(Received for publication, October 7, 1996, and in revised form, November 15, 1996)
From the Department of Pharmacology, University of California, San Diego, Cancer Center, La Jolla, California 92093-0636
Southern blot analysis has demonstrated that the
5 portion of the rabbit liver dexamethasone-inducible
UDP-glucuronosyltransferase (UGT) 2B13 RNA is related in sequence to a
family of UGT genes (Tukey, R. H., Pendurthi, U. R.,
Nguyen, N. T., Green, M. D., and Tephly, T. R. (1993) J. Biol. Chem. 268, 15260-15266). To identify these additional gene
transcripts, rabbit liver cDNA libraries were screened with a 5
conserved 330-base pair UGT2B13 cDNA fragment, resulting in the
isolation and characterization of several rabbit liver UGT cDNAs.
One such clone, called pGT11, encodes a putative glycoprotein that is
78% similar to rabbit UGT2B13. The new UGT has been designated
UGT2B16. The UGT2B16 gene is expressed as a single
4200-base RNA transcript that is regulated only in adult rabbits. The
predicted NH2-terminal 25 amino acids of UGT2B16 are
identical to that of rabbit liver UGT2B13, with the remainder of the
protein being 77% similar to UGT2B13. Expressed UGT2B16 protein in
COS-1 cells was active toward 4-hydroxybiphenyl, similar to that of
UGT2B13. However, UGT2B16 efficiently conjugated 4-hydroxyestrone
and 4-tert-butylphenol, substrates that are not efficiently
catalyzed by UGT2B13. To further characterize the structural domains of
UGT2B16 and UGT2B13, a series of chimeric cDNAs were constructed
that contained portions of both UGT2B16 and UGT2B13. Chimeric
2B163002B13531, which contained the
amino-terminal UGT2B16 amino acids 1-300 followed by amino acids
301-531 of UGT2B13, as well as chimeric
2B163582B13531 and
2B164342B13531 proteins, catalyzed the
glucuronidation of 4-hydroxyestrone, indicating that the carboxyl
terminus of UGT2B13 could substitute for those same regions on UGT2B16.
However, the replacement of the carboxyl end of UGT2B13 with
2B16300-531 or 2B16434-531 dramatically impaired the catalytic function of the chimeric proteins. These results
indicate that the carboxyl end of UGT2B13 plays an important role in
the functional and possible conformational state of the protein.
Glucuronidation is an important biochemical process of detoxification that leads to the removal of endogenous agents such as steroid hormones, bilirubin, and bile salts, in addition to thousands of xenobiotics and dietary by-products (1). The transfer of glucuronic acid from the cosubstrate UDP-glucuronic acid to the aglycones render the products water-soluble, a process that eliminates potential biological activities and facilitates excretion of the glucuronides from the cell. The UDP-glucuronosyltransferases (UGT,1 EC 2.4.1.17) are glycoproteins (2), which are channeled by a hydrophobic leader sequence (3) to the luminal surface of the endoplasmic reticulum (4). The diversity in the selection of structurally diverse compounds for glucuronidation results from a relatively large "superfamily" of UGTs, many of which have been identified through recombinant DNA techniques (5). Based upon overall structural similarities, the UGTs have been classified into either the UGT1 or UGT2 gene families (6, 7). All of the UGT1 proteins are transcribed from a single locus (8), with each protein containing a variable amino-terminal half and an identical carboxyl region. In contrast, the UGT2 proteins are believed to be transcribed from independent structural genes (9).
The UGT2 proteins share a high degree of similarity in their carboxyl regions with significant amino acid diversity occurring in the amino-terminal region of the proteins. Through the construction and expression of chimeric cDNA clones containing amino-terminal and carboxyl-terminal regions of different UGTs, it has been suggested (10) that the amino-terminal half is important in dictating which of the many different substrates will be selected for conjugation. Because of the high degree of similarity in the carboxyl portion of the UGTs, it has been proposed that this region dictates the conformational properties that underlies the binding of the cosubstrate UDP-glucuronic acid. Under this type of model, it is presumed that a substrate or aglycone binding pocket and a separate UDPGA domain interact to coordinate transfer of glucuronic acid to the facilitating substrate. Such a model would indicate that the UDPGA binding domain and possibly the secondary structure responsible for forming this region is closely related in all of the UGTs.
