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.