Tissue transglutaminase in cells of the vessel wall
This study investigated the distribution of tissue transglutaminase (TG2) antigen and activity in human umbilical vein endothelial cells (HUVEC) and peripheral blood derived monocytes. Both cell types produced and secreted TG2 with an approximate 50:50 distribution between cell associated and secreted TG2. TG2 antigen in HUVEC was principally distributed between cell lysates and extracellular matrix (ECM) samples, yet the activity of ECM samples was lower. The highest specific activity for both cell types was found in conditional medium. Monocyte cell lysates activity was consistently below the level of detection, suggesting that TG2 was inactive. TG2 antigen levels for both cell types were stable, both in quantity and distribution in the samples, in the presence of inflammatory type mediators. However, TG2 activity could be modified by inflammatory mediators. HUVEC treated with conditioned medium from monocytes and vice versa, both demonstrated stable antigen levels but the activity of secreted TG2 could be increased. This indicated that some factor secreted by monocytes augments TG2 activity the extracellular environment. TG2 secretion by the classical ER/Golgi route was investigated by synthesis of de novo radiolabelled TG2. Localisation of de novo synthesised TG2, from cells and from in vitro translation of TG2 in Xenopus oocyte extracts, demonstrated that TG2 was not found associated with ER/Golgi membranes. This indicated that TG2 is not a classically secreted protein and is therefore secreted from the cytosol by another mechanism. Local TG2 substrates were identified from 2D gel electrophoresis of HUVEC samples treated +/- calcium in the presence/absence of a TG specific inhibitor. These proteins were analysed by mass spectrometry. The nine proteins identified were keratin, albumin, actin, tropomyosin, HSP27, TCP-1-α, RhoGAP1, eIF3 and DPM synthase.