Department of Entomology, Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States.
J Proteome Res. 2012 Dec 7;11(12):5843-55. doi: 10.1021/pr3006167. Epub 2012 Nov 27.
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.
脂筏是真核细胞质膜中的微区。在其众多功能中，脂筏参与了由孔形成细菌毒素引起的细胞毒性，包括苏云金芽孢杆菌（Bt）Cry 毒素。我们从埃及伊蚊幼虫刷状缘膜囊泡（BBMV）中分离出脂筏，作为密度梯度上的去污剂抗性膜（DRM）部分。胆固醇、氨肽酶（APN）、碱性磷酸酶（ALP）和筏标记蛋白 flotillin 优先分配到脂筏部分。当杀蚊 Cry4Ba 毒素与 BBMV 预孵育时，Cry4Ba 定位于脂筏。基于一维凝胶电泳、胶内胰蛋白酶消化、然后进行液相色谱-质谱联用（geLC-MS/MS）的蛋白质组学方法鉴定出总共 386 种蛋白质。其中许多是典型的脂筏标记蛋白，包括 flotillins 和糖基磷脂酰肌醇（GPI）锚定蛋白。鉴定出的筏蛋白通过计算机注释了功能和理化特性。对 DRM 部分中鉴定出的蛋白质进行了与脂筏蛋白（GPI 锚定和豆蔻酰化或棕榈酰化）相关的等电点、分子量和预测的翻译后修饰的分布等参数分析。从功能角度来看，这项研究鉴定了与 Cry 毒素相互作用以及在蚊子中肠表达的膜相关蛋白有关的蛋白质，这些蛋白质可能与蚊子生物学和媒介管理有关。
