Izolacija in karakterizacija mutiranih različic erilizina A

Trčak, Anja (author), Sepčić, Kristina (mentor), Skočaj, Matej (comentor)

Protein erilizin A (EryA), ki ga proizvaja gliva kraljevi ostrigar (Pleurotus eryngii), prištevamo v proteinsko družino egerolizini (Pfam 06355; InterPro IPR009413). Zanj je značilno, da prepozna in se specifično veže na najbolj zastopan nevretenčarski sfingolipid, ceramid fosfoetanolamin. S pomočjo mutageneze v povezavi z verižno reakcijo s polimerazo smo v nukleotidni zapis za EryA uvedli točkovne mutacije in zamenjali pet posameznih aminokislinskih ostankov za alanin (različice EryA(W28A), EryA(E69A), EryA(T98A), EryA(W96A), EryA(K99A)) in enega za tirozin (različica EryA(A91Y)). Omenjene aminokislinske ostanke smo izbrali na podlagi predhodnih raziskav vezave različic egerolizinov ostreolizina A6 (OlyA6) in pleurotolizina A2 (PlyA2) na kombinacijo sfingomielina in holesterola, ter poravnave strukture egerolizina OlyA6 z modelom strukture EryA. Na podlagi poravnave smo sklepali, da sta vezavna žepa proteinov najverjetneje podobno zgrajena, zato smo pet mest za mutagenezo izbrali na podlagi variant OlyA6, ki so pri omenjenem proteinu imele ključno vlogo za vezavo proteina na membrano. EryA nima ohranjenega tirozinskega ostanka Y91, ki je prisoten pri proteinih OlyA6 in PlyA2 in naj bi skupaj s triptofanoma na mestu 28 in 96 tvoril vezavno mesto za lipide. Ker se EryA za razliko od OlyA6 in PlyA2 ne veže na komplekse sfingomielin/holesterol, smo sklepali, da odgovor za specifično interakcijo s ceramid fosfoetanolaminom leži prav tukaj. Uspešno smo izolirali rekombinantni protein EryA in njegovih pet različic. Dokazali smo, da imata različici EryA(A91Y) in EryA(T98A) z EryA primerljivo afiniteto do ceramid fosfoetanolamina v kombinaciji s holesterolom. Posledično imata kompleksa EryA(A91Y)/PlyB in EryA(T98A)/PlyB enako citolitično aktivnost, kot kompleks EryA/PlyB. Hkrati smo dokazali, da aminokislinski ostanek alanin na mestu 91 ni ključen za specifično vezavo proteina na ceramid fosfoetanolamin.

• Language: Slovenian
• Keywords: egerolizini, erilizin A, ceramid fosfoetanolamin, mutageneza s PCR
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2020
• PID: 20.500.12556/RUL-117719
• COBISS.SI-ID: 26795011
• Publication date in RUL: 23.07.2020

Secondary language

• Language: English
• Title: Isolation and characterization of mutated variants of Erylysin A

Abstract

Protein erylysin A (EryA) belongs to the protein family of aegerolysins (Pfam 06355; InterPro IPR009413) and is produced by the king oyster mushroom (Pleurotus eryngii). EryA specifically binds to the main invertebrate sphingolipid, ceramide phosphoethanolamine. Point mutations were introduced by mutagenesis to replace selected amino acid residues with alanine (protein variants EryA(W28A), EryA(E69A), EryA(T98A), EryA(W96A), EryA(K99A)) and one with tyrosine (protein variant EryA(A91Y)). These amino acid residues were selected on the basis of previous studies, on the binding of variants of aegerolysins ostreolysin A6 (OlyA6) and pleurotolysin A2 (PlyA2) to sphingomyelin, when combined with cholesterol, and their high degree of similarity with EryA. From the alignment visualization of structure OlyA6 with structure model EryA, we concluded that the protein binding pockets are most likely very similar. EryA does not have the preserved tyrosine residue Y91, which is present in OlyA6 and PlyA2 proteins. Together with tryptophan at site 28 and 96, Y91 is thought to form a lipid binding site. Unlike OlyA6 and PlyA2, EryA does not bind to sphingomylein/cholesterol complexes. Therefore, we concluded that the answer for specific EryA interaction with ceramide phosphoethanolamine could lie here. We successfully isolated the recombinant EryA protein and five variants thereof. We demonstrated that the variants EryA(A91Y) and EryA(T98A) have comparable affinity for binding to ceramide phosphoethanolamine, when combined with cholesterol, as EryA. Consequently, the EryA (A91Y)/PlyB and EryA(T98A)/PlyB complexes have the same cytolytic activity as the EryA/PlyB complex. At the same time, we have shown that amino acid residue of alanine at position 91 is not crucial for the specific binding of the EryA to ceramide phosphoethanolamine.

• Keywords: aegerolysins, erylysin A, ceramide phosphoethanolamine, PCR-directed mutagenesis