Gene Cloning、Expression&Structure And Functional Characterization Of Cathelicidins From Limnonectes Fragilis

Cathelicidins, one family of host defense peptides, are characterized by an N-terminal signal peptide, and a conserved cathelin domain followed by a highly divergent C-terminal mature peptide. The famile of cathelicidins is the bridge to connect the natural and specific immune system, which serve a critical role in vertebrate innate immune defense against invasion bacterial infection. They have been found from almost all vertebrates by far, including mammals, birds, reptiles and fish, but there are only few reports of cathelicidins in amphibians, In this paper, the cathelicidins from the frog skin of Limnonectes fragilis were firstly characterized and reported.Two cDNA sequences encoding the cathelicidins were cloned from the constructed skin cDNA library of L. fragilis. The full-length of these cDNA sequences were586bp and596bp, and deduced precursor peptides were composed of155and151amino acid residues, respectively. Blast analysis revealed that they had the same structure composition with other cathelicidins, including an N-terminal signal peptide, a conserved cathelin domain and a C-terminal mature peptide. Phylogenetic analysis revealed that L. fragilis cathelicidins clustered with cathelicidins from reptiles (snake) and birds (chicken, coturnix and pheasant) in phylogenetic tree, implying the same evolution relationship between L. fragilis and these species, which provides further proof for the studies of origin as well as structural diversity and functional differentiation evolutionary mechanism of cathelicidins.For different species, the processing of cathlicidin prepropeptide to release the cathelin and mature peptide is different. In addition, the mature peptide sequences released by protease are different. According to elastase-sensitive residues (-Val-represents the most common processing site in mammals cathelicidins) and trypsin-sensitive residues (-Lys-Arg-or-Arg-Arg-represents the most common processing site in amphibian antimicrobial peptides) in the cathelicin domain, the mature peptides of two L. fragilis cathelicidins were predicted, naming as Lf-CATH1/2and Lf-CATH1a/2a, respectively. Chemically synthesized Lf-CATH1/2exhibited no antibacterial activity against40experimental strains, while expressed Lf-CATH1a showed antibacterial activity against Staphyloccocus aureus. Chemically synthesized Lf-CATH1a exerted strong antimicrobial activity against S. aureus ATCC25923, IS090223, IS1307and Escherichia coli ATCC25922. Lf-CATHla displayed a negligible hemolytic activity and lectin activity against human erythrocytes. In addition, Lf-CATHla exhibits high antioxidant activity, lysing57.79%of DPPH radicals at160μg/ml. Taking into account the complex environment for the survival of amphibians, the results suggest that Lf-CATH1a may employ sophisticated mechanisms of action in host defense in addition to anti-microbial, such as antioxidant to protect the bare skin of amphibians.