Critical domains in the specific binding of radiolabelled Vip3Af insecticidal protein to brush border membrane vesicles from Spodoptera spp. and cultured insect cells

Applied and Environmental Microbiology ◽

10.1128/aem.01787-21 ◽

2021 ◽

Author(s):

Yudong Quan ◽

Maria Lázaro-Berenguer ◽

Patricia Hernández-Martínez ◽

Juan Ferré

Keyword(s):

Brush Border ◽

Binding Sites ◽

Brush Border Membrane ◽

Ex Vivo ◽

Specific Binding ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Cry Proteins ◽

Competition Binding

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis ). In this study, we have set up the conditions to analyze the specific binding of 125 I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but that Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine-mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125 I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding, and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125 I-DI-III. In addition, the truncated protein 125 I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo. IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for the ex vivo toxicity to Sf21 cells.

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Toxicity, Binding, and Permeability Analyses of FourBacillus thuringiensis Cry1 δ-Endotoxins Using Brush Border Membrane Vesicles of Spodoptera exigua and Spodoptera frugiperda

Applied and Environmental Microbiology ◽

10.1128/aem.65.2.457-464.1999 ◽

1999 ◽

Vol 65 (2) ◽

pp. 457-464 ◽

Cited By ~ 95

Author(s):

Ke Luo ◽

David Banks ◽

Michael J. Adang

Keyword(s):

Binding Site ◽

Spodoptera Frugiperda ◽

Brush Border ◽

Spodoptera Exigua ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Binding Assays ◽

Competition Binding

ABSTRACT The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua andSpodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with 125I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. 125I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles fromS. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.

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Binding Sites for Bacillus thuringiensis Cry2Ae Toxin on Heliothine Brush Border Membrane Vesicles Are Not Shared with Cry1A, Cry1F, or Vip3A Toxin

Applied and Environmental Microbiology ◽

10.1128/aem.02791-10 ◽

2011 ◽

Vol 77 (10) ◽

pp. 3182-3188 ◽

Cited By ~ 67

Author(s):

C. Gouffon ◽

A. Van Vliet ◽

J. Van Rie ◽

S. Jansens ◽

J. L. Jurat-Fuentes

Keyword(s):

Bacillus Thuringiensis ◽

Brush Border ◽

Binding Sites ◽

Brush Border Membrane ◽

Specific Binding ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Target Range ◽

Content Type ◽

Bt Toxins

ABSTRACTThe use of combinations ofBacillus thuringiensis(Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species,Heliothis virescens,Helicoverpa zea, andHelicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

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Potential differences influence amino acid/Na+ symport rates in larval Manduca sexta midgut brush-border membrane vesicles.

Journal of Experimental Biology ◽

10.1242/jeb.189.1.55 ◽

1994 ◽

Vol 189 (1) ◽

pp. 55-67

Author(s):

R Parthasarathy ◽

W R Harvey

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Manduca Sexta ◽

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Rate Determining Step ◽

Half Saturation Constant

The time-dependent fluorescence intensity of an intravesicular potential-sensitive dye was used to probe the real-time kinetics of potential difference (PD)-dependent amino acid/Na+ symport at pH9 into brush-border membrane vesicles obtained from larval Manduca sexta midgut. Neutral amino acids (alanine, proline) are symported at higher rates as the vesicles are hyperpolarized. The symport rates of acidic (glutamate) and basic (arginine) amino acids are almost PD-independent. The half-saturation constant of alanine is PD-independent between -108 and -78 mV, although the maximal symport velocity increases by half as the voltage is increased. Amino acid throughput is evidently enhanced as the relatively high transmembrane PDs (> 150 mV, lumen positive) measured in vivo are approached. The half-saturation concentrations of Na+ were in the range 15-40 mmol l-1 for most of the amino acids examined and increased with voltage for alanine. The Vmax observed as a function of cation or amino acid concentration increased as the vesicle was hyperpolarized in the case of leucine and alanine. The data support the hypothesis that carrier and substrates are at equilibrium inasmuch as substrate translocation seems to be the rate-determining step of symport.

