Investigations on plant Protease Inhibitors and their Interactions with Helicoverpa armigera mid gut Cysteine and Metalloproteases for effective control of Herbivory

Pest control strategies against Helicoverpa armigera using protease inhibitors relied on gut protease profile of the latter larval stage of the insect which is predominated by serine proteases. Little is known about the gut protease profile of the early larval instars. Specific substrates and inhibitors were used to detect gut proteases active in the 3rd, 4th, 5th and late 5th instar larvae reared on artificial diet. Analysis of gut protease profiles of different instars revealed differential expression of protease activity in different instars. There is also a statistically significant variation in the specific activity of trypsin, chymotrypsin, cysteine, carboxypeptidase-A and aminopeptidase-N across instars. In general, specific activity increased from 3rd to 5th instar and then decreased at the onset of pupation in late 5th instar. Optimum pH for proteolytic activity was pH 12 for the 3rd, 4th and 5th instar gut extracts. Level of inhibitors against papain, carboxypeptidase-A and aminopeptidase-N were estimated in 25 host plants and 25 non-host plants of H. armigera and compared. The non-host plants exhibited a statistically significant higher levels of cysteine protease inhibitors (P<0.05) compared to the host plants. A carboxypeptidase-A inhibitor and a papain inhibitor were purified from two non-host plants Oxalis corniculata and Withania somnifera with molecular weights 17 ± 1.7 kDa and 18.2 ± 1.8 kDa respectively. The inhibitors are thermally stable up to 75oC and are also active across a pH range from 2.6 to 10.6. Both inhibitors showed non-competitive inhibition. The carboxypeptidase-A inhibitor has a Ki = 0.031 to 0.095 μM and the papain inhibitor has a Ki = 0.024 to 0.16 μM. Both inhibitors showed high inhibition of H. armigera gut carboxypeptidase and cysteine proteases. These inhibitors were used together with Bowman-Birk inhibitor to study their individual as well as combined effects on H. armigera larvae. Mortality rate was highest (40%) and larval periods extended longest (39.5 ± 8.7 Days) when all the three inhibitors were used together. However, inhibition of carboxypeptidase proved to be key considering the low effective concentration of inhibitor required (3 IU/g of diet). This knowledge will help in formulating cost effective strategies of H. armigera control. Bioassays using synthetic inhibitors also showed similar results. EDTANa, a metalloprotease inhibitor retarded larval growth and a combination of PMSF and EDTA resulted in greater inhibition of gut protease activity than when either inhibitor was used alone. Several physicochemical factors influence the stability of peptidaseinhibitor interactions. The contributions of these factors on protease-inhibitor complexation and their effects on protease inhibition were investigated using cocrystallized complexes. The average number of hydrogen bonds and hydrophobic interactions are 17 ± 9 and 17 ± 2.7 each. Hydrogen bonds and hydrophobic interactions are the more significant non-covalent interactions and their numbers are highly correlated to interface sizes. The average interface size between peptidase and inhibitors is 1158 ± 668 Å2. Interface size also have a significant positive correlation to free energy of solvation (ΔiG), which indicates that larger interface sizes are generally more energetically favored for complexation. Shape complementarity moderately correlated to the empirically determined inhibition constants (Ki) in serine protease-inhibitor complexes. In cysteine proteaseinhibitor complexes, interface sizes are significantly correlated to the Ki. CONTENTS Page No. List of Tables i-ii List of Figures iii-iv