guanosine-triphosphate and Celiac-Disease

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Topics: Amino Acid Motifs; Binding Sites; Calcium; Celiac Disease; Cell Aggregation; Enzyme Inhibitors; Gliadin; Glutens; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; K562 Cells; Models, Biological; Molecular Docking Simulation; Peptide Fragments; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Glutamine gamma Glutamyltransferase 2; Protein Interaction Domains and Motifs; Transglutaminases

Human tissue transglutaminase (htTG) is one of the most important member within the transglutaminase family, enzymes that for their capacity of catalyzing post-translational modifications of proteins and peptides, rise an high interest for industrial applications. More recently, for its implication as the major autoantigen in the coeliac disease, availability of human tissue transglutaminase as recombinant form is required for accurate diagnostic tests. The aim of this study was to find an alternative and inexpensive source to produce human tissue transglutaminase. To date, plant systems are proposed as heterologous hosts to produce recombinant proteins for use in disease diagnosis and therapy. Here, we describe the stable expression of human tissue transglutaminase into Nicotiana tabacum cultured cells (cultivar Bright Yellow 2 (BY-2)). The recombinant enzyme was successfully expressed in different plant cell compartments and both apoplast (apo) and chloroplast (chl) purified proteins were shown to be catalytically active and able to bind GTP, a property possessed by the natural counterpart. Importantly, plant produced human tissue transglutaminase recognized autoantibodies in the serum of coeliac patients, suggesting possible applications in the diagnosis of coeliac disease.

Topics: Autoantibodies; Base Sequence; Celiac Disease; Cell Culture Techniques; Chromatography, Affinity; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Guanosine Triphosphate; Humans; Nicotiana; Polymerase Chain Reaction; Recombinant Proteins; Transglutaminases