Dataset Information

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer.

ABSTRACT: Transepithelial transport of proteins is an important step in the immune response to food allergens. Mammalian meat allergy is characterized by an IgE response against the carbohydrate moiety galactosyl-?-1,3-galactose (?-Gal) present on mammalian glycoproteins and glycolipids, which causes severe allergic reactions several hours after red meat consumption. The delayed reaction may be related to the processing of ?-Gal carrying proteins in the gastrointestinal tract. The aim of this study was to investigate how protein glycosylation by ?-Gal affects the susceptibility to gastric digestion and transport through the Caco-2 cell monolayer. We found that ?-Gal glycosylation altered protein susceptibility to gastric digestion, where large protein fragments bearing the ?-Gal epitope remained for up to 2 h of digestion. Furthermore, ?-Gal glycosylation of the protein hampered transcytosis of the protein through the Caco-2 monolayer. ?-Gal epitope on the intact protein could be detected in the endosomal fraction obtained by differential centrifugation of Caco-2 cell lysates. Furthermore, the level of galectin-3 in Caco-2 cells was not affected by the presence of ?-Gal glycosylated BSA (bovine serum albumin) (BSA-?-Gal). Taken together, our data add new knowledge and shed light on the digestion and transport of ?-Gal glycosylated proteins.

SUBMITTER: Krstic Ristivojevic M

PROVIDER: S-EPMC7461108 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer.

Krstić Ristivojević Maja M Grundström Jeanette J Apostolović Danijela D Radomirović Mirjana M Jovanović Vesna V Radoi Vlad V Kiewiet M B Gea MBG Vukojević Vladana V Ćirković Veličković Tanja T van Hage Marianne M

International journal of molecular sciences 20200811 16

Transepithelial transport of proteins is an important step in the immune response to food allergens. Mammalian meat allergy is characterized by an IgE response against the carbohydrate moiety galactosyl-α-1,3-galactose (α-Gal) present on mammalian glycoproteins and glycolipids, which causes severe allergic reactions several hours after red meat consumption. The delayed reaction may be related to the processing of α-Gal carrying proteins in the gastrointestinal tract. The aim of this study was to ...[more]

PMID: 32796496

Similar Datasets

Project description:IntroductionAlpha-gal syndrome (AGS) is characterized by delayed hypersensitivity to non-primate mammalian meat in people having specific immunoglobulin E (sIgE) to the oligosaccharide galactose-alpha-1,3-galactose. AGS has been linked to tick bites from Amblyomma americanum (Aa) in the U.S. A small animal model of meat allergy is needed to study the mechanism of alpha-gal sensitization, the effector phase leading to delayed allergic responses and potential therapeutics to treat AGS.MethodsEight- to ten-weeks old mice with a targeted inactivation of alpha-1,3-galactosyltransferase (AGKO) were injected intradermally with 50 μg of Aa tick salivary gland extract (TSGE) on days 0, 7, 21, 28, 42, and 49. Total IgE and alpha-gal sIgE were quantitated on Day 56 by enzyme-linked immunosorbent assay. Mice were challenged orally with 400 mg of cooked pork kidney homogenate or pork fat. Reaction severity was assessed by measuring a drop in core body temperature and scoring allergic signs.ResultsCompared to control animals, mice treated with TSGE had 190-fold higher total IgE on Day 56 (0.60 ± 0.12 ng/ml vs. 113.2 ± 24.77 ng/ml; p < 0.001). Alpha-gal sIgE was also produced in AGKO mice following TSGE sensitization (undetected vs. 158.4 ± 72.43 pg/ml). Further, sensitized mice displayed moderate clinical allergic signs along with a drop in core body temperature of ≥2°C as an objective measure of a systemic allergic reaction. Interestingly, female mice had higher total IgE responses to TSGE treatment but male mice had larger declines in mean body temperature.ConclusionTSGE-sensitized AGKO mice generate sIgE to alpha-gal and demonstrate characteristic allergic responses to pork fat and pork kidney. In keeping with the AGS responses documented in humans, mice reacted more rapidly to organ meat than to high fat pork challenge. This mouse model establishes the central role of tick bites in the development of AGS and provides a small animal model to mechanistically study mammalian meat allergy.

