No public access
master's thesis
Protein ostrelysine A interactions with liposomes composed by phosphatidylcholine, cholesterol and ceramide phosphoethanolamine

Ivana Pavlic (2016)
Sveučilište u Rijeci
Odjel za biotehnologiju
Metadata
TitleInterakcije proteina ostreolizina A s liposomima sastavljenim od fosfatidilkolina, kolesterola i ceramid fosfoetanolamina
AuthorIvana Pavlic
Mentor(s)Kristina Sepčić (thesis advisor)
Abstract
Ostreolizin A (u daljnjem tekstu OlyA) je protein niske molekulske mase (̴15 kDa) izoliran iz gljive Pleurotus ostreatus i pripada obitelji egerolizina. Poznato je da u kombinaciji s 59-kDa proteinom pleurotolizinom B (PlyB) tvori pore i da se specifično veže na liposome u čijem se sastavu nalaze sfingomijelin (SM) i kolesterol (Kol), ceramid fosfoetanolamin (CPE) i/ili CPE:Kol, zasićeni glicerofosfolipidi/Kol te da je za vezanje potrebno minimalno 30% kolesterola. Karakteristika OlyA je i vezanje na membranske splavi koje u sastavu također imaju sfingomijelin i kolesterol i time ima potencijalnu primjenu u označavanju membranskih domena bogatih navedenim lipidima. No, pošto je sfingomijelin glavni lipid kralješnjaka, a ceramid fosfoetanolamin beskralješnjaka i pošto se razlikuju jedino skupinama na glavi molekule odlučili smo ispitati interakcije OlyA s ceramid fosfoetanolaminom i kolesterolom i saznati količine ovih lipida potrebne za vezanje proteina. Pošto se CPE nalazi u membranama beskralješnjaka uočena je i potencijalna primjena kombinacije OlyA/PlyB u suzbijanju različitih insekata i parazita. U tu svrhu smo napravili lipidne filmove rotacionim uparivačem otapala te u prvom dijelu istraživanja preliminarnim testom SDS-PAGE elektroforeze ispitali vezanje OlyA. U ovom, ali i svim ostalim testovima, koristili smo liposome s različitim molnim udjelima CPE, Kol i 1-palmitoil-2-oleil-fosfatidilkolina (POPC), dok su liposomi POPC:Kol (1:1) služili kao negativna kontrola vezanja. Isti test vezanja OlyA, ali i kombinacije OlyA/PlyB smo ponovili i osjetljivijom metodom površinske plazmonske rezonancije koja prati intermolekularne interakcije u stvarnom vremenu. Pokazali smo da je za vezanje potrebno minimalno 5% CPE i više od 30% kol. U drugom djelu istraživanja proučavali smo permeabilizacijski učinak OlyA/PlyB proteinskog kompleksa na liposome punjene kalceinom i pokazali da se kalcein otpušta iz svih liposoma osim POPC:Kol (mol:mol, 1:1) koji su ionako negativna kontrola. Ovi rezultati ukazuju da smanjenje količine CPE i kolesterola u liposomima smanjuje i permeabilizacijsku aktivnost OlyA/PlyB. Također, stvaranje pora u liposomima načinjenim od lipida koji čine membranu parazita Toxoplasma gondii otkriva mogućnost korištenja OlyA/PlyB kompleksa u suzbijanju navedenog parazita. U posljednjem dijelu istraživanja razvili smo kolorimetrijski test za određivanje ugrađene količine CPE u liposome,te smo odredili količine CPE, POPC i kolesterola u liposomima, a pomoću metode dinamičkog sipanja svijetlosti odredili veličinu velikih unilamelarnih liposoma
Keywordsaegerolysin ostreolysin A ceramide phosphoetanolamine cholesterol
Parallel title (English)Protein ostrelysine A interactions with liposomes composed by phosphatidylcholine, cholesterol and ceramide phosphoethanolamine
Committee MembersKristina Sepčić (committee chairperson)
Sandra Kraljević Pavelić (committee member)
Nataša Poklar Ulrih (committee member)
Iztok Turel (committee member)
GranterSveučilište u Rijeci
Lower level organizational unitsOdjel za biotehnologiju
PlaceRijeka
StateCroatia
Scientific field, discipline, subdisciplineBIOTECHNICAL SCIENCES
Biotechnology
Study programme typeuniversity
Study levelgraduate
Study programmeBiotechnology in medicine
Academic title abbreviationmag. biotech. in med.
Genremaster's thesis
Language Croatian
Defense date2016-09-22
Parallel abstract (English)
Ostreolysin A (OlyA) is a low-molecular weight protein (̴15 kDa) isolated from fungus Pleurotus ostreatus and belongs to the aegerolysin family. It is known that in combination with a 59-kDa protein, pleurotolysin B (PlyB), it forms a pore and that it specifically binds to lipid vesicles that are comprised of sphingomyelin (SM) and cholesterol (Chol), ceramide phosphoethanolamine (CPE) and/or CPE:Chol and saturated glycerophospholipids/Chol. Furthermore, at least 30% cholesterol is necessary for binding. Another characteristic of OlyA is that it binds to membrane rafts composed of sphingomyelin and cholesterol and thus has a potential use in labelling membrane domains rich with the aforementioned lipids. Since sphingomyelin is the major lipid in mammals and in invertebrates that is ceramide phosphoethanolamine, and since these molecules differ only in their head groups, we decided to examine the interaction of OlyA with ceramide phosphoethanolamine and cholesterol, and to determine the quantity of these lipids necessary for binding. Since CPE is found in cell membranes of invertebrates a potential application of the combination Olya/PlyB in combating various insects and parasites. For this purpose, we produced lipid films with the rotary evaporator and in the first research stage we used the SDS-PAGE electrophoresis as a preliminary test to examine the binding of OlyA. In this and all subsequent tests, we used liposomes with varying molar fractions of CPE, Chol and palmitoyl-oleoyl-phosphatidylcholine (POPC), where POPC:Chol (mol:mol, 1:1) served as a negative control. The binding of OlyA, but also of the combination OlyA/PlyB, was further assayed with the sensitive surface plasmon resonance method that tracks intermolecular interactions in real time. We have shown that a minimum of 5% part CPE, and more than 30% part Chol is necessary for the OlyA binding.
Parallel keywords (Croatian)egerolizini ostreolizin A ceramid fosfoetanolamin kolesterol
Resource typetext
Access conditionNo public access
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:193:327324
CommitterLea Lazzarich