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Protein OlyA6 uvrščamo med egerolizine iz glivnega rodu ostrigarjev (Pleurotus), za katere je značilno, da se vežejo na nevretenčarski sfingolipid ceramid fosfoetanolamin (CPE). OlyA6 in njegova mutanta OlyA6 E69A se razlikujeta v eni sami aminoksilinski substituciji, kjer je glutamat na poziciji 69 zamenjan z alaninom. OlyA6 se veže na že prej formirane lipidne komplekse SM/Hol v membranah, OlyA6 E69A pa se lahko zaradi točkovne mutacije veže na lipidne membrane, ki vsebujejo SM, ki ni v kompleksu s Hol. V sklopu magistrske naloge smo protein OlyA6 E69A izolirali ter analizirali njegovo interakcijo z lipidnimi vezikli, ki vsebujejo CPE z ali brez dodanega holesterola. Prav tako smo preverili litičnost kompleksa OlyA6 E69A v prisotnosti proteinskega partnerja pleurotolizina B (PlyB) na lipidnih veziklih. Ugotovili smo, da se proteinski kompleks OlyA6 E69A/PlyB v primerjavi z OlyA6/PlyB bolje veže na lipidne vezikle, da za vezavo ne potrebuje Hol ter da jih učinkoviteje permeabilizira. Na vezavo in permeabilizacijo OlyA6 E69A vpliva tudi količina CPE ali SM, saj je vezava mutante boljša v primerih, ko je v membranah koncentracija teh lipidov višja. OlyA6 is an aegerolysin protein from the fungal genus Pleurotus, which binds to the sphingolipid enriched in invertebrates, ceramide phosphoethanolamine (CPE). OlyA6 and its mutant OlyA6 E69A differ in a single amino acid where glutamate at position 69 is replaced by alanine. OlyA6 binds to pre-formed SM/cholesterol lipid complexes in the membranes, while OlyA6 E69A can also bind to SM in cholesterol-free lipid membranes. Within this master thesis, the OlyA6 E69A protein was isolated and its interaction with CPE-containing lipid vesicles with or without cholesterol was analyzed. Using lipid vesicles, we also checked the lytic potential of OlyA6 E69A in combination with its protein partner pleurotolysin B (PlyB). We observed that protein complex OlyA6 E69A/PlyB binds better to vesicles, does not require cholesterol for binding, and permeabilizes the membranes more efficiently than the protein complex OlyA6/PlyB. The binding and permeabilization are also affected by the amount of CPE or SM, as mutant binding is better when higher amounts of these lipids are present in the membranes.

Potreba po iskanju novih bioinsekticidov je vedno večja. V tej magistrski nalogi smo testirali proteine egerolizinske družine iz bukovega ostrigarja, ki v kombinaciji s pleurotolizinom B, proteinskim partnerjem z domeno MACPF (ang. membrane-attack domain/perforin) deluje insekticidno na koloradskega in koruznega hrošča, ki spadata v družino lepencev (Chrysomelidae). Insekticidno delovanje je posledica vezave egerolizinskega partnerja na membranski sfingolipidni receptor ceramid fosfoetanolamin (CPE), ki je specifičen za nevretenčarje. Da bi preverili, ali so kompleksi na osnovi egerolizinov toksični tudi za druge hrošče iz družine lepencev, smo komplekse egerolizin/pleurotolizin B testirali še na dveh rastlinskih škodljivcih iste družine: na žitnem strgaču (Oulema melanopus) in na kapusovem bolhaču (Phyllotreta atra). Žuželke smo nabrali na netretiranih poljih in nastavili poskus tako, da smo z egerolizini in egerolizinskimi kompleksi obdelali liste, s katerimi so se žuželke prehranjevale. Naši testi so pokazali občutljivost žitnega strgača na izpostavitev proteinom, medtem ko osebki kapusovega bolhača za to niso bili dovzetni. Z analizo lipidnih ekstraktov žuželk smo preverili prisotnost egerolizinskega receptorja CPE pri žitnem strgaču in kapusovem bolhaču kakor tudi pri drugih rastlinskih škodljivcih (marmorirana smrdljivka, plodova vinska mušica, velika žitna uš, koruzni hrošč in koloradski hrošč). Prisotnost CPE smo dokazali v vseh vzorcih, a v različnih koncentracijah. Najvišjo koncentracijo CPE ima plodova vinska mušica, najmanjšo pa velika žitna uš. Primerjava lipidomskih analiz in testov toksičnosti je pokazala, da koncentracija CPE v membranah nima vloge pri delovanju egerolizinov in egerolizinskih kompleksov na žuželke. The need to find new bioinsecticides is continuously increasing. In this MSc thesis, we tested aegerolysin proteins from oyster mushrooms that had been found to exert insecticidal effects against two plant pests that belong to the Chrysomelidae family: the Colorado potato beetle and the Western corn rootworm. These aegerolysins bind to insect-specific membrane sphingolipid receptor ceramide phosphoethanolamine (CPE) and form transmembrane pores in insect cell membranes in concert with their protein partner pleurotolysin B, which bears a membrane-attack complex/perforin (MACPF) domain. In order to check whether these aegerolysin-based protein complexes are toxic also against other beetles from the Chrysomelidae family, we assessed their toxicity against the cereal leaf beetle (Oulema melanopus) and against the cabbage flea beetle (Phyllotreta atra). The insects were collected on non-treated fields and fed with leaves treated with aegerolysins and aegerolysin/pleurotolysin B mixtures. Our tests showed the sensitivity of cereal leaf beetle to these treatments, while cabbage flea beetles were not susceptible. We also analysed the presence of CPE in lipid extracts from cereal leaf beetle and cabbage flea beetle, as well as from other plant pests (brown marmorated stink bug, Drosophila suzukii, English Grain Aphid, Colorado potato beetle and Western corn rootworm). The presence of CPE was confirmed in all pests’ extracts, but in different concentrations. The highest CPE amount was detected in D. suzukii, and the lowest in English Grain Aphid. The comparison of lipidomic data and toxicity tests showed no correlation between the CPE membrane content and the toxic activity of aegerolysin-based complexes on tested insects.

