Specific Epitopes of Domains II and III of Bacillus thuringiensis Cry1Ab Toxin Involved in the Sequential Interaction with Cadherin and Aminopeptidase-N Receptors in Manduca sexta

Gómez, Isabel; Arenas, Iván; Benitez, Itzel; Miranda-Ríos, Juan; Becerril, Baltazar; Grande, Ricardo; Almagro, Juan C.; Bravo, Alejandra; Soberón, Mário

doi:10.1074/jbc.m604721200

Cited by 92 publications

(77 citation statements)

References 33 publications

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“…Then, toxin overlay binding competition assays in the presence of synthetic peptides were used to show that domain II loop 3 of Cry1Ab is an important epitope for interaction with the Bt-R 1 receptor. This interaction between the toxin and the anti-domain II loop 3 scFv antibody also promoted the formation of the pre-pore oligomer as a previously observed with an anti-domain II loop 2 scFv73 antibody [14]. The selected anti-loop 3 antibody (scFvL3-3) molecule lowered the toxicity of Cry1Ab to M. sexta larvae in bioassays.…”

Section: An Immune Rabbit Scfv Library-mapping the Binding Epitopes Osupporting

confidence: 69%

“…Finally, we found that scFvL3-3 and scFv73 antibodies preferentially recognized the monomeric toxin rather than the pre-pore structure, suggesting a conformational change in the domain II loop regions is recognized by these antibodies [14]. Overall, these results indicated that the first interaction of Cry1Ab with Bt-R 1 through domain II loop regions, promotes the formation of the pre-pore oligomeric structure with a subtle change in the structure of these exposed domain II loops; then the pre-pore interacts with APN through a different domain III region (β16) (Fig.…”

Section: An Immune Rabbit Scfv Library-mapping the Binding Epitopes Omentioning

confidence: 81%

“…Therefore we decided to construct a new scFv phage display library that would be enriched in scFv molecules that recognize Cry1Ab toxin. For this purpose, phage display libraries constructed from a host immunized with the target antigen favors selection of high-affinity, specific antibodies generated during immunization process [14]. A Cry1Ab-immune M13 phage-repertoire was constructed using antibody gene transcripts of bone marrow and spleen from a rabbit immunized with Cry1Ab toxin [14].…”

Section: An Immune Rabbit Scfv Library-mapping the Binding Epitopes Omentioning

confidence: 99%

“…The β16 and β22 from domain III play an important role in this interaction since a scFv-antibody that recognizes these regions (scFvM22) inhibited the interaction of the pre-pore oligomeric structure with APN and lowered the toxicity of Cry1Ab to M. sexta larvae in bioassays [14]. scFvM22 did not promote the in vitro formation of a pre-pore oligomeric structure.…”

Section: An Immune Rabbit Scfv Library-mapping the Binding Epitopes Omentioning

confidence: 99%

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

et al. 2008

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Phage display is an in vitro method for selecting polypeptides with desired properties from a large collections of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display singlechain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.

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Section: An Immune Rabbit Scfv Library-mapping the Binding Epitopes Osupporting

confidence: 69%

Section: An Immune Rabbit Scfv Library-mapping the Binding Epitopes Omentioning

confidence: 81%

Section: An Immune Rabbit Scfv Library-mapping the Binding Epitopes Omentioning

confidence: 99%

Section: An Immune Rabbit Scfv Library-mapping the Binding Epitopes Omentioning

confidence: 99%

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

et al. 2008

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“…Also, a scFv antibody that bound the domain III ␤16-␤22 region competed the binding of Cry1Ab to APN (10). However, the role of these binding regions in the interaction of Cry1Ab oligomer with APN has not been analyzed, nor whether the same regions are involved in the interaction with ALP.…”

Section: Analysis Of Binding Of Cry1abmentioning

confidence: 99%

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

Arenas

Bravo

Soberón

et al. 2010

Journal of Biological Chemistry

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Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins, a prepore oligomeric intermediate is formed after interaction with cadherin receptor. The Cry1A oligomer then interacts with glycosylphosphatidylinositol-anchored receptors. Two Manduca sexta glycosylphosphatidylinositol-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP), have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, because it was found in the first larval instars, whereas APN is induced later after the third larval instar. The binding of Cry1Ab oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity (apparent K d ‫؍‬ 0.6 nM), whereas the monomer showed weaker binding (apparent K d ‫؍‬ 101.6 and 267.3 nM for APN and ALP, respectively). Several Cry1Ab nontoxic mutants located in the exposed loop 2 of domain II or in ␤-16 of domain III were affected in binding to APN and ALP, depending on their oligomeric state. In particular monomers of the nontoxic domain III, the L511A mutant did not bind ALP but retained APN binding, suggesting that initial interaction with ALP is critical for toxicity. Our data suggest that APN and ALP fulfill two roles. First APN and ALP are initial receptors promoting the localization of toxin monomers in the midgut microvilli before interaction with cadherin. Then APN and ALP function as secondary receptors mediating oligomer insertion into the membrane. However, the expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action because Cry toxins are highly effective against the neonate larvae that is the target for pest control programs.The Cry toxins produced by Bacillus thuringiensis are used worldwide as effective biological control agents for many species of insects including agricultural and forest pests and several vectors of human and animal diseases. Its insecticidal property results from crystalline inclusions produced during sporulation that are formed by ␦-endotoxins known as Cry toxins (1).The Cry protein family is composed of more than 54 groups, among which the three-domain Cry family forms the major group, having members that show toxicity to different insect orders and to nematodes. The crystal structure has been solved for six three-domain Cry toxins and one protoxin that display different insect specificities (2). The activated Cry toxins are globular molecules consisting of a bundle of seven ␣-helices (domain I), a three-␤-sheet prism (domain II), and a ␤-sandwich (domain III) (3, 4). The N-terminal domain I is involved in oligomer formation, membrane insertion, and pore formation (5-8). Domain II and III are involved in the recogn...

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Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin‐like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins

Endo

Kobayashi²,

Hoshino³

et al. 2014

MicrobiologyOpen

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Directed evolution of a Cry1Aa toxin using phage display and biopanning was performed to generate an increased binding affinity to the Bombyx mori cadherin-like receptor (BtR175). Three mutant toxins (371WGLA374, 371WPHH374, 371WRPQ37425) with 16-, 16-, and 50-fold higher binding affinities, respectively, for BtR175 were selected from a phage library containing toxins with mutations in domain II loop 2. However, the observed toxicities of the three mutants against B. mori larvae and cultured cells expressing the BtR175 toxin-binding region did not increase, suggesting that increased binding affinity to cadherins does not contribute to the insecticidal activity. Affinity maturation of a Cry toxin to a receptor via directed evolution was relatively simple to achieve, and seems to have potential for generating a toxin with increased insecticidal activity.

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Specific Epitopes of Domains II and III of Bacillus thuringiensis Cry1Ab Toxin Involved in the Sequential Interaction with Cadherin and Aminopeptidase-N Receptors in Manduca sexta

Cited by 92 publications

References 33 publications

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

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

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

Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin‐like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins

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