Ivana Caputo

Mammalian transglutaminases. Identification of substrates as a key to physiological function and physiopathological relevance.

Transglutaminases form a large family of intracellular and extracellular enzymes that catalyse the Ca2+‐dependent post‐translational modification of proteins. Despite significant advances in our understanding of the biological role of most mammalian transglutaminase isoforms, recent findings suggest new scenarios, most notably for the ubiquitous tissue transglutaminase. It is becoming apparent that some transglutaminases, normally expressed at low levels in many tissue types, are activated and/or overexpressed in a variety of diseases, thereby resulting in enhanced concentrations of cross‐linked proteins. As applies to all enzymes that exert their metabolic function by modifying the properties of target proteins, the identification and characterization of the modified proteins will cast light on the functions of transglutaminases and their involvement in human diseases. In this paper we review data on the properties of mammalian transglutaminases, particularly as regards their protein substrates and the relevance of transglutaminase‐catalysed reactions in physiological and disease conditions.

Anti-tissue transglutaminase antibodies from coeliac patients inhibit transglutaminase activity both in vitro and in situ

Background and aims: Coeliac disease (CD) is a multifactorial disorder which has an autoimmune component characterised by the occurrence of disease specific autoreactive antibodies against the enzyme tissue transglutaminase (tTG). The aim of this study was to investigate whether binding of antibodies to the enzyme influences tTG activity. Methods: tTG activity was assayed in the presence of immunoglobulin A (IgA) and immunoglobulin G (IgG) purified from the serum of coeliac patients, CUB 7402 (an anti-tTG mouse monoclonal antibody), and human anti-tTG monoclonal antibodies derived from both intestinal lymphocytes from three patients with CD and from peripheral blood lymphocytes from healthy subjects. For our studies we used calcium treated and untreated recombinant human tTG. Furthermore, the effects of antibodies were determined by immunohistochemical detection of tTG activity in sections of human umbilical cord. Results: IgG and IgA from CD patients inhibited tTG activity in vitro in a dose dependent manner, with a different rate of inhibition among patients. The monoclonal antibody CUB 7402 and human monoclonal antibodies displayed a dose dependent inhibitory effect towards the catalytic activity of the enzyme, both in vitro and in situ. Preincubation of tTG with CaCl2 caused loss of the inhibitory effect due to CUB 7402 but not that caused by human monoclonal antibodies. Conclusions: Purified CD IgA, IgG, as well as human anti-tTG monoclonal antibodies inhibited the enzymatic activity of human tTG both in vitro and in situ.

Expression and enzymatic activity of small intestinal tissue transglutaminase in celiac disease

OBJECTIVES:The molecular and functional properties of small intestinal tissue transglutaminase are largely unknown despite growing interest because of its role in celiac disease (CD). In this study, we aimed to evaluate tissue transglutaminase expression and enzymatic activity in bioptic fragments obtained from the duodenum of untreated individuals with CD and from control subjects.METHODS:Analysis of tissue transglutaminase mRNA expression was performed by reverse transcription–polymerase chain reaction (RT-PCR). The presence of the enzyme in bioptic fragments as well as in homogenates from CD patients and controls was revealed by immunohistochemistry and Western blot, respectively, using the antitissue transglutaminase CUB 7402 clone. To evaluate in situ transglutaminase activity, sections of bioptic fragments were incubated in the presence of 5 mmol/L CaCl2 with 5-(biotinamido)pentylamine or, alternatively, with a biotinylated glutamine-containing hexapeptide (TVQQEL) and the biotinylated 31–43 A-gliadin–derived peptide.RESULTS:Tissue transglutaminase mRNA levels were 1.0-fold higher (p < 0.05) in CD patients than in controls. Immunohistochemistry and in situ demonstration of enzymatic activity in celiac mucosa clearly showed an increased expression of active tissue transglutaminase in the extracellular matrix of the subepithelial region and in the enterocytes. Staining of the biotinylated 31–43 A-gliadin peptide in the same area of tissue transglutaminase suggested the presence of lysine-donor substrates in intestinal mucosa.CONCLUSIONS:Tissue transglutaminase is more expressed and active in defined areas of the small intestinal mucosa from patients with CD. The presence in the celiac mucosa of proteins able to act as amine-donor substrates suggests that tissue transglutaminase–mediated post-translational modification of proteins cross-linked with gliadin peptides may represent a pathogenic mechanism of CD.

