THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 27, Issue of July 8, pp. 17960-17964, 1994 Printed in U.S.A.

Bilirubin UDP-glucuronosyltransferase 1 Is the Only Relevant Bilirubin Glucuronidating Isoform in Man*

(Received for publication, March 15, 1994, and in revised form, April 21, 1994)

Piter J. BosmaS, Jurgen Seppen, Bart Goldhoorn, Conny Bakker, and Ronald P. J. Oude Elferink From the Division of Gastroenterology, Academic Medical Centre, 1105 AZ Amsterdam, The Netherlands

Jayanta Roy Chowdhury and Namita Roy Chowdhuryl From the Marion Bessin Liver Research Center and the Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461

Peter L. M. Jansen From the Division of Gastroenterology and Hepatology, University Hospital of Groningen, NL-9713E2, Groningen, The Netherlands

Crigler-Najjar syndrome type I (CN-I) is caused by an inherited absence of UDP-glucuronosyltransferase ac- tivity toward bilirubin (B-UGT), resulting in severe non- hemolytic unconjugated hyperbilirubinemia. Based on the expression of cDNAs in COS cells, two UGT isoforms in human liver, B-UGT, and B-UGT,, have been reported to catalyze bilirubin glucuronidation. These isoforms, which are derived from a single gene, ugtl , have identi- cal carboxyl-terminal domains that are encoded by four consecutive exons shared by both isoforms. A critical lesion in any of these common exons should inactivate both B-UGT isoforms, giving rise to CN-I. The amino- terminal domains of the B-UGT isoforms are unique, each being encoded by a different 5’ exon. If both B-UGT isoforms contribute significantly to bilirubin glucu- ronidation, a mutation in one of these unique 5‘ exons should affect a single isoform, while the other isoforms should provide residual B-UGT activity. However, in two patients with CN-I, we found a mutation only in the unique exon of B-UGT,, the other exons being normal. To clarify this apparent paradox, we expressed the cDNA for each B-UGT isoform in COS cells and determined the specific B-UGT activity. These studies show that only B-UGT, has quantitatively significant catalytic activity. Furthermore, we show that the mutation in B-UGT, observed in each of the two CN-I patients inactivates B-UGT,. Together, the results indicate that B-UGT, is the only physiologically relevant isoform in bilirubin glucuronidation.

Glucuronidation of bilirubin, catalyzed by bilirubin UDP- glucuronosyltransferases (B-UGTs)’ is essential in the biliary

Grants RO1-DK 39137 (to N. R. C.), RO1-DK 46057 (to J. R. C.), and * This work was partly supported by National Institutes of Health

P3O-DK 41296 (Liver Research Core Center), a grant from the Najjar Fonds, The Netherlands (to C. B.), and Grant 6-FY93-0083 from the March-of-Dimes Birth Diseases Foundation (to J. R. C.). 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.

Research Center, Albert Einstein College of Medicine, 1300 Morris Park $ To whom correspondence should be addressed: Marion Bessin Liver

Ave., Bronx, NY 10461; Div. of Gastroenterology, FO-116 Academic Medical Center, Meibergdreef 9,1105 AZ Amsterdam, The Netherlands.

The abbreviations used are: B-UGT, bilirubin-UDP-glucuronosyl- transferase; CN-I, Crigler-Najjar syndrome type I; CN-11, Crigler- Najjar syndrome type 11; DOPC, deoxyphosphatidylcholine; ELISA, en- zyme-linked immunosorbent assay; MOPS, 4-morpholinepropane- sulfonic acid; HPLC, high performance liquid chromatography; nt, nucleotide(s1.

excretion of this potentially toxic metabolite. Severe inherited deficiency of hepatic B-UGT activity results in Crigler-Najjar syndrome, which is characterized by increased serum-unconju- gated bilirubin levels (1-3). Crigler-Najjar syndrome has been classified into type I (CN-I) and type I1 (CN-11) (4, 5). In CN-I, a virtual absence of B-UGT activity results in the lack of ex- cretion of bilirubin glucuronides in bile, and consequent, severe unconjugated hyperbilirubinemia which, if left untreated, leads to kernicterus and death. In CN-11, B-UGT activity is severely reduced, but not absent, which is reflected by a higher than normal ratio of bilirubin monoglucuronide to diglucu- ronide (6) and lower serum bilirubin levels than in CN-I. In most patients with CN-11, serum bilirubin levels are reduced by phenobarbital treatment, and the prognosis is much better than in CN-I.