Previous experiments in our laboratory utilizing cDNAs encoding UGT2B13 and UGT2B14 have demonstrated the existence of a large UGT2 family in rabbits (11). UGT2B13, which has an identical amino-terminal sequence to the purified rabbit liver estrone UGT, catalyzes the glucuronidation of small phenolic agents like 2-naphthol, as well as the bulkier phenols such as 4- and 2-hydroxybiphenyl. Interestingly, UGT2B13 has limited ability to conjugate steroids. The estrone UGT cDNA has not yet been identified. UGT2B13 is expressed constitutively as an adult RNA. However, in neonatal rabbits, UGT2B13 is expressed in a fashion concordant with the expression of rabbit CYP3A6, being induced in newborns with agents such as dexamethasone and macrolid antibiotics (11). Northern blot analysis also indicated the possibility that sequence-related UGT2B13 transcripts were regulated in a similar fashion to that of UGT2B13, suggesting that UGT2B13-like genes may be under similar modes of regulation. Combined, these results indicated that UGT2B13 could be used to selectively identify related rabbit UGT cDNAs. Since there appeared to be a high degree of nucleic acid sequence similarity to other regulated UGTs, experiments were undertaken to clone and characterize additional UGT2B13-like cDNAs, with the intent of examining how these genes are regulated as well as investigating the catalytic properties of the related proteins.
Restriction endonucleases, T4 polynucleotide
kinase, T4 DNA ligase, avian myeloblastosis virus reverse
transcriptase, and the RNase inhibitor RNasin were purchased from New
England Biolabs or Life Sciences (St. Petersburg, FL).
Oligo(dT)-cellulose was purchased from Boehringer Mannheim. Nitroplus
2000 nitrocellulose hybridization paper was purchased from Micron
Separations Inc (Westwood, MA). The Erase-a-Base DNA sequence kit was
obtained from Promega (Madison, WI), while DNA sequencing kits were
purchased from U. S. Biochemical Corp. DNA nick translation kits,
[-32P]dCTP (3000 Ci/mmol), [
-32P]ATP
(3000 Ci/mmol), and [
-35S]dATP (400 Ci/mmol) were
purchased from Amersham Corp. Uridine diphospho-D-[U-14C]glucuronic acid (225 mCi/mmol; ammonium salt) and Tran35S-label
([35S]L-methionine,
[35S]L-cysteine) were obtained from ICN
Radiochemicals (Irvine, CA). The eukaryotic expression vector pSVL was
purchased from Pharmacia Biotech Inc. Aglycone substrates, uridine
diphosphoglucuronic acid (ammonium salt), dexamethasone, rifampicin,
and formalin-fixed Staphylococcus aureus cells were obtained
from Sigma. Glass-backed linear K TLC plates with
preabsorbant strips were purchased from Whatman. All oligonucleotides
were manufactured with an Applied Biosystems model 380B DNA synthesizer
from the UCSD Cancer Center Molecular Biology Core laboratory.
The New Zealand
White adult rabbit liver-ZAP cDNA library, previously
constructed in this laboratory (11), was screened with a
32P-labeled 5
portion of the UGT2B13 cDNA that
corresponded to bases 111-442. All cDNA probes were labeled by
nick translation to an approximate specific activity of 1 × 108 cpm/µg of DNA. The libraries were plated on 100-mm LB
plates containing ampicillin at a density of 2-3 × 103 plaque-forming units/plate, and each filter annealed
with 5 × 106 cpm/ml of labeled DNA at 42 °C for
16 h in 50% deionized formamide, 5 × SSC (1 × SSC, 15 mM sodium citrate, pH 7.0, containing 150 mM
NaCl), 10 µg/ml sonicated salmon sperm DNA, and 20 mM
potassium phosphate buffer, pH 7.4. The filters were first washed at
room temperature in 2 × SSC containing 0.5% sodium dodecyl
sulfate (SDS) for 15 min, followed by washing in 0.1 × SSC, 0.5%
SDS for 2 h, with several changes of this wash solution. All of
the positive clones were picked and placed into 0.5 ml of water, and
rescreened as outlined. This procedure was repeated until each clone
was demonstrated to be 100% pure, as judged by positive hybridization. To evaluate the approximate size of each insert, 10 µl of each purified clone that had been placed in 0.5 ml of water was subjected to
polymerase chain elongation and replication (PCR) using T3 and T7
polymerase primers. Those PCR products that displayed a linear DNA size
of greater than 1600 bases were chosen for further studies. As outlined
by Stratagene, each recombinant
-ZAP cDNA clone was recovered as
a double stranded plasmid. Using both T3 and T7 primers, the plasmids
were sequenced through both their 5
- and 3
- regions, as outlined in
the protocols from the U. S. Biochemical Corp. sequencing kits. The
cDNA clone of interest, identified as pGT11, was selected for
further characterization.
