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• W1982202632 abstract "Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme. Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme. uridine-diphosphoglucuronate glucuronosyltransferases (prefix “h” indicates human) endoplasmic reticulum high-pressure liquid chromatography 1,6-bismaleimidohexane dimethyl 3,3′-dithiobispropionimidate base pair enzyme-linked immunosorbent assay human immunodeficiency virus Uridine-diphosphoglucuronate glucuronosyltransferases (UGTs)1 constitute a family of enzymes that detoxify numerous endogenous and exogenous substances by catalyzing the transfer of the glucuronic acid moiety of uridine diphosphoglucuronate to the aglycone substrates (1Roy Chowdhury J. Wolkoff A.W. Roy Chowdhury N. Arias I.M. Scriver R.C. Beaudet A.L. Valle D. Sly W.S. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Book Co., New York2001: 3063-3101Google Scholar). Based on structural homology, UGTs are classified into several families and subfamilies (1Roy Chowdhury J. Wolkoff A.W. Roy Chowdhury N. Arias I.M. Scriver R.C. Beaudet A.L. Valle D. Sly W.S. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Book Co., New York2001: 3063-3101Google Scholar, 2Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger A. Fournel-Gigleux S. Green M. Hum D. Iyanagi T. Lancet D. Louisot P. Magdalou J. Roy Chowdhury J. Ritter J. Schachter H. Tephly T.R. Tipton K.F. Nebert D.W. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (983) Google Scholar). UGT1A1, which mediates bilirubin glucuronidation (3Bosma P.J. Seppen J. Goldhoorn B. Bakker C. Oude Elferink R.P.J. Roy Chowdhury J. Roy Chowdhury N. Jansen P.L.M. J. Biol. Chem. 1994; 269: 17960-17964Abstract Full Text PDF PubMed Google Scholar), belongs to a subfamily that is expressed from the UGT1A locus at human chromosome 2q37 (4Van Es H.H.G. Bout A. Liu J. Anderson I. Duncan A.M.V. Bosma P.J. Oude Elferink R.P.J. Jansen P.L.M. Roy Chowdhury J. Schurr E. Cytogenet. Cell Genet. 1993; 63: 111-114Crossref PubMed Scopus (63) Google Scholar). The four exons (exons 2–5) located at the 3′-end of this locus are used in mRNAs for several UGT1A isoforms and encode the identical carboxyl-terminal halves of these enzymes (5Ritter J.K. Chen F. Sheen Y.Y. Tran H.M. Kimura S. Yeatman M.T. Owens I.S. J. Biol. Chem. 1992; 267: 3257-3261Abstract Full Text PDF PubMed Google Scholar). Upstream of these exons is a series of at least 12 unique exons, only one of which is used in the mRNA for a given isoform (5Ritter J.K. Chen F. Sheen Y.Y. Tran H.M. Kimura S. Yeatman M.T. Owens I.S. J. Biol. Chem. 1992; 267: 3257-3261Abstract Full Text PDF PubMed Google Scholar). The unique region exon encodes the amino-terminal domain of these enzymes, which imparts their aglycone substrate specificity (6Mackenzie P.I. J. Biol. Chem. 1990; 265: 3432-3435Abstract Full Text PDF PubMed Google Scholar). UGTs are concentrated in the endoplasmic reticulum (ER) (7Roy Chowdhury J. Novikoff P.M. Roy Chowdhury N. Novikoff A.B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2990-2994Crossref PubMed Scopus (103) Google Scholar). Except for a 21–26-amino acid carboxyl-terminal cytoplasmic tail, the enzymes are located within the ER lumen, predicting the need for transport of the sugar donor substrate, UDP-glucuronic acid, from the cytosol to the catalytic site within the ER lumen. UGT1A1 is encoded by exons 1A1 and 2–5. UGT1A1-mediated glucuronidation is essential for efficient biliary excretion of bilirubin. Genetic lesions within the coding region of UGT1A1 can abolish or markedly reduce UGT activity toward bilirubin, resulting in Crigler-Najjar syndrome type 1 or 2, respectively (8Kadakol A. Ghosh S.S. Sappal B.S. Sharma G. Roy Chowdhury J. Roy Chowdhury N. Hum. Mutat. 2000; 16: 297-306Crossref PubMed Scopus (309) Google Scholar), both of which are characterized by unconjugated hyperbilirubinemia (9Crigler J.F. Najjar V.A. Pediatrics. 