Monday, December 10, 2001 - 1:00 PM
0307

Isolation, identification and cloning of a novel Bacillus thuringiensis toxin-binding aminopeptidase from Manduca sexta

Anu Daniel1, Gang Hua2, David J. Banks1, and Michael J. Adang3. (1) University of Georgia, Department of Biochemistry and Molecular Biology, #432 Biological Sciences Building, University of Georgia, Athens, GA, (2) University of Georgia, Department of Entomology, #432 Biological Sciences Building, University of Georgia, Athens, GA, (3) University of Georgia, Departments of Biochemistry and Molecular Biology and Entomology, #432 Biological Sciences Building, University of Georgia, Athens, GA

Bacillus thuringiensis produces crystal (Cry) proteins during the sporulation phase of the life cycle. Cry toxins have been exploited commercially for their insecticidal properties. Cry toxins exhibit high levels of specificity in their interaction with target host making toxins key components in crop protection strategies. Upon ingestion by susceptible insects toxins are solubilized and activated in insect midgut. The activated toxin binds to brush border membrane proteins, aggregates and subsequently inserts into the membrane leading to pore formation. The toxin-induced pores create ion imbalance in cells causing cell lyses and insect mortality. Two classes of proteins in insect midgut including aminopeptidase Ns (APN) and cadherin-like proteins are shown to function as receptors for Cry toxins. A number of APNs from different insects have been identified as Cry toxin receptors. In M. sexta, a 120-kDa APN functions as a receptor for Cry1Ac. Recent studies revealed that mutations in domain III of Cry1Ac eliminated binding of Cry1Ac to 120-kDa APN. However, the mutant toxins retained toxicity to M. sexta. One such Cry1Ac-domain III mutant toxin, 509QNR513-AAA was used to isolate toxin-binding proteins from M. sexta brush border membrane vesicles. A 110-kDa protein was isolated by toxin-affinity chromatography and N-terminal amino acid sequence was TNLDEPKYR. The N-terminal sequence of 110-kDa protein shared ~87% sequence homology with a Heliothis virescens APN, indicating that the M. sexta 110-kDa protein was an APN. A cDNA encoding the APN gene was cloned from M. sexta midgut cDNAs. Sequence analysis revealed the 110-kDa APN was a novel M. sexta APN and shared ~72% identity with the H. virescens APN, ~68%identity with a Bombyx mori APN and ~70% identity with Helicoverpa punctigera APN2. Expression of the APN gene in insect cells is being attempted.

Species 1: Lepidoptera Sphingidae Manduca sexta (tobacco hornworm)
Keywords: Bacillus thuringiensis, aminopeptidase N

The ESA 2001 Annual Meeting - 2001: An Entomological Odyssey of ESA