Processing of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 by gut juices of various insect larvae

Ogiwara, Katsutoshi; Indrasith, Leslie S.; Asano, Shouji; Hori, Hidetaka

doi:10.1016/0022-2011(92)90084-h

Cited by 45 publications

(36 citation statements)

References 9 publications

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“…Processing of protoxins from Bt subsp. kurstaki strains HD-1 and HD-73 by midgut fluids from the smaller tea tortrix, Adoxophyes sp, B. mori, common cutworm, Spodoptera litura, diamondback moth, Plutella xylostella, housefly, Musca domestica resulted in polypeptides with different N-termini (Ogiwara et al, 1992). While most hydrolysates contained peptides beginning with arginine-28, those incubated with P. xylostella and S. litura gut proteases contained peptides initiating with leucine-46 and glycine-66 at the N-terminus, respectively.…”

Section: Icp Processingmentioning

confidence: 98%

Protease interactions with Bacillus thuringiensis insecticidal toxins

Oppert

1999

Arch Insect Biochem Physiol

111

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Section: Icp Processingmentioning

confidence: 98%

Protease interactions with Bacillus thuringiensis insecticidal toxins

Oppert

1999

Arch Insect Biochem Physiol

111

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“…These are found at the Cterminal end of helix ␣1 of Cry1Ac (46); between ␣1 and ␣2a of Cry1Ab (23) and Cry1Ac (35); within ␣2a of Cry1Ab (43) and Cry1Ac (35); between ␣2a and ␣2b of Cry1Ab (43) and Cry1Ac (35,46); between ␣2 and ␣3 of Cry4Ba (7); between ␣3 and ␣4 of Cry2Aa (3,45), Cry3Aa (10,12), and Cry9Ca (32); between ␣5 and ␣6 of Cry4Aa (66), Cry4Ba (2,68), and Cry9Aa (68); between ␤4-and ␤5-sheets of Cry11Aa (17,67); between ␤6 and ␤7r of Cry1Aa (48); within ␤7r of Cry1Ab (14); and between ␤9 and ␤10 of Cry1Ac (35). The functional significance of these cleavages remains unclear.…”

mentioning

confidence: 99%

Protease Inhibitors Fail To Prevent Pore Formation by the Activated Bacillus thuringiensis Toxin Cry1Aa in Insect Brush Border Membrane Vesicles

Kirouac

Vachon

Quievy

et al. 2006

Appl Environ Microbiol

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To investigate whether membrane proteases are involved in the activity of Bacillus thuringiensis insecticidal toxins, the rate of pore formation by trypsin-activated Cry1Aa was monitored in the presence of a variety of protease inhibitors with Manduca sexta midgut brush border membrane vesicles and by a light-scattering assay. Most of the inhibitors tested had no effect on the pore-forming ability of the toxin. However, phenylmethylsulfonyl fluoride, a serine protease inhibitor, promoted pore formation, although this stimulation only occurred at higher inhibitor concentrations than those commonly used to inhibit proteases. Among the metalloprotease inhibitors, o-phenanthroline had no significant effect; EDTA and EGTA reduced the rate of pore formation at pH 10.5, but only EDTA was inhibitory at pH 7.5. Neither chelator affected the properties of the pores already formed after incubation of the vesicles with the toxin. Taken together, these results indicate that, once activated, Cry1Aa is completely functional and does not require further proteolysis. The effect of EDTA and EGTA is probably better explained by their ability to chelate divalent cations that could be necessary for the stability of the toxin's receptors or involved elsewhere in the mechanism of pore formation.Bacillus thuringiensis is by far the most widely used alternative to chemical insecticides for the control of insect pests in forestry, agriculture, and public health. During sporulation, this bacterium produces insecticidal proteins that accumulate in the form of parasporal crystals. Following their ingestion by susceptible insect larvae, these protoxins are solubilized and converted to active toxins by midgut proteases. Activated toxins act by forming pores after binding to specific receptors at the surface of the insect midgut luminal membrane, leading to cell lysis, destruction of the epithelium, and death of the insect (52). In their activated form, all B. thuringiensis Cry toxins for which the crystal structure has been solved share a similar three-domain structure (7,21,24,33,34,44). Domain I is composed of a bundle of seven anti-parallel ␣-helices and is generally thought to be responsible for membrane insertion and pore formation, while domains II and III are mainly composed of ␤-sheets and involved in the binding, specificity, and stability of the toxin (24, 33, 52).Midgut proteases play an essential role in the activation of B. thuringiensis toxins (1, 6, 13). In the case of Cry1A toxins, the first 28 amino acid residues are removed from the N terminus and approximately half of the protoxin residues are removed from the C terminus (6). However, susceptibility of the activated toxins to further proteolysis in the midgut environment could possibly affect toxicity and explain apparent discrepancies that are occasionally observed between their in vitro and in vivo activities (15,41,49,60). For example, Cry1Ca can be completely degraded when incubated with midgut juice from advanced larval instars of Spodoptera littoralis (29). Synergisti...

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“…This relatively protease-resistant toxic core is derived from the N-terminal half of the protoxin by removal of 500 to 600 amino acid residues from the C terminus and the first 27 to 29 Nterminal residues (2,4,7,17,30,31,36,43). Activated Cry1Ac is generated by cleavages at R28 and K623 in the protoxin (4).…”

mentioning

confidence: 99%

“…Similarly, Keller et al (20) suggested that reduced sensitivity of fifth-instar larvae of Spodoptera littoralis to Cry1C could be attributed to increased degradation of the toxin in the less susceptible larvae. Ogiwara et al (31) compared the sites of proteolytic cleavage of the ␦-endotoxins of B. thuringiensis subsp. kurstaki HD-1 and HD-73.…”

mentioning

confidence: 99%

Role of Proteolysis in Determining Potency of Bacillus thuringiensis Cry1Ac δ-Endotoxin

Lightwood

Ellar

Jarrett³

2000

Appl Environ Microbiol

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Bacillus thuringiensis protein ␦-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac ␦-endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of ϳ56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of ϳ58, ϳ40, and ϳ20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.

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Processing of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 by gut juices of various insect larvae

Cited by 45 publications

References 9 publications

Protease interactions with Bacillus thuringiensis insecticidal toxins

Protease interactions with Bacillus thuringiensis insecticidal toxins

Protease Inhibitors Fail To Prevent Pore Formation by the Activated Bacillus thuringiensis Toxin Cry1Aa in Insect Brush Border Membrane Vesicles

Role of Proteolysis in Determining Potency of Bacillus thuringiensis Cry1Ac δ-Endotoxin

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