conserved among mammals and bony fish. Hepcidin peptides have been isolated from human urine and plasma [1,2] and the gill of hybrid striped bass [3]. Hepcidin mRNA has been found in mouse [4], rat, medaka, rainbow trout, Japanese flounder [5], winter flounder [6], long-jawed mudsucker [7], and Atlantic salmon [6]. The hepcidin peptides share a distinctive cystine bridge structure that is unique among other antimicrobial peptides. In humans and mice, hepcidin * Corresponding author. Tel.: þ1-619-543-5326; fax: þ 1-619543-3546. E-mail address: [email protected] (J.C. Burns). 0145-305X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2003.11.009
plays an important role in iron homeostasis. Hepcidin gene up-regulation was observed in mice with ironloading [4]. Over-expression of hepcidin from hepatic adenomas was found in a patient with refractory anemia [8] and mutations in the hepcidin gene were reported in two families with severe juvenile hemochromatosis [9]. Increased gene expression was also noted following lipopolysaccharide-challenge in mice [4] and bacterial challenge in white bass [3] and Atlantic salmon [6]. Although hepcidin expression is highest in the liver, hepcidin mRNA has also been detected in other organs such as spleen and pancreas in the mouse [10] and several fish species [6]. Thus, hepcidin, a multifunctional peptide important in both innate immunity and iron homeostasis, appears to be regulated through complex systems in response to diverse environmental stimuli.
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Use of an animal model such as zebrafish (Danio rerio) should help to elucidate further details of hepcidin expression and regulation. As a first step, we determined the sequence and organization of the zebrafish hepcidin gene and analyzed gene expression by challenge experiments with Escherichia (E.) coli and Streptococcus (S.) iniae, a hemolytic gram-positive coccus that causes fatal meningoencephalitis in both humans and fish [11,12]. Strains of S. iniae were shown to be potent pathogens in zebrafish [13] and to induce high levels of hepcidin gene expression in white bass [3].
This is the first report to identify cDNA, genes and upstream sequences of zebrafish hepcidin and to describe hepcidin gene expression following bacterial challenge.
2. Materials and methods 2.1. RNA sampling and cDNA sequencing Zebrafish were challenged with bacteria to attempt to induce zebrafish antimicrobial peptide genes, as
Fig. 1. Nucleotide sequence of zebrafish hepcidin cDNA and predicted amino acid sequence. Microsatellite (ATC) repeats are underlined in the 50 UTR. Binding sites for primers are shown with arrows (50 –30 ). Polyadenylation signal is underlined in the 30 UTR. The predicted organization of the peptide domains (signal peptide, prodomain, and mature peptide) is shown by gray boxes. The stop codon is indicated with an asterisk (*). Splicing sites for intron 1 and 2 are indicated with triangles.
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described previously [14]. Briefly, fish were injected with bacteria (approximately 106 cfu each), E. coli, INVF0 a(-) and S. iniae, K136-01 bB, a pathogenic strain that induced hepcidin expression in white bass liver [3], and had been isolated from the brain of white bass with fatal encephalomeningitis. Total RNA was extracted from abdominal organs including intestines and liver. Dozens of short, unidentified genomic sequences were in the Sanger zebrafish database that shared low similarity with the human hepcidin sequence (alignment score , 44). When converted into amino acid sequences, we found cysteine-rich regions in only three deposits, zfishG-a544c10.p1c, zfishCa2039d12.p1c, and zfishG-a550h10.p1c. We considered these as possible candidates for zebrafish hepcidin. Primers 246F and 275F were prepared to sequence 30 -mRNA (Fig. 1). RT-PCR products together with a poly-T primer were sequenced as described [14]. The 50 region of the mRNA was determined by 50 rapid amplification of cDNA ends (RACE) as described [14]. 2.2. Genomic DNA sequencing Inverse PCR was used to sequence 30 and 50 flanking sequences as described [14]. To obtain a continuous genomic sequence, a long PCR product that included both the hepcidin gene and upstream region was cloned and sequenced. A primer (47F) was designed from sequence 1.6 kb upstream from
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the transcription start site generated by inverse PCR and was used for long PCR together with rTth DNA polymerase XL (PE Applied Biosystems) and 567R, which binds near the 30 -terminal of the gene (Fig. 2). The long PCR products were cloned into TOPO TA vector and sequenced. Genomic DNA was tested for the presence of pseudogenes by PCR amplification with primer pairs spanning introns, 910F þ 205R, 910F þ 567R, and 234F þ 567R. The size of the PCR products was analyzed by agarose gel electrophoresis. 2.3. Bacterial challenge and gene expression analysis The first challenge experiment was performed by intra-muscular injection of either sterile Todd Hewitt broth (THB) or S. iniae, KST729 99aB (approximately 106 cfu each), a pathogenic strain isolated from white bass brain. Abdominal organs were dissected 1 day-post challenge. The second challenge experiment was conducted by intra-muscular injection of a mixture of E. coli and S. iniae (approximately 106 cfu each) or sterile THB. Tissue samples (abdominal organs, skin, gill, and heart) were dissected 3 days-post challenge. Zebrafish hepcidin mRNA and 18S rRNA were quantitated using kinetic RT-PCR as described [14]. Briefly, a primer pair, 234F þ 241R, was designed to span the second intron (1.5 – 1.6 kb) to amplify hepcidin cDNA (48 bp) but not genomic DNA (Figs. 1 and 2). Hepcidin cDNA was expressed as a copy number compared to the standard curve
Fig. 2. Organization of two zebrafish hepcidin genes and mRNA. Exons are depicted by boxes: open boxes, untranslated regions; striped boxes, regions encoding a signal peptide; stippled boxes, regions encoding a prodomain; filled boxes, regions encoding a mature hepcidin peptide. Binding sites for primers are shown with arrows (50 –30 ). GenBank accession numbers are shown in parentheses.
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generated by serial dilution of a positive control RTPCR product (509 bp) of known copy number generated by the primer pair 910F þ 567R. The copy number of hepcidin cDNA was normalized by arbitrary units of 18S for each sample. In this kinetic PCR system, a difference between two samples is significant when the ratio is four or greater [15]. Means of hepcidin levels from at least three fish were also compared using two standard deviations (SD) and 95th percentile confidence limits of the normal distribution.
3. Results 3.1. Zebrafish hepcidin cDNA sequence The 30 cDNA sequence from the challenged and control zebrafish samples were identical (Fig. 1). Careful visual examination of the 50 cDNA sequence in Chromas format revealed a mixture of multiple sequences that differed only by the number of microsatellite repeats with the tri-nucleotide ATC repeated 11 –16 times. A 50 RACE product cloned in TOPO TA vector is shown as a representative hepcidin cDNA sequence (GenBank accession no. AY363454) (Fig. 1). Despite the variability in the 50 UTR, all cDNAs shared an identical coding sequence for a 91 aa peptide. A stretch of eight cysteines, a signature for most hepcidins, was also found at the C-terminal. Since we did not observe any clinical signs of infection in zebrafish challenged with S. iniae, K136-01 bB, we used another strain of S. iniae, KST729 99aB, in subsequent challenge experiments. The strains were known to be equally pathogenic in bass. 3.2. Zebrafish hepcidin gene Two hepcidin genes were cloned, hepcidin 1 (GenBank accession no. AY363452) and hepcidin 2 (GenBank accession no. AY363453) (Fig. 2). Both genes consist of three exons and two introns, identical to hepcidin genes of white bass, mice, and humans [2 – 4]. The coding sequence of hepcidin 1 and the cDNA (Fig. 1) were identical. Three nucleotide substitutions in the coding sequences of hepcidin 2 resulted in two tandem aa substitutions, Leu55 – Ala56
substituted for Gln55 – Thr56. PCR products from genomic DNA with primer pairs spanning introns matched the expected sizes for hepcidin 1 and 2, thus eliminating the possibility of pseudogenes lacking introns (data not shown). 3.3. Upstream region of hepcidin gene The upstream regions (1.6 kb) for both hepcidin 1 and 2 were almost identical (98%) and contained extensive microsatellite repeats, 50 -TAAA-30 (2 1.6 to 2 1 kb upstream from the transcriptional start site). Thus, only 1.0 kb of the upstream region was searched for putative regulatory motifs by TFSEARCH [16]. Both upstream regions contained a TATA box (2 30) and several putative transcription factor binding sites for CAAT enhancer-binding protein (C/EBP)a, C/ EBPb, hepatocyte nuclear factor (HNF) 3b, HNF4, and NF-k B (Table 1). 3.4. Zebrafish hepcidin gene expression Quantitative analysis of the positive control RT-PCR product from hepcidin cDNA resulted in a linear standard curve with a dynamic range of 10– 107 copies. The melting temperatures ðTm Þ of the RT-PCR products were 78.6 8C for hepcidin and 80.5 8C for 18S. No detectable amplification of up to 10 ng of genomic DNA confirmed that the kinetic PCR result accurately reflected the quantitation of hepcidin cDNA as opposed to genomic DNA.
Table 1 Putative transcription factor bindings sites Transcription factors
Putative binding sites
TATA box C/EBPa C/EBPb HNF3b HNF4 NF-k B
230 2956, 2133 2956, 2969, 2147, 2133 2972, 2925, 2506, 2413, 2406, 2250, 2245 2108 2733
Putative transcription factor binding sites were predicted in the upstream sequence of hepcidin 1 (AY363452) using TFSEARCH, and expressed relative to the transcriptional starting site. C/EBP: CAAT enhancer-binding protein, HNF: hepatocyte nuclear factor, NF: nuclear factor.
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Fig. 3. Zebrafish hepcidin gene expression following bacterial challenge. Zebrafish hepcidin mRNA and 18S rRNA were quantitated by kinetic RT-PCR following intra-muscular injection of THB or bacteria. Hepcidin mRNA levels were normalized by 18S rRNA levels and expressed in arbitrary units. Fish challenged with THB, A; fish challenged with bacteria but remaining healthy, W; fish challenged with bacteria and clinically affected, X. In experiment 1, abdominal organs were tested 1 day-post challenge with THB or S. iniae. In experiment 2, abdominal organs, skin, gill, and heart were tested 3 days-post challenge with THB or a mixture of S. iniae and E.coli.
