a novel udp-glc-specific glucosyltransferase catalyzing the

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 20, Issue of July 15, pp. 15127-15135,1993 Printed in U. S. A .

A Novel UDP-Glc-specific Glucosyltransferase Catalyzing the Biosynthesis of 6-0-Glucosides of Bile Acids in Human Liver Microsomes*

(Received for publication, January 28, 1993)

Anna RadominskaSS, Joanna Little$, Jan S. Pyrekli, Richard R. Drake11 , Yuki IgariS, Sylvie Fournel-Gigleux**, Jacques Magdalou**, Brian Burchell$$, Alan D. Elbeinll, Gerard Siest**, and Roger Lester$ From the Departments of Slnternal Medicine and 11 Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkamas 72205, the VLife Sciences Mass Spectrometry Facility, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, the **Centre du Medicament, 54000 Nancy, France, and the $$Department of Biochemical Medicine, Ninewells Hospital and Medical School, University of Dundee, DDI 9SY Dundee, United Kingdom

Two active site-directed photoaffinity analogs, 5- [~-32P]azido-UDP-glucuronic acid and 5-[/3-32P]azido- UDP-glucose, were used for the characterization of UDP-sugar-utilizing enzymes in human liver microsomes. Both compounds were recognized by human microsomal proteins: major photolabeled bands of 50- 56 kDa were detected. Both photoincorporations were competitively decreased by increasing concentrations of either UDP-Glc or UDP-GlcUA, indicating a high affinity for both nucleotides. The patterns of photoaffinity labeling in the 50-56-kDa range by the two probes were significantly different, indicating the presence of different UDP-GlcUA- and UDP-Glc-specific enzymes of similar molecular mass. The presence of a UDP-Glc-dependent transferase was confirmed by the identification of an enzymatic activity catalyzing the formation of glucosides of the 6a-hydroxylated bile acid hyodeoxycholic acid (3a,6a-diOH (HDCA)) in the presence of UDP-Glc. The specific activity of 1.5-3.2 nmol/min/mg of protein was similar to that of 60- glucuronidation of HDCA. The apparent K , for UDP- Glc estimated with HDCA was 280 MM, and the formation of HDCA glucosides was strongly inhibited by UDP-GlcUA (apparent Ki = 7 p ~ ) . Evidence is presented that HDCA-specific UDP-glucuronosyltransferase (clone UGT2B4) expressed in V79 cells is not involved in glucosidation of HDCA and is not photolabeled with 5-[/3-32P]azido-UDP-Glc. Rigorous structure identification of the biosynthetic product proved that HDCA was glucosidated at the 6-position. Thus, this UDP-Glc-dependent activity catalyzing the biosynthesis of 6-0-glucosides of 6a-hydroxylated bile acids represents a new pathway in the metabolism of these bile acids.

* This work was supported in part by National Institutes of Health Grants DK-38678 (to A. R.) and HD-14198 (to R. L.), National Institutes of Health Postdoctoral Fellowship HL-08238 (to R. R. Drake), and a Veterans Administration grant (Biotransformation and Detoxification of Bile Acids in Liver Diseases) (to R. L.). Funds for the Kratos Concept 1H mass spectrometer and Varian NMR were provided by the University of Kentucky. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I To whom correspondence and reprints should be addressed Div. of Gastroenterology, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 567-1, Little Rock, AR 72205. Tel.: 501- 686-5414; Fax: 501-686-6248.

Several bile acid conjugation reactions have been identified, including amidation ( l ) , sulfation (2-4), glucuronidation (5- lo), and glucosidation (11-13). The three latter conjugation reactions play an important role in the elimination of poten- tially hepatotoxic bile acids. It has been shown that bile acid glucosides could be formed i n vitro (11, 14) and could be isolated from human urine (13). This bile acid glucosidation was not dependent on sugar nucleotide donor substrates, but utilized a lipophilic glucosyl donor, dolichyl phosphoglucose (Dol-P-Glc).’ Evidence was presented that chenodeoxycholic acid (3a,7a-diOH) (cDCA) and hyodeoxycholic acid (3a,6a- diOH (HDCA)) were glucosidated i n vitro at (2-3. I t was postulated that, in addition to the glycosyltransferase reactions involved in the biosynthesis of N-linked glycoproteins, there exists in human liver microsomes a sugar nucleotide- independent glucosyltransferase that catalyzes the transfer of glucose from glucosyl donors to the 3-hydroxyl of bile acids (11). This glucosyltransferase has been isolated from human liver microsomes and characterized (11).

Glucosidation of exogenous and endogenous compounds other than bile acids has been described previously. Mam- malian UDP-glucosyltransferases utilizing phenols and UDP- Glc as substrates have been demonstrated (15-17), and evidence of separate UDP-glucosyltransferase and UDP-glucuronosyltransferase for steroids has also been postulated (16). Glucosylated derivatives of bilirubin have been identified (18), and UDP-glucosyltransferase activity toward bilirubin has been demonstrated in rat liver (19). Burchell and Blanckaert (20) proposed a single bilirubin UDP-glucuronosyltransferase that could accept different sugar nucleotides. More recently, it has been postulated that hepatic esterification of bilirubin with glucuronic acid, glucose, and xylose depends on a single enzyme or involves, at least, a common subunit (21).

Since HDCA has been shown to serve as a substrate for the formation of both glucuronide and glucoside conjugates, the possibility of the participation of an HDCA-specific UDP- glucuronosyltransferase in the formation of HDCA glucosides has been investigated. Two human liver UDP-glucuronosyltransferase cDNA clones, UGT2B4 (22-24) and UGT2B1 (25), have been isolated and shown to encode isoenzymes active in the glucuronidation of HDCA. UGT2B1 has been shown to glucuronidate other steroid derivatives as well as

The abbreviations used are: Dol-P-Glc, dolichyl phosphoglucose; HDCA, hyodeoxycholic acid; DIDS, 4,4’-diisothiocyanostilbene-2,2’- disulfonic acid SITS, 4-acetamido-4’-isothiocyanostilbene-2,2’-disulfonic acid FAB-MS, fast atom bombardment mass spectrometry.

15127

15128 Biosynthesis of 6-0-Glucoside of HDCA

HDCA (25). Both of the cloned human UDP-glucuronosyltransferases (UGT2B4 and UGT2B1) glucuronidate HDCA with great efficiency.