1952; 10: 169-180PubMed Google Scholar). The inheritance of Crigler-Najjar syndrome types 1 and 2 generally follows an autosomal recessive pattern (9Crigler J.F. Najjar V.A. Pediatrics. 1952; 10: 169-180PubMed Google Scholar) because, in most heterozygous carriers, the wild-type enzyme expressed from the structurally normal allele is sufficient to keep plasma bilirubin concentrations within normal limits. However, the heterozygous carriers of certain human UGT1A1 (hUGT1A1) mutations exhibit mild to moderate hyperbilirubinemia, suggesting an autosomal dominant pattern of inheritance (10Koiwai O. Aono S. Adachi Y. Kamisako T. Yasui Y. Nishizawa M. Sato H. Hum. Mol. Genet. 1996; 5: 645-647Crossref PubMed Scopus (65) Google Scholar). It has been postulated that, in these cases, mutant hUGT1A1 may act as a dominant-negative protein. An essential component of this hypothesis is intermolecular association of hUGT1A1 molecules. Several previous observations have suggested the intermolecular association of some other UGT isoforms. Gel permeation chromatography of solubilized rat liver microsomal UGT isoforms with activity toward chenodeoxycholic acid (11Matern H. Matern S. Gerok W. J. Biol. Chem. 1982; 257: 7422-7427Abstract Full Text PDF PubMed Google Scholar) or phenols (12Gschaidmeier H. Bock K.W. Biochem. Pharmacol. 1994; 48: 1545-1549Crossref PubMed Scopus (41) Google Scholar) suggested that they exist in a larger than monomeric size. Radiation inactivation analysis of rat liver microsomes also suggested that UGT1A1 may exist as dimers and tetramers (13Peters W.H.M. Jansen P.L.M. Henk N. J. Biol. Chem. 1984; 259: 11701-11706Abstract Full Text PDF PubMed Google Scholar). However, these studies did not differentiate between the homodimerization of UGT isoforms and interaction between one UGT isoform and other isoforms or non-UGT proteins. In fact, interaction between rat liver testosterone-UGT and androsterone-UGT isoforms has been reported (14Matsui M. Nagai F. Anal. Biochem. 1980; 105: 141-146Crossref PubMed Scopus (13) Google Scholar). It has been proposed that heterodimerization of rat liver UGT2B1 with a UGT1A isoform may activate UDP-N-acetylglucosamine-stimulated UDP-glucuronic acid transport into the ER lumen (15Ikushiro S. Emi Y. Iyanagi T. Biochemistry. 1997; 36: 7154-7161Crossref PubMed Scopus (115) Google Scholar). The functional relevance of UGT dimerization was suggested by partial reconstitution of steroid-UGT activity upon coexpression of two catalytically inactive mutant forms of rat liver UGT2B1 (16Meech R. Mackenzie P.I. J. Biol. Chem. 1997; 272: 26913-26917Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). However, there is no direct evidence for homodimerization of human UGT1A1. Therefore, we have used a combination of gel permeation high-pressure liquid chromatography (HPLC), nearest-neighbor cross-linking analysis, and two-hybrid studies in yeast and mammalian systems to test the hypothesis that hUGT1A1 interacts with itself. We have also partially characterized the domain of hUGT1A1 that is required for such interaction. To express hUGT1A1 in a cell that does not express any other UGT isoform, we stably transduced skin fibroblasts from jaundiced Gunn rats, which lack the UGT1A subfamily of UGT isoforms, using a Moloney murine leukemia virus-based gene transfer system as previously described (17Seppen J. Tada K. Hellwig S. Bakker C.T.M. Prasad V.R. Roy Chowdhury N. Roy Chowdhury J. Bosma P.J. Oude Elferink R.P.J. Biochem. J. 1996; 314: 477-483Crossref PubMed Scopus (20) Google Scholar). The Gunn rat fibroblasts lack any other detectable UGT activity. These cells (termed GURF-hUGT1A1) express hUGT1A1 at a level comparable to that of normal human liver microsomes. To examine whether hUGT1A1 forms intermolecular disulfide