| Biodefense peptides mainly have the function of defense,such as resisting to microbial infections and attacks from predators or preys.Animals often take advantage of these peptides to protect themselves from many environmental risks,to reduce pain and recover as soon as possible when they are hurt.Anti-inflection andanalgesia are two of the most common defense mechanisms,which usually rely on anti-infection or analgesia substances,such as antimicrobial peptides and analgesic peptides.Antimicrobial peptides are defense peptides which play a critical role in protecting animals from the invasion of bacteria,viruses or fungi.The cationic antimicrobial peptide defensins are one of the largest and mostly studied antimicrobial peptides families.Up to now,more than 300 defensins have been identified.They represent in a range of organisms such as mammals,plants,bacteria and fungus,and provide immunologic defense to the organisms.In order to determine if there is antimicrobial peptide in urodela,we try to purify and characterize possible antimicrobial compounds from the skin secretions of the salamander,Cynops fudingensis.By the combination of Sephadex G-50 size-exclusion chromatography and revers phase high performance liquid chromatography,a novel antimicrobial peptide CFBD-1 was isolated and characterized from skin secretions of the salamander,C.fudingensis.The cDNA encoding CFBD-1 precursor was cloned from the skin cDNA library of C.fudingensis.The precursor was composed of three domains:a signal peptide of 17 residues,a mature peptide of 41 residues and a spacer peptide of 3 residues.There are six cysteines in the sequence of mature CFBD-1 peptide,which possibly form three disulfide-bridges.This peptide belongs to ?-defensin family based on its high sequence similarities with ?-defensins from other vertebrates.It is a new member of the beta defense family.This is the first time to find defensive antimicrobial peptides from urodela.For further research,we used genetic recombination to construct the prokaryotic expression system.The DNA sequence encoding mature CFBD-1 was cloned into the PET vector and the vector was transformed into Escherichia coli for recombinant expression to obtain the fusion protein with a His-tag.Recombined CFBD-1 was released from the fusion protein by formic acid cleavage and purified by the combination of Sephadex G-50 and RP-HPLC.CFBD-1 showed wide antimicrobial activities against Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Candida albicans.CFBD-1 had potential antimicrobial activities against the Gram-positive bacterium such as S.aureus and the MIC was 65 ?g/ml;It showed lower antimicrobial activities against the Gram-negative bacterium such as B.subtilis,E.coli and the MIC were 135 ?g/ml and 160 ?g/ml respectively;The MIC to the fungus C.albicans was 200 ?g/ml.Here,we also studied another kind of defensive peptide(spider analgesic peptide).A large number of spider toxins have been identified as voltage-gated ion channel toxins,one third of them were supposed to target on sodium channels.Cystein distribution of the spider toxins are very conserved,most of the disulfide connectivity to be C1-C4,C2-C5 and C3-C6,forming a typical inhibitor cystine knot motif.Haplopelma lividum is a kind of typical caved spider.The length of adult H.lividum is 130-150 mm.H.lividum lives in the tropical rain forests in Thailand and Myanmar,feeding on crickets,snails and other insects or small animals.H.lividum has strong toxicity.Attacking is a major defense strategy of Haplopelma lividum.It's painful when a man is bitten by H.lividum,implying that there may be neurotoxins in its venom.?-TRTX-H1la,a neurotoxin was screened from cDNA library of H.lividum.It has a conserved ICK motif.Synthesized and refolded?-TRTX-Hl1a was used for further research.Patch-clamp was used to screen the ion channel activities of ?-TRTX-Hl1a in the dorsal root ganglion neurons cells of SD rat.?-TRTX-Hlla had no function on voltage-gated potassium channels,calcium channels and tetrodotoxin-sensitive sodium channels(TTX-S),but could selectively inhibit tetrodotoxin-sensitive sodium channel(TTX-R)currents.The inhibition of TTX-R sodium channel currents in DRG by ?-TRTX-Hl1a was concentration-depended.The median effective inhibitory concentration(IC50)was 3.76±0.21 ?M.Pasmid containing sodium channel subtypes were transfected into HEK293T cells,and the selectivity of?-TRTX-Hl1a on sodium channel subtypes was tested.The toxin could selectively inhibit Nav1.8 currents and had no effect on other subtypes.?-TRTX-Hlla inhibited Nav1.8 channel with an IC50 value of 2.19±0.18 ?M.It did not change the voltage-current curve and the conductance voltage curve,and did not cause inactivation of the sodium channel.Four kinds of pain models(formalin-mediated licking model,tail flick model,acetic acid induced writhing model,and hot plate model)were used to evaluate the analgesic effect of?-TRTX-Hl1a.The results showed that ?-TRTX-Hl1a could reduce the acetic acid-induced writhing response in mice.It significantly prolonged the response time of mice in the heat pain flick and hot-plate models.Furthermore,it significantly reduced the licking numbers during the pain of both phase ? and phase ? in formalin-mediated licking model.The results indicated that ?-TRTX-Hl1a could significantly inhibit neuropathic pain and inflammatory pain.C.fudingensis is mainly distributed in wetland containing large amount of bacteria and fungi.In order to adapt to the living environment,C.fudingensi has developed antimicrobial peptides in its skin,such as defensin reported in this study.CFBD-1 is the first ?-defensin from C.fudingensis,which may have important functions for salamander to prevent microbial infection.?-TRTX-Hl1a is a toxin identified from H.lividum with the function of inhibiting sodium channel,which may help this spider to resist predators such as toads,frogs,lizards,and birds.These two defensive peptides have good antimicrobial and analgesic effects,respectively.This study is important for understanding of the survival strategies of the organisms.At the same time,it provides templates for the development of novel antimicrobial and analgesic drugs. |
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