EXPRESSION AND ROLE OF THE HUMAN LIVER UDP-GLUCURONOSYLTRANSFERASE UGT1-ASTERISK-6 ANALYZED BY SPECIFIC ANTIBODIES RAISED AGAINST A HYBRID PROTEIN PRODUCED IN ESCHERICHIA-COLI

Characterization of human UDP-glucuronyltransferases (UGTs) has been l imited by the unavailability of probes selective for each of several h ighly related isoforms. To better understand the role of this superfam ily in the metabolism of drugs and xenobiotics, we describe a molecula r/immunological strategy for discriminating the implication of each hu man isoenzyme in this process. Specific polyclonal antibodies were gen erated against the divergent amino-terminal domain of the UGT isoform UGT16 which is involved in the detoxification of nucleophilic compoun ds related to phenols and naphthols in human liver. The novel approach consists of the expression of a N-terminal UGT polypeptide fused to S taphylococcus aureus protein A in Escherichia coli and a single step p urification of the fusion protein by immunoaffinity chromatography. Im munoblot and immunoinhibition analysis showed that the antibodies rais ed against the fusion protein selectively recognized both the denatura ted and the native forms of UGT16, when expressed in V79 cell lines, but not three other recombinant UGT isoenzymes. In human liver microso mes, specific immunoinhibition analysis demonstrated that glucuronidat ion by UGT16 represented 20 to 50% of the total 1-naphthol UGT activi ty with a good correlation with the amount of protein selectively quan tified on immunoblot. The specific expression of UGT 1 6 was found to be significantly reduced in tumoral tissues but enhanced in cholestat ic livers, when compared with healthy hepatic tissues. Interestingly, in human kidney microsomes, antibodies revealed a high level of UGT16 expression on immunoblot and inhibited 1-naphthol glucuronidation up to 55%, indicating that this isoform is also expressed in kidney and e xtensively contributes to phenol glucuronidation in this tissue. (C) 1 994 Academic Press, Inc.