magistrsko delo
Alen Vukovič (Author), Peter Veranič (Reviewer), Nataša Resnik (Mentor), Matej Skočaj (Co-mentor)

Abstract

E69A je mutirana različica proteina OlyA6, ki ga proizvaja goba bukov ostrigar (Pleurotus ostreatus). Strukturno se E69A od OlyA6 razlikuje na 69. mestu, v točkovni mutaciji glutaminske kisline v alanin in se za razliko od OlyA6, ki prepozna zgolj komplekse sfingomielina in holesterola v membranskih raftih, veže tudi na prost sfingomielin. Namen magistrske naloge je bil preveriti vezavo E69A na membrane epitelijskih sesalskih celic MDCK in primerjati označbo z OlyA6. OlyA6 smo pridobili v rekombinantni obliki z zelenim fluorescenčnim označevalcem eGFP (OlyA6-eGFP) in E69A z rdečim fluorescenčnim označevalcem mCherry (E69A-mCherry) ter ju uporabili za označevanje MDCK celic. Z metodo fluorescenčne in konfokalne mikroskopije smo ugotovili, da E69A-mCherry označi plazmaleme celic pri nižji koncen-traciji in v krajšem času kot OlyA6-eGFP. S fizikalno fiksacijo, kjer je prišlo do naluknanja plazmalem, smo pokazali, da E69A označi tudi znotrajcelične membrane. V primeru odtegnitve celičnega holesterola z metil-beta-ciklodekstrinom E69A še vedno označi plazmaleme, kar pa ne velja za OlyA6, kjer do označbe ne pride. Nobeden od proteinov se ne veže na celice MDCK, če jih predhodno tretiramo s sfingomielinazo. Inkubacija celic z obema proteinoma je pokazala, da je delež kolokalizacije med OlyA6 in E69A razmeroma nizek, neodvisno od hkratnega ali zaporednega dodajanja proteinov celicam. Dokazali smo, da E69A označi področja na plazmalemi pri nižji koncentraciji in krajšem času inkubacije kot OlyA6 ter da oba proteina na plazmalemi zasedata skupna mesta v manjšem deležu kot različna mesta, iz česar sklepamo, da E69A označi več področij membran (raftni in neraftni sfingomielin) kot OlyA6.

Keywords

ostreolizin A6;holesterol;sfingomielin;eGFP;mCherry;označba membran;membranski rafti;celična linija MDCK;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL MF - Faculty of Medicine
Publisher: [A. Vuković ]
UDC: 576
COBISS: 146372099 Link will open in a new window
Views: 45
Downloads: 9
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Other data

Secondary language: English
Secondary title: Interaction of mutated variant of protein ostreolysin A6 E69A with biological membranes
Secondary abstract: E69A is a mutated version of the OlyA6 protein produced by the oyster mushroom (Pleurotus ostreatus). Structure of E69A differs from OlyA6 at position 69, in the point mutation of glutamic acid to alanine, and unlike OlyA6, which only recognizes sphingomyelin and cholesterol complexes in membrane rafts, it also binds to free sphingomyelin. The aim of the master's thesis was to verify the binding of E69A to the membranes of MDCK mammalian epithelial cells and to compare the labeling with OlyA6. OlyA6 was obtained in recombinant form with the green fluorescent marker eGFP (OlyA6-eGFP) and E69A with the red fluorescent marker mCherry (E69A-mCherry) and both were used to label MDCK cells. Using fluorescence and confocal microscopy, we revealed that E69A-mCherry labels the plasmalemma of cells at a lower concentration and in a shorter time than OlyA6-eGFP. By damaging the plasmalemma with physical fixation, we showed that E69A also labels intracellular membranes. The removal of cellular cholesterol with methyl-beta-cyclodextrin abol-ishes only OlyA6 labeling, but not E69A labeling. Neither E69A or OlyA6 binds to MDCK when cells were treated with sphingomyelinase. Incubation of cells with both proteins showed that the proportion of colocalization between OlyA6 and E69A was relatively low, regardless of the simultaneous or sequential addition of the proteins to the cells. We demonstrated that E69A labels the plasmalemma at a lower concentration and shorter incubation time than OlyA6, and that both proteins occupy common sites on the plasmalemma in a smaller proportion than different sites, from which we conclude that E69A labels several areas of membranes (raft and non-raft sphingomyelin) than OlyA6.
Secondary keywords: ostreolysin A6;cholesterol;sphingomyelin eGFP;mCherry;membrane labeling;membrane rafts;MDCK cell line;master thesis;Citologija;Univerzitetna in visokošolska dela;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za biologijo
Pages: 1 spletni vir (1 datoteka PDF (IX, 55 str.))
ID: 18219988