The C-terminal protoxin region of Bacillus thuringiensis Cry1Ab toxin has a functional role in binding to GPI-anchored receptors in the insect midgut.

Bacillus thuringiensis Cry toxins are used worldwide for controlling insects. Cry1Ab is produced as a 130-kDa protoxin that is activated by proteolytic removal of an inert 500 amino-acid-long C-terminal region, enabling the activated toxin to bind to insect midgut receptor proteins, leading to its membrane insertion and pore formation. It has been proposed that the C-terminal region is only involved in toxin crystallization, but its role in receptor binding is undefined. Here we show that the C-terminal region of Cry1Ab protoxin provides additional binding sites for alkaline phosphatase (ALP) and aminopeptidase N (APN) insect receptors. ELISA, ligand blot, surface plasmon resonance, and pulldown assays revealed that the Cry1Ab C-terminal region binds to both ALP and APN but not to cadherin. Thus, the C-terminal region provided a higher binding affinity of the protoxin to the gut membrane that correlated with higher toxicity of protoxin than activated toxin. Moreover, Cry1Ab domain II loop 2 or 3 mutations reduced binding of the protoxin to cadherin but not to ALP or APN, supporting the idea that protoxins have additional binding sites. These results imply that two different regions mediate the binding of Cry1Ab protoxin to membrane receptors, one located in domain II-III of the toxin and another in its C-terminal region, suggesting an active role of the C-terminal protoxin fragment in the mode of action of Cry toxins. These results suggest that future manipulations of the C-terminal protoxin region could alter the specificity and increase the toxicity of B. thuringiensis proteins.

Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta.

Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC-MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.

Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum.

BACKGROUND: IslA4 is a truncated single domain protein derived from the inulosucrase IslA, which is a multidomain fructosyltransferase produced by Leuconostoc citreum. IslA4 can synthesize high molecular weight inulin from sucrose, with a residual sucrose hydrolytic activity. IslA4 has been reported to retain the product specificity of the multidomain enzyme. RESULTS: Screening experiments to evaluate the influence of the reactions conditions, especially the sucrose and enzyme concentrations, on IslA4 product specificity revealed that high sucrose concentrations shifted the specificity of the reaction towards fructooligosaccharides (FOS) synthesis, which almost eliminated inulin synthesis and led to a considerable reduction in sucrose hydrolysis. Reactions with low IslA4 activity and a high sucrose activity allowed for high levels of FOS synthesis, where 70% sucrose was used for transfer reactions, with 65% corresponding to transfructosylation for the synthesis of FOS. CONCLUSIONS: Domain truncation together with the selection of the appropriate reaction conditions resulted in the synthesis of various FOS, which were produced as the main transferase products of inulosucrase (IslA4). These results therefore demonstrate that bacterial fructosyltransferase could be used for the synthesis of inulin-type FOS.