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The human cDNA clone, UDPGTh-2, encoding a liver UDP-glucuronosyltransferase (transferase) was isolated from a lambda gt11 cDNA library by hybridization to the mouse transferase cDNA clone, UDPGTm-1 (Kimura, T., and Owens, I. S. (1987) Eur. J. Biochem.168, 515-521). The two clones have nucleotide sequence identities in the coding region of 74%. UDPGTh-2 encodes a 529-amino acid protein with an NH2 terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. There are three potential asparagine-linked glycosylation sites at residues 67, 68, and 315. In order to establish substrate specificity, the clone was inserted into the pSVL vector (pUDPGTh-2) and expressed in COS-1 cells. The presence of a transferase with Mr congruent to 52,000 in transfected cells cultured in the presence of [35S]methionine was shown by immunocomplexed products with goat antimouse transferase IgG (Mackenzie, P. I., Hjelmeland, L. H., and Owens, I. S. (1984) Arch. Biochem. Biophys. 231, 487-497) and protein A-Sepharose and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The transferase is a glycoprotein as indicated by a shift in Mr congruent to 3000-4000 when expressed in the presence of tunicamycin. Sixty potential substrates were tested using cells transfected with pUDPGTh-2. The order of relative substrate activity was as follows: 4-hydroxyestrone greater than estriol greater than 2-hydroxyestriol greater than 4-hydroxyestradiol greater than 6 alpha-hydroxyestriol greater than 5 alpha-androstane-3 alpha,11 beta,17 beta-triol = 5 beta-androstane-3 alpha,11 beta,17 beta-triol. There were only trace amounts of glucuronidation of 2-hydroxyestrone and 2-hydroxyestradiol, and, in contrast to other cloned transferases, no glucuronidation of either the primary estrogens/androgens (estrone, 17 beta-estradiol/testosterone, androsterone) or any of the exogenous substrates tested. A Lineweaver-Burk plot of the effect of 4-hydroxyestrone concentration on the velocity of glucuronidation shows an apparent Km of 13 microM. The unique specificity of this transferase for 3,4-catechol estrogens and estriol suggests it may play an important role in regulating the level and activity of these potent and active estrogen metabolites. |
