Bivalent Sequential Binding Model of a Bacillus thuringiensis Toxin to Gypsy Moth Aminopeptidase N Receptor

Jenkins, Jeremy L.; Lee, Mi Kyong; Valaitis, Algimantas P.; Curtiss, April; Dean, Donald H.

doi:10.1074/jbc.275.19.14423

Cited by 127 publications

(148 citation statements)

References 64 publications

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“…Mutagenesis studies of Cry1Ab and Cry1Ac loop 3 previously performed indicated that this amino acid region was important for binding with M. sexta BBMV and toxicity (40,41). In addition, alanine substitutions of loop 3 residues in Cry1Ab and Cry1Ac toxins showed a correlative effect on APN binding, suggesting that their effects on toxicity were due to defects in this binding (22,23). However, it was also shown that loop 3 is the cognate bind- (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Also, recent reports indicate that the region ␤16 -␤22 located in domain III of Cry1Aa and Cry1Ab toxins is involved in the interaction with B. mori and M. sexta APNs, respectively (16,21). In addition, domain II is involved in Cry1A toxin interaction with APN because mutations in domain II loop regions that affected toxicity were shown to affect binding of Cry1Ac or Cry1Ab to M. sexta and Lymantria dispar APNs (22,23). It is important to mention that these binding studies were performed with monomeric toxins, and these mutations could also affect binding to the cadherin receptor.…”

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confidence: 99%

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Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Pacheco

Gómez

Arenas

et al. 2009

Journal of Biological Chemistry

126

135

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Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R 1 ) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R 1 promotes cleavage of the amino-terminal end, including helix ␣-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R 1 fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.Bacillus thuringiensis is a bacterium that produces crystalline inclusions formed by insecticidal proteins, called Cry toxins, during the sporulation phase of growth. Cry toxins are toxic to different insect orders as well as to other invertebrates, such as nematodes, mites, and protozoa (1). Cry toxins have been used worldwide in the control of insect pests in agriculture, either as transgenic crops or as spray formulations.The molecular mechanism proposed to describe the action of Cry1A toxins, which are active against different lepidopteran insect species, involves several steps. After larval ingestion of the crystalline inclusions, these are solubilized in midgut lumen and activated by proteases releasing a toxic 65-kDa fragment that binds, in a sequential manner, with at least two receptors located in midgut microvilli. The first interaction occurs with cadherin protein (Bt-R 1 2 in the case of Manduca sexta). This interaction promotes further proteolytic processing of the N-terminal end, including helix ␣-1 of the toxin, resulting in the formation of a prepore oligomeric structure (2). The oligomer has higher affinity to secondary receptors, which are anchored by glycosylphosphatidylinositol, such as aminopeptidase-N (APN) or alkaline phosphatase in the case of M. sexta or Heliothis virescens, respectively (3, 4). Glycosylphosphatidylinositol-anchored receptors are located in specific membrane regions called lipid rafts, where the oligomer inserts into the membrane-forming pores, disrupting the osmotic equilibrium and leading to cell death (1, 5). Although this mechanism of action is g...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Pacheco

Gómez

Arenas

et al. 2009

Journal of Biological Chemistry

126

135

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“…Also, we analyzed the binding of Cry1Ab mutants located in regions of domain II and domain III previously characterized as APN-binding epitopes (27,28). Our data suggest that both APN and ALP are functional receptor molecules that bind the oligomeric structure of Cry1Ab toxin with high affinity, facilitating membrane insertion and pore formation, and we also found that ALP may a have a predominant and more important role in Cry1Ab toxicity at earlier stages of larval development.…”

mentioning

confidence: 88%

“…The BBMV proteins that were solubilized with CHAPS as stated above were concentrated by Amicon YM-50 ultrafiltration, and a 2-ml aliquot was applied to a HR 5/10 Mono Q (GE Healthcare) column equilibrated with 20 mM Tris-HCl pH 8.5 buffer containing 2 mM MgCl 2 , 2 mM KCl (28,30). The column was eluted with a gradient of NaCl (0.5-1 M) with a flow rate of 1 ml/min for 40 min.…”

Section: Methodsmentioning

confidence: 99%

Role of Alkaline Phosphatase from Manduca sexta in the Mechanism of Action of Bacillus thuringiensis Cry1Ab Toxin

Arenas

Bravo

Soberón

et al. 2010

Journal of Biological Chemistry

155

157

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Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins, a prepore oligomeric intermediate is formed after interaction with cadherin receptor. The Cry1A oligomer then interacts with glycosylphosphatidylinositol-anchored receptors. Two Manduca sexta glycosylphosphatidylinositol-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP), have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, because it was found in the first larval instars, whereas APN is induced later after the third larval instar. The binding of Cry1Ab oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity (apparent K d ‫؍‬ 0.6 nM), whereas the monomer showed weaker binding (apparent K d ‫؍‬ 101.6 and 267.3 nM for APN and ALP, respectively). Several Cry1Ab nontoxic mutants located in the exposed loop 2 of domain II or in ␤-16 of domain III were affected in binding to APN and ALP, depending on their oligomeric state. In particular monomers of the nontoxic domain III, the L511A mutant did not bind ALP but retained APN binding, suggesting that initial interaction with ALP is critical for toxicity. Our data suggest that APN and ALP fulfill two roles. First APN and ALP are initial receptors promoting the localization of toxin monomers in the midgut microvilli before interaction with cadherin. Then APN and ALP function as secondary receptors mediating oligomer insertion into the membrane. However, the expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action because Cry toxins are highly effective against the neonate larvae that is the target for pest control programs.The Cry toxins produced by Bacillus thuringiensis are used worldwide as effective biological control agents for many species of insects including agricultural and forest pests and several vectors of human and animal diseases. Its insecticidal property results from crystalline inclusions produced during sporulation that are formed by ␦-endotoxins known as Cry toxins (1).The Cry protein family is composed of more than 54 groups, among which the three-domain Cry family forms the major group, having members that show toxicity to different insect orders and to nematodes. The crystal structure has been solved for six three-domain Cry toxins and one protoxin that display different insect specificities (2). The activated Cry toxins are globular molecules consisting of a bundle of seven ␣-helices (domain I), a three-␤-sheet prism (domain II), and a ␤-sandwich (domain III) (3, 4). The N-terminal domain I is involved in oligomer formation, membrane insertion, and pore formation (5-8). Domain II and III are involved in the recogn...

