The biological role of proteins of aegerolysin family is not defined but most probably they participate in development and defence of the organisms that produce them. Since their targets are specific lipids, aegerolysins show potential to be used as insecticides or as markers of specific membrane domains, so called membrane rafts. Some of the known lipid targets of aegerolysins are ceramide phosphoetanolamine (CPE), CPE combined with cholesterol (Chol) and the combination of sphingomyelin with Chol. With the purpose of better understanding their biological role, we tried to find new membrane targets of three recombinant aegerolysins, ostreolysin A (OlyA) from Pleurotus ostreatus, nigerolysin A2 (NigA2) from Aspergillus niger and RahU from Pseudomonas aeruginosa. During the extraction, lipids of Sf9 insect cells (derived from the moth Spodoptera frugiperda) were separated into two distinct phases: lipids of polar phase and lipids of nonpolar phase. We performed several binding tests and discovered that the three aegerolysins bind to the lipids of the nonpolar phase. Liquid chromatography- electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) showed that phosphoetanolamine (PE) and phosphocholine (PC) are the two most abundant lipids in nonpolar phase.However lipids of this phase also contain several CPE species. and the three aegerolysins bound to different ones. We performed a lipid dot blot assay on a broad range of lipid extracts from different organisms, i. e. yeasts, filamentous fungi and bacteria. While the binding of aegerolysins was observed to lipids of nonpolar phases of yeast and filamentous fungi, we also discover that the chosen aegerolysins bind to the lipids of the polar phase of bacterial lipids. Those may represent new potential targets, but further investigations are required.
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