In this work, we have used HDCA, a Ga-hydroxylated bile acid, as a substrate to s tudy the human glucosylation pathway, the properties of the enzyme system involved, and its rela- tionship to the bile acid 3-0-glucosylation (11) and 6-0- glucuronidation (10, 26, 27) systems. Photoaffinity labeling of human microsomes with the photoaffinity analogs 5- [B-32P]azido-UDP-GlcUA (28) and 5-[@-3ZP]azido-UDP-Glc has been used to differentially photolabel UDP-glucuronosyltransferases and related enzymes of 50-56 kDa. This work shows the presence in human liver microsomes of a UDP- Glc-dependent enzyme activity that catalyzes the 6-0-glucosylation of the 6a-hydroxylated bile acid HDCA. It is further shown that this in vitro reaction is not catalyzed by at least one cloned expressed UDP-glucuronosyltransferase that re- quires UDP-GlcUA as the glucuronate donor (29). Specifi- cally, cloned UGT2B4 expressed in V79 cells is not involved in the 6-0-glucosylation of HDCA. This reaction also is not carried out by the UDP-Glc-independent system described previously (11,14). Therefore, the presence of a new bile acid- specific UDP-glucosyltransferase(s) activity, with a molecular mass in the range of 50-56 kDa and involved in bile acid detoxification, is proposed.

MATERIALS AND METHODS

5-[j3-92P]Azido-UDP-Glc and 5-[@-32P]azido-UDP-GlcUA were synthesized and purified as previously described (28, 30). 32Pi was from ICN. Dolichol phosphate, Brij 58, sugar nucleotides, saccharolactone, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 4-acetamido-4'-isothiocyanostilbene-2,2'-~sulfonic acid (SITS) were from Sigma. [~arboxy-~~C]Lithocholic acid (3a-OH) (58 mCi/mmol) was obtained from Amersham Corp. Unlabeled or tritium-labeled HDCA, murideoxycholic acid (3a,s@-diOH), and chenodeoxycholic acid were purchased or synthesized as described previously (10, 31) and checked for chemical purity by TLC and gas-liquid chromatography. Amphomycin was obtained from Dr. A. D. Elbein (Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences). Plastic-backed cellulose thin-layer plates were from Kodak. Human liver autopsy samples 2 and 4-6 were obtained through the Organ Procurement Program (University of Rochester) following donation of other organs for transplantation. Human liver samples 1 and 14-22 were from the liver bank at the University of Groningen (Groningen, The Netherlands). The liver samples were macroscopically normal. Human liver autopsy samples 2 and 4-6 were described in a previous report (4). Additional new samples from the following subjects were used in these studies: sample 1, 13-year-old female (car accident); sample 14, 57-year-old female (cerebral bleeding); sample 15, 56-year-old male (cerebral bleeding); sample 16, 56- year-old male (car accident); sample 17, 17-year-old male (car accident); sample 18, 13-year-old female (car accident); sample 19, 12- year-old female (cerebral bleeding); sample 20, 36-year-old male (anoxia); sample 21, 41-year-old male (cerebral bleeding); and sample 22,32-year-o1d female (anoxia). Intact human liver microsomes were prepared as previously described (10); mannose 6-phosphate latency determinations were done as previously described (32).

The apparent kinetic parameters K,,,, V,,, and Ki from the primary data were estimated using a computer program (Enzyme Kinetics, J. Stanislawski, Trinity Software) based on the method of Lineweaver and Burk (33).

cDNA Expression-The cDNA clone UGT2B4 was subcloned into the mammalian expression vector pKCRH2 (22). The recombinant plasmid was cotransfected in V79 cells with a neomycin resistance vector, thereby allowing the selection of resistant colonies in G418 medium (24). The complete characterization of the resulting recombinant cell line has been described (24). V79 control or recombinant cells were cultured in Dulbecco's modified Eagle's medium (GIBCO, Cergy-Pontoise, France) supplemented with 10% Nu-serumR (Tebu, Le Perray-en-Yvelines, France), 100 units/ml penicillin, and 0.1 mg/ ml streptomycin. The cells were replated at -1 X lo6 cells/lO-cm diameter dish and harvested 2 days later. The cells were rinsed in ice-cold phosphate-buffered saline (pH 7.4) (GIBCO), suspended and

centrifuged twice at 1000 X g in this buffer, and stored as pellets at -80 "C. For membrane preparation, the cell pellet was resuspended in 250 mM sucrose and 1 mM Tris (pH 7.4), sonicated three times for 30 s (40 V), and gently homogenized in a Dounce homogenizer. The lysate was then centrifuged at 12,000 X g for 20 min, and the resulting supernatant was centrifuged again at 100,000 X g for 60 min to pellet the membrane fraction.

Photoaffinity Labeling-Human liver microsomes (50 pg of protein) were incubated for 10 min in the presence of 0.05% Triton X-100 at 37 "C in 250 mM HEPES (pH 7.0) and 12.5 mM MgC12 in a total volume of 20 p1. Either 5-[@-32P]azido-UDP-Glc or 5-[@-32P]azido- UDP-GlcUA (40 p ~ , 2-5 mCi/pmol) was added and allowed to equilibrate for 20 s, followed by UV irradiation with a hand-held lamp (UVP-11, 254 nm; Ultraviolet Products, Inc.) for 90 s at room temperature. For competition experiments, the appropriate unlabeled competing nucleotide was included in the reaction mixture. Reactions were terminated and processed for SDS-polyacrylamide gel electro- phoresis as previously described (28). Protein was separated on 10% SDS-polyacrylamide gels (34), followed by autoradiography for 1-2 days. Autoradiographs were quantified using an E-C Apparatus den- sitometer. In some cases, the separated proteins were transferred from the gel to nitrocellulose, and Western blot analysis was performed by the method of Towbin et al. (35). Blotted proteins were then examined by using a pig anti-4-nitrophenol UDP-glucuronosyltransferase antiserum, which was a generous gift of Dr. A. Dannen- berg (The New York Hospital, Cornel1 Medical Center).

Enzyme Assays-Bile acid UDP-glucuronosyltransferase and UDP-glucosyltransferase activities were measured with radioactive bile acids as substrates and UDP-GlcUA or UDP-Glc as the sugar donor as previously described (8, 10). Bile acids were dissolved and added in methanol (1.6% final concentration) or prepared in the form of mixed micelles with Brij 58 as described in detail in Ref. 8. For assays with intact microsomal proteins, the bile acid substrate mixed with radiolabeled substrate (0.1 mM final concentration, 0.1 pCi) was incubated in a total of 60 pl containing 100 mM HEPES/NaOH (pH 7.0 or 6.5), 5 mM MgCIZ, 5 mM saccharolactone, and 50 pg of freshly prepared microsomal proteins. For assays using solubilized microsomes, microsomal proteins were incubated with Brij 58 or Triton X- 100 (60 pg of detergent/mg of protein) in the same incubation medium. Reactions were started by addition of the appropriate UDP- sugar a t a concentration of 4 mM under standard assay conditions. After 10 min at 37 "C, the reaction was stopped by addition of 20 pl of ethanol, and 60 pl of the mixture was directly applied to the preadsorbent layer of 19-channeled silica gel TLC plates (J. T. Baker Inc.). The glucuronidated and glucosylated bile acids and the unreacted substrates were separated by two TLC developments in chloroform/methanol/glacial acetic acid/water (65:25:2:4, v/v). Hy- droxyl- and carboxyl-linked glucuronides and glucosides are clearly separated under these conditions. Radioactive compounds were lo- calized on TLC plates by autoradiography at -80 "C. Zones corresponding to the glucuronide and/or glucoside bands were scraped into scintillation vials, and radioactivity was measured by liquid scintillation counting (Rackbeta Model 1214, Pharmacia LKB Biotechnol- ogy Inc.). In some experiments, unlabeled bile acids were incubated with radiolabeled UDP-sugars to verify that the product was indeed a glucuronide or glucoside. These assays were carried out as described above with final concentrations of 0.1 mM unlabeled bile acid and 4 mM [gluc~ronyl-'~C]UDP-GlcUA (specific activity, 300 mCi/mmol; Du Pont-New England Nuclear) or [gluc~se-l-~H]UDP-Glc (specific activity, 7.8 Ci/mmol; Du Pont-New England Nuclear). The glucuronidation/glucosidation activity of transfected V79 cells was assayed as described above for the human liver microsomal preparations. Assay mixtures containing the same components, 50 pg of V79 cell homogenate proteins, or 1 mg of human liver microsomal proteins were incubated at 37 'C for 2 h.

Apparent K,,, values for UDP-GlcUA and UDP-Glc were determined under the conditions described above with a constant [3H] HDCA concentration of 0.1 mM, while the concentrations of UDP- GlcUA were varied from 0.01 to 4.0 mM and those of UDP-Glc from 0.05-4 mM. For studies of the inhibition of bile acid glucosylation by UDP-GlcUA, the incubations contained 0.1 mM [3H]HDCA, 0.05-0.5 mM UDP-Glc, and 0-0.1 mM UDP-GlcUA. Apparent Ki values were calculated from secondary plots of apparent KJV,. values uersus the concentration of inhibitor. The best fitting lines were obtained by linear least-squares regression analysis.

Structure Identification of Biosynthetic HDCA Glucoside-Prepar- ative scale syntheses were carried out to prepare the larger amounts of HDCA glucoside necessary for structure determination. The reac-

Biosynthesis of 6-0-Glucoside of HDCA 15129

tion mixture (containing 10 mg of microsomal protein, 0.15 mM [3H] HDCA (specific activity, -800 dpm/nmol), 2.5 mM UDP-Glc, 100 mM HEPES/NaOH (pH 6.5), 5 mM MgC12, and 5 mM saccharolactone in a final volume of 25 ml) was incubated at 37 “C for 60 min. Incubations were stopped by the addition of 90 ml of ice-cold 0.1 M glycine/trichloroacetate buffer (pH 2.8). Partial purification of products was accomplished by solid-phase extraction on Bond Elut Cla cartridges (Varian-Analytichem), followed by preparative TLC, either in an underivatized form or as the methyl ester acetate derivatives, as previously described (10). ’H NMR spectra were recorded in CDC13 with a Varian 300-MHz instrument and referenced to the CHC13 signal (7.26 ppm). Electron impact and negative fast atom bombardment mass spectrometries (negative FAB-MS) were carried out using a Kratos Concept 1H instrument.

RESULTS

Photolabeling of Human Liver Microsomes and Immunore- cognition of Photolabeled UDP-glucosyltransferases-Human liver microsomes (mannose 6-phosphate latency, 85%) photolabeled with either 5-[32P]azido-UDP-GlcUA or 5-[32P] azido-UDP-Glc (Fig. 1A) resulted in different patterns of photoincorporation into proteins with molecular masses of 50-56 kDa, the molecular mass range shown previously to contain UDP-glucuronosyltransferases (28). Photoincorpor- ations with both probes could be competitively decreased by increasing concentrations of UDP-GlcUA and UDP-Glc (Fig. L4 and Table I). To verify whether the 50-56-kDa proteins photolabeled with both photoprobes were indeed UDP-glucuronosyltransferases, an antiserum known to recognize UDP-glucuronosyltransferases was used in Western Blot analyses of photolabeled vesicles (Fig. 1B). The proteins recognized by the antibody also photoincorporate 5-[32P] azido-UDP-Glc and 5-[32P]azido-UDP-GlcUA in the 50-56- kDa region as was shown for rat liver microsomal preparations in Ref. 36 (data not shown). The 37-kDa band represents photolabeling of the protein UDP-g1ucose:dolichyl-phosphate glucosyltransferase. The radioactive band at 66 kDa, labeled

[32P]5-azido-UDPGlcA

with 5-[32P]azido-UDP-Glc, has been shown previously to be phosphoglucomutase (36). Other photolabeled bands have not been identified at present. Most of these were not protected by unlabeled UDP-sugars, indicating nonspecific binding of the photoprobes.

Incubation of Human Liver Microsomes with UDP-sugars and Bile Acids Including 6a/6@-Dihydroxylated Compounds- In experiments presented in Table 11, the formation of 3H- labeled bile acid-sugar conjugates by human liver microsomes incubated with UDP-Glc, UDP-GlcUA, UDP-Gal, or UDP- Xyl and four bile acids is shown. The results on HDCA 6-0- glucuronide formation are included in Table I1 for the purpose of comparison and as a check on the viability of the human microsomal preparations. The products behaved as monogly- cosides on TLC in chloroform/methanol/glacial acetic acid/ water (65:25:2:4, v/v). No evidence for the formation of dig- lycosides was obtained with either sugar nucleotide (no product more polar than the monoglucoside was detected by TLC), and no conjugates were formed when UDP-Glc or UDP- GlcUA was omitted from the incubation medium. The amounts of HDCA glucuronide formed were only slightly larger than those of HDCA glucoside. Other bile acids tested yielded only glucuronide conjugates. The reactions were specific for the human microsomal enzyme(s): similar experiments with microsomal fractions derived from Sprague-Daw- ley rat livers resulted exclusively in glucuronide formation (Table 111). The formation of HDCA glucoside was almost totally dependent on the presence of detergent. The treatment of human microsomes with the optimum concentrations of Brij 58 (0.6 mg of detergent/mg of protein) resulted in a 12- fold and an almost 6-fold increase in the formation of 6-0- glucuronide and 6-0-glucoside of HDCA, respectively. Man- nose 6-phosphate latency in intact microsomes routinely prepared for these experiments was 85% and was totally abolished in the presence of 0.05% detergent.

[32P]5-azido-UDPGlc

“GT+ {

GPDS-b { 1 2 3 4 5 6 7 1 2 3 4 5 6 7

-49.5

-32.5

B -49.5

FIG. 1. Photoaffinity labeling and Western blot analysis of human liver microsomal proteins. Human intact microsomes (50 mg) were preincubated for 10 min at room temperature with Triton X-100 (0.05% final concentration) and competing nucleotide. Incubation mixture was then added, and the photoaffinity labeling was done as described under “Materials and Methods.” A, autoradiograph of Western blot of human liver sample 22 proteins photolabeled with either 40 PM 5-[32P]azido-UDP-GlcUA (left panel) or 40 pM 5-[32P]azido-UDP-Glc (right panel). Lane I, control incubations without detergent or UV irradiation; lune 2, control incubations without detergent, but with UV irradiation; lune 3, control incubations with both detergent and UV irradiation; lunes 4 and 5, incubations with detergent and UV irradiation plus 0.1 and 0.2 mM UDP-GlcUA, respectively; lunes 6 and 7, incubations with detergent and UV irradiation plus 0.1 and 0.2 mM UDP-Glc, respectively. The photolabeled protein identified as GPDS is the enzyme UDP-g1ucose:dolichyl-phosphate glucosyltransferase; the higher molecular mass protein (-60 kDa) photolabeled only with 5-[32P]azido-UDP-Glc has been identified as phosphoglucomutase. UGT, UDP- glucuronosyltransferase. B, Western blot analysis of photolabeled human liver sample 22 proteins shown in A. Primary antibody was an anti- p-nitrophenol UDP-glucuronosyltransferase antiserum. Proteins recognized by the antiserum also photoincorporated 5-[32P]azido-UDP- GlcUA and 5-[32P]azido-UDP-Glc. The results shown are from a representative experiment. Mannose 6-phosphate latency of intact microsomes was 85%.

15130 Biosynthesis of 6-0-Glucoside of HDCA TABLE I

Quuntitation of the photoaffinity labeling of UDP-glucuronosyltransferase: effect of unlabeled nucleotides on photoincorporation of 5-[p-32PI azido-UDP-GlcUA and 5-[p-32Plazido-UDP-Glc in detergent-treated rat liuer microsomes

The autoradiograph shown in Fig. lA was analyzed by densitometry. The intensity of the bands corresponding to 50-56 kDa in lane 3 of each panel was assigned the value of 100%. Photoaffinity labeling was carried out as described under “Materials and Methods” with 40 p~ 5-[@-32P]5-azido-UDP-GlcUA (5-NaUDP-GlcUA) and 5-[@-32P]azido-UDP-Glc (5-N3UDP-Glc). The description of the experiments is presented in the legend of Fig. 1. The results presented are from a representative experiment. Mannose 6-phosphate latency of the intact microsomes was 85%.

Lane Conditions Triton X-100 Photoincorporation (0.05%) 5-NzUDP-GlcUA 5-NJJDP-Glc

1 -uv 2 +uv 3 +uv 4 +UV, +0.1 mM UDP-GlcUA 5 +UV, +0.2 mM UDP-GlcUA 6 +UV, +0.1 mM UDP-Glc 7 +UV, +0.2 mM UDP-Glc

% of control 0 0

23 16 100 100 36 10 16 0 34 9 26 4

TABLE I1 Conjugation of bile acids with different sugar nucleotides by human liuer microsomes

Human liver sample 22 microsomes (50 pg of protein) were incubated at 37 “C in the presence or absence of detergent with radiolabeled bile acids and unlabeled sugar nucleotides as described under “Materials and Methods.” The reactions were stopped by the addition of 20 p1 of ethanol, and separation and analysis of the radioactive products were performed as described under “Materials and Methods.” Values are means f S.D. of four determinations.

Sugar nucleotide Brij 58 (0.05%) in incubation

medium

None - UDP-GlcUA + UDP-Glc - + UDP-GlcNAc -

+ -

+ UDP-Xyl - + UDP-Gal - +

Bile acid UDP-glycosyltransferase activity

HDCA MDCA” CDCA (3o,Ga-diOH) (Ba,G@-diOH) (3a,7a-diOH)

LA (3a-OH)

3a-OH-specific Carboxyl-specific

NA NA

0.17 f 0.10 2.1 f O.lOb

0.21 f 0.06 1.50 f 0.08‘

NA NA 0.01

0.61 k 0.07’ 0.01

0.26 f 0.02’

NA NA 0.01

0.26 f O.Olb NA NA NA NA NA NA NA NA

nmol/mg/min NA NA NA NA NA NA

0.06

NA 0.18 f 0.05‘

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA 0.01

0.13 f 0.04d NA NA NA NA NA NA NA NA

a MDCA, murideoxycholic acid; CDCA, chenodeoxycholic acid LA, lithocholic acid; NA, no activity. Identified as 6-0-glucuronide. Identified as 3-0-glucuronide. Identified as carboxyl-linked glucuronide.

e Identified as 6-0-glucoside. ‘Structure not determined.

TABLE I11 Conjugation of bile acids with different sugar nucleotides by Sprague-Dawley rat liuer microsomes

Sprague-Dawley rat liver microsomes (50 pg of protein) were incubated at 37 “C in the presence of detergent with radiolabeled bile acids and unlabeled sugar nucleotides as described under “Materials and Methods.” The reactions were stopped by the addition of 20 pl of ethanol, and separation and analysis of the radioactive products were performed as described under “Materials and Methods.” Values are means f S.D. of at least four determinations. See Footnote a in Table I1 for definitions of abbreviations.

Bile acid UDP-glycosyltransferase activity

Sugar nucleotide HDCA (Ba,Ga-&OH) in incubation

MDCA (3a,G@-diOH) CDCA (3a,7a-diOH) LA (3a-OH)

medium Hydroxyl- Carboxyl- Hydroxyl- Carboxyl- Hydroxyl- Carboxyl- Hydroxyl- Carboxyl- specific specific specific specific specific specific specific specific

nmol/mg/min None NA NA NA NA NA NA NA NA UDP-GkUA 0.43 0.08 0.34 f 0.08 0.35 f 0.17 0.84 f 0.31 0.77 f 0.18 0.27 f 0.03 8.06 f 1.28 0.86 2 0.16 UDP-Glc NA NA NA NA NA NA NA NA UDP-GlcNAc NA NA NA NA NA NA NA NA


No HDCA conjugation was detected when 4 mM UDP- GlcNAc was present during incubation. However, inclusion of 4 mM UDP-Gal and UDP-Xyl in the assay incubation medium resulted in significant formation of the appropriate conjugates (Table 11). The structural identification of the bile acid conjugates produced by incubation in the presence of UDP-Gal and UDP-Xy1 was not undertaken.