Two cDNAs, B-UGT, and B-UGT,, have been reported to express B-UGT activity upon transfection into COS cells (7). It has been proposed that the more abundant isoform, B-UGT,, is responsible for at least 80% of total hepatic B-UGT activity (8). A single complex gene, ugtl (9, lo), located at the telomeric end of chromosome 2 (ll), expresses by differential exon usage B-UGT,, B-UGT,, and two other UGT isoforms, HLUG Pl (12) and HLUG P2 (13), with activity toward simple phenols. The 3‘ regions of mRNAs of these isoforms are identical and are de- rived from four consecutive exons (exons UGT1.2 through UGT1.5) at the 12-kilobase 3’ domain of the ugtl gene. The 5’ region of ugt l , spanning at least 100 kilobases, contains a se- ries of unique exons, designated UGTlAl to UGTlGl, each encoding the specific NH,-terminal domain of one isoenzyme. Lesions in any of the shared exons, UGT1.2 to UGT1.5, affect B-UGT,, B-UGT,, and the two phenol-UGTs, thereby simulta- neously inactivating all these isoforms. Such lesions should cause CN-I. This concept is supported by the finding of se- quence abnormalities in the common region exons in five pa- tients with CN-I (10, 14-16).

Genetic lesions in the unique region of B-UGT,, encoded by UGTlAl, should affect only B-UGT,, and B-UGT, and the phe- nol-UGTs should be normal. If B-UGT, were to contribute sig- nificantly to B-UGT activity, these patients should have re- sidual hepatic B-UGT activity mediated by B-UGT,, thereby resulting in CN-11. Consistent with this model, a mutation in exon UGTlAl was found in a kindred with CN-I1 (17). How- ever, van Es et al. (18) described two CN-I patients, in whom phenol-UGTs were normal, indicating that the genetic lesions were not located in the common region exons. To determine the molecular mechanism of the absence of B-UGT activity, in the presence of normal phenol-UGT activity, we determined the sequence of all B-UGT-encoding exons of these patients. Both

17960

Human Liver Bilirubin UDP-glucuronosyltransferases 17961

patients, C and D (181, were found to be homozygous for mu- tations in the unique region exon of B-UGT, (UGTlAl). In one patient, the unique region exon of B-UGT, (UGTlDl) had a normal sequence, while in the other, a common polymorphism was observed.

The existence of two catalytically active B-UGT isoforms in the liver is discrepant with the finding that a mutation affect- ing only one form (i.e. B-UGT,) results in the absence of hepatic B-UGT activity. To determine the mechanism of this apparent inconsistency, we have expressed both B-UGT cDNAs in COS cells and have compared their specific B-UGT activity. In ad- dition, we have examined the effect of the mutations identified in B-UGT, in patients C and D on the expression of B-UGT activity in COS cells.

EXPERIMENTAL PROCEDURES Materials-The expression vector, pSVK3, and DEAE-dextran were

obtained from Pharmacia (Uppsala, Sweden). Optimem medium, Taq polymerase, and restriction enzymes were purchased from Life Tech- nologies, Inc. Chloroquine diphosphate was obtained from FLUKAChe- mie AG, Buchs, Germany, and bilirubin and cell dissociation solution were purchased from Sigma. Goat anti-rabbit or anti-mouse alkaline phosphatase-conjugated immunoglobulins were purchased from Bio- Rad Laboratories. Endoglycosidase F, UDP-glucuronic acid, and Triton X-I00 were obtained from Boehringer Mannheim. DOPC was purchased from Avanti Polar Lipids.

Antibodies-Amouse monoclonal antibody, WP1, directed toward the common carboxyl-terminal region of all UGTs encoded by the ugtl gene (19), and a rabbit polyclonal antiserum, P6215, raised against immu- nopurified human UGTs (18), were used for immunodetection of the B-UGT isoforms and quantification of expressed recombinant B-UGTs by enzyme-linked immunosorbent assay (ELISA).

Amplification and Sequence Determination of E-UGT Exons-Exon UGTlDl (encoding the NH,-terminal domain of B-UGT,), exon UGTlAl (encoding the NH,-terminal domain of B-UGT,), and the com- mon region exons (UGT1.2 to UGT1.5) were amplified by polymerase chain reaction using oligomers directed at intron sequences flanking each exon as described (10). Thirty cycles of amplification were per- formed at 95 "C for 1 min, 56 "C for 1.5 min, and 72 "C for 3 min using a Gene ATAQ Controller (Pharmacia). The final extension step was at 72 "C for 30 min. Sequences of the amplicons were determined by the dideoxy chain termination method (20).

Construction ofB-UGT Expression Vectors-cDNAs encoding B-UGT, and B-UGT, were cloned in the EcoRI and ApaI restriction sites of pSVK3. Mutations were introduced in the B-UGT, cDNA by site-di- rected mutagenesis as described (21). All B-UGT plasmids were isolated using Qiagen columns (Qiagen Inc., Studio City, CA). Nucleotide se- quences of coding regions of wild-type and mutated forms of B-UGT, and B-UGT,, and their flanking plasmid sequences were determined to confirm the expected sequences.

