©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Altered Coding for a Strictly Conserved Di-glycine in the Major Bilirubin UDP-glucuronosyltransferase of a Crigler-Najjar Type I Patient (*)

(Received for publication, August 3, 1994; and in revised form, November 17, 1994)

Marco Ciotti Matthew T. Yeatman Ronald J. Sokol (1)(§) Ida S. Owens (¶)

From the Section on Genetic Disorders of Drug Metabolism, Human Genetics Branch NICHHD, National Institutes of Health, Bethesda, Maryland 20892-1830 and Pediatric Liver Center, Children's Hospital, Denver, Colorado 80218

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The characterization (Ritter, J. K., Chen, F., Sheen, Y. Y., Tran, H. M., Kimura, S., Yeatman, M. T., and Owens, I. S. (1992) J. Biol. Chem. 267, 3257-3261) of the single-copy UGT1 gene complex locus encoding both bilirubin and phenol UDP-glucuronosyltransferases (transferase) has been critical to the determination of genetic defects in Crigler-Najjar patients. The complex (UGT1A-UGT1M) codes for at least two bilirubin, three bilirubin-like, and eight phenol transferase isozymes. In the 5` region, a minimum of 13 different exons 1, each with an upstream promoter, are arrayed in series with 4 common exons in the 3` region of the locus. Each exon 1 encodes the amino terminus of a transferase, and the common exons encode the common carboxyl terminus of each isoform. Although a deleterious mutation in a common exon inactivates the entire locus, a deleterious mutation in an exon 1, as we report here for the UGT1A gene in a Crigler-Najjar Type I patient, affects the amino terminus of that single isoform. Recessively inherited mutant alleles for the predominant bilirubin isozyme, the HUG-Br1 protein, substituted Arg for Gly at codon 276 (G276R) in exon 1 of UGT1A abolishing a conserved di-glycine. The mutant HUG-Br1-G276R protein expressed in COS-1 cells had no detectable bilirubin glucuronidating activity at either pH 7.6 or 6.4. Although each of the bilirubin-type isozymes contains a conserved peptide between residues 270 and 288, all UDP-glucuronosyltransferases contain a di-glycine at approximately position 276-277, making it strictly conserved. Structure-function relationship was studied by site-directed mutations of the HUG-Br1 cDNA; G276A, G276Q, G276E, G276I, and P270G mutants were inactive, and V275I- and P285G-altered transferases expressed normal activity. Conservation of residues between the related baculoviral ecdysone UDP-glucosyltransferase and the UDP-glucuronosyltransferases confirms the critical role of the Gly-276 as well as other residues.


INTRODUCTION

The heme metabolite, bilirubin IXalpha, causes jaundice (icterus) in human and animal models when excess serum levels of the compound are sustained as a result of a deficiency in its detoxification, a condition that often leads to serious neurotoxicity. The failure of detoxification is critical, because 200-400 mg of the metabolite are produced daily, primarily from the normal turnover of senescent red blood cells. The continuous high production of the compound represents biochemical pressure for biosystems to maintain an efficient system(s) for its clearance. Special chemical properties of the nascent metabolite lead to its low clearance. Under physiological conditions, the compound has two different trios of internal hydrogen bonding of the carboxyl group of a propionic acid side chain to the amino groups and the lactam oxygen of a pyrrole ring in the opposite half of the molecule (1) generating a chemical form that is both hydrophobic and has a high affinity for tissues of the central nervous system. The UDP-glucuronosyltransferase-catalyzed covalent linkage of this heme derivative to glucuronic acid disrupts the bonds creating the water-soluble bilirubin glucuronides, which are highly excretable. Glucuronidation, creating both monoconjugates and a diconjugate, is the single efficient mechanism in humans for detoxifying this metabolite. When severe unconjugated hyperbilirubinemia persists, the metabolite forms deposits (kernicterus) in the central nervous system (1) and, eventually, lethal neurotoxicity. Children with the inheritable CN-I (^1)disease have no detectable bilirubin UDP-glucuronosyltransferase (transferase) activity and usually succumb in early childhood. Two other inheritable syndromes, CN Type II and Gilbert's, are characterized by reduced bilirubin transferase activity and intermediate levels of unconjugated hyperbilirubinemias, both of which are far less life-threatening than CN-I.

Recently, two human bilirubin transferase cDNAs, HUG-Br1 and HUG-Br2, were cloned and characterized(2) . The two encoded isoforms, generating identical amino acid sequences in the carboxyl-terminal regions but unique in the amino-terminal regions, were each shown to produce the two monoconjugates and the diconjugate of bilirubin(2) . Further, two phenol-metabolizing isoforms described in the literature also share the same carboxyl terminus(3, 4) . The substrate specificities of the bilirubin and the phenol isoforms with the shared carboxyl region indicate that the unique amino terminus determines acceptor-substrate selection.

