title: Molecular cloning and characterization of two novel hepcidins from orange-spotted grouper, Epinephelus coioides.
abstract: Orange-spotted grouper, Epinephelus coioides is one of the most important economic species of marine-cultured fish in China and Southeast Asia countries. However, very little information of the innate immune mechanisms against microbial pathogens is available in grouper, Epinephelus sp. Hepcidin, as an antimicrobial peptide (AMP), is a very important component in the innate immune system and widespread in fish. In this study, two novel types of hepcidin gene (designated EC-hepcidin1 and EC-hepcidin2) were cloned from E. coioides. They consist of open reading frames (ORFs) of 267 bp and 263 bp encoding the putative peptides of 88 and 87 amino acids, respectively. The homologous identity of deduced amino acid sequences between EC-hepcidin1 and EC-hepcidin2 is up to 79%, and predicted mature regions of both them have four cysteines residues. Genomic DNAs of both EC-hepcidin1 and EC-hepcidin2 consist of three exons and two introns. RT-PCR results showed that EC-hepcidin1 transcript was most abundant in liver and less in stomach. However, the transcript of EC-hepcidin2 was only detected in liver. The expressions of both EC-hepcidins were up-regulated by microbial and viral challenges, and iron overload, respectively, and EC-hepcidin1 was more responsive. The growth of Gram-negative bacterium of Vibrio vulnificus and Gram-positive bacterium of Staphylococcus aureus was inhibited by synthetic EC-hepcidins, and EC-hepcidin1 displayed stronger antimicrobial activity. The replication of Singapore grouper iridovirus (SGIV) was inhibited in the EC-hepcidin1 and EC-hepcidin2 over-expressed stable transfected fish cell lines (GS/pcDNA-Hep1, GS/pcDNA-Hep2) indicative of the antiviral activity of EC-hepcidins. These data should offer important information on the antimicrobial and antiviral roles of EC-hepcidins, and will be help to the better understanding of molecular mechanisms of grouper innate immunity.
authors.affiliation: State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 West Xingang Road, Guangzhou 510275, PR China.
authors.forename: Jing-Geng
authors.initials: JG
authors.lastname: Zhou
authors.forename: Jing-Guang
authors.initials: JG
authors.lastname: Wei
authors.forename: Dan
authors.initials: D
authors.lastname: Xu
authors.forename: Hua-Chun
authors.initials: HC
authors.lastname: Cui
authors.forename: Yang
authors.initials: Y
authors.lastname: Yan
authors.forename: Zheng-Liang
authors.initials: ZL
authors.lastname: Ou-Yang
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Huang
authors.forename: You-Hua
authors.initials: YH
authors.lastname: Huang
authors.forename: Qi-Wei
authors.initials: QW
authors.lastname: Qin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000276:immunology; D001424:Bacterial Infections / Q000276:immunology / Q000662:veterinary; D001483:Base Sequence; D001492:Bass / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D004266:DNA Virus Infections / Q000276:immunology / Q000662:veterinary; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D023061:Gene Order; D064451:Hepcidins; D017994:Iridovirus / Q000276:immunology; D007501:Iron / Q000494:pharmacology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000276:immunology; D016415:Sequence Alignment
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D000998:Antiviral Agents; D064451:Hepcidins; D012333:RNA, Messenger; D007501:Iron