Characterization and functional analysis of liver-expressed antimicrobial peptide-2 (LEAP-2) in Pelodiscus sinensis

Liver-expressed antimicrobial peptide-2 (LEAP-2) is a cationic antimicrobial peptide with two disulfide bonds, and it has been shown to play an important role in the host defence in fish, amphibians, birds and mammals. Here, we characterize LEAP-2 from Pelodiscus sinensis and identify its function. The deduced precursor of PsLEAP-2 was composed of 77 amino acids (aa), including a 22-aa signal peptide, a 15-aa pro-domain with a typical cleavage site ((RXXR37)-X-34), and a 40-aa mature peptide with four conserved cysteine residues that form two disulfide bonds (Cys(54, 60, 65, 70)), PsLEAP-2 was highly conserved in evolution, and the precursor/mature peptide of PsLEAP-2 showed 42%-62%/57%-72% sequence identity with LEAP-2 sequences from other known tetrapods. Expression analysis data showed that PsLEAP-2 was only expressed in the liver. RT-qPCR results showed that the mRNA expression level of PsLEAP-2 was induced after challenge with Edwardsiella tarda (E. tarda) or soft-shelled turtle iridovirus (STIV). In freshly isolated liver cells, induced expression of PsLEAP-2 was only observed at 24 h after stimulation with LPS or poly(I:C). Epithelioma papulosum cyprinid (EPC) cells transfected with the LEAP-2-Flag plasmid exhibited a reduced intake of E. tarda and reduced viral yield of STIV compared with cells transfected with the Flag plasmid. These results suggest that PsLEAP-2 plays an important role in the immune defence against bacterial and viral infection.