Purification, Characterization, Molecular Cloning Andfunction Analysis Of Antimicrobial Peptides From Skin Of Limnonectes Kuhlii

The discovery of antimicrobial peptides(AMPs) in 1980s the last century attracted our attention as a novel antibiotics because of its advantages than antibiotics such as high speed of killing bacteria,broad antimicrobial spectrum, different mode of action and hard to occur resistance to drug.Therefore AMPs have been considered as potential substitution for antibiotics.By a two-step protocol including one step Sephadex G-50 and one step of RP-HPLC,five novel antimicrobial peptides (temporin-LKl,rugosin-LKl, rugosin-LK2,gaegurin-LKl,and gaegurin-LK2) were purified and characterized from Kuhl’s wart frog skin secretions, Limnonectes kuhlii. They shared obvious similarity to temporin, rugosin, and gaegurin antimicrobial peptide family, respectively. Their amino acid sequences were determined by Edman degradation and mass spectrometry, and further confirmed by cDNA cloning. Nine cDNA sequences encoding precursors of these five purified antimicrobial peptides and other four hypothetical antimicrobial peptides were cloned from the skin cDNA library of L. kuhlii. The deduced precursors were composed of a signal and propiece domain and a mature domain. Most of them showed strong antimicrobial activities against bacteria and fungi.The rate of synonymous and nonsynonymous nucleotide substitutions were calculated in rugosin and gaegurin antimicrobial peptide families, and they displayed different nucleotide substitution patterns, indicating they might undergo different selecting pressures. By comparing the secondary structure of antimicrobial peptide with that from Rana nigrovittata, the difference in antimicrbial spectrum between L. kuhlii and Rana nigrovittata related to the amphipathy of the peptide.Little work was reported about antimicrobial peptides from the genus of Limnonectes. L. kuhlii belongs to Ranidae, dicroglossine, genus Limnonectes. It was a widely distributed frog in tropical regions, such as Yunnan province of China, Vietnam,Myanmar,and Thailand. Our current work identified and characterized three families of antimicrobial peptides including one member of temporin, four members of gaegurin, and four members of rugosin. Temporin> gaegurins,and rugosinswere common antimicrobial peptides in amphibians belonging to the genus Rana. These results indicated that L. kuhlii shared similar antimicrobial peptide families with other Ranidae frogs. Most of antimicrobial peptides of temporins had 13-16 aa but temporin-LKl found in this work had 17 aa. In addition, there were four phenylalanines in the sequence of temporin-LKl, which was much more than that of other temporins (one or two). Such special structure might provide novel templates or leading structures to design antimicrobial agents.