Following the generation of a partial
restriction enzyme map for pGT11, internal cDNA fragments were
individually subcloned into pBluescript and sequenced using primers
that recognized the T3 and T7 promoter regions. Approximately 40% of
the cDNA was sequenced in this fashion. In addition, a series of
clones with progressively overlapping deletions from both the 5 and 3
directions were constructed by removing portions of the cDNA by
exonuclease III and S1-nuclease digestion, as outlined
utilizing the Erase-a-Base kit supplied by Promega. When sequencing the
sense strand, pGT11 was first digested with AccI to
linearize the plasmid, followed by protection of the 5
protruding ends
with
-phosphorothioate nucleotides. The protected cDNA was then
digested with EcoRI prior to treatment with exonuclease III
and S1-nuclease. When sequencing the antisense strand,
pGT11 was digested with SpeI, the 5
protruding ends
protected and then the linearized plasmid digested with SmaI prior to exonuclease III and S1-nuclease digestion. During
digestion, an aliquot from each digestion was removed and treated with
Klenow DNA polymerase in the presence of dNTPs to create blunt ends. Each sample was then ligated with T4 DNA ligase and used to transform competent Escherichia coli JM109 cells. Colonies were
prepared using conventional miniplasmid preparations, and each clone
sequenced using both T3 and T7 primers. These two methods assured that
each strand of the cDNA was sequenced at least twice. The
generation of a linear DNA sequence was assembled using the Shotgun
merge program from the Microgene Sequence Analysis Program
(Beckman).
Total RNA was extracted from liver
using guanidine HCl (12), and polyadenylated RNA was purified by
twice-repeated chromatography on oligo(dT)-cellulose. For primer
extension of polyadenylated RNA, an antisense oligonucleotide
(5-TTCATCATTGCTATTAACTAA-3
) corresponding to bases 166-186 of pGT11
UGT2B16) and an antisense oligonucleotide (5
-TTCATTGTTGGAACCAATTAC-3
)
corresponding to bases 204-224 of the UGT2B13 RNA (11) were employed.
Approximately 100 µg of each oligonucleotide was end-labeled with
[
-32P]ATP and T4 DNA polynucleotide kinase, and
labeled product was purified through Sephadex G-50 columns packed in
9-inch Pasteur pipettes with 0.15 M Tris-HCl, pH 8.0, containing 10 mM MgCl2. For each reaction,
2 × 105 cpm of labeled oligonucleotide was added to
2.5 µg of mRNA in buffer containing 40 mM PIPES, pH
6.4, 1 mM EDTA, 0.4 M NaCl, and 80% formamide
in a total volume of 100 µl, and incubated at 80 °C for 10 min and
then at 25 °C overnight. After annealing, 10 µl of 3 M
sodium acetate, pH 7.0, and 275 µl of ethanol were added. The
precipitate was resuspended in 25 µl of 50 mM Tris-HCl, pH 7.6, 60 mM KCl, 10 mM MgCl2, 1 mM dNTPs, 1 mM dithiothreitol, 1 unit/µl
placental RNase inhibitor, 50 µg/ml actinomycin D, and 1 unit/µl
avian myeloblastosis virus reverse transcriptase, and the reaction
incubated at 42 °C for 2 h. The mRNA was then digested with
RNase A, and the extension products extracted with phenol/chloroform (1:1) and precipitated with 2.5 volumes of ethanol. The pellets were
resuspended in 5 µl of 80% formamide containing 10 mM
Tris-HCl, pH 8.0, and 1 mM EDTA, boiled, and the sizes
determined by electrophoresis in 6% polyacrylamide-DNA sequencing
gels. To determine the exact length of the extended products, a
parallel DNA sequencing reaction was included in the gel.
Approximately 2.5 µg of rabbit
mRNA was electrophoresed in 1% denaturing agarose gels containing
formaldehyde and transferred to nitrocellulose filters. Inserts from
the cDNA clones were labeled by nick translation with
[-32P]dCTP to a specific activity of >1 × 108 cpm/µg DNA. The filters were prehybridized for 2 h at 42 °C and then hybridized for 16 h with 107
cpm/ml of labeled insert in 6 × SSC, 5 × Denhardt's
solution (1 × Denhardt's solution: 0.02% Ficoll (type 400),
0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin (fraction
V)), 0.1% SDS, 100 µg/ml denatured salmon sperm DNA, and 50%
formamide. The filters were washed twice at room temperature in 2 × SSC and 0.1% SDS and then at 55 °C in 0.1 × SSC and 0.1%
SDS for 1 h with several changes of washing buffer.