Parodontoza je ena izmed najpogostejših (vnetnih) bolezni moderne družbe. Za razvoj bolezni so s svojimi virulentnimi dejavniki ključne predvsem parodontalne bakterije, med katere uvršamo tudi bakteriji Tannerella forsythia in Porphyromonas gingivalis. Z namenom iskanja inovativnih načinov diganostike zgodnjih oblik, obvladovanja ali zdravljenja parodontalne bolezni, smo iz bakterij T. forsythia in P. gingivalis izolirali lipide in jim z lipidomskimi analizami določili lastnosti. Rezultati so pokazali prisotnost različnih oblik manj znanega lipida ceramid fosfoetanolamina. Ker je omenjeni lipid v membranah sesalskih celic prisoten v zelo nizkih količinah, bi lahko predstavljal lipidno vrsto, ki jo v ustni votlini najdemo izključno ob prisotnosti parodontalne bolezni in je v korelaciji s povečananim številom parodontalnih bakterij. V magistrski nalogi smo preverjali ali je egerolizin erilizin A primerno molekularno orodje za detekcijo ceramid fosfoetanolamina v umetnih lipidnih sistemih in to tudi potrdili. S kombinacijo erilizina A in njegovega proteinskega partnerja pleurotolizina B smo s testom permeabilizacije veziklov, ki so bili sestavljeni iz celokupnih bakterijskih lipidov, dodatno potrdili vezavo erilizina A na bakterijske lipide ter pokazali, da kombinacija proteinov erilizina A in pleurotolizina B tvori pore v umetnih bakterijskih veziklih in posledično povzroči njihovo lizo. Glede na dobljene rezultate lahko zaključimo, da je (i) erilizin A potencialna molekula za detekcijo ceramid fosfoetanolamina v umetnih lipidnih membranah, sestavljenih iz celokupnega lipidnega ekstrakta parodontalnih bakterij P. gingivalis in T. forsythia in da (ii) erilizin A v kombinaciji s partnerjem pleurotolizinom B izkazuje litični potencial, kar bi lahko izkoristili v zdravljenju parodontalne bolezni. Periodontal disease is one of the most common (inflammatory) diseases nowadays. Responsible organisms for the development of the disease are periodontal bacteria, such as Tannerella forsythia and Porphiromonas gingivalis. To investigate new possibilities in diagnosis and treatment of periodontal disease, we extracted lipids from T. forsythia and P. gingivalis and performed lipidomic analysis. The results showed the presence of several isoforms of the lipid ceramide phosphoethanolamine. This lipid is almost absent in mammalian membranes therefore, it could be used for detecting pariodontal bacteria as their membranes contain ceramide phosphoethanolamine. On the other hand, proteins from the aegerolysin family emerged as suitable molecules for labeling ceramide phosphoethanolamine. Therefore, we have used aegerolysin erylysin A and successfully confirmed the presence of ceramide phosphoethanolamine in lipid extracts of bacteria T. forsythia and P. gingivalis. The binding of erylysin A to vesicles composed of total lipid extracts from T. forsythia and P. gingivalis was also confirmed with the permeabilization test by using erylysin A and its protein partner pleurotolysin B. Based on the obtained results we can conclude that erylysin A is a promising molecule for detection of ceramide phosphoethanolamine in lipid vesicles obtained from the total lipid extracts from periodontal bacteria P. gingivalis and T. forsythia. In combination with pleurotolysin B, erilysin A could also represent a potential solution for the treatment of periodontal disease.