Dansfection of COS-7 Cells-COS-7 cells in 162-cm2 flasks were incubated for 4 h at 37 "C in air containing 10% CO, with 9 ml of Optimem medium containing 2 mg/ml DEAE-dextran, 100 1" chloro- quine, and 10 pg of plasmid DNA. After removing the medium, the cells were treated for 2.5 min with 10% dimethyl sulfoxide in 20 mM Nap,, pH 7.4, containing 150 mM NaC1. Subsequently, the cells were rinsed twice with Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and grown for 2 to 3 days in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Life Technologies, Inc.).

Isolation of Microsomes-COS cells were harvested using the cell dissociation solution according to the manufacturer's protocol. m e r washing twice with 250 mM mannitol, 5 mM MOPS, 2 mM EGTA, pH 7.0 ("E), cells were kept in MME on ice for 15 min and then disrupted by 25 up and down strokes of a Dounce glass homogenizer on ice. Subse- quent procedures were performed a t 4 "C. Cell debris was removed by centrifugation (5 min at 5000 xg), and the supernatant was centrifuged (80,000 x g for 60 min) to pellet microsomes. Microsomes were sus- pended in 50 mM HEPES, pH 7.8, containing 2 mM EDTA and 20% glycerol (v/v), and stored a t -20 "C. Human liver microsomes were isolated as described (19) and resuspended and stored as above.

Immunotransblot-Proteins were resolved by SDS-10% polyacrylam- ide gel electrophoresis performed according to Laemmli (22) and elec- troblotted to nitrocellulose membranes (23). B-UGTs were visualized using the monoclonal (WP1) or rabbit polyclonal (P6215) antibody against B-UGTs. Alkaline phosphatase-conjugated goat anti-mouse or

anti-rabbit immunoglobulins were used as the secondary antibodies (18).

Quantification of E-UGT Isoforms by ELISA-B-UGT isoforms ex- pressed in the COS cells were quantified by a standard "sandwich" ELISA. In brief, microtiter plates were coated with the monoclonal antibody, WP1 (diluted 1 in 4000), and blocked with 1% bovine serum albumin, and COS cell microsomal samples (solubilized with 1% Triton X-100, which was then diluted with 0.1% Triton X-100) were applied. After binding, the plate was washed with 0.05% Tween 20 in phosphate- buffered saline and incubated with the polyclonal antibody, P6215, at 1500 dilution in phosphate-buffered saline containing 0.1% Triton X-100. The plates were then washed with 0.05% Tween 20 in phosphate- buffered saline. The bound polyclonal antibody was detected with horse- radish peroxidase-conjugated goat anti-rabbit immunoglobulin using 0.1 mg/ml 3,3',5,5'-tetramethylbenzidine, 0.0036% H,O, in 0.1 M NaH- COOH, pH 5.5, and quantified using an ELISA reader (Medgenix Di- agnostics BV, Soesterberg, The Netherlands). UGT concentrations in the samples were calculated using immunopurified human liver micro- somal UGTs as standard.

Deglycosylation-B-UGTs were denaturated by boiling for 5 min in 0.5% NaDodSO,. N-Linked carbohydrates were removed by incubation with 0.25 unit of endoglycosidase F per 50 pg of microsomal protein for 16 h according to the manufacturer's protocol.

Bilirubin UDP-glucuronosyltransferase Activity Assay-Small unila- mellar vesicles were prepared by evaporating deoxyphosphatidylcho- line (DOPC) (60 mg/3 ml chloroform) under a stream of nitrogen to form a thin film. Remaining traces of chloroform were removed in uacuo. The lipid film was hydrated with 6 ml of 10 mM HEPES, pH 7.8, containing 150 mM NaCl and vortexed for 5 min under argon to form multilameIlar vesicles, which were converted to small unilamellar vesicles by 1-s pulses of a probe sonicator for 30 min. Titanium particles were removed by centrifugation (4000 x g for 10 rnin), and liposomes were stored at 4 "C under argon for up to 2 weeks.

Microsomes were preincubated with the small unilamellar DOPC vesicles for 30 min a t 4 "C. Bilirubin solutions were made fresh by mixing 2.9 mg of bilirubin in 1.25 ml of 0.05 N NaOH, until the solution was optically clear, and adding 3.75 ml of 100 mM Tris-HC1, pH 7.8. Incubations were performed at 37 "C in a total volume of 400 pl under argon in the dark for 1 to 5 h. Concentrations used were: 50 mM Tris- HCI, pH 7.8,5 rn MgCl,, 1 mM glucaro-l,4-lactone, 75 1" bilirubin, 3.5 mM UDP-glucuronic acid, and 2.5 mg/ml DOPC vesicles. After adding a dash of ascorbic acid, the reaction was stopped by extraction of bilirubin and bilirubin glucuronides as follows. HC1-glycine (prepared by adding glycine to 0.4 N HCI to pH 1.8) and 1 and 1.4 ml of chlorofordethanol ( l : l , v/v) were added to the incubation mixture. After vigorous shaking, the mixture was centrifuged at 1800 x g for 5 min, and the lower colored phase was transferred to a glass tube. Chloroform was evaporated un- der a stream of nitrogen, and the samples were stored under argon in the dark at -20 "C for not longer than 2 days. Time needed for extrac- tion was less than 30 min, and all manipulations were performed in dimmed light. Bilirubin glucuronides were analyzed using reverse phase HPLC (24). Samples were dissolved in chloroform/ethanol ( l : l , v/v), and, immediately, 20 pl was injected onto a C-18 column (Chrom- spher 5C18, Chrompack, Middelburg, The Netherlands) and eluted as described. The eluate was monitored at 450 nm with a diode array detector. Spectra of the peaks were examined to confirm their identity. Amounts of conjugated bilirubin were determined by integration of areas under the respective peaks.

RESULTS

CN-I Patients-Patients C and D had serum bilirubin con- centrations of 400-440 VmoMiter. Serum bilirubin levels did not respond to phenobarbital administration (18). No B-UGT activity was detectable in liver microsomes from either patient. In contrast, UGT activity toward 4-nitrophenol was normal. Immunoblot analysis using W1, a monoclonal antibody di- rected to the common carboxyl-terminal domains of all ugtl- encoded isoforms, showed the presence of several immunoreac- tive UGT isoforms in both patients.

Nucleotide Sequence of B-UGTencoding Exons-Each exon encoding the two B-UGT isoforms was amplified using genomic DNA as template by polymerase chain reaction and sequenced directly. No sequence abnormalities were found in the common region exons (UGT1.2 through UGT1.5) of either patient. In exon UGTlAl, encoding the unique region of B-UGT,, one mu-

Human Liver Bilirubin UDP-glucuronosyltransferases 17962

[MWI

165.16. - 69.000 - 43.300 -

deglycosyl8ted Marker Mi8 E- 1 E-2 Mrk Mi8 B-I B-2

"--

10.300 -

mOl30ClOn81 8b FIG. 1. Immunoblot. Microsomal fractions were prepared from the

liver homogenate of a normal subject (Mic ) and from COS-7 cells trans- fected with wild-type B-UGT, (B-1) or B-UGT, plasmids. In each lane, 50 pg of protein was loaded. Deglycosylation was performed by incuba- tion overnight with 0.25 unit of endoglycosidase F per 50 pg of micro- somal protein. After electrophoresis on NaDodS04-10% polyacrylamide gels, the proteins were electroblotted to nitrocellulose sheets. Immuno- detection was performed with W P l , a monoclonal antibody directed toward the common COOH regions of all enzyme isoforms encoded by ugtl . The migration of prestained M , markers (Mrh) is shown.

tation was identified in each patient. In patient C, n t 508-510 were deleted. This 3-nt deletion is predicted to result in the absence of amino acid 170 of B-UGT,, a phenylalanine residue. In patient D, there was a C to T transition at nt 826, resulting in a glycine to arginine shift a t amino acid residue 276 of

Expression of B-UGT Isoforms in COS Cells-Three days after transfection, immunofluorescence was performed with WP1. Both B-UGT isoforms were visualized within the cells with a distribution pattern suggesting localization in the endo- plasmic reticulum (not shown). An immunoblot was performed using WP1 on microsomal fractions isolated from COS cells transfected with B-UGT, or B-UGT, or from human liver (Fig. 1). In human liver microsomes two bands were detected with M, = 52,000 and 56,000. A single band was detected in micro- somes isolated from COS cells transfected with B- UGT, ( M , = 52,000) or B-UGT, (M, = 56,000). These bands were of similar intensity, indicating that the two isoforms were expressed a t approximately the same level. The M, values of deglycosylated B-UGT,, B-UGT,, and all other UGT1-encoded isoforms in hu- man liver are identical, indicating that the differences in ap- parent M, are caused by glycosylation. In nontransfected COS cells and in COS cells transfected with the Escherichia coli P-galactosidase gene, no immunoreactive band was detected with WP1 (not shown).

The expressed B-UGT isoforms were quantified using ELISA. Both isoforms were expressed equally well. Concentra- tions of B-UGT, and B-UGT, in the corresponding transfected COS cells were 15.3 pg/mg and 10.3 pg/mg of microsomal pro- tein, respectively.

