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Title: A study of extracellular and intracellular tissue transglutaminase activity.
Author: Nicholas, Benjamin Leo.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 1999
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Tissue-type transglutaminase (tTG) is the most ubiquitously distributed of its enzyme family, displaying a wide range of substrates. It is a multifunctional enzyme which acts as a GTP binding protein, a stabilizer of extracellular matrix proteins, and as an effector enzyme in the apoptotic cell death pathway. Recent evidence to suggest that tTG is inactive within the healthy cell has raised questions as to the exact conditions necessary for intracellular and extracellular activity of the enzyme. To investigate the role of tTG in extracellular and intracellular processes, three model cell lines displaying various levels of intracellular tTG via sense and antisense transfection with the human cDNA for tTG were examined for the effects of altered intracellular tTG levels on cell morphology. The two fibroblast cell lines examined (Met B and Swiss 3T3) did not display any alterations in cell morphology with vastly increased intracellular tTG. The endothelial cell line ECV304 however, showed a reduction in cell spreading with reduced intracellular tTG, concommitant with collapse of the microtubule network, an effect duplicated by inactivation of extracellular tTG using a monoclonal antibody (CUB7402) to neutralize its activity. Reduced cell spreading was accompanied by the loss of polymerization of extracellular fibronectin and the loss of incorporation of a biotinylated tTG substrate into high molecular weight extracellular matrix material. Using a specially adapted in-vitro technique (the FN-cell ELSA), cell surfacerelated tTG activity was found to be reduced in tTG antisense clones of ECV304 cells, and increased in tTG sense clones of Swiss 3T3 cells. This activity could also be neutralized by pre-incubating cells with the anti-tTG monoclonal antibody CUB7402. This assay also revealed that the majority of extracellular tTG activity detected using standard techniques was present due to leakage resulting from cell manipulation such as trypsinization from culture surfaces and mechanical shear forces, or activation of the pre-existing matrix-bound enzyme through trypsin treatment, however a portion of the extracellular activity was attributable to the slow externalisation of the enzyme from the intact cell. An examination of intracellular tTG crosslinking events was made using an electropermeabilization metod, where it was observed that tTG became activated t form SDS-insoluble "shells" in the presence of high intracellular Ca2+ These shells contained cytoskeletal elements and probably also some trapped genomic DNA, implying a function for these shells as cell-sized "scars" preventing loss of cell contents upon mechanical rupture of the cells. A similar tTG activation was observed to occur in mechanically injured cells in-vitro and in-vivo, this time involving a re-distribution of tTG activity to the extracellular matrix from the intracellular compartment. During fibrosis of rat kidneys induced by subtotal nephrectomy (Snx), tTG and s(y-glutamyl)lysine were observed by immunofluorescence microscopy to be increased in renal tubule cells. With increasing tissue damage, c(Y-glutamyl)lysine was also observed in the extracellular renal interstitial space, suggesting a role for tTG in the stabilization of extracellular matrix following tissue damage. The release of tTG into the extracellular space may be due to a lack of sufficient clearance of apoptotic bodies resulting in secondary necrosis and subsequent release of the enzyme.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available