In the first challenge experiment, only one of four fish challenged with S. iniae developed decreased activity. Abdominal organs from this fish yielded high levels of hepcidin mRNA with a 700-fold increase as compared to the THB-challenged fish (Fig. 3). The other three fish remained normal despite the bacterial injection, and the hepcidin expression level was not significantly different from the levels in THBchallenged fish. In the second experiment (Fig. 3), the levels of hepcidin expression in different organs following challenge with S. iniae and E. coli were determined. One of four zebrafish again developed difficulty in swimming by 3 days post-challenge. In this ill-appearing fish, the hepcidin expression level was significantly higher in abdominal organs, skin, and heart (16–43-fold increase) compared to THB-challenged fish.
4. Discussion We report here the molecular characterization of zebrafish hepcidin, which shares striking similarity in gene organization and amino acid sequences with hepcidins from humans, mice, and other fish. Hepcidin prepropeptides from several different animals share a high degree of homology (Fig. 4). Signal P [17] predicted a cleavage site between Ala24 and Val25. The resulting signal peptide has a basic residue (Lys2) at the N-terminal end followed
by a hydrophobic region rich in Val and Ala, which is typical for a signal peptide. Interestingly, a signal peptide cleavage site for white bass preprohepcidin was predicted between Ala24 and Val25 [3], suggesting a conserved mechanism for proteolytic cleavage of the hepcidin signal peptide among fish species. The aa substitution located in the prodomain of hepcidin 2 (Gln55 –Thr56) results in conservative aa substitutions and would be unlikely to have an affect on the function of the prodomain. Since the nucleotide substitutions found in hepcidin 2 were not found among the cDNA sequences, we conclude that hepcidin 1 or hepcidin 1-like genes with various numbers of ATC repeats serve as the major source of transcripts in the tissues and under the conditions we employed. A stretch of eight cysteines, a signature for most mature hepcidins was found at the C-terminal. There are three processing sites in human hepcidin [2] and a single processing site in striped bass hepcidin [3] (Fig. 4). By analogy to bass hepcidin, zebrafish mature hepcidin may be 20 aa starting at Leu72. If this is the case, the predicted zebrafish mature hepcidin with four disulfide bridges is calculated to have a MW of 2330.92 Da and to be positively charged (pI of 8.94) [18]. The two independent bacterial challenge experiments in zebrafish showed increased hepcidin expression in fish that developed signs of infection. In Drosophila and other insects, rapid, transient gene transcription of the antimicrobial peptides after septic injury is mediated through Toll-like receptors
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Fig. 4. Similarity of hepcidin prepropeptide amino acid sequences between zebrafish and other animals. Identical or similar amino acid residues are shaded. A. salmon, Atlantic salmon; mudsucker, long-jawed mudsucker; w. flounder, winter flounder. Predicted cleavage site for signal peptide of zebrafish hepcidin and white bass hepcidin are shown ( # ). Mature peptide processing site for white bass and human are shown (O). SwissProt and GenBank accession numbers are shown in parentheses.
that recognize an array of microbial components (see Refs. [19,20] for review). In white bass [3] and Atlantic salmon [6], high levels of hepcidin expression were observed after a bacterial challenge and the bacteria were recovered from the fish, thus documenting established infection. However, these experiments did not address whether the trigger for
gene induction was exposure to microbial components or active infection. The lack of reproducible induction may be due to unpredictable severity of the infection and inflammatory response, which may vary among individual zebrafish. In humans, IL-6 appears to be the main cytokine that induces hepcidin expression
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in vivo [22]. In zebrafish, the homolog for IL-6 has not yet been identified. To assess if inflammation induces hepcidin expression in zebrafish, turpentine might be used as a pro-inflammatory agent, since it has been shown to induce inflammation and hepcidin expression in mice [23]. Additional experiments to explore the similarities in hepcidin gene regulation among mice, humans, and zebrafish should include anemia, iron-loading and hypoxia as variables. Putative binding sites for the transcription factors C/EBPa and C/EBPb found in mouse [4] and white bass [3] hepcidin promoters were also found in the upstream regions of hepcidin genes in zebrafish. The transcription factors, C/EBPa and C/EBPb, were shown to activate both human and mouse hepcidin promoters [21]. Thus the zebrafish model may be useful in studies of this signaling pathway. Although hepcidin expression was originally found predominantly in the liver of mammals and fish, zebrafish hepcidin expression was detected in abdominal organs, skin, and heart. Recently, hepcidin gene expression was detected in esophagus, heart, and stomach in winter flounder, and two hepcidin genes were differentially expressed in response to bacterial challenge in the skin, spleen, digestive tract, posterior kidney, and muscle of Atlantic salmon [6]. Hepcidin gene expression has also been noted in the spleen and pancreas of ironoverloaded mice [10]. In summary, we sequenced genes, cDNAs, and upstream regions of zebrafish hepcidin, a homologue of hepcidins from humans and many other fish. Study of this important gene will be facilitated in the zebrafish animal model.