Ostreolizin A (OlyA) iz gobe bukov ostrigar (Pleurotus ostreatus) uvrščamo v proteinsko družino egerolizinov. OlyA prepozna in se veže na membranske domene obogatene s holesterolom in sfingomielinom, t.i. lipidne rafte ter na ceramid fosfoetanolamin (CPE), sfingolipid, ki je specifičen za nevretenčarje in nekatere po Gramu negativne bakterije. Zaradi omenjenih lastnosti imajo OlyA in drugi egerolizini velik biotehnološki potencial, ki se kaže tudi v klinični diagnostiki in v kmetijstvu za zatiranje škodljivcev, saj lahko nekateri egerolizini v kombinaciji s partnerskim proteinom delujejo kot tvorci por s hemolitičnim in citolitičnim delovanjem. Za uspešno uporabo egerolizinov in razvijanje potencialnih aplikacij, je ključnega pomena poznavanje njihovih tarč in specifik vezave. Z metodo točkovnega nanosa smo preverili interakcijo fluorescenčno označenega egerolizina (OlyA-mCherry) s komponentami biofilmov različnih bakterijskih sevov in ugotovili, da v biofilmu teh sevov ni tarč, ki bi jih OlyA prepoznal in na katere bi se vezal. Z metodo točkovnega nanosa in metodo prenosa far-eastern smo preverili interakcijo OlyA-mCherry s polarnimi in nepolarnimi lipidi iz bakterij, kvasovk, nitastih gliv in ene vrste arhej. Očitno interakcijo smo dokazali le z nepolarnimi lipidi bakterije Prevotella sp. Signal je bil zaznan tudi v primeru interakcije OlyA s polarno in nepolarno frakcijo lipidov iz P. aeruginosa EXB L-1125, vendar so ti rezultati nezanesljivi. Na podlagi rezultatov lahko z veliko verjetnostjo ponovno potrdimo specifičnost vezave OlyA s CPE in odsotnost dodatnih membranskih tarč pri veliki večini testiranih mikroorganizmov. Pokazali smo, da OlyA ne interagira s komponentami bakterijskega biofilma in prav tako ne s polarnimi oz. nepolarnimi lipidi iz testiranih bakterij, kvasovk, nitastih gliv in ene vrste arhej. Dobljeni rezultati omogočajo nadaljevanje razvoja uporabe egerolizinov v različnih biotehnoloških aplikacijah. Ostreolysin A (OlyA) from oyster mushroom (Pleurotus ostreatus) is a member of the aegerolysin protein family. OlyA recognizes and binds to membrane domains enriched in cholesterol and sphingomyelin, so-called lipid rafts, and to ceramide phosphoethanolamine (CPE), a sphingolipid specific for invertebrates and some Gram negative bacteria. Due to previously mentioned features, OlyA and other aegerolysins have great biotechnological potential which is particulary shown in fields of clinical diagnostics and agriculture. Some of the aegerolysins, combined with their partner proteins, can act as pore formers and cam exert hemolytic and cytolytic effects. The key to successful use of aegerolysins and development of potential applications is in identifying their targets and in understanding their binding specificity. Using the dot blot method, we tested the interaction between fluorescently-labelled aegerolysin (OlyA-mCherry) and biofilm components of different strains of bacteria. We discovered bacterial biofilms do not contain targets that OlyA would recognize and bind. Using the dot blot and the far-eastern blot method, we examined the interaction of OlyA-mCherry with polar and non-polar lipids in bacteria, yeasts, filamentous fungus and in one species of archaea. The only apparent interaction was observed with non-polar lipids of a bacterium Prevotella spp. A signal was also shown in the case of OlyA interaction with polar and non-polar lipid fraction in P. aeruginosa EXB L-1125 however, these results are not reliable. According to the results we can, with great probability, confirm the specificity of OlyA interaction with CPE and the absence of additonal membrane targets in the majority of the tested microorganisms. We demonstrated that OlyA does not interact with bacterial biofilm components, nor with polar and non-polar lipids in bacteria, yeasts, filamentous fungus and archaea. The results thus allow further development of the use of aegerolysins in different biotechnological applications.

Egerolizini iz gliv ostrigarjev ostreolizin A6 (OlyA6), pleurotolizin A2 (PlyA2) in erilizin A (EryA) interagirajo s ceramid fosfoetanolaminom (CPE), membranskim lipidom specifičnim za nevretenčarje, še posebej za žuželke. Omenjeni lipid se v membranah višjih organizmov vključno s sesalci nahaja v sledovih ali ga sploh ni. Egerolizini, skupaj s proteinskim partnerjem pleuorotolizinom B (PlyB), ki ga proizvaja ista goba, tvorijo transmembranske pore in posledično delujejo toksično na žuželke, ki v svojih membranah vsebujejo omenjeni receptorski lipid. Takšni proteinski kompleksi (egerolizinski proteini + proteinski partner PlyB) kažejo toksičen učinek proti ličinkam dveh, v ekonomskem smislu, najbolj pomembnih rastlinskih škodljivcev - koruznega in koloradskega hrošča. Toksičnost omenjenih proteinskih kompleksov je primerljiva s toksičnostjo proteinskih kristalnih toksinov (proteinov Cry). Omenjene proteine bi lahko uporabili kot biopesticide za zatiranje koloradskega in koruznega hrošča. Aegerolysins from the oyster mushroom ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA) interact with the ceramide phosphoethanolamine (CPE), a membrane lipid specific to invertebrates, especially insects. This lipid is found only in trace amounts, or is completelly absent in the membranes of higher organisms, including mammals. Aegerolysins, together with their partnering protein pleurotolysin B (PlyB) produced by the same mushroom, form transmembrane pores, and therefore have a toxic effect on insects that contain the lipid receptor in their membranes. Such protein complexes (aegerolysin proteins + PlyB) show a toxic effect against larvae of the two economically most important plant pests - the Western Corn rootworm and Colorado potato beetle. The toxicity of these protein complexes is comparable to the toxicity of proteinaceous crystal toxins (Cry proteins). These proteins could be used as biopesticides to control the Colorado potato beetle and Western Corn rootworm.

