Characterization and functional analysis of three novel liver-expressed antimicrobial peptide 2 (LEAP-2) in Glyptothorax zanaensis

Abstract

Liver-expressed antimicrobial peptide 2 (LEAP-2) is an important component of innate immunity, a well-conserved, evolutionarily ancient host defensive mechanism. In this study, three novel full-length LEAP-2 cDNAs were identified and characterized from G. zanaensis. The deduced precursors of GzLEAP-2A, GzLEAP-2B and GzLEAP-2 C, are composed of 94, 74, and 83 amino acids, which possess four, three, and four conserved cysteine residues in the mature peptide, respectively. GzLEAP-2 s are highly conserved in evolution, with GzLEAP-2A and GzLEAP-2 C showing a close topologic relationship with LEAP-2 from Ictalurus furcatus and Danio rerio, respectively. Quantitative real-time polymerase chain reaction analysis indicated that GzLEAP-2 s ubiquitously existed in all examined tissues, with higher mRNA expression levels observed in muscle, intestine, and liver. Antimicrobial assay showed that synthetic GzLEAP-2A and GzLEAP-2B displayed specific antimicrobial activity against aquatic strains, whereas GzLEAP-2 C did not show any detectable antimicrobial activity. All GzLEAP-2 s were found to possess potent 2,2-diphenyl-1-picrythydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)+ radical scavenging activities, and high doses of GzLEAP-2B and GzLEAP-2 C pre-treatment significantly promoted macrophage phagocytic activity. Moreover, no toxicity of GzLEAP-2 s against tested cells was observed at the effective concentrations. The distinct tissue distribution and pleiotropic roles of GzLEAP-2 s suggest a sophisticated and multilevel antimicrobial defensive network of G. zanaensis.