Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N

ABSTRACT In order to test our hypothesis that Bacillus thuringiensis delta-endotoxin Cry1Ca domain III functions as a determinant of specificity for Spodoptera exigua, regardless of the origins of domains I and II, we have constructed by cloning and in vivo recombination a collection of hybrid proteins containing domains I and II of various Cry1 toxins combined with domain III of Cry1Ca. Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ea, and Cry1Fa all become more active against S. exigua when their domain III is replaced by (part of) that of Cry1Ca. This result shows that domain III of Cry1Ca is an important and versatile determinant of S. exigua specificity. The toxicity of the hybrids varied by a factor of 40, indicating that domain I and/or II modulate the activity as well. Cry1Da-Cry1Ca hybrids were an exception in that they were not significantly active against S. exigua or Manduca sexta, whereas both parental proteins were highly toxic. Incidentally, in a Cry1Ba-Cry1Ca hybrid, Cry1Ca domain III can also strongly increase toxicity for M. sexta.

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Ser170 of Bacillus thuringiensis Cry1Ab δ-endotoxin becomes anchored in a hydrophobic moiety upon insertion of this protein into Manduca sexta brush border membranes

BMC Biochemistry ◽

10.1186/1471-2091-10-25 ◽

2009 ◽

Vol 10 (1) ◽

pp. 25 ◽

Cited By ~ 7

Author(s):

Oscar Alzate ◽

Craig F Hemann ◽

Cristina Osorio ◽

Russ Hille ◽

Donald H Dean

Keyword(s):

Bacillus Thuringiensis ◽

Manduca Sexta ◽

Brush Border ◽

Brush Border Membranes ◽

Hydrophobic Moiety

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Bacillus thuringiensis delta-endotoxin and larval Manduca sexta midgut brush-border membrane vesicles act synergistically to cause very large increases in the conductance of planar lipid bilayers.

Journal of Experimental Biology ◽

10.1242/jeb.198.1.91 ◽

1995 ◽

Vol 198 (1) ◽

pp. 91-96 ◽

Cited By ~ 1

Author(s):

F G Martin ◽

M G Wolfersberger

Keyword(s):

Bacillus Thuringiensis ◽

Lipid Bilayers ◽

Manduca Sexta ◽

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Phospholipid Bilayers ◽

Brush Border Membrane Vesicles ◽

Delta Endotoxin ◽

Manduca Sexta Larvae

Brush-border membrane vesicles prepared from midguts of Manduca sexta larvae were incorporated into planar phospholipid bilayers. Addition of Bacillus thuringiensis delta-endotoxin to the buffered salt solutions bathing these bilayers resulted in large irreversible increases in conductance. At pH 9.6, the smallest toxin-dependent increase in bilayer conductance observed was 13 nS. Similar conductance increases were never observed in the absence of delta-endotoxin or in delta-endotoxin-treated bilayers not containing components of insect brush-border membranes.

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The Receptor for Bacillus Thuringiensis Cry 1Ac Delta-endotoxin in the Brush Border Membrane of the Lepidopteran Helicoverpa armigera is Aminopeptidase N

Journal of Biological Sciences ◽

10.3923/jbs.2001.782.784 ◽

2001 ◽

Vol 1 (8) ◽

pp. 782-784 ◽

Cited By ~ 5

Author(s):

Kausar Malik . ◽

Tariq Mahmood . ◽

S. Riazuddin .

Keyword(s):

Bacillus Thuringiensis ◽

Helicoverpa Armigera ◽

Brush Border ◽

Brush Border Membrane ◽

Aminopeptidase N ◽

Delta Endotoxin ◽

Helicoverpa Armígera

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The Bacillus thuringiensis Cry1Ac toxin-induced permeability change in Manduca sexta midgut brush border membrane vesicles proceeds by more than one mechanism

Journal of Cell Science ◽

10.1242/jcs.110.24.3099 ◽

1997 ◽

Vol 110 (24) ◽

pp. 3099-3104

Author(s):

J. Carroll ◽

M.G. Wolfersberger ◽

D.J. Ellar

Keyword(s):

Bacillus Thuringiensis ◽

Manduca Sexta ◽

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Aminopeptidase N ◽

Insecticidal Toxin ◽

Cry1ac Toxin ◽

Posterior Midgut

Aminopeptidase N purified from whole Manduca sexta midgut binds the Cry1Ac insecticidal toxin from Bacillus thuringiensis and this binding is inhibited by N-acetylgalactosamine (GalNAc). We have examined the membrane permeabilising activity of the Cry1Ac toxin using brush border membrane vesicles (BBMV) prepared from the anterior (A-BBMV) and posterior (P-BBMV) subregions of the M. sexta midgut. A toxin mixing assay demonstrated a faster rate of toxin activity on P-BBMV than on A-BBMV. In the presence of GalNAc this rapid activity on P-BBMV was reduced to the rate seen with A-BBMV. GalNAc had no effect on the rate of A-BBMV permeabilisation by Cry1Ac. Aminopeptidase N assays of A- and P-BBMV demonstrated that this Cry1Ac binding protein is concentrated in the posterior midgut region of M. sexta. It therefore appears that there are two mechanisms by which Cry1Ac permeabilises the M. sexta midgut membrane: a GalNAc-sensitive mechanism restricted to the posterior midgut region, probably involving aminopeptidase N binding, and a previously undetected mechanism found in both the posterior and anterior regions.

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Denaturation of Either Manduca sexta Aminopeptidase N or Bacillus thuringiensis Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.68.5.2106-2112.2002 ◽

2002 ◽

Vol 68 (5) ◽

pp. 2106-2112 ◽

Cited By ~ 21

Author(s):

Anu Daniel ◽

Sreedhara Sangadala ◽

Donald H. Dean ◽

Michael J. Adang

Keyword(s):

Bacillus Thuringiensis ◽

Manduca Sexta ◽

Brush Border ◽

Binding Proteins ◽

Membrane Vesicles ◽

Aminopeptidase N ◽

Toxin Binding ◽

Cry Toxin ◽

Domain I ◽

Ligand Blot

ABSTRACT The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with 125I-labeled Cry1Ac, Cry1Ac mutant 509QNR-AAA511 (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both 125I-Cry1Ac and 125I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots 125I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, 125I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. 125I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured 125I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) 125I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.

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