Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates
Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates
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DOI：
10.1096/fj.15-272112
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发表时间：
2015-09-01
期刊：
影响因子：
4.8
通讯作者：
Kobayashi, Toshihide
中科院分区：
文献类型：
--
作者：
Bhat, Hema Balakrishna;Ishitsuka, Reiko;Kobayashi, Toshihide
神经酰胺磷酸乙醇胺（CPE）是一种鞘磷脂类似物，是无脊椎动物和寄生虫中的主要鞘脂，而在哺乳动物细胞中仅存在痕量。在这项研究中，蘑菇衍生的蛋白质的aegerolysin家族-pleurotolysin A2（PlyA 2; K-D = 12 nM），ostreolysin（Oly; K-D = 1.3 nM），erylysin A（EryA; K-D = 1.3 nM）-强烈相关的CPE/胆固醇（Chol）-含有膜，而他们的低亲和力鞘磷脂/Chol排除建立的结合动力学。结合特异性通过多层脂质体结合试验、支持性双层试验和固相研究确定，针对一系列中性和带负电荷的脂质类与Chol或磷脂酰胆碱1：1混合。与磷脂酰乙醇胺无交叉反应。只有PlyA 2也与CPE，独立的胆固醇含量（K-D = 41 μ M），使其成为一个合适的工具，用于可视化的脂质印迹实验和生物样品中的固醇营养缺陷型生物体的CPE。通过荧光成像在果蝇幼虫的CNS中（通过PlyA 2）和在寄生虫布氏锥虫的血流形式中（通过EryA）的CPE富集的可视化证明了作为用于检测和可视化CPE的有效工具的嗜热菌溶素家族蛋白的多功能性。
Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family-pleurotolysin A2 (PlyA2; K-D = 12 nM), ostreolysin (Oly; K-D = 1.3 nM), and erylysin A (EryA; K-D = 1.3 nM)-strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K-D = 41 mu M), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.