Fish, the lowest vertebrate group, typically possess a weaker acquired immune response as compared to mammals. Therefore, the innate immune response to infectious invaders is generally agreed to be more vital to fish than mammals (Shi and Camus, 2006;Magnadottir, 2010). Antimicrobial peptides (AMPs) play crucial roles as the first line of defense against invading microbes, and diverse AMPs with different structures and charges are involved in the defense pathway in an interactive or coordinated fashion (Cuesta et al., 2008; Rajanbabu and Chen, 2011).
Hepcidin, a recently discovered AMP, is the first identified from human urine (Park et al., 2001).Similar to a number of naturally occurring AMPs,hepcidins are cysteine-rich and form multiple disulfide bonds in the β-sheet structures (Shi and Camus, 2006). Many previous studies have shown that hepcidin is central in the cross talk between innate immunity and iron homeostasis in vertebrates,although the primary role of mammalian hepcidins is now believed to be hormonal, serving as a negative regulator of iron homeostasis (Vyoral and Petrak, 2005; Hugman, 2006; Atanasiu et al., 2007). The function of mammalian hepcidin is believed to be conserved in fish. Previous studies support this hypothesis, showing that fish hepcidin peptides (both natural and synthetic versions) have potent antimicrobial activities (Lauth et al., 2005; Huang et al., 2007); the transcription of fish hepcidin genes are rapidly activated by experimental challenges using live bacteria, lipopolysaccharides (LPS), poly I:C, and viral suspensions (Shike et al., 2002; Douglas et al., 2003a; Hirono et al., 2005; Chiou et al., 2007); and fish hepcidins are significantly stimulated by iron overload and can regulate mammalian ferroportin, a known iron exporter in enterocytes and macrophages(Huang et al., 2007; De Domenico et al., 2008; Cho et al., 2009).
Unlike most mammals, having only one genomic copy of hepcidin (
The Javanese ricefish, or medaka (
Fish specimen and rearing conditions
Fish specimens used in this study were laboratorypropagated at the Institute of Marine Living Modified Organisms (IMLMO, PKNU). The conditions for general fish culture have been described previously by Cho et al. (2010) and Song et al. (2010). Fish were fed with Artemia nauplii (INVE Aquaculture Inc.,Salt Lake City, UT, USA) and artificial diets of flounder larvae (150-500 ㎛ in diameter, 50% crude protein; Woosung Feed Corp., Daejeon, Korea) on an ad libitum basis. Water temperature was maintained at 25±1°C, and dissolved oxygen was maintained at 5±1 ppm throughout the experiment. The daily water exchange rate was approximately 20%.
Nucleic acid preparation and cDNA library construction
Genomic DNA was isolated from fin tissue using conventional sodium dodecyl sulfate/proteinase K digestion, followed by phenol/chloroform extraction and ethanol precipitation. Total RNAs from various tissues were purified using the RNeasy Mini Kit(Qiagen, Hilden, Germany) according to the manufacturer’s instructions, including the DNase treatment step. The cDNA library was constructed from total RNA purified from whole body
Isolation of hepcidin genomic and cDNA genes
Based on sequence information from previously characterized
[Table 1.] Oligonucleotide primers used in this study
Oligonucleotide primers used in this study
Bioinformatic sequence analysis
Sequence homology of
Tissue distribution assay of hepcidin transcripts
Basal expression patterns of
Bacterial challenge and mRNA expression assay
To examine differential modulation of
Differences in the basal expression levels of hepcidin transcripts across tissues were assessed by ANOVA, followed by Duncan’s multiple range test.Transcriptional modulation in response to bacterial challenge was expressed as the fold change of hepcidin transcripts in bacteria-challenged groups compared to non-challenged controls. Differences in expression levels between challenged and non-challenged groups were assessed using Student’s
Characteristics of O. javanicus hamp1 sequences
Based on the genomic PCR isolation, the
Characteristics of the O. javanicus hamp2 sequences
[Fig. 1.] Nucleotide and deduced amino acid sequences of Oryzias javanicus hepcidin isoform hamp1. Coding region is indicated by bold uppercase letters while non-coding region by lower case letters. Stop codon (tga) is indicated by an asterisk. Deduced amino acid sequence in the singlet code is provided below the nucleotide sequence. Amino acid number and theoretical isoelectric point (pI) value for each of three regions (signal peptide proregion and mature peptide) are also indicated. The variable repetitive numbers for adenines (in the 5′-UTR) and cytosines (in the intron II) are observed. Two adenine-guanine sequences in the junction site between intron I and exon II are boxed which is a potential target site for alternative splicing to produce 90 or 91 aa-encoded mRNA species. The Gln missed in the 90 aa-encoded mRNA is noted in the parenthesis. In the 3′-UTR an insertion/deletion of five nucleotides (ggata) was found among different cDNA clones and mRNAs processed with two different polyadenylation signals (aataaa; underlined and bolded) were also detected. The sites observed for the poly(A)+ tailing are indicated by vertical arrows. UTR untranslated region.
before the poly(A)+ tail. The amino acid sequence of the OJHAMP2 polypeptide (the whole preproprotein)was predicted to have a negative overall charge(pI=5.70). The preproprotein OJHAMP2 had two potential cleavage sites: one between Ala24 and Val25(cleavage of the signal peptide), and the other between Arg64 and Glu65 (conversion of the proprotein to the mature peptide). The pI values for the three regions were 5.75 (for the signal peptide; 24 aa), 5.64 (proregion; 40 aa), and 6.03 (mature peptide; 25 aa). The OJHAMP2 mature peptide also had eight cysteine residues.
