Gene Cloning, Identification And Characterization Of Novel Antimicrobial Peptides With Multiple Functions From Skin Of Frog, Nanoorana Parkeri

Xizang plateau frog (Nanorana parkeri) captured in Lhasa, Tibet, China, is mainly found in China and Nepal. Its sole habitat is the eastern and southern Tibet plateau in subtropical zone, where there is strong ultraviolet radiation and long duration of sunshine. Considering its harsh living environment, the frog's innate defense against microbes and environmental stress was investigated. In current study, three antimicrobial peptides (AMPs) were purified and characterized from the skin secretion of N. parkeri. The coding cDNA sequences were also cloned from the skin cDNA library of N. parkeri. By structural characterization, two peptides were identified belonging to Japonicin-1 family, and named as Japonicin-1Npa (FLLFPLMCKIQGKC) and Japonicin-1Npb (FVLPLVMCKILRKC). The third peptide isolated named Parkerin with a unique sequence of GWANTLKNVAGGLCKITGAA did not show similarity to any known amphibian AMPs. Parkerin is a brand new amphibian AMP family, and firstly reported here. Multi-functions of three AMPs were examined (antimicrobial, antioxidant, MCD, hemolytic etc). Parkerin was the only one showing broad-spectrum antimicrobial activity against Gram -positive, Gram-negative bacteria and fungus, and has stronger antioxidant activity. Both japonicin-1NPa and japonicin-1NPb have relatively narrow range of their antimicrobial list, therefore, only have resistance for Gram-positive bacteria, moreover, only a limited part of free radicals can be eliminated. Otherwise, Three AMPs all had very little hemolytic activity on blood cells. And they were showing to promote mast cell degranulation. Their solution structures determined by CD and antimicrobial mechanisms investigated by SEM are very well consistent with their functional characters. Current result suggests that these novel multi-functional AMPs could play an important role in defending N. parkeri against environmental oxidative stress and pathogenic microorganisms, which may partially reveal the ecological adaptation of these plateau-living amphibians.