The modulatory effects of flavonoids on the enzymatic activities of transglutaminase2

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Alshintari, M., 2019. The modulatory effects of flavonoids on the enzymatic activities of transglutaminase2. PhD, Nottingham Trent University.

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Abstract

To date, a gluten-free diet is the only accepted form of therapy for coeliac disease (CD). Because of the important role of transglutaminase2 enzyme in the initiation of coeliac disease (CD).This study was aimed at the identification of TG2 inhibitors from natural sources, as a potential intervention in CD therapy. The natural products chosen for this study were dietary flavonoids. Kaempferol, morin, and quercetin displayed inhibition on hr TG2 (human recombinant transglutaminase 2) and in fewer extent gplTG2 (Guinea pig transglutaminase2) amine incorporation activity. However, the majority of flavonoids showed a trend of inhibitions (>50% of inhibition at 1.25, 12.5 and125µM) on the cross-linking activity of hrTG2.The flavonoids used did not show a significant inhibitory effect on the amount of ammonia released in the deamidating activity of hrTG2. A novel method for purification of flavonoids from food extracts was developed, using Immobilized Metal Ion Affinity Chromatography (IMAC). In addition, a BCA assay was optimised in a novel quantification method using quercetin as a standard flavonoid. In addition, to the pure flavonoids, flavonoids were extracted from different food samples. In the TG2 amine incorporation activity, all of food extracts display a significant inhibition effect towards the hrTG2 and gplTG2 (20-50% of inhibition). While in the TG2 cross-linking activity, the majority of food extracts did display an inhibition effect on the gpl TG2 cross-linking activity (50-70% of inhibition), but only the Strawberry and kale extract had an effect on hrTG2 activity (40-50% of inhibition). In the deamidation assay, no food extracts showed inhibition. A cell-based fluorescence assay confirmed the TG2 inhibitory characteristic of kaempferol on HT29 cell lines. This was among the first studies to examine the effects of flavonoids on the activity of TG2. The inhibition of transglutaminase 2 (TG2) deamidating activity can be considered as a potential therapeutic target in the treatment of coeliac disease (CD). All current TG2 assays measure amine incorporation, none can specifically measure TG2 deamidating activity because there would be little or no deamidation in the presence of an amine. A novel deamidating assay has been developed using o-pthaldehyde (OPA) to measure the ammonia released in deamidation. This assay demonstrated that the TG2 deamidation of Vicia faba storage proteins and gliadin peptides was enhanced by presence calcium and inhibited by EDTA.

Item Type:	Thesis
Creators:	Alshintari, M.
Date:	August 2019
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	24 Jun 2020 07:30
Last Modified:	24 Jun 2020 07:30
URI:	https://irep.ntu.ac.uk/id/eprint/40091