Characterization of the zebrafish Ugt repertoire reveals a new class of drug-metabolizing UDP glucuronosyltransferases

Article abstract of DOI:10.1124/mol.113.091462

The zebrafish genome contains a gene superfamily of 40 Ugt genes that can be divided into Ugt1, Ugt2, and Ugt5 families. Because the encoded zebrafish UDP glucuronosyltransferase (UGT) proteins do not display orthologous relationships to any of the mammalian and avian UGT enzymes based on molecular phylogeny, it is difficult to predict their substrate specificity. Here, we mapped their tissue-specific expression patterns. We showed that the zebrafish UGT enzymes can be glycosylated. We determined their substrate specificity and catalytic activity toward diverse aglycone substrates. Specifically, we measured mRNA levels of each of the 40 zebrafish Ugt genes in 11 adult tissues and found that they are expressed in a tissue-specific manner. Moreover, functional analyses with the donor of UDP glucuronic acid (UDPGA) for each of the 40 zebrafish UGT proteins revealed their substrate specificity toward 10 important aglycones. In particular, UGT1A1, UGT1A7, and UGT1B1 displayed good glucuronidation activities toward most phenolic aglycones (4-methylumbelliferone, 4-nitrophenol, 1-naphthol, bisphenol A, and mycophenolic acid) and the two carboxylic acids (bilirubin and diclofenac). Importantly, some members of the UGT5, a novel UGT family identified recently, are capable of glucuronidating multiple aglycones with the donor cofactor of UDPGA. In particular, UGT5A5, UGT5B2, and UGT5E1 glucuronidate phenols and steroids with high specificity toward steroid hormones of estradiol and testosterone and estrogenic alkylphenols 4-tert-octylphenol. These results shed new insights into the mechanisms by which fish species defend themselves against vast numbers of xenobiotics via glucuronidation conjugations and may facilitate the establishment of zebrafish as a model vertebrate in toxicological, developmental, and pathologic studies. Copyright

Full text of DOI:10.1124/mol.113.091462

1521-0111/86/1/62–75$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.113.091462
Mol Pharmacol 86:62–75, July 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Characterization of the Zebrafish Ugt Repertoire Reveals a New
Class of Drug-Metabolizing UDP Glucuronosyltransferases s
Yuanming Wang, Haiyan Huang, and Qiang Wu
Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems
Biomedicine, and State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of
Medicine, and Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X
Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Received December 24, 2013; accepted April 11, 2014
ABSTRACT
The zebrafish genome contains a gene superfamily of 40 Ugt aglycones. In particular, UGT1A1, UGT1A7, and UGT1B1 displayed
genes that can be divided into Ugt1, Ugt2, and Ugt5 families. good glucuronidation activities toward most phenolic aglycones
Because the encoded zebrafish UDP glucuronosyltransferase (4-methylumbelliferone, 4-nitrophenol, 1-naphthol, bisphenol A,
(UGT) proteins do not display orthologous relationships to any of and mycophenolic acid) and the two carboxylic acids (bilirubin
the mammalian and avian UGT enzymes based on molecular and diclofenac). Importantly, some members of the UGT5, a novel
phylogeny, it is difficult to predict their substrate specificity. UGT family identified recently, are capable of glucuronidating mul-
Here, we mapped their tissue-specific expression patterns. We tiple aglycones with the donor cofactor of UDPGA. In particular,
showed that the zebrafish UGT enzymes can be glycosylated. UGT5A5, UGT5B2, and UGT5E1 glucuronidate phenols and ste-
We determined their substrate specificity and catalytic activity roids with high specificity toward steroid hormones of estradiol
toward diverse aglycone substrates. Specifically, we measured and testosterone and estrogenic alkylphenols 4-tert-octylphenol.
mRNA levels of each of the 40 zebrafish Ugt genes in 11 adult These results shed new insights into the mechanisms by which
tissues and found that they are expressed in a tissue-specific fish species defend themselves against vast numbers of xeno-
manner. Moreover, functional analyses with the donor of UDP biotics via glucuronidation conjugations and may facilitate the
glucuronic acid (UDPGA) for each of the 40 zebrafish UGT establishment of zebrafish as a model vertebrate in toxicological,
proteins revealed their substrate specificity toward 10 important developmental, and pathologic studies.
bioavailability (Wu et al., 2011; Meech et al., 2012). In
mammals, UGTs are encoded by two gene clusters: Ugt1 and
Ugt2 (Mackenzie et al., 2005; Owens et al., 2005). The
Introduction
With a high-quality reference genome assembled and
a repertoire of molecular genetics tools available, the zebra-
mammalian Ugt1 clusters are organized in a tandem array
fish has become a powerful vertebrate model for human
of multiple, highly similar, variable exons followed by a single
biology and diseases (Stegeman et al., 2010; Howe et al.,
set of four constant exons (Ritter et al., 1991; Emi et al., 1995;
2013). The zebrafish has also emerged as a popular toxico-
logical model for drug discovery, endocrinology, and environ-
mental chemical defense studies (Stegeman et al., 2010;
James, 2011; Peterson and Macrae, 2012; Tokarz et al., 2013).
