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Title: Characterisation of neural transglutaminases and their potential roles in organophosphate toxicity
Author: Howden, Richard
ISNI:       0000 0001 3582 6361
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2006
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The aims of this work were to characterize neural transglutaminases (TGases) and to determine the effects that phenyl saligenin phosphate (PSP) had on their activity. Calcium dependent TGase activity was detected in both cytosol and purified mitochondrial fractions from porcine brain. Peptides with high glutamine content were found to be better substrates for brain cytosol TGase mediated crosslinking activity, compared to TVQQEL with low glutamine content. This was not the case for guinea pig liver tissue transglutaminase (tTGase), suggesting that brain and liver TGases have different substrate specificities. Crossreactivity with monoclonal anti-tTGase antibodies ID 10 and Cub 7402 determined the molecular weight of brain cytosolic TGase to be 55 kDa, which may lack a C-terminal P-barrel 2 domain, as GTP did not inhibit the transamidating activities in the cytosolic fraction. Analysis of purified porcine brain mitochondria localized TGase activity to the outer membrane, where it is likely to be covalently bound by a lipid attachment. An apparent molecular weight shift on SDS-PAGE following TVQQEL crosslinking suggested that the cytoskeletal protein tubulin was a potential substrate for mitochondrial associated TGase, while the electrophoretic migration of intermediate filament proteins was not affected by TGase activity. The molecular weight of mitochondria associated TGase (shown by crossreactivity with IDIO and Cub 7402) was determined to be 75 kDa, which matched that determined for guinea pig liver (tissue) transglutaminase separated and probed in parallel. PSP treatment was shown to induce a significant increase in the activity of the 55 kDa cytosolic form. By contrast, the activity of mitochondrial associated TGase activity and purified guinea pig liver TGase were significantly decreased in the presence of PSP, showing that the 75 kDa enzyme was affected by PSP in a different way to that of the shorter TGase studied. This also indicates that PSP is capable of interacting directly with the 75 kDa tTGase. In the presence of sub lethal concentrations of PSP shown to inhibit axon formation in differentiating N2a neuroblastoma cells, TGase mediated crosslinking activity in cell cytosol extracts, also displaying the 55 kDa TGase, was shown to be significantly increased compared to untreated controls, suggesting that the inhibition of axon formation may be caused by an upregulation of in-situ TGase activity. The in situ activity of a 75 kDa mitochondrial associated TGase was not significantly altered in differentiating N2a cells in the presence of PSP, although levels of protein expression were significantly decreased. No significant change in the level of cytosolic TGase expression showed that PSP was able to increase transamidating activity by mechanisms other than increasing protein levels in situ. Finally, indirect immunofluorescence staining showed that, although TGase appeared to be localised increasingly to the cell neurites following PSP treatment, the amount of TGase crosslink product (N®-(L-y-glutamyl)-L-lysine) was shown to increase in the cell body of differentiating N2a cells. The cyto skeletal protein tubulin was not shown to be significantly disrupted following PSP exposure. These results are of particular interest, for both soluble and mitochondrial associated TGase activity have been shown to be disrupted in neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease and Huntington's disease. These conditions share many of the symptoms that are also displayed in organophosphate induced neurotoxicity, suggesting disrupted TGase activity may be a possible target for organophosphate toxicity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available