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Indirect coupling of urate and p-aminohippurate transport to sodium in human brush-border membrane vesicles

AJP Renal Physiology ◽

10.1152/ajprenal.1996.270.1.f61 ◽

1996 ◽

Vol 270 (1) ◽

pp. F61-F68 ◽

Cited By ~ 10

Author(s):

F. Roch-Ramel ◽

B. Guisan ◽

L. Schild

Keyword(s):

Brush Border ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Indirect Coupling ◽

Sodium Gradient ◽

Beta Hydroxybutyrate ◽

Sodium Cotransport ◽

Stimulation Of

[14C]urate and p-[14C]aminohippurate (PAH) uptake by human brush-border membrane vesicles (BBMV) were measured in the presence of an inwardly oriented sodium gradient. No direct sodium cotransport was observed. Indirect [14C]urate coupling to sodium transport was demonstrated by cis-stimulation of [14C]urate with nicotinate or pyrazinoate (PZA) in the extravesicular medium but not by adding lactate, alpha-ketoglutarate, or beta-hydroxybutyrate. Indirect sodium coupling of [14C]PAH uptake was observed only when alpha-ketoglutarate was added to the extravesicular medium, a mechanism similar to that of basolateral membranes. The ability for PZA (and nicotinate) to cis-stimulate urate uptake was correlated with a high apparent affinity for the urate/anion exchanger. In urate-loaded vesicles, for identical medium concentrations, [14C]PZA uptake via the urateanion exchanger was 10 times higher than [14C]lactate uptake. Such high PZA affinity for the urate exchanger, working in parallel with PZA sodium cotransport can account for the stimulation of urate reabsorption by PZA in vivo.

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Cholera toxin facilitates calcium transport in jejunal brush border vesicles

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y86-094 ◽

1986 ◽

Vol 64 (5) ◽

pp. 568-574 ◽

Cited By ~ 5

Author(s):

David D. Maenz ◽

G. W. Forsyth

Keyword(s):

Cholera Toxin ◽

Brush Border ◽

Brush Border Membrane ◽

Equilibrium Dialysis ◽

Membrane Vesicles ◽

Second Messenger ◽

Brush Border Membrane Vesicles ◽

Ionophore A23187

Cholera toxin is very well characterized in terms of the activation of adenylate cyclase. In some systems, however, this cyclase activation does not seem to account for all of the physiological responses to the toxin. On the premise that cholera toxin may also exert effects through other second messenger compounds we have studied the effect of cholera toxin on the rate of Ca2+ movement across the membrane of intestinal brush border vesicles. Increasing concentrations of cholera toxin progressively accelerated the passive uptake of Ca2+ into, and the efflux of Ca2+ from, an osmotically active space in brush border membrane vesicles. This effect of cholera toxin was saturable by excess Ca2+ and was relatively specific, as the toxin did not affect vesicle permeability to an uncharged polar solute. The toxin had two high affinity Ca2+ binding sites on the A subunit as measured by equilibrium dialysis. Ca2+ transport facilitated by cholera toxin was temperature dependent, required the holotoxin, and could be inhibited by preincubation of the toxin with excess free ganglioside GM1.This increased rate of Ca2+ influx caused by the in vitro addition of cholera toxin to brush border membrane vesicles may have physiological significance as it was comparable to rates observed with the Ca ionophore A23187. Similar effects occurring in vivo could permit cholera toxin to increase cytoplasmic Ca2+ concentrations and to produce accompanying second messenger effects.