Protein erilizin B (EryB) uvrščamo med proteine z domeno MACPF (Pfam PF01823). Sintetizira ga goba Pleurotus eryngii, njegova biološka vloga pa je še neznana. Njegov homolog je protein pleurotolizin B (PlyB), ki pa ga sintetizira goba Pleurotus ostreatus. Gobe iz tega rodu prav tako sintetizirajo proteine egerolizine, ki s proteini z domeno MACPF, tvorijo porotvorne komplekse. Dokazano je, da imajo egerolizini specifično afiniteto za vezavo na membrane, sestavljene iz kombinacij sfingomielina (SM) in holesterola ali pa ceramid fosfoetanolamina (CPE) in holesterola, proteini z domeno MACPF pa to vezavo ojačajo in skupaj z egerolizini tvorijo dvokomponentne transmembranske pore. V magistrski nalogi smo EryB sintetizirali v bakteriji Escerichia coli, ga uspešno izolirali in v kombinaciji z egerolizini iz gliv rodu Pleurotus (erilizin A, ostreolizin A6, pleurotolizin A2) preverili njegovo aktivnost z uporabo površinske plazmonske resonance, testom sproščanja kalceina in testom hemolize. Ugotovili smo, da EryB, podobno kot njegov homolog PlyB, tvori dvokomponentne komplekse z egerolizini in da so le ti porotvorni. Kompleksi egerolizin/EryB so manj uspešni pri tvorbi por, v primerjavi s kompleksi egerolizin/PlyB, za kar so najverjetneje odgovorni različni aminokislinski ostanki proteinov z domeno MACPF, ki prepoznajo egerolizine. 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.

Protein erilizin A (EryA), ki ga proizvaja gliva kraljevi ostrigar (Pleurotus eryngii), prištevamo v proteinsko družino egerolizini (Pfam 06355 InterPro IPR009413). Zanj je značilno, da prepozna in se specifično veže na najbolj zastopan nevretenčarski sfingolipid, ceramid fosfoetanolamin. S pomočjo mutageneze v povezavi z verižno reakcijo s polimerazo smo v nukleotidni zapis za EryA uvedli točkovne mutacije in zamenjali pet posameznih aminokislinskih ostankov za alanin (različice EryA(W28A), EryA(E69A), EryA(T98A), EryA(W96A), EryA(K99A)) in enega za tirozin (različica EryA(A91Y)). Omenjene aminokislinske ostanke smo izbrali na podlagi predhodnih raziskav vezave različic egerolizinov ostreolizina A6 (OlyA6) in pleurotolizina A2 (PlyA2) na kombinacijo sfingomielina in holesterola, ter poravnave strukture egerolizina OlyA6 z modelom strukture EryA. Na podlagi poravnave smo sklepali, da sta vezavna žepa proteinov najverjetneje podobno zgrajena, zato smo pet mest za mutagenezo izbrali na podlagi variant OlyA6, ki so pri omenjenem proteinu imele ključno vlogo za vezavo proteina na membrano. EryA nima ohranjenega tirozinskega ostanka Y91, ki je prisoten pri proteinih OlyA6 in PlyA2 in naj bi skupaj s triptofanoma na mestu 28 in 96 tvoril vezavno mesto za lipide. Ker se EryA za razliko od OlyA6 in PlyA2 ne veže na komplekse sfingomielin/holesterol, smo sklepali, da odgovor za specifično interakcijo s ceramid fosfoetanolaminom leži prav tukaj. Uspešno smo izolirali rekombinantni protein EryA in njegovih pet različic. Dokazali smo, da imata različici EryA(A91Y) in EryA(T98A) z EryA primerljivo afiniteto do ceramid fosfoetanolamina v kombinaciji s holesterolom. Posledično imata kompleksa EryA(A91Y)/PlyB in EryA(T98A)/PlyB enako citolitično aktivnost, kot kompleks EryA/PlyB. Hkrati smo dokazali, da aminokislinski ostanek alanin na mestu 91 ni ključen za specifično vezavo proteina na ceramid fosfoetanolamin. Protein erylysin A (EryA) belongs to the protein family of aegerolysins (Pfam 06355 InterPro IPR009413) and is produced by the king oyster mushroom (Pleurotus eryngii). EryA specifically binds to the main invertebrate sphingolipid, ceramide phosphoethanolamine. Point mutations were introduced by mutagenesis to replace selected amino acid residues with alanine (protein variants EryA(W28A), EryA(E69A), EryA(T98A), EryA(W96A), EryA(K99A)) and one with tyrosine (protein variant EryA(A91Y)). These amino acid residues were selected on the basis of previous studies, on the binding of variants of aegerolysins ostreolysin A6 (OlyA6) and pleurotolysin A2 (PlyA2) to sphingomyelin, when combined with cholesterol, and their high degree of similarity with EryA. From the alignment visualization of structure OlyA6 with structure model EryA, we concluded that the protein binding pockets are most likely very similar. EryA does not have the preserved tyrosine residue Y91, which is present in OlyA6 and PlyA2 proteins. Together with tryptophan at site 28 and 96, Y91 is thought to form a lipid binding site. Unlike OlyA6 and PlyA2, EryA does not bind to sphingomylein/cholesterol complexes. Therefore, we concluded that the answer for specific EryA interaction with ceramide phosphoethanolamine could lie here. We successfully isolated the recombinant EryA protein and five variants thereof. We demonstrated that the variants EryA(A91Y) and EryA(T98A) have comparable affinity for binding to ceramide phosphoethanolamine, when combined with cholesterol, as EryA. Consequently, the EryA (A91Y)/PlyB and EryA(T98A)/PlyB complexes have the same cytolytic activity as the EryA/PlyB complex. At the same time, we have shown that amino acid residue of alanine at position 91 is not crucial for the specific binding of the EryA to ceramide phosphoethanolamine.

Plasma membranes of axon-wrapping glial cells develop specific cylindrical bilayer membranes that surround thin individual axons or axon bundles. Axons are wrapped with single layered glial cells in lower organisms whereas in the mammalian nervous system, axons are surrounded with a characteristic complex multilamellar myelin structure. The high content of lipids in myelin suggests that lipids play crucial roles in the structure and function of myelin. The most striking feature of myelin lipids is the high content of galactosylceramide (GalCer). Serological and genetic studies indicate that GalCer plays a key role in the formation and function of the myelin sheath in mammals. In contrast to mammals, Drosophila lacks GalCer. Instead of GalCer, ceramide phosphoethanolamine (CPE) has an important role to ensheath axons with glial cells in Drosophila. GalCer and CPE share similar physical properties: both lipids have a high phase transition temperature and high packing, are immiscible with cholesterol and form helical liposomes. These properties are caused by both the strong headgroup interactions and the tight packing resulting from the small size of the headgroup and the hydrogen bonds between lipid molecules. These results suggest that mammals and Drosophila wrap axons using different lipids but the same conserved principle.

Oyster mushrooms (Pleurotus spp.) have recently been shown to produce insecticidal bi-component protein complexes based on the aegerolysin proteins. A role for these proteins is thus indicated for defence and protection of the mushroom, and we propose their use as new environmentally friendly bioinsecticides. These aegerolysin-based protein complexes permeabilise artificial lipid vesicles through aegerolysin binding to an insect-specific sphingolipid, ceramide phosphoethanolamine (CPE), and they are cytotoxic for the Spodoptera frugiferda (Sf9) insect cell line. Tandem mass spectrometry analysis of the Sf9 lipidome uncovered lipids not previously reported in the literature, including in particular C14 sphingosine-based CPE molecular species, which comprised similar to 4 mol% of the whole lipidome. Further analysis of the lipid binding specificity of an aegerolysin from P. ostreatus, ostreolysin A6 (OlyA6), to lipid vesicles composed of commercial lipids, to lipid vesicles composed of the total lipid extract from Sf9 cells, and to HPLC-separated Sf9 cell lipid fractions containing ceramides, confirmed CPE as the main OlyA6 receptor, but also highlighted the importance of membrane cholesterol for formation of strong and stable interactions of OlyA6 with artificial and natural lipid membranes. Binding assays performed with glycan arrays and surface plasmon resonance, which included invertebrate-specific glycans, excluded these saccharides as potential additional OlyA6 receptors.

Quantitatively and rapidly analyzing lipids is necessary to elucidate their biological functions. Herein, we developed a quantitative method for various lipid classes using supercritical fluid chromatography (SFC) coupled with a charged aerosol detector (CAD), providing high-throughput data analysis to detect a large number of molecules in each lipid class as one peak. Applying the CAD was useful for analyzing lipid molecules in the same lipid class with a constant response under the same mobile phase composition. First, we optimized the washing method for the diethylamine column, achieving baseline separation of lipid classes while maintaining good peak shapes. In addition, the CAD conditions (organic solvent evaporation and numerical correction of the CAD data) were optimized to improve the signal-to-noise ratio. We used an internal standard (ceramide phosphoethanolamine d17:1–12:0), which did not coelute with the lipid classes and showed high extraction efficiency. Based on a quantitative analysis of HepG2 cells, the concentration of lipid classes detected by CAD was adequate compared with that obtained by triple-quadrupole MS (QqQMS) in a previous study because the deviations of the concentrations were 0.6- to 2.3-fold. These results also supported the quantitative performance of SFC-QqQMS developed in our previous report.