The cDNA that encodes UGT2B16 does not contain the first 7 amino acids of the leader sequence. To assure that UGT2B16 chimeric protein contains a functional leader sequence, the leader sequence that encodes UGT2B13 was used to replace the same region on the pGT11 cDNA. Using the p2B13 cDNA (11), a AmaI/BglII fragment was isolated and cloned into the same location in the pGT11 cDNA. The 186-base pair UGT2B13 insert replaced the leader sequence encoded by pGT11, and the first 34 amino acids. The entire coding sequence was then removed by digesting the plasmid with XhoI and BlnI, and then cloning this fragment into the XhoI and XbaI sites in the eukaryotic expression vector pSVL. This vector is identified as p2B16.SVL. From previous work, the expression of UGT2B13 was carried out using p2B13.SVL (11).
Using p2B13.SVL and p2B16.SVL, a number of UGT2B13 and UGT2B16 chimeric cDNAs were constructed. Chimeric cDNAs were constructed by using the restriction enzyme sites SacI, BamHI, or AseI to create exchanges at codons 300, 358, or 434, respectively. For the SacI site exchange, both plasmids were digested with SacI and the small and large fragments were recovered from 1% low melting agarose gels. The large SacI fragments contained linearized pSVL vector in addition to coding region that spanned amino acids 1-300, while the smaller fragments encoded the COOH-terminal end of the UGTs. The large SacI fragments from one UGT plasmid were ligated with the smaller SacI fragments from the other UGTs, generating plasmids p2B16S13 and p2B13S16. Plasmid p2B13S16 encodes a complete transcript covering amino acids 1-300 of UGT2B13 and 301-531 of UGT2B16, and the expressed protein is identified as 2B133002B16531, while plasmid p2B16S13 encodes the chimeric protein 2B163002B13531.
A BamHI restriction enzyme site at codon 358 is
present in both p2B16.SVL and p2B13.SVL along with a single
BamHI site that is located 3 of the cDNA. Plasmids
p2B13.SVL and p2B16.SVL were each digested with BamHI and
the fragments purified, followed by ligation of the 3
BamHI
fragment from p2B13 with the 5
portion of p2B16. This construct,
p2B16Ba13, encoded a chimeric protein called
2B163582B13531 that encoded UGT2B16 from
amino acids 1 to 358 and UGT2B13 from amino acids 359 to 531. After
digesting p2B13S16 with BamHI and purifying the 5
cDNA/vector fragment, the 3
BamHI fragment from p2B13
was mixed with this fragment in a ligation reaction to generate plasmid
p2B13S16Ba13, which encodes the chimeric protein
2B133002B163582B13531.
The pSVL vector contains two AseI sites, and there is a
single AseI site in both p2B13.SVL and p2B16.SVL at codon
434. Both plasmids were digested with AseI under conditions
that generated partial restriction enzyme digestion, and the different
fragments removed from low melting agarose. A p2B13 fragment that
contains a portion of the vector and the 5 portion of the cDNA was
ligated together with the 3
portion of p2B16. A similar ligation was constructed with the 5
portion of p2B16 and the 3
portion of p2B13.
These plasmids are identified as p2B16A13 and p2B13A16, and encode
chimeric proteins that are identified as
2B164342B13531 and
2B134342B16531, respectively. The proper
orientation, chimeric nature, and the absence of mutations at the
ligation site were confirmed by restriction map analysis and partial
DNA sequence analysis. All of the pSVL cDNAs were transfected and
expressed in COS-1 cells as described previously (13).
After
48 h of transfection, COS-1 cells from a single 35-mm tissue
culture dish were washed three times with Hank's balanced salt
solution and incubated for 4 h in methionine-free modified Eagle's medium supplemented with 100 µCi/ml
Tran35S-label. The cells were washed twice in
phosphate-buffered saline and the cells lysed on ice in 600 µl of
RIPA solution containing 1% Triton X-100, 1% sodium deoxycholate, 1%
SDS, 150 mM NaCl, 1 mM phenylmethanesulfonyl
fluoride, and 50 mM Tris-HCl, pH 7.5. Cellular DNA was
broken by shearing through a 25-gauge needle and was removed with other
cellular debris by centrifugation at 16,000 × g. The
cleared supernatant was incubated for 4 h at 4 °C with 22 µg
of sheep anti-rabbit UGT1A62 IgG (13),
followed by 80 µl of a 10% suspension of formalin-fixed S. aureus cells. After washing the cells four times in RIPA buffer and once in TSA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl), the S. aureus cells were suspended in
100 µl of loading buffer containing 63 mM Tris-Cl, pH
6.8, 2% SDS, 5% glycerol, 5% -mercaptoethanol, and 0.02%
bromphenol blue, and the radiolabeled antigens were released by boiling
the sample for 5 min, followed by removal of the cells by
centrifugation. The immunoprecipitated proteins were analyzed by 8%
SDS-polyacrylamide gel electrophoresis. The gel was soaked with 1 M sodium salicylate for 30 min before drying, and the
radiolabeled bands detected by fluorography at
70 °C overnight.
UGT activities were
determined according to the method of Bansal and Gessner (14), as
modified (11). Transfected COS-1 cells were homogenized in 5 volumes of
ice-cold 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2. In a total volume of 100 µl of 100 µg of cell extract, 20 µM UDPGA, and 0.04 µCi of
UDP-[14C]glucuronic acid, 10 µg of phosphatidylcholine,
100 µM substrate in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 were incubated at
37 °C for 1 h, and then extracted with 200 µl of 100%
ethanol. The protein was removed by centrifugation at 10,000 rpm for 10 min in an Eppendorf centrifuge, and the supernatant dried and resuspended in 30 µl of methanol for application to a Whatman glass-backed linear K TLC plate. Chromatography was performed in a
mixture of 1-butanol/acetone/acetic acid/water (35:35:10:20). The TLC
plate was dried, sprayed with a thin layer of EN3HANCE, and
exposed to x-ray film at 70 °C. The appropriate portions of the
TLC cellulose that represented the glucuronides were scraped into glass
scintillation vials and dissolved in 0.5 ml of water and then 12 ml of
Ecolite scintillation liquid was added. The vials were shaken and
counted in a Beckman scintillation counter.
Southern
blot analysis demonstrated that the 5 portion of the
dexamethasone-inducible 4-hydroxybiphenyl UGT2B13 RNA is related in
sequence to additional UGT genes (11). In attempts to
identify additional UGT2B13-like RNA transcripts, a 5
portion of the
UGT2B13 cDNA covering bases 111-442 was employed as a probe to
screen a rabbit liver cDNA library constructed in
ZAP. Our
initial screen resulted in the purification of over 20
ZAP cDNA
clones. Each cDNA was initially characterized by PCR analysis to
assure that the size of the inserts were at least 1600 bases, the
minimum length needed to encode the 530-amino acid UGTs. A second
screen was then conducted to determine the DNA sequence at both the 5
and 3
locations of the inserts to assure that each recombinant would
encode a full-length protein. From these initial screens, one cDNA,
designated as plasmid pGT11, was selected and further characterized by
DNA sequence analysis.Clone pGT11 is 2832 base pairs in length (Fig.
1). There exists an open reading frame that encodes a
protein of 523 amino acids, followed by a 3
-untranslated region of
1260 bases. There is no predicted 5
-untranslated region or
identifiable 3
RNA termination signals, indicating that the original
RNA transcript is larger than the cloned cDNA. When the predicted
amino acid sequence encoded by pGT11 was examined for similarity with
the other rabbit UGTs, it displays 78% similarity to UGT2B13 (Fig.
2) and 74% similarity to UGT2B14 (not shown). Based
upon the high degree of similarity to the rabbit UGT2B sequences and
the guidelines for classifying new UGTs (7), the predicted protein that
is encoded by pGT11 has been designated as UGT2B16.
Expression of UGT2B16 Transcripts in Rabbit Liver
To examine
the relative levels of RNA expression of UGT2B16 RNA as well as
transcript size, primer extension analysis was conducted with RNA
isolated from both neonatal and adult rabbits. Since there exists such
a high degree of similarity in the 5 portion of pGT11 with the UGT2B13
RNA, the first divergent region between the two RNAs was selected to
identify antisense oligonucleotides, as indicated under "Experimental
Procedures." Primer extension with the UGT2B16 oligonucleotide
generated transcripts of 224 and 227 bases, while the UGT2B13
oligonucleotide generated a single transcript of 256 bases (Fig.
3). These results indicate that there may exist multiple
transcriptional start sites on the gene for UGT2B16, or that
the oligonucleotide is priming not only UGT2B16 RNA but a transcript
with similar DNA sequence. The size of the UGT2B16 transcripts indicate
that the cDNA cloned is approximately 40 bases short of a
full-length transcript. Results from primer extension using the UGT2B13
oligonucleotide suggest that the previously cloned RNA (11) is
approximately 32 bases short of full length. The genes that encode both
UGT2B16 and UGT2B13 are transcriptionally active
under constitutive conditions only in adult rabbits.
In examining the expression of UGT2B16, Northern blot
analysis was conducted using two cDNA fragments as probes, one that spanned the coding region and the other that encompassed just the
noncoding 3 region of the cDNA. As demonstrated in Fig.
4A, the noncoding region of UGT2B16
identified a major 4.2-kb RNA transcript that begins to be expressed in
15 day old neonatal rabbits but exists primarily as an adult RNA
transcript. When the 5
coding region was used, the 4.2-kb fragment and
several other transcripts were also identified. This would be expected since the coding region shares significant homology to other rabbit UGT
RNAs, such as UGT2B13. To examine this possibility, the same Northern
blot was stripped and reprobed with the 5
portion of the UGT2B13
cDNA. Two distinct transcripts, primarily expressed in adult RNA,
are present as 4 and 1.9 kb, and migrate in the same location as two of
the transcripts identified with the 5
portion of the UGT2B16 cDNA.
This result indicates that the 4- and 1.9-kb transcripts identified
with the 5
portion of UGT2B16 insert encode the UGT2B13 RNA. Another
transcript, migrating at 2.2 kb and which is also identified with the
5
portion of the UGT2B16 cDNA, most likely represents another
UGT2B gene product.
Although UGT2B13 is expressed primarily in adults, we had
previously demonstrated that this gene was regulated in a fashion similar to that of CYP3A6, being induced in neonatal rabbits with dexamethasone and macrolid antibiotics (11). To determine if UGT2B16 is regulated by these same inducers, RNA isolated
from neonatal rabbits treated with dexamethasone was used to quantitate the levels of UGT2B16 RNA. As shown in Fig. 4B,
dexamethasone had no effect on the ability to induce the 4.2-kb
transcript that encodes UGT2B16 RNA. However, when the 5 coding region
of the UGT2B16 cDNA was used as a probe, it did detect induction of
the UGT2B13 RNA transcripts in addition to the unidentified 2.2-kb RNA.
Induction of UGT2B13 and CYP3A6 were verified in duplicate Northern
blots using the respective cDNAs as probes. It should be noted that
while CYP3A6 is usually expressed in adult rabbits, it exhibits
variable levels of expression (15). The sample used in Fig. 4 was from
an animal that exhibited a low level of expression. Although UGT2B16
and UGT2B13 RNAs are related in sequence, these results indicate that
the cis-acting regulatory elements associated with the
induction of the UGT2B13 gene are not conserved in the UGT2B16 gene.
The predicted amino acid sequence of UGT2B16 is 523 amino acids with approximately 8 amino acids missing in the leader sequence when compared with UGT2B13 (Fig. 2). Since the leader sequence and the first 25 amino acids are identical between UGT2B16 and UGT2B13, a 186-base pair fragment of the UGT2B13 cDNA that encoded all of the leader sequence was used to exchange this same region of the UGT2B16 cDNA, generating a cDNA that encoded the additional 7 amino acids plus the initiation methionine (see "Experimental Procedures" for details). This chimeric UGT2B16 cDNA was then transferred to the plasmid pSVL and transfected into COS-1 cells to examine protein expression and catalytic activity.
The predicted amino-terminal end of the processed UGT2B16 is identical
to UGT2B13 and the amino-terminal sequence of the purified rabbit liver
estrone UGT. After confirming by pulse-chase analysis with
[35S]methionine/cysteine-labeled protein that UGT2B16 and
UGT2B13 are synthesized in COS-1 cells (Fig. 6B), UGT
activity was examined in whole cell extracts using substrates catalyzed
by the rabbit liver UGTs, as shown in Fig. 5. Expressed
UGT2B16 catalyzes the glucuronidation of bulky phenols such as 2- and
4-hydroxybiphenyl, as well as smaller phenolic compounds like
2-naphthol. The bulky phenols are also conjugated by UGT2B13. However,
UGT2B16 was able to glucuronidate 4-tert-butylphenol, a
bulky phenol that did not serve as an efficient substrate for expressed
UGT2B13. Interestingly, there were two substrates that showed strict
selectivity to either UGT2B13 or UGT2B16. Octylgallate, a large
nonplanar phenol was conjugated only by UGT2B13, while 4-hydroxyestrone
served as a substrate for expressed UGT2B16. Neither UGT2B13 nor
UGT2B16, which contain an amino-terminal region identical to that of
the rabbit liver estrone UGT, were able to conjugate estrone.
The Expression of Chimeric UGT2B16 and UGT2B13
The largest degree of divergence between the two proteins resides at amino acids 64-228, with overall amino acid similarity from amino acids 1-64 and 229-530 being greater than 85%. It has been proposed that the substantial variation in amino acid sequence observed in the amino-terminal half of the transferases contributes to the diversity in substrate specificity (10). Since UGT2B16 and UGT2B13 share the ability to glucuronidate some common substrates, yet also show selective substrate specificity, the variable regions between these two proteins may underlie the ability of UGT2B16 and UGT2B13 to display differences in catalytic activity. To examine this possibility, regions of UGT2B16 and UGT2B13 cDNAs were exchanged and expressed in COS-1 cells to generate chimeric proteins that encoded the different regions of the two proteins.
UGT2B16, UGT2B13, and the chimeric proteins that are expressed in COS-1 cells are schematically shown in Fig. 6A. With 2B16, switches were made at amino acids Ser-300, Trp-358, and Val-434, while with 2B13 switches were made at amino acids Ser-300 and Val-434. In addition, amino acids Ser-300 through Trp-358 of 2B13 were replaced with 2B16. Protein synthesis of each chimeric protein, evaluated in COS-1 cells following pulse-chase with 35S-amino acids, indicates that the chimeric cDNAs produce the appropriate size protein and that each is synthesized in similar abundance (Fig. 6B). Based upon immunoreactivity of newly synthesized protein, the UGT chimeric proteins appear to be of the appropriate molecular weight, indicating that stable protein is encoded by the transcripts of each transfected chimeric cDNA.
With each construct, glucuronidation activity from transfected COS-1
cells was evaluated with 4-hydroxyestrone, octylgallate, and
4-hydroxybiphenyl, as shown in Fig. 7 (A-C).
4-Hydroxyestrone is a selective substrate for UGT2B16, and octylgallate
serves as a selective substrate for UGT2B13. The expression of UGT2B16 and those UGT2B16 chimeric proteins that share UGT2B13 carboxyl portions were catalytically active in the presence of
4-hydroxyesterone. There are 36 amino acid differences between UGT2B16
and UGT2B13 in the carboxyl region between Ser-300 and Asp-531, but
switching this region on UGT2B16
(2B163002B13531) actually makes a more efficient protein in the glucuronidation of 4-hydroxyestrone when compared to that of the parent UGT2B16. Chimeric
2B163582B13531 and
2B164342B13531 were as functional as the parent
UGT2B16. Since all of the chimerics that contained 2B16 in the amino
terminus were functional toward the glucuronidation of
4-hydroxyestrone, an activity that is not supported by UGT2B13, these
results indicate that the amino-terminal region of UGT2B16 supports the
structural requirements necessary for the specific glucuronidation of
4-hydroxyestrone.
Since both UGT2B16 and UGT2B13 catalyze the glucuronidation of some common substrates such as 4-hydroxybiphenyl (Fig. 5), it was anticipated that the replacement of the carboxyl region of UGT2B13 with that of UGT2B16 would have little impact on catalytic activity toward 4-hydroxybiphenyl. The cDNAs that encoded chimeric proteins 2B133002B16531 and 2B134342B16531 efficiently led to the production of immunoprecipitable UGTs, as displayed in Fig. 6B. However, when these expressed proteins were examined for catalytic activity, the UGT2B13 chimerics were not capable of conjugating 4-hydroxybiphenyl (Fig. 7B). In addition, these two chimerics were inactive with other substrates that were readily conjugated by UGT2B13, such as octylgallate (Fig. 7C). Even the replacement of amino acids 300-358 from UGT2B13 in this same region of UGT2B16 (chimeric 2B133002B163582B13531) dramatically interrupted the glucuronidation activity toward octylgallate. While there are 15 amino acid differences between UGT2B13 and UGT2B16 from Val-434 to Asp-531, substituting UGT2B13 with the UGT2B16 sequences completely inactivates the protein. It would appear that rigid structural requirements for the sequences between Val-434 and Asp-531 are needed to maintain UGT2B13 in an active conformation.
The UGT2B family in rabbits is composed of the UGT2B13, UGT2B14, and the 4-hydroxyestrone UGT2B16. Enzyme purification and NH2-terminal analysis of estrone UGT demonstrates that the NH2-terminal sequence is identical to that of both UGT2B13 and UGT2B16, but the mRNA encoded by the estrone UGT has not yet been identified. Northern blot analysis indicates that UGT2B16 is encoded by a large RNA of approximately 4.2 kb, and is not inducible in neonatal rabbits in a fashion similar to that of P4503A6.