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Specific binding of Bacillus thuringiensis Cry1Ea toxin, and Cry1Ac and Cry1Fa competition analyses in Anticarsia gemmatalis and Chrysodeixis includens

Scientific Reports ◽

10.1038/s41598-019-54850-3 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Yolanda Bel ◽

Marc Zack ◽

Ken Narva ◽

Baltasar Escriche

Keyword(s):

Bacillus Thuringiensis ◽

Brush Border ◽

Binding Sites ◽

Brush Border Membrane ◽

Specific Binding ◽

Anticarsia Gemmatalis ◽

Binding Studies ◽

Velvetbean Caterpillar ◽

Competition Binding ◽

Chrysodeixis Includens

AbstractAnticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper) are two important defoliation pests of soybeans. In the present study, we have investigated the susceptibility and brush border membrane-binding properties of both species to Bacillus thuringiensis Cry1Ea toxin. Bioassays performed in first-instar larvae demonstrated potent activity against both soybean pests in terms of mortality or practical mortality. Competition-binding studies carried out with 125Iodine-labelled Cry1Ea, demonstrated the presence of specific binding sites on the midgut brush border membrane vesicles (BBMV) of both insect species. Heterologous competition-binding experiments indicated that Cry1Ea does not share binding sites with Cry1Ac or Cry1Fa in either soybean pest. This study contributes to the knowledge of Cry1Ea toxicity and midgut binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ea with other Bt proteins aimed at controlling lepidopteran pests in soybeans.

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Lack of cisplatin-ranitidine kinetic interactions: In vivo study in children, and in vitro study using dog renal brush border membrane vesicles

Life Sciences ◽

10.1016/s0024-3205(98)00217-3 ◽

1998 ◽

Vol 62 (25) ◽

pp. PL387-PL392 ◽

Cited By ~ 1

Author(s):

Shinya Ito ◽

Sheila Weitzman ◽

Julia Klein ◽

Mark Greenberg ◽

Robert Lau ◽

...

Keyword(s):

Brush Border ◽

Brush Border Membrane ◽

In Vitro Study ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

In Vivo Study ◽

Renal Brush Border Membrane ◽

Renal Brush Border

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Iron uptake from transferrin and lactoferrin by rat intestinal brush-border membrane vesicles

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1990.258.4.g535 ◽

1990 ◽

Vol 258 (4) ◽

pp. G535-G541 ◽

Cited By ~ 7

Author(s):

H. Kawakami ◽

S. Dosako ◽

B. Lonnerdal

Keyword(s):

Brush Border ◽

Brush Border Membrane ◽

Specific Binding ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Human Lactoferrin ◽

Affinity Constant ◽

Bovine Lactoferrin ◽

Intestinal Brush Border Membrane ◽

Intestinal Brush Border

Interaction of 59Fe-labeled rat transferrin, human lactoferrin, and bovine lactoferrin with rat small intestinal brush-border membrane vesicles was investigated with the use of a rapid filtration technique. Specific binding of 59Fe-labeled rat transferrin and bovine lactoferrin to brush-border membrane vesicles from suckling and adult rats was identified. In contrast, no binding of human lactoferrin occurred. The presence of transferrin receptors on the brush-border membrane of suckling rats was confirmed by immunoblotting, and the molecular mass of the receptor was 96 kDa under nonreducing conditions. Scatchard plot analysis indicated 2.4 x 10(14) binding sites/mg of membrane protein with an affinity constant (Ka) of 4.9 x 10(6) M-1 for rat milk transferrin and 2.2 x 10(14) bi

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Study of the Bacillus thuringiensis Cry1Ia Protein Oligomerization Promoted by Midgut Brush Border Membrane Vesicles of Lepidopteran and Coleopteran Insects, or Cultured Insect Cells

Toxins ◽

10.3390/toxins12020133 ◽

2020 ◽

Vol 12 (2) ◽

pp. 133 ◽

Cited By ~ 1

Author(s):

Ayda Khorramnejad ◽

Mikel Domínguez-Arrizabalaga ◽

Primitivo Caballero ◽

Baltasar Escriche ◽

Yolanda Bel

Keyword(s):

Bacillus Thuringiensis ◽

Growth Phase ◽

Brush Border ◽

Brush Border Membrane ◽

Insect Cells ◽

3D Structure ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Host Susceptibility ◽

Cry Proteins

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.

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