B-UGT Activity of Human Liver Microsomes or Wild-type B-UGTs Expressed in COS Cells-B-UGT assays were per- formed with 150 pg of human liver microsomes at 37 "C for 2 h. The products were identified and quantified by HPLC (Fig. 2.4 ). In human liver microsomes, bilirubin was glucuronidated a t

B-UGT,.

450 pmol/mg of microsomal protein per min. Incubation for 2 h at 37 "C, with 100 pg of microsomes from B-UGT,-transfected COS cells, resulted in the formation ofbilirubin glucuronides a t 360 pmol/mg of microsomal protein per min (Fig. 2C). In con- trast, similar incubations with B-UGT,-transfected COS cell microsomes did not result in detectable bilirubin glucuronide formation (not shown). When the amount of microsomes was increased 6-fold and the incubation time was increased to 5 h, HPLC analysis showed the presence of minor amounts of bili- rubin monoglucuronide as 0.25 pmol/mg of microsomal protein per min (Fig. 2E, please note the difference in scale). Based on the concentration of the expressed B-UGT isoforms in the COS cells, as determined by ELISA, specific activity of B-UGT, was 23 pmol/pg of the expressed enzyme. The specific activity of B-UGT, was lower by 3 orders of magnitude (24 fmol/pg of enzyme). When UDP-glucuronic acid was omitted from the re- action mixture, no bilirubin conjugates were detectable with B-UGT, (Fig. 2 0 ) or B-UGT,-transfected (Fig. 2 F ) COS cell microsomes. No bilirubin glucuronidating activity was detected in nontransfected COS cells or in cells transfected with the @galactosidase gene (not shown).

Recently, Ritter et al. (25) have reported that the pH opti- mum of B-UGT is 6.4, when using a low ionic strength buffer. To evaluate whether our assay conditions were optimal, we compared both methods. Human liver microsomes (150 pg) were incubated for 2 h a t 37 "C according to the method de- scribed by Ritter et al. (25) (Fig. 2B). Under these conditions, the rate of bilirubin glucuronidation was 7 pmol/mg of hepatic microsomal protein per min, which is only 1 to 2% of the rate obtained a t conditions used in this paper. Incubation of micro- somes from COS cells transfected with B-UGT, according to the protocol of Ritter et al. (7) did not result in the formation of detectable bilirubin glucuronides (not shown).

Expression and Activity of Mutated Forms of B-UGT, in COS Cells-We have identified mutations in exon UGTlAl of two CN-I patients. To determine the effect of these mutations on B-UGT activity, we introduced these mutations into B-UGT, cDNA by site-directed mutagenesis. The mutated forms and the wild-type B-UGT, were expressed in COS cells. Immunoblot of microsomal fractions isolated from the transfected cells was performed with a monoclonal antibody, WP1 (Fig. 3A) or a polyclonal antibody, P6215 (Fig. 3B). The B-UGT, with the 3-nt deletion (lesion found in patient C) (lane C) migrated with an M, similar to that of the wild-type B-UGT, ( M , = 52,000). The form of B-UGT, that had a C to T shift a t n t 826 (lesion in patient D) (lane D) exhibited a slightly slower migration rate, possibly because of differences in glycosylation. The intensity of bands detected for both mutated forms was reduced compared to that of the wild-type, indicating a higher expression of the wild-type in COS cells. No immunoreactive band was observed in the control lane (COS cells transfected with P-galactosidase gene) with either antibody.

B-UGT activities of the mutated forms and of the wild-type B-UGT, were determined as described. To correct for the re- duced expression of the mutated forms of B-UGT,, 5-fold higher amounts of microsomes were used. No bilirubin glucuronidat- ing activity was detectable for either mutated form, despite prolongation of incubation times up to 4 h. Considering the sensitivity of the method, the results show that the B-UGT activity of the mutated forms is less than 0.1% of the activity of the wild-type form (not shown).

DISCUSSION

Patients with CN-I have no detectable hepatic B-UGT activ- ity and excrete only traces of bilirubin glucuronides in bile. If both B-UGT, and B-UGT, significantly contribute to bilirubin glucuronidation, an absence of B-UGT activity would imply

