Izolacija in karakterizacija proteina VaaMPIII-3 iz modrasovega strupa

Požek, Kity (author), Križaj, Igor (mentor), Župunski, Vera (co-mentor)

Strup modrasa (Vipera ammodytes ammodytes) je kompleksna mešanica farmakološko aktivnih molekul. Med najbolj zastopane strupne proteine sodijo metaloproteinaze. VaaMPIII-3 je prvi predstavnik novega podrazreda metaloproteinaz P-IIIe. Sestavljen je iz dveh nekatalitičnih domen – skrajšane domene, podobne disintegrinom (D'), in domene, bogate s cisteini (C). Toksini s podobno domensko zgradbo med drugim vplivajo na strjevanje krvi. Lastnosti in delovanje VaaMPIII-3 še niso bili znani, zato smo se ga namenili izolirati in okarakterizirati ter raziskati njegov vpliv na strjevanje krvi. V sklopu magistrskega dela smo razvili in optimizirali postopek za izolacijo VaaMPIII-3 iz surovega modrasovega strupa. Potrdili smo, da ima VaaMPIII-3 prost cisteinski ostanek, ki omogoča vezavo na tiolno sefarozo in izolacijo s kovalentno kromatografijo. Pogoje izolacije smo prilagodili ugotovitvam o optimalni sestavi pufrskih raztopin za shranjevanje VaaMPIII-3 in se tako izognili izgubam zaradi obarjanja. Izolirali smo mešanico šestih izooblik VaaMPIII-3, z izoelektričnimi točkami med 4,5 in 5,1. Do homogenosti smo očistili prevladujočo izoobliko z izoelektrično točko 4,5. Prosti cistein smo lokalizirali na mestu 6, v manjši meri pa tudi na mestu 19 v molekuli VaaMPIII-3. Ugotovili smo, da se VaaMPIII-3 po razporeditvi disulfidnih vezi v domeni D' razlikuje od metaloproteinaz razreda P-IIIa. Pripravili smo tridimenzionalni strukturni model VaaMPIII-3. Ugotovili smo, da ima VaaMPIII-3 podoben vpliv na strjevanje krvi kot drugi proteini z enako domensko zgradbo. Zavrl je agregacijo trombocitov, sproženo z ADP, kolagenom ali arahidonsko kislino, ni pa imel vpliva na aglutinacijo trombocitov, sproženo z ristocetinom. V bakteriji E. coli smo izrazili rekombinantni protein VaaMPIII-3, ga izolirali, okarakterizirali in njegove lastnosti primerjali z naravnim. Molekulski masi naravnega in rekombinantnega VaaMPIII-3 se razlikujeta za približno 4 kDa, kar gre na račun N-glikozilacije prvega. Rekombinantni VaaMPIII-3 ima izoelektrično točko pri pH 5,1 in je pravilno zvit. Enako kot naravni protein je zavrl agregacijo trombocitov, sproženo z ADP. Kaže, da je rekombinantna oblika VaaMPIII-3 primeren nadomestek za raziskovanje bioloških učinkov naravnega VaaMPIII-3, kar bo vsekakor pospešilo pojasnjevanje njegove vloge v delovanju kačjega strupa in ugotavljanje njegove terapevtske uporabnosti.

• Language: Slovenian
• Keywords: modras (Vipera ammodytes ammodytes), strup, metaloproteinaze, disintegrinom podobni/s cisteini bogati proteini, inhibitorji agregacije trombocitov
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-130294
• COBISS.SI-ID: 82704899
• Publication date in RUL: 13.09.2021

Secondary language

• Language: English
• Title: Isolation and characterization of the VaaMPIII-3 protein from the venom of the nose-horned viper

Abstract

The venom of the nose-horned viper (Vipera ammodytes ammodytes) is a complex mixture of pharmacologically active molecules. Among the most abundant venom proteins are metalloproteinases. VaaMPIII-3 is the first member of a new subclass, P-IIIe, of metalloproteinases. It consists of two non-catalytic domains – the truncated disintegrin like domain (D') and the Cys-rich domain (C). Toxins with similar domain structure affect blood coagulation. The properties and functions of VaaMPIII-3 are yet unexplored. Our aim was to isolate and characterize this protein and discover its effect on blood. In this work, we developed and optimized the procedure to isolate VaaMPIII-3 from the nose-horned viper venom. We confirmed that VaaMPIII-3 has a free Cys that enables its binding to thiol sepharose and isolation by covalent chromatography. We adjusted the isolation conditions according to our findings on the optimal buffer solutions for storage of VaaMPIII-3 to avoid loss of the protein by aggregation. We isolated a mixture of six isoforms of VaaMPIII-3 with isoelectric points between 4.5 and 5.1, and also purified the predominant isoform with isoelectric point 4.5. We localized the free Cys at position 6, while a small portion of the protein had a free cysteine at position 19. We demonstrated that the disulfide pattern of the VaaMPIII-3's D' domain is distinct from that of class P-IIIa metalloproteinases. We constructed a 3D homology model of the structure of VaaMPIII-3 to predict its key structural features. We showed that VaaMPIII-3 affects blood coagulation in a similar manner to other proteins with similar domain structure. It inhibited ADP-, collagen-, or arachidonic acid-induced platelet aggregation. However, it had no effect on ristocetin-induced platelet agglutination. We produced a recombinant VaaMPIII-3 in E. coli, determined its properties, and compared them with its natural form. Their molecular masses differ by about 4 kDa, as the natural protein is highly N-glycosylated. The non-glycosylated recombinant VaaMPIII-3 has an isoelectric point of 5.1 and is correctly folded. Its inhibitory effect on ADP-induced platelet aggregation is equal to that of its natural form. Therefore, recombinant VaaMPIII-3 is suitable for use in the discovery of other biological activities of VaaMPIII-3. These studies will help to understand its role in snake venom and determine its potential for therapeutic use.

• Keywords: nose-horned viper (Vipera ammodytes ammodytes), venom, metalloproteinases, disintegrin-like/cysteine-rich proteins, platelet aggregation inhibitors