Binding of Pleurotus aegerolysins to invertebrate-specific sphingolipid ceramide phosphoethanolamine (CPE) and the formation of transmembrane pores in association with pleurotolysin B (PlyB) suggest the possibility of using aegerolysin/PlyB complexes as novel biopesticides as well as producing genetically modified crops resistant to from an economical point of view important pests. Erylysin A (EryA) is the only known fungal aegerolysin that specifically interacts with CPE-enriched membranes. By isolating the single point mutant, EryA E69A, we sought to eliminate cholesterol-specific binding and increase the affinity for CPE. The mutant could not be obtained so its further characterization was not possible. According to previous research, the OlyA6/PlyB complex has the strongest lytic activity of all aegerolysin/PlyB complexes studied and an even stronger insecticidal activity could be shown by using the OlyA6 E69A mutant. Both versions of the protein do not exhibit lytic activity in the absence of the PlyB protein partner, while in combination with PlyB they are both lytic even at low concentrations when cholesterol (Chol) is present in the membranes. The binding affinity of the mutant to Chol-free vesicles is increased compared to the wild type protein, while the OlyA6 E69A/PlyB complex showed no greater permeabilization of biological membranes compared to the OlyA6/PlyB complex. We confirmed that OlyA6 E69A recognizes different CPE conformations in biological membranes, confirming that this mutant could be biotechnologically very important as many insects are auxotrophic for Chol.
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