20.500.12556/RUL-187075 Priprava in karakterizacija izbranih aktinoporinom podobnih proteinov iz polžev rodu Conus Preparation and characterization of selected actinoporin-like proteins from snails of the genus Conus Aktinoporini (AP) so porotvorni toksini ožigalkarjev, ki sodelujejo pri lovu plena, prebavi in obrambi ter tvorijo transmembranske pore z vezavo na sfingomielin v celičnih membranah. Zaradi specifičnih membranskih interakcij imajo pomemben biotehnološki potencial v diagnostiki, terapijah in tehnologiji nanopor. Aktinoporinom podobni proteini (ALP) predstavljajo evolucijsko sorodno skupino, med katere sodijo tudi konoporini iz stožčastih polžev. V tej magistrski nalogi smo prvič rekombinantno izrazili ter biofizikalno in biokemijsko opredelili konoporina Eb2 in Eb3 iz vrste Conus ebraeus. Kljub izraziti strukturni podobnosti z AP smo ugotovili, da zaradi sprememb v ključnih vezavnih mestih specifično ne prepoznavata sfingomielina in ne izkazujeta porotvorne aktivnosti. V mediteranski klapavici Mytilus galloprovincialis poleg ALP-ja mitiporina-1 (MYTP1) najdemo tudi mitilektine, himerne proteine z lektinsko in proaerolizinsko domeno, ki specifično prepoznavajo glikane in tvorijo pore v obliki β-sodčka. Za podrobnejšo opredelitev njihove funkcije smo raziskali mitilektina MytiLec-3 in OrbiLec-1. Ugotovili smo, da je MytiLec-3 hemolitičen ter se specifično veže na glikanske epitope LacNAc, LacdiNAc in α-Gal, medtem ko OrbiLec-1 ne izkazuje vezave na glikane, niti hemolitične aktivnosti. S krio-elektronsko mikroskopijo smo v nativni membrani eritrocitov eksperimentalno potrdili nonamerno poro MytiLec-3, ki smo jo predhodno napovedali z uporabo modela AlphaFold3. Naši rezultati razkrivajo funkcionalno raznolikost ALP-jev in mitilektinov ter poudarjajo njihov potencial za uporabo v diagnostiki, biosenzoriki in farmaciji. Actinoporins (AP) are pore-forming toxins produced by cnidarians that participate in prey capture, digestion and defense by forming transmembrane pores through specific binding to sphingomyelin-containing cell membranes. Due to their unique membrane interacting properties, they hold considerable potential for applications in diagnostics, therapeutics and nanopore technologies. Actinoporin-like proteins (ALP) represent an evolutionary related group that also inludes conoporins from cone snails. In this master's thesis, we recombinantly expressed and biophysically and biochemically characterized conoporins Eb2 and Eb3 from Conus ebraeus for the first time. Despite their pronounced structural similarity to APs, we found that substitutions at key binding sites prevent specific recognition of sphingomyelin and abolish pore-forming activity. In the Mediterranean mussel Mytilus galloprovincialis, in addition to the ALP mytiporin-1 (MYTP1), mytilectins are also present. These chimeric proteins contain lectin domain and proaerolysin-like domain, enabling the recognition of specific glycans and the formation of β-barrel pores. To further elucidate their function, we investigated mytilectins MytiLec-3 and OrbiLec-1. We demonstrated that MytiLec-3 is hemolytic and specifically binds the glycan epitopres LacNAc, LacdiNAc and α-Gal, whereas OrbiLec-1 exhibits neither glycan-binding nor hemolytic activity. Using cryo-electron microscopy, we experimentally confirmed the presence of a nonameric MytiLec-3 pore in native erythrocyte membranes, which we had previously predicted using AlphaFold3. Our findings reveal the functional diversity of ALPs and mytilectins and highlight their potential applications in diagnostics, biosensing and pharmaceutical research. aktinoporinom podobni protein konoporini mitilektini porotvorni protein actinoporin-like protein conoporin mytilectin pore-forming protein true false false [U. Zupančič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-09 07:18:10 2026-09-09 07:18:15 2026-09-10 04:30:37 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 577.112:594.347(043.2) 287562 290476291 61282.pdf 61282.pdf 1 C6DF4B4F8F50DE5C6CDB3CD45A4B9BAB febfd6468ab8c69ddadef001f48e8236a7df6d40e1fff48f1d0bcaa92d0ab5e2 d6bf903c-ac0d-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=246511 Biotehniška fakulteta 0 0 0