Subsequently, the novel UGT1 gene complex locus encoding two bilirubin, three bilirubin-like, and one phenol transferase isozyme was uncovered and characterized(5) . The locus, located on chromosome 2 (6) , was shown to span at least 95 kb with 4 common (shared) exons in the 3` region, encoding the identical 245-amino acid carboxyl terminus of the six isoforms. In the 5` region, 6 exons 1 arranged in a tandem array, each having its own promoter element in the proximal upstream position, were shown to exist encoding the entire 288 residues of the unique amino-terminal region of an isoform. A schematic of the locus with nested primary transcripts copied from this gene complex is shown in Fig. 1(5) . Extension (^2)of the 5` region of the locus shows that at least 13 exons 1 exist in this complex, expanding the locus by more than 150 kb. Based on the exon arrangement in conjunction with the distribution of consensus RNA splice-site sequences described in the original version of the complex, it is predicted that at least 13 independently regulated and overlapping primary transcripts are synthesized and that the lead exon 1 in each transcript is spliced to the 4 common exons. The model proposes that a primary transcript can generate only the mature mRNA corresponding to its lead exon 1. Predictably, a deleterious mutation in any of the common exons inactivates the entire complex, whereas a similar mutation in any exon 1 affects that single isoform (see Fig. 1). This prediction has been borne out in a study (7) using microsomes isolated from the explanted livers of several CN-I individuals who underwent transplant surgery. The results revealed two phenotypes as follows: defective bilirubin isozyme activity with either normal or severely defective phenol transferase activity. In this report, we demonstrate an alteration in exon 1 of UGT1A, which selectively inactivates the HUG-Br1 protein, the major bilirubin isozyme, by disrupting a conserved di-glycine present in all UDP-glucuronosyltransferase isoforms reported in the literature, as well as a related UDP-glucosyltransferase.


Figure 1: Schematic of the UGT1 locus and its predicted primary transcripts. A, in the 3` region, the UGT1 locus contains common exons 2-5 encoding the carboxyl-terminal 245 amino acid residues of each isoform specified by this locus, and, in the 5` region, each of seven exons 1 (1A-1G) encodes the unique amino-terminal portion (285-289 residues) of an isoform. The schematic (not drawn to scale) represents 107 kb of the locus. A promoter element (bentarrows) is located upstream of each exon 1; thus, it is predicted that alternative transcriptional initiation generates nested primary transcripts as shown in B. Due to the presence of RNA donor splice sites at each 3` exon-intron junction of the exons 1, both acceptor/donor splice sites in exons 2-4, and only an acceptor site in exon 5, it is predicted that the lead exon 1 in each transcript is differentially spliced to common exons 2-5. A, arrows below the exons represent deleterious mutations detected in Crigler-Najjar Type I patients. Missense (arrows with circles) mutations (G276R, G308E(31) , S375F(31) ), deletion (arrows with squares) mutations (codon-170(9) , 13-bp deletion (10) ), or conversion (arrows with squares) (Q331, R341, and Q357 to stop codons(32, 33, 34) ) mutations were shown to either totally abolish bilirubin transferase activity or generate a pH-sensitive mutation or were predicted to critically alter the major bilirubin transferase protein structure.




EXPERIMENTAL PROCEDURES

Materials

UDP-glucuronic acid, bilirubin, and chloroquine were from Sigma. [^14C]UDP-glucuronic acid was from DuPont-NEN; [alpha-P]deoxycytidine triphosphate was from Amersham Corp.; [S]methionine was from ICN Biomedicals (Costa Mesa, CA). Restriction enzymes and other reagents used in molecular biology techniques were from New England Biolabs (Beverly, MA), Pharmacia LKB Biotechnology Inc., Boehringer Mannheim, IBI Biochemicals (New Haven, CT), U. S. Biochemical Corp., or Life Technologies, Inc. Amplitaq polymerase was from Perkins-Elmer. The pSVL vector, the oligo-labeling kit, and DEAE-Dextran were from Pharmacia, and COS-1 cells were from the American Type Tissue Culture Collection (Rockville, MD). The Bluescript plasmids and XL-1 Blue cells were from Stratagene (La Jolla, CA). Tissue culture supplies were from Mediatech (Washington, DC) or Biofluids (Rockville, MD).

Isolation of Genomic DNA from the Liver of a CN-I Patient and from Lymphocytes of the Parents

A specimen of the explanted liver from a CN-I patient, SS, was obtained from The Children's Hospital, Denver, CO. The frozen section was minced in 0.5 M EDTA, pH 8.0, and genomic DNA was isolated as described(8) . Genomic DNA was isolated from whole blood taken from the parents and an unrelated normal female as described previously(9) .

