Cloning and characterization of the Cry1Ac-binding alkaline phosphatase (HvALP) from Heliothis virescens

Perera, Omaththage P.; Willis, Jonathan D.; Adang, Michael J.; Jurat-Fuentes, Juan Luís

doi:10.1016/j.ibmb.2009.01.006

Cited by 53 publications

(49 citation statements)

References 32 publications

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“…It was observed that the purified refolded ALP exhibited an apparent specific activity of 0.30 Ϯ 0.02 mol/min/mg toward the cleavage of the small organic phosphate pNPP, albeit at lower activity compared to the AamALP expressed in Sf9 cells (0.85 Ϯ 0.08 mol/min/mg [7]). However, the results of our study are not consistent with the results of another study of the lepidopteran-specific Cry1Ac-binding ALPs from Heliothis virescens; in that study, the E. coli-expressed ALP clones showed no detectable enzymatic activity toward BCIP/NBT substrates (17). The authors suggested that the loss of phosphatase activity of the expressed ALPs is due to their misfolding or posttranslational modifications (17), although it remains to be clearly elucidated whether the denaturing conditions performed on SDS-PAGE abolished the cloned ALP activity or whether additional refolding steps are needed.…”

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confidence: 99%

“…Our data are in agreement with the results of Hua et al (10), which also suggested that an E. coli-expressed Anopheles gambiae ALP isoform (Ag-ALP1 t ) is not glycosylated and thus, that its counterpart toxin, Cry11Ba, was supposed to recognize the polypeptide part rather than a saccharide portion. However, these findings are inconsistent with the binding of the lepidopteran-specific Cry1Ac toxin to ALPs from Helicoverpa armigera and Heliothis virescens larvae where N-acetylgalactosamine on the receptor is essential for toxin binding (14,17). …”

Section: Downloaded Frommentioning

confidence: 88%

“…Of particular interest, ALPs (EC 3.1.3.1; phosphomonoester hydrolases), ubiquitous enzymes widely found in microorganisms and animal kingdoms, are a highly conserved family of largely distributed cell surface and secreted metalloenzymes; many are GPI-anchored glycoproteins (for a review, see reference 13). To date, various ALPs from mosquitoes and other insect species have been reported as functional Cry toxin receptors, including Cry11Aa receptors from A. aegypti (8,9), Cry11Ba receptors from A. aegypti (12) and Anopheles gambiae (10), a Cry1Ab receptor from Manduca sexta (3), and Cry1Ac receptors from Helicoverpa armigera (14) and Heliothis virescens (17).…”

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

Thammasittirong

Dechklar

Leetachewa

et al. 2011

Appl Environ Microbiol

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Glycosylphosphatidylinositol-linked alkaline phosphatase (GPI-ALP) from the epithelial membrane of the larval midgut of Aedes aegypti was previously identified as a functional receptor of the Bacillus thuringiensis Cry4Ba toxin. Here, heterologous expression in Escherichia coli of 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 [K d ] of ϳ14 nM) which is comparable to that calculated from kinetic parameters (dissociation rate constant [k off ]/binding constant [k on ]). Altogether, the data presented here of the E. coli-expressed ALP from A. aegypti retaining high-affinity toxin binding support our notion that glycosylation of this receptor is not required for binding to its counterpart toxin, Cry4Ba.

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confidence: 99%

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confidence: 88%

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

Thammasittirong

Dechklar

Leetachewa

et al. 2011

Appl Environ Microbiol

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“…Nevertheless, it has been shown that Cry1Ac relies on N-acetylgalactosamine interaction for binding to APN1 (16), and recombinant MsALP produced in E. coli is not glycosylated, explaining the low affinity of binding of Cry1Ac to this MsALP. In Heliothis virescens, Cry1Ac binding to ALP also relies on sugar interaction (32).…”

Section: Discussionmentioning

confidence: 99%

Differential Role of Manduca sexta Aminopeptidase-N and Alkaline Phosphatase in the Mode of Action of Cry1Aa, Cry1Ab, and Cry1Ac Toxins from Bacillus thuringiensis

Flores‐Escobar

Rodríguez-Magadán

Bravo

et al. 2013

Appl Environ Microbiol

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Aminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium of Manduca sexta have been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles of M. sexta showed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned from M. sexta midgut RNA and expressed in Escherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP-or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting that B. thuringiensis subsp. kurstaki produces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.

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“…kurstaki strain HD73 producing Cry1Ac toxin was 121 obtained from the Bacillus Genetic Stock Center (BGSC, Columbus, OH). Bacterial 122 culturing, toxin activation and purification were as described elsewhere (Perera et al, 2009). accepted if they could be established at greater than 95% probability as specified by the 213 Peptide Prophet algorithm (Keller et al, 2002).…”

Section: Bacterial Toxins 120mentioning

confidence: 99%

Arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication

Castagnola

Jackson

Perera

et al. 2017

Preprint

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Cloning and characterization of the Cry1Ac-binding alkaline phosphatase (HvALP) from Heliothis virescens

Cited by 53 publications

References 32 publications

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

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

Differential Role of Manduca sexta Aminopeptidase-N and Alkaline Phosphatase in the Mode of Action of Cry1Aa, Cry1Ab, and Cry1Ac Toxins from Bacillus thuringiensis

Arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication

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