Gaudry, C.A., 1998. Tissue transglutaminase: a new secretory protein. PhD, Nottingham Trent University.
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Abstract
The protein cross-linking enzyme tissue transglutaminase is believed to be involved in the processing and stabilisation of extracellular matrix structures. The major aim of this study was to investigate the secretory mechanism of the enzyme which lacks a typical leader sequence. Shorter studies gave some understanding of its involvement in tumour growth and cell death mechanisms.
Preliminary investigations indicated that the enzyme was not found in the culture media of cell lines expressing high intracellular levels of the tissue transglutaminase (tTG) protein. Subsequent immunohistochemistry (immunocytochemistry) indicated a cell surface localisation for the enzyme which colocalised with fibronectin during the early stages of fibril assembly.
In order to track the enzyme fusion constructs were made whereby tTG was fused to reporter proteins (bacterial enzyme β-galactosidase, Green Fluorescent Protein, GFP and a Protein Kinase Ce peptide, s-tag). One of the fusion constructs to β-galactosidase had the proposed fibronectin binding site (the first seven N-terminus amino acids) removed. Using these constructs it was established that the ECM protein fibronectin was involved in the externalisation of the enzyme. The GFP fusion constructs allowed live cell observations and lead to the conclusion that there is a probable involvement of cytoskeleton structures in the intracellular distribution and trafficking of the tissue transglutaminase.
Immunogold studies using transmission electron microscopy were conducted to provide further evidence of the subcellular distribution of the enzyme and to define its extracellular localisation. Label corresponding to the tissue transglutaminase antigen was found in the overall extra cellular matrix structures showing that the enzyme progresses in those structures once secreted from the cells and its co-localisation with fibronectin was identified both extra and intracellularly.
A further study was undertaken to define the subcellular localisation of tTG during cell injury induced by electropermeabilisation. Using fluorescein cadaverine incorporation into proteins by the enzyme as a measure of crosslinking, tTG was shown to be first activated in cytoplasmic structures. When the injury was sustained for 20min and with increasing concentrations of calcium up to 2mM entering the cells there was evident crosslinking activity of the enzyme in nuclear structures. These results suggest the involvement of tissue transglutaminase in the crosslinking of intracellular components thus preventing their leakage into the extra-cellular environment in the context of cell injury /death.
The β-galactosidase fusion constructs were also used to stably transfect a well characterised hamster fibrosarcoma cell line (MetB) for an in vivo study conducted in Syrian hamsters. Results showed that tumours originating from the cell line transfected with the tissue transglutaminase-β-galactosidase construct were slower to grow than controls (MetB cells and MetB cells transfected with β-galactosidase) and that the tissue transglutaminase was selected against during tumour progression.
Item Type: | Thesis | ||||
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Creators: | Gaudry, C.A. | ||||
Date: | 1998 | ||||
ISBN: | 9781369323580 | ||||
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Divisions: | Schools > School of Science and Technology | ||||
Record created by: | Linda Sullivan | ||||
Date Added: | 02 Oct 2020 11:37 | ||||
Last Modified: | 29 Sep 2023 12:59 | ||||
URI: | https://irep.ntu.ac.uk/id/eprint/41107 |
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