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1.
Biochemistry ; 40(13): 3869-81, 2001 Apr 03.
Article in English | MEDLINE | ID: mdl-11300766

ABSTRACT

UDP-glucuronosyltransferase (UGT) enzymes belonging to the UGT2B subfamily catalyze the transfer of glucuronic acid to a large number of endogenous compounds, particularly steroids, to facilitate their elimination from target cells. A novel human UGT2B cDNA of 1666 bp was isolated and encodes a 529-amino acid protein named UGT2B28 type I. Glucuronidation assays demonstrated that UGT2B28 type I catalyzes the conjugation of endogenous and exogenous compounds. The tissue distribution of UGT2B28 revealed the expression of the type I transcript in the liver, breast, and LNCaP cells. Two other UGT2B cDNAs were isolated, and sequence analysis led to the identification of two truncated UGT2B28 species. UGT2B28 type II differs from type I by a deletion of 308 bp in the cofactor binding domain, whereas UGT2B28 type III lacks 351 bp in the putative substrate binding domain. All UGT2B28 isoforms are encoded by a single UGT2B28 gene which has a genomic organization similar to that of the other UGT2B genes characterized thus far. Although no substrates could be identified for the shorter isoforms, the three subtypes were shown to be located in the endoplasmic reticulum and the perinuclear membrane, demonstrating that the missing domains are not required for the subcellular localization of these UGT2B proteins. However, all the domains remain necessary for observing glucuronidation activity. The expression of UGT2B28 type I in the breast and liver suggests a role of this enzyme in the androgen and estrogen metabolism in these tissues.


Subject(s)
DNA, Complementary/chemistry , DNA, Complementary/isolation & purification , Glucuronosyltransferase/genetics , Glucuronosyltransferase/isolation & purification , Hymecromone/analogs & derivatives , Alternative Splicing , Amino Acid Sequence , Androstane-3,17-diol/metabolism , Androsterone/metabolism , Base Sequence , Bile Acids and Salts/metabolism , Cell Line , Cyst Fluid/chemistry , Cyst Fluid/metabolism , Enzyme Activation/genetics , Estradiol/metabolism , Female , Fibrocystic Breast Disease/chemistry , Fibrocystic Breast Disease/metabolism , Glucuronides/metabolism , Glucuronosyltransferase/chemistry , HeLa Cells , Humans , Hymecromone/metabolism , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/isolation & purification , Molecular Sequence Data , Organ Specificity/genetics , RNA, Messenger/metabolism , Subcellular Fractions/enzymology , Testosterone/metabolism
2.
Endocrinology ; 142(2): 778-87, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11159850

ABSTRACT

Androgens and estrogens play major roles in cell differentiation, cell growth, and peptide secretion in steroid target tissues. In addition to the binding of these hormones to their receptors, formation and metabolism are important in the action of steroids. Metabolism of the potent steroid hormones includes glucuronidation, a major pathway of steroid elimination in liver and several steroid target tissues. Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs), which transfer the polar moiety from UDP-glucuronic acid to a wide variety of endogenous compounds, including steroid hormones. The UGT superfamily of enzymes is subdivided into two families, UGT1 and UGT2, on the basis of sequence homology. To date, six UGT2B proteins have been isolated, namely UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, all of which have been demonstrated to be active on steroid molecules, except for UGT2B10 and UGT2B11, for which no substrate was found. The relative activity of these enzymes on steroidal compounds remains unknown due to variable levels of UGT2B expression in different in vitro cell line models and various conditions of the enzymatic assays. Comparison of the glucuronidation rates of these enzymes requires a unique system for UGT2B protein expression, protein normalization, and enzymatic assays. In this study we have stably expressed UGT2B4, UGT2B7, UGT2B15, and UGT2B17 in the HK293 cell line, which is devoid of steroid UGT activity; characterized their kinetic properties relative to UGT protein expression; determined their transcript and protein stabilities; and established extensively their tissular distributions. UGT2B7 was demonstrated to glucuronidate estrogens, catechol estrogens, and androstane-3alpha,17beta-diol more efficiently than any other human UGTB isoform. UGT2B15 and UGT2B17 showed similar glucuronidation activity for androstane-3alpha,17beta-diol (30% lower than that of UGT2B7), whereas UGT2B17 demonstrated the highest activity for androsterone, testosterone, and dihydrotestosterone. UGT2B4 demonstrates reactivity toward 5alpha-reduced androgens and catechol estrogens, but at a significantly lower level than UGT2B7, 2B15, and 2B17. Cycloheximide treatment of stably transfected HK293 cells demonstrated that the UGT2B17 protein is more labile than the other enzymes; the protein levels decrease after 1 h of treatment, whereas other UGT2B proteins were stable for at least 12 h. Treatment of stable cells with actinomycin D reveals that UGT2B transcripts are stable for 12 h, except for the UGT2B4 transcript, which was decreased by 50% after the 12-h incubation period. Tissue distribution of the UGT2B enzymes demonstrated that UGT2B isoforms are expressed in the liver as well as in several extrahepatic steroid target tissues, namely, kidney, breast, lung, and prostate. This study clearly demonstrates the relative activities and the major substrates of human steroid-metabolizing UGT2B enzymes, which are expressed in a wide variety of steroid target tissues.


