Identification, characterization, cloning and expression of a Heliothis virescens 110 kDa aminopeptidase N that binds Bacillus thuringiensis Cry1Ac and Cry1Fa [delta]-endotoxins
Banks, David Jay
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The initial goal of this research was to determine whether Cry1Ac and Cry1Fa ä -endotoxins from the insecticidal bacterium Bacillus thuringiensis (Bt) shared binding proteins in the lepidopteran insect pest species, Heliothis virescens. The identification of molecules that bind Bt toxins is important, because they have been demonstrated to catalyze pore formation in vitro. Further, these binding proteins have been predicted to catalyze toxin mode of action in vivo. I demonstrated that Cry1Ac and Cry1Fa toxins shared 110, 120 and 170 kDa binding proteins. This evidence suggests that the cross-resistance between Cry1Ac and Cry1Fa in the Cry1Ac resistant H. virescens strain YHD2 is likely related to toxin recognition of common binding proteins.|During the early phase of my research, I discovered that while the 120 and 170 kDa binding proteins had previously been identified as aminopeptidase N (APN) molecules, the 110 kDa binding protein had been described, but remained unidentified. I subsequently identified the 110 kDa binding protein as an APN using N-terminal amino acid sequence analysis, APN antibody, and PCR of an internal region of total H. virescens cDNA. With the cooperation of Juan Luis Jurat Fuentes, we established that toxin recognition of the 110 kDa APN was not mediated by N-acetylgalactosamine (GalNAc). This finding was important, because previous research had indicated that the Cry1Ac domain III mutant 509 QNR 511 -AAA that contained an ablated GalNAc binding pocket remained toxic to H. virescens. This suggsted that Cry1Ac toxin mode of action was not entirely GalNAc dependent, as previous research had indicated. Further, these results indicated that Cry1Ac toxicity was likely mediated by a molecule that displayed GalNAc independent toxin recognition. Since the 110 kDa APN fulfilled this criteria, we predicted that it likely mediated toxicity in vivo. In order to address this prediction, I then cloned the 110 kDa APN gene, and expressed it in Drosophila S2 cells. Microscopy results showed that rhodamine-labeled Cry1Ac toxin bound to some 110 kDa APN transfected cells, but not to cells transcfected with vector-alone. However, microscopy and flow cytometry results both showed that cells expressing the 110 kDa APN remained viable after exposure to toxin, measured as a function of membrane integrity. This indicates that the 110 kDa APN does not mediate cytotoxicity when expressed in S2 cells. Alternatively, it remains possible that 110 kDa APN mediated toxin mode of action may not be dependent on pore formation, but rather on intercellular phenomena that remain unidentified.