Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid
Hema Balakrishna Bhat1, Reiko Ishitsuka1, Takehiko Inaba1
1*Lipid Biology Laboratory, RIKEN, and Laboratory of Molecular Membrane Neuroscience, Brain Science Institute, RIKEN, Saitama, Japan; Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan; Yukiguni Maitake Co. Ltd., Niigata, Japan; Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan; Graduate School of Life and Environmental Sciences and Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan; **Department of Environmental Science and Education, Faculty of Human Life Science, Tokyo Kasei University, Tokyo, Japan; and Institut National de la Santé et de la Recherche Médicale Unité 1060, Université Lyon 1, Villeurbanne, France.
Abstract:
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 μM), 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.