Anti-transglutaminase antibodies in non-coeliac children suffering from infectious diseases.

Anti‐transglutaminase antibodies are the diagnostic markers of coeliac disease. A role is suggested for infectious agents in the production of anti‐transglutaminase antibodies. The aim was to measure positive anti‐transglutaminase antibody levels in children with infectious diseases and to compare immunological and biological characteristics of the anti‐transglutaminase antibodies derived from these children with that from coeliac patients. Two hundred and twenty‐two children suffering from infectious diseases were enrolled prospectively along with seven biopsy‐proven coeliacs. Serum samples were tested for anti‐transglutaminase antibodies and anti‐endomysium antibodies; positive samples were tested for coeliac‐related human leucocyte antigen (HLA)‐DQ2/8 and anti‐viral antibodies. Purified anti‐transglutaminase antibodies from the two study groups were tested for urea‐dependent avidity, and their ability to induce cytoskeletal rearrangement and to modulate cell‐cycle in Caco‐2 cells, using phalloidin staining and bromodeoxyuridine incorporation assays, respectively. Nine of 222 children (4%) tested positive to anti‐transglutaminase, one of whom also tested positive for anti‐endomysium antibodies. This patient was positive for HLA‐DQ2 and was diagnosed as coeliac following intestinal biopsy. Of the eight remaining children, two were positive for HLA‐DQ8. Levels of anti‐transglutaminase returned to normal in all subjects, despite a gluten‐containing diet. Purified anti‐transglutaminase of the two study groups induced actin rearrangements and cell‐cycle progression. During an infectious disease, anti‐transglutaminase antibodies can be produced temporarily and independently of gluten. The infection‐triggered anti‐transglutaminase antibodies have the same biological properties as that of the coeliacs, with the same in‐vivo potential for damage.

Tissue transglutaminase in celiac disease: role of autoantibodies.

In celiac disease (CD), gluten, the disease-inducing toxic component in wheat, induces the secretion of IgA-class autoantibodies which target tissue transglutaminase (tTG). These autoantibodies are produced in the small-intestinal mucosa, and, during gluten consumption, they can also be detected in patients’ serum but disappear slowly from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The finding of IgA deposits on extracellular tTG in the liver, kidney, lymph nodes and muscles of patients with CD indicates that tTG is accessible to the gut-derived autoantibodies. Although the specific autoantibody response directed against tTG is very characteristic in celiac patients, their role in the immunopathology of the celiac mucosal lesion is a matter of debate. Here we report a brief summary of anti-tTG antibody effects demonstrating that these antibodies are functional and not mere bystanders in the disease pathogenesis. In fact, they inhibit intestinal epithelial cell differentiation, induce intestinal epithelial cell proliferation, increase epithelial permeability and activate monocytes and disturb angiogenesis.

Gliadin Peptides Induce Tissue Transglutaminase Activation and ER-Stress through Ca2+ Mobilization in Caco-2 Cells

Background Celiac disease (CD) is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG) seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca2+ homeostasis and tTG activity. Methods/Principal Findings We studied Ca2+ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31–43 and 57–68 rapidly mobilized Ca2+ from intracellular stores. Specifically, peptide 31–43 mobilized Ca2+ from the endoplasmic reticulum (ER) and mitochondria, whereas peptide 57–68 mobilized Ca2+ only from mitochondria. We also found that gliadin peptide-induced Ca2+ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31–43, but not peptide 57–68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31–43, but not of peptide 57–68, induces the expression of both genes. Conclusions By inducing Ca2+ mobilization from the ER, peptide 31–43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31–43 and 57–68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin–tTG crosslinking in enterocytes and specialized antigen-presenting cells.