Polymerase Chain Reaction, Subcloning, and Sequencing of PCR Products

In order to localize the mutation that inactivated the bilirubin transferase in the CN-I patient, the 4 common exons were amplified as described(9) . Exon 1 of the UGT1A gene, which encodes the HUG-Br1 cDNA, was amplified as a 990-bp fragment by using the primer set PAG4 (5`-AACCTCTGGCAGGAGCAAAG-3`) and PAG5 (5`-CTCAGAATGCTTGCTCAGC-3`). The fragment contained the coding part along with 101 bp of 3`-flanking intron sequence and was subcloned into the SmaI site of pBluescript II SK. A single clone was completely sequenced as described previously (2) and found to be identical to the normal, with the exception of a point mutation (GC) at codon 276 converting a Gly to an Arg (G276R). Allele-specific PCR amplification as described below verified that the genome of the patient was homozygous for the altered codon. The sequence data from the subclones of the 4 common exons were identical to data from a normal individual.

Construction of Wild-type or Mutant pHUG-Br1 Expression Units

The construction of the pSVL-based expression unit (pHUG-Br1) for the normal HUG-Br1 protein has already been described (2) . The pHUG-Br1-G276R unit that encodes the G276R conversion was constructed in the pSVL-based HUG-Br1 unit. The G to C mutant was made by PCR amplification with the independent generation of the appropriate point mutation in both the sense and antisense strand. Independent PCR reactions were with a primer set, with a change in the sense primer UGT1-276S (5`-GGTTTTTGTTCGTGGAATCAAC-3`) used with antisense primer, PXAS6 (5`-TAAACACCATGGGAACC-3`) to generate a 321-bp fragment, and a second reaction with a base change in the antisense primer UGT1A-276AS (5`-GTTGATTCCACGAACAAAAACC-3`) was used with the sense primer, P2S4 (5`-CTGTGCGACGTGGTTTA-3`) to generate a 174-bp fragment. The interchanged hybridizing fragment generated by combining the 321- and 174-bp PCR products was used as a primer in a reaction with the primer set, PXAS6 and P2S4. The reaction generated a 413-bp PCR product that contained the codon 276 (TGG to TCG) point mutation. The fragment was digested with EcoRI and BstEII and ligated into the EcoRI/BstEII digested wild-type expression unit, pSVL-HUG-Br1, with the 385-bp fragment specifying Arg instead of Gly at codon 276, replacing the original 385-bp fragment. The replaced segment, including the ligation sites of the pHUG-Br1-G276R-construct, was sequenced to ensure that no other changes occurred in the reading frame. Similarly, other missense mutations in the pHUG-Brl unit were made in this region with the following degenerate primers: G276E, sense (5`-GGTTTTTGTTGAAGGAATCAAC-3`) and antisense (5`-GTTGATTCCTTCAACAAAAACC-3`); G276A, sense (5`-GGTTTTTGTTGCCGGAATCAAC-3`) and antisense (5`-GTTGATTCCGGCAACAAAAACC-3`); G276I, sense (5`-GGTTTTTGTTATCGGAATCAAC-3`) and antisense (5`-GTTGATTCCGATAACAAAAACC-3`); G276Q, sense (5`-GGTTTTTGTTCAGGGAATCAAC-3`) and antisense (5`-GTTGATTCCCTGAACAAAAACC-3`); P270G, sense (5`-AGGCCCATCATGGGTAATATG-3`) and antisense (5`-CATATTACCCATGATGGGCCT-3`); V275I, sense (5`-GGTTTTTATCGGTGGAATCAAC-3`) and antisense (5`-GTTGATTCCACCGATAAAAACC-3`); P285G, sense (5`-CTTCACCAAAATGGTCTATCC-3`) and antisense (5`-GGATAGACCATTTTGGTGAAG-3`) and, finally, the outside primer set for all mutations was sense (P2S4) and antisense (PXAS6).

