UBC Theses and Dissertations
Structure-function relationship of uridine diphosphate glucuronosyltransferases (UGTs) Liu, Yuejian
PURPOSE Uridine diphosphate glucuronosyltransferases (UGTs) are Phase II conjugation enzymes that are critical for vertebrate detoxication. They catalyze the glucuronidation biotransformations of numerous endo- and xenobiotics to facilitate their clearance and elimination and are important for hormonal regulation. Glucuronidation-related disorders include congenital syndromes, excessive chemical toxicities, adverse drug reactions, and dysregulated hormonal status. Since the 1980s it has been known that UGT proteins can be expressed but not fully active, yet little is known about how the UGT protein structure regulates its activity and function, because of the lack of a complete structure. We hypothesized that translational and post-translational mechanisms may play a role in regulating the activity and function of human UGTs. Aim 1 and 2 focused on UGT isoform 1A6, one of the most important isoforms in endo- and xenobiotic metabolism. Aim 3 studied the co-expression of major human hepatic UGT isoforms to gain an insight on their co-regulation in vivo. METHODS N-glycosylation variants of human UGT1A6 were established to study the influence of N-glycosylation on their expression, activity, cellular function and localization. HEK293 expressed UGT1A6-(His)6 and Sf9-insect expressed soluble UGT1A6 were purified and their activity and latency were characterized. The co-expression between 7 UGTs was investigated based on a western blot database collected from healthy liver donors, aged fetal (20 weeks) – 87 years. GENERAL CONCLUSIONS N-glycosylation is an important regulator of human UGT1A6 in expression, activity, and cellular function. Different forms of N-glycosylation were observed for UGT1A6 between HEK293 cells and human liver microsomes, showing a caveat of using recombinant cell lines to study UGT structure/function. Sf9-insect expressed soluble UGT1A6 is a good candidate for structure analysis due to sufficient yield and activity after purification. The influence of membranes on UGTs may be different from traditional beliefs as the data suggested alamethicin and Brij 58 can directly activate the enzyme. Lastly, expression of major human hepatic UGTs was significantly correlated and correlations were affected by age. The UGTs may be co-regulated in the human liver so single-isoform systems may have limitations on reflecting UGT activity in vivo.
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