Acknowledgements This research was supported in part by the Advanced Technology Program from Department of Commerce to Kent SeaTech Corp with UCSD as a subcontract. DNA sequencing was performed by the Molecular Pathology Shared Resource, University of California, San Diego Cancer Center, which is funded in part by National Cancer Institute, Cancer Center Support Grant No. #5P0CA23100-16.
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References [1] Krause A, Neitz S, Magert H-J, Schulz A, Forssmann W-G, Schulz-Knappe P, Adermann K. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 2000;480:147–50. [2] Park CH, Valore EV, Waring AJ, Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 2001;276:7806–10. [3] Shike H, Lauth X, Westerman ME, Ostland VE, Carlberg JM, Van Olst JC, Shimizu C, Bulet P, Burns JC. Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge. Eur J Biochem 2002;269:2232–7. [4] Pigeon C, Iiyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, Loreal O. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 2001;276:7811–9. [5] Inoue S, Nam B-H, Hirono I, Aoki T. A survey of expressed genes in Japanese flounder (Paralichthys olivaceus) liver and spleen. Mol Mar Biol Biotechnol 1997;6:376–80. [6] Douglas SE, Gallant JW, Liebscher RS, Dacanay A, Tsoi SC. Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish. Dev Comp Immunol 2003;27: 589 –601. [7] Gracey AY, Troll JV, Somero GN. Hypoxia-induced gene expression profiling in the euryoxic fish Gillichthys mirabilis. Proc Natl Acad Sci USA 2001;98:1993–8. [8] Weinstein DA, Roy CN, Fleming MD, Loda MF, Wolfsdorf JI, Andrews NC. Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease. Blood 2002;100:3776– 81. [9] Roetto A, Papanikolaou G, Politou M, Alberti F, Girelli D, Christakis J, Loukopoulos D, Camaschella C. Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis. Nat Genet 2003;33:21–2. [10] Ilyin G, Courselaud B, Troadec M-B, Pigeon C, Alizadeh M, Leroyer P, Brissot P, Loreal O. Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co-inducibility during iron overload. FEBS Lett 2003;542:22–6. [11] Weinstein MR, Litt M, Kertesz DA, Wyper P, Rose D, Coulter M, McGeer A, Franklam R, Ostach C, Billey BM, Borczyk A, Low DE. Invasive infection due to a fish pathogen, Streptococcus iniae. N Engl J Med 1997;337:589–94. [12] Fuller JD, Bast DJ, Nizet V, Low DE, de Azavedo JC. Streptococcus iniae virulence is associated with a distinct genetic profile. Infect Immun 2000;69:1994–2000. [13] Neely MN, Pfeifer JD, Caparon M. Streptococcus-zebrafish model of bacterial pathogenesis. Infect Immun 2002;70:3904–14. [14] Lauth X, Shike H, Burns JC, Westerman M, Ostland VE, Carlberg JM, VanOlst JC, Nizet V, Taylor SW, Shimizu C, Bulet P. Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass. J Biol Chem 2002;277:5030–9. [15] Kang JJ, Watson RM, Fisher ME, Higuchi R, Gelfand DH, Holland MJ. Transcript quantitation in total yeast cellular RNA using kinetic PCR. Nucl Acid Res 2000;28:e2.
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[16] Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, Podkolodny NL, Kolchanov NA. Databases on transcriptional regulation: TRANSFAC, TRRD, and COMPEL. Nucl Acid Res 1998;26:362–7. [17] Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 1997; 10:1 – 6. [18] Guruprasad K, Reddy BVB, Pandit MW. Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng 1990;4:155–61. [19] Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol 2000;12:13 –19.
[20] Underhill DM, Ozinsky A. Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol 2002;14:103–10. [21] Courseland B, Pigeon C, Inoue Y, Inoue J, Gonzalez FJ, Leroyer P, Gilot D, Boudjema K, Guguen-Guillouzo C, Brissot P, Loreal O, Ilyin G. C/EBPa regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism. J Biol Chem 2002;277:41163–70. [22] Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003; 101:2461–3. [23] Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, Beaumont C, Kahn A, Vaulont S. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. Clin Invest 2002;110:1037–44.
Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates Hiroko Shike, Chisato Shimizu, Xavier Lauth, Jane C. Burns* Department of Pediatrics, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, San Diego, CA 92093-0830, USA Received 9 September 2003; revised 20 November 2003; accepted 29 November 2003
Abstract Hepcidin is an antimicrobial peptide and iron-regulatory molecule that is conserved among vertebrates. Mutations or overexpression of the human hepcidin gene have been found in patients with hemochromatosis and refractory anemia. To further understand the function and regulation of hepcidin, animal models are needed. We sequenced cDNA, genes and upstream regions of zebrafish hepcidin and analyzed gene expression by kinetic PCR. Zebrafish hepcidin genes consist of two introns and three exons that encode a prepropeptide (91 amino acids). The amino acid sequences and gene organization were remarkably conserved between zebrafish and other species. Elevated gene expression was observed in abdominal organs, skin, and heart in fish that developed signs of infection following bacterial injection. Zebrafish may be a suitable model organism for further study of hepcidin gene regulation. q 2003 Elsevier Ltd. All rights reserved. Keywords: Fish; Streptococcus iniae; Infection; Hepcidin; Bacterial challenge
1. Introduction Hepcidin is an antimicrobial peptide and ironregulatory molecule primarily expressed in liver, and conserved among mammals and bony fish. Hepcidin peptides have been isolated from human urine and plasma [1,2] and the gill of hybrid striped bass [3]. Hepcidin mRNA has been found in mouse [4], rat, medaka, rainbow trout, Japanese flounder [5], winter flounder [6], long-jawed mudsucker [7], and Atlantic salmon [6]. The hepcidin peptides share a distinctive cystine bridge structure that is unique among other antimicrobial peptides. In humans and mice, hepcidin * Corresponding author. Tel.: þ1-619-543-5326; fax: þ 1-619543-3546. E-mail address: [email protected] (J.C. Burns). 0145-305X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2003.11.009
plays an important role in iron homeostasis. Hepcidin gene up-regulation was observed in mice with ironloading [4]. Over-expression of hepcidin from hepatic adenomas was found in a patient with refractory anemia [8] and mutations in the hepcidin gene were reported in two families with severe juvenile hemochromatosis [9]. Increased gene expression was also noted following lipopolysaccharide-challenge in mice [4] and bacterial challenge in white bass [3] and Atlantic salmon [6]. Although hepcidin expression is highest in the liver, hepcidin mRNA has also been detected in other organs such as spleen and pancreas in the mouse [10] and several fish species [6]. Thus, hepcidin, a multifunctional peptide important in both innate immunity and iron homeostasis, appears to be regulated through complex systems in response to diverse environmental stimuli.
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Use of an animal model such as zebrafish (Danio rerio) should help to elucidate further details of hepcidin expression and regulation. As a first step, we determined the sequence and organization of the zebrafish hepcidin gene and analyzed gene expression by challenge experiments with Escherichia (E.) coli and Streptococcus (S.) iniae, a hemolytic gram-positive coccus that causes fatal meningoencephalitis in both humans and fish [11,12]. Strains of S. iniae were shown to be potent pathogens in zebrafish [13] and to induce high levels of hepcidin gene expression in white bass [3].
This is the first report to identify cDNA, genes and upstream sequences of zebrafish hepcidin and to describe hepcidin gene expression following bacterial challenge.
2. Materials and methods 2.1. RNA sampling and cDNA sequencing Zebrafish were challenged with bacteria to attempt to induce zebrafish antimicrobial peptide genes, as
Fig. 1. Nucleotide sequence of zebrafish hepcidin cDNA and predicted amino acid sequence. Microsatellite (ATC) repeats are underlined in the 50 UTR. Binding sites for primers are shown with arrows (50 –30 ). Polyadenylation signal is underlined in the 30 UTR. The predicted organization of the peptide domains (signal peptide, prodomain, and mature peptide) is shown by gray boxes. The stop codon is indicated with an asterisk (*). Splicing sites for intron 1 and 2 are indicated with triangles.
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described previously [14]. Briefly, fish were injected with bacteria (approximately 106 cfu each), E. coli, INVF0 a(-) and S. iniae, K136-01 bB, a pathogenic strain that induced hepcidin expression in white bass liver [3], and had been isolated from the brain of white bass with fatal encephalomeningitis. Total RNA was extracted from abdominal organs including intestines and liver. Dozens of short, unidentified genomic sequences were in the Sanger zebrafish database that shared low similarity with the human hepcidin sequence (alignment score , 44). When converted into amino acid sequences, we found cysteine-rich regions in only three deposits, zfishG-a544c10.p1c, zfishCa2039d12.p1c, and zfishG-a550h10.p1c. We considered these as possible candidates for zebrafish hepcidin. Primers 246F and 275F were prepared to sequence 30 -mRNA (Fig. 1). RT-PCR products together with a poly-T primer were sequenced as described [14]. The 50 region of the mRNA was determined by 50 rapid amplification of cDNA ends (RACE) as described [14]. 2.2. Genomic DNA sequencing Inverse PCR was used to sequence 30 and 50 flanking sequences as described [14]. To obtain a continuous genomic sequence, a long PCR product that included both the hepcidin gene and upstream region was cloned and sequenced. A primer (47F) was designed from sequence 1.6 kb upstream from
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the transcription start site generated by inverse PCR and was used for long PCR together with rTth DNA polymerase XL (PE Applied Biosystems) and 567R, which binds near the 30 -terminal of the gene (Fig. 2). The long PCR products were cloned into TOPO TA vector and sequenced. Genomic DNA was tested for the presence of pseudogenes by PCR amplification with primer pairs spanning introns, 910F þ 205R, 910F þ 567R, and 234F þ 567R. The size of the PCR products was analyzed by agarose gel electrophoresis. 2.3. Bacterial challenge and gene expression analysis The first challenge experiment was performed by intra-muscular injection of either sterile Todd Hewitt broth (THB) or S. iniae, KST729 99aB (approximately 106 cfu each), a pathogenic strain isolated from white bass brain. Abdominal organs were dissected 1 day-post challenge. The second challenge experiment was conducted by intra-muscular injection of a mixture of E. coli and S. iniae (approximately 106 cfu each) or sterile THB. Tissue samples (abdominal organs, skin, gill, and heart) were dissected 3 days-post challenge. Zebrafish hepcidin mRNA and 18S rRNA were quantitated using kinetic RT-PCR as described [14]. Briefly, a primer pair, 234F þ 241R, was designed to span the second intron (1.5 – 1.6 kb) to amplify hepcidin cDNA (48 bp) but not genomic DNA (Figs. 1 and 2). Hepcidin cDNA was expressed as a copy number compared to the standard curve
Fig. 2. Organization of two zebrafish hepcidin genes and mRNA. Exons are depicted by boxes: open boxes, untranslated regions; striped boxes, regions encoding a signal peptide; stippled boxes, regions encoding a prodomain; filled boxes, regions encoding a mature hepcidin peptide. Binding sites for primers are shown with arrows (50 –30 ). GenBank accession numbers are shown in parentheses.
