Penetration of a Single Domain of Bacillus thuringiensis Cry1Ie-Domain I to a Lipid Membrane In vitro

Guo, Shuangshuang; Li, Jie; Chen, Zhen; He, Kanglai

doi:10.1016/s2095-3119(13)60589-4

Cited by 3 publications

(7 citation statements)

References 23 publications

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“…Domain I and domain III were dissolved in alkaline solution of Na 2 CO 3 as previously reported for domain I [ 30 ]. IE648 and domain I was purified as indicated in our previous report [ 14 , 30 ] and the pure samples were shown in Fig 1D and 1E . Domain II protein was dissolved using the solubilization solutions of the Inclusion Body Solubilization and Refolding Kit.…”

Section: Resultsmentioning

confidence: 99%

“…A Ni-agarose column was used to purify these protein fragments as described in Materials and Methods. Domain I was eluted with 100 mM imidazole as indicated in our previous report [ 30 ], domain II with Na 2 CO 3 buffer (pH 10.2) ( Fig 1F , lane 3), and domain III with 50 mM imidazole ( Fig 1G , lanes 5 and 6).…”

Section: Resultsmentioning

confidence: 99%

“…In this work we used a truncated version of cry1Ie gene, containing the first 648 amino acid residues of Cry1Ie, cloned in vector pET-21b (Novagen) and named pET1Ie-648 [ 30 ]. This recombinant plasmid was expressed in E .…”

Section: Methodsmentioning

confidence: 99%

“…coli BL21 (DE3) containing the corresponding recombinant plasmid, induced by 0.4 mM IPTG at 20°C for 20 h. The three domains were all expressed mostly as inclusions. Purification of domain I was described in a previous report [ 30 ]. The inclusion of domain III was dissolved by 50 mM Na 2 CO 3 , pH 10.2 as same as domain I. Domain II was dissolved by using the solubilization solutions of Inclusion Body Solubilization and Refolding Kit.…”

Section: Methodsmentioning

confidence: 99%

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Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer

et al. 2015

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The insecticidal IE648 toxin is a truncated Cry1Ie protein with increased toxicity against Asian corn borer (ACB). Cry toxins are pore-forming toxins that disrupt insect midgut cells to kill the larvae. However, the peritrophic membrane (PM) is an important barrier that Cry toxins must cross before binding to midgut cells. Previously, it was shown that Cry toxins are able to bind and accumulate in the PM of several lepidopteran insects. Binding of IE648 toxin to PM of ACB was previously reported and the goal of the current work was the identification of the binding region between Cry1Ie and the PM of ACB. Homologous competition binding assays showed that this interaction was specific. Heterologous competition binding assays performed with different fragments corresponding to domain I, domain II and domain III allowed us to identify that domain III participates in the interaction of IE648 with the PM. Specifically, peptide D3-L8 (corresponding to Cry1Ie toxin residues 607 to 616), located in an exposed loop region of domain III is probably involved in this interaction. Ligand blot assays show that IE648 interact with chitin and PM proteins with sizes of 30, 32 and 80 kDa. The fact that domain III interacts with proteins of similar molecular masses supports that this region of the toxin might be involved in PM interaction. These data provide for the first time the identification of domain III as a putative binding region between PM and 3D-Cry toxin.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer

et al. 2015

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“…Moreover, the sequence and structural alignments of Cry1Id with its closest Cry proteins’ homologues suggested that Cry1Id domain I can be involved in membrane penetration and pore formation (Dehury et al 2013 ). Similarly, domain I of Cry1Ie protein can insert into lipid monolayers, whereas maintaining this penetration needed all three domains (Guo et al 2014 ). These results besides our findings point to the importance of Cry1Ia domain I in toxicity to lepidopterans.…”

Section: Discussionmentioning

confidence: 99%

Activation of Bacillus thuringiensis Cry1I to a 50 kDa stable core impairs its full toxicity to Ostrinia nubilalis

Khorramnejad

Bel

Talaei-Hassanloui

et al. 2022

Appl Microbiol Biotechnol

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Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O. nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O. nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O. nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products. Key points • Processing Cry1I to a 50 kDa stable core impairs its full toxicity to O. nubilalis • Partially processed Cry1Ia protoxin retains the toxicity of protoxin vs O. nubilalis • Protoxin and its final processed forms compete for the same functional binding sites

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Possible Insecticidal Mechanism of Cry41-Related Toxin against Myzus persicae by Enhancing Cathepsin B Activity

Zhao

Zhang

et al. 2020

J. Agric. Food Chem.

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Cry toxins produced by Bacillus thuringiensis are well known for their high insecticidal activities against Lepidoptera, Diptera, and Coleoptera; however, their activities against Aphididae are very low. Recently, it has been reported that a Cry41-related toxin exhibited moderate activity against the aphid Myzus persicae, and thus, it is highly desirable to uncover its unique mechanism. In this paper, we report that Cathepsin B, calcium-transporting ATPase, and symbiotic bacterial-associated protein ATP-dependent-6-phosphofructokinase were pulled down from the homogenate of M. persicae as unique proteins that possibly bound to Cry41related toxin. Cathepsin B has been reported to cleave and inactivate antiapoptotic proteins and plays a role in caspase-initiated apoptotic cascades. In this study, Cathepsin B was expressed in Escherichia coli and purified, and in vitro interaction between recombinant Cathepsin B and Cry41-related toxin was demonstrated. Interestingly, we found that addition of Cry41-related toxin obviously enhanced Cathepsin B activity. We propose a model for the mechanism of Cry41-related toxin as follows: Cry41-related toxin enters the aphid cells and enhances Cathepsin B activity, resulting in acceleration of apoptosis of aphid cells.

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Penetration of a Single Domain of Bacillus thuringiensis Cry1Ie-Domain I to a Lipid Membrane In vitro

Cited by 3 publications

References 23 publications

Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer

Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer

Activation of Bacillus thuringiensis Cry1I to a 50 kDa stable core impairs its full toxicity to Ostrinia nubilalis

Possible Insecticidal Mechanism of Cry41-Related Toxin against Myzus persicae by Enhancing Cathepsin B Activity

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