linkage, GURF-hUGT1A1 cells were disrupted by sonication, and microsomes were prepared as described (17Seppen J. Tada K. Hellwig S. Bakker C.T.M. Prasad V.R. Roy Chowdhury N. Roy Chowdhury J. Bosma P.J. Oude Elferink R.P.J. Biochem. J. 1996; 314: 477-483Crossref PubMed Scopus (20) Google Scholar). The microsomal proteins (25 μg/lane) were resolved by SDS-10% polyacrylamide gel electrophoresis with or without pretreatment with 100 mm dithiothreitol and with or without heating the sample with SDS. Immunotransblot analysis was performed using a monoclonal antibody (WP1) against the human UGT1A group of proteins (18Seppen J. Jansen P.L.M. Oude Elferink R.P.J. Protein Expression Purif. 1995; 6: 149-154Crossref PubMed Scopus (3) Google Scholar). Microsomes derived from GURF-hUGT1A1 cells were suspended in 50 mmHEPES (pH 7.8) containing 2% n-octyl glucoside. After gentle stirring on ice for 1 h, the preparation was subjected to centrifugation (100,000 × g for 60 min). The soluble proteins in the supernatant were diluted with 50 mm HEPES (pH 7.8) to reduce the concentration of n-octyl glucoside to 0.2% (critical micellar concentration of n-octyl glucoside is 0.6%). The solubilized proteins (0.5 ml) were applied to a gel permeation HPLC column (I-250, two columns in tandem; Waters Associates, Milford, MA), and the proteins were eluted isocratically at 1 ml/min with 50 mm HEPES (pH 7.8) containing 0.1%n-octyl glucoside. The fractions (0.5 ml) were analyzed by immunoblotting using the WP1 monoclonal antibody. The immunoreactive bands were quantified by laser densitometry. To determine whether hUGT1A1 interacts with itself within the ER membrane, we performed “nearest-neighbor” analysis using two different cross-linkers. For generating intermolecular disulfide bonds, 10% homogenates of GURF-hUGT1A1 cells were prepared in 20 mm Tris-HCl (pH 8.0) containing 0.25 m sucrose using a glass homogenizer by 30 up-and-down strokes of a motorized Teflon pestle. Microsomes were prepared as described (17Seppen J. Tada K. Hellwig S. Bakker C.T.M. Prasad V.R. Roy Chowdhury N. Roy Chowdhury J. Bosma P.J. Oude Elferink R.P.J. Biochem. J. 1996; 314: 477-483Crossref PubMed Scopus (20) Google Scholar) and suspended in 50 mm Tris-HCl (pH 7.4) containing 0.25 mm sucrose and 5 mmMgCl2. The microsomal suspension (containing 100 μg of protein) was treated with 1,6-bismaleimidohexane (BMH; Pierce) in dimethyl sulfoxide at a final concentration of 1 or 2 mm. The cross-linking reaction was carried out at 25 °C for 60 min. The reaction was quenched with 20 mm 2-mercaptoethanol. To determine whether the chemical cross-linking involves the cytoplasmic tail or the part of the protein within the microsomal lumen, a membrane-impermeable inhibitor, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (referred to as stilbenedisulfonate maleimide below; Molecular Probes, Inc.), was used. For this experiment, the microsomal preparation was preincubated with 2 mm stilbenedisulfonate maleimide for 60 min at 25 °C before the cross-linking with BMH as described above. The protein samples were then subjected to SDS-4–10% gradient polyacrylamide gel electrophoresis, followed by immunoblot analysis with WP1 (15Ikushiro S. Emi Y. Iyanagi T. Biochemistry. 1997; 36: 7154-7161Crossref PubMed Scopus (115) Google Scholar). To evaluate the extent to which the microsomes were sealed, the degree of latency of UGT1A1 activity in the microsomal preparation was determined. For this purpose, the enzyme activity toward bilirubin was determined in the presence or absence of 0.17%n-octyl glucoside. As BMH is a relatively ineffective cross-linker at alkaline pH, to determine the effect of hydrogen ion concentration on intermolecular binding of hUGT1A1, we used dimethyl 3,3′-dithiobispropionimidate (DTBP, Pierce), which cross-links amino groups and is effective at a wide pH range. To generate intermolecular bonds, GURF-hUGT1A1 microsomes were suspended in 50 mm Tris-HCl containing 0.25 mm sucrose and 5 mm MgCl2 at pH 7.4 or 9.0. The microsomal suspension (containing 100 μg of protein) was treated with DTBP at a final concentration of 2, 5, or 10 mm. The cross-linking reaction was carried out at 22 °C (optimum temperature) for 60 min. The reaction was stopped by adding 0.5 m glycine (pH 7.0). SDS-4–10% gradient polyacrylamide gel electrophoresis and immunoblot analysis was performed as described (19Briggs M.M. Capaldi R.A. Biochemistry. 