Project description:The α-gal epitope is a carbohydrate antigen which appeared early in mammalian evolution and is synthesized in large amounts by the glycosylation enzyme α1,3galactosyltransferase (α1,3GT) in non-primate mammals, lemurs, and New-World monkeys. Ancestral Old-World monkeys and apes synthesizing α-gal epitopes underwent complete extinction 20-30 million years ago, and their mutated progeny lacking α-gal epitopes survived. Humans, apes, and Old-World monkeys which evolved from the surviving progeny lack α-gal epitopes and produce the natural anti-Gal antibody which binds specifically to α-gal epitopes. Because of this reciprocal distribution of the α-gal epitope and anti-Gal in mammals, transplantation of organs from non-primate mammals (e.g., pig xenografts) into Old-World monkeys or humans results in hyperacute rejection following anti-Gal binding to α-gal epitopes on xenograft cells. The in vivo immunocomplexing between anti-Gal and α-gal epitopes on molecules, pathogens, cells, or nanoparticles may be harnessed for development of novel immunotherapies (referred to as "α-gal therapies") in various clinical settings because such immune complexes induce several beneficial immune processes. These immune processes include localized activation of the complement system which can destroy pathogens and generate chemotactic peptides that recruit antigen-presenting cells (APCs) such as macrophages and dendritic cells, targeting of antigens presenting α-gal epitopes for extensive uptake by APCs, and activation of recruited macrophages into pro-reparative macrophages. Some of the suggested α-gal therapies associated with these immune processes are as follows: 1. Increasing efficacy of enveloped-virus vaccines by synthesizing α-gal epitopes on vaccinating inactivated viruses, thereby targeting them for extensive uptake by APCs. 2. Conversion of autologous tumors into antitumor vaccines by expression of α-gal epitopes on tumor cell membranes. 3. Accelerating healing of external and internal injuries by α-gal nanoparticles which decrease the healing time and diminish scar formation. 4. Increasing anti-Gal-mediated protection against zoonotic viruses presenting α-gal epitopes and against protozoa, such as Trypanosoma, Leishmania, and Plasmodium, by vaccination for elevating production of the anti-Gal antibody. The efficacy and safety of these therapies were demonstrated in transgenic mice and pigs lacking α-gal epitopes and producing anti-Gal, raising the possibility that these α-gal therapies may be considered for further evaluation in clinical trials.

Project description:Objective:In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Methods: Human Caco-2 cells were cultured for 10 days in culture flasks or for 14 days on transwell inserts in either glucose-containing or galactose-containing medium. Mitochondria were visualized and cellular respiration and levels of oxidative phosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletion was induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability was assessed using transepithelial electrical resistance (TEER) and fluorescein flux. Gene expression and cellular distribution of tight junction proteins were analyzed. Results: Caco-2 cells cultured in galactose-containing, but not in glucose-containing, medium showed increased mitochondrial connectivity, oxygen consumption rates and levels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP, rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability. In-depth studies with PA showed a six fold decrease in cellular ATP and revealed increased gene expression of tight junction proteins (TJP) 1 and 2, occludin, and claudin 1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearly redistributed from the cellular membrane into the cytoplasm, while the others were not (TJP1, occludin) or slightly (claudin 2, actin) affected. In vivo studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an in vitro model mimicking the oxidative in vivo situation. Conclusions: Well-functioning mitochondria are essential for maintaining cellular energy status and monolayer integrity of galactose grown Caco-2 cells. Energy depletion-induced Caco-2 monolayer permeability may be facilitated by changes in the distribution of claudin 7.

Dataset Information

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer.

Publications

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets