Tissue transglutaminase is an important player at the surface of human endothelial cells: Evidence for its externalization and its colocalization with the beta(1) integrin

We recently reported [J. Cen Sci, 110, 2461-2472 (1997)] that reduced expre ssion of tissue transglutaminase (tTgase, type II) in human endothelial cel l line ECV304 led to impaired cell spreading and adhesion; however, there i s no immunocytochemical evidence for its presence and specific location at the surface of these cells. In this report we have stably transfected the s ame cell line with the cDNA for human tTgase which has been tagged at the C -terminus of the encoded protein with a 12-amino-acid peptide from protein kinase C epsilon. Using antibodies directed against this epitope tag peptid e we show for the first time using immunogold electron microscopy and fluor escent immunocytochemistry the presence of cell surface-related tTgase. In cells undergoing attachment and cell spreading the enzyme appears to be con centrated at cell adhesion points which are rich in beta(1) integrin, sugge sting that these areas may be the initial focal points for enzyme externali zation. In more spread and. confluent cells the enzyme appears more diffuse ly distributed along the basal membrane, with increased concentrations foun d at areas of cell-cell and cell-substratum contact. These findings strengt hen the argument for the enzyme's role in cell-matrix interactions, (C) 199 9 Academic Press.