Binding of Phylogenetically Distant Bacillus thuringiensis Cry Toxins to a Bombyx mori Aminopeptidase N Suggests Importance of Cry Toxin's Conserved Structure in Receptor Binding

1999 ◽  
Vol 39 (1) ◽  
pp. 14-20 ◽  
Author(s):  
Ayaka Shinkawa ◽  
Katsuro Yaoi ◽  
Tomoyuki Kadotani ◽  
Morikazu Imamura ◽  
Nobuo Koizumi ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Bombyx Mori ◽  
Receptor Binding ◽  
Aminopeptidase N ◽  
Cry Toxins

scholarly journals Aminopeptidase N from Bombyx Mori as a Candidate for the Receptor of Bacillus Thuringiensis Cry1Aa Toxin

1997 ◽  
Vol 246 (3) ◽  
pp. 652-657 ◽  
Author(s):  
Katsuro Yaoi ◽  
Tomoyuki Kadotani ◽  
Hisanaga Kuwana ◽  
Ayaka Shinkawa ◽  
Tsuyoshi Takahashi ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Bombyx Mori ◽  
Aminopeptidase N

scholarly journals Location of the Bombyx mori Aminopeptidase N Type 1 Binding Site on Bacillus thuringiensis Cry1Aa Toxin

2005 ◽  
Vol 71 (7) ◽  
pp. 3966-3977 ◽  
Author(s):  
Shogo Atsumi ◽  
Eri Mizuno ◽  
Hirotaka Hara ◽  
Kazuko Nakanishi ◽  
Madoka Kitami ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Bacillus Thuringiensis ◽  
Binding Site ◽  
Bombyx Mori ◽  
Epitope Mapping ◽  
Aminopeptidase N ◽  
Cysteine Substitution ◽  
Fab Fragments ◽  
Domain Iii

ABSTRACT We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.

scholarly journals Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Blanca I. García-Gómez ◽  
Sayra N. Cano ◽  
Erika E. Zagal ◽  
Edgar Dantán-Gonzalez ◽  
Alejandra Bravo ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Receptor Binding ◽  
Insecticidal Activity ◽  
Insect Pests ◽  
Biotechnological Applications ◽  
Cry Proteins ◽  
Cry Toxins ◽  
Content Type ◽  
Hsp90 Chaperone ◽  
Protease Degradation

ABSTRACT Bacillus thuringiensis Cry proteins are pore-forming insecticidal toxins with specificity against different crop pests and insect vectors of human diseases. Previous work suggested that the insect host Hsp90 chaperone could be involved in Cry toxin action. Here, we show that the interaction of Cry toxins with insect Hsp90 constitutes a positive loop to enhance the performance of these toxins. Plutella xylostella Hsp90 (PxHsp90) greatly enhanced Cry1Ab or Cry1Ac toxicity when fed together to P. xylostella larvae and also in the less susceptible Spodoptera frugiperda larvae. PxHsp90 bound Cry1Ab and Cry1Ac protoxins in an ATP- and chaperone activity-dependent interaction. The chaperone Hsp90 participates in the correct folding of proteins and may suppress mutations of some client proteins, and we show here that PxHsp90 recovered the toxicity of the Cry1AbG439D protoxin affected in receptor binding, in contrast to the Cry1AbR99E or Cry1AbE129K mutant, affected in oligomerization or membrane insertion, respectively, which showed a slight toxicity improvement. Specifically, PxHsp90 enhanced the binding of Cry1AbG439D protoxin to the cadherin receptor. Furthermore, PxHsp90 protected Cry1A protoxins from degradation by insect midgut proteases. Our data show that PxHsp90 assists Cry1A proteins by enhancing their binding to the receptor and by protecting Cry protoxin from gut protease degradation. Finally, we show that the insect cochaperone protein PxHsp70 also increases the toxicity of Cry1Ac in P. xylostella larvae, in contrast to a bacterial GroEL chaperone, which had a marginal effect, indicating that the use of insect chaperones along with Cry toxins could have important biotechnological applications for the improvement of Cry insecticidal activity, resulting in effective control of insect pests. IMPORTANCE Bacillus thuringiensis took advantage of important insect cellular proteins, such as chaperones, involved in maintaining protein homeostasis, to enhance its insecticidal activity. This constitutes a positive loop where the concentrations of Hsp90 and Hsp70 in the gut lumen are likely to increase as midgut cells burst due to Cry1A pore formation action. Hsp90 protects Cry1A protoxin from degradation and enhances receptor binding, resulting in increased toxicity. The effect of insect chaperones on Cry toxicity could have important biotechnological applications to enhance the toxicity of Cry proteins to insect pests, especially those that show low susceptibility to these toxins.

cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori

1999 ◽  
Vol 1444 (1) ◽  
pp. 131-137 ◽  
Author(s):  
Katsuro Yaoi ◽  
Kazuko Nakanishi ◽  
Tomoyuki Kadotani ◽  
Morikazu Imamura ◽  
Nobuo Koizumi ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Bombyx Mori ◽  
Cdna Cloning ◽  
Aminopeptidase N ◽  
Cloning And Expression ◽  
Toxin Binding

scholarly journals ATP-Binding Cassette Subfamily a Member 2 Is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, But Not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins

Toxins ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 104 ◽  
Author(s):  
Xiaoyi Li ◽  
Kazuhisa Miyamoto ◽  
Yoko Takasu ◽  
Sanae Wada ◽  
Tetsuya Iizuka ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Bombyx Mori ◽  
Cell Swelling ◽  
Cross Resistance ◽  
Atp Binding ◽  
Transcription Activator ◽  
Cry Toxins ◽  
Toxin Resistance ◽  
Functional Receptor ◽  
Atp Binding Cassette

Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the ABCA2 sequence in Bombyx mori using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239T cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1Ca, Cry1Da, Cry1Fa or Cry9Aa toxins.

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