Characterization and functional analysis of liver-expressed antimicrobial peptide-2 (LEAP-2) from golden pompano Trachinotus ovatus (Linnaeus 1758)

Abstract

The liver-expressed antimicrobial peptide-2 (LEAP-2) is an important component of the innate immune defense system and plays an important role in resisting the invasion of pathogenic microorganisms. In this study, LEAP-2 from golden pompano (Trachinotus ovatus) was characterized and its expression in response to Photobacterium damselae was investigated. The full-length LEAP-2 cDNA was 1758 bp, which comprised a 5′-UTR of 250 bp, an ORF of 321 bp, and a 3′-UTR of 1187 bp, encoding 106 amino acids. LEAP-2 consisted of a conserved saposin B domain and four conserved cysteines that formed two pairs of disulphide bonds. The genomic organization of LEAP-2 was also determined and shown to consisted of three introns and two exons. The predicted promoter region of ToLEAP-2 contained several putative transcription factor binding sites. Quantitative real-time (qRT-PCR) analysis indicated that LEAP-2 was ubiquitously expressed in all examined tissues, with higher mRNA levels observed in the muscle, liver, spleen, and kidney. After P. damselae stimulation, the expression level of LEAP-2 mRNA was significantly upregulated in various tissues of golden pompano. In addition, SDS-PAGE showed that the molecular mass of recombinant LEAP-2 expressed in pET-32a was approximately 23 kDa. The purified recombinant protein showed antibacterial activity against Gram-positive and Gram-negative bacteria. Luciferase reporters were constructed for five deletion fragments of different lengths from the promoter region (−1575 bp to +251 bp), and the results showed that L3 (−659 bp to +251 bp) presented the highest activity, and it was therefore defined as the core region of the LEAP-2 promoter. The seven predicted transcription factor binding sites were deleted by using PCR technology, and the results showed that the mutation of the USF transcription factor binding site caused the activity to significantly decrease. The results indicate that golden pompano LEAP-2 potentially exhibits antimicrobial effects in fish innate immunity.