1977; 16: 73-77Crossref PubMed Scopus (53) Google Scholar). Site-directed mutagenesis was done to introduce point mutations into hUGT1A1 cDNA. This was performed based on the method of Deng and Nickoloff (20Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1078) Google Scholar) with some minor modifications of the protocols (TransformedTMmutagenesis kit, CLONTECH). Site-directed mutagenesis was performed on plasmid pSVK3, which contains the entire hUGT1A1 coding region (21Kadakol A. Sappal B.S. Ghosh S.S. Lowenheim M. Chowdhury A. Chowdhury S. Santra A. Arias I.M. Roy Chowdhury J. Roy Chowdhury N. J. Med. Genet. 2001; 38: 244-248Crossref PubMed Google Scholar). The mutagenized clones were primarily selected by XbaI digestion. The entire 2.2-kilobase pair mutagenized hUGT1A1 cDNA was excised by digestion with NotI and XhoI and subcloned into the pcDNA3.1/Zeo(+) expression vector (Invitrogen). Introduction of the desired mutation was confirmed by nucleotide sequence determination by the dideoxy chain termination method (22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 74: 5463-5467Crossref Scopus (52229) Google Scholar, 23Gantla S. Attavar P. Sengupta K. Roy Chowdhury N. Epicentre Forum. 1995; 2: 3Google Scholar, 24Gantla S. Bakker C.T.M. Deocharan B. Thumala N.R. Zweiner J. Sinaasappel M. Roy Chowdhury J. Bosma P.J. Roy Chowdhury N. Am. J. Hum. Genet. 1998; 62: 585-592Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To delete a segment of hUGT1A1 (amino acids 152–180) that spans a membrane-embedded helix, we generated an expression plasmid with in-frame deletion of nucleotides 454–540. A 453-base pair (bp) segment of the UGT1A1 cDNA including the translation initiation codon (ATG) was amplified from plasmid pcDNA3.1/Zeo(+)-hBUGT (21Kadakol A. Sappal B.S. Ghosh S.S. Lowenheim M. Chowdhury A. Chowdhury S. Santra A. Arias I.M. Roy Chowdhury J. Roy Chowdhury N. J. Med. Genet. 2001; 38: 244-248Crossref PubMed Google Scholar) using the following amplimers: sense, 5′-CGGGATCCGATGGCTGTGGAGTCCCAGGGCGGAC-3′ (a BamHI linker (italic) was added at the 5′-end for subsequent cloning into the mammalian two-hybrid vectors); and antisense, 5′-GTCCGTCAGCATGACATCAAAGCTG-3′. The amplicon was end-repaired with Klenow and digested with BamHI. Another 300-bp fragment (nucleotides 541–841) was amplified using the following amplimers: sense, 5′-TTTGAGGCTACCCAGTGCCCCAA-3′; and antisense, 5′-GCAGTTGATTCCACCAAC-3′. The 300-bp amplicon was digested with EcoRI. The 166-bp 5′-fragment of the amplicon was blunt-ended and ligated to the 3′-end of the 453-bp amplicon to generate a 619-bp fragment. The parental plasmid, pcDNA3.1/Zeo(+), was digested with EcoRI to excise a 707-bp segment from the 5′-end of the UGT1A1 coding region. The 619-bp fragment generated by ligation of the two polymerase chain reaction amplicons was ligated to the EcoRI site of pcDNA3.1/Zeo(+). The resulting UGT1A1 cDNA with the deletion of nucleotides 454–540 was cloned in-frame at the BamHI and XhoI sites of the mammalian two-hybrid vectors. COS-7 cells, grown in 100-mm dishes to 50–60% confluency, were transfected with pcDNA3.1/Zeo(+) containing normal or mutagenized hUGT1A1 sequences and a ZeocinTM resistance gene using DEAE-dextran (Amersham Pharmacia Biotech) as described (21Kadakol A. Sappal B.S. Ghosh S.S. Lowenheim M. Chowdhury A. Chowdhury S. Santra A. Arias I.M. Roy Chowdhury J. Roy Chowdhury N. J. Med. Genet. 