Toward celiac-safe foods - Investigation of the interaction between transglutaminase 2 and gluten
Celiac disease, a chronic autoimmune enteropathy, may develop in genetically predisposed individuals upon ingestion of gluten proteins found in wheat, barley, and rye. Overall prevalence of celiac disease is increasing and it currently affects around 1% of the population. The types and severity of symptoms of celiac disease show high variability, leading to many sufferers remaining undiagnosed. The only available treatment is to follow a strict gluten-free diet, but gluten-free alternatives are less available, more expensive, and often have lower nutritional and sensorial quality.
This thesis work examined the interactions between the intestinal enzyme transglutaminase 2 and gluten peptides. Transglutaminase 2 plays a significant role in disease initiation and progression, and is the main autoimmune target in developed celiac disease. A method for measuring the interaction between transglutaminase 2 and gluten was developed and tested in studies on sourdough fermentation of wheat flour and bread. Transglutaminase 2-mediated transamidation of gluten was assayed and the extent of available binding motifs for transglutaminase 2 in α2-gliadin, considered the most immunogenic part of gluten, was assessed using an ELISA-based method. The results showed that lactic acid fermentation, which is not specifically tailored to degrade gluten, cannot sufficiently prevent transglutaminase 2 interaction with gluten or decrease the extent of available binding motifs for transglutaminase 2 on α2-gliadin. In studies investigating the possibility to block specific binding motifs for transglutaminase 2 on gluten peptides, using molecules suitable as food additives, binding to α2-gliadin was computationally simulated and promising candidates were identified. These candidates were analyzed in vitro for the ability to prevent transglutaminase 2-mediated transamidation and deamidation of gliadin. Ascorbyl palmitate was found to interact with α2-gliadin in computer simulations and effectively reduced gliadin interaction with transglutaminase 2 in vitro. The cytotoxicity profile of ascorbyl palmitate, in combination with gliadin, was evaluated in Caco-2 cell cultures by determining cell survival, direct cytotoxicity, inflammatory mediators, and cell layer integrity, and no negative effects were found. In ancillary studies of human ileostomy contents after ingestion of raw and extruded gluten-containing products, degradation products of α-gliadin were identified and the effect of extrusion on digestion was investigated. Preliminary results indicate that protein digestibility was decreased after intake of the extruded product, but the effect on α-gliadin digestion needs further evaluation. However, the majority of α-gliadin seems to be undigested after in vivo digestion in both products.
The interaction between transglutaminase 2 and gluten is crucial for celiac disease and in this thesis work, several strategies for preventing this have been explored. Ascorbyl palmitate has been shown to effectively prevent this interaction in vitro and is thus a promising candidate for creating cereal-based foods potentially safe for celiacs.