Uloga glavnih toksičnih komponenti otrova poskoka (Vipera ammodytes) u njegovoj imunogenosti s posebnim osvrtom na hemoragične metaloproteinaze

doctoral thesis

• Title (english): Role of the major toxic components from Vipera ammodytes venom in its immunogenicity with special emphasis on hemorrhagic metalloproteinases
• Author: Tihana Kurtović
• Mentor: Beata Halassy (mentor); MBZ: 223834
• Committee member: Jasmina Rokov Plavec (član povjerenstva)
• Committee member: Beata Halassy (član povjerenstva); MBZ: 223834
• Committee member: Anica Horvat Knežević (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2013, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 577 - Biochemistry. Molecular biology. Biophysics

Abstract

Otrov poskoka (Vipera a. ammodytes) se koristi u proizvodnji protuotrova protiv ugriza europskih zmija otrovnica. O strukturnim i funkcionalnim osobitostima njegovih glavnih toksičnih komponenti, amoditoksina (Atx-a) i hemoragina, postoje brojni literaturni podaci. Međutim, njihova imunogenost, tj. uloga u stvaranju zaštitnih antitijela sposobnih neutralizirati toksične učinke otrova, nije poznata. Stoga je cilj istraživanja bio pripremiti serume definirane specifičnosti imunizacijom kunića s pročišćenim komponentama – Atx-om A, ukupnom hemoraginskom frakcijom, odnosno kombinacijom Atx-a i hemoragina kako bi ispitali njihov zaštitni doprinos u neutralizaciji letalne toksičnosti inducirane cijelim otrovom. Prema rezultatima primijenjenog ED50 testa, jedine regulatorno priznate metode za procjenu potencije protuotrova protiv ugriza europskih zmija otrovnica, potpuno funkcionalna anti-Atx antitijela iskazuju iznimno slabu zaštitnu moć, dok ona specifična za ukupnu hemoraginsku frakciju uopće ne osiguravaju protekciju, iako su u životinjskom pokusnom modelu sama po sebi dostatna za neutralizaciju lokalnog hemoragičnog učinka. U kontekstu hemoragičnog efekta kao dominantne kliničke manifestacije otrovanja u ljudi za koju su zaslužne Zn2+-ovisne metaloproteinaze, cilj rada bio je i sustavno istražiti strukturu i funkciju potpuno novog reprezentativnog predstavnika skupine nazvanog amoditagin. Na njegovom primjeru je ispitan i doprinos antitijela specifičnih za jedan izdvojeni protein hemoragičnog fenotipa u neutralizaciji ukupne hemoragičnosti eksprimirane od strane otrova poskoka. Amoditagin je glikozilirani, blago kiseli heterodimer od 108 kDa s multidomenskom organizacijom jedinstvenom za pripadnike P-IIIc podgrupe koji je okarakteriziran kao enzim široke proteolitičke specifičnosti. Novoizolirani protein djeluje i kao jaki hemoragin. Prema rezultatima proizašlima iz ispitivanja njegove supstratne specifičnosti, hemoragičnost amoditagina najvjerojatnije je povezana s proteolitičkim efektom kojeg iskazuje na pojedine komponente bazalne membrane te faktore zgrušavanja krvi. Serum specifičan za amoditagin se pokazao dostatnim za neutralizaciju hemoragične aktivnosti cijelog otrova, što sugerira da bi indukcija nastanka anti-amoditagin antitijela mogla predstavljati bitni čimbenik u dobivanju terapeutski djelotvornog protuotrova.

Abstract (english)

The venom of horn-nosed viper (Vipera a. ammodytes) has been used as an antigen for the production of antivenom against European vipers. In contrast to the detailed knowledge on structural and functional properties of ammodytoxins and hemorrhagins as its main toxic components, almost nothing has been known about their immunogenicity. Our research goal was to prepare the rabbit antisera containing antibodies specific for both classes of the pathology-inducing venom constituents and to evaluate the involvement of the obtained antibodies in neutralisation of the whole venom toxicity in mice. According to ED50 test, the only regulatory approved assay for the estimation of antivenom potency, fully functional antibodies specific for ammodytoxins participate in neutralisation of the whole venom's lethal toxicity only partially. Antibodies specific for hemorrhagins are not protective at all, despite their sufficiency in neutralisation of the local hemorrhagic effect in vivo. Furthermore, since hemorrhage as the prevalent clinical manifestation of envenoming in human is due to action of Zn2+-dependent metalloproteinases, structural and functional characterisation of the group representative, named ammodytagin, was of special research interest. Also, role of antibodies specific for individual protein exhibiting hemorrhagic phenotype in neutralisation of the whole venom hemorrhagicity was defined. Ammodytagin is a glycosylated, slightly acidic heterodimer of 108 kDa with multidomain organisation unique for members of P-IIIc subgroup which is characterised as enzyme of broad proteolytic specificity. The novel protein also acts as a strong hemorrhagin. Investigation of a substrate specificity revealed that hemorrhagic activity of ammodytagin might be the result of its involvement in cleavage of basal membrane components and depletion of coagulation factors in blood circulation. Finally, antiserum raised against ammodytagin was able to completely neutralise the hemorrhagic activity of the whole venom, suggesting it might be one of the key molecules towards which effective antivenom should be directed.

• Keywords: otrov poskoka; toksične komponente; amoditoksini; hemoragini; zaštitna moć; Zn2+-ovisna metaloproteinaza
• Keywords (english): horn-nosed viper venom; toxic components; ammodytoxins; hemorrhagins; protective efficacy; Zn2+-dependent metalloproteinase
• Language: croatian
• URN:NBN: urn:nbn:hr:217:225807
• Study programme: Title: Biology; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija (doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-05-19 09:01:02