Namen magistrske naloge je bil pridobiti egerolizinske proteine OlyA6, PlyA2 in EryA in njihov partnerski protein PlyB z domeno MACPF. Navedeni egerolizin v kompleksu s proteinom PlyB lahko tvori transmembransko neselektivno poro v membranah z ustrezno lipidno sestavo. Kombinacija EryA/PlyB je selektivno aktivna za membrane, ki vsebujejo ceramid fosfoetanolamin in holesterol, medtem ko OlyA6/PlyB in PlyA2/PlyB delujeta tudi na membrane, sestavljene iz sfingomielina in holesterola. Ker naj bi bil ceramid fosfoetanolamin, skupaj s holesterolom, prisoten v membranah določenih žuželk v dovolj visokih koncetracijah za delovanje egerolizinskih kompleksov, smo toksičnost teh testirali na plodovi vinski mušici (Drosophila suzukii), nimfah velike žitne uši (Sitobion avenae), larvah voščene vešče (Galleria mellonella) in larvah hrošča mokarja (Tenebrio molitor). Izkazalo se je, da omenjeni organizmi niso občutljivi na porotvorne komplekse, za kar je lahko odgovorna (1) odsotnost ceramid fosfoetanolamina v membranah žuželk, saj zanje ni neposredno dokazano, da ga imajo, (2) proteolitična razgradnja egerolizinskih kompleksov s črevesnimi encimi, (3) neoptimalen pH za delovanje egerolizinskih kompleksov v črevesju, (4) nedostopnost ustreznih lipidnih domen zaradi izvenceličnega matriksa ali neposrednih stereokemičnih ovir in/ali (5) odsotnost še neznanega koreceptorja, ki je v živih sistemih potreben za dokončno delovanje egerolizinskega kompleksa. The purpose of this master thesis was to obtain aegerolysin proteins OlyA6, PlyA2 and EryA, and their MACPF-partnering protein PlyB. Combinations of the aegerolysins with PlyB are known to form non-selective transmembrane pores on membranes of suitable lipid composition (ceramide phosphoethanolamine or spingomyelin in combination with cholesterol). EryA/PlyB is selective for combination of ceramide phosphoethanolamine and cholesterol, while OlyA6/PlyB and PlyA2/PlyB can also perforate membranes composed of sphingomyelin and cholesterol. Since ceramide phosphoethanolamine, together with cholesterol, is present in certain insect membranes in high enough concentrations for activity of aegerolysin complexes, we decided to evaluate their toxicity on spotted wing Drosophila (Drosophila suzukii), English grain aphid nymphs (Sitobion avenae), mealworm larvae (Tenebrio molitor) and greater wax moth larvae (Galleria mellonella). Results showed that none of the tested animals are susceptible to aegerolysin-based complexes. The reasons might include a (1) lack of ceramide phosphoethanolamine in membranes of these animals since there is no direct evidence of its presence in these particular insects, (2) proteolytic degradation of pore-forming proteins by gut enzymes, (3) unsuitable pH in guts for activity of aegerolysin complexes, (4) inaccessibility of appropriate lipid domains because of extracellular matrix or stereochemical obstructions or (5) absence of yet unknown coreceptor that is essential for definitive function of aegerolysins complexes in living systems.

