Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide

Tersch, M A Von; Slatin, Stephen L.; Kulesza, Caroline; English, Leigh

doi:10.1128/aem.60.10.3711-3717.1994

“…The Cyt toxin family is distinct from the Cry family in a number of ways [1,3]. Only the Cyt toxins cause the cytolysis of a variety of eukaryotic cells and red blood cells (RBCs) in itro, even though both activated Cyt and Cry toxins can form pores in lipid bilayers [4,5].…”

Section: Introductionmentioning

confidence: 99%

Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis

Promdonkoy

¹

,

Ellar

²

2000

Biochemical Journal

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To investigate the membrane pore structure of Cyt2Aa1 toxin from Bacillus thuringiensis, 14 single-cysteine substitutions of the toxin were constructed. Five of these mutants (L172C, V186C, L189C, E214C and L220C) yielded characteristic products when processed by proteinase K; other mutants were degraded by this enzyme. Mutants that yielded characteristic proteolysed products and wild-type toxin were labelled with polarity-sensitive acrylodan (6-acryloyl-2-dimethylaminonaphthalene) at the thiol group of cysteine residues. A green-blue shift in the emission spectra was observed with all labelled toxins on transfer from an aqueous solution into a solution containing membranes or liposomes from red blood cells. These results suggested that the label moved into the hydrophobic environment of the membrane or became buried within hydrophobic regions of the protein oligomers. Digestion of membrane-bound labelled toxin with proteinase K did not cause a significant decrease in emission intensity from any of the labelled mutants. This suggests that L172C, V186C, L189C, E214C and L220C are inserted into the membrane and are therefore protected from proteolysis. In contrast, a marked decrease in emission intensity was observed when membrane-bound labelled wild-type toxin was digested with proteinase K. This suggests that Cys-19 does not insert into the membrane. Fluorimetric analysis of delipidated pore complexes suggests that L172C, V186C, L189C and E214C point towards the lipid in the membrane, whereas L220C is either within the hydrophobic environment of the protein oligomers or exposed to the membrane lipids. Most of the Cys-19 from wild-type molecules is enclosed within the hydrophobic pockets of the protein oligomers.

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“…The Cyt toxin family is distinct from the Cry family in a number of ways [1,3]. Only the Cyt toxins cause the cytolysis of a variety of eukaryotic cells and red blood cells (RBCs) in itro, even though both activated Cyt and Cry toxins can form pores in lipid bilayers [4,5].…”

Section: Introductionmentioning

confidence: 99%

Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis

Promdonkoy

¹

2000

View full text Add to dashboard Cite

To investigate the membrane pore structure of Cyt2Aa1 toxin from Bacillus thuringiensis, 14 single-cysteine substitutions of the toxin were constructed. Five of these mutants (L172C, V186C, L189C, E214C and L220C) yielded characteristic products when processed by proteinase K; other mutants were degraded by this enzyme. Mutants that yielded characteristic proteolysed products and wild-type toxin were labelled with polarity-sensitive acrylodan (6-acryloyl-2-dimethylaminonaphthalene) at the thiol group of cysteine residues. A green-blue shift in the emission spectra was observed with all labelled toxins on transfer from an aqueous solution into a solution containing membranes or liposomes from red blood cells. These results suggested that the label moved into the hydrophobic environment of the membrane or became buried within hydrophobic regions of the protein oligomers. Digestion of membrane-bound labelled toxin with proteinase K did not cause a significant decrease in emission intensity from any of the labelled mutants. This suggests that L172C, V186C, L189C, E214C and L220C are inserted into the membrane and are therefore protected from proteolysis. In contrast, a marked decrease in emission intensity was observed when membrane-bound labelled wild-type toxin was digested with proteinase K. This suggests that Cys-19 does not insert into the membrane. Fluorimetric analysis of delipidated pore complexes suggests that L172C, V186C, L189C and E214C point towards the lipid in the membrane, whereas L220C is either within the hydrophobic environment of the protein oligomers or exposed to the membrane lipids. Most of the Cys-19 from wild-type molecules is enclosed within the hydrophobic pockets of the protein oligomers.

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“…However, the three domains together make the insertion more stable, as observed for IE648, albeit with a lower critical insertion pressure than that of domain I alone. Von Tersch M. A. et al demonstrated that domain I of Cry3B2 is sufficient to induce ion leakage from phopholipid vesicles, but the efflux produced by the native toxin was greater than that produced by the domain I peptide (Von Tersch et al 1994). Walters et al also reported that the putative -helix bundle from domain I of Cry1Ac formed channels (Walters et al 1993).…”

Section: Discussionmentioning

confidence: 99%

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

Guo

¹

,

Li

²

,

Chen

³

et al. 2014

Journal of Integrative Agriculture

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Domain I of the activated Crystal protein from Bacillus thuringiensis has a seven α-helix bundle structure, which is responsible for membrane channel formation in its insecticidal mechanism. Cry1Ie is toxic to Asian corn borer and plays important roles in insect biological control. The domain I from Cry1Ie has been expressed and purified in its normal conformation, as embedded in the full length homologous toxin structure. The membrane insertion ability of this single domain was compared with the full length homologous toxin using a monolayer insertion experiment. The results indicated that the Cry1Ie-domain I had the ability to insert into the lipid monolayer, and this ability is greater than that of the IE648 toxin. However, the state of insertion is not stable and remains for only a short period of time. The Cry1Ie-domain I plays no role in receptor binding as it had a nonspecific binding with the brush border membrane vesicles of the Asian corn borer.

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Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide

Cited by 63 publications

References 33 publications

Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis

Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis

Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis

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

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