Effect of Rothia mucilaginosa enzymes on gliadin (gluten) structure, deamidation, and immunogenic epitopes relevant to celiac disease.

Rothia mucilaginosa, a natural microbial inhabitant of the oral cavity, cleaves gluten (gliadin) proteins at regions that are resistant to degradation by mammalian enzymes. The aim of this study was to investigate to what extent the R. mucilaginosa cell-associated enzymes abolish gliadin immunogenic properties. Degradation of total gliadins and highly immunogenic gliadin 33-mer or 26-mer peptides was monitored by SDS-PAGE and RP-HPLC, and fragments were sequenced by liquid chromatography and electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS). Peptide deamidation by tissue transglutaminase (TG2), a critical step in rendering the fragments more immunogenic, was assessed by TG2-mediated cross-linking to monodansyl cadaverine (MDC), and by a +1-Da mass difference by LC-ESI-MS. Survival of potential immunogenic gliadin epitopes was determined by use of the R5 antibody-based ELISA. R. mucilaginosa-associated enzymes cleaved gliadins, 33-mer and 26-mer peptides into smaller fragments. TG2-mediated cross-linking showed a perfect inverse relationship with intact 33-mer and 26-mer peptide levels, and major degradation fragments showed a slow rate of MDC cross-linking of 6.18 ± 2.20 AU/min compared with 97.75 ± 10.72 and 84.17 ± 3.25 AU/min for the intact 33-mer and 26-mer, respectively, which was confirmed by reduced TG2-mediated deamidation of the fragments in mass spectrometry. Incubation of gliadins with Rothia cells reduced R5 antibody binding by 20, 82, and 97% after 30 min, 2 h, and 5 h, respectively, which was paralleled by reduced reactivity of enzyme-treated 33-mer and 26-mer peptides in the R5 competitive ELISA. Our broad complementary approach to validate gluten degrading activities qualifies R. mucilaginosa-associated enzymes as promising tools to neutralize T cell immunogenic properties for treatment of celiac disease.

Immunoreactivity of antibodies against transglutaminase-deamidated gliadins in adult celiac disease.

BACKGROUND: The significance of the presence of anti-gliadin antibodies in patients affected by celiac disease is still unclear. It is hypothesized that gliadin deamidation, catalysed by transglutaminase, plays a role in favoring the antigen presentation. AIM: To determine the immunoreactivity of anti-gliadin antibodies from untreated celiac patients to transglutaminase deamidated gliadins. MATERIALS AND METHODS: Gliadins from wheat flour underwent enzymatic digestion and were deamidated or cysteamine-transamidated by transglutaminase. Immunoreactivity of anti-gliadin antibodies from untreated adult celiac patients sera was evaluated by means of a competitive enzyme-linked immunosorbent assay (ELISA) method. RESULTS: Gliadin deamidation increased antibodies immunoreactivity from 25% to 50% while cysteamine incorporation into the gliadin peptides resulted in an immunoreactivity decrease. CONCLUSIONS: Increased immunoreactivity of transglutaminase deamidated gliadins tested with anti-gliadin antibodies from untreated adult celiac patients supports the hypothesis of a pivotal role of gliadin deamidation in the pathomechanism of celiac disease.

Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease.

The action of tissue Transglutaminase (TGase) on specific protein-bound glutamine residues plays a critical role in numerous biological processes. Here we provide evidence for a new role of this enzyme in the common, HLA-DQ2 (and DQ8) associated enteropathy, celiac disease (CD). The intestinal inflammation in CD is precipitated by exposure to wheat gliadin in the diet and is associated with increased mucosal activity of TGase. This enzyme has also been identified as the main target for CD-associated anti-endomysium autoantibodies, and is known to accept gliadin as one of its few substrates. We have examined the possibility that TGase could be involved in modulating the reactivity of gliadin specific T cells. This could establish a link between previous reports of the role of TGase in CD and the prevailing view of CD as a T-cell mediated disorder. We found a specific effect of TGase on T-cell recognition of gliadin. This effect was limited to gliadin-specific T cells isolated from intestinal CD lesions. We demonstrate that TGase mediates its effect through an ordered and specific deamidation of gliadins. This deamidation creates an epitope that binds efficiently to DQ2 and is recognized by gut-derived T cells. Generation of epitopes by enzymatic modification is a new mechanism that may be relevant for breaking of tolerance and initiation of autoimmune disease.

Effect of gamma-interferon on binding of gliadin and other food peptides to the human intestinal cell line HT-29.

gamma-Interferon is one of the main cytokines released during activation of intestinal lymphocytes in coeliac patients. The question has never been addressed whether gamma-interferon influences binding of gliadin and other food peptides to human enterocytes. Therefore, the human intestinal epithelial cell line HT-29 was cultured with gliadin, casein, beta-lactoglobulin and ovalbumin, with or without gamma-interferon, and peptide binding to cells was determined by flow cytometry and fluorescence microscopy. gamma-Interferon stimulated gliadin binding by a factor of 4. Binding was saturable with half maximal binding at 0.15 mg/ml. For maximal binding, an incubation of at least 24 h was necessary. gamma-Interferon increased binding of beta-lactoglobulin and casein, too, but inhibited that of ovalbumin. Binding of gliadin was inhibited by the other peptides. Under the conditions of ongoing mucosal inflammatory reactions and release of gamma-interferon, enhanced binding may trigger intestinal lymphocytes, increase secretion of cytokines and thus induce a vicious circle.

Peptic-tryptic digests of gliadin: contaminating trypsin but not pepsin interferes with gastrointestinal protein binding characteristics.

For many years, peptic-tryptic digests of gliadin, known as Frazer's fraction III, have been used in investigations of gliadin effects. Potential contamination by the proteases pepsin and trypsin, however, was not considered. To investigate the influence of contaminating proteases on binding of gliadin peptides to rat small intestinal brush border membranes we compared binding characteristics of different gliadin digests. Binding of biotinylated probes was studied in dot blots and Western blots with an enhanced chemiluminescence system. In gliadin peptide preparations only contaminating trypsin, but not pepsin, was detectable by specific antisera. Digestion with insoluble proteases attached to cross-linked beaded agarose yielded gliadin peptides free of contaminating pepsin and trypsin. These peptides bound 30% less to brush border membranes. Using these peptides, there was no trypsin-typical binding pattern to low molecular mass membrane proteins in contrast to peptide preparations which contained contaminating trypsin. In conclusion contaminating trypsin might alter gliadin peptide binding characteristics by direct binding to brush border membranes and by interfering with interactions between gliadin peptides and brush border membranes.