Cloning, Expression And Function Of A Hepcidin Antimicrobial Peptide Gene From Carp (cyprinus Carpio L.)

Hepcidin is a cysteine-rich cationic antimicrobial peptide (AMP), which plays an important role in host innate immune response and iron regulation. A great many of hepcidin genes have been isolated from mammalians and teleosts. Here we report the Cloning and identification of a hepcidin gene from the liver of carp.A full length hepcidin cDNA was amplified from the liver of carp, using RT-PCR and RACE. The complete cDNA is 647 bases and contains a 46 bp 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 276 bp encoding a protein of 91 amino acids, and an 325 bp 3'-UTR with a polyadenylation signal ATTAAA sequence appearing at 275bp and the poly(A) tail at 303 bp downstream of stop codon TGA.The hepcidin gene of carp is 1216 bp in length, including two introns (180 bp and 785 bp) and three exons (133 bp, 90 bp and 424 bp). The first exon contains partial 5'-UTR, the signal peptide and part of the prodomain. The prodomain extends from exon 1 to exon 3 which also encodes the mature peptide and the 3'-UTR. The organisations with three exons and two introns of the known hepcidin genes are quite conservative in humans, mice and fish.The deduced amino acid sequence from carp hepcidin cDNA was highly similar to hepcidins of other fish species and mammals. The deduced amino acid sequence for hepcidin consists of 91amino acids and consists of three domains: signal peptide (24 amino acids), prodomain (42 amino acids) and mature peptide (25 amino acids). The signal peptide cleavage site of the deduced peptide was predicted between Ala 24 and Val 25. The mature peptide region was predicted, consisting of 25 amino acid residues at the C-terminus of ORF. The predicted mature hepcidin, with a MW of 2893.47 Da and pI of 8.34, share eight cysteines at the identical conserved position.Carp hepcidin was found to be structurally similar to the zebrafish and human hepcidins, being comprised of a twistedβ-sheet structure with a small hydrophobic patch, stabilized by three disulfide bonds on alternating sides of theβ-sheet, and a rare vicinal disulfide bond at the top of a flexible loop structure.A sequence alignment of carp hepcidin peptide and some other known and predicted hepcidin peptides was performed at the amino acid level. The deduced amino acid sequence from carp hepcidin cDNA was highly similar to other hepcidins, most of which share eight cysteines at the identical conserved position. However, there were several hepcidin sequences with 6 or 7 cysteine residues in the mature peptide. Alanine (A) and valine (V) residues abound in the hepcidin signal peptide part of fish, but leucine-rich residues are in the signal peptide part of amphibian and mammalian hepcidins.To determine the phylogenetic relations between vertebrate hepcidins, a phylogentic tree was constructed with the amino acid sequence of these hepcidins, indicating that two clusters were present: (1) mammalian and amphibian hepcidins, (2) fish hepcidins. It is obvious that the carp hepcidin is more similar to that of zebrafish than those of other species.RT-PCR demonstrated that hepcidin transcripts were highly abundant in liver, abundant in head kidney, spleen, skin, foregut, hindgut, oral epithelial, gonad and whole blood, less abundant in gill and undetectable in muscle.The kinetics of hepcidin expression after stimulation was similar in liver, head kidney, foregut and hindgut although the fold induction was different in these tissues. The highest induction level in liver, head kidney and hindgut was apparently at hour 6 and was followed by a decline until day 7 when the hepcidin expression in In-L.ang-injected fish was comparable to the control fish. In foregut, the highest induction level was at day 3.The liver cDNA of carp was served as the template to amplify the cDNA of the hepcidin mature peptide. The product of amplification was treated by double endonuclease digestion (NcoⅠa nd XhoⅠ) and linked to pET-32a(+) expression vector. The fusion protein was expressed in E.coli AD494 (DE3) with IPTG induction. SDS-PAGE results showed that a protein with a molocular weight of 20.21 kD was expressed within the host strain after lysis with sonieation, and the expressed fusion protein existed in the dissolved state was much more than that in the form of inclusion bodies.The hepcidin was eleaved from the fusion protein by enterokinase and used for antimicrobial assay. The expression produet displayed antibacterial activity against Gram-negative E.coli. Thus the recombination of hepeidin will facilitate the functional study for hepcidin in future.