Abstract
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.
Highlights
UDP-glucuronosyltransferase was isolated from a Xgt 11 cDNA library by hybridization to the mouse transferase cDNA clone, UDPGT,1
Assay for the Expression and Glycosylation of the UDP-Glucuronosyltransferase Encoded by UDPGTh-2 in COS-1 Cells-At the end of the transfection period, cells either received no further treatment or were treated with tunicamycin (1.0 rg/ml) for the incubation periods indicated in the legend to Fig. 3
Isolation of the Human Liver Clone, UDPGT,-2--The cDNA clone hybridized at high stringency to the mouse liver clone, UDPGT,1 [30], upon screening a human liver Xgtll cDNA library
Summary
Materials-UDP-glucuronic acid and all aglycones tested for substrate activity were from Sigma, Aldrich, and Fluka. [“CIUDP-. The purified plasmid was combined with the carrier, Lipofectino, and added to the cell culture dishes according to the manufacturer’s instructions. Assay for the Expression and Glycosylation of the UDP-Glucuronosyltransferase Encoded by UDPGTh-2 in COS-1 Cells-At the end of the transfection period, cells either received no further treatment or were treated with tunicamycin (1.0 rg/ml) for the incubation periods indicated in the legend to Fig. 3. Control cultures (mock-transfected) and control IgG reactions were carried out as indicated in the legend to Fig. 3 Both control and anti-mouse transferase IgG were used to immunocomplex solubilized, tritiated microsomes [23] from untreated C57BL/6N mice. Acid-Appropriate control and transfected COS-1 cells were assayed for glucuronidating activity according to a published procedure [39], but with modifications. Plates were exposed to x-ray film for 21 days to obtain prints of the results
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