Identification And Validation Of The Target Protein Of Insecticidal Dihydroagarofuran Sesquiterpene Polylesters Compounds

Discovery of novel targets is the basis for innovative development of new pesticides. Identification of the target proteins for naturally occurring insecticidal lead compounds can provide new targets and ideas for design and development of novel pesticides. A series of insecticidal dihydroagarofuran sesquiterpene polyolesters compounds which have insecticidal activity have been isolated and identified from the root bark of Celastrus angulatus, but their mode of action is still unknown. In order to provide new targets and ideas for developing novel pesticides that can act on digestive system, this dissertation mainly focuses on identification and validation of the target protein of these insecticidal dihydroagarofuran sesquiterpene polyolesters compounds using oriental armyworm(Mythimna separata Walker) as a model insect. The main methods and results are as follows:1. Affinity chromatography and LC/Q-TOF-MS were combined to isolate and identify the Celangulin V binding proteins from the larval midgut brush border membrane vesicle(BBMV) extracts of M. separata. Celangulin V, a representative of the insecticidal dihydroagarofuran sesquiterpene polyolesters compounds, was first modified at its C-6 postiton with an aminoacetic acid and then coupled to the CNBr activated Sepharosed 4B via the added aminoacetic acid group to form the affinity matrix for separation of the Celangulin V bingding proteins from the larval midgut BBMV extracts of M. separata. A total of eleven Celangulin V binding proteins were isolated through affinity chromatography. They were identified by LC/Q-TOF-MS as follows: Zinc finger protein, Transmembrane protein 1, Thioredoxin peroxidase(TPx), Glyceraldehyde 3-phosphate dehydrogenase(GAPDH), SUMO E3 ligase Ran BP2, actin, APN3, V-ATPase subunit a, B and H. Based on the functions of these binding proteins and the toxicological symtoms of M. separata larval caused by Celangulin V, we speculated that the putative target proteins of the insecticidal dihydroagarogarofun sesquiterpene polylesters compounds could be V-ATPase and/or APN3.2. Force-feeding bioassay was conducted to test the insecticidal activity of the 12 tested dihydroagarofuran sesquiterpene polyolesters compounds against the 5th instar larvae of M. separata. Among the 12 compounds, Celangulin V-6-N-methylisatoic anhydride ester, Celangulin V-6-isobutyric acid ester, Celanglin V-6-ketone, NW57 and NW62 didn’t show stomach poisoning activity against the 5th instar larvae of M. separata even at the concentration of 668.45 μg/g. But Wilforine, Celangulin V, Celangluin V-6-α-aminopropanoic acid ester, Celangulin V-6-aminoacetic acid ester, NW69, NW03 and NW70 were all toxic to the 5th instar larvae of M.separata and had a LD50 value of 0.597, 679.479, 33.605, 767.811, 309.559, 379.286 and 86.271 μg/g respectively. Enzymatic assays found that none of the 12 dihydroagarofuran sesquiterpene polyolesters compounds inhibited the larval midgut APN activity of M. separata. In contrast, the 12 dihydroagarofuran sesquiterpene polyolesters compounds differentially repressed the larval midgut V-ATPase activity of M.separata. The inhibition rates of V-ATPase activity by Celangulin V, Celangluin V-6-α-aminopropanoic acid ester, Celangulin V-6-aminoacetic acid ester, Celangulin V-6-Isobutyric acid ester, Celanglin V-6-ketone, Celangulin V-6-N-Methylisatoic anhydride ester, NW69, NW70, NW03, NW57, NW62 and Wilforine were 14.19%, 19.83%, 12.72%, 12.30%, 6.72%, 24.04%, 12.63%, 29.64%, 33.98%, 1.9%, 6.90% and 54.78% respectively. Correlation analysis revealed a positive relationship between the larval stomach toxicities of the 12 compound and their inhibition rates of V-ATPases activity. These results suggest that V-ATPase is the target enzyme of the insectidal dihydroagarofuran sesquiterpene polyolesters compounds.3. Due to the function importance of V-ATPase subunit H, the full-lengted c DNA sequence of this subunit was cloned by RACE technology from the larval midgut of M.separata.The full-length c DNA sequence of V-ATPase subunit H is 1807 bp long in total, containing a 5’-UTR of 136 bp, an open reading frame(ORF) of 1425 bp and a 3’-UTR of 246 bp. The 1425 bp ORF encodes a protein of 474 amino acids, with a calculated molecular weight of 54.8 KDa and an estimated isoelectric point of 6.26.4. The V-ATPase subunit H was heterologously expressed in E. coli and purified. Microscale thermophoreis(MST) was employed to measure the binding abilities of the 12 insecticidal dihydroagarofuran sesquiterpene polyolesters compounds with the purified V-ATPase subunit. Celangulin V-6-isobutyric acid ester, Celanglin V-6-ketone, Celangulin V-6-N-Methylisatoic anhydride ester, NW57 and NW62 were unable to bind to the purified V-ATPase subunit H. In contrast, Celangulin V, Celangluin V-6-α-aminopropanoic acid ester, Celangulin V-6-aminoacetic acid ester, NW69, NW70, NW03 and Wilforine were acapable of binding to the purified V-ATPase subunit H, with a dissociation constant(KD value, which was calculated through the binding curves of each compound) of 253, 144, 156, 164, 186, 203 and 59 μM respectively. The KD value of these compounds correlated well with their inhibiton rates of V-ATPases activity and their larval stomach toxicities. These results indicate that V-ATPase subunit H is the target protein of the insecticidal dihydroagarofuran sesquiterpene polyolesters compounds.5. The three-dimensional(3D) structure of V-ATPase subunit H of yeast(Saccharomyces cerevisiae) was used as the template to construct the 3D model of the V-ATPase subunit H from midgut of M. separata larvae by using Homology Modeling of Discovery Studio software. A total of 18 potentical binding sites in the 3D model of M. separata V-ATPase subunit H were predicted by receptor cavities and by using the binding site of yeast V-ATPase sunbunit H. Molecular docking of all the 12 tested insecticidal dihydroagarofuran sesquiterpene polyolesters compounds into each of the 18 potential binding sites generated a total of 18 docking scores per compound. Correlation analysis of the resulted docking scores of the 12 compouds and their binding KD values with the purified M.separata V-ATPase subunit H suggested that Site 17, which is located at the C-terminal of H subunit, was the most possible active site for the insecticidal dihydroagarofuran sesequiterpene polyolesters compounds. The amino acid residues of Site 17 contain Cys, His, Trp, Ser, Lys, Ala, Glu, Gly and so on.