Purification,identification And Functional Studies Of Defensive Peptides From The Skin Secretions Of Tree Frog Theloderma Kwangsiensis And The Venom Of Scorpion Mesobuthus Martensii

Biodefense peptides have the function of defense,such as resisting to microbial infections and attacking to predators or preys.Animals often take advantage of these peptides to protect themselves from environmental risks.Antimicrobial peptides are defense peptides which play a critical role in protecting animals from the invasion of bacteria,viruses or fungi.Defensins are one of the major and most studied family of antimicrobial peptides(AMPs).Up to now,more than 300 defensins have been reported across procaryotic and eukaryotic kingdoms.But defensins are seldom found in amphibian,a major family for isolating novel AMPs.A novel defensin(Defensin-TK)was isolated and characterized from skin secretions of the tree frog Theloderma kwangsiensis.The cDNA encoding Defensin-TK precursor was cloned from the skin cDNA library of T.kwangsiensis.The precursor was composed of three domains,a signal peptide of 16 residues,a spacer peptide of 1 residues and a mature peptide of 42 residues.The deduced mature peptide of Defensin-TK shared the highest identity with the salamander(Cynops fudingensis)defensin CFBD-1.The six conserved cysteines which form intramolecular disulfide bonds of defensins also existed in the deduced mature peptide.The theoretical pI of the mature peptide of Defensin-TK was 4.83.Phylogenetic analysis indicated that Defensin-TK was closely related to fish β-defensin.Defensin-TK showed potent and broad-spectrum antimicrobial activity against Gram-negative Escherichia coli ATCC25922(MIC,12 μg/mL),Bacillus dysenteriae(MIC,3 μg/mL),Gram-positive bacterium Staphylococcus aureus ATCC2592(MIC,3 μg/mL)and fungus Candida albicans ATCC2002(MIC,6 μg/mL).In addition,Defensin-TK exerted a low hemolytic activity on human red cells(1.5%at a concentration of 50 μg/mL).These results suggested Defensin-TK might play an important role in host defense mechanisms of the tree frog,and might be a promising candidate for development of novel antimicrobial agents.Scorpion venoms are highly complex mixtures of peptides and other substances.Polypeptides contained in scorpion venom appear to have evolved primarily for subduing prey and also play a highly effective role in defense.As a consequence,scorpion venoms possess a wide spectrum of bioactivities and be deemed to as an important source of natural compounds.Mesobuthus martensii is one of traditional medicine in China.Out of the two hundred proteins identified in M.martensii,only a few peptide toxins have been found so far.Herein,a combinational approach based upon RNA sequencing and Liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS)was employed to explore the venom peptides in M.martensii.A total of 153 proteins were identified from the scorpion venom,26 previously known and 127 newly identified Of the novel toxins,97 proteins exhibited sequence similarities to known toxins,and 30 were never reported.Combining peptidomic and transcriptomic analyses,the peptide sequence of BmKKx1 was reannotated and four disulfide bridges were confirmed within it.In light of the comparison of conservation and variety of toxin amino acid sequences,highly conserved and variable regions were perceived in 24 toxins that were parts of two sodium channel and two potassium channel toxins families.Taking all of this evidences together,the peptidomic analysis on M martensii indeed identified numerous novel scorpion peptides,expanded our knowledge towards the venom diversity,and afforded a set of pharmaceutical candidates.Based on the hints from peptidomics research,a sodium channel inhibitor,BmKaTX15,have been isolated and identified from M martensii venoms by chromatographic technique.BmKaTX15 exhibits a potent inhibitory function with an IC50 of 5×10-3 μM for TTX-R sodium channels.The results of conductance-voltage(G-V)relationship and steady-state inactivation suggested BmKaTX15 may be action on the pore of TTX-R Nav channel.The properties suggests BmKaTX15 may be a valuable tools for understanding the structure-function relationship of sodium channels and for clinical drug development.