Zhang et al., 2004; Mackenzie et al., 2005; Owens et al., 2005;
Li and Wu, 2007), similar to the organization of the pro-
tocadherin (Pcdh) clusters (Wu, 2005). Each Ugt1 variable
exon encodes a signal peptide and the amino-terminal
The UDP-glucuronosyltransferases (UGTs) are an important aglycone-recognition domain. The constant exons encode
a highly conserved donor-binding domain and the carboxyl-
terminal endoplasmic reticulum–anchoring transmembrane seg-
ment. Alternative splicing from each of the multiple variable first
exons to the common set of downstream constant exons generates
the enormous molecular diversity required for metabolizing
diverse small lipophilic chemicals. In addition, characterization
of the genetic variability of the UGT1 gene cluster in different
human populations has revealed ethnic specific diversity of
UGT1 haplotypes and linkage blocks (Maitland et al., 2006;
Saeki et al., 2006; Thomas et al., 2006; Ménard et al., 2009;
Yang et al., 2012). The mammalian Ugt2 clusters include three
class of diverse phase II drug-metabolizing and detoxification
enzymes that affect the drug toxicity as well as its oral
This work was supported by the National Natural Science Foundation for
the Youth of China [Grant 81302861]; the Program of Shanghai Subject Chief
Scientist to Q.W.; the Ministry of Science and Technology of China [Grant
2009CB918700]; the National Natural Science Foundation of China [Grants
31171015 and 30970669]; and the Science and Technology Commission of
Shanghai Municipality [Grant 09PJ1405300].
Y.W. and H.H. contributed equally to this work.
dx.doi.org/10.1124/mol.113.091462.
s
This article has supplemental material available at molpharm.aspetjournals.
org.
ABBREVIATIONS: BPA, bisphenol A; DMSO, dimethylsulfoxide; E2, b-estradiol; Endo H, endoglycosidase H; HEK, human embryonic kidney;
HPLC, high-performance liquid chromatography; MPA, mycophenolic acid; 4-MU, 4-methylumbelliferone; 4-NP, 4-nitrophenol; RT-PCR, reverse
transcriptase-polymerase chain reaction; t-OP, 4-tert-octylphenol; UDPGA, UDP glucuronic acid; UGT, UDP glucuronosyltransferase; E2-17G,
b-estradiol 17-b-D-glucuronide; E2-3G, b-estradiol 3-b-D-glucuronide.
62

Glucuronidation Activity of the Zebrafish UGT Superfamily
63
Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA).
Taq DNA polymerase and ribonuclease-free DNase I were purchased
from Takara (Otsu, Japan). Avian myeloblastosis virus reverse
transcriptase and pGEM-T Easy vector were purchased from Promega
(Fitchburg, WI). Restriction endonucleases, T4 DNA ligase, and
endoglycosidase H (Endo H) were purchased from the New England
Biolabs (Ipswich, MA). Cell culture medium, fetal bovine serum, and
trypsin were purchased from Thermo Fisher Scientific (Waltham, MA).
The high-performance liquid chromatography (HPLC)–grade methanol
and acetonitrile were purchased from Merck (Darmstadt, Germany).
Expression Profiling. We designed an isoform-specific primer
pair for each member of the zebrafish Ugt repertoire (Supplemental
Table 1). The polymerase chain reaction (PCR) product for each
zebrafish Ugt gene spans at least one intervening intron to avoid
contamination from the genomic DNA. The specificity of all of the
primer pairs was confirmed by sequencing the amplified PCR product
for each member of the zebrafish Ugt superfamily. Adult zebrafish
(Danio rerio) of about 6 months old were purchased from the Institute
of Biochemistry and Cell Biology, Shanghai Institute of Biological
Sciences, Chinese Academy of Sciences. Total RNA was isolated from
various tissues of the adult zebrafish with the TRIzol reagent
according to the manufacturer’s instructions. The zebrafish Ugt
Ugt2a genes and about a dozen Ugt2b genes (Mackenzie et al.,
2005; Owens et al., 2005). The mammalian UGT enzymes
catalyze the glucuronidation of a vast number of lipophilic
endobiotics and xenobiotics with the UDP glucuronic acid
(UDPGA) as a donor cofactor (Meech et al., 2012).
Glucuronidation has long been proven to be an important
detoxification pathway for organic pollutants in fish species
(Dutton, 1980; Stegeman et al., 2010). Glucuronidation by
phase II enzymes converts endogenous lipophilic metabolites
into hydrophilic compounds, which are easily excreted in
urine (James, 2011). For example, fish glucuronidate one or
both propionic groups of the pigmental bilirubin and excrete
the conjugated bilirubin through bile (Dutton, 1980; Clarke
et al., 1992). To date, limited information is available on the
specific piscine UGT isoforms that are responsible for the
glucuronidation of various xenobiotics and endobiotics (Wu
et al., 2011; Meech et al., 2012). We recently reported
identification of the complete repertoire of 40 Ugt genes and
5 Ugt pseudogenes in the zebrafish and found that teleosts
have many more Ugt genes than mammals (Huang and Wu,
2010). Specifically, zebrafish Ugt genes can be divided into cDNAs were amplified from total RNA by reverse transcriptase-
polymerase chain reaction (RT-PCR). Twenty microliters of total
RNA was treated with ribonuclease-free DNase I at 37°C for 30
minutes. The first-strand cDNA was synthesized using 1 mg of DNase
I–treated RNA and 0.5 mg of random primers in the presence of the
avian myeloblastosis virus reverse transcriptase in a final volume
of 20 ml. One microliter of the RT product was added to a mixture
containing 1ꢀ PCR buffer, 250 mM dNTP, 0.25 mM isoform-specific
primers, and 1 unit of Taq DNA polymerase in a total volume of 20 ml
PCR reaction. The PCR conditions were 94°C for 3 minutes; 35 cycles
of 94°C, 30 seconds; 57°C, 30 seconds; 72°C, 40 seconds; followed by
a final extension at 72°C for 7 minutes. The zebrafish b-actin was used
as a control. The PCR product was electrophoresed on a 2% agarose
gel and visualized by ethidium bromide staining under UV light.
