title: Proteome analysis of Cry4Ba toxin-interacting Aedes aegypti lipid rafts using geLC-MS/MS.
abstract: Lipid rafts are microdomains in the plasma membrane of eukaryotic cells. Among their many functions, lipid rafts are involved in cell toxicity caused by pore forming bacterial toxins including Bacillus thuringiensis (Bt) Cry toxins. We isolated lipid rafts from brush border membrane vesicles (BBMV) of Aedes aegypti larvae as a detergent resistant membrane (DRM) fraction on density gradients. Cholesterol, aminopeptidase (APN), alkaline phosphatase (ALP) and the raft marker flotillin were preferentially partitioned into the lipid raft fraction. When mosquitocidal Cry4Ba toxin was preincubated with BBMV, Cry4Ba localized to lipid rafts. A proteomic approach based on one-dimensional gel electrophoresis, in-gel trypsin digestion, followed by liquid chromatography-mass spectrometry (geLC-MS/MS) identified a total of 386 proteins. Of which many are typical lipid raft marker proteins including flotillins and glycosylphosphatidylinositol (GPI)-anchored proteins. Identified raft proteins were annotated in silico for functional and physicochemical characteristics. Parameters such as distribution of isoelectric point, molecular mass, and predicted post-translational modifications relevant to lipid raft proteins (GPI anchorage and myristoylation or palmitoylation) were analyzed for identified proteins in the DRM fraction. From a functional point of view, this study identified proteins implicated in Cry toxin interactions as well as membrane-associated proteins expressed in the mosquito midgut that have potential relevance to mosquito biology and vector management.
authors.affiliation: Department of Entomology, Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States.
authors.forename: Krishnareddy
authors.initials: K
authors.lastname: Bayyareddy
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhu
authors.forename: Ron
authors.initials: R
authors.lastname: Orlando
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Adang
mesh_terms: D000330:Aedes / Q000737:chemistry* / Q000201:enzymology; D000469:Alkaline Phosphatase / Q000737:chemistry; D000626:Aminopeptidases / Q000737:chemistry; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry*; D007526:Isoelectric Point; D007814:Larva / Q000737:chemistry / Q000201:enzymology; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D008871:Microvilli / Q000737:chemistry; D017830:Octoxynol; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D011956:Receptors, Cell Surface / Q000737:chemistry; D012995:Solubility; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D003902:Detergents; D004731:Endotoxins; D017261:Glycosylphosphatidylinositols; D006460:Hemolysin Proteins; D008565:Membrane Proteins; D020543:Proteome; D011956:Receptors, Cell Surface; C106354:flotillins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D017830:Octoxynol; D000469:Alkaline Phosphatase; D000626:Aminopeptidases