Isolation and partial characterization of gypsy moth BTR‐270, an anionic brush border membrane glycoconjugate that binds <i>Bacillus thuringiensis</i> Cry1A toxins with high affinity

Valaitis, Algimantas P.; Jenkins, Jeremy L.; Lee, Mi Kyong; Dean, Donald H.; Garner, Karen J.

doi:10.1002/arch.1028

Cited by 72 publications

(49 citation statements)

References 54 publications

(48 reference statements)

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“…In nematodes, glycolipids are believed to be an important class of Cry toxin receptors (60). Other putative receptors include alkaline phosphatases (ALPs) (38,85,86), a 270-kDa glycoconjugate (176), and a 252-kDa protein (73). In the following sections, each receptor class will be discussed with a particular focus on toxin-receptor binding interactions and the ability of receptors to confer toxin susceptibility.…”

Section: Figmentioning

confidence: 99%

Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

Pigott

Ellar

2007

Microbiol Mol Biol Rev

600

598

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SUMMARY Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death.

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Section: Figmentioning

confidence: 99%

Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

Pigott

Ellar

2007

Microbiol Mol Biol Rev

600

598

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“…Binding partners for Bt Cry toxins in Lepidoptera have included the GPI-anchored molecules, aminopeptidase N (APN) and alkaline phosphatase (ALP), and cadherin-like molecules. Other binding partners for Cry toxins include a glyco-conjugate receptor, V-ATP synthase subunit and actin (Krishnamoorthy et al, 2007;Valaitis et al, 2001). Further binding investigations for both the cadherin-like and GPI-anchored proteins have led to proposals that both protein groups interact with Cry toxins to form pores.…”

Section: Introductionmentioning

confidence: 99%

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

Angelucci

Barrett-Wilt²,

Hunt

et al. 2008

Insect Biochemistry and Molecular Biology

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Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) δ-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.

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“…The protease-activated form of the toxin binds to receptors on the surface of the insect brush border membrane. Several receptors implicated in binding to the toxin include cadherins (8,9), alkaline phosphatase, and one or more forms of aminopeptidases (10,11), glycolipids (12,13), and glycoproteins (14). The receptor-bound toxin has been proposed to undergo conformational changes (15,16) before or after inserting into the membrane to form ion channels.…”

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confidence: 99%

All Domains of Cry1A Toxins Insert into Insect Brush Border Membranes

Nair

Dean

2008

Journal of Biological Chemistry

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A critical step in understanding the mode of action of insecticidal crystal toxins from Bacillus thuringiensis is their partitioning into membranes and, in particular, the insertion of the toxin into insect brush border membranes. The Umbrella and Penknife models predict that only ␣-helix 5 of domain I along with adjacent helices ␣-4 or ␣-6 insert into the brush border membranes because of their hydrophobic nature. By employing fluorescent-labeled cysteine mutations, we observe that all three domains of the toxin insert into the insect membrane. Using proteinase K protection assays, steady state fluorescence quenching measurements, and blue shift analysis of acrylodanlabeled cysteine mutants, we show that regions beyond those proposed by the two models insert into the membrane. Based on our studies, the only extended region that does not partition into the membrane is that of ␣-helix 1. Bioassays and voltage clamping studies show that all mutations examined, except certain domain II mutations in loop 2 (e.g. F371C and G374C), which disrupt membrane partitioning, retain their ability to form ion channels and toxicity in Manduca sexta larvae. This study confirms our earlier hypothesis that insertion of crystal toxin does not occur as separate helices alone, but virtually the entire molecule inserts as one or more units of the whole molecule.Insecticidal crystal proteins produced by Bacillus thuringiensis are of great commercial potential in the field of agriculture and health (1) by targeting a wide spectrum of crop pests and vectors of human diseases. Cry1A toxins are active against lepidopteran insects, which include agricultural pests. The toxins are produced by the bacterium in the stationary phase as inactive crystal protoxins (1). Activation of the 130-kDa protoxin to a 65-kDa active toxin occurs in the alkaline environment of the lepidopteran midgut. Crystal structures of the active toxin show that the toxin has three domains that are conserved through all the crystal toxins (2-7). Domain I is an ␣-helical bundle made of seven antiparallel ␣-helices. Domain II is a globin-like, wedge-shaped prism made of antiparallel ␤-sheets ending in predominant ⍀-loops, and domain III is a lectin-like ␤-sandwich. The protease-activated form of the toxin binds to receptors on the surface of the insect brush border membrane. Several receptors implicated in binding to the toxin include cadherins (8, 9), alkaline phosphatase, and one or more forms of aminopeptidases (10, 11), glycolipids (12, 13), and glycoproteins (14). The receptor-bound toxin has been proposed to undergo conformational changes (15, 16) before or after inserting into the membrane to form ion channels.Studies focused on insertion of Cry1A toxins into the insect membrane have limited their studies to two ␣-helices of domain I of Cry1A toxins, ␣-helix 4 and 5 based on the theoretical Umbrella model of insertion proposed early in the 1980s (17). There has been little analysis of the other regions of the toxin. Several studies using nonspecific proteases to det...

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Isolation and partial characterization of gypsy moth BTR‐270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

Cited by 72 publications

References 54 publications

Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

All Domains of Cry1A Toxins Insert into Insect Brush Border Membranes

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