Mapping the Epitope in Cadherin-like Receptors Involved inBacillus thuringiensis Cry1A Toxin Interaction Using Phage Display

Abstract

In susceptible lepidopteran insects, aminopeptidase N and cadherin-like proteins are the putative receptors for Bacillus thuringiensis (Bt) toxins. Using phage display, we identified a key epitope that is involved in toxin-receptor interaction. Three different scFv molecules that bind Cry1Ab toxin were obtained, and these scFv proteins have different amino acid sequences in the complementary determinant region 3 (CDR3). Binding analysis of these scFv molecules to different members of the Cry1A toxin family and to Escherichia coli clones expressing different Cry1A toxin domains showed that the three selected scFv molecules recognized only domain II. Heterologous binding competition of Cry1Ab toxin to midgut membrane vesicles from susceptible Manduca sexta larvae using the selected scFv molecules showed that scFv73 competed with Cry1Ab binding to the receptor. The calculated binding affinities (K d ) of scFv73 to Cry1Aa, Cry1Ab, and Cry1Ac toxins are in the range of 20 -51 nM. Sequence analysis showed this scFv73 molecule has a CDR3 significantly homologous to a region present in the cadherin-like protein from M. sexta (Bt-R 1 ), Bombyx mori (Bt-R 175 ), and Lymantria dispar. We demonstrated that peptides of 8 amino acids corresponding to the CDR3 from scFv73 or to the corresponding regions of Bt-R 1 or Bt-R 175 are also able to compete with the binding of Cry1Ab and Cry1Aa toxins to the Bt-R 1 or Bt-R 175 receptors. Finally, we showed that synthetic peptides homologous to Bt-R 1 and scFv73 CDR3 and the scFv73 antibody decreased the in vivo toxicity of Cry1Ab to M. sexta larvae. These results show that we have identified the amino acid region of Bt-R 1 and Bt-R 175 involved in Cry1A toxin interaction.

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

“…Cry1Ab crystals were purified by sucrose gradients and protoxin solubilized in 50 mM Na 2 CO 3 (pH 10.5), 0.2% b-mercaptoethanol at 37°C for 2 h [18]. The oligomeric and monomeric forms of Cry1Ab toxin were produced by incubating Cry protoxin for 1 h with scFv73 antibody in a mass ratio of 1:4 and digested with midgut juice (5%) for 1 h at 37°C.…”

The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

“…In addition the binding characteristics of this mutant was analyzed, showing that mutant R99E was able to bind to BBMV with similar high affinity as the wild type, since binding competition of biotinylatedCry1Ab toxin with unlabelled mutant protein was identical to that of the unlabelled wild type toxin [21]. Specifically the binding interaction with Bt-R 1 was not affected, since ligand blots and ELISA binding competition assay of the mutant R99E to a Bt-R 1 protein fragment that contains all the Cry1A binding sites characterized so far (CR7 to CR12 ) [14,15,19] showed that it bound to Bt-R 1 protein fragment with the same K D as the wild type Cry1Ab toxin [21]. It is important to mention that the procedure of ELISA competition assay permits the determination of true association rate constants in solution, and several reports have shown agreement in the determination of K D values by these assays and those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, fluorescence transfer or surface plasmon resonance) [11,12,17].…”

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

“…A synthetic human scFv library-selection of an scFv molecule that mimics a cadherin receptor Our first attempt to characterize the amino acid epitopes involved in the interaction of Cry toxins with their receptor molecules was with the lepidopteran insect Manduca sexta and the Cry1Ab toxin [10]. At that time, two M. sexta proteins that bind Cry1A toxins had been cloned and characterized, a 210 kDa cadherin protein known as Bt-R 1 and a 120 kDa glycosylphosphatidy-linositol (GPI) anchored aminopeptidase N (APN) [21,33].…”

“…For the panning procedure, we immobilized Cry1Ab toxin in immunotubes and after several panning rounds of selection we identified three scFv-phages that bound specifically to Cry1Ab toxin. Using ligand blots assays and by competing the binding of Cry1Ab toxin with M. sexta brush border membrane vesicles (BBMV) blotted proteins, we identified one scFv (scFv73) that competed the binding of Cry1Ab with the 210 kDa Bt-R 1 molecule but not with the 120 kDa APN [10]. Antibody scFv73 inhibited the toxicity of Cry1Ab toxin in bioassays.…”

“…Antibody scFv73 inhibited the toxicity of Cry1Ab toxin in bioassays. Sequence analysis of the CDR3 region of scFv73 led to the identification of a eight amino acid region ( 869 HITDTNNK 876 ) in cadherin repeat 7 of Bt-R 1 [10]. Synthetic peptides corresponding to the Bt-R 1 amino acid region or to scFv73 CDR3 competed the binding of Cry1Ab with Bt-R 1 [10].…”