Recombinant UGT Production. We expressed each of the 40
zebrafish recombinant UGT proteins by subcloning the corresponding
cloned cDNA from the pGEM vector into the expression vector
pcDNA3 (Huang and Wu, 2010). Briefly, the zebrafish Ugt open
reading frame from each of the 40 pGEM-T plasmids (Huang and Wu,
2010) was subcloned with primers (Supplemental Table 2) into the
expression vector pcDNA3 containing a myc tag and confirmed by
sequencing. It has been shown that the C-terminal tag does not
interfere with the glucuronidating activity of the recombinant human
UGT protein (Zhang et al., 2012). For recombinant zebrafish UGT
production, human embryonic kidney (HEK)293T cells were trans-
fected at 90% confluency in a 10-cm dish with 12 mg of experimental or
mock plasmids using Lipofectamine 2000. After 6 hours, the medium
was changed. The cells were then grown in the high-glucose
Dulbecco’s modified Eagle medium supplemented with 10% fetal
bovine serum, 4 mM L-glutamine, and 100 mg/ml penicillin/
streptomycin in a humidified incubator with 5% CO2 at 37°C. The
cells were harvested by scraping and were washed in a phosphate-
buffered saline 48 hours after transfection. The cell suspension was
subsequently centrifuged at 500g for 5 minutes. The pellet was
resuspended with phosphate buffer (0.1 M, pH 7.4) containing
dithiothreitol and phenylmethylsulfonyl fluoride. The resuspended
cells were lysed by sonication using an ultrasonic processor Uibra cell
(Sonics & Materials, Newton, CT). Five hundred microliter suspension
Ugt1, Ugt2, and Ugt5 families. Both Ugt1 and Ugt2 clusters
are duplicated and organized into variable and constant
regions. Every Ugt1 and Ugt2 variable exon is separately
spliced to a common set of downstream constant exons in
respective clusters (Huang and Wu, 2010). In addition, there
are a set of 18 zebrafish Ugt5 genes whose entire open reading
frames are encoded by single large exons. These Ugt5 genes
are found in the genomes of teleosts and amphibians but not
in mammals and may arise from retrotransposition (Huang
and Wu, 2010). The expression patterns and functions of none
of these zebrafish Ugt genes are known.
Here we report the expression patterns and catalytical
functions of the entire zebrafish Ugt repertoire. In particular,
we characterized the glucuronidation functions of each of the
40 recombinant zebrafish UGT proteins toward ten important
aglycone substrates, including endogenous hormones or me-
tabolites (estradiol, testosterone, and bilirubin), exogenous
drugs (4-methylumbelliferone, mycophenolic acid, and diclofenac),
as well as environmental toxins (4-nitrophenol, 1-naphthol,
4-tert-octylphenol, and bisphenol A). We demonstrated for the
first time that members of the UGT5 family proteins are true
glucuronosyltransferase enzymes utilizing UDPGA as the
donor substrate. These results have important implications
on the catalytic mechanisms by which zebrafish phase II drug-
metabolizing UGT enzymes metabolize endobiotics and xeno-
biotics. Our findings should also facilitate the development of
the zebrafish as a model organism for drug screening and
environment monitoring.
Materials and Methods
4-Methylumbelliferone (4-MU), diclofenac sodium salt, 4-methyl-
umbelliferyl b-D-glucuronide hydrate, 4-nitrophenyl b-D-glucuronide,
b-estradiol 3-b-D-glucuronide sodium salt (E2-3G), b-estradiol 17-b-D-
glucuronide sodium salt (E2-17G), UDPGA, and dimethylsulfoxide of cells was sonicated for four trains each of 5-second bursts separated by
(DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). 1-Naphthol at least 1 minute of cooling on ice. Lysates were centrifuged at 12,000g
b-D-glucuronide was purchased from Santa Cruz Biotech (Santa Cruz, for 10 minutes. The supernatant fraction was aliquoted, and the
CA). b-Estradiol (E2), testosterone, and bilirubin were purchased from aliquots were frozen at 280°C until used. The protein concentration was
Adamas-beta (Shanghai, China). 4-tert-Octylphenol (t-OP) was pur- determined using the Bradford method with bovine serum albumin as
chased from Tokyo Chemical Industry (Tokyo, Japan). Mycophenolic the standard.
acid (MPA), 4-nitrophenol (4-NP), 1-naphthol, and bisphenol A (BPA)
Western Blot. The relative expression levels of individual
were purchased from Sangon (Shanghai, China). TRIzol reagent and recombinant UGT proteins were evaluated using the Western blot

64
Wang et al.
analyses. Each of the 40 recombinant zebrafish UGT proteins was
separated by 10% SDS-PAGE and transferred onto a nitrocellulose
membrane. The membranes were then probed with a mouse anti-myc
antibody (Millipore; 1:2000 dilution) or a mouse anti-actin antibody
(Abgent; 1:5000 dilution). A goat anti-mouse IgG antibody (1:10,000
dilution) conjugated with IR dye 800CW (Biosciences, San Jose, CA)
was subsequently used as the second antibody. The expression of each
UGT protein was visualized and quantified using the Odyssey
Infrared Imaging system (LI-COR Biosciences, Lincoln, NE).