Human Liver Bilirubin UDP-glucuronosyltransferases 17963

FIG. 2. HPLC profiles of reaction products. 150 pg of human liver micro- somes were incubated for 2 h at 37 "C with 100 p~ bilirubin, 50 mM "is-HCI, pH 7.8, 5 mM MgCI,, 16.6 mM glucaro-1,4-lac- tone, 3.5 mw UDP-glucuronic acid, and 2.5 mg/ml DOPC small unilamellar vesicles (A) or with 100 p~ bilirubin, 5 mM MgCI,, 16.6 mM glucaro-1,4-lactone, 10 mM Nap,, pH 6.4, and 1.41 M UDP-glucu- ronic acid, with 5% dimethyl sulfoxide ( B ) . B-UGT,-transfected COS cell micro- somes (100 pg) were incubated for 2 h a t 37 "C with bilirubin as described under "Experimental Procedures," with ( C ) or without (D) UDP-glucuronic acid. B-UGT,-transfected COS cell micro- somes, 600 pg, were incubated for 5 h a t 37 "C as described with ( E ) or without (F, please note the difference in scale) UDP- glucuronic acid. Extraction of bilirubin

chlorofodethanol (l:l), and samples and its glucuronides was performed with

were analyzed on a reverse phase HPLC (Spivak and Carey). Peaks: 1 = unconju- gated bilirubin; 2 = bilirubin monoglucu- ronide; 3 = bilirubin diglucuronide.

1 w

90 A 80 7 0 60 50 40

20 30

10

0 - 1 0

- l ~ ~ ' ' . ' ' . ' . . ' ~ l . ~ . . . . . . . . I 0 2 4 6 8 10 12 14 1 6 1 8 20 22 0 2 4 6 8 10 12 14 1 6 1 8 20 22

retentlon tlme (mln)

A lMrl Con B1 C D

105 + 70 +

t 52 43 -+

28 + 6 lMrl Cnn B1 C D

105 + 70 + 43 + t 52

28 + FIG. 3. Immunoblots of wild-type and mutated B-UGT,. Micro-

somal fractions were prepared from COS cells transfected with: E. coli P-galactosidase (Con), wild-type B-UGT, ( B l ) , B-UGT, with a deletion of phenylalanine 170 (C), or B-UGT, with a glycine to arginine shift a t amino acid 276 (D). In each lane, 50 pg of microsomal protein was loaded. Electrophoresis and transblotting were performed as in Fig. 1. Immunodetection was done with wP1 (A) or P6215, a polyclonal anti- body that recognizes all ugtl-encoded enzymes ( B ) . Migration of prestained M, marker proteins is indicated.

that both isoforms are defective. Since the carboxyl-terminal domain of all UGT isoforms derived from the ugtl gene are encoded by four common 3' exons, a lesion in any of these exons

can simultaneously inactivate all these isoforms. In several CN-I patients, mutations were found in the common region (10, 14-16), explaining the absence of B-UGT activity, as well as inactivation of the phenol-UGT isoforms.

In contrast to patients with CN-I, patients with CN-I1 have a n incomplete deficiency of B-UGT activity and significant amounts of bilirubin glucuronides are excreted in bile. In most CN-I1 cases, serum bilirubin concentrations are reduced by phenobarbital administration, presumably owing to the induc- tion of the residual B-UGT activity. In one kindred with CN-11, a mutation in the unique region exon of B-UGT, (UGTlAl) was found (17). Since this exon is not shared by other UGT isoforms, B-UGT, should not be affected by this mutation. If it is assumed that both B-UGT isoforms significantly contribute to hepatic B-UGT activity, the presence of a normal B-UGT, isoform could explain the residual bilirubin glucuronidating activity.

However, contrary to the above notion, the two CN-I patients studied in this paper (patients C and D) had lesions in the unique region exon of B-UGT,. The unique region exon of B-UGT, had a normal sequence in patient C and contained only a common, probably functionally inconsequential, polymor- phism in patient D. Similarly, a single nonsense mutation in the unique region exon of B-UGT, was found in another CN-I patient of Japanese origin (261, who had no abnormality in the sequence of the unique region exon of B-UGT,. In all these patients, hepatic B-UGT activity was undetectable (18, 26). These observations cast doubts about the bilirubin glucu- ronidating activity of B-UGT,. To explore this further, we ex- pressed B-UGT, and B-UGT, in COS cells and compared their specific bilirubin glucuronidating activity. Our results show that although both isoforms were expressed equally well in the COS cells, the specific activity of B-UGT, was less than 0.1% that of B-UGT,. This very low catalytic activity of B-UGT, explains the finding that a genetic lesion in B-UGT, alone is

17964 Human Liver Bilirubin UDP-glucuronosyltransferases

sufficient to reduce hepatic bilirubin glucuronidating activity to undetectable levels and cause CN-I.

