Strukturna i imunološka karakterizacija aktivnog i inhibiranog aktinidina, cistein proteaze iz kivija (Actinidia deliciosa Liang, Ferguson)

Abstract

Alergije na hranu su IgE antitelima posredovane imunološke reakcije na komponente hrane,najčešće proteine. Razvijaju se nakon primarne senzitizacije preko gastrointestinalnogtrakta, i za sada nije poznat mehanizam senzitizacije ovim putem. Smatra se da bi alergeniproteini hrane morali da prođu kroz gastrointestinalni trakt barem delimično očuvanestrukture kako bi doveli do senzitizacije. Pored nerazjašnjenog mehanizma senzitizacije,alergije na hranu predstavljaju i izazov na polju dijagnostike, dok je jedina do sada potpunouspešna metoda terapije strogo izbegavanje unosa alergene namirnice.Tokom svega 30-tak godina od svog prvog pojavljivanja na svetskom tržištu pa do danasplod kivija je postao jedna od omiljenijih namirnica u ljudskoj ishrani, ali je istovremenodospeo i među deset najčešćih uzročnika alergije na hranu. Aktinidin, cistein proteaza izpapainske familije, je najzastupljeniji protein pulpe kivija i smatra se glavnim alergenimmolekulom ovog izvora.U oviru ove disertacije ispitivane su strukturne i imunološke osobine aktinidina u ciljurasvetljavanja puta oralne senzitizacije i poboljšanja dijagnostike alergije na kivi. Pokazanoje da aktinidin prečišćen iz svežeg ploda kivija pod nativnim uslovima predstavlja smešuaktivnog i neaktivnog enzima. Između proteolitički aktivnog i neaktivnog aktinidina postojirazlika u elektroforetskoj pokretljivosti, sposobnosti vezivanja IgE u imunoblotu iobrascima reagovanja pacijenata u kožnim probama. Pokazano je da samo aktivni enzimpreživljava uslove simuliranog želudačnog i crevnog soka i da nakon ovih tretmanapokazuje očuvanu imunogenost i proteolitičku aktivnost. Uočene razlike između aktivnog iinhibiranog oblika aktinidina najverovatnije su posledica razlika u strukturi enzima kojenastaju prilikom inhibicije, a ova pretpostavka je potkrepljena rezultatima CDspektroskopije i fluorimetrije...

Food allergy is an IgE mediated reaction of the immune system to food components, mostcommonly to food proteins. Food allergies develop after primary sensitization through thegastrointestinal tract and the mechanism behind this route of sensitization has not yet beenelucidated. It has been postulated that in order to sensitize the organism through an oralroute, food allergens must reach the gastrointestinal mucosa in at least a partially intactform in order to interact with the immune system. In addition to the unclear mechanism ofsensitization, food allergies also present a challenge in the field of diagnostics, while theonly effective therapy method to date is the strict avoidance of allergenic substances.During the last 30 years, since its first appearance on the world market, kiwifruit hasbecome one of the more popular components of human nutrition, and yet, at the same time,it has emerged as one of the ten most frequent elicitors of allergic reactions to food.Actinidin, a cysteine protease from the papain family, is the most abundant protein in thepulp of kiwifruit and is considered the major allergen of this fruit.In this thesis the structural and immunological characteristics of actinidin were examined inorder to shed light on the mechanism of oral sensitization and to aid in the improvement ofkiwifruit allergy diagnosis. It was shown that actinidin purified from fresh kiwifruit undernative conditions represents a mixture of the active and inactive enzyme. Differences inelectrophoretic mobility, IgE binding in immunoblot and patient response in skin prick testswere shown for proteolytically active and inhibited actinidin. It was demonstrated that onlythe active enzyme survives conditions of simulated gastric and intestinal fluid whilepreserving its immunogenicity and proteolytic activity. The observed differences betweenthe active and inhibited form of actinidin are most likely a consequence of differences in the conformation of the enzyme that arise upon inhibition, a hypothesis supported by resultsfrom CD spectroscopy and fluorimetry...