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Title: The use of antisense-mediated gene silencing to study tissue transglutaminase in ECV304 cells
Author: Jones, Richard A.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2001
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Tissue transglutaminase (tTGase) is a widespread multifunctional enzyme that catalyses protein cross-linldng and nucleotide hydrolysis. The complex nature of the tTGase protein has led the enzyme to be implicated in many aspects of cellular function, but a definitive biological role for tTGase has not been demonstrated. In order to study tTGase function in ECV304 cells, cells were stably transfected with tTGase antisense constructs and two clones were isolated with reductions in tTGase activity of 70 and 90%. These clones exhibited a number of phenotypic differences from their high tTGase-expressing counterparts that were related to their extracellular matrix (ECM) processing. Antisense clones exhibited an impaired cell spreading ability, reduced adhesive strength, and impaired ECM deposition in serum-free conditions. Analysis of cell surface-specific tTGase activity on a range of ECM proteins indicated that cell spreading ability could be correlated to extracellular activity, and fibronectin was the preferred substrate when compared to collagens type I, III and IV and laminin. However, labeling of live cells with fluorescein cadaverine indicated numerous other γ-glutamyl substrates were present on the cell surface. Analysis of in situ tTGase activity also suggested the enzyme was inactive in the cytoplasmic compartment of healthy cells. Adhesion blockade experiments in control cells with an inactivating anti-tTGase monoclonal antibody, and addition of exogenous tTGase to antisense cells indicated that the cell surface enzyme was present in a specific location and configuration on the cell surface. The findings suggest that the primary role of tTGase in ECV304 is related to the binding and organisation of the ECM.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Cell; Cellular function; Enzymes