<i>Bacillus thuringiensis</i> Cry4Ba Insecticidal ToxinExploits Leu<sup>615</sup> in Its C-Terminal Domain to Interact with a Target Receptor—<i>Aedes aegypti</i> Membrane-Bound Alkaline Phosphatase
In addition to the receptor-binding domain (DII), the C-terminal domain (DIII) of three-domain Cry insecticidal δ-endotoxins from <i>Bacillus thuringiensis</i> has been implicated in target insect specificity, yet its precise mechanistic role remains unclear. Here, the 21 kDa high-purity...
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MDPI AG
2021-08-01
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| Series: | Toxins |
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| Online Access: | https://www.mdpi.com/2072-6651/13/8/553 |
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| author | Anon Thammasittirong Sutticha Na-Ranong Thammasittirong Chompounoot Imtong Sathapat Charoenjotivadhanakul Somsri Sakdee Hui-Chun Li Siriporn Okonogi Chanan Angsuthanasombat |
| author_facet | Anon Thammasittirong Sutticha Na-Ranong Thammasittirong Chompounoot Imtong Sathapat Charoenjotivadhanakul Somsri Sakdee Hui-Chun Li Siriporn Okonogi Chanan Angsuthanasombat |
| author_sort | Anon Thammasittirong |
| collection | DOAJ |
| description | In addition to the receptor-binding domain (DII), the C-terminal domain (DIII) of three-domain Cry insecticidal δ-endotoxins from <i>Bacillus thuringiensis</i> has been implicated in target insect specificity, yet its precise mechanistic role remains unclear. Here, the 21 kDa 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—<i>Aedes aegypti</i> 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 65 kDa 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 (<i>K</i><sub>d</sub>) of ≈1.1 × 10<sup>−7</sup> 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., Glu<sup>522</sup>, Asn<sup>552</sup>, Asn<sup>576</sup>, and Leu<sup>615</sup>, 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 <i>A. aegypti</i> 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-Leu<sup>615</sup> 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. |
| first_indexed | 2024-03-10T08:19:38Z |
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| id | doaj.art-78a3865ced004eafa98de3fad89a33bb |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-03-10T08:19:38Z |
| publishDate | 2021-08-01 |
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| series | Toxins |
| spelling | doaj.art-78a3865ced004eafa98de3fad89a33bb2023-11-22T10:04:37ZengMDPI AGToxins2072-66512021-08-0113855310.3390/toxins13080553<i>Bacillus thuringiensis</i> Cry4Ba Insecticidal ToxinExploits Leu<sup>615</sup> in Its C-Terminal Domain to Interact with a Target Receptor—<i>Aedes aegypti</i> Membrane-Bound Alkaline PhosphataseAnon Thammasittirong0Sutticha Na-Ranong Thammasittirong1Chompounoot Imtong2Sathapat Charoenjotivadhanakul3Somsri Sakdee4Hui-Chun Li5Siriporn Okonogi6Chanan Angsuthanasombat7Microbial Biotechnology Unit, Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University, Nakhon Pathom 73140, ThailandMicrobial Biotechnology Unit, Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University, Nakhon Pathom 73140, ThailandFaculty of Science and Technology, Prince of Songkla University, Pattani 94000, ThailandBacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Salaya Campus, Mahidol University, Nakorn Pathom 73170, ThailandBacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Salaya Campus, Mahidol University, Nakorn Pathom 73170, ThailandDepartment of Biochemistry, School of Medicine, Tzu Chi University, Hualien 97004, TaiwanResearch Center of Pharmaceutical Nanotechnology, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, ThailandBacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Salaya Campus, Mahidol University, Nakorn Pathom 73170, ThailandIn addition to the receptor-binding domain (DII), the C-terminal domain (DIII) of three-domain Cry insecticidal δ-endotoxins from <i>Bacillus thuringiensis</i> has been implicated in target insect specificity, yet its precise mechanistic role remains unclear. Here, the 21 kDa 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—<i>Aedes aegypti</i> 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 65 kDa 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 (<i>K</i><sub>d</sub>) of ≈1.1 × 10<sup>−7</sup> 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., Glu<sup>522</sup>, Asn<sup>552</sup>, Asn<sup>576</sup>, and Leu<sup>615</sup>, 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 <i>A. aegypti</i> 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-Leu<sup>615</sup> 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.https://www.mdpi.com/2072-6651/13/8/553Cry4Ba mosquito-active toxinenzyme-linked immunosorbent assayGPI-anchored alkaline phosphatasehomology-based modelingmolecular dockingreceptor binding |
| spellingShingle | Anon Thammasittirong Sutticha Na-Ranong Thammasittirong Chompounoot Imtong Sathapat Charoenjotivadhanakul Somsri Sakdee Hui-Chun Li Siriporn Okonogi Chanan Angsuthanasombat <i>Bacillus thuringiensis</i> Cry4Ba Insecticidal ToxinExploits Leu<sup>615</sup> in Its C-Terminal Domain to Interact with a Target Receptor—<i>Aedes aegypti</i> Membrane-Bound Alkaline Phosphatase Toxins Cry4Ba mosquito-active toxin enzyme-linked immunosorbent assay GPI-anchored alkaline phosphatase homology-based modeling molecular docking receptor binding |
| title | <i>Bacillus thuringiensis</i> Cry4Ba Insecticidal ToxinExploits Leu<sup>615</sup> in Its C-Terminal Domain to Interact with a Target Receptor—<i>Aedes aegypti</i> Membrane-Bound Alkaline Phosphatase |
| title_full | <i>Bacillus thuringiensis</i> Cry4Ba Insecticidal ToxinExploits Leu<sup>615</sup> in Its C-Terminal Domain to Interact with a Target Receptor—<i>Aedes aegypti</i> Membrane-Bound Alkaline Phosphatase |
| title_fullStr | <i>Bacillus thuringiensis</i> Cry4Ba Insecticidal ToxinExploits Leu<sup>615</sup> in Its C-Terminal Domain to Interact with a Target Receptor—<i>Aedes aegypti</i> Membrane-Bound Alkaline Phosphatase |
| title_full_unstemmed | <i>Bacillus thuringiensis</i> Cry4Ba Insecticidal ToxinExploits Leu<sup>615</sup> in Its C-Terminal Domain to Interact with a Target Receptor—<i>Aedes aegypti</i> Membrane-Bound Alkaline Phosphatase |
| title_short | <i>Bacillus thuringiensis</i> Cry4Ba Insecticidal ToxinExploits Leu<sup>615</sup> in Its C-Terminal Domain to Interact with a Target Receptor—<i>Aedes aegypti</i> Membrane-Bound Alkaline Phosphatase |
| title_sort | i bacillus thuringiensis i cry4ba insecticidal toxinexploits leu sup 615 sup in its c terminal domain to interact with a target receptor i aedes aegypti i membrane bound alkaline phosphatase |
| topic | Cry4Ba mosquito-active toxin enzyme-linked immunosorbent assay GPI-anchored alkaline phosphatase homology-based modeling molecular docking receptor binding |
| url | https://www.mdpi.com/2072-6651/13/8/553 |
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