REDUCED EXPRESSION OF TISSUE TRANSGLUTAMINASE IN A HUMAN ENDOTHELIAL-CELL LINE LEADS TO CHANGES IN CELL SPREADING, CELL-ADHESION AND REDUCED POLYMERIZATION OF FIBRONECTIN

Tissue transglutaminase (tTgase, type II) is a Ca2+-dependent GTP bind ing protein which crosslinks proteins via epsilon(gamma-glutamyl)lysin e bridges. Although essentially a cytosolic enzyme there is increasing evidence to suggest the enzyme is externalised where it may play a ro le in extracellular matrix organisation. To investigate the function o f this enzyme in a human umbilical endothelial cell line ECV304 tTgase expression was reduced in these cells by up to 90% by stable transfec tion with a 1.1. kb antisense construct in the plasmid vector pSG5. Tw o clones showing a reduction in expression of tTgase activity of 70 an d 90% have been isolated and characterised. These clones show a number of phenotypic differences when compared to the parent cell line and t he transfected controls which include reduced cell spreading and a dec reased adhesion of cells on different substrata as measured by their s usceptibility to removal by trypsin. Reduced cell spreading in the ant isense transfected clones was accompanied by a decrease in the crossli nking of fibronectin into polymeric multimers which could be correlate d to the amount of tTgase externalised by cells. A novel assay was dev eloped to measure externalised tTgase activity which is cell mediated, inhibited by preincubation of cells with anti-tTgase antibody and rel ies on the incorporation of biotinylated cadaverine into fibronectin. The results of these experiments suggest that externalised tTgase may play a key role in a number of cell behavioural patterns which might b e related to the enzymes ability to bind and crosslink fibronectin.