Tissue Transglutaminase Is an Important Player at the Surface of Human Endothelial Cells: Evidence for Its Externalization and Its Colocalization with the  1 Integrin C. A. Gaudry,* E. Verderio,* R. A. Jones,* C. Smith,† and M. Griffin* ,1 *Department of Life Sciences, Nottingham Trent University, Nottingham, United Kingdom; and †Unilever Research, Colworth House, Bedford, United Kingdom We recently reported [J. Cell Sci. 110, 2461–2472 (1997)] that reduced expression of tissue transglutami- nase (tTgase, type II) in human endothelial cell line ECV304 led to impaired cell spreading and adhesion; however, there is no immunocytochemical evidence for its presence and specific location at the surface of these cells. In this report we have stably transfected the same cell line with the cDNA for human tTgase which has been tagged at the C-terminus of the en- coded protein with a 12-amino-acid peptide from pro- tein kinase C epsilon. Using antibodies directed against this epitope tag peptide we show for the first time using immunogold electron microscopy and fluo- rescent immunocytochemistry the presence of cell surface-related tTgase. In cells undergoing attach- ment and cell spreading the enzyme appears to be concentrated at cell adhesion points which are rich in  1 integrin, suggesting that these areas may be the initial focal points for enzyme externalization. In more spread and confluent cells the enzyme appears more diffusely distributed along the basal membrane, with increased concentrations found at areas of cell– cell and cell–substratum contact. These findings strengthen the argument for the enzyme’s role in cell– matrix interactions. © 1999 Academic Press Key Words: tissue transglutaminase; -tagged pro- tein; cell surface; cell adhesion;  1 integrin. INTRODUCTION Transglutaminases are a group of protein cross-link- ing enzymes which polymerize proteins into high-mo- lecular-weight aggregates via intramolecular (-glu- tamyl)lysine bonds. In higher vertebrates they are dependent on Ca 2+ for their activity and have a diverse range of functions, localizations, and structures [for review, see 1, 2]. The most ubiquitous of this family of enzymes is tissue transglutaminase (tTgase, type II; EC 2.3.2.13) which unlike the other transglutaminases is a GTP–GDP binding enzyme [3, 4]. In the intracel- lular environment, binding of GTP and GDP prevents Ca 2+ activation of the enzyme [5], unless GTP and GDP are depleted and/or Ca 2+ levels approach those present in the extracellular environment. Given this scenario, there is mounting evidence to indicate that tTgase may have an important role at the cell surface and in the extracellular matrix (ECM) where the cross-linking of ECM proteins such as fibronectin, collagen, and os- teonectin [6 –10] is believed to be important in their deposition and stabilization. In human endothelial cells it has been suggested that its primary role may lie in the stabilization of the basement membrane [11–13]. More recently, by using antisense technology, we have also shown that the endothelial cell tTgase may have a more dynamic role in both cell spreading and cell ad- hesion, which appears to depend on the ability of the enzyme to polymerize fibronectin at the cell surface [14]. However, despite these cited functions for the en- zyme, direct immunocytochemical evidence to indicate its presence and specific location at the cell surface of endothelial cells is still lacking, as conventional immu- nocytochemical techniques do not discriminate be- tween the cytoplasmic pool of enzyme and exterior cell surface-specific tTgase. In this article we show for the first time that by transfecting human endothelial cells with a novel epitope-tagged tTgase construct the tagged enzyme may be clearly detected at the cell surface. Moreover, in cells undergoing cell attachment and spreading the enzyme is clearly codistributed with the  1 inte- grin at cell adhesion sites, while in more confluent cells the enzyme is more diffusely spread along the basal membrane of these cells, with concentration of 1 To whom correspondence and reprint requests should be ad- dressed at Department of Life Sciences, Nottingham Trent Univer- sity, Clifton Lane, Clifton, Nottingham NG11 8NS, UK. Fax: 0115 948 6636. E-mail: martin.griffin@ntu.ac.uk. Experimental Cell Research 252, 104 –113 (1999) Article ID excr.1999.4633, available online at http://www.idealibrary.com on 104 0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.