Izolacija mutirane različice proteina ostreolizina A6, OlyA6 E69A, in njena interakcija z umetnimi lipidnimi membranami : magistrsko delo

Cerjak, Nastja (author), Sepčić, Kristina (mentor), Panevska, Anastasija (co-mentor), Skočaj, Matej (thesis defence commission member), Turk, Tom (thesis defence commission member)

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.

• Language: Slovenian
• Keywords: ceramid fosfoetanolamin, lipidna membrana, ostreolizin A6, OlyA6 E69A, sfingomielin
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [N. Cerjak]
• Year: 2022
• Number of pages: XI, 48 str.
• PID: 20.500.12556/RUL-134838
• UDC: 577:543.384(043.2)
• COBISS.SI-ID: 97120259
• Publication date in RUL: 04.02.2022

Secondary language

• Language: English
• Title: Isolation of a mutated version of the ostreolysin A6 protein, OlyA6 E69A, and its interaction with artificial lipid membranes : magistrski študij - 2. stopnja

Abstract

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.

• Keywords: ceramide phosphoethanolamine, lipid membrane, ostreolysin A6, OlyA6 E69A, sphingomyelin