Abstract
Celangulin V (CV) is a compound isolated from Celastrus angulatus Max that has a toxic activity against agricultural insect pests. CV can bind to subunits a, H, and B of the vacuolar ATPase (V-ATPase) in the midgut epithelial cells of insects. However, the mechanism of action of CV is still unclear. In this study, the soluble complex of the V-ATPase A subunit mutant TSCA which avoids the feedback inhibition by the hydrolysate ADP and V-ATPase B subunit were obtained and then purified using affinity chromatography. The H+K+-ATPase activity of the complex and the inhibitory activity of CV on ATP hydrolysis were determined. The results suggest that CV inhibits the ATP hydrolysis, resulting in an insecticidal effect. Additionally, the homology modeling of the AB complex and molecular docking results indicate that CV can competitively bind to the AB complex at the ATP binding site, which inhibits ATP hydrolysis. These findings suggest that the AB subunits complex is one of the potential targets for CV and is important for understanding the mechanism of interaction between CV and V-ATPase.
Highlights
Celastrus angulatus Maxim is China’s traditional insecticidal plant and is widely distributed in the Yellow River and Yangtze River basins [1]
Celangulin V (CV) is a natural product isolated from the plant and has been found to act on the midgut cells of the oriental armyworm larvae by immunoelectron microscopy [2,3]
Celangulin V is a representative compound of this class of natural products which predominantly acts on the plasma membrane and endomembrane system of insects’ intestinal midgut cells, causing perforation of the insectile midgut
Summary
Celastrus angulatus Maxim is China’s traditional insecticidal plant and is widely distributed in the Yellow River and Yangtze River basins [1]. An analysis of the digestive enzyme activity showed that there was no significant change in the activities of protease, amylase, or lipase in the midgut of poisoned insects, compared with normal insects This suggests that CV mainly acts on the plasma membrane of the midgut cell and its intima system [5]. The mechanism of how the binding of CV to the H subunit affects the holo V-ATPase activities, including proton pump activity and ATP hydrolysis, is not yet clear. A subunit of Thermococcus thermophilus (T. thermophilus) revealed that the mutation of serine 232 to alanine and threonine 235 to serine of the A subunit can reduce ADP feedback inhibition without affecting ATP hydrolysis activity [11]. The complex of subunits A and B can be reconstructed in vitro, and its ATP hydrolysis activity can be measured. The ATP hydrolysis activity of the TSCA–B complex and the inhibition of CV to ATP hydrolysis were measured, and the binding of CV was further explored and proved by computer-aided molecular simulation
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