Reduced expression of tissue transglutaminase in a human endothelial cell line leads to changes in cell spreading, cell adhesion and reduced polymerisation of fibronectin.
Tissue transglutaminase (tTgase, type II) is a Ca2+-dependent GTP binding protein which crosslinks proteins via (epsilon)((gamma)-glutamyl)lysine bridges. Although essentially a cytosolic enzyme there is increasing evidence to suggest the enzyme is externalised where it may play a role in extracellular matrix organisation. To investigate the function of this enzyme in a human umbilical endothelial cell line ECV304 tTgase expression was reduced in these cells by up to 90% by stable transfection with a 1.1. kb antisense construct in the plasmid vector pSG5. Two clones showing a reduction in expression of tTgase activity of 70 and 90% have been isolated and characterised. These clones show a number of phenotypic differences when compared to the parent cell line and the transfected controls which include reduced cell spreading and a decreased adhesion of cells on different substrata as measured by their susceptibility to removal by trypsin. Reduced cell spreading in the antisense transfected clones was accompanied by a decrease in the crosslinking of fibronectin into polymeric multimers which could be correlated to the amount of tTgase externalised by cells. A novel assay was developed to measure externalised tTgase activity which is cell mediated, inhibited by preincubation of cells with anti-tTgase antibody and relies 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 be related to the enzymes ability to bind and crosslink fibronectin.
Jones
R A
RA
Department of Life Sciences, Nottingham Trent University, Nottingham, UK.
Nicholas
B
B
Mian
S
S
Davies
P J
PJ
Griffin
M
M
D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D019921:Biotinylation; D015152:Blotting, Northern; D002103:Cadaverine / Q000378:metabolism; D002448:Cell Adhesion / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D048429:Cell Size / Q000276:immunology; D002999:Clone Cells / Q000201:enzymology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000276:immunology; D005353:Fibronectins / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D019204:GTP-Binding Proteins; D006801:Humans; D011108:Polymers / Q000378:metabolism; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D016372:RNA, Antisense; D014162:Transfection; D011503:Transglutaminases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D011108:Polymers; D016372:RNA, Antisense; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D002103:Cadaverine