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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?-(?-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.

SUBMITTER: Lexhaller B

PROVIDER: S-EPMC7198585 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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

Lexhaller Barbara B Ludwig Christina C Scherf Katharina Anne KA

Scientific reports 20200504 1

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<sup>ε</sup>-(γ-glutamyl)-lysine isopeptide bonds. The resulting TG2-glute ...[more]

PMID: 32367038

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Project description:Celiac disease (CD) is triggered by ingestion of gluten-containing cereals such as wheat, barley, rye and in some cases oat. The only way for affected individuals to avoid symptoms of this condition is to adopt a gluten-free diet. Thus, gluten-free foodstuffs need to be monitored in order to ensure their innocuity. For this purpose, commercial immunoassays based on recognition of defined linear gluten sequences are currently used. These immunoassays are designed to detect or quantify total gluten regardless of the cereal, and often result in over or underestimation of the exact gluten content. In addition, Canadian regulations require a declaration of the source of gluten on the label of prepackaged foods, which cannot be done due to the limitations of existing methods. In this study, the development of new antibodies targeting discrimination of gluten sources was conducted using synthetic peptides as immunization strategy. Fourteen synthetic peptides selected from unique linear amino acid sequences of gluten were bioconjugated to Concholepas concholepas hemocyanin (CCH) as protein carrier, to elicit antibodies in rabbit. The resulting polyclonal antibodies (pAbs) successfully discriminated wheat, barley and oat prolamins during indirect ELISA assessments. pAbs raised against rye synthetic peptides cross-reacted evenly with wheat and rye prolamins but could still be useful to successfully discriminate gluten sources in combination with the other pAbs. Discrimination of gluten sources can be further refined and enhanced by raising monoclonal antibodies using a similar immunization strategy. A methodology capable of discriminating gluten sources, such as the one proposed in this study, could facilitate compliance with Canadian regulations on this matter. This type of discrimination could also complement current immunoassays by settling the issue of over and underestimation of gluten content, thus improving the safety of food intended to CD and wheat-allergic patients.

Project description:The consumption of wheat, rye, and barley may cause adverse reactions to wheat such as celiac disease, non-celiac gluten/wheat sensitivity, or wheat allergy. The storage proteins (gluten) are known as major triggers, but also other functional protein groups such as α-amylase/trypsin-inhibitors or enzymes are possibly harmful for people suffering of adverse reactions to wheat. Gluten is widely used as a collective term for the complex protein mixture of wheat, rye or barley and can be subdivided into the following gluten protein types (GPTs): α-gliadins, γ-gliadins, ω5-gliadins, ω1,2-gliadins, high- and low-molecular-weight glutenin subunits of wheat, ω-secalins, high-molecular-weight secalins, γ-75k-secalins and γ-40k-secalins of rye, and C-hordeins, γ-hordeins, B-hordeins, and D-hordeins of barley. GPTs isolated from the flours are useful as reference materials for clinical studies, diagnostics or in food analyses and to elucidate disease mechanisms. A combined strategy of protein separation according to solubility followed by preparative reversed-phase high-performance liquid chromatography was employed to purify the GPTs according to hydrophobicity. Due to the heterogeneity of gluten proteins and their partly polymeric nature, it is a challenge to obtain highly purified GPTs with only one protein group. Therefore, it is essential to characterize and identify the proteins and their proportions in each GPT. In this study, the complexity of gluten from wheat, rye, and barley was demonstrated by identification of the individual proteins employing an undirected proteomics strategy involving liquid chromatography-tandem mass spectrometry of tryptic and chymotryptic hydrolysates of the GPTs. Different protein groups were obtained and the relative composition of the GPTs was revealed. Multiple reaction monitoring liquid chromatography-tandem mass spectrometry was used for the relative quantitation of the most abundant gluten proteins. These analyses also allowed the identification of known wheat allergens and celiac disease-active peptides. Combined with functional assays, these findings may shed light on the mechanisms of gluten/wheat-related disorders and may be useful to characterize reference materials for analytical or diagnostic assays more precisely.

Project description:Celiac disease (CD) is a chronic inflammation of the small intestine triggered by the ingestion of gluten in genetically predisposed individuals. Tissue transglutaminase (TG2) is a key factor in the pathogenesis of CD, because it catalyzes both the deamidation of specific glutamine residues and the formation of covalent Nε-(γ-glutamyl)-lysine isopeptide crosslinks resulting in TG2-gluten peptide complexes. These complexes are thought to activate B cells causing the secretion of anti-TG2 autoantibodies that serve as diagnostic markers for CD, although their pathogenic role remains unclear. To gain more insight into the molecular structures of TG2-gluten peptide complexes, we developed a reciprocal proteomic analysis strategy, which facilitates the comprehensive identification of isopeptides in a complex protein hydrolysate. The workflow consists of four steps: (1) performance of the model reaction between TG2 and gluten peptide(s) followed by tryptic hydrolysis, clean-up and untargeted nLC-MS/MS analysis (2), prediction of tryptic TG2-derived lysine peptides that serve as potential isopeptide modifications (3) the search for isopeptides by configuring the TG2-modifications in MaxQuant and search against the α-gliadin fasta file and (4) the reciprocal search for isopeptides by configuring the gluten peptide(s) as modification in MaxQuant and search against the TG2 fasta file. After verification by manual data curation and visualization, this strategy enabled the identification of 26 different isopeptides involving 19 TG2 lysine residues, only six of which were known previously. Experiments with different TG2-gluten peptide molar ratios revealed that K-425, K-590, K-600 and K-649 were the most preferred lysine residues involved in isopeptide crosslinking. Further, expanding the model system to three gluten peptides allowed the localization of the preferred glutamine crosslinking sites. The described strategy is generally applicable to any hydrolysate containing isopeptides and these new insights into the structure of TG2-gluten peptide complexes may help clarify the role of extracellular TG2 in CD autoimmunity and in other inflammatory diseases.

Dataset Information

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

Publications

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

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