Characterization and Kinetic Properties of HDCA Glucosi- dation by Human UDP-glucosyltransferases-The conditions for HDCA glucosidation in human liver microsomes were optimized by varying the concentrations of [3H]HDCA (from 0.01 to 0.1 mM), UDP-Glc (from 0.025 to 4.2 mM), and microsomal protein (from 10 to 200 Fg) and the length of the incubation time (from 1 to 30 min). The optimum conditions were established to be 0.1 mM HDCA (prepared in Brij 58 micelles), 4 mM UDP-Glc, 0.8 mg of protein/ml, and 10 min of incubation at 37 “C.

Two-substrate kinetic analysis of HDCA glucuronidation and glucosylation reactions as a function of the concentrations of UDP-GlcUA and UDP-Glc a t several fixed concentrations of HDCA gave the double-reciprocal plots from which the apparent K, values for UDP-GlcUA and UDP-Glc were calculated (Table IV). Both enzymatic activities appeared to obey Michaelis-Menten kinetics, with K, values of 127 and 280 p~ for UDP-glucuronosyltransferase and UDP-glucosyltransferase, respectively, when assayed under optimum standard conditions with HDCA as a second substrate. The utilization ratios (KJVmax) were calculated for the two sugar nucleotides and HDCA and are shown in Table IV. It can thus be concluded that the turnover rate is better for the glucuronidation than the glucosylation reaction. Both UDP- sugar nucleotides were evaluated as potential inhibitors of the glucuronidation or glucosylation reaction, and the inhibition constant (K;) and the kinetic mode of the inhibition for the glucosylation reaction were determined. Addition of various amounts of UDP-GlcUA changed only the apparent K, and did not affect the V,,,, suggesting competitive inhibition. The slope of K,,,/V,,, uersus the concentration of inhibitor was linear, suggesting pure competitive inhibition. The calculated apparent K; was 7.0 p ~ . The inhibition of the HDCA glucuronidation reaction by UDP-Glc was less pronounced at a UDP-Glc concentration of 1 mM, the glucuronidation rate was only 40% inhibited. The detailed kinetics of this inhibition reaction were not evaluated.

Biosynthesis and Structure Determination of HDCA Gluco- side-To obtain amounts of the glucoside large enough for structure determination, preparative reactions were carried out with the same human microsomal preparations used in the analytical incubations. After the 60-min incubation, 35- 45% of the initial radioactivity was present as a product more polar than HDCA; the proportion of product did not increase with longer incubation times. The reaction rate was 2.2-2.6

TABLE IV Apparent kinetic constants (K, and V,,,J of human microsomal

UDP-glycosyltransferases determined in the presence of UDP-GlcUA and UDP-Glc

Apparent K, and Vmax values were determined at 0.01-4 mM UDP- Glc and 0.05-4 mM UDP-GlcUA and a constant concentration (0.1 mM, 0.1 pci) of [3H]HDCA. Results are means ( n = 2-3) of determinations carried out on microsomal preparations from liver sample 22.

nucleotide Apparent K m Vmax Sugar Utilization ratio

( K ” / V m a x )

P M nmol/mg/min UDP-Glc 280.0 k 17.1 1.8 k 0.09 UDP-GlcUA

6.6 127.0 3.8 29.2

nmol/min/mg of protein for the single metabolite formed. The glucoside of HDCA was purified as described under “Materials and Methods.” The underivatized product was used for the initial characterization by TLC, and after deriv- atization this was followed by a rigorous determination of structure by ‘H NMR, electron impact mass spectrometry, and negative FAB-MS.

Analysis of the CIS extract of the incubation mixture by negative FAB-MS (Fig. 2 A ) using glycerol as the matrix showed two distinct ions at m/z 391 and 553. The same two ions were observed in the spectrum obtained from the crys- talline biosynthetic product (Fig. 2B) purified by preparative TLC in chloroform/methanol/glacial acetic acidlwater (65:25:2:4). These ions corresponded to (M - HI- ions of HDCA (unreacted substrate) and its glucoside, respectively. The weak ion at m/z 391 was evidently due to the elimination of the sugar moiety from the purified glucoside.

The site of glucosidation was established by characterizing the corresponding methyl ester acetate derivative. The electron impact mass spectrum of this derivative (spectrum not shown) showed a series of ions resulting from the successive loss of acetic acid from the molecular ion at m/z 778 (not recorded) and at m/z 718, 658, 598, and 538. Ions at m/z 603, 543, and 483 corresponded to the combined eliminations of the side chain (115 atomic mass units) and acetic acid. Other ions at m/z 430,415,371,370, and 255 resembled those of the methyl ester of dihydroxylated bile acids, while ions at m/z 331, 229, 169, and 109 corresponded to the acetylated glucose moiety.

Obviously, apart from indicating glucosidation a t a hydroxyl group, neither negative FAB-MS of the free glucoside nor electron impact mass spectrometry of its methyl ester acetate derivative could be used for the identification of the site of sugar attachment. This information, however, was clearly provided by the 300-MHz NMR spectrum, obtained using a long acquisition time, of the methyl ester acetate derivative (Fig. 3). General structural features expected for an HDCA glucoside derivative were confirmed by the presence of five singlets of the 0-acetyl groups (2.079, 2.022, 2.012, 2.002, and 1.905); one methoxyl group singlet (3.67); and three signals of the methyl groups of the HDCA moiety at 0.635 (singlet, Me - 18), 0.908 (singlet, Me - 19), and 0.904 (doublet, J = 6.5 Hz, Me - 21). These signals were close to those observed previously for the methyl ester acetate of HDCA 6- 0-glucuronide (singlet, 0.63; singlet, 0.90; doublet, 0.90) (10).

Most important, this ‘H NMR spectrum showed multiplets of seven glucosyl protons: H-1’ at 4.555, d, J1z,2, = 8.0 Hz; H- 2’ at 4.950, dd, J2r,3. = 9.5 Hz; H-3’ at 5.195, dd, J3r,4. = 9.5 Hz; H-4’ a t 5.070, dd, J4,,5f = 9.5 Hz; and H-5’ multiplet at 3.682 and H-6’A and H-6’B at 4.13 and 4.26, respectively, dd, J5,,6z = 2.0 Hz, J5’,6’A = 4.7 Hz, and J5,6’B = 12.0 Hz, respectively. From these data, the P-D-glucoside structure and attachment at a hydroxyl (as opposed to the carboxyl) group were confirmed. For comparison, the H-1’ signal of the above- mentioned HDCA glucuronide was observed at 4.60 as a doublet (J19,2f = 7.6 Hz).