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“…Su tamaño varía de 90-170 kDa y están unidas a la membrana celular, en concreto a las regiones denominadas "balsas lipídicas", mediante anclajes de glicosil fosfatidil inositol (GPI) (Pigott y Ellar, 2007). Las APNs han sido identificadas como sitios de unión de toxinas Cry en varias especies de lepidópteros (Candas et al, 2003;Jenkins et al, 2000;Knight et al, 1994;Luo et al, 1997;McNall y Adang, 2003;Yaoi et al, 1997) El conocimiento de la secuencia y la estructura de algunas proteínas Cry (ver apartado 1.2.1.3) ha permitido adquirir mucha información acerca de la participación de cada uno de los dominios en el mecanismo de acción. El dominio II es el dominio estructuralmente más variable en las toxinas estudiadas (Boonserm et al, 2005), por ello es considerado un factor determinante de la especificidad de las toxinas Cry (Pigott y Ellar, 2007).…”

Section: Segunda Faseunclassified

Estrategias para la utilización de la bacteria entomopatógena Bacillus thuringiensis (Berliner) en el control de Ceratitis capitata (Wiedemann)

Quist¹,

José²

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AgradecimientosEl trabajo que aquí se presenta ha sido desarrollado en la Unidad Asociada de Entomología IVIA-CIB, gracias a la concesión de una beca/contrato predoctoral del Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) y a la financiación del proyecto de investigación CICYT-AGL2006 3346/AGR.Ante todo, quiero agradecer muy sinceramente el apoyo recibido de mis dos directores de tesis, Pedro Castañera y Joel González. Pedro, gracias por haber hecho posible esta oportunidad de sumergirme en el campo de la entomología agrícola, en todo momento me has facilitado mucho las cosas y has sabido orientar cabalmente el curso de esta investigación (una tesis "de riesgo", como hábilmente calificaste). Joel, agradezco profundamente tu forma de haber dirigido mi trabajo. Como tú bien me hiciste ver desde el primer momento, ha parecido más una colaboración que una dirección. Hemos trabajado codo con codo y compartido el día a día, has sido un sabio mentor y me has abierto los ojos al mundo de esta curiosa bacteria, Bacillus thuringiensis, que, sin que sepamos exactamente por qué, destina gran parte de sus recursos a producir proteínas que matan insectos. Bueno, nosotros también hemos destinado gran parte de nuestros recursos a producir este trabajo, valoro mucho tu implicación y espero que tú lo hayas vivido de una forma tan provechosa como yo mismo.Agradezco también a los doctores Alberto Urbaneja, Félix Ortego y Salvador Herrero su excelente y altruista revisión de esta tesis. Vuestros comentarios han permitido enriquecer sustancialmente este documento.Quiero expresar mi reconocimiento a todas aquellas personas que han participado directamente en esta tesis: Bea, Inma, Helga, Nacho, Vicky, Sandra, Marta, Francesc y Houda, que me han ayudado a nivel técnico en el IVIA; y Ángel, Nathalie, Bea, Marisa, Carolina y Lola que hicieron lo propio en el CIB. Agradezco también los consejos, el apoyo o simplemente la compañía que me han brindado todos los miembros de las instituciones vinculadas a este trabajo: la unidad de entomología del IVIA y el laboratorio de interacción planta-insecto del CIB, así como el laboratorio de Lucas Sánchez y los servicios de proteómica y microscopía de dicho centro. Sin ánimo de pareceros un tanto escueto en agradecimientos "personalizados", os considero, uno a uno, de un capital humano incalculable y espero que nuestros caminos se crucen a menudo en el futuro.Por último, a mi familia y amigos, y en especial a Daph, por estar siempre en la primera línea…muchas gracias! RESÚMENESResumen La mosca mediterránea de la fruta, Ceratitis capitata (Wiedemann) (Diptera:Tephritidae), es considerada la principal plaga de la fruticultura a nivel mundial. Actualmente, la vigencia del control químico como medida prioritaria en el manejo de esta plaga está seriamente comprometida, tanto por restricciones legales (prohibición de materias activas) y sociales (tendencias de consumo) como por limitaciones intrínsecas del método (resistencias). Por ello, el desarrollo e implementación de mét...

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Bivalent Sequential Binding Model of a Bacillus thuringiensis Toxin to Gypsy Moth Aminopeptidase N Receptor

Cited by 127 publications

References 64 publications

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Role of Alkaline Phosphatase from Manduca sexta in the Mechanism of Action of Bacillus thuringiensis Cry1Ab Toxin

Estrategias para la utilización de la bacteria entomopatógena Bacillus thuringiensis (Berliner) en el control de Ceratitis capitata (Wiedemann)

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