Vezava izbranih fluorescenčno označenih egerolizinov, ostreolizina A in erilizina A na in vitro kulturo nediferenciranih živčnih celic in človeške celice ustne sluznice

Gregorčič, Tanja (avtor), Sepčić, Kristina (mentor), Skočaj, Matej (komentor)

Egerolizini so družina proteinov, katerih pomembna lastnost je vezava na specifične lipide in lipidne membranske mikrodomene, zato so primerno orodje za označevanje ali tarčno uničevanje specifičnih celičnih vrst. Ostreolizin A (OlyA) je egerolizin iz užitne gobe Pleurotus ostreatus. Za OlyA je značilno, da se specifično veže na naravne in umetne lipidne membrane, ki so obogatene s sfingomielinom in holesterolom. Pred kratkim so iz gobe Pleurotus eryngii izolirali protein erilizin A (EryA), ki se veže na ceramid fosfoetanolamin (CPE), sfingolipid prisoten v plazmalemi žuželk, v nekaterih parazitih in bakterijah. V okviru diplomskega dela smo preverili sposobnost vezave rekombinantnih fluorescenčno označenih egerolizinov OlyA-eGFP in EryA-mCherry na celice človeške ustne sluznice. Ugotovili smo, da se protein OlyA-eGFP veže na plazmalemo celic človeške ustne sluznice, medtem ko se EryA-mCherry na iste celice, za katere je znano, da ne vsebujejo CPE, ni vezal. Proučevali smo tudi vezavo OlyA-eGFP in EryA-mCherry na kulturo nediferenciranih živčnih celic, katerih plazmalema naj bi poleg prevladujočega sfingomielina in holesterola vsebovala tudi nizko količino CPE. Ugotovili smo, da se OlyA-eGFP veže na plazmalemo teh celic in da je torej primeren membranski označevalec s sfingomielinom in holesterolom obogatenih domen plazmaleme tudi nediferenciranih živčnih celic, medtem ko se protein EryA-mCherry zaradi verjetno prenizke količine CPE v plazmalemi nediferenciranih živčnih celic na te celice ni vezal in torej ni primerna molekula za označevanje CPE v plazmalemi.

• Jezik: Slovenski jezik
• Ključne besede: ostreolizin A
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: PEF - Pedagoška fakulteta
• Leto izida: 2017
• PID: 20.500.12556/RUL-95820
• COBISS.SI-ID: 11725641
• Datum objave v RUL: 22.09.2017

GREGORČIČ, Tanja, 2017, Vezava izbranih fluorescenčno označenih egerolizinov, ostreolizina A in erilizina A na in vitro kulturo nediferenciranih živčnih celic in človeške celice ustne sluznice [na spletu]. Diplomsko delo. [Dostopano 2 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=95820

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: binding of fluorescently labelled aegerolysins ostreolysin A and erylysin A to in vitro culture of non-differentiated neurons and oral mucose cells

Izvleček

Members of the aegerolysins protein family are unique in their binding to specific lipids and lipid membrane domains. They can be used as specific membrane markers, or as tools for targeting specific cell types. Ostreolysin A (OlyA) is an aegerolysin from the edible oyster mushroom Pleurotus ostreatus. OlyA was shown to bind to biological and artificial lipid membranes enriched with sphingomyelin and cholesterol. Recently discovered erylysin A (EryA) from the mushroom Pleurotus eryngii specifically binds to ceramide phosphoethanolamine (CPE), a sphingolipid found in higher quantities in cell membranes of insects, some parasites and bacteria. We tested the lipid-binding activity of recombinant fluorescently-labelled aegerolysins OlyA-eGFP and EryA-mCherry to oral mucose cells. We found that OlyA-eGFP binds to plasma membrane of oral mucose cells, but the binding of EryA-mCherry on plasma membrane of oral mucose cells, which do not contain CPE, was not shown. We tested the binding specifity of OlyA-eGFP in EryA-mCherry on in-vitro culture of non-differntiated neurons, whose plasma membrane is enriched with sphingomyelin and cholesterol, and also contain trace amount of CPE. We found that OlyA-eGFP binds on plasma membrane of these cells and therefore could be used as a specific membrane marker for membrane rafts in neural cells. The binding of EryA-mCherry on non-differntiated neurons was not detected – probably because of too low amount of CPE in their plasma membranes - and thus EryA could not be used as specific membrane marker for CPE in these cells.

• Ključne besede: ostreolysin A