Napovedovanje navzkrižne reaktivnosti med proteini kravjega mleka in črevesne mikrobiote s pomočjo primerjav tridimenzionalnih struktur z orodjem Dali

Nemec, Oskar (avtor), Tavčar, Eva (mentor), Vidak, Marko (komentor)

Alergijske bolezni so škodljive specifične imunske reakcije na običajno neškodljive snovi. Med pogostimi vrstami so alergije na hrano, ki predstavljajo globalni zdravstveni problem, ki prizadene okoli 10 % svetovnega prebivalstva, pri čemer najnovejši podatki kažejo trend naraščanja. Alergene sestavine v hrani so običajno proteini. Pogost pojav pri alergijah na hrano je navzkrižna reaktivnost (CR), pri kateri imunski mehanizmi, občutljivi na primarni alergen, prepoznajo in reagirajo na sekundarne alergene zaradi prisotnosti navzkrižno reaktivnih epitopov. Metode za identifikacijo CR vključujejo eksperimentalne tehnike in bioinformacijske analize, ki temeljijo na iskanju podobnosti med alergenimi proteini s poravnavo aminokislinskih zaporedij (npr. BLAST) ali naprednejšimi orodji za kartiranje epitopov (npr. CrossReact). Uporaba orodij za določanje podobnosti terciarnih oz. 3D struktur, kot je Dali, pa je še premalo raziskana, prav tako tudi morebitna CR med prehranskimi antigeni in črevesno mikrobioto, ključnim dejavnikom alergij na hrano. V magistrski nalogi smo želeli pokazati, da je orodje Dali uporabno za napovedovanje CR ter da imajo alergeni kravjega mleka in proteini, s katerimi potencialno navzkrižno reagirajo, večjo podobnost na ravni terciarnih struktur kot na ravni zaporedij. Rezultati analiz z orodjem Dali kažejo splošno korelacijo med podobnostjo terciarnih in primarnih struktur, vendar obstajajo izjeme, pri katerih proteini z nizko podobnostjo zaporedij izkazujejo visoko podobnost terciarnih struktur in obratno. Po validaciji orodja smo želeli identificirati proteine črevesne mikrobiote, ki so strukturno podobni alergenom kravjega mleka, ter oceniti njihov potencial za CR. Zaradi omejene razpoložljivosti strukturnih podatkov in dokazov za CR v literaturi smo analizo izvedli le za mlečne alergene Bos d 4, Bos d 5 in Bos d 6. S pomočjo orodja Dali smo preiskali zbirki PDB in AF-DB ter identificirali številne podobne proteine iz mikrobiote, vendar na podlagi teh rezultatov ne moremo z gotovostjo presoditi verjetnosti za nastanek CR. Okvirno lahko določimo le, da je verjetnost za nastanek CR s proteini mikrobiote nižja pri Bos d 6 in višja pri Bos d 4 in 5. Na podlagi našega dela ugotavljamo, da bi pri nadaljnjih raziskavah primerjava struktur z orodji, kot je Dali, lahko služila kot začetno presejalno orodje za identifikacijo kandidatov s splošno strukturno podobnostjo, s katerimi bi nato izvedli nadaljnje bioinformacijske analize na ravni epitopov ter eksperimentalne validacije imunološke odzivnosti.

• Jezik: Slovenski jezik
• Ključne besede: navzkrižna reaktivnost, alergija na kravje mleko, hrana, proteini, bioinformatika
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-170133
• Datum objave v RUL: 02.07.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Prediction of cross-reactivity between cow's milk proteins and gut microbiota using three-dimensional structural comparisons with the Dali tool

Izvleček

Allergic diseases are harmful specific immune reactions to normally harmless substances. Among the common types are food allergies, a global health problem affecting about 10% of the world’s population, with recent data showing an increasing trend. The allergenic components in food are usually proteins. A common phenomenon in food allergies is cross-reactivity (CR), in which immune mechanisms sensitive to the primary allergen recognize and react to secondary allergens due to the presence of cross-reactive epitopes. Methods for identifying CR include experimental techniques and bioinformatic analyses based on searching for similarities between allergenic proteins by aligning amino acid sequences (e.g., BLAST) or more advanced tools for epitope mapping (e.g., CrossReact). The use of tools for determining the similarity of tertiary or 3D structures, such as Dali, is still underexplored, as is the potential CR between dietary antigens and the gut microbiota, a key factor in food allergies. In this Master’s thesis, we aimed to demonstrate that the Dali tool is useful for predicting CR and that cow's milk allergens and proteins with which they may cross-react share greater similarity at the tertiary structural level than at the sequence level. The results of analyses with the Dali tool show a general correlation between tertiary and primary structure similarity, but there are exceptions where proteins with low sequence similarity show high tertiary structure similarity and vice versa. After tool validation, we aimed to identify gut microbiota proteins that are structurally similar to cow's milk allergens and assess their potential for CR. Due to the limited availability of structural data and evidence for CR in the literature, the analysis was performed only for milk allergens Bos d 4, Bos d 5, and Bos d 6. Using the Dali tool, we examined the PDB and AF-DB databases and identified numerous similar proteins from the microbiota, but based on these results, we cannot conclusively determine the probability of CR occurring. We can only roughly estimate that the probability of CR with microbiota proteins is lower for Bos d 6 and higher for Bos d 4 and 5. Based on our work, we conclude that in future studies, comparison of structures using tools like Dali could serve as an initial screening tool for identifying candidates with general structural similarity, with further bioinformatic analyses at the epitope level and experimental validations of immune responsiveness.

• Ključne besede: cross-reactivity, cow’s milk allergy, food, proteins, bioinformatics