Substrate specificity of transglutaminases for gluten peptides

The human immune system has developed intricate mechanisms to protect the body by discriminating between infectious agents and self. Unfortunately, in some cases these mechanisms can be bypassed and immune responses may be elicited by antigens derived from self. The loss of tolerance to autoantigens may lead to the development of autoimmune diseases. Autoimmune diseases are chronic inflammatory diseases of unknown etiology where both genetic and environmental factors play a role. Human leukocyte antigens (HLA) have been shown to be the most important susceptibility factor for several of the autoimmune diseases, what strongly suggests the involvement of T cells. In many autoimmune diseases the self antigen causing the disease is not known. This is the case for rheumatoid arthritis, multiple sclerosis and type 1 diabetes. For celiac disease however, the trigger of the disease is known to be gluten. Interestingly, although gluten was known to be the environmental factor for years, it was not until a decade ago that a posttranslational modification of gluten mediated by an enzyme called tissue transglutaminase 2 (TG2) was discovered to be critical for the disease. The posttranslationally modified gluten peptides bind the disease associated HLA-molecules with a higher affinity than the unmodified gluten peptides, what results in a multifaceted T-cell response. Thus, posttranslational modifications of self-antigens, or in this case; of food antigens normally tolerated by the body, is one way in which novel epitopes are created that are not tolerated by the immune system. Interestingly, immune responses directed towards enzymatically modified self-antigens were also reported for other autoimmune diseases, e.g. against citrullinated proteins in rheumatoid arthritis and methylated and phosphorylated proteins in systemic lupus erythematosus. As the importance of posttranslational modifications of gluten peptides is acknowledged in celiac disease, further research in this field may be relevant also for other autoimmune diseases. In this thesis, we have used mass spectrometry-based strategies to investigate the posttranslational gluten modifications catalyzed by the transglutaminase enzymes implicated in gluten sensitive diseases. The main focus has been on TG2 and its important role in T-cell epitope selection in celiac disease

Different binding motifs of the celiac disease-associated HLA molecules DQ2.5, DQ2.2, and DQ7.5 revealed by relative quantitative proteomics of endogenous peptide repertoires

Celiac disease is caused by intolerance to cereal gluten proteins, and HLA-DQ molecules are involved in the disease pathogenesis by presentation of gluten peptides to CD4+ T cells. The α- or β-chain sharing HLA molecules DQ2.5, DQ2.2, and DQ7.5 display different risks for the disease. It was recently demonstrated that T cells of DQ2.5 and DQ2.2 patients recognize distinct sets of gluten epitopes, suggesting that these two DQ2 variants select different peptides for display. To explore whether this is the case, we performed a comprehensive comparison of the endogenous self-peptides bound to HLA-DQ molecules of B-lymphoblastoid cell lines. Peptides were eluted from affinity-purified HLA molecules of nine cell lines and subjected to quadrupole orbitrap mass spectrometry and MaxQuant software analysis. Altogether, 12,712 endogenous peptides were identified at very different relative abundances. Hierarchical clustering of normalized quantitative data demonstrated significant differences in repertoires of peptides between the three DQ variant molecules. The neural network-based method, NNAlign, was used to identify peptide-binding motifs. The binding motifs of DQ2.5 and DQ7.5 concurred with previously established binding motifs. The binding motif of DQ2.2 was strikingly different from that of DQ2.5 with position P3 being a major anchor having a preference for threonine and serine. This is notable as three recently identified epitopes of gluten recognized by T cells of DQ2.2 celiac patients harbor serine at position P3. This study demonstrates that relative quantitative comparison of endogenous peptides sampled from our protein metabolism by HLA molecules provides clues to understand HLA association with disease.Fil: Bergseng, Elin. University of Oslo; NoruegaFil: Dørum, Siri. University of Oslo; NoruegaFil: Arntzen, Magnus Ø.. University of Oslo; NoruegaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Nygård, Ståle. University of Oslo; NoruegaFil: Buus, Søren. Universidad de Copenhagen; DinamarcaFil: de Souza, Gustavo A.. University of Oslo; NoruegaFil: Sollid, Ludvig M.. University of Oslo; Norueg

Experimental and numerical study of the fragmentation of expanding warhead casings by using different numerical codes and solution techniques

AbstractThere has been increasing interest in numerical simulations of fragmentation of expanding warheads in 3D. Accordingly there is a pressure on developers of leading commercial codes, such as LS-DYNA, AUTODYN and IMPETUS Afea, to implement the reliable fracture models and the efficient solution techniques. The applicability of the Johnson–Cook strength and fracture model is evaluated by comparing the fracture behaviour of an expanding steel casing of a warhead with experiments. The numerical codes and different numerical solution techniques, such as Eulerian, Lagrangian, Smooth particle hydrodynamics (SPH), and the corpuscular models recently implemented in IMPETUS Afea are compared. For the same solution techniques and material models we find that the codes give similar results. The SPH technique and the corpuscular technique are superior to the Eulerian technique and the Lagrangian technique (with erosion) when it is applied to materials that have fluid like behaviour such as the explosive and the tracer. The Eulerian technique gives much larger calculation time and both the Lagrangian and Eulerian techniques seem to give less agreement with our measurements. To more correctly simulate the fracture behaviours of the expanding steel casing, we applied that ductility decreases with strain rate. The phenomena may be explained by the realization of adiabatic shear bands. An implemented node splitting algorithm in IMPETUS Afea seems very promising

