scholarly journals Bacillus thuringiensis Cry4Ba Insecticidal ToxinExploits Leu615 in Its C-terminal Domain to Interact with a Target Receptor—Aedes aegypti Membrane-Bound Alkaline Phosphatase

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 553
Author(s):  
Anon Thammasittirong ◽  
Sutticha Na-Ranong Thammasittirong ◽  
Chompounoot Imtong ◽  
Sathapat Charoenjotivadhanakul ◽  
Somsri Sakdee ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Receptor Binding ◽  
Sandwich Elisa ◽  
Full Length ◽  
Terminal Domain ◽  
Binding Analysis ◽  
Membrane Bound ◽  
Larval Toxicity

In addition to the receptor-binding domain (DII), the C-terminal domain (DIII) of three-domain Cry insecticidal d-endotoxins from Bacillus thuringiensis has been implicated in target insect specificity, yet its precise mechanistic role remains unclear. Here, the 21kDa high-purity isolated DIII fragment derived from the Cry4Ba mosquito-specific toxin was achieved via optimized preparative FPLC, allowing direct rendering analyses for binding characteristics toward its target receptor—Aedes aegypti membrane-bound alkaline phosphatase (Aa-mALP). Binding analysis via dotblotting revealed that the Cry4Ba-DIII truncate was capable of specific binding to nitrocellulose-bound Aa-mALP, with a binding signal comparable to its 65kDa Cry4Ba-R203Q full-length toxin. Further determination of binding affinity via sandwich ELISA revealed that Cry4Ba-DIII exhibited a rather weak binding to Aa-mALP with a dissociation constant (Kd) of ≈1.1 ×10−7 M as compared with the full-length toxin. Intermolecular docking between the Cry4Ba-R203Q active toxin and Aa-mALP suggested that four Cry4Ba-DIII residues, i.e., Glu522, Asn552, Asn576, and Leu615, are potentially involved in such toxin–receptor interactions. Ala substitutions of each residue (E522A, N552A, N576A and L615A) revealed that only the L615A mutant displayed a drastic decrease in biotoxicity against A. aegypti larvae. Additional binding analysis revealed that the L615A-impaired toxin also exhibited a reduction in binding capability to the surface-immobilized Aa-mALP receptor, while two bio-inactive DII-mutant toxins, Y332A and F364A, which almost entirely lost their biotoxicity, apparently retained a higher degree of binding activity. Altogether, our data disclose a functional importance of the C-terminal domain of Cry4Ba for serving as a potential receptor-binding moiety in which DIII-Leu615 could conceivably be exploited for the binding to Aa-mALP, highlighting its contribution to toxin interactions with such a target receptor in mediating larval toxicity.

The C-terminal domain of the toxic fragment of a Bacillus thuringiensis crystal protein determines receptor binding

1991 ◽  
Vol 5 (11) ◽  
pp. 2799-2806 ◽  
Author(s):  
G. Honée ◽  
D. Convents ◽  
J. Rie ◽  
S. Jansens ◽  
M. Peferoen ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Receptor Binding ◽  
Crystal Protein ◽  
Terminal Domain

scholarly journals Aedes aegypti Membrane-Bound Alkaline Phosphatase Expressed in Escherichia coli Retains High-Affinity Binding for Bacillus thuringiensis Cry4Ba Toxin

2011 ◽  
Vol 77 (19) ◽  
pp. 6836-6840 ◽  
Author(s):  
Anon Thammasittirong ◽  
Manasave Dechklar ◽  
Somphob Leetachewa ◽  
Kusol Pootanakit ◽  
Chanan Angsuthanasombat
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Nitrilotriacetic Acid ◽  
Affinity Column ◽  
Dot Blot ◽  
Content Type ◽  
Binding Characteristics ◽  
High Affinity

ABSTRACTGlycosylphosphatidylinositol-linked alkaline phosphatase (GPI-ALP) from the epithelial membrane of the larval midgut ofAedes aegyptiwas previously identified as a functional receptor of theBacillus thuringiensisCry4Ba toxin. Here, heterologous expression inEscherichia coliof the cloned ALP, lacking the secretion signal and GPI attachment sequences, and assessment of its binding characteristics were further investigated. The 54-kDa His tag-fused ALP overexpressed as an inclusion body was soluble when phosphate buffer (pH 7.5) was supplemented with 8 M urea. After renaturation in a nickel-nitrilotriacetic acid (Ni-NTA) affinity column, the refolded ALP protein was able to retain its phosphatase activity. This refolded ALP also showed binding to the 65-kDa activated Cry4Ba toxin under nondenaturing (dot blot) conditions. Quantitative binding analysis using a quartz crystal microbalance revealed that the purified ALP immobilized on a gold electrode was bound by the Cry4Ba toxin in a stoichiometry of approximately 1:2 and with high affinity (dissociation constant [Kd] of ∼14 nM) which is comparable to that calculated from kinetic parameters (dissociation rate constant [koff]/binding constant [kon]). Altogether, the data presented here of theE. coli-expressed ALP fromA. aegyptiretaining high-affinity toxin binding support our notion that glycosylation of this receptor is not required for binding to its counterpart toxin, Cry4Ba.

