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Title: Causes and consequences of altered O-glycosylation in colon epithelial cells
Author: Tam, Benjamin Armstrong
ISNI:       0000 0004 2670 7864
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2008
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Changes of cellular glycosylation, such as the increased expression of the oncofetal Thomsen-Freidenreich antigen (galactoseB1-3N-acetylgalactosaminea-,TF), are common in cancer and pre-cancerous conditions. The glycosylation changes observed in colon cancer can be induced by prevention of the nonnal acidification of the Golgi in colonocytes probably through fragmentation of the Golgi apparatus. This might in turn be triggered by bacterial-epithelial interactions or by consequential inflammation. It seems highly likely that the altered mucosal glycosylation seen in the inflamed, pre-malignant and malignant epithelium leads to recruitment of 'nonpathogenic' bacteria. It would also result in increased recruitment of dietary TFbinding lectins. The edible mushroom (Agaricus bisporus) lectin (ABL), that binds TF and sialyl-TF, has an anti-proliferative effect that relates to its internalisation -and inhibition of NLS-dependent nuclear protein import mediated via its interaction with an N-tenninally truncated fonn of the stress glycoprotein Orp150. This thesis sets out to characterise Orp150 glycosylation and investigate further the role of Orp150 in nuclear protein import, and to observe whether bacteria alter mucosal glycosylation by inducing a disorganisation ofthe Golgi apparatus. In this study, immunoblotting ofprotein extracts from HT29 colon cancer cells showed the presence of two Orp150 isofonns of which the lower molecular weight isofonn is stress-inducible by glucose starvation; selective membrane penneabilisation with digitonin showed release of a constitutive Orp150 isofonn in the released cytoplasm. Lectin affinity purification of protein extracts from a glucose-starved cell preparation with Jacalin yielded selective extraction of the constitutive Orp150 fonn suggesting differential glycosylation of the two Orp150 isofonns. siRNAOrp150 treatment of HT29 cells resulted in 63±5% reduction of Orp150 expression and subsequently 61% reduction (% nuclear fluorescence, without heat stress 46±4, after heat stress 58±3, siOrp150 + heat stress 51±3, P
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral