Monitoring of in vitro bioavailability and uptake of glycosylated food allergens using cell-based models

Abstract

extensive engagement of researchers in the elucidation of the mechanisms underlying processes of food digestion, allergen transport, and uptake by the immune cells and its effector immune responses. The development of the in vitro assays and cell-based models has allowed bridging the problem of food allergy research and the respect of the ethical norms in used research procedures. The red meat allergy is a novel type of food allergy characterized by the production of an IgE antibody against the carbohydrate galactose-α-1,3-galactose (α- Gal). Glycoproteins from non-primate mammals are rich with an α-Gal as posttranslational modification. Also, red meat allergy is characterized by the delayed onset of symptoms which may be related to the mechanism and the fate of α-Gal carrying proteins in the human gastrointestinal tract. Furthermore, the uptake, processing, and mechanisms of presentation of α-Gal by the immune cells are still unknown. Therefore this doctoral dissertation aimed to investigate how protein glycosylation by α-Gal affects their susceptibility to gastric digestion, does α-Gal conjugated to proteins affects their transport through the Caco-2 cell monolayer, which mimics the gastrointestinal layer, and to examine the influence of α-Gal epitopes on the protein surface on their uptake and processing by immature monocyte-derived dendritic cells (iMDDCs). The study revealed that the presence of the α-Gal glycosylation on protein surface had an impact on their susceptibility to gastric digestion and the digestion pattern of the obtained protein fragments upon pepsinolysis. Prolonged survival, up to 2h of digestion, was characteristic of the large proteins fragments bearing the α-Gal epitope. Importantly, transport through the Caco-2 monolayer of proteins conjugated to α-Gal was hampered in comparison to unconjugated proteins. Furthermore, differential centrifugation of Caco-2 cell lysates upon transport experiments revealed that α-Gal could be detected on the intact protein in the endosomal fraction of the cells...

Alergije na hranu su rastući problem u ljudskoj populaciji širom sveta i rešavanje ovog problema zahteva opsežno angažovanje istraživača u rasvetljavanju mehanizama uključenih u procese varenja hrane, transporta alergena i njihovog unosa od strane imunih ćelija odgovornih za efektorske mehanizme imunoloških odgovora. Ovo je nezamislivo bez razvoja in vitro testova i ćelijskih modela koji premošćuju problem istraživanja alergija na hranu uz poštovanja etičkih normi u korišćenim istraživačkim metodama. Novu vrstu alergije na hranu, alergiju na crveno meso, karakteriše sinteza imunoglobulina E kao odgovor na prisustvo šećera galaktoza-α-1,3-galaktoza (α-Gal), koji je prisutan na površini glikoproteina primata. Takođe, alergiju na crveno meso karakteriše odložena pojava simptoma što može biti rezultat promena u mehanizmu obrade proteina koji nose α-Gal u gastrointestinalnom traktu čoveka. Dalje, unos, obrada i mehanizmi prezentacije α-Gal šećera od strane imunih ćelija još uvek nisu poznati. Stoga ciljevi ove doktorske disertacije su ispitivanje kako α-Gal glikozilacija proteina utiče na njihovu digestiju od strane pepsina, da li α-Gal glikozilacija proteina utiče na njihov transport kroz monosloj Caco-2 ćelija, koji oponaša gastrointestinalni epitel, kao i ispitivanje uticaja α-Gal glikozilacije na površini proteina na njihov unos i obradu od strane nezrelih dendritičnih ćelijama kultivisanih iz monocita (iMDDC). Iz dobijenih rezultata moze se zaključiti da prisustvo α-Gal glikozilacije na površini proteina utiče na njihovu podložnost na digestiju a najviše na obrazac dobijenih fragmenata proteina nakon pepsinolize. Veliki fragmenti proteina koji nose α-Gal prisutni su čak i do 2 sata digestije. Takođe, važno je istaći da je transport α-Gal glikozilovanih proteina kroz Caco-2 monosloj otežan u poređenju sa neglikozilovanim proteinima. Dalje, diferencijalnim centrifugiranjem lizata aco-2 ćelija nakon transcitoze pokazalo je da je α-Gal prisutan na intaktnim proteinima u endozomalnim frakcijama ćelija...