Endoglycosidase H Treatment. The supernatant fraction of the
cell lysates containing recombinant zebrafish UGT proteins was
adjusted to 2 mg/ml with a glycoprotein denaturing buffer and denatured
by heating at 100°C for 10 minutes. The denatured proteins (10 mg) were
incubated with 250 U of Endo H in 50 mM sodium citrate buffer
(pH 5.5) at 37°C for 1 hour and then subjected to 8% SDS-PAGE
and Western blot analyses.
Glucuronidation Activity Assay. The glucuronidation assay was
performed as previously described (Uchaipichat et al., 2004) with minor
modifications. Briefly, 100 ml of the assay mixture contained 0.1 M
phosphate buffer (pH 7.4), 5 mM MgCl2, 200 mg of lysates containing
recombinant proteins, and the tested aglycone substrate. The lysate of
HEK293T cells transfected with empty vector was used as negative
mock controls. After preincubating the assay mixture at 30°C for
3 minutes in a thermomixer (Eppendorf, Hamburg, Germany) with
automatic shaking, the reaction was initiated by the addition of 5 mM
UDPGA. Incubation was continued for 30–120 minutes before being
terminated by adding 100 ml of ice-cold methanol or acetonitrile. Both
incubation times and protein concentrations in the assays were within
the linear range of individual UGT activities. After centrifugation at
14,000g for 5 minutes, the supernatant was subjected to an HPLC
column (COSMOSIL 5C18-AR-II; Nacalai Tesque, Kyoto, Japan) and
analyzed for the presence of glucuronide conjugates in an HPLC
machine (Prominence LC-20A from Shimadzu, Kyoto, Japan) with a UV
or fluorescence detector under the conditions shown in Table 1. The
limits of detection of the HPLC assays were determined at signal-
to-noise ratios of 3 (Supplemental Table 3). The identities of the
glucuronide conjugates were confirmed by running HPLC with 4-MU
b-D-glucuronide, 4-NP b-D-glucuronide, 1-naphthol b-D-glucuronide,
E2 3-b-D-glucuronide, E2 17-b-D-glucuronide, or with the glucuroni-
dated products using mouse liver microsomes with or without
UDPGA (data not shown). In particular, the identity of the mono-
and diglucuronidated bilirubin was also confirmed by a validation
assay by using the recombinant human UGT1A1 proteins. Finally,
the bilirubin glucuronidating activity of the recombinant zebrafish
UGT1B7 was confirmed by three control experiments: with mock
lysates, without bilirubin, or without UDPGA. The activities were
normalized to the relative expression levels of individual UGT
proteins with UGT1A1 set as 1.0. All of the glucuronidation reactions
were performed at least three times.
For the initial screening of the zebrafish UGT activity, each of the
40 recombinant zebrafish UGT was incubated with one of ten
aglycone substrates (500 mM 4-MU, 2500 mM 4-NP, 2500 mM
1-naphthol, 200 mM BPA, 500 mM t-OP, 1000 mM MPA, 500 mM E2,
500 mM testosterone, 150 mM bilirubin, or 2500 mM diclofenac). The
glucuronidation assays were performed as described above but with
a longer incubation time of 12 hours. When estradiol, testosterone,
bilirubin, bisphenol A, or diclofenac was used as a substrate, DMSO
was added to the reaction mixture at a final concentration of 10%. This
concentration of DMSO does not inhibit the enzymatic activity.
Kinetic Analysis. For kinetic measurements, we used at least
seven different aglycone concentrations and adjusted the enzyme
concentration so that no more than 5% of the substrate was consumed
during the reactions. In the case of 4-MU, the aglycone concentrations
were between 7.5 and 1000 mM for UGT1A7 and between 2.5 and
150 mM for UGT1B1. In the case of 4-NP, the aglycone concentrations
were between 100 and 10,000 mM for UGT1A1, between 100 and
5000 mM for UGT1A7, and between 50 and 10,000 mM for UGT1B1. In
the case of 1-naphthol, the aglycone concentrations were between

Glucuronidation Activity of the Zebrafish UGT Superfamily
65
5 and 1000 mM for UGT1A1, between 2.5 and 150 mM for UGT1B1,
and between 150 and 7500 mM for UGT5A5. In the case of BPA, the
aglycone concentrations were between 25 and 500 mM. In the case of
t-OP, the aglycone concentrations were between 25 and 1000 mM for
UGT5A5 and between 50 and 1000 mM for UGT5B2. In the case of
MPA, the aglycone concentrations were between 50 and 2500 mM for
UGT1A1 and between 25 and 750 mM for UGT1B1. In the case of E2,
the aglycone concentrations were between 10 and 500 mM for UGT1B1
and between 5 and 250 mM for UGT5E1. In the case of testosterone, the
aglycone concentrations were between 10 and 1000 mM. In the case of
diclofenac, the aglycone concentrations were between 50 and 5000 mM.
All of the assays were carried out in duplicates.
The kinetic constants were estimated using nonlinear fitting of
experimental data to the following kinetic equations with GraphPad
Prism 6 (GraphPad Software, La Jolla, CA).