Expression of Wild-type pHUG-Br1 and the pHUG-Br1 Mutants in COS-1 Cells

COS-1 cells were plated in 100-mm dishes at 10^6 cells and grown to 90% confluency in 24 h in Dulbecco's modified Eagle's medium (DMEM) with Hepes buffer and 4% fetal calf serum (FCS). pHUG-Br1, or each of the pHUG-Br1 mutants, was transfected into cells using DEAE-dextran as the carrier as described (10) . Cultures were washed twice with sterile phosphate-buffered saline (PBS) (10) and exposed to 10 ml of DMEM without serum. Each expression unit was precombined with DEAE-dextran as follows: 80 µl of DEAE-dextran (100 mg/ml in Tris-buffered saline) was added to 500 µl of Tris-buffered saline containing 25 µg of plasmid DNA. The 580-µl mixture was added to the culture dish. (The DEAE-dextran was presterilized by passage through a 0.2-µ pore diameter sterile filter unit). After 3 h of incubation, the cultures were washed twice with 6 ml of PBS and incubated 2-3 h in 10 ml of DMEM (4% FCS) containing 200 µg/ml chloroquine. Cells were washed twice with PBS after removing the chloroquine medium and incubated for 72 h in 15 ml of regular DMEM (4% FCS).

Radiolabeling of Wild-type and Mutant Bilirubin Transferase Expressed in COS-1 Cells Transfected with Wild-type or Mutant pHUG-Br1

The transferase isozymes, synthesized in COS-1 cell as described above, were radiolabeled during the final 4 h of the incubation as described previously(10) . Goat anti-mouse UDP-glucuronosyltransferase IgG was added to the solubilized labeled cellular extract and processed for SDS-gel electrophoresis as described (11) ; the dried gel was exposed to x-ray film for an autoradiograph.

Assay for the Expression of Bilirubin Transferase Activity

Bilirubin transferase activity was measured by using COS-1 cells transfected with either the wild-type or a mutant pHUG-Br1 cDNA expression unit. The glucuronidation assay previously described (11, 12) was adapted to determine bilirubin glucuronidation; optimum conditions were 1.41 mM [^14C]UDP-glucuronic acid (1.41 µCi/µM), 100 µM bilirubin, 5.0 mM MgCl(2), 16.6 mM saccharic acid 1,4-lactone, and either 33 mM triethanolamine, pH 7.6, or 20 mM sodium phosphate, pH 6.4, in a total volume of 100 µl. Cell homogenates (used fresh or stored at -70 °C in PBS) were spun at 10,000 rpm in a microcentrifuge for 15 min to remove harvesting or storage buffer and were resuspended in the reaction buffer containing MgCl(2) and saccharic acid 1,4-lactone; cellular protein was then treated with CHAPS (0.7 mg/mg protein). The pH of the mixture was monitored for stability over 10-20 min and adjusted, if necessary. Detergent-treated cell homogenate was added to the reaction vessel followed by [^14C]UDP-glucuronic acid; the contents were gassed with nitrogen and capped tightly. Under low light, bilirubin solubilized in fresh dimethyl sulfoxide was added to the reaction, regassed, capped immediately, and incubated in the dark. All subsequent manipulations and data analyses were carried out by TLC and scanned using the Ambis Radioanalytical Imaging System II as already described (10, 11) . TLC plates were exposed to x-ray film to generate radiographs.

Determination of Inheritance of the Defective Alleles-Because the point mutation at codon 276 did not alter a restriction enzyme site, allele-specific PCR amplification was carried out with DNA from an unaffected individual, both parents of SS, and that from SS using either the unaltered sense primer APS2 (5`-CCCAATATGGTTTTTGTTGGT-3`) or the mutant sense primer APS3 (5`-CCCAATATGGTTTTTGTTGCT-3`) with the antisense primer PAG6 (5`-GACAGACTCAAACCTAGGAGTC-3`). The primers are designed such that the penultimate base in the normal or mutant sense primer hybridized to the target base (i.e. the point mutation), and either set is predicted to generate a 209-bp fragment with the appropriate genomic DNA.


RESULTS

Identification of a Codon-276 Missense Mutation in Exon 1 of the UGT1A Gene of a CN-I Patient

Exon 1 of UGT1A and common exons 2-5 at the UGT1 locus in CN-I patient, SS, were amplified, subcloned, and sequenced as described (2) . Exons 2-5 contained normal sequence (data not shown). The exon 1 contained normal sequence except for the point mutation (G C) converting a Gly to an Arg at codon 276 as shown in Fig. 2(right panel, solid circle) and generating a UGT1A-G276R mutant allele when compared with the sequence of the normal UGT1A allele (left panel).


Figure 2: Comparison of nucleotide sequences in the HUG-Br1-cDNA in a normal genome and that from SS. Autoradiograms of Sanger nucleotide sequencing reactions of normal and CN-I patient, SS, DNA are shown. Plasmid DNA containing the entire 990-bp exon 1 of UGT1A was sequenced using the Bluescript primers, as well as synthetic ones designed to provide overlap data at 250-base intervals as described under methods. The sequence ladders were generated by electrophoresis through a 6% denaturing polyacrylamide gel and were transferred onto blotting paper, dried, and exposed to x-ray film to generate the autoradiograms shown. A, C, T, and G are lanes corresponding to separate reactions with added dideoxy derivatives of ATP, CTP, TTP, and GTP, respectively. The solidcircle indicates the nucleotide C substitution in the genome of the CN-I patient, SS.



Comparison of Amino Acid Sequences between Residues 270 and 288 in Bilirubin and Bilirubin-like Transferase Isozymes from Human and Rat

Because the exon 1 of UGT1A-G276R specifying the mutant HUG-Br1 protein (Fig. 3, line 1) encodes an Arg, which abolishes a di-glycine at position 276-277 in patient SS (line 1), it was of interest to compare the structure in this region in all bilirubin and bilirubin-like isozymes. HUG-Br1 protein is the major bilirubin isoform in humans; the HUG-Br2, encoded by UGT1D, is the less abundant bilirubin isozyme(2) . Exon 1 of both UGT1C and UGT1E are 90% identical to that for UGT1D in the coding and at least 200 bp of flanking 5` and 3` sequences; the encoded isoforms are, therefore, designated bilirubin-like(5) . A report on a rat bilirubin transferase clone, bil UDPGT, has also appeared(13) . The deduced amino acid sequences in the region flanking codon 276 of UGT1A for these isoforms are compared in Fig. 3. The di-glycine at position 276-277 is conserved in all of these bilirubin-related isozymes. In addition, there is extensive conservation at other positions in this region, which is quite nonpolar with potentially titratable positively charged groups. Among 19 amino acid residues, there are 4 alpha-helix breaking residues (2 Pro and the di-glycine), 5 polar amide-containing residues (271, 279, 283, 284, and 288), and 7 large nonpolar residues (272-275, 278, 281, and 286). This region has been designated micro-region (MR-B). A similar micro-region A with a conserved di-phenylalanine at residues 170-171 has been described(10) .


Figure 3: Comparison of the amino acid sequences between positions 270 and 288 of bilirubin or bilirubin-like isozymes from human and rat. The amino acid sequences represent positions between 270 and 288 of the predominant human bilirubin isozyme encoded by the normal UGT1A(5) (HUG-Br1 cDNA, line2) gene and its mutant allele (UGT1A-G276R, line1) with the conversion of a Gly-276 to an Arg as described in the legend to Fig. 2. The region between amino acid 270 and 288 is aligned with the corresponding one in the bilirubin isozyme encoded by UGT1D(5) (HUG-Br2 cDNA, line4), two human bilirubin-like isozymes encoded by UGT1C and UGT1E(5) , (lines3 and 5, respectively), and a rat bilirubin isozyme (13) (Rat Bil UDPGT cDNA). The continuous line encompasses conserved residues between the isoforms. Shaded residues proved to be essential; underlined residues were replaceable. Position 270 in the UGT1A-encoded isoform is position 271 for all other isoforms.



Immunocomplexes of [S]Methionine-labeled Transferases Specified by the pHUG-Br1 or Each Mutant pHUG-Br1 Expression Unit

To establish whether the expression units, pHUG-Br1 and pHUG-Br1-G276R, specify bilirubin transferases after transfection into COS-1 cells, [S]methionine-labeled and solubilized cell homogenates were immunocomplexed with goat anti-mouse UDP-glucuronosyltransferase immunoglobulin(11) . The cDNA in the pHUG-Br1 expression unit, as well as that in the pHUG-Br1-G276R, encodes a protein with three potential Asn-linked glycosylation sites. We previously demonstrated (10) that the HUG-Br1 protein has a molecular mass of 52 kDa. Fig. 4shows that migration of the mutant protein (lane 3) in the SDS gel was similar to that for the wild type; it is predicted that both mature forms contain 508 residues from a consideration of the possible signal-peptide cleavage sites(14) . Fig. 4also demonstrates that there was equal or more of the mutant protein than of the wild type. Control cells (Fig. 4, lanes 1 and 4) did not contain a similarly radiolabeled protein.


Figure 4: Immunocomplexes of pHUG-Br1- and mutants of pHUG-Br1 transfected COS-1 cells following [S]methionine labeling. Cells were transfected with the pHUG-Br1 or each of the mutant pHUG-Br1, radiolabeled with [S]methionine, solubilized, and immunocomplexed with goat anti-mouse UDP-glucuronosyltransferase immunoglobulin as described under ``Experimental Procedures.'' It has been established (10) that the HUG-Br1 cDNA encodes a 52-kDa protein when expressed in COS-1 cell.



