EC-proHep3

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.

Anti-lipopolysaccharide factors (ALFs), the potential antimicrobial peptides that bind and neutralize lipopolysaccharide (LPS), are common effectors of innate immunity in crustaceans. In this study, a novel isoform of ALFs (SpALF5) was isolated from the hemocytes of mud crab Scylla paramamosain. The full-length 975bp SpALF5 contains a 375bp open reading frame (ORF) encoding 125 amino acids. Although SpALF5 exhibits a low degree of nucleotide homology with other reported ALFs, it contains the conserved amino acid sequence with a signal peptide and a LPS-binding domain including two conservative cysteine residues. The genomic organization of SpALF5 consists of four exons and three introns, with each intron containing one or more tandem repeats. Unlike most of ALFs mainly distributed in crab hemocytes, SpALF5 transcript was predominantly observed in the brain, muscle and skin, while barely detected in the hemocytes in our study. In situ hybridization assay also showed that SpALF5 mRNA was localized in brain, muscle and skin tissues of mud crab. Further, SpALF5 transcript was significantly up-regulated after challenge with LPS, polyinosinic polycytidylic acid (PolyI:C) (with the except of that in brain), Vibrio parahemolyticus or white spot syndrome virus (WSSV). The recombinant SpALF5 protein showed a varying degree of binding activity towards bacteria and fungus. Moreover, in vitro, the recombinant SpALF5 revealed a strong antimicrobial activity against Gram-negative bacteria (V. parahemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) and fungus (Sacchromyces cerevisiae), but could only inhibited the growth of some Gram-positive bacteria like Staphylococcus aureus. The results suggest that SpALF5 is a potent immune protector and plays an important role in immune defense against invading pathogens in S. paramamosain.

C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell-cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys(71), Cys(152), Cys(167), Cys(175)) involved in the formation of disulphide bridges and the potential Ca(2+)/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca(2+)-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca(2+)-dependent manner.