2001; 38: 244-248Crossref PubMed Google Scholar). Total protein in the cell lysates was quantitated by enzyme-linked immunosorbent assay (ELISA; see below), and an equal amount of protein from different transfected cell lysates was subjected to immunotransblot analysis using WP1 (3Bosma P.J. Seppen J. Goldhoorn B. Bakker C. Oude Elferink R.P.J. Roy Chowdhury J. Roy Chowdhury N. Jansen P.L.M. J. Biol. Chem. 1994; 269: 17960-17964Abstract Full Text PDF PubMed Google Scholar, 21Kadakol A. Sappal B.S. Ghosh S.S. Lowenheim M. Chowdhury A. Chowdhury S. Santra A. Arias I.M. Roy Chowdhury J. Roy Chowdhury N. J. Med. Genet. 2001; 38: 244-248Crossref PubMed Google Scholar). The immunoreactive bands were visualized using a chemiluminescent substrate (Pierce). Expressed hUGT1A1 in each cell lysate was quantitated by sandwich ELISA as described (21Kadakol A. Sappal B.S. Ghosh S.S. Lowenheim M. Chowdhury A. Chowdhury S. Santra A. Arias I.M. Roy Chowdhury J. Roy Chowdhury N. J. Med. Genet. 2001; 38: 244-248Crossref PubMed Google Scholar) with minor modifications. Briefly, ELISA plates were coated overnight with the WP1 antibody (1:4000 dilution), blocked with phosphate-buffered saline containing 3% bovine serum albumin and 5% fetal calf serum, and then overlaid with lysates of the transfected cells. A rabbit antibody against a synthetic peptide corresponding to a unique region of hUGT1A1 (polyclonal antibody 136) was applied at a 1:500 dilution. The detection system consisted of horseradish peroxidase-conjugated goat anti-rabbit IgG. Absorbance at 405 nm was determined using an ELISA plate reader. Based on ELISA quantitation, equal amounts of hUGT1A1 from the various cell lysates were assayed for UGT activity toward bilirubin in the presence of 80 μmbilirubin, 4.4 mm UDP-glucuronic acid, and dioleoylphosphatidylcholine as described (3Bosma P.J. Seppen J. Goldhoorn B. Bakker C. Oude Elferink R.P.J. Roy Chowdhury J. Roy Chowdhury N. Jansen P.L.M. J. Biol. Chem. 1994; 269: 17960-17964Abstract Full Text PDF PubMed Google Scholar). In brief, after incubation at 37 °C for 4 h, the reaction was stopped by adding 0.4 m glycine HCl (pH 1.8), and the pigments were extracted in chloroform/ethanol (1:1, v/v) (3Bosma P.J. Seppen J. Goldhoorn B. Bakker C. Oude Elferink R.P.J. Roy Chowdhury J. Roy Chowdhury N. Jansen P.L.M. J. Biol. Chem. 1994; 269: 17960-17964Abstract Full Text PDF PubMed Google Scholar). The solvents were evaporated under a stream of nitrogen. The pigments were dissolved in dimethyl sulfoxide, mixed with an equal volume of methanol, and analyzed by reverse-phase HPLC using a Waters μBondapak C18 column as described (25Roy Chowdhury J. Roy Chowdhury N. Wu G. Shouval R. Arias I.M. Hepatology. 1981; 1: 622-627Crossref PubMed Scopus (50) Google Scholar). Formation of bilirubin glucuronides was calculated from the electronically integrated areas under the bilirubin glucuronide peaks. In the yeast two-hybrid system, two physically functional domains are necessary, a DNA-binding domain and a transactivation domain. A 2.2-kilobase pair fragment spanning the entire coding region of hUGT1A1 was amplified from plasmid pcDNA3.1/Zeo(+)-hBUGT (21Kadakol A. Sappal B.S. Ghosh S.S. Lowenheim M. Chowdhury A. Chowdhury S. Santra A. Arias I.M. Roy Chowdhury J. Roy Chowdhury N. J. Med. Genet. 