Protein erylysin B (EryB) is a protein with the MACPF domain (Pfam PF01823). It is produced by the mushroom Pleurotus eryngii. Its biological role is still unknown. Pleurotolysin B (PlyB), a homologue of EryB, is synthesized by Pleurotus ostreatus. Mushrooms of this genus also synthesize proteins called aegerolysins, which form pore-forming complexes with proteins that have MACPF domain. Aegerolysins have been shown to have a specific binding affinity for membranes, composed of sphingomyelin (SM) and cholesterol or ceramide phosphoetanolamine (CPE) and cholesterol. Proteins with MACPF domain further enhance this binding and form two-component transmembrane pores in concert with aegerolysins. As a part of this master's thesis, we expressed EryB in bacteria Escerichia coli. We successfully isolated it and tested its functionality in combination with Pleurotus aegerolysins erylysin A, ostreolysin A6, and pleurotolysin A2. Tests included surface plasmon resonance, calcein-release test and hemolysis test. We found that EryB, like its homologue PlyB, forms two-component complexes with aegerolysins and that these complexes are pore-forming. Aegerolysin/EryB complexes are less effective in pore formation than aegerolysin/PlyB complexes, most likely due to different aminoacids at some key sites, that are responsible for binding of proteins with MACPF domain to aegerolysins.
|