Enzymatic Strategies to Detoxify Gluten: Implications for Celiac Disease

Celiac disease is a permanent intolerance to the gliadin fraction of wheat gluten and to similar barley and rye proteins that occurs in genetically susceptible subjects. After ingestion, degraded gluten proteins reach the small intestine and trigger an inappropriate T cell-mediated immune response, which can result in intestinal mucosal inflammation and extraintestinal manifestations. To date, no pharmacological treatment is available to gluten-intolerant patients, and a strict, life-long gluten-free diet is the only safe and efficient treatment available. Inevitably, this may produce considerable psychological, emotional, and economic stress. Therefore, the scientific community is very interested in establishing alternative or adjunctive treatments. Attractive and novel forms of therapy include strategies to eliminate detrimental gluten peptides from the celiac diet so that the immunogenic effect of the gluten epitopes can be neutralized, as well as strategies to block the gluten-induced inflammatory response. In the present paper, we review recent developments in the use of enzymes as additives or as processing aids in the food biotechnology industry to detoxify gluten.

New Therapeutic Strategies for Coeliac Disease: Tissue Transglutaminase as a Target

Coeliac disease is a multifactorial disease characterized by a dysregulated immune response to ingested wheat gluten and related cereal proteins. With an incidence of about 1% of the general population, it is considered the most common food intolerance disorder. The mainstay of coeliac disease treatment is strict lifelong adherence to a gluten-free diet. Elimination of gluten and related proteins from the diet leads to clinical and histological improvement. However, some patients do not respond to dietary therapy and others have poor dietary compliance. This has prompted the search for a therapy alternative to a gluten-free diet. Tissue transglutaminase is a crucial factor in coeliac disease because it promotes the gluten-specific T-cell response and is also the target of the autoimmune response. Tissue transglutaminase induces changes in gluten, which in turn, cause the generation of a series of gluten peptides that bind to HLA-DQ2 or DQ8 molecules with high affinity. The resulting HLA-DQ2 (DQ8)-gluten peptide interaction triggers the proinflammatory T cell response. Tissue transglutaminase is also involved in other non-T-cell-mediated biological activities of gliadin peptides. For these reasons, tissue transglutaminase is a potential target for therapeutic intervention. In this paper we review the state-of-the-art of tissue transglutaminase inhibition, and examine known and new-generation inhibitors and their activity in in vitro and in vivo models. We also examine their potential as therapeutic tools for coeliac disease.

Celiac anti-tissue transglutaminase antibodies interfere with the uptake of alpha gliadin peptide 31-43 but not of peptide 57-68 by epithelial cells

Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31-43 (but not peptide 57-68) uptake by cells, thereby impairing the ability of p31-43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31-43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.

Surface water disinfection by chlorination and advanced oxidation processes: Inactivation of an antibiotic resistant E. coli strain and cytotoxicity evaluation.

The release of antibiotics into the environment can result in antibiotic resistance (AR) spread, which in turn can seriously affect human health. Antibiotic resistant bacteria have been detected in different aquatic environments used as drinking water source. Water disinfection may be a possible solution to minimize AR spread but conventional processes, such as chlorination, result in the formation of dangerous disinfection by-products. In this study advanced oxidation processes (AOPs), namely H2O2/UV, TiO2/UV and N-TiO2/UV, have been compared with chlorination in the inactivation of an AR Escherichia coli (E. coli) strain in surface water. TiO2 P25 and nitrogen doped TiO2 (N-TiO2), prepared by sol-gel method at two different synthesis temperatures (0 and -20°C), were investigated in heterogeneous photocatalysis experiments. Under the investigated conditions, chlorination (1.0 mg L(-1)) was the faster process (2.5 min) to achieve total inactivation (6 Log). Among AOPs, H2O2/UV resulted in the best inactivation rate: total inactivation (6 Log) was achieved in 45 min treatment. Total inactivation was not observed (4.5 Log), also after 120 min treatment, only for N-doped TiO2 synthesized at 0°C. Moreover, H2O2/UV and chlorination processes were evaluated in terms of cytotoxicity potential by means of 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenylte-trazolium colorimetric test on a human-derived cell line and they similarly affected HepG2 cells viability.