2001; 38: 244-248Crossref PubMed Google Scholar) using sense and antisense amplimers containing BamHI and XhoI linkers, respectively. The amplicon was digested with BamHI and XhoI and cloned in-frame into the BamHI and SalI sites of the “bait” vector pSH2.1 so that hUGT1A1 is expressed as a fusion protein with the bacterial Lex DNA-binding domain. The nucleotide sequence of the insert was determined to confirm the fidelity of the amplification. The hUGT1A1 cDNA was also cloned in-frame into the BamHI and SalI sites of the “prey” vector Gal4-GADNOT so that hUGT1A1 is expressed as a fusion protein with the Gal4 transactivator protein (activation domain). The constructs (20 μg each) were cotransfected into yeast strain CTY. In this yeast strain, the LexA operator is engineered into the 5′-flanking region of the Escherichia coli β-galactosidase reporter gene (lacZ). The transformed cells were plated on selective dropout agar plates without histidine and leucine. After growing the yeast cells for 3–4 days at 30 °C, the colonies were blotted onto nitrocellulose membranes and stained for E. coli β-galactosidase activity with the 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside substrate (X-gal; Stratagene, La Jolla, CA). Interaction of the proteins expressed by the bait and prey constructs brings the Gal4 activation domain into proximity with LexA (DNA-binding domain), resulting in the transcription of LacZ and the appearance of blue colonies. HIV-1 integrase protein (IN) cloned into both the bait and prey vectors was used as a positive control. As a negative control, we used a bait construct expressing hUGT1A1 in combination with a prey construct without any fusion sequence or a bait construct without any fusion sequence in combination with a prey construct expressing hUGT1A1 (26Fields S. Song O. Nature. 1989; 340: 245-247Crossref PubMed Scopus (4793) Google Scholar). In the mammalian two-hybrid assay system, the reporter plasmid pFR-Luc contains a synthetic promoter with five tandem repeats of the yeast Gal4 binding sites that control expression of the luciferase gene. This reporter gene expression occurs as a result of reconstitution of a functional transcription factor by the association of two hybrid proteins. A 2.2-kilobase pair DNA segment containing the full-length hUGT1A1 cDNA was cloned in-frame into the BamHI and XbaI sites of the bait vector pCMV-BD so that hUGT1A1 is expressed as a fusion protein with the DNA-binding domain of yeast Gal4. The test sequence (wild-type or mutagenized hUGT1A1 or human UGT1A6) was cloned in-frame into the BamHI and XhoI sites of the pCMV-AD vector, where it is expressed as a fusion protein with the transcriptional activation domain of mouse NF-κB. The various coding sequences cloned into the prey vector are listed in Table I. These two constructs were cotransfected into COS-7 cells, cultured at 50% confluence, along with plasmid pFR-Luc. Positive interaction of the proteins expressed from the bait and prey vectors brings the NF-κB activation domain into proximity with the Gal4 DNA-binding domain, thereby activating the expression of luciferase. As a positive control, the coding regions of simian virus 40 large T antigen and p53 were cloned into the bait and prey vectors, respectively (27Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.L. Merriam S. Du M.Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2000; 275: 41082-41086Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Luciferase activity was assayed according to the manufacturer’s protocol (Stratagene). To determine whether the mutagenized forms of hUGT1A1 express stable proteins, the bait and prey plasmid constructs were transfected into COS-7 cells by the DEAE-dextran method (21Kadakol A. Sappal B.S. Ghosh S.S. Lowenheim M. Chowdhury A. Chowdhury S. Santra A. Arias I.M. Roy Chowdhury J. Roy Chowdhury N. J. Med. Genet. 