Human hepatic UDP-glucuronosyltransferase (UGT) biotransformations of endogenous and exogenous compounds are well established. The role of the intestine, continually exposed to potentially toxic dietary components, drugs and metabolites secreted in bile, has not been as well studied. The long term goals of this proposal are to identify and characterize intestinal UGTs of the 2B family and to compare them with hepatic UGT2B enzymes, with primary emphasis on steroid-directed isoenzymes. The central hypothesis to be tested is that the human intestine is involved in the biotransformation of endogenous and xenobiotic compounds and functions as part of the total detoxification mechanism. Specifically, it is postulated that intestinal tissue is an active site of biotransformation of steroid hormones, some bile acid (BA) and retinoids. The intestine will be screened for specific mRNA using sequence-specific probes. Parallel experiments will be carried out to identify UGT2B enzymatic activities and proteins. A functional comparison of human recombinant UGT2B4, 2B7 and novel 2B isoforms will be carried out in relation to glucuronidation of steroid hormones, BA and retinoids. This grant also proposes to define, for both hepatic and intestinal UGT2B isoforms, the structural characteristics of the substrate binding sites which confer unique differences in substrate specificity. The hypothesis to be tested is that UGT substrate selectivity is dictated by a subset of amino acid residues in the variable N-terminal domain of the protein. Enzymatic assays, photoaffinity labeling, cDNA cloning, expression and purification of recombinant protein, proteolytic mapping of active sites and mutagenesis are among the techniques that will be used. To approach these problems, two specific aims are proposed: 1. Identify and characterize human intestinal UGT2B isoforms and compare them with human hepatic UGTs. 2. Localize the UGT2B substrate binding sites and identify the residues that determine their unique substrate specificities. It is anticipated that these studies will lead to novel concepts regarding the effect of intestinal UGT expression on detoxification of drugs and dietary constituents and its impact on hepatic and intestinal diseases. Knowledge of the molecular basis of substrate specificity should have important implications for prediction of biotransformation pathways, inhibitor design and inter-individual differences in metabolism of endogenous and xenobiotic compounds.

R01DK056226

1999-09-01

2004-08-31