Identification of Isopeptides Between Human Tissue Transglutaminase and Wheat, Rye, and Barley Gluten Peptides

Abstract

Celiac disease (CD) is a chronic immune-mediated enteropathy of the small intestine, which is triggered by the ingestion of storage proteins (gluten) from wheat, rye, and barley in genetically predisposed individuals. Human tissue transglutaminase (TG2) plays a central role in the pathogenesis of CD, because it is responsible for specific gluten peptide deamidation and covalent crosslinking, resulting in the formation of N ε-(γ-glutamyl)-lysine isopeptide bonds. The resulting TG2-gluten peptide complexes are assumed to cause the secretion of anti-TG2 autoantibodies, but the underlying mechanisms are only partly known. To gain more insight into the structures of these complexes, the aim of our study was to identify TG2-gluten isopeptides. With the use of discovery-driven as well as targeted nanoscale liquid chromatography tandem mass spectrometry, we detected 29 TG2-gluten isopeptides in total, involving seven selected TG2 lysine residues (K205, K265, K429, K468, K590, K600, K677). Several gluten peptides carried known B-cell epitopes and/or T-cell epitopes, either intact 9-mer core regions or partial sequences, as well as sequences bearing striking similarities to already known epitopes. These novel insights into the molecular structures of TG2-gluten peptide complexes may help clarify their physiological relevance in the initiation of CD autoimmunity and the role of anti-TG2 autoantibodies. Celiac disease (CD) is defined as a chronic immune-mediated inflammatory disorder of the small intestine initiated by the storage proteins (gluten) of wheat, rye and barley in genetically predisposed subjects 1. The ingestion of gluten causes villous atrophy, lymphocyte infiltration and the stimulation of CD4 + T cells against gluten epitopes in CD patients. These epitopes are presented by the human leukocyte antigen (HLA) class II alleles HLA-DQ2.5, HLA-DQ2.2 and HLA-DQ8 of the major histocompatibility complex (MHC) expressed on B cells and antigen-presenting cells. The presentation of gluten peptides leads to the activation of CD4 + T cells, which are the main effector cells for immunologic processes 2,3. Human tissue transglutaminase (TG2), a Ca 2+-dependent protein-glutamine γ-glutamyltransferase (EC 2.3.2.13), is ubiquitously expressed and catalyses the deamidation of glutamine residues or the crosslinking reaction (transamidation) between a glutamine and a lysine residue to form a covalent N ε-(γ-glutamyl)-lysine isopeptide bond 4. The TG2-mediated deamidation converts certain glutamine residues to glutamic acid residues by releasing ammonia and incorporating water. This leads to an introduction of negative charges in gluten peptides following a distinct pattern, e.g., the glutamine residues in the sequences QXP, QXXF(Y/W/M/L/I/V) or QXPF(Y/W/M/L/I/V), where X designates any other amino acid except P, are preferentially targeted 5. This introduction of negatively charged amino acids increases the binding affinity of gluten peptides to the HLA molecules and enhances their antigenicity in CD patients 6. During transamidat...

Basophil Activation to Gluten and Non-Gluten Proteins in Wheat-Dependent Exercise-Induced Anaphylaxis

“…The samples were shaken for 45 min at 37°C in the dark. The solutions were evaporated to dryness ( 37 , 40 ).…”

“…Digestion was performed by adding pepsin [750 µl, 0.2 mg/ml in 0.15 mol/L HCl, pH 2, enzyme/substrate (E:S) ratio of 1:20 (w/w)] to the alkylated residues and shaking for 60 min at 37 • C. After the peptic digest, the pH was adjusted to 6.5 with PBS (50 mmol/L). Then, trypsin and/or chymotrypsin [E:S of 1:20 for T or C, E:S of 1:40 for TC (w/w)] were added and the samples were hydrolyzed for 120 min at 37 • C. For TC digestion, TC was added to the alkylated residues [1 ml, 0.12 mg/ml T/C in 0.1 mol/L TRIS-HCl-buffer, E:S of 1:50 (w/w)] followed by incubation for 16 h at 37 • C. The digestions were stopped by heating for 10 min at 95 • C (37,40). TLY digestion [E:S of 1:20 (w/w)] was carried out in TRIS-HCl CaCl 2 buffer (0.2 mol/L TRIS, 0.5 mmol/L CaCl 2 • 2H 2 O, pH 6.5) at 37 • C for 16 h. The reaction was stopped with formic acid (FA) (41)(42)(43).…”

The Two Faces of Wheat

“…Typical features of CD-active peptides are high contents of proline (P) residues and a left-handed polyproline II helical conformation that protects from enzymatic degradation as well as high contents of glutamine (Q) residues that serve as substrates for deamidation or transamidation by human tissue transglutaminase (TG2) (107). Having reached the lamina propria, gluten peptides with QXP or QXXJ motifs (X, any amino acid, J, hydrophobic amino acid) are specifically deamidated (Q → E, introduction of a negatively charged glutamic acid residue) or transamidated by TG2 (either to itself or to other lysine donors) (108,109), whereas QP and QXXP motifs are left unmodified (110). Then, gluten peptides are bound to the heterodimeric HLA-DQ2 or -DQ8 receptors on the surface of APCs.…”

Co-factors, Microbes, and Immunogenetics in Celiac Disease to Guide Novel Approaches for Diagnosis and Treatment

“…18 In CeD, active TG2 mediates the cross-linking of donor gluten peptides that are rich in glutamine to extracellular matrix proteins 19 and to itself (autocatalysis). 20,21 Importantly, in the absence of an acceptor protein, TG2 deamidates specific glutamine residues in the gluten peptides, resulting in their higher affinity binding to HLA-DQ2 or -DQ8 molecules on antigenpresenting cells (APCs). 22,23 Gluten-specific T cells recognize the APC-bound peptides and are activated, leading to expansion of pro-inflammatory gluten-specific CD4þ T helper 1 (T H 1) cells that centrally contribute to inflammation of the lamina propria, villous atrophy, and epithelial damage.…”