Subject(s)
Glucuronosyltransferase/chemistry , Glucuronosyltransferase/metabolism , Multigene Family/physiology , Steroids/metabolism , Cell Line , Enzyme Stability , Glucuronosyltransferase/genetics , Humans , Kinetics , RNA, Messenger/metabolism , Tissue Distribution
3.
Biochem Biophys Res Commun ; 272(2): 616-21, 2000 Jun 07.
Article in English | MEDLINE | ID: mdl-10833461

ABSTRACT

Glucuronidation is a major pathway involved in the metabolism of drugs and numerous endogenous compounds, such as bile acids and steroid hormones. The enzymes responsible for this conjugation reaction are UDP-glucuronosyltransferases (UGT). Among the UGT2B subfamily, UGT2B7, a UGT enzyme present in the liver and several steroid target tissues, is an important member since it conjugates a large variety of compounds including estrogens, androgens, morphine, AZT, and retinoic acid. Although this enzyme is well characterized, the gene encoding the UGT2B7 protein and its promoter region remain unknown. In this article, we report the genomic organization and the promoter region of the human UGT2B7 gene. To isolate this gene, a P-1 artificial chromosome (PAC) library was screened with a full length UGT2B7 probe and a clone of approximately 100 kb in length was isolated. In addition to the UGT2B7 gene, this PAC contains two other UGT2B genes previously characterized, namely UGT2B26P and UGT2B27P. The UGT2B7 gene is composed of six exons spanning approximately 16 kb, with introns ranging from 0.7 to 4.2 kb. The 5'-flanking region of the human UGT2B7 gene contains several potential cis-acting elements such as Oct-1, Pbx-1, and C/EBP. Only one TATA-box at nucleotide -106 was found within the first 500 nucleotides relative to the adenine base of the initiator ATG codon. Characterization of the UGT2B7 gene provides insight into the organization and regulation of this important metabolic gene.


Subject(s)
Exons/genetics , Glucuronosyltransferase/genetics , Introns/genetics , Promoter Regions, Genetic/genetics , Base Sequence , Cloning, Molecular , Conserved Sequence/genetics , Gene Expression Profiling , Humans , Molecular Sequence Data , Pseudogenes/genetics , Response Elements/genetics , TATA Box/genetics
4.
J Mol Biol ; 295(3): 489-504, 2000 Jan 21.
Article in English | MEDLINE | ID: mdl-10623541

ABSTRACT

Glucuronidation is a major pathway of androgen metabolism and is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. UGT2B15 and UGT2B17 are 95% identical in primary structure, and are expressed in steroid target tissues where they conjugate C19 steroids. Despite the similarities, their regulation of expression are different; however, the promoter region and genomic structure of only the UGT2B17 gene have been characterizedX to date. To isolate the UGT2B15 gene and other novel steroid-conjugating UGT2B genes, eight P-1-derived artificial chromosomes (PAC) clones varying in length from 30 kb to 165 kb were isolated. The entire UGT2B15 gene was isolated and characterized from the PAC clone 21598 of 165 kb. The UGT2B15 and UGT2B17 genes are highly conserved, are both composed of six exons spanning approximately 25 kb, have identical exon sizes and have identical exon-intron boundaries. The homology between the two genes extend into the 5'-flanking region, and contain several conserved putative cis-acting elements including Pbx-1, C/EBP, AP-1, Oct-1 and NF/kappaB. However, transfection studies revealed differences in basal promoter activity between the two genes, which correspond to regions containing non-conserved potential elements. The high degree of homology in the 5'-flanking region between the two genes is lost upstream of -1662 in UGT2B15, and suggests a site of genetic recombination involved in duplication of UGT2B genes. Fluorescence in situ hybridization mapped the UGT2B15 gene to chromosome 4q13.3-21.1. The other PAC clones isolated contain exons from the UGT2B4, UGT2B11 and UGT2B17 genes. Five novel exons, which are highly homologous to the exon 1 of known UGT2B genes, were also identified; however, these exons contain premature stop codons and represent the first recognized pseudogenes of the UGT2B family. The localization of highly homologous UGT2B genes and pseudogenes as a cluster on chromosome 4q13 reveals the complex nature of this gene locus, and other novel homologous UGT2B genes encoding steroid conjugating enzymes are likely to be found in this region of the genome.


Subject(s)
Chromosomes, Human, Pair 4 , Glucuronosyltransferase/genetics , Isoenzymes/genetics , Multigene Family , Pseudogenes , Amino Acid Sequence , Base Sequence , Cell Line , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Exons , Humans , In Situ Hybridization, Fluorescence , Introns , Molecular Sequence Data , Open Reading Frames , Promoter Regions, Genetic
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