The Preferred Substrates for Transglutaminase 2 in a Complex Wheat Gluten Digest Are Peptide Fragments Harboring Celiac Disease T-Cell Epitopes

BACKGROUND: Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2). In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten. METHODS: A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation. RESULTS: We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized. CONCLUSION: TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease

Serologic Assay for Diagnosis of Celiac Disease Based on a Patient-Derived Monoclonal Antigliadin Antibody

Manuscript title Serodiagnostic of celiac disease: Patient derived monoclonal anti-gliadin antibody harnessed in a novel inhibition assay Background & Aims Patients with celiac disease can be identified based on the detection of serum antibodies to deamidated gliadin peptides (DGPs). Recombinant human monoclonal antibodies (hmAb) against gliadin are produced by cloning antibody genes from single IgA-producing plasma cells isolated from lesions of patients with celiac disease. We developed an assay to identify patients with celiac disease based on the ability of antibodies from their serum to inhibit the binding of a gliadin-specific hmAb (1002-1E03) to a specific peptide antigen (inhibition assay). Methods We selected 2 peptides (a 34-mer and a 26-mer) found in ω-gliadins and low-molecular-weight glutenins that had been identified as specific targets of the hmAb 1002-1E03 from a digest of gliadin treated by transglutaminase 2. These peptides contained repeat sequence motifs; their interaction with hmAb 1002-1E03 was assessed in an amplified luminescent proximity homogeneous inhibition assay. We also tested peptides we created that included 3 repeated sequence motifs. Serum samples from untreated patients diagnosed with celiac disease (n = 106) and 2 control groups (198 blood donors, 151 patients with Crohn’s disease) were analyzed using the assay, as well as in conventional commercial assays that measure IgA against transglutaminase 2 (TG2) or IgG against DGP. Results In our inhibition assays, the 34-mer peptide showed the best results, and identified patients with celiac disease with 86.8% sensitivity and 98.6% specificity. Its diagnostic accuracy was comparable with that of commercial anti-DGP IgG (sensitivity, 87.9%; specificity, 98.0) and anti-TG2 IgA (sensitivity, 81.1%; specificity, 98.9) assays, and it detected most of the patients with anti-TG2 IgA-negative celiac disease without a significant decrease in specificity. Combined use of the anti-ω34 and the anti-TG2 assays produced specificity and sensitivity values of 95.3% and 98.0%, respectively. Conclusions We developed an antigliadin inhibition assay that identifies patients with celiac disease with high levels of specificity and sensitivity. It may prove useful as an adjunct to the current assay for anti-TG2 IgA

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells

Characterization of the Small Intestinal Lesion in Celiac Disease by Label-Free Quantitative Mass Spectrometry

Global characterization of tissue proteomes from small amounts of biopsy material has become feasible because of advances in mass spectrometry and bioinformatics tools. In celiac disease (CD), dietary gluten induces an immune response that is accompanied by pronounced remodeling of the small intestine. Removal of gluten from the diet abrogates the immune response, and the tissue architecture normalizes. In this study, differences in global protein expression of small intestinal biopsy specimens from CD patients were quantified by analyzing formalin-fixed, paraffin-embedded material using liquid chromatography–mass spectrometry and label-free protein quantitation. Protein expression was compared in biopsy specimens collected from the same patients before and after 1-year treatment with gluten-free diet (n = 10) or before and after 3-day gluten provocation (n = 4). Differential expression of proteins in particular from mature enterocytes, neutrophils, and plasma cells could distinguish untreated from treated CD mucosa, and Ig variable region IGHV5-51 expression was found to serve as a CD-specific marker of ongoing immune activation. In patients who had undergone gluten challenge, coordinated up-regulation of wound response proteins, including the CD autoantigen transglutaminase 2, was observed. Our study provides a global and unbiased assessment of antigen-driven changes in protein expression in the celiac intestinal mucosa

Identified T-cell epitopes in wheat gluten and their observed T-cell response in celiac disease patients.

<p>Glutamine residues expected to be targeted by TG2 are given in bold.</p>a<p>Peptide PQPQLPYPQPQLPY harboring both epitopes, DQ2-α-II and DQ2-α-III, was tested.</p>b<p>Peptide LQPQQPFPQQPQQPYPQQPQ harboring both epitopes, DQ2-γ-III/DQ8-γ-I and DQ2-γ-VI, was tested.</p>c<p>Note that this peptide is not identical to peptide QQPPFSQQQQQPLPQ which was previously known as the “Glt-17” epitope. Peptide QQPPFSQQQQQPLPQ has previously been tested for T-cell response in CD patients: 3/20 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014056#pone.0014056-Vader2" target="_blank">[17]</a>.</p><p>♦Identified TG2 peptide substrates that harbor incomplete 9mer core binding regions.</p