Multiple sequence alignment of mature peptides with their representative orthologs
The mature peptide sequence of OJHAMP1 was aligned with 40 orthologs compiled from nine orders(Fig. 3). Fish HAMP1s possessed either 25 or 26 aa,displaying a cationic pI range from 7.73 to 8.94 (Fig.3). They shared a relatively high degree of sequence homology, with the eight conserved Cys residues in all the active sequences, except one isoform from Atlantic salmon
[Fig. 2.] Nucleotide and deduced amino acid sequences of Oryzias javanicus hepcidin isoform hamp2. Coding region is indicated by bold uppercase letters while non-coding region by lower case letters. Stop codon (tga) is indicated by an asterisk. Deduced amino acid sequence in the singlet code is provided below the nucleotide sequence. Amino acid number and theoretical isoelectric point (pI) value for each of three regions (signal peptide proregion and mature peptide) are also indicated. A putative polyadenylation signal (aataaa) is underlined and bolded while the site for poly(A)+ tailing is indicated by vertical arrow.
residues at positions 3 (His), 20 (Gly), 22 (Gly), and 27 (Phe) (alignment positions including gaps in Fig.3). Moreover, the sequence identities across the species were roughly in agreement with the known taxonomic appraisal of the species, although we did not reconstruct the molecular phylogeny in detail. As shown in the alignment, teleostean HAMP1 represented a well conserved N-terminal motif(QSHL/I). Exceptions were the ISHI signals found in a few species belonging to Pleuronectiformes and the QIHL observed in one of the three isoforms recovered from Atlantic salmon. However, these substitutions did not change the pI values of the first 5 aa essential for hormonal function of HAMP1. All the HAMP1s had identical pI values (6.74) in this region.
Alignment of the major HAMP2 sequences (only HAMP2s having eight clearly conserved Cys residues, aligned in Fig. 4) revealed that teleostean HAMP2s were more variable and heterogeneous than HAMP1s in both the length and charge of the mature peptide(Fig. 4). The HAMP2 sequences recovered from species belonging to the superorder Acanthopterygii and many of species from Perciformes possess multiple HAMP2 copies. The fish HAMP2 mature peptides are predicted to have amino acid lengths ranging from 21 aa (empirically identified from the bass species
[Fig. 3.] Multiple alignment of Oryzias javanicus mature HAMP1 peptide along with its teleostean orthologs.Seven clearly conserved Cys residues are noted by asterisks on the top while four conserved non-cysteine residues by open circle. One Cys residue conserved in most species except one isoform from Atlantic salmon (S.salar) was indicated by a closed square on the top. Predicted amino acid number (aa) and theoretical isoelectric point (pI) value are provided at the end of each sequence. For the accession code for O. niloticus (TH2-3) refer to Huang et al. (2007).
estimated in the two
Basal expression patterns of hamp1 and hamp2 in adult tissues
Using the RT-PCR assay,
Differential expression of ojhamp1 and ojhamp2 resulting from bacterial challenge
After bacterial challenge with
[Fig. 4.] Multiple alignment of Oryzias javanicus mature HAMP2 peptide along with its teleostean orthologs in a quantitative mode. Eight clearly conserved Cys residues are noted by asterisks on the top. Putative RXKR or RRXR cleavage sites are also shown and boxed based on the prediction using the ProP Server. Cleavage sites based on the bioinformatic prediction or experimental isolation (from the hybrid bass M. chrysops) are indicated by vertical arrows. Predicted amino acid number (aa) and theoretical isoelectric point (pI) values for whole predicted mature peptide (pI-w) and the five N-terminal amino acids (pI-n) are provided at the end of each sequence.
both tissues. The
Teleosts, which belong to Acanthopterygii, are thought to possess at least two copies of
[Fig. 5.] Representative reverse transcription-PCR gels showing the tissue distribution of Oryzias javanicus hamp1 and hamp2 transcripts. Abbreviations for tissues are brain (B) eye (E) fin (F) gill (G) intestine (I) kidney (K) liver (L) muscle (M) ovary(O) and testis (T). A gel for 18S rRNA normalization control is also shown on the top.
a similar alternative splicing pattern has been reported in hepcidin isoforms from the rockbream
Based on the multiple sequence alignments of the mature hepcidin peptides, fish HAMP1s have highly conserved characteristics in their amino acid lengths, as well as in the pI values. However, in contrast to the HAMP1 group, the teleost HAMP2s are highly variable in these parameters across species and isoforms. Previous studies have claimed that fish
[Fig. 6.] Differential expression of Oryzias javanicus hamp1 (A) and hamp2 (B) in the liver and kidney in response to the bacterial challenge using Edwardsiella tarda. Real-time RT-PCR assay was performed at 24 or 48 h post challenge (HPC) and the gene modulations are defined as fold changes of transcript levels relative to non-challenged control.Mean±SDs with different letters (a-c or w-z) are significantly different based on ANOVA (P<0.05) while the significant up- or down-regulations from the control level are noted by asterisks based on the student’s t-test (P<0.05).
the mature peptide, similar to mammalian HAMPs.The negatively charged N-terminal region (usually 5 aa prior to the first conserved Cys) is known to be essential for its interaction with ferroportin (Nemeth et al., 2006). The QSHLS motif of the zebrafish(
Tissue distribution patterns of fish hepcidin transcripts are known to be species-specific, unlike the liver-exclusive or predominant patterns found in most mammalian species. In this study, both
During immersion treatment with
In summary, two hepcidin paralogs (