Pomembna lastnost egerolizinov je vezava na različne lipide, lipidne vezikle ali lipidne dvosloje. Egerolizini lahko oblikujejo pore sami ali v kombinaciji s partnerskim proteinom z domeno MACPF (angl. membrane attack complex/perforin). Zaradi tega so uporabni kot označevalci lipidov in primerni za zatiranje škodljivih organizmov. Eden izmed pomembnih vezavnih partnerjev egerolizinov je ceramid fosfoetanolamin (CPE), sfingolipid, ki je najbolj zastopan v membranah nevretenčarjev (predvsem žuželk) in praživali. V sklopu magistrskega dela smo se osredotočili na tri egerolizine: nigerolizin A2 (NigA2) iz nitaste glive Aspergillus niger, pleurotolizin A2 (PlyA2) iz gobe Pleurotus eryngii in RahU iz bakterije Pseudomonas aeruginosa z namenom bolje opredeliti njihove vezavne lastnosti na lipidne vezikle, sestavljene iz različnih deležev CPE in holesterola (Hol) ali drugih sterolov. To smo preverili s testom sedimentacije in s površinsko plazmonsko resonanco. S testom sproščanja kalceina iz različnih lipidnih veziklov, smo spremljali permeabilizacijsko aktivnost PlyA2 v kombinaciji s partnerskim proteinom pleurotolizinom B (PlyB). Odkrili smo, da je PlyA2 najprimernejši marker za označevanje membran, ki vsebujejo CPE, medtem ko sta NigA2 in RahU za to manj primerna. Za uspešno vezavo PlyA2 na lipidne vezikle je potrebnih vsaj 5 molskih % CPE in 30 molskih % Hol. Prav tako se dani egerolizin veže na lipidne vezikle sestavljene iz CPE in različnih sterolov (najmočnejša vezava je bila zaznana v primeru 7-dehidroholesterola in β-sitosterola). Pokazali smo tudi, da PlyA2 v kombinaciji s PlyB permeabilizira membrane lipidnih veziklov, ki vsebujejo CPE in različne sterole, kar nakazuje na možnost uporabe teh dveh proteinov za zatiranje škodljivih organizmov. Important feature of aegerolysins is binding to various lipids, lipid vesicles or lipid bilayers. Aegerolysins can form pores directly or in combination with a partnering protein containing a MACPF (membrane attack complex/perforin) domain. They can therefore be used as specific lipid markers and for the pest control. One of the important binding partners of aegerolysins is ceramide phosphoethanolamine (CPE), a sphingolipid that is very abundant in the membranes of the invertebrates (particularly insects) and protozoa. Within M. Sc. Thesis we focused on three aegerolysins: nigerolysin A2 (NigA2) from the filamentous fungus Aspergillus niger, pleurotolysin A2 (PlyA2) from the mushroom Pleurotus eryngii and RahU from the bacterium Pseudomonas aeruginosa. Our aim was to determine their binding characteristics to the lipid vesicles composed of different molar ratios of CPE and cholesterol (Chol) or other sterols using sedimentation assay and surface plasmon resonance. Monitoring the calcein release from various lipid vesicles, the permeabilization activity of aegerolysin PlyA2 in combination with its partnering protein, pleurotolysin B (PlyB), was tested. We have discovered that PlyA2 is the most appropriate marker for labeling membranes containg CPE, while NigA2 and RahU are less suitable. For the successful binding of PlyA2 to lipid vesicles, at least 5 mol % of CPE and 30 mol % of Chol are required. In addition, PlyA2 binds to lipid vesicles composed of CPE and various sterols (the strongest binding was detected in the case of 7-dehydrocholesterol and β-sitosterol). We have also shown that PlyA2 in combination with PlyB permeabilizes membranes of lipid vesicles containing CPE and various sterols, indicating their potential to be used as pest control agents.

Ceramide phosphoethanolamine (CPE) is the major sphingolipid in invertebrates and in some bacterial species. It has been also detected in mammalian cells, although only in trace amounts. Complete understanding of the biophysical and physiological relevance of CPE is still lacking, and its biological role is still an open question. CPE differs in its biosynthetic mechanisms from sphingomyelin, due to the specific CPE synthase in invertebrates. In contrast to well-established sphingomyelin/cholesterol interactions that result in the formation of ordered membrane domains, the formation of ordered CPE/cholesterol domains is not favored. CPE might be crucial for the early development of Drosophila melanogaster, and it might be involved in the developmental stages of Trypanosoma brucei. As a Bacteroidetes-associated sphingolipid, CPE might also be involved in maintenance of these bacteria in their ecological niches. Therefore, efficient detection of CPE in biological systems is needed to better define its distribution and biological role(s).

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, sphingomyelin synthase-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmatic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with glutamic acid permitting SMS-catalyzed CPE production and aspartic acid confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.

International audience; Pore-forming toxins (PFTs) represent a unique class of highly specific lipid-binding proteins. The cytotoxicity of these compounds has been overcome through crystallographic structure and mutation studies, facilitating the development of non-toxic lipid probes. As a consequence, non-toxic PFTs have been utilized as highly specific probes to visualize the diversity and dynamics of lipid nanostructures in living and fixed cells. This review is focused on the application of PFTs and their non-toxic analogs as tools to visualize sphingomyelin and ceramide phosphoethanolamine, two major phosphosphingolipids in mammalian and insect cells, respectively. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16- and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine, but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma’s, but only 3% of the host’s, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine, but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host’s immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen.

Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family—pleurotolysin A2 (PlyA2; K(D) = 12 nM), ostreolysin (Oly; K(D) = 1.3 nM), and erylysin A (EryA; K(D) = 1.3 nM)—strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K(D) = 41 μM), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.