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generated by serial dilution of a positive control RTPCR product (509 bp) of known copy number generated by the primer pair 910F þ 567R. The copy number of hepcidin cDNA was normalized by arbitrary units of 18S for each sample. In this kinetic PCR system, a difference between two samples is significant when the ratio is four or greater [15]. Means of hepcidin levels from at least three fish were also compared using two standard deviations (SD) and 95th percentile confidence limits of the normal distribution.
3. Results 3.1. Zebrafish hepcidin cDNA sequence The 30 cDNA sequence from the challenged and control zebrafish samples were identical (Fig. 1). Careful visual examination of the 50 cDNA sequence in Chromas format revealed a mixture of multiple sequences that differed only by the number of microsatellite repeats with the tri-nucleotide ATC repeated 11 –16 times. A 50 RACE product cloned in TOPO TA vector is shown as a representative hepcidin cDNA sequence (GenBank accession no. AY363454) (Fig. 1). Despite the variability in the 50 UTR, all cDNAs shared an identical coding sequence for a 91 aa peptide. A stretch of eight cysteines, a signature for most hepcidins, was also found at the C-terminal. Since we did not observe any clinical signs of infection in zebrafish challenged with S. iniae, K136-01 bB, we used another strain of S. iniae, KST729 99aB, in subsequent challenge experiments. The strains were known to be equally pathogenic in bass. 3.2. Zebrafish hepcidin gene Two hepcidin genes were cloned, hepcidin 1 (GenBank accession no. AY363452) and hepcidin 2 (GenBank accession no. AY363453) (Fig. 2). Both genes consist of three exons and two introns, identical to hepcidin genes of white bass, mice, and humans [2 – 4]. The coding sequence of hepcidin 1 and the cDNA (Fig. 1) were identical. Three nucleotide substitutions in the coding sequences of hepcidin 2 resulted in two tandem aa substitutions, Leu55 – Ala56
substituted for Gln55 – Thr56. PCR products from genomic DNA with primer pairs spanning introns matched the expected sizes for hepcidin 1 and 2, thus eliminating the possibility of pseudogenes lacking introns (data not shown). 3.3. Upstream region of hepcidin gene The upstream regions (1.6 kb) for both hepcidin 1 and 2 were almost identical (98%) and contained extensive microsatellite repeats, 50 -TAAA-30 (2 1.6 to 2 1 kb upstream from the transcriptional start site). Thus, only 1.0 kb of the upstream region was searched for putative regulatory motifs by TFSEARCH [16]. Both upstream regions contained a TATA box (2 30) and several putative transcription factor binding sites for CAAT enhancer-binding protein (C/EBP)a, C/ EBPb, hepatocyte nuclear factor (HNF) 3b, HNF4, and NF-k B (Table 1). 3.4. Zebrafish hepcidin gene expression Quantitative analysis of the positive control RT-PCR product from hepcidin cDNA resulted in a linear standard curve with a dynamic range of 10– 107 copies. The melting temperatures ðTm Þ of the RT-PCR products were 78.6 8C for hepcidin and 80.5 8C for 18S. No detectable amplification of up to 10 ng of genomic DNA confirmed that the kinetic PCR result accurately reflected the quantitation of hepcidin cDNA as opposed to genomic DNA.
Table 1 Putative transcription factor bindings sites Transcription factors
Putative binding sites
TATA box C/EBPa C/EBPb HNF3b HNF4 NF-k B
230 2956, 2133 2956, 2969, 2147, 2133 2972, 2925, 2506, 2413, 2406, 2250, 2245 2108 2733
Putative transcription factor binding sites were predicted in the upstream sequence of hepcidin 1 (AY363452) using TFSEARCH, and expressed relative to the transcriptional starting site. C/EBP: CAAT enhancer-binding protein, HNF: hepatocyte nuclear factor, NF: nuclear factor.
