# Towards Understanding the Function of Aegerolysins
## 2 Aegerolysins
### Fungal Aegerolysins from Basidiomycota--Agaricomycotina
#### 2.1.1. Aegerolysins from Pleurotus ostreatus
However, as in the case of **Ply**, it was later revised that native isolate of **Oly** (**OlyA**) was contaminated with **PlyB**, which was responsible for the observed cytolytic effects [72]. Ostreolysin A6 (**OlyA6**), which has 78% identity (31 amino acids substitution) with **OlyA**[11], was expressed as recombinant protein in 2013 [72]. First experiments showed that **OlyA6** specifically bind to SM/Chol-rich lipid membranes and has very similar binding properties to **PlyA**[72]. Recently, **OlyA6** was confirmed to bind to SM/Chol lipid complexes and not to free SM (PDB ID: 6 MY) [73]. **OlyA6** was later fused to the fluorescent protein mCherry, and was used as a lipid raft marker [74]. **OlyA6**-mCherry was shown to be non-toxic to vertebrate cells and, due to its stability and small size, it became an important tool for studying lipid rafts [74]. **OlyA6**-mCherry stained lipid rafts in plasma membranes in live and fixed Madin-Darby canine kidney (MDCK) cells did not colocalize with other raft markers, that sense individual raft-residing lipids, and did not bind to intracellular membranes, and its binding was abolished by methyl-\(\beta\)-cyclodextrintr or sphingomyelinase pretreatment [74]. However, it has been observed that excessive concentrations of **OlyA6** or **Oly6**-mCherry can lead to membrane vesiculation [72; 74; 75]. In addition, **OlyA6**-mCherry applied to live MDCK cells was internalized via caveolin-dependent pathway and was detected near the Golgi complex after 90 min [74]. Similar to the combination of **PlyA** and **PlyB** or **OlyA** and **PlyB**, the mixtures of **OlyA6** and **PlyB** form transmembrane pores,  
Figure 1: Pore-forming pleurotolysin complex of _Pleurotus ostreatus_. The hetero 39-mer structure of the pore was solved (PDB ID: 4V2T) [60]; it consists of 26 aegypti molecules PlyA and 13 MACPF protein molecules PlyB; the size of the pore is 8 \(\times\) 10 nm. (**a**) Bottom view; (**b**) Top view; PlyA, pleurotolysin A—in grey, magenta, and orange; PlyB, pleurotolysin B—in cian. Presentation of the structure by PyMOL [62]. leading to cell lysis [72]. The combination of lipid raft sensing **OlyA6** and **PlyB** can be used as anticancer treatment for cancers with increased concentration of lipid rafts in their membranes as is the case in urothelial cancer cells [76]. Recently, it was revealed that **OlyA6** has another high affinity lipid receptor, ceramide phosphoethanolamine (CPE), which is specific for cell membranes of invertebrates and some bacteria [77; 78; 79]. **OlyA6** binds 1000-fold more strongly to vesicles composed of CPE and Chol than to vesicles composed of SM/Chol [80], and it also binds to membranes with physiological concentrations (5 mol%) of CPE [78; 79]. In addition to labelling lipid rafts, **OlyA6** can also be used as a marker to label CPE in insect cells and tissues [79; 80; 81]. Furthermore, **OlyA6**/**PlyB** protein mixtures permeabilized lipid vesicles containing \(\leq\)5 mol% CPE and are toxic to CPE-containing Sf9 cells, as well as to the larvae of Colorado potato beetle (_Leptinotaxis decmelinenta_ [PERSON], 1824) and western corn rootworm (_Diabrotica virgifera_ virgifera_ [PERSON], 1868), and thus have been proposed as new environmentally friendly biopesticides [10; 79] with low toxicity for non-target organisms [82] and no toxicity for humans after ingestion due to their fast decomposition by mammalian digestive enzymes [83]. It must be noted, however, that the intravenous application of the native **OlyA6**/**PlyB** isolate in rodents resulted in a cardiorespiratory arrest and can induce severe histopathological changes in kidneys, lungs, and heart [84; 85]. The half lethal dose (LD50) in mice is 1.17 mg/kg [86]. Very recently, another lipid receptor of **OlyA6**, ceramide aminoethylphosphonate (CAEP), a CPE analogue of marine invertebrates such as mollusks and cnidarians, was identified [87]. Moreover, **OlyA6**/**PlyB** complexes triggered strong lysis of CAEP-lipid containing vesicles. In 2017, another aegerolysin from _P.