Separation, Purification And Identification Of L-amino Acid Oxidase From Coral Snake

In our country,snake used as medicinal materials to treat many diseases has a long developmental history.Bungarus multicinctus commonly known as multibanded krait,belongs to chordate phyla,reptile,squamata,elapidae,bungarus.Chinese medicine believes that B.multicinctus,sweet and salty in flavor,warm in nature,toxic,has the ability of reducing rheumatism,arresting convulsion,smoothening energy channel and relieving itching.Snake venom secreted from the venom gland tissue contains a number of enzymes,non-enzyme proteins,and other small molecules,which display a broad spectrum of biological and pharmacological properties,and is characteristic as strong toxic.B.multicinctus contains a variety of active ingredients,and the main component is neurotoxin.Treat the tonifying disease with poisonous agents is a traditional medical therapeutics,and studies have shown that snake venom exhibited a variety of biologically activities,including depressurization,analgesia,anti-inflammatory and anticancer action.So far,some of the ingredients with some modification have been made into medicines to treat diseases in clinical.In recent years,more and more researchers have shifted emphasis to explore the component and function of peptides in animal drug with the increasing matureness of high-throughput sequencing technology and bioinformatics analysis technology,and animal drugs have become the source of finding and developing natural medicine.Therefore,it is of theoretical importance and practical significance to explore the active substances and pharmacological activity of B.Multicinctus.In this study,a transcriptome library was constructed by the Illumina HiseqTM 2500 technology and analyzed the bioinformation by some softwares.Trinity software was used to carry out the de-novo assembly,and 63,947 high quality unigenes(>200 bp)with total 64,485,567 bp nucleotides were obtained,the longest unigene was 23,902 bp,the shortest length was 200 bp,the mean length was 1008 bp,and the N50 length was 1806 bp.Search the five database for gene functional annotation by Blastx.The results showed that 21,900(34.25%)unigenes had similar sequences in the NR database,and the venom of B.multicinctus was rich in neurotoxin.Meanwhile,17,987(28.13%),13,963(21.84%),6981(10.92%)and 15,964(24.96%)unigenes were annotated successfully in the SWISSPROT,KOG,KEGG and GO database,respectively.The massive unigenes of B.multicinctus provided important information of its components and laid the foundation for the molecular cloning of functional genes and study of medicinal value.In the present study,a new kind of LA AO from B.multicinctus snake venom,named Bm-LAAO,was purified using a three-step chromatography procedure of size-exclusion and ion-exchange chromatography,and provided highly homogeneous by RP-HPLC and SDS-PAGE.4.4 mg of LAAO protein was purified per 500 mg with 0.88%recovery of venom protein.The total activity obtained was 297.81 U,and the specific activity was 67.70 U/mg.This purified sample was subjected to MALDI-TOF mass fingerprinting analysis,and these identified sequences covered 33.5%of the protein sequence deduced from the mRNA of Bm-LAAO in the NCBI.Bm-LAAO is a glycosylated flavoprotein presented an apparent molecular weight of 62.5 kDa under reducing conditions in SDS-PAGE.Unlike other known SV-LAAOs which mainly displayed their maximum enzyme activity at 37℃,Bm-LAAO had an optimal temperature at 45℃ with an optimal pH at 8.4.Kinetics studies showed that the enzyme displayed a catalytic preference for hydrophobic L-amino acids and did not catalyze the oxidative deamination of D-amino acids.The best substrates were L-Met,L-Leu and L-Phe.The enzymatic activity decreased by 54.3%after deglycosylation suggesting that the glycosylation had great effects on LAAO activity.Storage temperature was also important for activity,and-80℃ was the relatively best temperature.A cDNA of 1551 bp was obtained,encoding a mature protein with 498 amino acid residues through PCR.A model based on sequence homology was automatically built using crystal structure of Gloydius halys venom LAAO as template to predict the three-dimensional structure of Bm-LAAO.MTT assay was used to detect the cytotoxicity effects of Bm-LAAO on tumor cells and normal cells viability,and the apoptosis-inducing effects of Bm-LAAO on the tumor cells were observed and determined by microscope and flow cytometric method.In the eleven kinds of cancer cells detected,it had the strongest effect on proliferation inhibition of MGC-823 gastric cancer cells,and the IC50 value was 314 ng/ml.The IC50 values of Bm-LAAO in BT474,MDA-MB-231 and MCF-7 were 378ng/ml,1213ng/ml and 1480ng/ml,respectively.However,the IC50 in MCF-10A human normal breast cells was 13,556 ng/ml,which was 35.86,11.18 and 9.16 times of that in above human breast cancer cells respectively.IC50 values indicated that Bm-LAAO exhibited significantly higher selective cytotoxic activity against cancer cells compared to normal cells.When cultured with Bm-LAAO and catalase,cell viability recovered by 75%,suggesting this biological effect may largely result from hydrogen peroxide produced during the oxidation reaction of the substrate.Flow cytometry method indicated Bm-LAAO presented apoptosis-inducing effect on the tumor cell lines MGC-803 and MCF-7 in a dose-dependent manner,whereas this effect was largely inhibited in the presence of catalase,indicating the apoptosis induced by LAAO may also be related to the produce of H2O2.In summary,the transcriptome library of B.Multicinctus was constructed and analyzed,and a kind of L-amino acid oxidase,named Bm-LAAO,was purified from the venom of B.Multicinctus.The antitumor effects and cell apoptosis-inducing activities of the enzyme were preliminarily studied.The mechanisms of action need further investigation,because better knowledge of the mechanisms will allow us to consider the use of Bm-LAAO as models for therapeutic agents.