Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity

Aroonkesorn, Aratee; Pootanakit, Kusol; Katzenmeier, Gerd; Angsuthanasombat, Chanan

doi:10.1016/j.bbrc.2015.04.026

Cited by 22 publications

(16 citation statements)

References 32 publications

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“…gambiae (Zhang et al, 2008) and Aedes aegypti (Aroonkesorn et al, 2015;Chen et al, 2009;Chen et al, 2013). The apparent sizes of several identified APNs suggested proteolytic degradation and/or formation of dimers with high-molecular weight.…”

Section: Discussionmentioning

confidence: 99%

Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae

Rezende

Romão

Batista

et al. 2017

Insect Biochemistry and Molecular Biology

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A binary mosquitocidal toxin composed of a three-domain Cry-like toxin (Cry48Aa) and a binary-like toxin (Cry49Aa) was identified in Lysinibacillus sphaericus. Cry48Aa/Cry49Aa has action on Culex quinquefasciatus larvae, in particular, to those that are resistant to the Bin Binary toxin, which is the major insecticidal factor from L. sphaericus-based biolarvicides, indicating that Cry48Aa/Cry49Aa interacts with distinct target sites in the midgut and can overcome Bin toxin resistance. This study aimed to identify Cry48Aa/Cry49Aa ligands in C. quinquefasciatus midgut through binding assays and mass spectrometry. Several proteins, mostly from 50 to 120 kDa, bound to the Cry48Aa/Cry49Aa toxin were revealed by toxin overlay and pull-down assays. These proteins were identified against the C. quinquefasciatus genome and after analysis a set of 49 proteins were selected which includes midgut bound proteins such as aminopeptidases, amylases, alkaline phosphatases in addition to molecules from other classes that can be potentially involved in this toxin's mode of action. Among these, some proteins are orthologs of Cry receptors previously identified in mosquito larvae, as candidate receptors for Cry48Aa/Cry49Aa toxin. Further investigation is needed to evaluate the specificity of their interactions and their possible role as receptors.

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Section: Discussionmentioning

confidence: 99%

Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae

Rezende

Romão

Batista

et al. 2017

Insect Biochemistry and Molecular Biology

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“…More recently, we have shown that the stability of two receptor-binding hairpins (i.e., β 2 -β 3 and β 4 -β 5 within Cry4Ba-DII) contributed by hydrogen bonding between Thr 328 -Thr 369 side-chains is important for toxin binding to Cyt2Aa2—an alternative receptor for the Cry4Ba toxin [ 17 , 18 ]. Furthermore, we have succeeded in identifying two different types of Cry4Ba receptors from Aedes mosquito larvae, i.e., glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase and GPI-anchored aminopeptidase N (APN) [ 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

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

Thammasittirong

Imtong

Sriwimol

et al. 2019

Toxins

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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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“…Insect M1 peptidases are mainly expressed in the gut, where they play important intermediate roles in protein digestion [ 111 ] as well as host-pathogen interactions. Membrane-bound aminopeptidases in the gut are receptors for Bacillus thuringiensis toxins in several insect species [ 112 – 114 ]. Aminopeptidases have also been detected in other insect tissues, such as the fat body [ 115 ], salivary glands [ 116 ], and Malpighian tubes [ 116 ].…”

Section: Resultsmentioning

confidence: 99%

Next Generation Sequencing Identifies Five Major Classes of Potentially Therapeutic Enzymes Secreted byLucilia sericataMedical Maggots

Franta

Vogel

Lehmann

et al. 2016

BioMed Research International

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Lucilia sericata larvae are used as an alternative treatment for recalcitrant and chronic wounds. Their excretions/secretions contain molecules that facilitate tissue debridement, disinfect, or accelerate wound healing and have therefore been recognized as a potential source of novel therapeutic compounds. Among the substances present in excretions/secretions various peptidase activities promoting the wound healing processes have been detected but the peptidases responsible for these activities remain mostly unidentified. To explore these enzymes we applied next generation sequencing to analyze the transcriptomes of different maggot tissues (salivary glands, gut, and crop) associated with the production of excretions/secretions and/or with digestion as well as the rest of the larval body. As a result we obtained more than 123.8 million paired-end reads, which were assembled de novo using Trinity and Oases assemblers, yielding 41,421 contigs with an N50 contig length of 2.22 kb and a total length of 67.79 Mb. BLASTp analysis against the MEROPS database identified 1729 contigs in 577 clusters encoding five peptidase classes (serine, cysteine, aspartic, threonine, and metallopeptidases), which were assigned to 26 clans, 48 families, and 185 peptidase species. The individual enzymes were differentially expressed among maggot tissues and included peptidase activities related to the therapeutic effects of maggot excretions/secretions.

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Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity

Cited by 22 publications

References 32 publications

Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae

Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae

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

Next Generation Sequencing Identifies Five Major Classes of Potentially Therapeutic Enzymes Secreted byLucilia sericataMedical Maggots

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