The 6-0 location of the P-D-glucosyl moiety was unambiguously determined by observing the characteristic 3P-H broad multiplet a t 4.690, i.e. corresponding to the fifth acetyl located at 3a-H, and the 6P-H multiplet at 3.980, considerably higher than in HDCA methyl ester diacetate (3P-H at 4.68 and 6p- H at 5.12).

6-0-Glucuronidation and 6-0-Glucosidation in Different H u m a n Liuers-One of the goals of this study was to determine whether the HDCA-specific UDP-glucuronosyltransferases were also responsible for the glucosidation of HDCA in

15132 Bbsynthesk of 6-@Glucoside of HDCA

A.

M l.,..LJ , , , , , , ! , ,A:,T ; , , , I , , - , , , , , , I,,, ( , , , , , , ,

250 3w 353 4M) 450 500 550 wo -m 100-

B. 553

80-

80-

40- 391

250 300 350 150 500 550 800 BY)

FIG. 2. Negative FAB-MS spectra. A, negative FAB spectrum of the incubation mixture extract (see “Resulta” for details) measured without further purification in glycerol as the matrix. Ions at m/z 275, 367, and 453 correspond to the matrix; ions at m/z 391 and 483 correspond, in part, to (M - H)- and (M - H, glycerol)- of hyodeoxycholic acid; and the ion at m/z 553 corresponds to (M - H)- of hyodeoxycholic acid glucoside. B, negative FAB spectrum of hyodeoxycholic acid glucoside purified by preparative TLC (see “Results” for details). Diagnostic ions are as described for A.

~ ~ ~ ~ ~ , ~ ~ ~ ~ , ~ ~ ~ ~ , ~ ~ ~ ~ , . . , ~ , ~ ~ ~ ~ ~ ~ ~ ~ ~ , ~ ~ ~ ~ , . ~ ~ ~ , ~ ~ ’ ’ , ~ ~ ~ ’ , 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 P W

FIG. 3. 3 O O ” H z proton NMR spectrum of hyodeoxycholic acid glucoside methyl ester pentmeetate. The spectrum was measured in CDC13; 7792 transienta were accumulated in -8 h. Line broadening of 1 Hz was used in the processing. Signals a and b are due to water and aliphatic hydrocarbon impurities. See “Results” for all assignments. A, full spectrum; B, expanded 3.6-5.4 ppm region; C, expanded 1.9-2.1 ppm region.

human liver. UDP-glucuronosyltransferase and UDP-glucosyltransferase activities toward HDCA in the presence of UDP-GlcUA or UDP-Glc were assayed in microsomal preparations from 14 human livers (Fig. 4). In individual livers, UDP-GlcUA-dependent transferase activity did not parallel UDP-Glc-dependent transferase activity (correlation coeffi- cient ( r ) = 0.34). The UDP-glucosyltransferase activity varied between 0.8 and 3.2 nmol/min/mg of microsomal protein.

Evidence That HDCA Glucosidation Is UDP-Glc-dependent and Does Not Involve Endogenous Dolichyl Phsphglucose- To gain evidence as to whether the 6-0-glucosidation of HDCA involves endogenous Dol-P-Glc, the effect of the antibiotic amphomycin and other inhibitors was tested. The experiments were performed with detergent-treated microsomes only. When human liver microsomes were incubated with UDP-GlcUA or UDP-Glc in the presence and absence of varying concentrations of amphomycin (0.5-20 pg) and 5 mM Caz+, there was no significant difference in the formation of either conjugate of HDCA, and inhibition of their synthesis was not observed in the concentration range of 0.5-5 pg (Table V). At high concentrations (10 and 20 pg), significant inhibi-

tion of both reactions was observed. The effect of other inhibitors on the formation of HDCA

6-0-glucuronides and 6-0-glucosides was also tested. The nonpenetrating compounds DIDS and SITS were the most effective inhibitors. A significant decrease in the formation of HDCA glucosides (residual activity, 30% at 10 p~ DIDS and 56% at 10 p~ SITS) and HDCA glucuronides (residual activity, 41% at 10 p~ DIDS and 68% at 10 p~ SITS) was observed (Table V). In both cases, HDCA glucosylation was more affected than glucuronidation.

Evidence That Human UGTZB4 Expressed in V79 Cells Is Not Involved in HDCA Glucosidation-A human liver UDP- glucuronosyltransferase cDNA clone (UGT2B4) has been shown previously to encode an enzyme active in the glucuronidation of HDCA (22-24). Transfection of V79 cells with the UGT2B4 cDNA, performed as described previously (24), resulted in the biosynthesis of a protein with a molecular mass of 52 kDa. This protein was identified as a UDP-glucuronosyltransferase by immunoblotting using polyclonal anti-rat liver antibodies (37) and was not present in control V79 cell homogenates. The glucuronidating and glucosidating activi-

Biosynthesis of 6-0-Glucoside of HDCA 15133 A “ . R -0.34

0 1 2 3 4 5 6

Giucuronidation activity (nmoilmg x rnin)

FIG. 4. Comparison of UDP-glucosyltransferase and UDP- glucuronosyltransferase activities of 14 human microsomal preparations. UDP-sugar-specific HDCA glucuronidation/glucosylation reactions (with 50 pg of microsomal proteins) were carried out as described under “Materials and Methods.”

TABLE V Effect of inhibitors on the utilization of UDP-Glc and UDP-GlcUA

by human liver microsomes for the formation of P’H/HDCA 6-0-glucosides from [RHJHDCA

Incubations were carried out as described under “Materials and Methods” with [‘HIHDCA (0.1 mM, 0.1 pCi) and UDP-GlcUA or UDP-Glc (4.0 mM). Preincubations in the presence or absence of amphomycin, DIDS, and SITS were performed a t room temperature for 10 min. Amphomycin was added in water, and the other reagents were prepared as methanol solutions and evaporated under nitrogen. Results are means of two determinations carried out on microsomal preparations from liver sample 22.

