Abstract
PurposeTo investigate the role of thioredoxin (TRX), a novel regulator of extracellular transglutaminase 2 (TG2), in celiac patients IgA (CD IgA) mediated TG2 enzymatic activation.MethodsTG2 enzymatic activity was evaluated in endothelial cells (HUVECs) under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA) were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study.ResultsWe have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity.ConclusionsAltogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.
Highlights
Transglutaminase 2 (TG2) is a ubiquitously expressed highly complex multifunctional protein with enzymatic, cell adhesion, cell signaling and G-protein activities [1,2]
In order to establish whether celiac patients IgA (CD IgA) increases the endothelial cell extracellular transamidating activity of TG2 in a time- and/or dose-dependent manner we performed an in situ TG2 activity assay with confluent Human umbilical vein endothelial cells (HUVECs) monolayers starved O/N
With CD IgA extracellular TG2 enzymatic activity was already detectable after 3h of treatment
Summary
Transglutaminase 2 (TG2) is a ubiquitously expressed highly complex multifunctional protein with enzymatic, cell adhesion, cell signaling and G-protein activities [1,2]. These functions of TG2 are regulated by other proteins and cofactors, including Ca2+ and GTP, and by the availability of these modulators in different tissue compartments [3,4]. TG2 catalyzes the formation of isopeptide bonds (ε-γ-glutamyl- lysine crosslink) between a selected glutamine residue on one substrate and a lysine residue on another. In the absence of adequate concentrations of any suitable amine substrate, TG2 deamidates the targeted glutamine side chain, resulting in its conversion to glutamate [6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
