Alternative RNA splicing of leucocyte tissue transglutaminase in coeliac disease.

Tissue transglutaminase is a ubiquitous and multifunctional protein that contributes to several processes such as apoptosis/survival, efferocytosis, inflammation and tissue repairing under physiological and pathological conditions. Several activities can be associated with well-established functional domains; in addition, four RNA alternative splice variants have been described, characterized by sequence divergences and residues deletion at the C-terminal domains. Tissue transglutaminase is recognized as the central player in the physiopathology of coeliac disease (CD) mainly through calcium-dependent enzymatic activities. It can be hypothesized that differential regulation of tissue transglutaminase splice variants expression in persons with CD contributes to pathology by altering the protein functionality. We characterized the expression pattern of RNA alternative splice variants by RT-PCR in peripheral cells from patients with CD under free gluten diet adhesion; we considered inflammatory parameters and specific antibodies as markers of the stage of disease. We found significant higher expression of both the full length and the shortest C-truncated splice variants in leucocytes from patients with CD in comparison with healthy individuals. As tissue transglutaminase expression and canonical enzymatic activity are linked to inflammation, we studied the RNA expression of inflammatory cytokines in peripheral leucocytes of persons with CD in relation with splice variants expression; interestingly, we found that recently diagnosed patients showed significant correlation between both the full length and the shortest alternative spliced variants with IL-1 expression. Our results points that regulation of alternative splicing of tissue transglutaminase could account for the complex physiopathology of CD.

Characterization of catalytic and non-catalytic activities of EgGST2-3, a heterodimeric glutathione transferase from Echinococcus granulosus.

Glutathione transferases (GSTs) perform several catalytic and non-catalytic roles in the defense against toxicities of electrophile compounds and oxidative stress, and therefore are involved in stress-response and cell detoxification. Previously, we have provided evidence indicating that EgGST2 and EgGST3, two phylogenetically distant Echinococcus granulosus GSTs, can naturally form a heterodimeric structure (EgGST2-3). In the present work, the recombinant heterodimer GST (rEgGST2-3) is characterized. Hence, rEgGST2-3 was able to conjugate GSH to three substrates: 1-chloro-2,4-dinitrobenzene (CDNB, general substrate for GSTs), 1,2-dichloro-4-nitrobenzene (specific substrate for mammalian Mu class) and trans,trans-deca-2,4-dienal (reactive carbonyl). The canonical activity was considerably reduced by all the conventional inhibitors (cybacron blue, triphenylthin chloride and bromosulfophthalein) and by other inhibitors (ellagic acid, alizarin and chenodeoxycholic acid). Besides this, rEgGST2-3 activity was inhibited by a number of anthelmintic drugs, where the halogenated phenolic drugs (mainly bithionol and hexachlorophene) acted as stronger inhibitors, suggesting they may bind to the EgGST2-3. Moreover, rEgGST2-3 exhibited glutathione-peroxidase activity, and its specific constant (kcat/KM) was calculated. Finally, rEgGST2-3 displayed the ability to bind non-substrate molecules, particularly anthelmintic drugs, suggesting that ligandin activity may have potential to act as a passive protection parasite mechanism. Overall, the rEgGST2-3 behavior was shown to be both complementary and redundant to that reported for rEgGST1, another characterized GST from E. granulosus. It would be appropriate that different enzymes in the same organism do not have exactly the same functional properties to develop a better adaptation to life in the host.