2001; 38: 244-248Crossref PubMed Google Scholar), and the cell lysates were analyzed by immunotransblotting using WP1.Table IcDNAs cloned into the bait and prey vectorsBaitPreyIntermolecular associationhUGT1A1hUGT1A1+hUGT1A1hUGT1A6−hUGT1A1C127Y hUGT1A1+hUGT1A1F171Y hUGT1A1+hUGT1A1L175E hUGT1A1±hUGT1A1C223Y hUGT1A1±hUGT1A1L519 XhUGT1A1+hUGT1A1K530 X hUGT1A1+hUGT1A1hUGT1A1-(Δ152–180)−Positive (+), absent (−), or markedly reduced (±) intermolecular associations are shown. Open table in a new tab Positive (+), absent (−), or markedly reduced (±) intermolecular associations are shown. To examine whether some mutant forms of hUGT1A1 can inhibit the function of wild-type hUGT1A1, we performed cotransfection experiments. A series of plasmids expressing wild-type hUGT1A1 and several mutant forms of hUGT1A1 were prepared. The plasmids were transfected into COS-7 cells singly (10 μg/75-cm2 plate) or in pairs (5 μg each/75-cm2 plate) using DEAE-dextran as the transfection vehicle (21Kadakol A. Sappal B.S. Ghosh S.S. Lowenheim M. Chowdhury A. Chowdhury S. Santra A. Arias I.M. Roy Chowdhury J. Roy Chowdhury N. J. Med. Genet. 2001; 38: 244-248Crossref PubMed Google Scholar). To confirm the expression of transfected plasmids, immunotransblot experiments were performed using WP1. The expressed total hUGT1A1 proteins were quantified by sandwich ELISA as described above. UGT activity toward bilirubin was determined by a sensitive HPLC method as described above (25Roy Chowdhury J. Roy Chowdhury N. Wu G. Shouval R. Arias I.M. Hepatology. 1981; 1: 622-627Crossref PubMed Scopus (50) Google Scholar). One mutagenized form of hUGT1A1 (C127Y) exhibited marked inhibition of the activity of the wild-type enzyme upon coexpression in COS-7 cells (see “Results”). To determine whether the wild-type and mutagenized forms were coexpressed, we extracted the total RNA from the lysate of the transfected COS-7 cells. A 646-bp segment (nucleotides 320–1066) was amplified by reverse transcription-primed polymerase chain reaction using the following amplimers: sense, 5′-GCGTGTGATCAAAACATACAA-3′; and antisense, 5′-GCCACTTAACAAGTATCGTGTTG-3′. The sequence of the amplimer was determined by the dideoxy chain termination method using the cycle sequencing procedure as described (22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 74: 5463-5467Crossref Scopus (52229) Google Scholar, 23Gantla S. Attavar P. Sengupta K. Roy Chowdhury N. Epicentre Forum. 1995; 2: 3Google Scholar, 24Gantla S. Bakker C.T.M. Deocharan B. Thumala N.R. Zweiner J. Sinaasappel M. Roy Chowdhury J. Bosma P.J. Roy Chowdhury N. Am. J. Hum. Genet. 1998; 62: 585-592Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). COS-7 cells were transfected with wild-type or C127Y mutant hUGT1A1, and the expressed proteins were quantified by ELISA as described above. Homogenates of the cells (10%) in 20 mmTris-HCl (pH 8.0) containing 0.25 m sucrose were prepared in a glass homogenizer by 30 up-and-down strokes of a motorized Teflon pestle. UGT1A1 activity was determined in the homogenates of the transfected cells by two different methods. First, to determine whether the C127Y mutant contained in unperturbed microsomes can inhibit the activity of the wild-type enzyme present in separate microsomal vesicles, the enzyme activity in the cell homogenates was determined in the absence of any detergent to avoid membrane perturbation. For this assay, the enzyme was activated by the addition of 0.2 mmUDP-N-acetylglucosamine to the incubation mixture. UGT1A1 activity toward bilirubin was determined in homogenates of COS-7 cells transfected with the wild-type or mutant form and also in a mixture of the two homogenates. Second, to determine whether dissolution of the microsomal membranes would permit the interaction of the two forms, resulting in the inhibition of enzyme activity, we incubated the cell homogenates with 1% n-octyl glucoside on ice for 15 min before determining the UGT1A1 activity toward bilirubin at a final detergent concentration of 0.17%. To e" @default.
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• W1982202632 title "Homodimerization of Human Bilirubin-Uridine-diphosphoglucuronate Glucuronosyltransferase-1 (UGT1A1) and Its Functional Implications" @default.
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