Volume 133, Issue 3, Pages 780-789 (September 2007) Transamidation of Wheat Flour Inhibits the Response to Gliadin of Intestinal T Cells in Celiac Disease  Carmen Gianfrani, Rosa A. Siciliano, Angelo M. Facchiano, Alessandra Camarca, Maria F. Mazzeo, Susan Costantini, Virginia M. Salvati, Francesco Maurano, Giuseppe Mazzarella, Gaetano Iaquinto, Paolo Bergamo, Mauro Rossi  Gastroenterology  Volume 133, Issue 3, Pages 780-789 (September 2007) DOI: 10.1053/j.gastro.2007.06.023 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 tTG-mediated modifications of the α-gliadin p56-68 inhibited IFN-γ production in iTCLs from patients with celiac disease. (A) MALDI-TOF mass spectrum of the peptide cross-linked to lysine; satellite ions are due to sodium and potassium adducts. (B) MS/MS spectrum of the doubly charged ion originated from the peptide cross-linked to lysine; the peptide sequence and the fragmentation pattern are reported. (C) Sequences of native (Q65), tTG-deamidated [(E65)], and cross-linked [(Q65-K), (Q65-K-CH3)] p56-68 peptides, and IFN-γ production of peptide-responsive iTCLs from patients with celiac disease, expressed as percentage of (E65) responses (mean ± SD of triplicate cultures; n = 5). *Statistically different from (E65) (ANOVA test). Results are representative of 3 independent experiments. Gastroenterology 2007 133, 780-789DOI: (10.1053/j.gastro.2007.06.023) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Modeling of DQ2-(p56-68) interaction. (A) Bar graphs of the energies of interaction and free binding energies computed for each peptide-DQ2 complex. (Top) Van der Waals (white bars) and electrostatic contributions (grey bars) to the energy of interaction. (Bottom) Free binding energies. (B) Detailed view of the molecular interaction between DQ2 and p56-68 (E65) (top) and p56-68 (Q65-K) peptide (bottom). The C-terminal portion of the peptide is shown as a stick representation, and the amino acids of DQ2 (see labels) involved in H-bonds with the modified amino acid of peptide are shown as a ball and stick representation (atom colors: green, carbon; red, oxygen; blue, nitrogen). Gastroenterology 2007 133, 780-789DOI: (10.1053/j.gastro.2007.06.023) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Effect of tTG-mediated transamidation of gliadin on the IFN-γ response of iTCLs. (A) IFN-γ production (pg/mL) of 12 iTCLs isolated from patients with celiac disease. Results are expressed as mean ± SD of triplicate cultures. (B) Percentages of IFN-γ production; results shown in A are expressed as a percentage of the response induced by PT-gliadin + tTG and reported as the mean of all 12 iTCLs. *Different from PT-gliadin + tTG; #different from medium; §different from PT-gliadin + tTG + K (ANOVA test). Gastroenterology 2007 133, 780-789DOI: (10.1053/j.gastro.2007.06.023) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Inhibition of gliadin activity following treatment of wheat flour with mTG and lysine methyl ester. (A) Coomassie staining of mTG preparation and undigested gliadin isolated from differently treated wheat flours (left) and Western blot analysis of equivalent protein amount (right). (B) IFN-γ production (pg/mL) of iTCLs following stimulation with PT-gliadin isolated from treated flour (mild conditions) and deamidated in vitro with tTG. Results are expressed as mean ± SD of triplicate cultures. (C) Percentages of IFN-γ production; results shown in B are expressed as a percentage of the response induced by PT-gliadin isolated from untreated flour and reported as the mean of all 12 iTCLs. *Statistically different from untreated (ANOVA test). Gastroenterology 2007 133, 780-789DOI: (10.1053/j.gastro.2007.06.023) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Effect of mTG-mediated transamidation of flour on the gliadin-specific cytokine pattern. IFN-γ, IL-2, IL-4, and IL-10 levels (pg/mL) of iTCLs isolated from patients CD280900, CD090401, and CD230204 stimulated in vitro with tTG-treated PT-gliadin extracted from different flour preparations. Results are expressed as mean ± SD of triplicate cultures. Gastroenterology 2007 133, 780-789DOI: (10.1053/j.gastro.2007.06.023) Copyright © 2007 AGA Institute Terms and Conditions