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Fig. 3. Zebrafish hepcidin gene expression following bacterial challenge. Zebrafish hepcidin mRNA and 18S rRNA were quantitated by kinetic RT-PCR following intra-muscular injection of THB or bacteria. Hepcidin mRNA levels were normalized by 18S rRNA levels and expressed in arbitrary units. Fish challenged with THB, A; fish challenged with bacteria but remaining healthy, W; fish challenged with bacteria and clinically affected, X. In experiment 1, abdominal organs were tested 1 day-post challenge with THB or S. iniae. In experiment 2, abdominal organs, skin, gill, and heart were tested 3 days-post challenge with THB or a mixture of S. iniae and E.coli.
In the first challenge experiment, only one of four fish challenged with S. iniae developed decreased activity. Abdominal organs from this fish yielded high levels of hepcidin mRNA with a 700-fold increase as compared to the THB-challenged fish (Fig. 3). The other three fish remained normal despite the bacterial injection, and the hepcidin expression level was not significantly different from the levels in THBchallenged fish. In the second experiment (Fig. 3), the levels of hepcidin expression in different organs following challenge with S. iniae and E. coli were determined. One of four zebrafish again developed difficulty in swimming by 3 days post-challenge. In this ill-appearing fish, the hepcidin expression level was significantly higher in abdominal organs, skin, and heart (16–43-fold increase) compared to THB-challenged fish.
4. Discussion We report here the molecular characterization of zebrafish hepcidin, which shares striking similarity in gene organization and amino acid sequences with hepcidins from humans, mice, and other fish. Hepcidin prepropeptides from several different animals share a high degree of homology (Fig. 4). Signal P [17] predicted a cleavage site between Ala24 and Val25. The resulting signal peptide has a basic residue (Lys2) at the N-terminal end followed
by a hydrophobic region rich in Val and Ala, which is typical for a signal peptide. Interestingly, a signal peptide cleavage site for white bass preprohepcidin was predicted between Ala24 and Val25 [3], suggesting a conserved mechanism for proteolytic cleavage of the hepcidin signal peptide among fish species. The aa substitution located in the prodomain of hepcidin 2 (Gln55 –Thr56) results in conservative aa substitutions and would be unlikely to have an affect on the function of the prodomain. Since the nucleotide substitutions found in hepcidin 2 were not found among the cDNA sequences, we conclude that hepcidin 1 or hepcidin 1-like genes with various numbers of ATC repeats serve as the major source of transcripts in the tissues and under the conditions we employed. A stretch of eight cysteines, a signature for most mature hepcidins was found at the C-terminal. There are three processing sites in human hepcidin [2] and a single processing site in striped bass hepcidin [3] (Fig. 4). By analogy to bass hepcidin, zebrafish mature hepcidin may be 20 aa starting at Leu72. If this is the case, the predicted zebrafish mature hepcidin with four disulfide bridges is calculated to have a MW of 2330.92 Da and to be positively charged (pI of 8.94) [18]. The two independent bacterial challenge experiments in zebrafish showed increased hepcidin expression in fish that developed signs of infection. In Drosophila and other insects, rapid, transient gene transcription of the antimicrobial peptides after septic injury is mediated through Toll-like receptors
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Fig. 4. Similarity of hepcidin prepropeptide amino acid sequences between zebrafish and other animals. Identical or similar amino acid residues are shaded. A. salmon, Atlantic salmon; mudsucker, long-jawed mudsucker; w. flounder, winter flounder. Predicted cleavage site for signal peptide of zebrafish hepcidin and white bass hepcidin are shown ( # ). Mature peptide processing site for white bass and human are shown (O). SwissProt and GenBank accession numbers are shown in parentheses.
that recognize an array of microbial components (see Refs. [19,20] for review). In white bass [3] and Atlantic salmon [6], high levels of hepcidin expression were observed after a bacterial challenge and the bacteria were recovered from the fish, thus documenting established infection. However, these experiments did not address whether the trigger for
gene induction was exposure to microbial components or active infection. The lack of reproducible induction may be due to unpredictable severity of the infection and inflammatory response, which may vary among individual zebrafish. In humans, IL-6 appears to be the main cytokine that induces hepcidin expression
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in vivo [22]. In zebrafish, the homolog for IL-6 has not yet been identified. To assess if inflammation induces hepcidin expression in zebrafish, turpentine might be used as a pro-inflammatory agent, since it has been shown to induce inflammation and hepcidin expression in mice [23]. Additional experiments to explore the similarities in hepcidin gene regulation among mice, humans, and zebrafish should include anemia, iron-loading and hypoxia as variables. Putative binding sites for the transcription factors C/EBPa and C/EBPb found in mouse [4] and white bass [3] hepcidin promoters were also found in the upstream regions of hepcidin genes in zebrafish. The transcription factors, C/EBPa and C/EBPb, were shown to activate both human and mouse hepcidin promoters [21]. Thus the zebrafish model may be useful in studies of this signaling pathway. Although hepcidin expression was originally found predominantly in the liver of mammals and fish, zebrafish hepcidin expression was detected in abdominal organs, skin, and heart. Recently, hepcidin gene expression was detected in esophagus, heart, and stomach in winter flounder, and two hepcidin genes were differentially expressed in response to bacterial challenge in the skin, spleen, digestive tract, posterior kidney, and muscle of Atlantic salmon [6]. Hepcidin gene expression has also been noted in the spleen and pancreas of ironoverloaded mice [10]. In summary, we sequenced genes, cDNAs, and upstream regions of zebrafish hepcidin, a homologue of hepcidins from humans and many other fish. Study of this important gene will be facilitated in the zebrafish animal model.
