Comprehensive Detection of Isopeptides between Human Tissue Transglutaminase and Gluten Peptides

Lexhaller, Barbara; Ludwig, Christina; Scherf, Katharina Anne

doi:10.3390/nu11102263

Cited by 15 publications

(19 citation statements)

References 34 publications

(60 reference statements)

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“…These seven peptides (FLKNAGR, WKNHGCQR, ISTKSVGR, LAEKEETGMAMR, DLYLENPEIKIR, QKR, AVKGFR, lysine residue involved in crosslink formation highlighted in bold) containing the lysine residues K205, K265, K429, K468, K590, K600 and K677 from the TG2 amino acid sequence were selected as possible crosslinking sites. The lysine residues K590, K600 and K677 had previously been identified by Fleckenstein et al 8 and the lysine residues K205, K265, K429 and K468 additionally by Lexhaller et al 15 . K590, K600 and K677 were known as preferred TG2 crosslinking sites also for TG2 self-multimerization 13 , while K205, K265, K429 and K468 were involved in the formation of isopeptides with high identification scores 15 .…”

Section: Resultsmentioning

confidence: 82%

“…These complexes were hydrolysed with trypsin followed by solid phase extraction (SPE) for clean-up of the isopeptide/peptide mixture and subsequent discovery-driven nanoscale liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis (Fig. 1b) 15 . The GPT blank controls without addition of TG2 were used to create customized protein databases (Table S1) for each GPT that were applied in the proteomics software MaxQuant (MQ) 20 .…”

Section: Resultsmentioning

confidence: 99%

“…In-depth studies about the formation of covalent TG2-gluten peptide complexes showed that six lysine residues of TG2 were involved in crosslinking with two different gluten peptides 8 or even with TG2 molecules to create covalent TG2-TG2-multimers 13 . In addition, a reciprocal proteomics strategy using an α-gliadin-derived model peptide recently allowed the identification of 34 isopeptides involving 20 different lysine residues of TG2 15 . It is also known that, when confronted with a complex gluten peptide mixture, TG2 preferentially crosslinks peptides containing known CD-active T-cell epitopes to an acyl acceptor substrate such as 5-biotinamido-pentylamine 16 .…”

mentioning

confidence: 99%

“…It is also known that, when confronted with a complex gluten peptide mixture, TG2 preferentially crosslinks peptides containing known CD-active T-cell epitopes to an acyl acceptor substrate such as 5-biotinamido-pentylamine 16 . However, there is no information as to which gluten peptides are good substrates for crosslinking to TG2, because all studies so far have worked only with selected gluten-derived model peptides and not with physiologically relevant enzymatic gluten hydrolysates due to their extreme heterogeneity 8,15 .…”

mentioning

confidence: 99%

“…Therefore, the aim of our study was to apply our recently developed reciprocal mass spectrometric approach, including discovery-driven mass spectrometry 15 and additional targeted proteomics, to complex gluten hydrolysates that had been incubated with TG2 and identify TG2-gluten isopeptides. We used well-characterized gluten protein types (GPTs) of wheat, rye and barley 17 and extended our analysis strategy with additional confirmation of isopeptide identities by parallel reaction monitoring (PRM) LC-MS/MS as follow-up measurements.…”

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confidence: 99%

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

Lexhaller

Ludwig

Scherf

2020

Sci Rep

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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...

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Section: Resultsmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Lexhaller

Ludwig

Scherf

2020

Sci Rep

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Cross‐linking reactions in food proteins and proteomic approaches for their detection

Renzone

Arena

Scaloni

2021

Mass Spectrometry Reviews

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Various protein cross‐linking reactions leading to molecular polymerization and covalent aggregates have been described in processed foods. They are an undesired side effect of processes designed to reduce bacterial load, extend shelf life, and modify technological properties, as well as being an expected result of treatments designed to modify raw material texture and function. Although the formation of these products is known to affect the sensory and technological properties of foods, the corresponding cross‐linking reactions and resulting protein polymers have not yet undergone detailed molecular characterization. This is essential for describing how their generation can be related to food processing conditions and quality parameters. Due to the complex structure of cross‐linked species, bottom‐up proteomic procedures developed to characterize various amino acid modifications associated with food processing conditions currently offer a limited molecular description of bridged peptide structures. Recent progress in cross‐linking mass spectrometry for the topological characterization of protein complexes has facilitated the development of various proteomic methods and bioinformatic tools for unveiling bridged species, which can now also be used for the detailed molecular characterization of polymeric cross‐linked products in processed foods. We here examine their benefits and limitations in terms of evaluating cross‐linked food proteins and propose future scenarios for application in foodomics. They offer potential for understanding the protein cross‐linking formation mechanisms in processed foods, and how the inherent beneficial properties of treated foodstuffs can be preserved or enhanced.

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Identification of the initial reactive sites of micellar and non-micellar casein exposed to microbial transglutaminase

Duerasch

Konieczny

Henle

2022

Eur Food Res Technol

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To investigate the influence of the internal micellar structure on the course of enzymatic cross-linking especially in the initial phase of the reaction, casein micelles isolated from raw milk via ultracentrifugation were incubated with microbial transglutaminase (mTG) in comparison with non-micellar sodium caseinate. Reactive lysine and glutamine residues were identified using a label-free approach, based on the identification of isopeptides within tryptic hydrolysates by targeted HRMS as well as manual monitoring of fragmentation spectra. Identified reactive sites were furthermore weighted by tracking the formation of isopeptides over an incubation time of 15, 30, 45 and 60 min, respectively. Fifteen isopeptides formed in the early stage of mTG cross-linking of caseins were identified and further specified concerning the position of lysine and glutamine residues involved in the reaction. The results revealed lysine K176 and glutamine Q175 of β-casein as the most reactive residues, which might be located in a highly flexible region of the molecule based on different possible reaction partners identified in this study. Except for the isopeptide αs1 K34–αs2 Q101 in sodium caseinate (SC), all reactive sites were detected in micellar and in non-micellar casein, indicating that the initial phase of enzymatic cross-linking is not affected by micellar aggregation of caseins. Graphical abstract

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Comprehensive Detection of Isopeptides between Human Tissue Transglutaminase and Gluten Peptides

Cited by 15 publications

References 34 publications

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

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

Cross‐linking reactions in food proteins and proteomic approaches for their detection

Identification of the initial reactive sites of micellar and non-micellar casein exposed to microbial transglutaminase

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