Identification And Validation Of The Target Protein Of Insecticidal Periplocoside Compounds

Periploca sepium Bunge,a member of the Asclepiadaceae family,is also known as the beiwujia,xiangjiapi or yangjiaotao,and distribute in most regions of China.In China,as a herbal material,has extensive applications in the treatment of human diseases,such as anti-inflammation,cardiac,anticancer,etc.In recent years,the active constituents of glycoside were found in root bark extractive of P.sepium in our laboratory,such as periplocoside A,P,T,and NW.Further studies showed that the site of action of periplocoside compounds is the midgut of Mythimna separata larvae,and its most typical symptom is abdominal swelling.So far,except Bt toxins and Celangulin,only P.sepium is another insecticidal plant that acts on insect midgut in domestic and foreign studies.But,the poisoning symptom of insects caused by P.sepium is completely different from Bt toxins and Celangulin.Up to now,the insecticidal mechanism of the periplocosides on insects is still unclear.In this study,the target proteins of periplocoside compounds were identified through quantitative differences proteomics technology using M.separata as model insect.Identified putative target proteins were studied systematically through the technology of molecular biology,bioinformatics,etc.The main results are as follows:1.Based on quantitative differential proteomic technique（2-DE）,the differentially expressed proteins in BBMV from the M.separata midguts was obtained after PSP treatment.Eleven differentially expressed proteins（DEPs）were identified by the matrix assisted laser desorption ionization flight of time/flight of time mass spectrometry（MALDI-TOF/TOF）analysis,and respectively are transferrin,protein disulfide isomerase precursor,calreticulin,tropomyosin-2 isoform 4,V-type ATPase A subunit,diverged serine protease,trypsin-like protease,39S ribosomal protein L46,mitochondrial-like,farnesoic acid O-methyltransferase,aminopeptidase N1 and heat shock protein cognate 72.The cellular functions of these DEPs were analyzed by bioinformatics analysis.Based on the functions of these DEPs,the toxicological symtoms and histopathological results,we speculated that V-ATPase A subunit and aminopeptidase may be the putative initiation target of the insecticidal periplocoside compounds.2.The toxicity of six periplocoside compounds to 3^th M.separata larvae was measured by toxic leaf method,and results showed that PSP and PST showed obvious insecticidal activity,PSA,PSD and PSF revealed weak insecticidal activity and PSE had no insecticidal activity.The LC₅₀ value of PSA,PSD,PSF,PSP and PST at 24 h respectively were 28.68,22.44,21.31,1.60 and 1.23 mg/mL.The activity of aminopeptidase,alkaline phosphatase and V-ATPase in BBMV was determined by using six periplocoside compounds.The results indicated that six periplocosides have no significant inhibitory effect on aminopeptidase and alkaline phosphatase.The activity of V-ATPase was significantly inhibited by PSP and PST with a concentration dependence manner.Therefore,we preliminarily speculated that the initial binding site of insecticidal periplocoside compounds is located on the V-ATPase A subunit.3.The protein expression technology of Bac-to-Bac/BmNPV was applied to acquire the soluble expression protein of V-ATPase A subunit in M.separata midguts.The results showed that the eukaryotic expression system constructed by pBacPAK9 as expression vector is successful and the proteins were induced by this system is soluble in supernatant of cell lysis.Determination results of fluorescence quenching spectrophotometry showed that fluorescence quenching type of wild-type recombinant protein and periplocoside compounds belongs to the static quenching caused by interaction.The binding constant K_a of PSA,PSD,PSE,PSF,PSP and PST and wild-type recombinant protein respectively were 0.81×10⁵,1.42×10⁵,0.50×10⁵,1.62×10⁵,6.95×10⁵ and 4.4×10⁵ L·mol^-1.ITC curve showed that the K_a of PSA,PSD,PSE,PSF,PSP and PST and wild-type recombinant protein respectively were 1.86×10⁵,2.48×10⁵,1.35×10⁵,2.67×10⁵,9.25×10⁵ and 8.27×10⁵ L·mol^-1.These results indicated that insecticidal activity of periplocoside compounds has a significant correlation with the binding ability of wild type recombinant protein and periplocoside compounds.4.With 3D structure（PDB ID:5bw9.1）of Saccharomyces cerevisiae V-ATPase A subunit as template,homologous modeling of V-ATPase A subunit from M.separata midguts was conducted through the Swiss Model software.Based on the reliable homologous model,active binding cavities of V-ATPase A subunit were predicted and defined by SYBYL software,and two potential active binding cavities（Cavity A and B）were found.Then,the periplocoside compounds were docked flexibly with two binding cavities respectively,and we predicted that eight amino acid residues（Lys85、Arg171、Glu199、Gln207、Val208、Pro226、Gln242 and Arg400）were the important binding sites to combine periplocoside compounds.5.Two groups of mutations were designed for the eight important amino acid residues in active sites of V-ATPase A subunit.One of them was the alanine mutation,and respectively were K85A、R171A、E199A、Q207A、V208A、P226A、Q242A and R400A.The other group,according to the nature of the amino acids（acid,basic,polarity,charge,etc.）,was substituted for 8 amino acids and respectively were K85D、R171D、E199K、Q207T、V208G、P226S、Q242T and R400D.Based on eukaryotic expression system of Bac-to-Bac/BmNPV,16mutants were constructed successfully.All mutants were soluble in supernatant of the cell lysis.Ni-NTA His-Bind Resin column was used to obtain purified mutant proteins.6.The binding constants of PSP and PST with 8 alimonine mutants or 8 missense mutants were studied based on fluorescence quenching spectra and isothermal titration calorimetry（ITC）technology.Site-directed mutagenesis results indicated that the binding sites of periplocoside active compounds exist in V-ATPase A subunit of the M.separata midgut,and K85 and R171 in active cavity A may be the binding site for periplocoside insecticidal compounds.