Acknowledgements This research was supported in part by the Advanced Technology Program from Department of Commerce to Kent SeaTech Corp with UCSD as a subcontract. DNA sequencing was performed by the Molecular Pathology Shared Resource, University of California, San Diego Cancer Center, which is funded in part by National Cancer Institute, Cancer Center Support Grant No. #5P0CA23100-16.
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References [1] Krause A, Neitz S, Magert H-J, Schulz A, Forssmann W-G, Schulz-Knappe P, Adermann K. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 2000;480:147–50. [2] Park CH, Valore EV, Waring AJ, Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 2001;276:7806–10. [3] Shike H, Lauth X, Westerman ME, Ostland VE, Carlberg JM, Van Olst JC, Shimizu C, Bulet P, Burns JC. Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge. Eur J Biochem 2002;269:2232–7. [4] Pigeon C, Iiyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, Loreal O. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 2001;276:7811–9. [5] Inoue S, Nam B-H, Hirono I, Aoki T. A survey of expressed genes in Japanese flounder (Paralichthys olivaceus) liver and spleen. Mol Mar Biol Biotechnol 1997;6:376–80. [6] Douglas SE, Gallant JW, Liebscher RS, Dacanay A, Tsoi SC. Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish. Dev Comp Immunol 2003;27: 589 –601. [7] Gracey AY, Troll JV, Somero GN. Hypoxia-induced gene expression profiling in the euryoxic fish Gillichthys mirabilis. Proc Natl Acad Sci USA 2001;98:1993–8. [8] Weinstein DA, Roy CN, Fleming MD, Loda MF, Wolfsdorf JI, Andrews NC. Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease. Blood 2002;100:3776– 81. [9] Roetto A, Papanikolaou G, Politou M, Alberti F, Girelli D, Christakis J, Loukopoulos D, Camaschella C. Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis. Nat Genet 2003;33:21–2. [10] Ilyin G, Courselaud B, Troadec M-B, Pigeon C, Alizadeh M, Leroyer P, Brissot P, Loreal O. Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co-inducibility during iron overload. FEBS Lett 2003;542:22–6. [11] Weinstein MR, Litt M, Kertesz DA, Wyper P, Rose D, Coulter M, McGeer A, Franklam R, Ostach C, Billey BM, Borczyk A, Low DE. Invasive infection due to a fish pathogen, Streptococcus iniae. N Engl J Med 1997;337:589–94. [12] Fuller JD, Bast DJ, Nizet V, Low DE, de Azavedo JC. Streptococcus iniae virulence is associated with a distinct genetic profile. Infect Immun 2000;69:1994–2000. [13] Neely MN, Pfeifer JD, Caparon M. Streptococcus-zebrafish model of bacterial pathogenesis. Infect Immun 2002;70:3904–14. [14] Lauth X, Shike H, Burns JC, Westerman M, Ostland VE, Carlberg JM, VanOlst JC, Nizet V, Taylor SW, Shimizu C, Bulet P. Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass. J Biol Chem 2002;277:5030–9. [15] Kang JJ, Watson RM, Fisher ME, Higuchi R, Gelfand DH, Holland MJ. Transcript quantitation in total yeast cellular RNA using kinetic PCR. Nucl Acid Res 2000;28:e2.
754
H. Shike et al. / Developmental and Comparative Immunology 28 (2004) 747–754
[16] Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, Podkolodny NL, Kolchanov NA. Databases on transcriptional regulation: TRANSFAC, TRRD, and COMPEL. Nucl Acid Res 1998;26:362–7. [17] Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 1997; 10:1 – 6. [18] Guruprasad K, Reddy BVB, Pandit MW. Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng 1990;4:155–61. [19] Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol 2000;12:13 –19.
[20] Underhill DM, Ozinsky A. Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol 2002;14:103–10. [21] Courseland B, Pigeon C, Inoue Y, Inoue J, Gonzalez FJ, Leroyer P, Gilot D, Boudjema K, Guguen-Guillouzo C, Brissot P, Loreal O, Ilyin G. C/EBPa regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism. J Biol Chem 2002;277:41163–70. [22] Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003; 101:2461–3. [23] Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, Beaumont C, Kahn A, Vaulont S. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. Clin Invest 2002;110:1037–44.