Proteome Analysis of Cry4Ba Toxin-interacting <i>Aedes aegypti</i> Lipid Rafts using geLC–MS/MS

Bayyareddy, Krishnareddy; Zhu, Xiang; Orlando, Ron; Adang, Michael J.

doi:10.1021/pr3006167

Cited by 24 publications

(32 citation statements)

References 71 publications

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“…Also, this Cry4Ba-binding ALP from Ae. aegypti , and a number of other GPI-anchored proteins, are localized in cell membrane lipid rafts (Bayyareddy et al 2012). Moreover, Sf9 cells expressing this ALP protein become susceptible to the Cry4Ba toxin (Dechklar et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp. israelensis and jegathesan

2017

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Presently three major groups of proteins from Aedes aegypti, cadherin, alkaline phosphatases (ALP) and aminopeptidases N (APN), have been identified as Cry11Aa toxin receptors. To further characterize their role on toxicity, transgenic mosquitoes with silenced Aedes cadherin expression were previously generated and the role of cadherin in mediating the toxicity of four different mosquitocidal toxins (Cry11Aa, Cry11Ba, Cry4Aa and Cry4Ba) was demonstrated. Here, we investigated the role of another reported Cry11Aa receptor, ALP1. As with Aedes cadherin, this protein is localized in the apical cell membrane of distal and proximal gastric caecae and the posterior midgut. We also successfully generated transgenic mosquitoes that knockdowned ALP1 transcript levels using an inducible Aedes heat shock promoter, Hsp70A driving dsALP1RNA. Four different mosquitocidal toxins were used for larval bioassays against this transgenic mosquito. Bioassay results show that Cry11Aa toxicity to these transgenic larvae following a heat shock decreased (4.4 fold) and Cry11Ba toxicity is slightly attenuated. But Cry4Aa and Cry4Ba toxicity to ALP1 silenced larvae is unchanged. Without heat shock, toxicity of all four toxins does not change, suggesting this heat shock promoter is heat-inducible. Notably, transgenic mosquitoes with ALP1 knockdown are about 3.7 times less resistant to Cry11Aa toxin than those with Aedes cadherin knockdown. These results demonstrate that the ALP1 is an important secondary receptor for Cry11Aa and Cry11Ba, but it might not be involved in Cry4Aa and Cry4Ba toxicity.

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Section: Introductionmentioning

confidence: 99%

Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp. israelensis and jegathesan

2017

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“…In transcriptome analysis, AAEL008158 and AAEL008162 were significantly reduced in the resistant larvae midgut, implying these APNs are likely putative receptors of Cry11Aa. However, AAEL008162 may not be associated with Cry11Aa resistance in the G30 strain, since this APN bound Cry4Ba in Aedes lipid rafts (Bayyareddy et al, 2012) and only low cross resistance to Cry4Ba was observed here. On the other hand, AAEL008158 is a newly found putative receptor of Cry toxin and could be involved in Cry11Aa and Cry4Aa toxicity in mosquitoes.…”

Section: Discussionmentioning

confidence: 62%

“…In transcriptome analysis, AAEL013330 and AAEL015070 are likely putative Cry11Aa receptors, since both of these ALPs were significantly reduced in the resistant larvae midgut. In addition, AAEL015070 was previously found in Aedes lipid rafts where Cry4Ba localized (Bayyareddy et al, 2012) and was identified as a functional receptor of Cry4Ba (Dechklar et al, 2011; Thammasittirong et al, 2011). Because the G30 strain shows high resistance to Cry11Aa and low cross-resistance to Cry4Ba, this data implies the ALPs AAEL009077 and AAEL015070 may not be associated with Cry11Aa resistance in the G30 strain, since both of these ALPs bind Cry4Ba.…”

Section: Discussionmentioning

confidence: 96%

“…This ALP also binds Cry4Ba (Jiménez et al, 2012), but other ALPs also bind this toxin. Proteomic analysis showed Cry4Ba toxin was localized in lipid rafts from larval BBMV and bound three ALPs (AAEL003298, AAEL003313, and AAEL015070) (Bayyareddy et al, 2009; Bayyareddy et al, 2012). Moreover, the ALP (AAEL015070) bound Cry4Ba with high affinity and mediated Cry4Ba toxicity in Sf9 cells expressing the ALP (Dechklar et al, 2011; Thammasittirong et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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Alkaline phosphatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti

Lee

Aimanova

Gill

2014

Insect Biochemistry and Molecular Biology

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Bacillus thuringiensis subsp. israelensis (Bti) has been widely for the biological control of mosquito populations. However, the mechanism of Bti toxins is still not fully understood. To further elucidate the mechanism of Bti toxins, we developed an Aedes aegypti resistant strain that shows high-level resistance to Cry11Aa toxin. After 27 selections with Cry11Aa toxin, the larvae showed a 124-fold resistance ratio for Cry11Aa (strain G30). G30 larvae showed cross-resistance to Cry4Aa (66-fold resistance), less to Cry4Ba (13-fold), but not to Cry11Ba (2-fold). Midguts from these resistant larvae did not show detectable difference in the processing of the Cry11Aa toxin compared to that in susceptible larvae (WT). Brush border membrane vesicles (BBMV) from resistant larvae bound slightly less Cry11Aa compared to WT BBMV. To identify potential proteins associated with Cry11A resistance, not only transcript changes in the larval midgut were analyzed using Illumina sequencing and qPCR, but alterations of previously identified receptor proteins were investigated using immunoblots. The transcripts of 375 genes were significantly increased and those of 208 genes were down regulated in the resistant larvae midgut compared to the WT. None of the transcripts for previously identified receptors of Cry11Aa (Aedes cadherin, ALP1, APN1, and APN2) were altered in these analyses. The genes for the identified functional receptors in resistant larvae midgut did not contain any mutation in their sequences nor was there any change in their transcript expression levels compared to WT. However, ALP proteins were expressed at reduced levels (~40%) in the resistant strain BBMV. APN proteins and their activity were also slightly reduced in resistance strain. The transcript levels of ALPs (AAEL013330 and AAEL015070) and APNs (AAEL008158, AAEL008162) were significantly reduced. These results strongly suggest that ALPs and APNs could be associated with Cry11Aa resistance in Ae. aegypti.

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“…Ultracentrifugation and density gradient methods were employed to obtain total cell membranes from BeWo cells, brush border membranes from human term placenta, and lipid rafts using a Beckman L7-55 ultra-centrifuge (Beckman Coulter, Brea, CA) [27–29]. …”

Section: Methodsmentioning

confidence: 99%

Localization of the placental BCRP/ ABCG2 transporter to lipid rafts: Role for cholesterol in mediating efflux activity

et al. 2017

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Introduction The breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro. Methods BCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-β-cyclodextrin (MβCD, 5mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200μM, 48 h). Results and Discussion BCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MβCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MβCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts.

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Proteome Analysis of Cry4Ba Toxin-interacting Aedes aegypti Lipid Rafts using geLC–MS/MS

Cited by 24 publications

References 71 publications

Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp. israelensis and jegathesan

Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp. israelensis and jegathesan

Alkaline phosphatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti

Localization of the placental BCRP/ ABCG2 transporter to lipid rafts: Role for cholesterol in mediating efflux activity

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