CDNA Screening And Characterization Of Bioactive Peptides From Skin Of Pelophylax Nigromaculatus

The skin of amphibious animals is rich in the bioactive peptides, including antimicrobial peptides, protease inhibitors and antioxidant peptides. More than2,000kinds of bioactive peptides have been identified from Amphibian skin. In the same species, bioactive peptides often exhibit the obvious diversity, and their expressing profiles are different when the sample are collected from different regions.Dark-Spotted Frogs (Pelophylax nigromaculatus) are distributed in wide regions (from frigid belt to tropical zone, from plateau to basin) in China, Japan, Korea and the former Soviet Union. The extensive distribution of P. nigromaculatus indicates its powerful fitting ability to different environments. Until now, only two antimicrobial peptides, nigrocin1and nigrocin2, have been identified from this species.In this study, Dark-Spotted Frogs were collected from Kunming, Yunnan Province and Guiyang, Guizhou Province to verify whether there are more kinds of bioactive peptides and whether the pepeides are different in two regions. Nine bioactive peptides were identified by screening the cDNA library, five of them (nigrocin-1、nigrocin-2、pelophylaxin-2、esculentin-1P, and esculentin-2P) have been reported, the others (pelophylaxin-2GY, temporin-1GY, temporin-1KM, and antioxidin) haven’t been reported. Moreover, the expressing profiles are distinct in two regions.Six peptides (antioxidin-PN, pelophylaxin-2GY, temporin-1GY and temporin-1KM with or not the amidated C-terminus) were synthesized with the purity higher than95%to screen the potential bioactivity, including antimicrobial assay, protease inhibitory assay, antioxidant assay, and hemolytic activity determination, etc.The amidated temporin-1s showed antimicrobial activity, while the unamidated peptides had no antimicrobial activity. It suggests that amidated C-terminus is vital to their antimicrobial activity. Temporin-1GY and temporin-1KM have similar antimicrobial capability against tested bacteria. The MIC of temporin-1s for S. aureus and E.coli are6.25μg/mL and12.5μg/mL, respectively. Furthermore, C. albicans was sensitive to temporin-1KM with the MIC of12.5μg/mL. Pelophylaxin-2GY only has antibiotics activity to the Gram-negative bacteria E.coli, the MIC is12.5μg/mL. All three antimicrobial peptides can keep the activity well after incubating in50%serum for6h, but Pelophylaxin-2GY lost the activity after12h incubation, temporins lost the activity after18h incubation.The antioxidin-PN showed strong ABTS+free radical scavenging ability with dose-dependent and time-dependent manners. As shown in Fig.3, the inhibition percentage raised with the increase of antioxidin-PN concentration. At the concentration of100μg/mL, antioxidin-PN scavenged30%of ABTS+in15sec and nearly100%of ABTS+in10min. Even at a concentration as low as6.25μg/mL, more than30%of ABTS+free radical was scavenged in10min.All the synthesized peptides with bioactivity, pelophylaxin-2GY, temporin-1GY, temporin-1KM and antioxidin-PN, had no hemolytic activity even the concentration up to200μg/mL.In this study, nine bioactive peptides were identified from P. nigromaculatus. Four of them with antimicrobial activity or antioxidant activity are unreported. Two peptides, pelophylaxin-2GY and temporin-1GY, are found in the frogs from Guiyang. While the antioxidant peptide named antioxidin-PN and three other antimicrobial peptides, esculetin-1, esculetin-2and temporin-1KM, are present in the frog from Kunming. The distinguish of same species from different localities might rely on adaptive evolution according to the environment, such as the oxidative damage caused by ultraviolet rays, the infection of microorganisms, and so on.