N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin

Burton, Sarah L; Ellar, David J.; Li, Jade; Derbyshire, D.J.

doi:10.1006/jmbi.1999.2649

Cited by 184 publications

(170 citation statements)

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“…Thus we have shown that, similar to earlier results for binding of Cry1Ac to aminopeptidase N of M. sexta [14], this region of domain III is involved in receptor binding. Whereas the mutations in Cry1Ac had no apparent effect on toxicity against M. sexta in bioassays [14], the mutations reported here destroy activity against S. exigua. Activity against M. sexta is not affected, indicating that decreased stability of the mutant protein is not a likely explanation.…”

Section: Discussionsupporting

confidence: 90%

“…The location of a tryptophan residue, which was shown previously to also be involved in specificity for S. exigua, is also indicated [12]. Thus we have shown that, similar to earlier results for binding of Cry1Ac to aminopeptidase N of M. sexta [14], this region of domain III is involved in receptor binding. Whereas the mutations in Cry1Ac had no apparent effect on toxicity against M. sexta in bioassays [14], the mutations reported here destroy activity against S. exigua.…”

Section: Discussionsupporting

confidence: 89%

“…For instance, hybrid toxins containing domains I and II of Cry1Ea or Cry1Ab and domain III of Cry1Ca (G27 and H04 respectively) showed an increase in activity against Spodoptera exigua compared with Cry1Ab or Cry1Ea [10]. Furthermore, domain III was shown to be responsible for the N-acetylgalactosamine-inhibited binding of Cry1Ac to M. sexta aminopeptidase N [13,14].…”

Section: Introductionmentioning

confidence: 99%

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Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

Herrero

González‐Cabrera

Ferré

et al. 2004

Biochemical Journal

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Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541-544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis toxins.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 89%

Section: Introductionmentioning

confidence: 99%

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Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

Herrero

González‐Cabrera

Ferré

et al. 2004

Biochemical Journal

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“…In M. sexta, two proteins bind Cry1A toxins, a 120-kDa APN (13,43) and a 210-kDa cadherin-like protein (Bt-R 1 ) (15). Expression of these putative receptor proteins in heterologous cell lines did not render the cells sensitive to Cry1A toxins (11,15,46).…”

Section: Discussionmentioning

confidence: 99%

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

Gómez¹,

Oltean²,

Gill³

et al. 2001

Journal of Biological Chemistry

134

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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.

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“…Importantly, another GPI-anchored protein, an alkaline phosphatase, has been recognized as a Cry1Ac receptor in Heliothis virescens and M. sexta [22,23]. Alkaline phosphatase could also participate in facilitating the pre-pore membrane insertion into lipid rafts, explaining why Cry1Ac toxin mutations that were severely affected in APN binding are still active against M. sexta larvae [6,20]. In the pore forming model, the pre-pore oligomer is responsible for initiating cell death [3].…”

Section: Models Of the Mode Of Action Of Cry Toxins In Lepidopteran Imentioning

confidence: 99%

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

et al. 2008

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The insecticidal Cry toxins from Bacillus thuringiensis bacteria are pore-forming toxins that lyse midgut epithelial cells in insects. We have previously proposed that they form pre-pore oligomeric intermediates before membrane insertion.For formation of these oligomers coiled-coil structures are important, and helix α-3 from Cry toxins could form coiled-coils. Our data shows that different mutations in helix α-3 are affected in pore formation and toxicity. Mutants affected in toxicity bind Bt-R 1 receptor with a similar K D as the wild type toxin but do not form oligomers nor induce pore formation in planar lipid bilayers, indicating that the pre-pore oligomer is an obligate intermediate in the intoxication of Cry1Ab toxin and that interaction of monomeric Cry1Ab with Bt-R 1 is not enough to kill susceptible larvae.

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N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin

Cited by 184 publications

References 48 publications

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

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

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

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