Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet Bharani Srinivasan,

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Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet Bharani Srinivasan,

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Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet Bharani Srinivasan, PhD, Margarete Focke-Tejkl, PhD, Milena Weber, MSc, Sandra Pahr, PhD, Alexandra Baar, PhD, Raja Atreya, MD, Markus F. Neurath, MD, Harald Vogelsang, MD, Wolf-Dietrich Huber, MD, Rudolf Valenta, MD Journal of Allergy and Clinical Immunology Volume 136, Issue 6, Pages 1607-1618.e3 (December 2015) DOI: 10.1016/j.jaci.2015.04.040 Copyright © 2015 The Authors Terms and Conditions

Fig 1 Multiple sequence alignment of the GG1 amino acid sequence with amino acid sequences of prolamins from closely related cereals (spelt, rye, and barley) and distantly related rice and oat. Identical amino acids are represented by dots, and gaps are represented by dashes. The coloring of the amino acids is based on the domain architecture, as described in Fig E1, A. The open green and dark blue rectangles represent peptides 4 and 18, respectively. The T-cell epitope described by Molberg et al24 has been boxed in light blue. The conserved cysteines are represented by open red rectangles. Percentages of sequence identities between GG1 and respective prolamins are indicated in bold italics in the bottom right corner. Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions

Fig 2 Biochemical and structural characterization of rGG1. A, Coomassie-stained SDS-PAGE containing rGG1 separated under reducing (left lane) and nonreducing conditions (right lane). Molecular weight markers (in kilodaltons) are indicated on the left margin. B, Circular dichroism analysis of purified rGG1. The spectra are expressed as mean residue ellipticities (y-axis) at given wavelengths (x-axis). Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions

Fig 3 IgA reactivity to rGG1 and gliadin extract (e). A, Sera from patients with CD, patients with IDs, healthy persons, patients with IgE-associated allergies, and AGA+/EMA− subjects were tested for IgA reactivity to rGG1 and gliadin extract (e) by means of ELISA. The results are shown as a scatter plot, and OD values corresponding to the amounts of bound antibodies are displayed on the y-axes. The horizontal lines within groups indicate the median, and the dotted horizontal lines indicate the cutoff values based on the Youden index (P values between 2 groups are indicated to denote significance). B, Receiver operating characteristic (ROC) curves comparing IgA reactivity with rGG1 and gliadin extract (e) as CD-specific markers. Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions

Fig 4 Antibody reactivities of patients with CD on a GFD. Sera obtained from 6 patients with CD “before and after” a GFD (A) and from 18 patients with CD on a GFD (B) were tested for IgA reactivity to rGG1, tTG2, and DGP, as well as for IgG against DGP. OD values and units per milliliter corresponding to bound antibody levels are shown on the y-axes. Cutoff values for each assay are indicated by horizontal dashed lines (P values between 2 groups are indicated to denote significance). Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions

Fig 5 Epitope mapping of GG1 using synthetic overlapping peptides. IgA (A) and IgG (B) reactivity of patients with active CD (n = 8) and healthy persons (n = 3) to GG1-derived peptides (Fig 1) or to rGG1 (x-axes) by using ELISA. The results are shown as box plots of OD values for each group, where 50% of the values are within the boxes, lines within the boxes indicate median values, and open circles and stars indicate outliers and extremes, respectively. Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions

Fig 6 Detection of GG1-like proteins in cereal and bread extracts. Protein extracts from wheat, rye, spelt, barley, oat, rice, and maize (A) and extracts from the crumb and crust of (I) common bread (I), rolls (II), rye bread (III), and gluten-free bread (GFB; B) were blotted onto nitrocellulose membrane and probed with preimmune serum (left panels) or rabbit anti-rGG1 antibodies (right panels). Molecular weights in kilodaltons are shown on the left margins. Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions

Fig 7 Immunoblot detection of GG1 after peptic and chymotryptic digestion. A, Silver-stained SDS-PAGE gels of undigested rGG1 (lane a) and rGG1 digested with pepsin for 30 (lane b), 60 (lane c), and 120 (lane d) minutes. Lanes e to i contain rGG1 samples, which after 120 minutes of peptic digestion were further digested with chymotrypsin for 30 (e), 60 (f), 120 (g), 240 (h), or 1200 (i) minutes. Molecular weights are indicated as kilodaltons. B-D, Identically prepared gels were blotted onto nitrocellulose and then probed with rabbit anti-rGG1 antibodies (right panel) or the corresponding preimmune serum (Pre-Ig; left panel) in Fig 7, B; with rabbit anti–peptide 4 antibodies (right panel) or the preimmune serum (left panel) in Fig 7, C; or with rabbit anti–peptide 18 antibodies (right panel) or the preimmune serum (left panel) in Fig 7, D. Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions

Fig E1 Schematic representation of GG1. A, Depiction of the domain architecture of GG1. The signal peptide is followed by a short N-terminal nonrepetitive domain (I), a highly variable Pro- and Gln-rich repetitive domain (II), a cysteine-rich nonrepetitive domain (III), a Gln-rich region (IV), and the C-terminal nonrepetitive domain containing 2 conserved cysteine residues (V). B, Sequence of synthetic GG1 peptides. Displayed are peptide numbers, amino acid sequences, positions of the peptides in the full length GG1, and their lengths. Asterisks indicate peptides that have not been tested. The coloring of the letters is based on the domain architecture, as described in Fig E1, A. Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions

Fig E2 MALDI-TOF analysis of purified rGG1. The m/z ratio is provided on the x-axis, and the signal intensity (in arbitrary units) is shown on the y-axis. Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions

Fig E3 IgG reactivity of preimmune sera (Pre-Ig) and immune sera from rabbits immunized with rGG1, peptide 4, or peptide 18 of GG1-derived peptides (Fig E1) with GG1-derived peptides or rGG1 (x-axes) as tested by using ELISA. OD values (y-axis) correspond to the amount of bound antibodies. Journal of Allergy and Clinical Immunology 2015 136, 1607-1618.e3DOI: (10.1016/j.jaci.2015.04.040) Copyright © 2015 The Authors Terms and Conditions