Recently, Ritter et al. (25) have reported identical sequence findings in patient C. No B-UGT activity could be detected in liver microsomes of this patient. Only 12% of the pigments excreted in bile consisted of bilirubin monoconjugates, the re- mainder being unconjugated bilirubin. considering that only a minor amount of unconjugated bilirubin is excreted in bile (27), the biliary concentration of bilirubin monoconjugates was ex- tremely low in this patient. However, the excretion of even minute amounts of bilirubin monoconjugates suggests the pres- ence of some residual bilirubin glucuronidating activity. Ritter et al. (25) have proposed that this residual activity is probably derived from B-UGT,. Our data do not completely exclude this possibility, because B-UGT, does have some, albeit extremely low, glucuronidating activity. The presence of small amounts of bilirubin monoconjugates in bile has been reported in CN-I (6, 28,291. Homozygous Gunn rats, in which both B-UGT isoforms are truncated and inactivated because of a premature stop codon in the common region (30-321, traces of bilirubin mono- conjugates are excreted in bile (33). Formation of these small amounts of monoconjugates may be catalyzed by other enzyme systems that may have a low affinity for bilirubin and may catalyze the formation of non-glucuronide conjugates. Minor amounts of bilirubin glucuronides may also be contributed by UGT isoforms that are products of genes other than ugtl . For- mation of these minuscule amounts of bilirubin conjugates is clearly inadequate to prevent CN-I. Therefore, the presence of small amounts of bilirubin monoconjugates in bile of patients with CN-I does not indicate the presence of a physiologically significant level of residual B-UGT activity provided by

Ritter et al. (7) reported that both B-UGT forms had bilirubin glucuronidating activity. However, the expressed recombinant enzymes were not quantified in that study. Therefore, specific activities of the two isoforms cannot be compared from these results. By directly quantifying the two isoforms expressed in COS cells, we have shown that the specific activity of B-UGT, is less than 0.1% of that of B-UGT,, indicating that this isoform does not significantly contribute to bilirubin glucuronidation. Recently, Ritter et al. (25) reported that a low ionic strength buffer results in an increased B-UGT activity at pH 6.4. To determine whether a higher B-UGT activity can be obtained at other assay conditions, we assayed B-UGT activity by the method described by Ritter et al. (25). Under these assay con- ditions, bilirubin glucuronidation by liver microsomes occurred at a rate that was only 1 to 2% of that observed under our conditions. These results indicate that the method of Ritter et al. (25) is not optimal for measuring B-UGT activity.

We found that the mutant B-UGT,, containing the deletion of a phenylalanine residue 170 (patient C), is catalytically inac- tive. Recently, this patient was studied by Ritter et al. (25), who also expressed this mutated B-UGT, form in COS cells and have reported it to be normally active at pH 7.4. This difference in activity observed may again be caused by the difference in the assay method. As mentioned earlier, the specific activity for the wild-type B-UGT, using our assay is at least 50-fold higher. Thus, the absence of activity of mutated B-UGT, measured by us is unlikely to be caused by insensitivity of our assay. Fur- thermore, the absence of activity of this mutated form is con- sistent with the absence of detectable activity in liver micro- somes of this patient at pH 7.4 (18).

Considered together, our data show that, in man, B-UGT, is the only isoform with significant bilirubin glucuronidating ac- tivity. Furthermore, B-UGT, is expressed at a much higher level in the liver than is B-UGT, (7 ,8) . The combination of low

B-UGT,.

level of expression and low catalytic activity makes B-UGT, a physiologically insignificant contributor to bilirubin glucu- ronidation. Genetic lesions in the unique or common region of B-UGT, that completely inactivate this isoform will result in CN-I, even when they do not affect B-UGT,. Such mutations can occur in the unique or common region of the molecule. Mutations that cause partial inactivation of B-UGT, may result in CN- 11. This type of mutation can also occur in the unique region (16) or the common region (34). The very low enzyme activity of B-UGT, toward bilirubin strongly suggests that bil- irubin is not the primary substrate of this enzyme. There are several B-UGT,-like exons in the ugtl gene (8). The physiologi- cal role of B-UGT,, as well as that of the other B-UGT,-like isoforms, remains to be elucidated.

1.

2. 3.

4. 5.

6.

7.

8. 9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21. 22. 23.

24. 25.

26.

27.

28.

29. 30.

31.

32.

33.

34.

REFERENCES Fevery, J., Van Damme, B., Michiels R., De Grootte, J., and Heirwegh, K. P. M.

Crigler, J. F., and Najjar, V. A. (1952) Pediatrics 10, 169-180 Roy Chowdhury, J., Lahiri, P., and Roy Chowdhury, N. (1990) in Principles and

Practice ofMedical Genetics (Emery, A. E. H., and Rimoin, D. L., eds) 2nd Ed, pp. 1135-1164, Churchill-Livingstone, Edinburgh

(1972) J. Clin. Znuest. 51, 2482-2492

Arias, I. M. (1962) J. Clin. Inuest. 41, 2233-2245 Arias, I. M., Gartner, L. M., Cohen, M., Ezzer, J. B., and Levi, A. J. (1969) Am.

Fevery, J., Blanckaert, N., Heirwegh, K. P. M., Preaux, A,”., and Berthelot, P.

