Effect of pH on the Pore Forming Activity and Conformational Stability of Ostreolysin, a Lipid Raft-Binding Protein from the Edible MushroomPleurotus ostreatus^,

Abstract

Ostreolysin, a pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is a member of the aegerolysin protein family, a novel group of small acidic proteins found in bacteria, molds, mushrooms, and plants. It binds to lipid rafts and interacts specifically with cholesterol-rich lipid domains. In this study, ostreolysin was classified as a single-domain all-beta-structured protein on the basis of cDNA sequencing. pH-induced and thermally induced unfolding of ostreolysin was studied by means of CD, UV absorption, and intrinsic tryptophan fluorescence to characterize conformational transitions associated with its functional properties, i.e., binding to lipid membranes, pore forming activity on lipid vesicles, and hemolysis. At 25 degrees C and between pH 6 and 9, ostreolysin adopted a monomeric and thermodynamically stable nativelike conformation, characterized by rigid tertiary structure and predominantly beta-sheet secondary structure. Between pH 2 and 3, the protein underwent an irreversible transition to a partially unfolded, molten globule-like state which bound ANS, and exhibited disrupted tertiary structure and enhanced non-native alpha-helical structure. Functional studies showed that, unlike colicins and some other bacterial pore-forming toxins, the acid-induced molten globule-like state of ostreolysin is not relevant for lipid binding and pore formation. Instead, the compact native state was necessary for binding to cholesterol/sphingomyelin multilamellar vesicles, optimally in the pH range from 6 to 7, and for pore formation and hemolysis, maximally between pH 7 and 8.

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

“…Due to the absence of suffi ciently homologous protein templates, the PlyA2 model was generated via an iterative of the folding state as well as the binding mechanism between members of the aegerolysin and actinoporin families has been proposed, based on circular dichroism (CD) and fl uorescence spectra ( 45 ). Resubmission of PlyA2 sequence aligned to FraC produced the second PlyA2 model ( 44 ).…”

Toward Elucidating the Membrane Topology of Helix Two of the Colicin E1 Channel Domain

“…Colicins can be grouped into the following three categories based on their mode of action: (i) membrane depolarization via formation of ion-conducting channels (2), (ii) inhibition of protein (3) or peptidoglycan synthesis (4); and (iii) DNA degradation (5). Because of their ability to cross the Gram-negative bacterial membrane, colicins have become a model for studying bacterial protein import (6) as well as protein unfolding and folding (7,8), membrane insertion (9,10), and pore formation (11).…”

Characterization of a 62-Kilodalton Acidic Phospholipid-Binding Protein Isolated from the Edible Mushroom Pleurotus ostreatus

“…13,17) Pleurotolysin A specifically bound to SM/cholesterol vesicles and formed a transmembrane pore in concert with pleurotolysin B. Ostreolysin was also isolated as a 16-kDa hemolytic protein from the same mushroom, which was suggested to specifically recognize a cholesterol-rich lipid domain. [18][19][20] In the present study, we detected a 62-kDa protein (p62) from P. ostreatus and showed that p62 binds to lipid vesicles containing acidic phospholipids, especially phosphatidylglycerol (PG). PC (POPC), egg yolk phosphatidylethanolamine, bovine brain SM, bovine brain PS, bovine liver phosphatidylinositol, egg yolk phosphatidic acid, egg yolk PG, and bovine heart cardiolipin were purchased from Sigma (St. Louis, MO, U.S.A.).…”