Purification and characterization of a trypsin-like digestive enzyme from spruce budworm (Chroristoneura fumiferana) responsible for the activation of δ-endotoxin from Bacillus thuringiensis

Milne, R.; Kaplan, Harvey

doi:10.1016/0965-1748(93)90040-y

Cited by 98 publications

(58 citation statements)

References 28 publications

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“…the midguts of many Lepidoptera species (Applebaum 1985;Terra and Ferriera 1994). Some studies confirmed serine proteinases in the midgut activate protoxins, thereby mediating Bt toxicity (Milne and Kaplan 1993;Martínez-Ramírez and Real 1996), while they also may play a concurrent role in Bt resistance caused by degradation of toxins by gut proteinases in resistant strains (Forcada et al 1996;Keller et al 1996). Although the elevated gut proteinases may degrade Bt toxin, we believe the accumulation of toxic proteins in transgenic poplar resulted in high mortality of the sixth instars and low adult oviposition of L. dispar.…”

Section: Discussionmentioning

confidence: 80%

Plant-Toxin Interactions in Transgenic Bt Cotton and their Effect on Mortality of <I>Helicoverpa armigera</I> (Lepidoptera: Noctuidae)

Olsen¹,

Daly²

2000

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Section: Discussionmentioning

confidence: 80%

Plant-Toxin Interactions in Transgenic Bt Cotton and their Effect on Mortality of <I>Helicoverpa armigera</I> (Lepidoptera: Noctuidae)

Olsen¹,

Daly²

2000

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“…The battery of midgut proteases that an insect possesses is therefore likely to be a major determinant of toxin potency. The midgut lumina of lepidopteran insect larvae have been shown to contain a variety of alkaline proteases, mainly members of the serine protease class, that exhibit predominantly trypsinlike and chymotrypsinlike protease activities (9,19,20,28). Such midgut proteases are likely to be responsible for ␦-endotoxin activation.…”

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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“…The major component of crystals toxic to lepidopteran larvae is a 130-kDa protein (protoxin) (2). On ingestion, the protoxin is acted on by a trypsin-like gut protease and converted to a 65-kDa toxin derived from the N-terminal region of the protein (3)(4)(5). The toxin binds to receptors on the brush border membrane (6 -8) and is inserted into the membrane, leading to disruption of membrane function with subsequent larval death (9,10).…”

mentioning

confidence: 99%

Role of DNA in the Activation of the Cry1A Insecticidal Crystal Protein from Bacillus thuringiensis

Clairmont

Milne

Pham

et al. 1998

Journal of Biological Chemistry

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The Cry1A insecticidal crystal protein (protoxin) from six subspecies of Bacillus thuringiensis as well as the Cry1Aa, Cry1Ab, and Cry1Ac proteins cloned in Escherichia coli was found to contain 20-kilobase pair DNA. Only the N-terminal toxic moiety of the protoxin was found to interact with the DNA. Analysis of the crystal gave approximately 3 base pairs of DNA per molecule of protoxin, indicating that only a small region of the Nterminal toxic moiety interacts with the DNA. It is proposed that the DNA-protoxin complex is virus-like in structure with a central DNA core surrounded by protein interacting with the DNA with the peripheral ends of the C-terminal region extending outward. It is shown that this structure accounts for the unusual proteolysis observed in the generation of toxin in which it appears that peptides are removed by obligatory sequential cleavages starting from the C terminus of the protoxin. Activation of the protoxin by spruce budworm (Choristoneura fumiferana) gut juice is shown to proceed through intermediates consisting of protein-DNA complexes. Larval trypsin initially converts the 20-kilobase pair DNA-protoxin complex to a 20-kilobase pair DNAtoxin complex, which is subsequently converted to a 100-base pair DNA-toxin complex by a gut nuclease and ultimately to the DNA-free toxin.Bacillus thuringiensis (Bt) 1 deposits a proteinaceous crystal during sporulation (1). The major component of crystals toxic to lepidopteran larvae is a 130-kDa protein (protoxin) (2). On ingestion, the protoxin is acted on by a trypsin-like gut protease and converted to a 65-kDa toxin derived from the N-terminal region of the protein (3-5). The toxin binds to receptors on the brush border membrane (6 -8) and is inserted into the membrane, leading to disruption of membrane function with subsequent larval death (9, 10). An unusual feature is that activation of the protoxin appears to occur by a sequential series of proteolytic cleavages, starting at the C terminus and proceeding toward the N terminus until the protease-stable toxin is generated (11, 12). As there is no known protein structural motif that would give rise to such a proteolytic process, this phenomenon is indicative of either a novel type of protein structure or the presence of an additional structural component that confers this property to the protein.An unexpected finding was that a 20-kbp heterologous DNA fragment is intimately associated with the crystals from B. thuringiensis subsp. kurstaki HD73 (13). The DNA is not susceptible to nuclease attack unless the protoxin is removed or proteolyzed to toxin. The active toxin is not associated with DNA; however, evidence was obtained which indicated that the DNA was involved in the generation of toxin from the crystal protein. At present, the nature of the interaction of the Bt protein with DNA and the role of the DNA in the generation of toxin are unknown. The present investigation was undertaken to determine the role of the DNA in the structure and function of the Bt crystal protein. EXPERIMENTAL...

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Purification and characterization of a trypsin-like digestive enzyme from spruce budworm (Chroristoneura fumiferana) responsible for the activation of δ-endotoxin from Bacillus thuringiensis

Cited by 98 publications

References 28 publications

Plant-Toxin Interactions in Transgenic Bt Cotton and their Effect on Mortality of <I>Helicoverpa armigera</I> (Lepidoptera: Noctuidae)

Plant-Toxin Interactions in Transgenic Bt Cotton and their Effect on Mortality of <I>Helicoverpa armigera</I> (Lepidoptera: Noctuidae)

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

Role of DNA in the Activation of the Cry1A Insecticidal Crystal Protein from Bacillus thuringiensis

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