Relative activity

UDP-GlcUA UDP-Glc

%

Addition

None 100 100 Amphomycin (+2 mM CaC1,)

0.5-5 pg 100 100 10.0 pg 78 61 20.0 pg 56 31

SITS 5.0 p M 85 18 10.0 p M 68 56 50.0 pM 61 31 100.0 p M 45 19

5.0 p M 68 41 10.0 p M 41 30 50.0 p M 16 2 100.0 p M 12 0

DIDS

ties of recombinant V79 cells and control microsomes from human liver were assayed by thin-layer chromatography. [3H] HDCA was shown to be both glucuronidated and glucosylated by human liver microsomes (Fig. 5, lanes 2 and 3 ) , and the reactions were totally dependent on the presence 01 UDP- sugars. Recombinant UGT2B4 enzymes from several independent cell preparations exhibited activity toward UDP- GlcUA ( laws 4, 8, and l o ) , but not toward UDP-Glc (lanes 5 , 9, and 11). No UDP-glucuronosyltransferase activity was observed in the control V79 cell homogenates ( laws 6 and 7). The UGT2B4 enzyme was successfully photolabeled with 5- [@-32P]azido-UDP-GlcUA, but not with 5-[@-32P]azido-UDP- Glc (data not shown). This strongly suggests that the

IJHIHDCA

PHIHDCA -

1 2 3 4 5 6 7 8 9 1 0 1 1

FIG. 5. Autoradiograph of TLC plate showing HDCA-specific UDP-glucuronosyltransferase and UDP-glucosyltransferase activities in human liver sample 22 microsomes and control V 7 9 homogenates and several V 7 9 homogenates transfected with UGT2B4 cDNA. HDCA glucuronidation/glucosylation reactions (with 5 pg of microsomal and 50-60 pg of VS9 homogenate proteins) were carried out as described under “Materials and Methods.” The [‘HIHDCA substrate concentration was 0.1 mM, and the sugar nucleotide substrate concentration was 4.0 mM. Lanes 1-3, human liver microsomes; lanes 4, 5, and 8-11, various V79 homogenates transfected with UGT2B4 cDNA; lanes 6 and 7, control V79 homogenates. Lane 1, no nucleotide; lanes 2, 4 , 6 , 8, and 10, UDP-GlcUA; lanes 3 .5 , 7, 9, and Zl, UDP-Glc.

UGT2B4 glucuronic acid-binding site does not accept UDP- Glc as a substrate.

DISCUSSION

The characterization and purification of human membrane- associated UDP-glucuronosyltransferases is difficult due to their instability after solubilization from their membrane environment. This was also observed with the human HDCA UDP-Glc-specific transferase. Therefore, the strategy in the identification and characterization of this enzyme(s) has fo- cused on photoaffinity labeling of microsomal preparations in conjunction with conventional biochemical approaches. These experiments served a dual purpose: 1) to differentiate between HDCA glucuronidation and glucosylation and 2) to demon- strate whether the formation of the 6-0-glycosides of HDCA was carried out by enzymes different from those involved in the formation of chenodeoxycholic acid 3-0-glucosides (11, 14).

In an earlier study using the two active site-directed photoaffinity analogs 5-[/3-”2P]azido-UDP-GlcUA and 5-[@-32P] azido-UDP-Glc, cross-photolabeling of rat liver microsomal proteins in the 52-56-kDa range was observed, and these proteins are likely to be UDP-glucuronosyltransferases (28, 36). The patterns of photoaffinity labeling of the microsomal proteins in the 52-56-kDa range by the two probes were

15134 Biosynthesis of 6-0-Glucoside of HDCA

distinctly different (36), indicating, as one possibility, the presence of different UDP-GlcUA- and UDP-Glc-specific enzymes of similar molecular mass. In addition, the photoaffinity analogs were used to characterize the photolabeled rat liver proteins as well as to elucidate the subcellular localization of their active sites (36). In this study, the use of the same photoprobes has allowed us to characterize human UDP- GlcUA- and UDP-Glc-specific transferases. As with rat microsomal preparations, cross-photolabeling with 5-[32P]azido- UDP-GlcUA and 5-[32P]azido-UDP-Glc of human liver microsomal proteins of 50-56 kDa was observed. Both photoincorporations could be competitively decreased by increasing concentrations of unlabeled UDP-GlcUA and UDP-Glc (Fig. 1). The cross-photolabeling and the competitive cross-inhibition of the photoincorporation into 50-56-kDa proteins with both analogs can be explained in two ways: 1) high affinity of both binding sites for both photoprobes and/or 2) the presence, in addition to the UDP-glucuronosyltransferases, of UDP-Glc-glucosyltransferases in the 50-56-kDa area. Both possibilities were investigated, resulting in identification of a bile acid UDP-Glc-specific transferase catalyzing the biosynthesis of 6-0-glucosides of HDCA.

The results in Table I1 show that of the bile acids tested, only HDCA forms glucosides when incubated with human liver microsomes in the presence of UDP-Glc. As observed previously with UDP-GlcUA (lo), only 6-0-conjugates were formed. The structure identification, which was rigorously determined by spectral methods (electron impact mass spectrometry, negative FAB-MS, and 'H NMR), established the biosynthetic product unambiguously as the 6-0-P-D-glucoside of HDCA. The fact that the formation of galactosides and xylosides was detected in this study (Table 11) indicated that HDCA was a good acceptor for these sugars. However, the possibility that the formation of these glycosides was carried out by distinct enzymes, e.g. a UDP-xylose-specific transferase, remains to be elucidated, as does the identification of the position of HDCA conjugation with these sugars. Studies defining the kinetic behavior of the HDCA-specific glucosyltransferase showed that the enzyme obeyed simple Michaelis- Menten kinetics. The effect of UDP-GlcUA on glucosidation of HDCA was tested and found to be inhibitory. A double- reciprocal plot suggested a competitive inhibition for UDP- GlcUA, which is not surprising due to the structural similar- ities of these two sugars.

The formation of glucosides of bilirubin with UDP-GlcUA, UDP-Glc, and UDP-Xyl has been described in rat liver microsomes (38). I t was postulated that these reactions depend on a single enzyme or on enzymes that at least share a common subunit. The data presented in Fig. 4 suggest that the formation of the 6-0-glucuronides and 6-0-glucosides of HDCA may not be the result of a single enzyme since the ratios of activities for UDP-GlcUA and UDP-Glc were very different in the 14 livers studied. These livers were found to exhibit large (up to 400%) interindividual variations in glucoside formation. No obvious correlation of UDP-sugar-dependent glucosyltransferase activity with the age or sex of the liver donors was found.

