Development of immunochemical biology tools for the study of TG2 function and mapping of the antibody reactivity in serum samples

BAYARDO M; DI NIRO R; FERRARA F; MARZARI R; SBLATTERO D; CHIRDO F.G

Praga, República Checa

Workshop; 19th Meeting of the European Working Group on Prolamin Analysis and Toxicity; 2005

European Working Group on Prolamin Analysis and Toxicity

Introduction Different roles have been described for transglutaminase 2 (TG2) in cell biology. This enzyme takes part in some critical functions such as cell signalling, apoptosis, cell migration, among others. The crosslinking of extracellular matrix components allows tissue repairing and remodelling and it was one of the first enzymatic activities described for TG2. TG2 has two, close located, binding regions: one binds Ca+2 ions and the second acts as GTP binding/GTPase site. These forms present different, and mutually exclusive, conformations which determine, according to instructions given by the microenvironment (presence of Ca+2 ions or GTP/GDP) and the localisation (intracellular, membrane bound or extracellular) different functional properties (Fesus and Piacentini, 2002). Besides, the experiments carried out by Dieterich et al. (1997) identified tissue transglutaminase as the main autoantigen detected in the antiendomisium determination. Although the pathogenic role for anti-TG2 antibodies has not been definitely proven, detection in serum of anti-TG2 specific antibodies is routinarily used in serological assays for coeliac disease detection (Dieterich et al., 1998; Sulkanen et al., 1998; Sblattero et al., 2000). For screening purposes, the use of TG2 in serological assays led to a relevant technical advantage compared with the anti-endomisium antibody determination by indirect immunofluorescence on monkey esophagous sections. To identify the epitopes recognised by anti-TG2 antibodies from serum samples, deletion mutants of TG2 were produced by different groups. Using this strategy, Seissler et al. (2001) found that main reactivity is observed in the N terminal region (aminoacid position: 1-281) and in the C terminal region (aminoacid position: 473-687). Similar results were obtained by Nakachi et al (2004) who reported the main reactivity in the N terminal region (aminoacid position: 1-89) and in the C terminal region (aminoacid position: 401-491). Sblattero et al. (2002) showed that main reactivity can be localised in the Core domain (aminoacid position: 140-376) but immunorecognition depends on the presence of N terminal or C terminal regions. On the contrary, Tiberti et al. (2003) demonstrated that main reactivity can be observed in the C terminal region (position: 227 - 687 and/ or  473 - 687). All these reports described recognition of conformational epitopes. Consequently, N terminal and C terminal domains appears as the regions mainly recognised by serum antibodies from coeliac patients but the immunodominant epitopes are still poorly defined. The aim of this work was to develop molecular biology and immunochemical tools which allow the study of TG2 function and map the antibody reactivity in serum samples