In addition, Fig. 4shows (lanes 6-12) that the COS-1 cells transfected with other mutant cDNAs synthesized similar amounts of protein to that generated by the wild-type cDNA (lane 5); those mutant proteins contained a Glu (G276E), Gln (G276Q), Ala (G276A), or Iso (G276I) instead of Gly-276 or contained a Gly-270 (P270G) or -285 (P285G) instead of Pro. All molecular masses are similar to that of the wild-type protein (lane 5).

Bilirubin Transferase Activity Generated by the pHUG-Br1, the pHUG-Br1-G276R Mutant, and Other Related pHUG-Brl Mutants in COS-1 Cells

To determine the effect of the natural substitution of an Arg-276 on activity, we carried out bilirubin glucuronidation assays with pHUG-Br1- and pHUG-Br1-G276R-transfected COS-1 cell homogenates as described under ``Experimental Procedures.'' Assays conducted at pH 6.4 are usually 3-fold more productive than those at pH 7.6. The results are shown as an autoradiogram of the x-ray film-exposed TLC plates and with a summary of the total counts incorporated into bilirubin ^14C-glucuronide (Fig. 5. The wild-type protein generated slightly more product at pH 6.4 than at pH 7.6, and the mutant HUG-Br1-G276R protein encoded by the genome of the CN-I patient lacked activity at both pH values. Because the disrupted di-glycine was conserved in each bilirubin or bilirubin-related transferase, it was of interest to examine the effect of a negatively charged, a large polar, or a small or large nonpolar amino acid at that position. The results (Fig. 5, Experiment 2, lines 2-4) indicate that as with the positively charged Arg-276, no basal activity is present with either of the 276-mutant proteins. This complete absence of activity is in contrast to the low level of activity at both pH values with the HUG-Br-Delta170 mutant protein, which is missing a Phe at position 170(10) .


Figure 5: Effect of pH on the formation of bilirubin glucuronides using either pHUG-Br1- or mutants of pHUG-Br1-transfected COS-1 cell homogenates. Bilirubin glucuronidation was carried out using 1.41 mM [^14C]UDP-glucuronic acid (1.4 µCi/µmol) with either 20 mM sodium phosphate, pH 6.4, or 33 mM triethanolamine, pH 7.6. The reactions, carried out as described under ``Experimental Procedures,'' contained 0.3 mg of protein and were incubated for 16 h at room temperature. The samples were processed and analyzed as indicated under ``Experimental Procedures.''



Furthermore, we examined whether Gly (HUG-Br1-P270G and -P285G) could substitute for the conserved Pro in positions 270 and 285. The HUG-Br1-P270G isozyme was completely inactive, whereas the HUG-Br1-P285G mutant was active at a level comparable with that for the wild-type (Fig. 5).

The neutral substitution of Iso-275 for Val (HUG-Br1-V275I), as predicted from its presence at that position in all the other bilirubin-related transferases (Fig. 3), generated activity comparable with the wild type (Fig. 5).

Inheritance of the UGT1A-276 Allele by CN-I Patient, SS

The inheritance of the GC point mutation at codon 276 by CN-I patient, SS, was determined by allele-specific PCR amplification of normal, parental, and proband genomic DNA. Fig. 6shows that the 209-bp fragment was generated with either the combination of normal DNA and normal sense primer or proband DNA and mutant sense primer. In contrast, the parental DNA samples supported the synthesis of the fragment with both the normal and mutant primers, indicating that both progenitors are heterozygous for the same point mutation.


Figure 6: Inheritance of the UGT1A-276 allele by the CN-I patient. Genomic DNA from a normal individual, the CN-I proband, and both parents were PCR-amplified to give a 209-bp fragment as described under ``Experimental Procedures.'' The DNA from the normal individual generated product with only the normal sense primer (lanes8 and 9, whereas DNA from the proband supported DNA synthesis with only the mutant primer (lanes2 and 3). The parental DNA (lanes 4-7) supported synthesis with both primers, indicating that both are heterozygous for the same point mutation.



Comparison of the Conserved Peptides in Micro-region B of UGT1-encoded versus Non-UGT1-encoded Transferase Isoforms

