Abstract
Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten.
Highlights
Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat
For the wheat flour and gluten modified with K-C2H5, proteins extracted in the gliadin fraction showed a decrease in their retention time, being in accordance with a decrease in hydrophobicity[21] due to the introduction of positive charges resulting from the modification of glutamine residues with K-C2H5 (Fig. 1c,d)
The higher susceptibility of gliadins to the chemical modification by the action of microbial transglutaminase (mTG) is certainly related with their high content of glutamine residues[22] and due to the fact they are present essentially as monomeric proteins23. mTG treated flour and gluten presented a modification in the reversed phase-high performance liquid chromatography (RP-HPLC) profile of gliadins, but the modifications observed are substantially different from that observed when flour and gluten were treated in the presence of K-C2H5 and n-butylamine, showing that the modifications observed by the action of mTG in the presence of the amine nucleophile are not entirely due to the cross-linking action of mTG (Fig. 1g)
Summary
The exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. We present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. A key step in the pathogenesis of CD occurs when certain gluten peptides, namely those derived from glutamine-rich gliadins, are deamidated by tissue tranglutaminase (TG2), increasing their affinity for HLA-DQ2 or HLA-DQ8. This generates a more vigorous CD4+ T-helper 1 T cell activation, which can result in intestinal mucosal inflammation, malabsorption, and numerous secondary symptoms and autoimmune diseases[2,4]. Wheat is the only known cereal whose flours after mixing with water and kneading are able to form an unique viscoelastic dough due to the formation of a three dimensional network interconnected by disulphide bonds, UCIBIO-REQUIMTE, Chemistry Department, Faculty of Science and Technology, University NOVA of Lisbon, Caparica, www.nature.com/scientificreports/
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