(Received for publication, August 3, 1994; and in revised form, November 17, 1994)
From the
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.
The heme metabolite, bilirubin IX, 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 (
)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 ()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.
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.
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.
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.
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).
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 [C]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).
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.
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.
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.
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 -hairpin loop.
-Hairpins
connect antiparallel
-strands (30) or helical and
-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
-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. ()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.