Ritter, J. K., Crawford, J. M., and Owens, I. S. (1991) J. B i d . Chem. 266,

Owens, J. S., and Ritter, J. K. (1992) Pharmacogenetics 2, 93-100 Ritter, J. K., Chen F., Sheen,Y. Y., Tran, H. M., Kimura, S., Yeatman, M. T., and

Bosma, P, J., Roy Chowdhury, N., Goldhoorn, B. G., Hofker, M. H., Oude Owens, I. S. (1992) J. Bid . Chem. 267, 3257-3261

Elferink, R. P. J., Jansen, P. L. M., and Chowdhury, J. (1992) Hepatology (Baltimore) 15, 941-947

Van Es, H. H. G., Bout, A., Liu, J., Duncan, A. M. V., Anderson, L., Bosma, P. J., Oude Elferink, R., Jansen, P. L. M., Roy Chowdhury, J., and Schurr, E.

Harding, D., Fournel-Gigleux, S., Jackson, M. R., and Burcheil, B. (1988)Proc. (1993) Cytogenet. Cell Genet. 63, 114-116

Wooster, R., Sutherland, L., Ebner, T., Clarke, D., Da CNZ e Silva, O., and Natl. Acad. Sci. U. S. A. 85,838143385

Bosma, P. J., Roy Chowdhury, J., Huang, T., Oude Elferink, R. P. J., van Es, H. Burchell, B. (1991) Biochem. J. 278, 465469

H. G., Lederstein, M., Whithington, P. E, Jansen, P. L. M., and Roy Chowdhury, N. (1992) FASEB J . 6,2859-2863

Ritter, J. K., Yeatman, M. T., Ferreira, P., and Owens, I. S. (1992) J. Clin. Znuest. 90, 150-155

Moghrabi, N., Clark, D., Burcheil, B., andBoxer, M. (1993)Am. J. Hum. Genet. 53,722-729

Bosma, P. J., Goldhoorn, B., Oude Elferink, R. P. J., Sinaasappel, M., Oostra, B. A,, and Jansen, P. L. M. (1993) Gastroenterology 105,216220

Van Es, H. H. G., Goldhoorn, B. G., Paul-Abrahamse, M., Oude Elferink, R. P. J., and Jansen P. L. M. (1990) J. Clin. Znuest. 85, 1199-1205

Peters, W. H. M., Allebes, W. A,, Jansen, P. L. M., Poeis, L. G., and Capel, P. J. A. (1987) Gastroenterology 93, 162-169

Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467

Deng, W. P., and Nickoloff J . A. (1992) Anal. Biochem. 200,8148 Laemmli, U. K. (1970) Nature 227,680-685 Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sei. U. S. A.

Spivak, W., and Carey, C. (1985) Biochem. J . 225,787-805 Ritter, J. K.,Yeatman, M. T., Kaiser, C., Gridelli, B., and Owens, I. S. (1993) J.

Aono, S., Yamada Y., Keino, H., Sasaoka, Y., Nakagawa, T., Yazawa, T., Biol. Chem. 268, 23573-23579

Kaufman, S. S., Wood, R. P., Shaw, B. W., Jr., Markin, R. S., Rosenthai, P., Mimura, S., Koiwa, O., and Sato, H. (1993) Zbratology 48, 520, Abstr. C08

Gridelli, B., and Vanderhoof, J. A. (1986) Hepatology (Baltimore) 6, 1259- 1262

Duhamel G., Blanckaert N., and Metreau, J . M., Preaw, A. M., Bouvry, M.,

Sinaasappel, M., and Jansen, P. L. M. (1991) Gastroenterology 100, 783-789 Fevery, J., and Berthelot, P. (1984) J. Hepatol. 1, 47-53

ElAwady, M., Roy Chowdhury, J., Kesari, K., Van Es, H., Jansen, P. L. M., Lederstein, M., Arias, I. M., and Roy Chowdhury, N. (1990) J. Biol. Chem. 265,10752-10758

Roy Chowdhury, J., Huang, T., Kasari, K., Lederstein, M., Arias, I. M., and Roy Chowdhury, N. (1991) J. Biol. Chem. 266,18294-18298

Sato, H., Aono, S., Kashiwamata, S., and Koiwai, 0. (1991) Biochem. Biophys. Res. Commun. 177, 1161-1164

Blanckaert, N., Fevery, J., Heinvegh, K. P. M., and Compernolle, E (1977) Biochem. J. 164,237-249

Moghrabi, N., Clarke, D. J., Boxer, M., and Burcheil, B. (1993) Genomics 18, 171-173

J. Med. 47,395409

(1977) J. Clin. Znuest. 60,970-979

1043-1047

76,4350-4354