Isolation,Identification And Functional Study Of Bioactive Peptides From Oriental Fire-bellied Toad,Bombina Orientalis

Objective: This study aims to molecular clone,isolate and identify the active peptides from the skin secretions of Oriental Fire-bellied toad,Bombina orientalis(B.orientalis),and further study their biological function,which will provide theoretical foundation and experimental basis for developing such kind of anticancer and antimicrobial drug candidates.Methods: 1.Total RNA was isolated from the skin of B.orientalis using column extraction method,and then the c DNA library was constructed by reverse transcription with a SMARTer?RACE kit.Primers were designed,and the c DNA library was amplified by RT-PCR.The amplified DNA fragments were cloned using a p GEM-T Easy Vector System,and the selected DNA sample was sequenced.2.Sequences were edited and manipulated using Chromas and Vector-NTI software programs.The nucleotide sequence and deduced amino acid sequence data were retrieved and compared with sequences deposited in the EMBL-EBI,NCBI and Swiss Prot databases using the BLASTn and BLASTp programs.Sequence alignments were performed using Align X software.3.RP-HPLC was used to isolate the peptide fragments from the toad skin secretions.Mass spectrometry was used to analyze the molecular weight and primary structure peptides.4.The deduced active peptides from the skin secretions of Oriental fire-bellied toad were synthesized separately by solid-phase synthesis method.The secondary structure of each peptide was predicted through software modeling by I-TASSER online server.The helical wheel plots and a few physicochemical properties of peptides such as hydrophobicity,hydrophobic moments,net charge(Z)were calculated using Heliquest web server.CD spectroscopy was employed to further assess the secondary structures of both peptides.5.The antimicrobial activity of the synthetic peptides was assessed by minimal inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)assays.A safety evaluation and the cytotoxic effect of individual peptides were evaluated by haemolytic assay.6.MTT assay was used to determine the inhibitory effect of peptides on the proliferation of three human hepatoma cell lines(Hep G2,SK-HEP-1 and Huh7) and human prostate cancer cell line(PC3),human lung cancer line(A549),and human melanoma cell line(A375).Result: 1.A c DNA library was successfully constructed using the Orient-bellied toad skin total RNA as a template.A sense primer was designed according to a highly conserved segment of the 5'-untranslated region of bombinin precursor-encoding c DNAs from Bombina species in the Genebank.By employing the RACE/PCR strategy,a novel full-length c DNA was cloned from the skin secretion-derived c DNA library of B.orientalis.2.The novel full-length c DNA encoded two bombinins.One of the encoded bombinins was a bombinin H-type peptide with novel primary structure,and this peptide was named as Bombinin H-BO.The other was a previously reported bomninin like peptide(BLP-7)from this species.3.BLP-7 and Bombinin H-BO were isolated from the toad skin secretion by using reverse-phase HPLC.The primary structures of BLP-7(GIGGALLS AGKSALKGLAKGLAEHFAN-NH2)and Bombinin H-BO(IIGPVLGLIGKA LGGLL-NH2)were identified by using an electrospray mass spectrometry.4.Two peptides were synthesized by solid-phase synthesis method.The peptides were both purified to > 95% purity.The secondary structures and physicochemical properties prediction revealed that BLP-7 contained ?-helical domain representing 74.07% of the structure with two positive charges,while Bombinin H-BO adopted ?-helix representing 64.71% of the peptide with one positive charge.Both peptides are amphiphilic.5.BLP-7 displayed a strong growth inhibitory activity against all three tested microorganisms.The MIC of BLP-7 was 6.3 ?M for E.coli and S.aureus,and 12.5 ?M for C.albicans.After treatment with maximum concentration(159.7 ?M)of Bombinin H-BO,the inhibitory rates against all three tested microorganisms were approximately 50%.BLP-7 exited no hemolytic activity at concentration of 50.2 ?M.Bombinin H-BO caused 38% of the red blood cells hemolysis at the concentration of 80.0 ?M,and up to 85% at the concentration of 159.7 ?M.6.BLP-7 and Bombinin H-BO inhibited three human hepatpma cells and several other human cancer cells proliferation in a dose-dependent manner.Among them,the inhibitory effect of BLP-7 and Bombinin H-BO on human lung cancer A549 cells was the most significant,with IC50 as 0.244 ?M and 0.597 ?M After 48 h treatment,respectively.Conclusion: 1.Two bombinin peptides,a bombinin like peptide(BLP-7)and a novel bombinin H type peptide(named as Bombinin H-BO),were identificated and characterized from the skin secretion of Oriental fire-bellied toad,B.orientalis by combination techniques of molecular cloning,HPLC and MS spectrometry.The structure of Bombinin H-BO was reported for the first time.2.Both BLP-7 and Bombinin H-BO showed potent antimicrobial activities and obvious anticancer activities in vitro,which was considered with potential utility value.Their specific antimicrobial and anti-tumor mechanism remains to be further studied.