E69A is a mutated version of the OlyA6 protein produced by the oyster mushroom (Pleurotus ostreatus). Structure of E69A differs from OlyA6 at position 69, in the point mutation of glutamic acid to alanine, and unlike OlyA6, which only recognizes sphingomyelin and cholesterol complexes in membrane rafts, it also binds to free sphingomyelin. The aim of the master's thesis was to verify the binding of E69A to the membranes of MDCK mammalian epithelial cells and to compare the labeling with OlyA6. OlyA6 was obtained in recombinant form with the green fluorescent marker eGFP (OlyA6-eGFP) and E69A with the red fluorescent marker mCherry (E69A-mCherry) and both were used to label MDCK cells. Using fluorescence and confocal microscopy, we revealed that E69A-mCherry labels the plasmalemma of cells at a lower concentration and in a shorter time than OlyA6-eGFP. By damaging the plasmalemma with physical fixation, we showed that E69A also labels intracellular membranes. The removal of cellular cholesterol with methyl-beta-cyclodextrin abol-ishes only OlyA6 labeling, but not E69A labeling. Neither E69A or OlyA6 binds to MDCK when cells were treated with sphingomyelinase. Incubation of cells with both proteins showed that the proportion of colocalization between OlyA6 and E69A was relatively low, regardless of the simultaneous or sequential addition of the proteins to the cells. We demonstrated that E69A labels the plasmalemma at a lower concentration and shorter incubation time than OlyA6, and that both proteins occupy common sites on the plasmalemma in a smaller proportion than different sites, from which we conclude that E69A labels several areas of membranes (raft and non-raft sphingomyelin) than OlyA6.
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