Tissue transglutaminase: mechanism of secretion and role in cell migration and extracellular matrix stabilisation

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Balklava, Z., 2002. Tissue transglutaminase: mechanism of secretion and role in cell migration and extracellular matrix stabilisation. PhD, Nottingham Trent University.

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Abstract

Increasing evidence suggests that tissue transglutaminase (tTGase, type II) is externalised from cells where it may play a key role in cell attachment and spreading and in the stabilisation of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the mechanism of enzyme secretion are not fully understood. The role of tTGase in cell attachment and migration was investigated using stably transfected fibroblast cell lines, which express tTGase in its active and inactive (Cys277Ser mutant) state. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface but only the clone overexpressing the catalytically active tTGase deposited the enzyme into the ECM and in the cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme in the ECM or secrete it into the cell culture medium. Similarly lack of tTGase secretion was observed when cells were transfected with tTGase mutated at Tyr274 (Tyr274Ala), the proposed site for the cis/trans peptide bond within the tTGase active site domain. However, inhibition of tTGase activity with a range of competitive substrates or the active site directed inhibitor Rob283 did not affect enzyme secretion. All together these data suggest that the tertiary conformation, which depends on Cys277 and Tyr274 may be essential for tTGase exteraalisation. These results also indicate that tTGase regulates cell motility as a novel cell surface adhesion protein rather than a matrix cross-linking enzyme. They also demonstrate further important insights into the mechanism of externalisation of the enzyme into the ECM.

Gene expression patterns were analysed in cells inducible for the overexpression of catalytically active and inactive (Cys277Ser mutant) tTGase and no widespread differences in gene expression were observed. Although only part of the genome was analysed, the obtained results suggested that any changes in cell behaviour are likely to be the direct effect of tTGase. However, further analysis is necessary.

Increased secretion of tTGase into the ECM did not affect the ECM turnover rate. However, addition of exogenous catalytically active tTGase to the fibroblasts culture medium resulted in an increased ECM deposition, but did not increase its stability to digestion by microbial collagenase and trypsin.

Item Type:

Thesis

Creators:

Balklava, Z.

Date:

2002

ISBN:

9781369314588

Identifiers: