Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation.
Lipid rafts are characterized by their insolubility in nonionic detergents such as Triton X-100 at 4 degrees C. They have been studied in mammals, where they play critical roles in protein sorting and signal transduction. To understand the potential role of lipid rafts in lepidopteran insects, we isolated and analyzed the protein and lipid components of these lipid raft microdomains from the midgut epithelial membrane of Heliothis virescens and Manduca sexta. Like their mammalian counterparts, H. virescens and M. sexta lipid rafts are enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins. In H. virescens and M. sexta, pretreatment of membranes with the cholesterol-depleting reagent saponin and methyl-beta-cyclodextrin differentially disrupted the formation of lipid rafts, indicating an important role for cholesterol in lepidopteran lipid rafts structure. We showed that several putative Bacillus thuringiensis Cry1A receptors, including the 120- and 170-kDa aminopeptidases from H. virescens and the 120-kDa aminopeptidase from M. sexta, were preferentially partitioned into lipid rafts. Additionally, the leucine aminopeptidase activity was enriched approximately 2-3-fold in these rafts compared with brush border membrane vesicles. We also demonstrated that Cry1A toxins were associated with lipid rafts, and that lipid raft integrity was essential for in vitro Cry1Ab pore forming activity. Our study strongly suggests that these microdomains might be involved in Cry1A toxin aggregation and pore formation.
Zhuang
Meibao
M
Environmental Toxicology Graduate Program, Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521, USA.
Oltean
Daniela I
DI
Gómez
Isabel
I
Pullikuth
Ashok K
AK
Soberón
Mario
M
Bravo
Alejandra
A
Gill
Sarjeet S
SS
D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins; D019921:Biotinylation; D015153:Blotting, Western; D002784:Cholesterol / Q000378:metabolism; D003505:Cyclodextrins / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins; D015151:Immunoblotting; D007313:Insecta; D007930:Leucine / Q000737:chemistry; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D018402:Manduca; D021962:Membrane Microdomains / Q000737:chemistry*; D008564:Membrane Potentials; D017830:Octoxynol / Q000494:pharmacology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D047392:beta-Cyclodextrins
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003505:Cyclodextrins; D003902:Detergents; D004731:Endotoxins; D006460:Hemolysin Proteins; D008055:Lipids; D047392:beta-Cyclodextrins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C108732:methyl-beta-cyclodextrin; D017830:Octoxynol; D002784:Cholesterol; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D007930:Leucine