Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Ac toxin

1999 ◽  
Vol 29 (6) ◽  
pp. 527-535 ◽  
Author(s):  
Karen J Garner ◽  
Shiv Hiremath ◽  
Kirsten Lehtoma ◽  
Algimantas P Valaitis
Keyword(s):  
Bacillus Thuringiensis ◽  
Gypsy Moth ◽  
Complete Sequence ◽  
Aminopeptidase N ◽  
Sequence Characterization ◽  
Cry1ac Toxin

scholarly journals Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment

1998 ◽  
Vol 333 (3) ◽  
pp. 677-683 ◽  
Author(s):  
Matthew A. COOPER ◽  
Joe CARROLL ◽  
Emma R. TRAVIS ◽  
Dudley H. WILLIAMS ◽  
David J. ELLAR
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Model Membrane ◽  
Aminopeptidase N ◽  
Lipid Monolayer ◽  
Affinity Constant ◽  
Second Phase ◽  
High Affinity ◽  
Cry1ac Toxin ◽  
Initial Binding

The Bacillus thuringiensis Cry1Ac δ-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein–protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles.

scholarly journals Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment

1998 ◽  
Vol 335 (3) ◽  
pp. 711-711 ◽  
Author(s):  
M. A. COOPER ◽  
J. CARROLL ◽  
E. R. TRAVIS ◽  
D. H. WILLIAMS ◽  
D. J. ELLAR
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Model Membrane ◽  
Aminopeptidase N ◽  
Cry1ac Toxin

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

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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