Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say)

Rausell, Carolina; García-Robles, Inmaculada; Sánchez, Jorge; Muñóz-Garay, Carlos; Martínez‐Ramírez, Amparo C.; Real, M. Dolores; Bravo, Alejandra

doi:10.1016/j.bbamem.2003.11.004

Cited by 82 publications

(77 citation statements)

References 36 publications

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“…4). These data are in agreement with previous reports which showed that the oligomeric structure of Cry1Ab toxin is membrane insertioncompetent and demonstrates higher pore-formation activity than the monomeric toxin [5,7,9]. It is important to mention that the increased pore-formation activity of the oligomeric structure of the Cry1A toxins could also be due to the increased affinity of the oligomers to the membrane and to the APN receptor [4] and not only because the monomeric structure of these toxins is less active.…”

Section: Cry1casupporting

confidence: 92%

“…PMSF was added to a final 1 mM concentration to stop proteolysis. The oligomeric structure of these toxins was produced by incubation of 100 lg of each Cry protoxin for 15 min at 25°C with 10 lg of BBMV isolated from M. sexta midgut tissue in the absence of protease inhibitors; the reaction was stopped with 1 mM PMSF, and samples were centrifuged (20 min at 12,000 x g) [5,9]. Protein concentration was determined in the supernatant by Bradford assay using bovine serum albumin as standard.…”

Section: Preparation Of Insecticidal Crystal Proteinsmentioning

confidence: 99%

“…The Cry1Ab pre-pore is soluble at low concentrations, displays new hydrophobic surfaces with respect to the monomeric form and is highly resistant to heat denaturalization [8]. However, the formation of Cry oligomeric structures has been demonstrated only for Cry1Ab and Cry3 toxins [5,7,9]. In this work, we analyzed the oligomer formation of different Cry1 toxins and correlated the presence of their oligomeric structures with high pore-formation activity.…”

Section: Introductionmentioning

confidence: 99%

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Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

et al. 2006

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Abstract. The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K + permeability. A fluorometric assay performed with 3,3¢-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K + -selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

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Section: Cry1casupporting

confidence: 92%

Section: Preparation Of Insecticidal Crystal Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

et al. 2006

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“…Cry1Ca [18,30], Cry1Da, Cry1Ea, Cry1Fa [30]; coleopteran specific, Cry3Aa, Cry3Ba, Cry3Ca [37], and dipteran specific, Cry4Ba [26] and Cry11Aa (Pérez and Bravo, unpublished results). Protein samples containing Cry1Ab pre-pore oligomer were more toxic to M. sexta larvae than protein samples containing only monomeric Cry1Ab toxin [13].…”

Section: Final Remarksmentioning

confidence: 89%

“…After proteolytic activation of Cry1A protoxin by insect midgut proteases, the activated toxin binds to the Bt-R 1 receptor, and this interaction facilitates additional cleavage of the N-terminal end of the toxin, eliminating helix α-1, resulting in the formation of a pre-pore oligomeric structure [13]. The oligomeric structure has been observed in several Cry toxins [1,13,18,26,30,33,36,37,40]. The pre-pore binds to a second receptor, the GPI-anchored aminopeptidase, due to its higher affinity to this receptor [3], facilitating the insertion of the oligomer into membrane lipid rafts and forming pores [3,46].…”

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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IDENTIFICATION OF A CALMODULIN‐BINDING SITE WITHIN THE DOMAIN I OF BACILLUS THURINGIENSISCry3Aa TOXIN

Ochoa-Campuzano

Sánchez

García-Robles

et al. 2012

Arch Insect Biochem Physiol

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Bacillus thuringiensis Cry3Aa toxin is a coleopteran specific toxin highly active against Colorado Potato Beetle (CPB).We have recently shown thatCry3Aa toxin is proteolytically cleaved by CPBmidgut membrane associated metalloproteases and that this cleavage is inhibited by ADAMmetalloprotease inhibitors. In the present study, we investigated whether the Cry3Aa toxin is a calmodulin (CaM) binding protein, as it is the case of several different ADAMshedding substrates. In pull‐down assays using agarose beads conjugated with CaM, we demonstrated that Cry3Aa toxin specifically binds to CaMin a calcium‐independent manner. Furthermore, we used gel shift assays and 1H NMRspectra to demonstrate that CaMbinds to a 16‐amino acid synthetic peptide corresponding to residues N256‐V271 within the domain I of Cry3Aa toxin. Finally, to investigate whether CaM has any effect on Cry3Aa toxin CPBmidgut membrane associated proteolysis, cleavage assays were performed in the presence of the CaM‐specific inhibitor trifluoperazine. We showed that trifluoperazine significantly increased Cry3Aa toxin proteolysis and also decreased Cry3Aa larval toxicity.

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Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say)

Cited by 82 publications

References 36 publications

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

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

IDENTIFICATION OF A CALMODULIN‐BINDING SITE WITHIN THE DOMAIN I OF BACILLUS THURINGIENSISCry3Aa TOXIN

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