Sphingolipids are vital components of cellular membranes that provide mechanical stability and play key roles in signal transduction, cell recognition and molecular sorting. They are synthesized from ceramide, a potent mediator of programmed cell death. Hence, cells face the dilemma of how to generate sufficient amounts of sphingolipids without killing themselves in the process. Ceramides are produced in the endoplasmic reticulum (ER) and transported to the Golgi for conversion to sphingomyelin (SM), the major sphingolipid in human cells. Cloning of the Golgi-resident SM synthase (SMS) uncovered a family of SMS enzymes. We found that one family member, SMSr, synthesizes the SM analogue ceramide phosphoethanolamine (CPE) in the ER. SMSr produces only trace amounts of CPE but blocking its catalytic activity causes a substantial rise in ER ceramide levels and triggers a mitochondrial pathway of apoptosis. SMSr-depleted cells become resistant to apoptosis upon disruption of ER-mitochondria contact sites or metabolic conversion of mitochondrial ceramides, indicating that apoptosis is triggered by mistargeting of ER ceramides to mitochondria. We also found that SMSr carries a SAM domain involved in polymeric protein-protein interactions. While SAM is dispensable for SMSr-catalysed CPE production, its removal proved sufficient to trigger ER ceramide accumulation and cell death. Our findings indicate that SMSr is an ER-resident ceramide sensor with a crucial role in protecting cells against ceramide-induced apoptosis. Since ceramide is intimately involved in the regulation of cancer cell-growth and survival, SMSr provides a legitimate target for modulating drug-induced cell death in tumors.

Sphingolipids are vital components of cellular membranes that provide mechanical stability and play key roles in signal transduction, cell recognition and molecular sorting. They are synthesized from ceramide, a potent mediator of programmed cell death. Hence, cells face the dilemma of how to generate sufficient amounts of sphingolipids without killing themselves in the process. Ceramides are produced in the endoplasmic reticulum (ER) and transported to the Golgi for conversion to sphingomyelin (SM), the major sphingolipid in human cells. Cloning of the Golgi-resident SM synthase (SMS) uncovered a family of SMS enzymes. We found that one family member, SMSr, synthesizes the SM analogue ceramide phosphoethanolamine (CPE) in the ER. SMSr produces only trace amounts of CPE but blocking its catalytic activity causes a substantial rise in ER ceramide levels and triggers a mitochondrial pathway of apoptosis. SMSr-depleted cells become resistant to apoptosis upon disruption of ER-mitochondria contact sites or metabolic conversion of mitochondrial ceramides, indicating that apoptosis is triggered by mistargeting of ER ceramides to mitochondria. We also found that SMSr carries a SAM domain involved in polymeric protein-protein interactions. While SAM is dispensable for SMSr-catalysed CPE production, its removal proved sufficient to trigger ER ceramide accumulation and cell death. Our findings indicate that SMSr is an ER-resident ceramide sensor with a crucial role in protecting cells against ceramide-induced apoptosis. Since ceramide is intimately involved in the regulation of cancer cell-growth and survival, SMSr provides a legitimate target for modulating drug-induced cell death in tumors.

ABBREVIATIONS ]. INTRODUCTION II. PHOSPHOSPHINGOLIPIDS A. Phosphonosphingolipids I. Species distribution B. Ceramide phosphoethanolamine IlL GLYCOSPHINGOLIPIDS A. Nomenclature for GSL of arthropods and molluscs B. Preparation and purification C. Glycosphingnlipids in molluscs 1. Bivalves 2. Sea snails 3. Sea bare D. Glycosphingolipids in arthropods 1. Insects 2. Crustacea E. Glycosphingnlipids in parasites F. Glycosphingnlipids in annelids G. Glycosphingolipids in echinoderms IV. IMMUNOLOGICAL STUDIES A. Antibodies against mannose-containing lipids (mannolipids) from freshwater bivalves and insects 1. Anti-Ml2Cer antibody 2. Anti-Ml4Cer antibody 3. Anti-V3~,GalNAc3Me,V4flGIcA4Me-MIsCer antibody 4. Anti-Ar3Cer antibody B. Antibodies against PnGSL from sea hare, Aplysia kurodui I. Anti-SGL-II antibody 2. Anti-FGL-Ilb antibody C. Antibody against ganglioside from sea urchin Eggs 93 ACKNOWLEDGEMENTS REFESmNCm 25 26 26 26 26 26 28 29 29 30 30 32 32 33 35 37 37 38 40 40

Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an equimolar mixture of bovine buttermilk CPE (milk CPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (diC18:1 PC) tends to form tubules and helical ribbons, while pure milk CPE mainly exhibits amorphous aggregates and, at low frequency, straight needles. Negative staining electron microscopy indicated that helices and tubules were composed of multilayered 5–10 nm thick slab-like structures. Using different molecular species of PC and CPE, we demonstrated that the acyl chain length of CPE but not of PC is crucial for the formation of tubules and helices in equimolar mixtures. Incubation of the lipid suspensions at the respective phase transition temperature of CPE facilitated the formation of both tubules and helices, suggesting a dynamic lipid rearrangement during formation. Substituting diC18:1 PC with diC18:1 PE or diC18:1 PS failed to form tubules and helices. As hydrated galactosylceramide (GalCer), a major lipid in mammalian myelin, has been reported to spontaneously form tubules and helices, it is believed that the ensheathment of axons in mammals and Drosophila is based on similar physical processes with different lipids.