TCDB is operated by the Saier Lab Bioinformatics Group

1.C.97 The Pleurotolysin Pore-Forming (Pleurotolysin) Family

Pleurotolysin, a sphingomyelin-specific cytolysin consisting of A (17 kDa) and B (59 kDa) components from the basidiomycete Pleurotus ostreatus, assembles into a transmembrane pore complex. Sakurai et al. 2004 cloned complementary and genomic DNAs encoding pleurotolysin, and studied pore-forming properties of recombinant proteins. Recombinant pleurotolysin A lacking the first methionine was purified as a 17-kDa protein with sphingomyelin-binding activity. The cDNA for pleurotolysin B encoded a precursor consisting of 523 amino acid residues, of which N-terminal 48 amino acid residues were absent in natural pleurotolysin B. Mature and precursor forms of pleurotolysin B were expressed as insoluble 59- and 63-kDa proteins, respectively. Although neither recombinant pleurotolysin A nor B alone was hemolytically active at higher concentrations of up to 100 mg/ml, they cooperatively assembled into a membrane pore complex on human erythrocytes and lysed the cell.

Tomita et al. 2004 purified pleurotolysin, a sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied pore-formation. Pleurotolysin caused leakage of potassium ions from human erythrocytes and formed membrane pores with a functional diameter of 4-5 nm. Pleurotolysin-induced lysis of human erythrocytes was inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B.  Pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes.

Another two-component hemolysin, erylysin A and B (EryA and EryB), was isolated from an edible mushroom, Pleurotus eryngii (Shibata et al. 2010). Hemolytic activity was exhibited only by the EryA and EryB mixture. EryA showed one band on SDS-PAGE while EryB showed two bands at 15 kDa (EryB1) and 37 kDa (EryB2).  At pH 7.2, EryA exists as a homodimer whereas EryB exists as a heterodimer of B1 and B2. CD spectrum analysis showed T(m) values of 47°C and 37°C for EryA and EryB, respectively. EryB was particularly unstable.

While Pleurotolysin B is in the MACPF superfamily (1.C.39), Pleurotolysin A is in the Aegerolysin superfamily. Several members of the Aegerolysin family have been used as tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates but not in animals (Bhat et al. 2015).  It may be distantly related to members of the Equinatoxin Family (1.C.38). 

Proteins with membrane-attack complex/perforin (MACPF) domains have a variety of biological roles, including defence and attack, organismal development, and cell adhesion and signalling. The distribution of these proteins in fungi appears to be restricted to some Pezizomycotina and Basidiomycota species only, in correlation with the aegerolysins. These two protein groups coincide in only a few species, and they operate as cytolytic bi-component pore-forming agents (Ota et al. 2014). Representative proteins include pleurotolysin B, which has a MACPF domain, and the aegerolysin-like protein pleurotolysin A, and the very similar ostreolysin A, which have been purified from oyster mushroom (Pleurotus ostreatus). These act in concert to perforate natural and artificial lipid membranes with high cholesterol and sphingomyelin contents. The complex has a 13-meric rosette-like structure with a central lumen that is ~4-5 nm in diameter. The opened transmembrane pore is non-selectively permeable to ions and smaller neutral solutes, and is a cause of cytolysis of a colloid-osmotic type. The biological significance of these proteins for the fungal life-style has been discussed (Ota et al. 2014).

The aegerolysin family consists of several bacterial and eukaryotic aegerolysin-like proteins. It has been found that aegerolysin and ostreolysin are expressed during formation of primordia and fruiting bodies and possibly play a role in the initial phase of fungal fruiting. The bacterial members of this family are expressed during sporulation. Ostreolysin is cytolytic to various erythrocytes and tumor cells because of pore formation (Berne et al. 2002; Berne et al. 2002; Berne et al. 2009). 

In this TC family, both constituents of pleurotolysin and ostreolysin (A and B) are included under TC#s 1.C.97.1.1 and 1.2, respectively.  However, homologues of Pleurotolysin B are found under TC #s 1.C.97.1.3 - 1.9 while homologues of Pleurotolysin A are found under TC #s 1/C/97.2.1 - 2.4 and 3.1 - 3.8.  Pleurotolysins A are not homologous to Pleurotolysins B.  While some homologues depend on the presence of both constituents for pore formation, as noted for both pleurotolysin and ostreolysin, some homologues of both A and B can  form pores without the other.

This family belongs to the: MACPF Superfamily.

References associated with 1.C.97 family:

Berne, S., I. Krizaj, F. Pohleven, T. Turk, P. Macek, and K. Sepcić. (2002). Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting. Biochim. Biophys. Acta. 1570: 153-159. 12020804
Berne, S., K. Sepcić, G. Anderluh, T. Turk, P. Macek, and N. Poklar Ulrih. (2005). Effect of pH on the pore forming activity and conformational stability of ostreolysin, a lipid raft-binding protein from the edible mushroom Pleurotus ostreatus. Biochemistry 44: 11137-11147. 16101298
Berne, S., L. Lah, and K. Sepcić. (2009). Aegerolysins: structure, function, and putative biological role. Protein. Sci. 18: 694-706. 19309687
Bhat HB., Ishitsuka R., Inaba T., Murate M., Abe M., Makino A., Kohyama-Koganeya A., Nagao K., Kurahashi A., Kishimoto T., Tahara M., Yamano A., Nagamune K., Hirabayashi Y., Juni N., Umeda M., Fujimori F., Nishibori K., Yamaji-Hasegawa A., Greimel P. and Kobayashi T. (2015). Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates. FASEB J. 29(9):3920-34. 26060215
Lukoyanova, N., S.C. Kondos, I. Farabella, R.H. Law, C.F. Reboul, T.T. Caradoc-Davies, B.A. Spicer, O. Kleifeld, D.A. Traore, S.M. Ekkel, I. Voskoboinik, J.A. Trapani, T. Hatfaludi, K. Oliver, E.M. Hotze, R.K. Tweten, J.C. Whisstock, M. Topf, H.R. Saibil, and M.A. Dunstone. (2015). Conformational Changes during Pore Formation by the Perforin-Related Protein Pleurotolysin. PLoS Biol 13: e1002049. 25654333
Ota, K., M. Butala, G. Viero, M. Dalla Serra, K. Sepčić, and P. Maček. (2014). Fungal MACPF-like proteins and aegerolysins: bi-component pore-forming proteins? Subcell Biochem 80: 271-291. 24798017
Sakurai, N., J. Kaneko, Y. Kamio, and T. Tomita. (2004). Cloning, expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus. Biochim. Biophys. Acta. 1679: 65-73. 15245918
Schlumberger S., Kristan KC., Ota K., Frangez R., Molgomicron J., Sepcic K., Benoit E. and Macek P. (2014). Permeability characteristics of cell-membrane pores induced by ostreolysin A/pleurotolysin B, binary pore-forming proteins from the oyster mushroom. FEBS Lett. 588(1):35-40. 24211835
Shibata, T., M. Kudou, Y. Hoshi, A. Kudo, N. Nanashima, and K. Miyairi. (2010). Isolation and characterization of a novel two-component hemolysin, erylysin A and B, from an edible mushroom, Pleurotus eryngii. Toxicon 56: 1436-1442. 20816689
Tomita, T., K. Noguchi, H. Mimuro, F. Ukaji, K. Ito, N. Sugawara-Tomita, and Y. Hashimoto. (2004). Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex. J. Biol. Chem. 279: 26975-26982. 15084605