A careful examination of the amino acids surrounding the di-glycine structure was made for all UDP-glucuronosyltransferase isozymes reported in the literature. Amino acid residues examined for Fig. 7(residues 269-280) were shifted slightly compared with Fig. 3in order to emphasize differences between the UGT1-encoded versus the non-UGT1-encoded transferases from human, rat, mouse, and rabbit. Non-UGT1 designates all isoforms not containing the characteristic common carboxyl terminus encoded at the UGT1 locus. A consensus sequence was developed from a tabulation of alternative residues appearing at certain positions as shown (Fig. 7). Fig. 7shows that isozymes encoded at the equivalent UGT1 locus specifying a common carboxyl terminus in these species contain a hydrophobic residue 273(2, 3, 13, 15, 16, 17, 18) (J. G. Lamb, P. Straub, and R. H. Tukey, GenBank Accessions U09101 and U09030), whereas isoforms encoded at other loci contain a negatively charged residue 273(11, 15, 19, 20, 21, 22, 23, 24, 25, 26, 27) (M. D. Green et al., GenBank Accession U06274). The results show that although this region is very similar in all isoforms, an isozyme can be categorized into one of two types based on this amino acid difference. The UGT1 locus also encodes an invariant Met-272 instead of Val, Phe, or Iso seen in the non-UGT1-encoded proteins. The Pro-Asn, Gly-Gly, and Cys at positions 270-271, 276-277, and 280, respectively, were invariant in every form examined. It is notable that the olfactory UGT(27) with Phe-272 and an Asp-273 resembles the non-UGT1 type.


Figure 7: A comparison of the micro-region B of UGT1-encoded versus non-UGT1 encoded UDP-glucuronosyltransferase isozymes in human, rat, mouse, and rabbit. The results of the comparison shown in Fig. 2were extended to compare all UGT1-encoded transferases reported from human, rat, mouse, and rabbit to all other transferases encoded at other loci in these four animal models. All isoforms were compared between amino acid residues 269 and 280. The UGT1-encoded isoforms(2, 3, 13, 15, 16, 17, 18) (GenBank Accessions U09101 and U09030) were tabulated (superscript) for the number of times that residue appears at that position for the 17 isoforms considered. The non-UGT1-encoded isoforms(11, 15, 19, 20, 21, 22, 23, 24, 25, 26, 27) (GenBank Accession U06274) were evaluated in a similar manner, where a total of 16 isoforms were evaluated. A consensus sequence for each type was derived (right), based on the most frequently appearing residue at a position. Residues in boldfacetype were invariant. Residue 273 is underlined.



Comparisons of Critical Amino Acids in Membrane-bound HUG-Br1 and Other Mammalian UDP-glucuronosyltransferase Isoforms with Soluble but Related Baculoviral UDP-glucosyltransferase

The fact that all UDP-glucuronosyltransferases are endoplasmic reticulum-bound and insoluble makes it imperative that we take advantage of related proteins with similar structures, when possible, to gain insight into the conformation of the membrane-bound enzymes, critical peptide structures, residues at the active center, the basis of missense versus a silent mutation in the bilirubin transferase, and the evolution of the novel UGT1 locus. Fig. 8shows a comparison of a HUG-Br1 (UGT1-type) and three (non-UGT1-type) UDP-glucuronosyltransferases from human, rat, and mouse with that of the related UDP-glucosyltransferase (EGT) from the baculovirus(28) . The sequence PSVQYLGGGIH, strikingly similar to the consensus sequences described for the non-UGT1 type in MR-B in Fig. 7, is present in the baculoviral ecdysone UDP-glucosyltransferase (Fig. 8, underline). Again, the Gly-Gly at the equivalent 276-277 position is conserved, and the Gln in the equivalent position of 273 makes the protein more like a non-UGT1 type. It can be seen that this protein shares many other segments of conservation with the mammalian UDP-glucuronosyltransferases (described below).


Figure 8: Comparison of HUG-BR1 and three non-UGT1-encoded UDP-glucuronosyltransferases from human, rat, and mouse to the related UDP-glucosyltransferases from the baculovirus. The HUG-Br1(2) , human HLUG P1(3) , rat UDPGTr-3 (22) , mouse UDPGTm-1(19) , and the EGT (28) proteins are aligned by the Pile-Up Program (GCG Inc., Madison, WI) and printed by using the Box Program. Representations are as follows: black-boxed letters, identity; gray-boxed, similarity; lower case letters, without identity or similarity; and dots, gaps. The underline shows the comparable MR-B regions (residues 270-280) in the aligned proteins. The arrows identify amino acid residues 273, 276, 294, 308, 336, 376, and 387 in the HUG-Br1 protein. The solid circle is the last residue (288) encoded by exon 1 for HUG-Br1.




DISCUSSION

In this study, we uncovered a recessive missense mutation in the unique exon 1 of the UGT1A gene encoding the HUG-Br1 protein of a CN-I patient, which defines a conserved MR-B in bilirubin-related UDP-glucuronosyltransferase proteins. The mutation converted Gly-276 to Arg disrupting a di-glycine at position 276-277, a structure within MR-B. After careful examination of other isoforms, it was discovered that the di-glycine peptide is strictly conserved in all UDP-glucuronosyltransferase isoforms, suggesting that it is pivotal to the secondary structure in this region of each transferase. The lack of activity by the various HUG-Br1-276 mutant proteins confirms that the Gly-Gly at 276-277 is indispensable. The inactive HUG-Br1-P270G mutant enzyme confirms a critical role for Pro-270, whereas the active P285G mutant demonstrates that conserved Pro-285 is replaceable.

