Structural ans immunological characterization of actinidin, a cysteine protease from kiwifruit (Actinidia celiciosa Liang, Ferguson)

Beograd : [M. M. Grozdanović]

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Prirodno-matematičke nauke - Biohemija / Life sciences - Biochemistry

Alergije na hranu su IgE antitelima posredovane imunološke reakcije na komponente hrane, najčešće proteine. Razvijaju se nakon primarne senzitizacije preko gastrointestinalnog trakta, i za sada nije poznat mehanizam senzitizacije ovim putem. Smatra se da bi alergeni proteini hrane morali da prođu kroz gastrointestinalni trakt barem delimično očuvane strukture kako bi doveli do senzitizacije. Pored nerazjašnjenog mehanizma senzitizacije, alergije na hranu predstavljaju i izazov na polju dijagnostike, dok je jedina do sada potpuno uspešna metoda terapije strogo izbegavanje unosa alergene namirnice. Tokom svega 30-tak godina od svog prvog pojavljivanja na svetskom tržištu pa do danas plod kivija je postao jedna od omiljenijih namirnica u ljudskoj ishrani, ali je istovremeno dospeo i među deset najčešćih uzročnika alergije na hranu. Aktinidin, cistein proteaza iz papainske familije, je najzastupljeniji protein pulpe kivija i smatra se glavnim alergenim molekulom ovog izvora. U oviru ove disertacije ispitivane su strukturne i imunološke osobine aktinidina u cilju rasvetljavanja puta oralne senzitizacije i poboljšanja dijagnostike alergije na kivi. Pokazano je da aktinidin prečišćen iz svežeg ploda kivija pod nativnim uslovima predstavlja smešu aktivnog i neaktivnog enzima. Između proteolitički aktivnog i neaktivnog aktinidina postoji razlika u elektroforetskoj pokretljivosti, sposobnosti vezivanja IgE u imunoblotu i obrascima reagovanja pacijenata u kožnim probama. Pokazano je da samo aktivni enzim preživljava uslove simuliranog želudačnog i crevnog soka i da nakon ovih tretmana pokazuje očuvanu imunogenost i proteolitičku aktivnost. Uočene razlike između aktivnog i inhibiranog oblika aktinidina najverovatnije su posledica razlika u strukturi enzima koje nastaju prilikom inhibicije, a ova pretpostavka je potkrepljena rezultatima CD spektroskopije i fluorimetrije...

Food allergy is an IgE mediated reaction of the immune system to food components, most commonly to food proteins. Food allergies develop after primary sensitization through the gastrointestinal tract and the mechanism behind this route of sensitization has not yet been elucidated. It has been postulated that in order to sensitize the organism through an oral route, food allergens must reach the gastrointestinal mucosa in at least a partially intact form in order to interact with the immune system. In addition to the unclear mechanism of sensitization, food allergies also present a challenge in the field of diagnostics, while the only 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 has become 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 the pulp of kiwifruit and is considered the major allergen of this fruit. In this thesis the structural and immunological characteristics of actinidin were examined in order to shed light on the mechanism of oral sensitization and to aid in the improvement of kiwifruit allergy diagnosis. It was shown that actinidin purified from fresh kiwifruit under native conditions represents a mixture of the active and inactive enzyme. Differences in electrophoretic mobility, IgE binding in immunoblot and patient response in skin prick tests were shown for proteolytically active and inhibited actinidin. It was demonstrated that only the active enzyme survives conditions of simulated gastric and intestinal fluid while preserving its immunogenicity and proteolytic activity. The observed differences between the 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 results from CD spectroscopy and fluorimetry...

Alergije na hranu, plod kivija, cistein peptidaze, aktinidin, digestibilnost proteina, E-64 inhibitor, konformacija proteina, koagulaciono sredstvo

Food allergy, kiwifruit, cysteine proteases, actinidin, protein digestibility, E-64 inhibitor, protein conformation, milk-clotting agent

577.112(043.3)

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