Research On The Interaction Of Destruxin A With Four Proteins In Bombyx Mori

Destruxin A(DA),a cyclic hexadepsipeptid toxin produced by Metarhizium anisopliae,has prominent anti-immunity and insecticidal effect including contact toxicity and antifeedant against insect.This study is mainly focus on DA and intended to find the binding(target)proteins in Lepidoptera model Bombyx mori for making mechanism of destruxins clearly.On the basis of drug affinity responsive target stability(DARTS),previously,our group isolated and identified hundreds of proteins.This study selected and verify that four proteins might interact or bind to DA,the mainly results are listed as follows.(1)Molecular dockingThe binding free energies of DA with BmPiwi,BmTudor-sn and BmAGO2 were-10.8577,-11.002 and-9.1947 kcal/mol respectively.DA might form hydrogen bond with BmPiwi,BmTudor-sn and BmAGO2 proteins.(2)Heterologous expressionThe fragments of BmPiwi,BmTudor-sn and BmAGO2 proteins were cloned by PCR,and then were ligated into pEASY and pFast-Bac1 vectors respectively.BmPiwi,BmTudor-sn and BmAGO2 proteins were successfully expressed in Bac-to-Bac baculovirus expression system in Spodoptera frugiperda Sf9 cell line.After purified by Ni-NTA affinity chromatography,high degree of concentration and purity recombinant proteins were collected.(3)Verification of the interaction of DA with proteins by Bio-Layer Interferometry(BLI)DA interacts or binds with BmTudor-sn protein.Basing on the interaction graph and kinetic parameter of DA and BmTudor-sn,there are positive correlation of concentration-dependent response on DA at 125?mol/L,250?mol/L and 500?mol/L while KD(M)value was491?mol/L.But the interaction is relative weak.Only in high concentration,can DA interact with BmPiwi protein.DA cannot interact with BmAGO2 or interact with it in critical point.(4)Verification of the interaction of DA with proteins by Insect two Hybrid(I2H)DA influences the interaction of BmPiwi,BmTudor-sn and BmAGO2 with its interacted proteins in vivo.After treated with concentration gradient at 2,0.2,0.02 and0.002 ?g/mL of DA,luminescent value of Sf9 cell line,transfected vector of I2 H,is inverse correlation with DA treatment which suggested that DA indeed hinder interaction of three protein pairs.In relative high concentration(2,0.2 ?g/mL)of DA,DA greatly affect interaction of protein pairs,and this effect relative weak at 0.02 ?g/mL of DA.Meanwhile,this effect is extremely weak at 0.002 ?g/mL of DA treatment.In addition,in relative low concentration(0.02 and 0.002 ?g/mL),DA hinder the interaction of pair BmTudor-sn-BmAGO1 much more.Meanwhile,after treated with DA at 0.02 ?g/mL in Sf9,Cys614 Ala,Lys649Ala and Lys875 Ala have significant differential in lumen value compared with BmPiwi and it's suggested that there are key animo acid sites between the interaction of DA with BmPiwi.In BmTudor-sn,the difference amino acid are Ser701 Ala,Leu704Ala and Leu708 Ala in 0.2?g/mL while Ser707 Ala and Leu704 Ala in 0.02 ?g/mL of DA.The experiment of BmAGO2 is in progress.Apparently,under different dosage of DA,differential mutants are not completely equivalent which suggested that the interplay of DA with target or binding proteins is dosage dependent.In addition,we conducted I2 H to compare the intercion of cyclophilin of silkworm(BmPPI)and human(HsPPIA)with DA and immunosuppressor CsA and FK506 and results showed that DA can interact with BmPPI and HsPPIA which indicated that DA indeed hava immunosuppression function.