Identification and molecular cloning of novel antimicrobial peptides from skin secretions of the Chinese bamboo leaf odorous frog (Odorrana versabilis) and the North American pickerel frog (Rana palustris)

摘要

Objective:Amphibian skin secretions are an abundant source of bioactive peptides,some of which could be developed as candidate drugs.Among these natural peptides,cytolytic peptides have attracted the most attention given that they might replace conventional antibiotics and help deal with the problem of microbial resistance.This study discovered two bioactive peptides,Brevinin-1-PLr and Nigrocin-2-OV,from two species frogs,the Chinese bamboo leaf odorous frog (Odorrana versabilis) and the North American pickerel frog (Rana palustris),respectively.Their antimicrobial,anticancer and hemolytic activities were also investigated.Methods:cDNA sequences encoding peptides were cloned from cDNA libraries constructed from the lyophilized secretions of the Chinese bamboo leaf odorous frog and the North American pickerel frog.By reversed-phase HPLC and MS/MS fragmentation sequencing,the encoded novel peptides,named Nigrocin-2-OV and Brevinin-1-PLr,were identified in skin secretions and their structures were confirmed.Replicates of both peptides were produced through solid phase peptide synthesis.Their antimicrobial and anticancer activity was studied against three types of microorganisms (Staphylococcus aureus,Candida albicans,and Escherichia coli) and five cancer cell lines (NCI-H157,PC-3,MDA-MB-435s,MCF-7,and U251MG).Their hemolytic activity was investigated using whole horse blood.Results:In this research,cDNA sequences encoding two novel 24-mer peptides were cloned from cDNA libraries constructed from the lyophilized skin secretions of the Chinese bamboo leaf odorous frog and the North American pickerel frog.Both of the peptides had the strongest inhibitory effect against C.albicans,and IC50 values against five cancer cell lines were all under 6 μM.Conclusions:Nigrocin-2-OV and Brevinin-1-PLr had the strong ability to inhibit the proliferation of studied microorganisms and tumor cell lines,with slight hemolytic activity.Compared with Brevinin-1-PLr,Nigrocin-2-OV exhibited higher antimicrobial and anticancer activity but slightly higher hemolytic activity.