Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein, pleurotolysin B

Abstract

The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.

Keywords: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Aegerolysin; C-terminally hexa-histidine tagged OlyA6; CDC; Cholesterol-dependent cytolysin; H(6)-OlyA6; H(6)-Δ48PlyB; Lipid-binding protein; Membrane attack complex; N-terminally hexa-histidine-tagged N-truncated precursor of PlyB (Acc. code AB177872, lacking 48 amino acids at N-terminus); N-terminally hexa-histidine-tagged OlyA6; OlyA6; OlyA6-H(6); POPC; Pleurotolysin; Pleurotus ostreatus; cholesterol-dependent cytolysin; nOlyA; nPlyB; native ostreolysin A; native pleurotolysin B; ostreolysin A6.