scholarly journals C-Type Lectin-20 Interacts with ALP1 Receptor to Reduce Cry Toxicity in Aedes aegypti

Toxins ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 390 ◽  
Author(s):  
Khadija Batool ◽  
Intikhab Alam ◽  
Guohui Zhao ◽  
Junxiang Wang ◽  
Jin Xu ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Survival Rate ◽  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Western Blot ◽  
Brush Border Membrane ◽  
Mosquito Control ◽  
Membrane Vesicles ◽  
Cry Toxins ◽  
Far Western Blot

Aedes aegypti is a crucial vector for human diseases, such as yellow fever, dengue, chikungunya, and Zika viruses. Today, a major challenge throughout the globe is the insufficient availability of antiviral drugs and vaccines against arboviruses, and toxins produced by Bacillus thuringiensis (Bt) are still used as biological agents for mosquito control. The use of Cry toxins to kill insects mainly depends on the interaction between Cry toxins and important toxin receptors, such as alkaline phosphatase (ALP). In this study, we investigated the function of A. aegypti C-type lectin-20 (CTL-20) in the tolerance of Cry toxins. We showed that recombinant CTL-20 protein interacted with both Cry11Aa and ALP1 by the Far-Western blot and ELISA methods, and CTL-20 bound to A. aegypti larval brush border membrane vesicles (BBMVs). Binding affinity of CTL-20 to ALP1 was higher than that of Cry11Aa to ALP1. Furthermore, the survival rate of A. aegypti larvae fed with Cry11Aa toxin mixed with recombinant CTL-20 fusion protein was significantly increased compared with that of the control larvae fed with Cry11Aa mixed with thioredoxin. Our novel results suggest that midgut proteins like CTLs may interfere with interactions between Cry toxins and toxin receptors by binding to both Cry toxins and receptors to alter Cry toxicity.

scholarly journals Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins

2012 ◽  
Vol 42 (9) ◽  
pp. 683-689 ◽  
Author(s):  
Alan I. Jiménez ◽  
Esmeralda Z. Reyes ◽  
Angeles Cancino-Rodezno ◽  
Leidy P. Bedoya-Pérez ◽  
Gustavo G. Caballero-Flores ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Functional Receptor

scholarly journals Reduced Levels of Membrane-Bound Alkaline Phosphatase Are Common to Lepidopteran Strains Resistant to Cry Toxins from Bacillus thuringiensis

PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e17606 ◽  
Author(s):  
Juan Luis Jurat-Fuentes ◽  
Lohitash Karumbaiah ◽  
Siva Rama Krishna Jakka ◽  
Changming Ning ◽  
Chenxi Liu ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Thuringiensis ◽  
Cry Toxins ◽  
Membrane Bound

scholarly journals The C-Terminal Domain of the Bacillus thuringiensis Cry4Ba Mosquito-Specific Toxin Serves as a Potential Membrane Anchor

Toxins ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 62
Author(s):  
Anon Thammasittirong ◽  
Chompounoot Imtong ◽  
Wilaiwan Sriwimol ◽  
Somsri Sakdee ◽  
Chanan Angsuthanasombat
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Tight Binding ◽  
Attenuated Total Reflection ◽  
Full Length ◽  
Proteinase K ◽  
Prolonged Treatment ◽  
Lipid Bilayer Membranes ◽  
Membrane Anchor ◽  
Terminal Domain

Although the C-terminal domain (DIII) of three-domain Cry insecticidal toxins from Bacillus thuringiensis has been implicated in various biological functions, its exact role still remains to be elucidated. Here, the 21-kDa isolated DIII fragment of the 65-kDa Cry4Ba mosquito-specific toxin was analyzed for its binding characteristics toward lipid-bilayer membranes. When the highly-purified Cry4Ba-DIII protein was structurally verified by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, it revealed the presence of a distinct β-sheet structure, corresponding to its structure embodied in the Cry4Ba crystal structure. Binding analysis via surface plasmon resonance (SPR) spectroscopy revealed that the 21-kDa Cry4Ba-DIII truncate displayed tight binding to immobilized liposome membranes in a two-step manner, exhibiting a dissociation rate constant (kd) comparable to the 65-kDa full-length toxin. Also similar to the Cry4Ba full-length toxin, its isolated DIII truncate was able to anchor a part of its molecule into the immobilized membrane as the SPR signal was still detected after prolonged treatment with proteinase K. However, unlike the full-length active toxin, the DIII truncate was unable to induce membrane permeability of calcein-loaded liposomes or ion-channel formation in planar lipid bilayers. Together, our present data have disclosed a pivotal role of C-terminal DIII in serving as a membrane anchor rather than a pore-forming moiety of the Cry4Ba mosquito-active toxin, highlighting its potential mechanistic contribution to the interaction of the full-length toxin with lipid membranes in mediating toxicity.

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