Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane

Tomimoto, Kazuya; Hayakawa, Tohru; Hori, Hidetaka

doi:10.1016/j.cbpb.2006.04.013

Cited by 27 publications

(34 citation statements)

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“…These studies did not address the fate of regions of the toxin other than the ␣-helices of domain I once the toxin is inserted. However, proteinase K protection studies (typically used for detecting the regions of membrane proteins inside lipid bilayer) have shown that a 60-kDa form (or higher molecular weight aggregate) of the toxin has been protected in membranes (15,18,19,31,35). This suggests that most of the toxin was likely to be embedded in the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…There has been little analysis of the other regions of the toxin. Several studies using nonspecific proteases to determine the presence of the toxin in the membrane (18,19) have shown that almost the entire toxin is protected from the protease, but the extent of partitioning of the toxin into the membrane remains unmeasured.Our current study spans the three domains of the toxin to identify specific regions that may be embedded into the insect membrane, including the regions of the toxin proposed by the Umbrella model and beyond. Biochemical protease protection assay and steady state fluorescence measurements using cysteine mutations in regions of three domains of Cry1Aa or Cry1Ab toxin show that all regions of the toxin studied except ␣-helix 1 are embedded into the membrane, although to different extents.…”

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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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Section: Discussionmentioning

confidence: 99%

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All Domains of Cry1A Toxins Insert into Insect Brush Border Membranes

Nair

Dean

2008

Journal of Biological Chemistry

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“…As with any water-soluble poreforming proteins, their toxic mechanism would involve protein-protein interactions, protein-membrane interactions, and a particular protein folding trail underlying the conformational transition from a stable water-soluble monomer to a membrane-inserted oligomeric form (Bayley Nature 2009) [35]. Among the proposed models for depicting the membrane-insertion and pore-formation stages (Knowles AIP 1994) [ [39], the "umbrella-like" model seems now to be generally accepted as the best description of the membrane-bound state of the three-domain Cry toxins. This model involves an insertion of helices 4 and 5 into the lipid bilayers as a helical hairpin structure, and in so doing the remaining helices spread apart on the membrane surface like the opening of an umbrella (Knowles AIP 1994) [11] (Gazit JBC 1995) [36].…”

Section: Insights Into the Mechanism Of Membrane Pore Formationmentioning

confidence: 99%

Mosquito Resistance to Bacterial Larvicidal Toxins

Wirth¹

2013

TOTNJ

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Abstract:The insecticidal character of the three-domain Cry -endotoxins produced by Bacillus thuringiensis during sporulation is believed to be caused by their capability to generate lytic pores in the target larval midgut cell membranes. This review describes toxic mechanisms with emphasis on the structural basis of pore formation by two closely related dipteran-specific toxins, Cry4Aa and Cry4Ba, which are highly toxic to mosquito larvae. One proposed toxic mechanism via an "umbrella-like" structure involves membrane penetration and pore formation by the 4-5 transmembrane hairpin. The lipid-induced -conformation of 7 could possibly serve as a lipid anchor required for an efficient insertion of the pore-forming hairpin into the bilayer membrane. Though current electron crystallographic data are still inadequate to provide such critical insights into the structural details of the Cry toxin-induced pore architecture, this pivotal evidence clearly reveals that the 65-kDa active toxin in association with the lipid membrane could exist in at least two different trimeric conformations, implying the closed and open states of a functional pore.

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“…After the interaction of the domains with receptors, several a-helices comprising domain I were suggested to penetrate the plasma membrane, causing pore formation . Recently, some important reports regarding the mechanism of pore formation have been published (Tomimoto et al, 2006;Groulx et al, 2009). According to their theory, the whole of domain I in Cry1A toxin was inserted into the midgut epithelial cell membrane during the irreversible phase of pore formation.…”

Section: Introductionmentioning

confidence: 99%

Investigation of physicochemical condition to stabilize phosphatidylcholine-liposome enclosing fluorescent calcein and its exploitation for analysis of pore formation with Cry1A toxins of Bacillus thuringiensis

Haginoya

Vaijayanthi

Pandian

et al. 2010

Appl. Entomol. Zool.

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Liposome was prepared using phosphatidylcholine (PC), and calcein, a fluorescent chemical, was simultaneously enclosed within the liposome (PC-Lipo). The stability of PC-Lipo in its ability to retain calcein was evaluated under various conditions. PC-Lipo lost stability at pH 6 and pH 11-13, but was stable in the range of pH 8.3-10. PC-Lipo was stable in the temperature range of 15-30ºC, but lost the stability acutely at 35ºC. Ionic strength, given as the concentration of NaCl, also affects its stability, and a higher concentration of NaCl, i.e., more than 150 mM, induced a higher leak of calcein from PC-Lipo. The optimal conditions to achieve stable PC-Lipo were employed to characterize the differences in pore formation with Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac toxins. These toxins were reacted with PC-Lipo under these optimal conditions, and the affinity and maximum speed of Cry1Ab to form pores on PC-Lipo was shown to be highest. Here we show these conditions and evidence of the usefulness of PC-Lipo to investigate the mode of action of Cry1A toxins.

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Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane

Cited by 27 publications

References 40 publications

All Domains of Cry1A Toxins Insert into Insect Brush Border Membranes

All Domains of Cry1A Toxins Insert into Insect Brush Border Membranes

Mosquito Resistance to Bacterial Larvicidal Toxins

Investigation of physicochemical condition to stabilize phosphatidylcholine-liposome enclosing fluorescent calcein and its exploitation for analysis of pore formation with Cry1A toxins of Bacillus thuringiensis

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