The Michaelis–Menten equation (eq. 1) (Michaelis et al., 2011;
Michaelis and Menten, 1913):
(Fig. 1, A–C). In addition, quantitative real-time PCR experi-
ments were performed for eight representative members of
the Ugt gene superfamily in four representative tissues. The
results were consistent with those from the isoform-specific
semiquantitative RT-PCR assays (Supplemental Fig. 1). The
specificity of each primer pair was confirmed by sequencing
the amplicons. b-Actin was used as an internal control (Fig.
1D).
Ugt1 Clusters. Members of the Ugt1a subfamily display
distinct tissue-specific expression patterns (Fig. 1A). Ugt1a1
is highly expressed in the liver, intestine, testis, and spleen
and at low levels in the brain, gill, ovary, and muscle. There
are low levels of expression of Ugt1a2 in the intestine and of
Ugt1a6 in the liver and intestine. Ugt1a3 and Ugt1a4 are
mainly expressed in the liver, intestine, and testis. In addition,
Ugt1a4 is expressed at low levels in the gill and spleen. Ugt1a5
is predominantly expressed in the gill and moderately in the
kidney and liver. Finally, Ugt1a7 is expressed at high levels in
the gill and intestine and at very low levels in the liver, kidney,
and testis. Interestingly, none of the Ugt1a subfamily is de-
tectable in the eye and heart (Fig. 1A).
Among members of the Ugt1b subfamily, Ugt1b1 is
expressed in all of the tissues examined (Fig. 1A). The ex-
pression patterns of Ugt1b2 to Ugt1b5 are very similar in the
fact that they are mainly expressed in the intestine, liver, and
kidney. In addition, Ugt1b2 is also expressed at low levels in
the muscle, and Ugt1b3 and Ugt1b4 are expressed at low
levels in the gill. Finally, Ugt1b7 is expressed at high levels in
the liver and at low levels in the brain, eye, spleen, intestine,
testis, and muscle (Fig. 1A).
Vmax ꢀ ½Sꢁ
n 5
(1)
K
m 1 ½Sꢁ
where n is the rate of reaction, Vmax is the maximum reaction rate, Km
is the substrate concentration at which the reaction rate is one-half of
Vmax, and [S] is the substrate concentration.
The substrate inhibition equation (eq. 2) (Luukkanen et al., 2005):
Vmax ꢀ ½Sꢁ
n 5
(2)
2
½Sꢁ
K
m 1 ½Sꢁ 1
Ki
where Ki is the constant describing the substrate inhibitory potency.
For data showing both sigmoidal kinetics and substrate inhibition
kinetics, the kinetic constants were calculated using a modified Hill
equation (eq. 3) (Hill, 1910; LiCata and Allewell, 1997):
Ugt2 Clusters. Compared with members of the Ugt1a and
Ugt1b subfamilies, most members of the Ugt2a subfamily are
expressed in a broad range of tissues except Ugt2a4, which is
only expressed in the liver (Fig. 1B). In particular, Ugt2a1,
Ugt2a3, and Ugt2a5 are expressed in all of the tissues
examined. Specifically, Ugt2a1 shows high levels of expres-
sion in all tissues except the brain and eye; Ugt2a3 is highly
expressed in the liver and intestine; Ugt2a5 is expressed at
high levels in all tissues. Ugt2a2 exhibits modest expression
in most tissues, including the intestine, liver, ovary, testis,
kidney, spleen, and muscle and low levels in the gill and heart.
Finally, Ugt2a6 is expressed in most tissues except the brain
and muscle (Fig. 1B).
Members of the Ugt2b subfamily are expressed at high
levels in the liver and intestine except Ugt2b6 (Fig. 1B). In
addition, Ugt2b1 is also expressed in the spleen and at low
levels in the testis and kidney. Ugt2b3 shows low levels of
expression in the testis. Ugt2b5 is expressed with high levels
in the kidney and testis and low levels in the gill and spleen.
Finally, Ugt2b6 is expressed at low levels in the heart, spleen,
liver, ovary, and testis (Fig. 1B).
ꢀ
ꢁ
2
½Sꢁ
V
max 1 Vi ꢀ
Ki
n 5
(3)
ꢀ
ꢁ
ꢀ
ꢁ
n
2
½Sꢁ
Ks
½Sꢁ
1 1
1
Ki
where Ks is the dissociation constant of the ES complex, Vi is the
reaction rate in the presence of inhibition, and n is the Hill coefficient.
Goodness of fitting to the three equations was evaluated on the
basis of standard deviations of the parameter estimates at 95%
confidence intervals, R2 values, and by visual inspection of the
Michaelis–Menten (Michaelis and Menten, 1913; Michaelis et al.,
2011) and Eadie–Hofstee (Eadie, 1942; Hofstee, 1959) plots.
Intrinsic clearance (CLint) was calculated as Vmax/Km for both
Michaelis–Menten kinetics and substrate inhibition kinetics. For
sigmoidal kinetics, maximum clearance (CLmax) was calculated with
the following equation (eq. 4) (Houston and Kenworthy, 2000):
Vmax
Ks
ðn 2 1Þ
1=n
CLmax 5
ꢀ
(4)
nðn 2 1Þ
Results
Ugt5 Clusters. The Ugt5 genes encode a novel family of
Tissue-Specific Expression Patterns of the Zebrafish UDP glucuronosyltransferases that have distinct genomic
Ugt Gene Repertoire. We recently identified the complete organization and only exist in lower vertebrates (Huang and
zebrafish Ugt gene repertoire and found that the zebrafish Wu, 2010). For members of the Ugt5a cluster, Ugt5a1 and
genome contains 40 putative functional genes and 5 pseudo- Ugt5a2 display similar expression profiles (Fig. 1C). In
genes (Huang and Wu, 2010). To characterize their tissue- addition to high levels of expression in the liver and intestine,
specific expression profiles, we measured the expression Ugt5a1 and Ugt5a2 are also expressed at low levels in the eye,
pattern of each member of the zebrafish Ugt superfamily in gill, kidney, spleen, and testis. Ugt5a3 is only expressed at low
a wide variety of tissues, including brain, eye, gill, heart, levels in the liver and intestine (Fig. 1C). Ugt5a4 is expressed
kidney, spleen, liver, intestine, ovary, testis, and muscle, by at high levels in the liver and intestine and at low levels in the
using an isoform-specific semiquantitative RT-PCR method gill and testis. Ugt5a5, the most ancient gene in the cluster

66
Wang et al.
Fig. 1. Tissue-specific expression profiles of the zebrafish Ugt gene repertoire. The expression of the zebrafish Ugt1 (A), Ugt2 (B), and Ugt5 (C) genes
was measured by semiquantitative RT-PCR analyses using isoform-specific primers spanning at least one intron. The expression of the zebrafish b-actin
gene (D) was used as a control. The position of each primer is indicated by an arrow, and their sequences are listed in the Supplemental Table 1. The
genomic organization of each cluster is shown above the respective panels. Yellow and green boxes represent variable exons and constant exons of Ugt1
and Ugt2, respectively. Red and orange boxes represent exons of Ugt5 and b-actin, respectively. Gray and blue boxes represent pseudogenes (p) or relics
(r) and noncoding exons, respectively. Variants u1 and u2 refer to alternative splicing of different 59 noncoding exons upstream of Ugt5b1 and Ugt5b2.
The gene names are shown on the left. The tissue sources are indicated above each lane.

Glucuronidation Activity of the Zebrafish UGT Superfamily
67
(Huang and Wu, 2010), is ubiquitously expressed in all tissues
(Fig. 1C).
Both Ugt5b1 and Ugt5b2 have two alternative upstream 59
noncoding exons (u1 and u2), and alternative splicing from
these two noncoding exons generate two variants for each
of Ugt5b1 and Ugt5b2 (Fig. 1C) (Huang and Wu, 2010).
Interestingly, we found that the two variants of Ugt5b1 and
Ugt5b2 have different expression patterns. The u1 variant of
Ugt5b1 is expressed at high levels in the gill and spleen and at
low levels in the heart, kidney, liver, intestine, ovary, and
testis. The u2 variant of Ugt5b1 is expressed at high levels in
the gill, kidney, and spleen and at low levels in the liver,
intestine, ovary, and muscle (Fig. 1C). The u1 variant of
Ugt5b2 is expressed at high levels in all of the tissues except
the heart. The u2 variant of Ugt5b2 is expressed at high levels
in the eye, gill, and kidney but at low levels in the ovary (Fig.
1C). This observation suggests that the two upstream
noncoding exons were differentially spliced in a tissue-
specific manner. The expression of Ugt5b3 is limited to the
intestine and liver, with a higher level in the intestine.
Finally, Ugt5b4 is expressed at high levels in the brain, eye,
gill, kidney, spleen, liver, and intestine, and at low levels in
the testis (Fig. 1C).
There are three members of the Ugt5c cluster (Huang and
Wu, 2010). Ugt5c1 is expressed at low levels in the brain,
spleen, intestine, and testis (Fig. 1C). Ugt5c2 is expressed at
high levels in the kidney, spleen, liver, intestine, and ovary
and at low levels in the eye and testis (Fig. 1C). Finally,
Ugt5c3 is highly expressed in all tissues except in the gill (Fig.
1C).
Other members of the Ugt5 family, including Ugt5d1,
Ugt5e1, Ugt5f1, Ugt5g1, and Ugt5g2, are nonclustered Ugt
genes (Huang and Wu, 2010). Ugt5d1 is expressed at high
levels in the liver and intestine and at low levels in the spleen,
testis, and ovary (Fig. 1C). Ugt5e1 is expressed in the eye, gill,
heart, kidney, and testis (Fig. 1C). Ugt5f1 and Ugt5g1 are
ubiquitously expressed in all tissues except the eye and spleen
(Fig. 1C). Interestingly, Ugt5g2 is only expressed at high
levels in the eye and at low levels in the gill (Fig. 1C).
Glucuronidation Activities of the Zebrafish UGT
Superfamily Proteins. Although glucuronidation is known
Fig. 2. Expression (A) and Endo H treatment (B) of recombinant zebrafish
UGT enzymes. Shown are Western blots of the recombinant zebrafish
UGT superfamily (A). Multiple bands were rendered to a single band after
the Endo H treatment (B). The name of each recombinant enzyme is
indicated on the top of each lane. The molecular weights of the markers
are indicated on the left of the panel.
to be an important pathway in fish species, to date no member (Figs. 3 and 4). The substrate (aglycone) concentrations used
of the zebrafish UGT superfamily has been shown to have to obtain the data shown in Table 2 were the same as that
catalytic glucuronidating activity (Dutton, 1980; James, used in the initial screening (see Materials and Methods).
2011). To investigate the biological function of individual The HPLC chromatograms of glucuronidation assays with
members of the UGT superfamily in zebrafish, we expressed members of the UGT1 and UGT2 families were shown in
each of the 40 recombinant UGT proteins in HEK293T cells. Fig. 3 for enzymes with strong activities toward specific
We confirmed the expression of recombinant proteins using substrates and in Supplemental Fig. 2 for the others. The
Western blot analysis (Fig. 2A). Interestingly, we observed results for UGT5 were shown in Fig. 4.
multiple bands for most UGT isoforms; however, treatment of
UGT1 Family. Some members of the UGT1 family have
individual UGTs with Endo H invariably resulted in a single strong glucuronidation activities toward most of the phenols
polypeptide band (Fig. 2B). This demonstrated that the (except t-OP) and carboxylic acids (Fig. 3; Supplemental Fig.
multiple bands are derived from different levels of the gly- 2; Table 2). Moreover, UGT1 is the only family with isoforms
cosylation of the same UGT enzyme.
that can conjugate bilirubin and BPA. However, no member of
We measured the glucuronidation activities of the zebrafish the UGT1 family can glucuronidate steroids (Fig. 3; Supple-
UGT enzymes, with UDP-glucuronic acid as the donor, toward mental Fig. 2; Table 2), except UGT1A1 and UGT1B1, which
ten commonly used aglycone substrates, including three small have trace glucuronidation activities toward b-estradiol at the
phenols (4-MU, 4-NP, 1-naphthol), three complex phenols 39-OH position (Fig. 3I; Supplemental Fig. 2D; Table 2).
(BPA, t-OP, and MPA), two hormone steroids (E2 and
Among the seven members of the UGT1A subfamily, UGT1A1
testosterone), and two carboxylic acids (bilirubin and diclofe- glucuronidates most of the substrates, except testosterone
nac) (Table 2) by using the HPLC system to separate the (Table 2), and is the most active isozyme involved in the
conjugated glucuronide from the respective aglycone substrate glucuronidation of 1-naphthol (Fig. 3A), BPA (Fig. 3B), MPA

68
Wang et al.
(Fig. 3C), and diclofenac (Fig. 3D). Multiple peaks appeared in
the HPLC chromatogram after diclofenac was conjugated by
recombinant zebrafish UGTs, probably because of the intra-
molecular rearrangement of acyl-glucuronides (Harada et al.,
2009). Moreover, UGT1A1 also has glucuronidation activities
toward the coumarin derivative 4-MU (Supplemental Fig.
2A), the small phenol 4-NP (Supplemental Fig. 2B), complex
phenol t-OP (Supplemental Fig. 2C), steroid E2 (Supplemen-
tal Fig. 2D), and endobiotic bilirubin (Supplemental Fig. 2E).
UGT1A3 has high glucuronidation activity toward MPA (Fig.
3E) and low activity toward 4-MU (Supplemental Fig. 2F),
4-NP (Supplemental Fig. 2G), 1-naphthol (Supplemental Fig.
2H), BPA (Supplemental Fig. 2I), t-OP (Supplemental Fig.
2J), bilirubin (Supplemental Fig. 2K), and diclofenac (Sup-
plemental Fig. 2L). UGT1A4 has a low activity toward 4-MU
(Fig. 3F) and trace activities toward 4-NP (Supplemental
Fig. 2M) and 1-naphthol (Supplemental Fig. 2N). Finally,
UGT1A7 has strong activities toward 4-MU (Fig. 3G) and
4-NP (Supplemental Fig. 2O) and weak activity toward
1-naphthol (Supplemental Fig. 2P). Its activity toward 4-MU
is the highest among all zebrafish UGTs (Table 2).
Among the six members of the UGT1B subfamily, UGT1B1
has glucuronidation activities toward 4-MU (Supplemental
Fig. 2Q), 4-NP (Fig. 3H), 1-naphthol (Supplemental Fig. 2R),
BPA (Supplemental Fig. 2S), t-OP (Supplemental Fig. 2T),
MPA (Supplemental Fig. 2U), E2 (Fig. 3I), and bilirubin
(Supplemental Fig. 2V). In particular, UGT1B1 has high
activities toward multiple phenols, including 4-MU, 4-NP,
1-naphthol, and MPA, with its activity toward 4-NP the
highest among the 40 zebrafish UGT isozymes (Table 2);
however, its activities toward E2 (39-OH) and t-OP are very
low (Table 2). Interestingly, UGT1B1 and two members of
UGT1A (1A1 and 1A3) conjugate a common set of aglycone
substrates, including all of the six phenols as well as bilirubin
(Table 2), consistent with the fact that these three genes are
close paralogs (Huang and Wu, 2010). UGT1B2 has trace
activities toward three phenols 4-MU (Supplemental Fig.
2W), 4-NP (Supplemental Fig. 2X), and 1-naphthol (Supple-
mental Fig. 2Y). Finally, UGT1B7 glucuronidates bilirubin at
the highest rate among all of the zebrafish UGTs, suggesting
its important role in bilirubin metabolism (Fig. 3J; Table 2).
UGT2 Family. Among members of the UGT2 family, we
found that UGT2B3 has low glucuronidation activities toward
three phenols: 4-MU (Supplemental Fig. 2Z), 4-NP (Supple-
mental Fig. 2AA), and 1-naphthol (Fig. 3K). Moreover,
UGT2B6 has low glucuronidation activities toward 4-MU
(Fig. 3L), 4-NP (Supplemental Fig. 2AB), and 1-naphthol
(Supplemental Fig. 2AC). Other members of the UGT2 family
do not have detectable glucuronidation activity toward the ten
substrates examined.