I t was demonstrated that Dol-P-Glc or synthetic lipophilic alkyl derivatives were involved in the nucleotide-independent formation of 3-0-glucosides of the bile acids chenodeoxycholic acid and HDCA (11-13). The antibiotic amphomycin has been shown to inhibit glycosylation reactions involving dolichyl phosphate (39). The inhibitory effect of the lipopeptide has been attributed to an interaction with dolichyl phosphate and Caz+ (40, 41). Table V shows that the 6-0-glucosidation of HDCA does not involve endogenous Dol-P-Glc by compar-

ison of the effect of the antibiotic amphomycin on pig liver Dol-P-Glc synthase (data not shown) and HDCA glucuronidation and glucosylation. In concentrations inhibitory for the formation of Dol-P-Glc, no inhibition of HDCA-specific conjugation reactions was observed. At inhibitor concentrations >10 pg, strong inhibition of both conjugation reactions was observed, probably due to nonspecific interactions of membrane phospholipids (obligatory for the glucuronidation reaction) with antibiotic.

Both DIDS and SITS had a strong inhibitory effect on HDCA glucoside and glucuronide formation (Table V). It is notewothy that the two inhibitors tested were found to be only slightly more inhibitory for glucosyltransferase than for glucuronosyltransferase. Since the DIDS/SITS inhibition of UDP-GlcUA and UDP-Glc utilization was studied in membrane vesicles disrupted with Triton X-100 or Brij 58, it appears that the reagents react with the luminally oriented binding sites of UDP-Glc-specific transferase and UDP- G1cUA:HDCA glucosyl- and glucuronosyltransferases. Our findings indicated that DIDS, a known inhibitor of anion transport in erythrocytes (42), of glucose 6-phosphate trans- membrane movement (43), and of the formation of lipid- linked saccharides from UDP-Glc and UDP-GlcNAc (44), had a pronounced active-site inhibitory effect on the formation of HDCA glucuronides and glucosides, most likely by interaction with the catalytic sites.

To determine whether a known human HDCA-specific UDP-glucuronosyltransferase was involved in HDCA glucosidation, the UDP-glucuronosyltransferase clone UGT2B4 was tested. Since human UDP-glucuronosyltransferases are not stable to purification, only two human UDP-glucuronosyltransferases have been purified to homogeneity (45, 46). One of these purified enzymes catalyzes the glucuronidation of HDCA and exhibits minor activity toward hyocholic acid (3a,Ga,lBa-triOH) and estradiol (46). More progress has been achieved in molecular biological studies of human UDP- glucuronosyltransferases. Thus far, the cDNAs encoding two forms of HDCA-glucuronidating transferases have been cloned and sequenced (22-25). Expression of these cDNAs in culture demonstrated that one form, UGT2B4, encodes a 529- amino acid polypeptide active in the glucuronidation of HDCA only (24). A second form, UGT2B1 (25), also glucuronidated HDCA, but was equally active toward 17-epiestriol and 3,4-catechol estrogens. HDCA is a dihydroxylated bile acid that contains a carboxylic acid side chain in addition to two hydroxyl groups at the 3a- and 6a-positions. Hepatic microsomal glucuronidation of this bile acid in rat liver microsomes has been shown to occur at any or all of these three positions (47). The question of which functional group of the bile acid moiety is glucuronidated by cloned and expressed human UDP-glucuronosyltransferase has not been elucidated. However, we have found that the conjugate of HDCA formed by human liver UGT2B4 expressed in the V79 cell system is a 6-O-glucuronide, and this has been confirmed by mass spectroscopic analysis.' As presented in Fig. 5, transfection of V79 cells with the UGT2B4 cDNA has been used for testing the glucosidation and glucuronidation reactions of HDCA. It was shown that this expressed human liver microsomal UDP- glucuronosyltransferase had the capacity to glucuronidate (but not glucosidate) HDCA. Also, UGT2B4 was photolabeled with 5-[3'P]azido-UDP-GlcUA, but not with 5-[3'P]azido- UDP-Glc. Therefore, we conclude that UGT2B4 is not involved in 6-0-glucosidation of HDCA.

With regard to the subcellular localization of the HDCA

T. Tagucki, S. Fournel-Gigleux, R. Habar, J. Magdalou, and G. Siest, manuscript in preparation.


UDP-Glc-specific transferase, the latency of enzymatic activity, and results of the photoaffinity labeling experiments involving protease treatment, the effects of detergents and of anion-specific inhibitors in sealed and solubilized vesicles (data not shown)3 indicate a luminal orientation of the HDCA UDP-Glc-specific transferase. The results raised the question of the nature of the mechanism of UDP-glucose transfer into the lumen of the vesicles. The possibility exists that a UDP- glucuronic acid transporter might, in the presence of excess UDP-Glc and in the absence of UDP-GlcUA, be capable of transporting UDP-Glc. A second alternative is that there are two separate transport proteins with an overlapping specific- ity, evidence for which has been observed in rat liver microsomes.'

The formation of the 6-0-glucosides of HDCA under the conditions used in this work has not been previously demonstrated. The observation that a 6-hydroxylated bile acid can form 6-0-glucosides in human liver microsomes is novel, but is not unexpected. Recently, the presence of a bile acid glucosyltransferase that uses lipophilic glucose donors instead of polar sugar nucleotides was demonstrated in human liver (11, 14). The fact that bile acid 3-0-glucosides are excreted in urine of normal humans in similar amounts compared with bile acid glucuronides suggests a physiological importance for glucosidation of bile acids (13). Our UDP-Glc-dependent mechanism of 6-0-glucoside formation represents an additional detoxification pathway of toxic bile acids in humans. Previous studies indicate that glucosylation of other substrates in uiuo is ordinarily a minor pathway of detoxification (15, 29). Data presented in this paper suggest that HDCA 6- 0-glucosylation may not be a minor pathway. In human microsomal preparations, the ratio of UDP-glucuronosyltransferase activity to UDP-glucosyltransferase activity (for human liver sample 22) is only 2.1:1.5 (Table 11). UDP- glucosyltransferase is subject to inhibition by UDP-GlcUA, but UDP-glucuronosyltransferase is also subject to inhibition by UDP-Glc. The results reported herein indicate an HDCA- specific UDP-glucosyltransferase activity. This activity may represent the presence of an enzyme that has both UDP- GlcUA and UDP-Glc as substrates or one that functions exclusively with UDP-Glc as a substrate. There is without doubt a large family of specific UDP-glucuronosyltransferases, some of which may also be able to utilize UDP-Glc to form glucosides. However, evidence presented herein indicates the presence of at least one UDP-Glc-specific glucosyltransferase.

Acknowledgment-We thank Dr. Jack P. Goodman for measure- ments of FAB spectra.

A. Radominska, J. Little, R. R. Drake, and R. Lester, manuscript

'A. Radominska, J. Little, S. Treat, R. Lester, and R. R. Drake, in preparation.

manuscript in preparation.

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