In comparing the MR-B of UGT1-encoded bilirubin and phenol-metabolizing transferases with that of all other non-UGT1-encoded isoforms, it became evident that similar consensus sequences exist for the two categories of genes, except all UGT1-encoded isoforms contain a large hydrophobic residue 273 and all non-UGT1 encoded isoforms contain a negatively or weakly charged residue 273. In effect, one could divide all isoforms into two classes (see Fig. 7) based on this amino acid difference. Although other regions of the unique portion of transferases are undoubtedly involved in substrate selection, residue 273 suggests that a hydrophobic versus a negative-charge distinction is made by this region in the selection process.

Comparison of both ``classes'' of UDP-glucuronosyltransferases was made to the soluble and secreted ecdysone UDP-glucosyltransferase encoded in the baculoviral genome (Fig. 8), which was previously reported (28) to have a 21% sequence identity to the three mammalian UDP-glucuronosyltransferases. The enzyme expressed in the insect-host inactivates ecdysone via its conjugation to glucose (donated by UDP-glucose), thus perpetuating the life cycle of the virus by blocking ecdysone-dependent molting of the insect. (The insoluble UDP-glucuronosyltransferases use UDP-glucuronic acid as the donor substrate to form glucuronides.) As pointed out in the original report (28) , the UDP-glucosyltransferase contains many short segments of conservation with respect to the UDP-glucuronosyltransferases, which prompted the appropriate experiments and the discovery of the function of the EGT protein(28) . It is interesting that this protein also contains the conserved di-glycine in the sequence, PSVQYLGGGIH (see Fig. 8, underline), at a similar position in the protein. This sequence resembles the two previously identified consensus sequences shown in Fig. 3and Fig. 7. The equivalent Gln-273, a large polar residue, in EGT (Fig. 8, underlined arrow) suggests a greater resemblance to the non-UGT1-encoded isoforms.

Based on analysis of crystallographic protein data, a di-glycine structure is a typical class 2 subset of a 2-residue beta-hairpin loop. beta-Hairpins connect antiparallel beta-strands (30) or helical and beta-strand secondary structures (29) in the folding and conformation of proteins. Although these structures often serve as recognition and binding sites for other molecules, they can incorporate catalytic residues(29) . Loops can accommodate insertions and deletions (designated indels) of residues without the loss of function (30) in a homologous protein series. Thus, these structures often give rise to a rich source of mutations(29) . Because the UDP-glucuronosyltransferase has significant sequence similarity to the UDP-glucosyltransferase, it is interesting to speculate whether the UDP-glucuronosyltransferases could have undergone a Gly deletion to a di-glycine in PNVDFVGGLHC compared with the EGT protein with a tri-glycine in PSVQYLGGGIH while maintaining what could be a beta-hairpin. Further, it suggests that a similar ancestral protein, resembling EGT, gave rise to the UDP-glucuronosyltransferases and that other versions of EGT-like proteins might exist in other organisms.

It is also interesting that conserved residues between the UDP-glucuronosyltransferases and EGT are mutated in CN patients. Equivalent residues with mutations are shown in Fig. 8(arrows) as follows: 276 (described here); 308 and 376(31) ; 294, 336, and 387. (^3)Each inactivates the HUG-Br1 protein, demonstrating that these conservations are critical both to an active mammalian UDP-glucuronosyltransferase system and, most likely, the EGT enzyme. The conservation between the two systems and the deleterious mutations seen in patients strongly suggest many similarly critical amino acids, secondary structures, and, perhaps, mechanisms of catalysis.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the General Clinical Research Branch, Division of Research Resources, NIH (Grant RR00069).

To whom correspondence should be addressed: National Institute of Health, Bldg. 10, Rm. 9S-242, Bethesda, MD 20892. Tel.: 302-496-6091.

(^1)
The abbreviations used are: CN, Crigler-Najjar; kb, kilobase(s); PCR, polymerase chain reaction; bp, base pair(s); DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonic acid; TLC, thin layer chromatography; MR-B, microregion.

(^2)
J. Cho, N. Gholami, M. Ciotti, S. Carvalho, and I. Owens, manuscript in preparation.

(^3)
M. Ciotti and I. S. Owens, manuscript in preparation.


ACKNOWLEDGEMENTS

We express our appreciation to Drs. Jeong Cho and Brian Moldover for help with executing the Pile-Up and Box Programs.


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