Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Abstract

Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R 1 ) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R 1 promotes cleavage of the amino-terminal end, including helix ␣-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R 1 fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.Bacillus thuringiensis is a bacterium that produces crystalline inclusions formed by insecticidal proteins, called Cry toxins, during the sporulation phase of growth. Cry toxins are toxic to different insect orders as well as to other invertebrates, such as nematodes, mites, and protozoa (1). Cry toxins have been used worldwide in the control of insect pests in agriculture, either as transgenic crops or as spray formulations.The molecular mechanism proposed to describe the action of Cry1A toxins, which are active against different lepidopteran insect species, involves several steps. After larval ingestion of the crystalline inclusions, these are solubilized in midgut lumen and activated by proteases releasing a toxic 65-kDa fragment that binds, in a sequential manner, with at least two receptors located in midgut microvilli. The first interaction occurs with cadherin protein (Bt-R 1 2 in the case of Manduca sexta). This interaction promotes further proteolytic processing of the N-terminal end, including helix ␣-1 of the toxin, resulting in the formation of a prepore oligomeric structure (2). The oligomer has higher affinity to secondary receptors, which are anchored by glycosylphosphatidylinositol, such as aminopeptidase-N (APN) or alkaline phosphatase in the case of M. sexta or Heliothis virescens, respectively (3, 4). Glycosylphosphatidylinositol-anchored receptors are located in specific membrane regions called lipid rafts, where the oligomer inserts into the membrane-forming pores, disrupting the osmotic equilibrium and leading to cell death (1, 5). Although this mechanism of action is g...

Role of Alkaline Phosphatase from Manduca sexta in the Mechanism of Action of Bacillus thuringiensis Cry1Ab Toxin

“…Interestingly, characterization of nontoxic domain II loop 3 mutants revealed an opposite phenotype when compared with the loop 2 mutations regarding the binding of monomeric or oligomeric Cry1Ab to APN. Domain II loop 3 mutants affected monomer binding to APN in contrast to the binding of the oligomeric structure that was not affected (38). Here we show that the monomeric structures of domain II loop 2 mutations had no effect on their binding to APN or ALP, whereas oligomer binding to both GPI-anchored molecules was greatly affected.…”

“…Recently we proposed that APN fulfills two roles in the mode of action of Cry1Ab: first as a low affinity and highly abundant binding site that locates monomeric toxin in the vicinity of the microvilli before cadherin binding and second as a secondary high affinity receptor that mediates insertion of the oligomeric Cry1Ab structure into the membrane, suggesting a ping-pong binding mechanism (38). Interestingly, characterization of nontoxic domain II loop 3 mutants revealed an opposite phenotype when compared with the loop 2 mutations regarding the binding of monomeric or oligomeric Cry1Ab to APN.…”

“…Cry1Ab-binding Regions Involved in APN and ALP Interaction-Domain II loop 2 and 3 mutants were previously shown to be affected in APN binding (27,37,38). Also, a scFv antibody that bound the domain III ␤16-␤22 region competed the binding of Cry1Ab to APN (10).…”

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

“…A number of functional Cry toxin receptors have been identified, including proteins and glycolipids (Pigott and Ellar, 2007), and it has been proposed that they may interact sequentially with the toxin (Pacheco et al, 2009). Proteinprotein and protein-carbohydrate interactions have been proposed between Cry toxin domains and receptors, and recognition of unique carbohydrate structures present in invertebrates has been proposed to explain specificity of Cry toxins to insects and nematodes but not to vertebrates (Griffitts et al, 2003).…”

Efficacy of genetically modified Bt toxins against insects with different genetic mechanisms of resistance

“…14,15 The signaling model suggests that after protease-activated monomeric toxins bind to cadherin, initiation of a magnesium-dependent signaling pathway causes cell death. 14,15 In contrast, a recent version of the pore formation model 21 proposes the following sequence of events: proteaseactivated monomers bind to glycosylphosphatidylinositol (GPI)-anchored proteins, including aminopeptidases and alkaline phosphatases. This interaction promotes binding of toxin monomers to cadherin, which facilitates protease cleavage of the N terminus of the toxin, including helix α-1 of domain I, inducing oligomerization of the toxin.…”