title: Characterization, evolution and functional analysis of the liver-expressed antimicrobial peptide 2 (LEAP-2) gene in miiuy croaker.
abstract: As an evolutionarily older defense strategy, the innate immune is the dominant immune system and provides a first line of antimicrobial host defense in teleost. Liver-expressed antimicrobial peptide-2 (LEAP-2) is a critical molecule of the innate immune system and plays a very important role in resistance against bacterial infections. We reported comprehensive analysis and characterization of LEAP-2 gene from miiuy croaker (Miichthys miiuy) in here. The complete cDNA of miiuy croaker LEAP-2 consists 2360 bp, including a 5' terminal untranslated region (UTR) of 170 bp, an open reading frame (ORF) of 312 bp, and a 3'-UTR of 1878 bp. Interestingly, two polyadenylation signals (AATTAAA) which may involve the stability, translation efficiency, or localization of an mRNA in a tissue were found in 3'-UTR. Genomic DNA of miiuy croaker LEAP-2 includes three exons and two introns, which is similar to LEAP-2 genes in other mammals and fish. The deduced 103 amino acids consist of signal peptide, prodomain and mature peptide. Four highly conserved cysteine residues involved two disulfide bridges in mature peptide. Real-time PCR results showed that LEAP-2 was ubiquitously expressed in all tissues and the expression level was highest in liver. Significantly, the expression levels were increased after infection with Vibrio anguillarum in liver and spleen. The antimicrobial activity analysis result of LEAP-2 in vitro indicated that LEAP-2 of miiuy croaker was effective in controlling Aeromonas hydrophila. In addition, we performed evolutionary analysis in order to estimate the selective constraints on the LEAP-2 gene. The result indicated that no positive selection exists in LEAP-2 gene sequences, which may be on account of irreplaceable function constrains. Meanwhile, we compared the structure of LEAP-2 with that of another Liver-expressed antimicrobial peptide (LEAP-1, also named HAMP), and found the LEAP-2 from miiuy croaker comprises of α-helix, β-sheet, and β-turn while the LEAP-1 of miiuy croaker only contains β-sheet.
authors.affiliation: Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan 316022, China.
authors.forename: Tianxing
authors.initials: T
authors.lastname: Liu
authors.affiliation: College of Animal Science and Veterinary Medicine, Jilin Agriculture University, Changchun 130118, China.
authors.forename: Yunhang
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan 316022, China.
authors.forename: Rixin
authors.initials: R
authors.lastname: Wang
authors.affiliation: Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan 316022, China. Electronic address: tianjunxu@163.com.
authors.forename: Tianjun
authors.initials: T
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001499:Bayes Theorem; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D019143:Evolution, Molecular; D005393:Fish Diseases / Q000235:genetics* / Q000276:immunology*; D005786:Gene Expression Regulation / Q000276:immunology; D008099:Liver / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010473:Perciformes; D010802:Phylogeny; D011487:Protein Conformation; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D012641:Selection, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000662:veterinary; D014735:Vibrio Infections / Q000276:immunology / Q000662:veterinary*
chemical_list: D023181:Antimicrobial Cationic Peptides; D018076:DNA, Complementary
keywords: Antimicrobial activity; Expression pattern; Liver-expressed antimicrobial peptide-2 (LEAP-2); Miiuy croaker (Miichthys miiuy); Molecular evolution