Molecular Cloning And Expression Of The Antibacterial Peptide Hepcidin Gene

It has been well documented that hepcidin is a key regulator of iron balance and recycling in mammalians. Hepcidin gene is conserved in vertebrates from fish to mammalians. It has been reported that many teleost fish present multiple copies(isoforms) of hepcidin. So far, hepcidins in fish have not been well studied. Particularly, the biological functions and mechanisms of gene expression of various isoforms of hepcidin in fish need to be elucidated. Mudskipper, a kind of amphibious fish, can switch between aquatic and terrestrial habits with different oxygen availability. They live in the mud with high possibility of exposure to various pathogens, implying the powerful immunity they may possess.In this study, hepcidins in great blue-spotted mudskipper(Boleophthalmus pectinirostris) were obtained. The gene structure, the 5' flanking sequence and the specific transcriptional regulatory elements were analyzed. The regulation of transcription and antimicrobial activities were studied as well to provide fundamental data to elucidate the biological function of two isoforms of hepcidin in mudskipper.Two isoforms of hepcidin in great blue-spotted mudskipper(Boleophthalmus pectinirostris), bpHep-1 and bpHep-2, were cloned. The deduced amino acids sequence of bpHep-1 contains 88 aa, including 22 aa of signal peptide, 40 aa of prodomain and 26 aa of mature peptide. Similarly, bpHep-2 contains 88 aa, including 24 aa of signal peptide, 42 aa of prodomain and 22 aa of mature peptide. The mature peptides of both bpHep-1 and bp Hep-2 contain 8 C ys, which can form four disulfide bonds to maintain the stability of the ?-sheet structure.Molecular phylogenetic analysis showed that topology of neighbor joining(NJ) tree based on amino acid sequences of hepcidin from vertebrates was consistent with phylogenetic tree of the teleost fish based on several nuclear genes.All hepcidins from the Ostarioclupeomorpha cluster within one clade which is a sister clade of Euteleostei, with high bootstrap value 88%. In clade Euteleostei, lower evolutionary status of Protacanthopterygii is a sister of clade Neoteleostei. Hepcidins in the clade Neoteleostei clusters into two subclades, Neo1 and Neo2 with lower bootstrap support value. bp Hep-1 and bpHep-2 were included in subclade Neo1 and Neo2 respectively. It is noted that hepcidins in subclade Neo1 has the putative iron regulatory sequence motif [Q-S/I-H-L/I-S/A]in N-terminal of the mature peptide, however hepcidins in subclade Neo2 lack the motif.The gene structures of both bpHep-1 and bp Hep-2 consist of two introns and three exons. Exon 1 contains the 5'untranslated region, the signal peptide and part of the prodomain. The prodomain extends from exon1 to the exon3 which encods the mature peptide and the 3'untranslated region. The full length of bp Hep-1 cDNA is 573 bp, containing an open reading frame of 267 bp, 5' untranslated region of 152 bp and 3'untranslated region of 154 bp. The full length of bp Hep-2 cDNA is 598 bp, containing an open reading frame of 267 bp, 5' untranslated region of 76 bp and 3'untranslated region of 74 bp. There is a TATA boxupstream of transcription start site in bpHep-1 and bpHep-2 gene sequences.The transcription factor binding sites of the two hepcidin isoforms were predicted using online bioinformatic tools. The results showed that therewere 23 transcription factor binding sites in the region of-1341 bp to+152bp of bp Hep-1 gene, 37 transcription factor binding sitesin the region of-1780 bp to +75bp of bp Hep-2 gene. Both bp Hep-1 gene and bp Hep-2 gene contain C/EBPalp, SP1, Oct-1, HNF-3, TBP, GR, and WT1 I-K. C/EBPalp has been proven to be an effective transactivation activator of mammalian hepcidins. HNF-3 is an important transcription factor in liver, suggesting that it plays a role in the transactivation activity in the expression of hepcidin. It is interesting to note that bp Hep-2 but not bpHep-1 contains NF-?B binding site. It is implied that bp Hep-2 may play a more important role in the innate immunity than bpHep-1 since NF-?B is involved in the signaling pathways of innate immune factor expression.The mRN A expression levels of bp Hep-1 and bp Hep-2 were studied in the tissues of liver, kidney, spleen, gill and intestinalin. Boleophthalmus pectinirostris could be detected with semi quantitative RT-PCR and fluorescence quantitative RT-PCR. Transcripts were detected in all of the tested tissues, with most abundance in liver of the fish. The expression level of bp Hep-2 was significantly higher than that of bp Hep-1 in liver(p<0.05). Iron overloading with 0.5mg/g of iron dextran injection upregulated the expression of bpHep-1 in intestine, head kidney and spleen(p<0.05), but no change was observed in liver and gill. The expression of bp Hep-2 increased significantly in gill, intestinal, head kidney and spleen under iron overloading(p<0.05). The LPS and poly(I:C) did not change t he transcription of the two hepcidin isoforms significantly. It is suggested that both bpHep-1 and bp Hep-2 may be involved in the regulation of iron metabolism in this species of fish.Fusion prokaryotic expression vectorsof bpHep-1 and bpHep-2 were constructed using vector p ET-SUMO. The constructs were transformed into E. coli BL21(DE3) and recombinant bp Hep-1 and bp Hep-2 were obtained. It was observed that recombinant bp Hep-1 and bp Hep-2 could inhibit the growth of E.coli and Staphyloccocus aureus at the concentration of 12.5?M.In conclusion, the cDNA and gene of two hepcidin isoforms in Boleophthalmus pectinirostris were cloned and analyzed in this study. The promoter and transcription factor binding sites were predicted. The tissue distributions of basal transcription levels of bp Hep-1 and bpHep-2 were studied. Iron overloading with 0.5mg/g of iron dextran injection upregulated the expression of bp Hep-1 and bp Hep-1 in tissue specific manner. Fusion prokaryotic expression vectors of bp Hep-1 and bp Hep-2 were constructed using vector pET-SUMO and recombinant bpHep1 and bpHep2 were obtained.