The expression of UGT2B16 has catalytic activity that overlaps that of
UGT2B13. Both enzymes glucuronidate small phenolic agents such as
2-naphthol and 4-methylumbelliferone, as well as bulky phenolic
compounds like 2-hydroxy- and 4-hydroxybiphenyl. The larger phenolic
agents are preferentially metabolized by UGT2B13. However, in contrast
to UGT2B13, UGT2B16 had significant catalytic activity toward the
polyhydroxylated estrogen 4-hydroxyestrone, but was catalytically
inactive toward estriol and 17-estradiol. In humans,
4-hydroxyestrone has been reported to be conjugated by UGT2B7, UGT2B8,
UGT2B9, and UGT2B11, while UGT2B8 and UGT2B11 also conjugate
4-hydroxybiphenyl. UGT2B16 is unable to conjugate hyodeoxycholic acid,
which also cannot be conjugated by UGT2B8 and UGT2B11. Based upon a
comparison of the catalytic activities with UGT2B16 and the human UGT2
enzymes, UGT2B16 is most similar to UGT2B8 and UGT2B11. However, the
4-hydroxyestrone UGT activity observed with UGT2B16 was equivalent to
the 4-tert-butylphenol UGT activity from expressed UGT2B16,
which has been reported to be conjugated only by human UGTHP4.
Therefore, UGT2B16 appears to have a much broader substrate specificity
range than any of the UGT2 proteins identified in humans.
The observation that UGT2B16 and UGT2B13 share substrate specificity toward 4-hydroxybiphenyl, while UGT2B16 is selective toward 4-hydroxyestrone and UGT2B13 is specific for octylgallate, provided us the opportunity to examine the ability of chimeric proteins containing portions of both UGTs to glucuronidate these substrates. The results suggest that the amino-terminal portion of the proteins, as reported previously (10), are important for aglycone selection. This was demonstrated by linking the amino-terminal region of UGT2B16 with the carboxyl region of UGT2B13 and demonstrating that these chimerics were active in the conjugation of 4-hydroxyestrone. In one instance, a more efficient enzyme was derived using the amino-terminal 300 amino acids of UGT2B16 with the carboxyl-terminal 231 amino acids of UGT2B13.
However, the construction of UGT2B13-UGT2B16 chimeric proteins indicates that the carboxyl region of the UGTs may play a critical role in catalytic activity. For example, when the same NH2-terminal region of UGT2B13 was spliced to the carboxyl region of UGT2B16, there was a complete loss of catalytic activity. Even the replacement of amino acids 300-358 of UGT2B13 with UGT2B16 drastically reduced catalytic activity. Although these proteins are efficiently manufactured in the cells (Fig. 6B), the replacement of virtually any portion of the carboxyl region of UGT2B13 with UGT2B16 impaired enzyme function. This is clearly shown with 2B134342B16531, where amino acid residues 434-531 of UGT2B13 have been replaced with the same region from UGT2B16. It has been suggested (16) that amino acids 352-408 may be important in the recognition of UDP-glucuronic acid. If one proposes that the amino-terminal region, for example amino acids 1-300, are critical for substrate specificity while the region from 352-408 is important for UDP-glucuronic acid binding, amino acids 434-531 would appear to play an important role in some aspect of conformational stability. This is supported in experiments using chimeric 2B134342B16531, which was shown to be inactive when compared to the proginator UGT2B13 in the glucuronidation of octylgallate.
Analysis of the carboxyl region of UGT2B13 and UGT2B16 by the method of Kyte and Doolittle (17) to identify membrane-spanning regions demonstrates that amino acids 495-519 are hydrophobic. This hydrophobic domain is flanked by two charged areas, which are characteristic of the halt-transfer signals of transmembrane proteins (18) as well as all UGTs (19), and plays a role in securing the protein in the membrane. Aside from the importance of amino acids 494-531 in positioning the protein in the membrane, this region may have a minor impact on substrate recognition and catalysis. With this assumption and based upon the negative catalytic activity of expressed 2B134342B16531, it would appear that amino acids 434-495 might be critical to UGT2B13 for activity. Interestingly, when UGT2B13 and UGT2B16 are aligned, there are conserved amino acid differences at positions 463, 468, and 488, and two nonconserved differences at Asn-443 and Arg-461. It may be that the nonconserved changes between UGT2B13 and 2B134342B16531 at Asn-443 and Arg-461 underlie the dramatic reduction in catalytic activity associated with 2B134342B16531.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U72742[GenBank].