UGT5 Family. In contrast to the variable and constant
genomic organization of the Ugt1 and Ugt2 families, the
zebrafish Ugt5 genes form a novel family with strikingly
distinct genomic organization. The entire open reading frame
of each Ugt5 gene (except Ugt5g2) is encoded by a single large
exon (Huang and Wu, 2010). In addition, they are the most
abundant Ugt genes in teleosts. Among the 17 zebrafish
UGT5s, we found that UGT5A4, UGT5A5, UGT5B2, UGT5C3,
and UGT5E1 have glucuronidation activities toward
at least one of the ten substrates (Fig. 4). None of the
UGT5s is capable of glucuronidating BPA and bilirubin
(Table 2).

Glucuronidation Activity of the Zebrafish UGT Superfamily
69
Fig. 3. High glucuronidation activities of zebrafish UGT1 and UGT2 enzymes. Shown are the HPLC chromatograms of strong glucuronidation activities
of UGT1A1 toward 1-naphthol (A), BPA (B), MPA (C), and diclofenac (D); UGT1A3 toward MPA (E); UGT1A4 toward 4-MU (F); UGT1A7 toward 4-MU
(G); UGT1B1 toward 4-NP (H) and E2 (I); UGT1B7 toward bilirubin (J); UGT2B3 toward 1-naphthol (K); and UGT2B6 toward 4-MU (L) with the mock
control in the right half of each panel. The glucuronide peak for each substrate is indicated by an arrow. The chemical structure for each substrate is
shown in the respective panel and the glucuronidation site is indicated in red color. Bilirubin-MG and bilirubin-DG refer to the mono- and di-
glucuronides of bilirubin, respectively. The mV (millivolt) or mAU (milli Absorbance Unit) represent the measurement unit used by the fluorescence or
UV detector, respectively.

70
Wang et al.
Fig. 4. UGT5 glucuronidation activities toward aglycone substrates with the donor UDPGA. Shown are the HPLC chromatograms of glucuronidation
activities of UGT5A toward t-OP (A); UGT5A5 toward 4-MU (B), 4-NP (C), 1-naphthol (D), t-OP (E), MPA (F), E2 (G), testosterone (H), and diclofenac (I);
UGT5B2 toward 4-MU (J), 4-NP (K), 1-naphthol (L), t-OP (M), and E2 (N); UGT5C3 toward 4-MU (O) and t-OP (P); UGT5E1 toward 4-MU (Q), 4-NP (R),
t-OP (S), E2 (T), and testosterone (U). The black or gray lines represent glucuronidation assays of recombination proteins or mock controls, respectively.
The chemical structures for t-OP, E2, and testosterone and their glucuronidation sites are shown in the respective panel. The glucuronide peak for each
substrate is indicated by an arrow. Some assays shared the same controls. The mV (millivolt) or mAU (milli Absorbance Unit) represent the
measurement unit used by the fluorescence or UV detector, respectively.

Glucuronidation Activity of the Zebrafish UGT Superfamily
71
We found that UGT5A4 has very low glucuronidation us from observing the substrate inhibition. However, the data
activity toward t-OP (Fig. 4A). We detected a small peak of fitted very well to the Michaelis–Menten equation (eq. 1)
the conjugated substrate t-OP b-D-glucuronide for the (Table 3; R2 5 0.9992) (Fig. 5N). The corresponding
recombinant UGT5A4 but not the mock in the HPLC glucuronidation kinetic parameters were calculated from the
chromatograms (see the inset in Fig. 4A). In addition, this appropriate models and listed in Table 3.
small peak disappeared when the donor UDPGA was opted
UGT1 Family. UGT1A1 displays comparably high turn-
out of the glucuronidation assay (data not shown). Thus, over rates (Vmax) toward 4-NP, 1-naphthol, MPA, and diclofenac
UGT5A4 has low but definitive glucuronidation activity (Table 3). However, the apparent Km values for 1-naphthol
toward t-OP.
and MPA are about one order of magnitude lower than those
UGT5A5, the most ancient (Huang and Wu, 2010) and for 4-NP and diclofenac. Thus, UGT1A1 has higher affinity
ubiquitously expressed member of the UGT5A subfamily (Fig. and glucuronidation efficiency (CLint) toward 1-naphthol
1C), is capable of conjugating phenols, steroids, and carboxylic and MPA (Table 3). Although UGT1A1 is the isozyme with
acids, including 4-MU (Fig. 4B), 4-NP (Fig. 4C), 1-naphthol the highest activity among all of the zebrafish UGTs toward
(Fig. 4D), t-OP (Fig. 4E), MPA (Fig. 4F), E2 (Fig. 4G), BPA (Table 2), its efficiency toward BPA is lower than that
testosterone (Fig. 4H), and diclofenac (Fig. 4I). We noted that toward 1-naphthol and MPA (Table 3).
when E2 was used as an aglycone substrate, two glucuronide
UGT1A7 conjugates 4-MU (CLint 5 6.01) more efficiently
peaks appeared (Fig. 4G). We confirmed that these two peaks than 4-NP (CLint 5 0.62). Its catalytic efficiency toward 4-NP
corresponded to E2-3G and E2-17G, respectively, by running is comparable to that of UGT1A1. For both UGT1A1 and
HPLC with commercial E2-3G and E2-17G as the standards UGT1A7 catalyzed reactions, substrate inhibition was not
(data not shown). The rate of glucuronidation at E2 (179-OH) is significant (Ki .. Km) (Table 3).
much higher than at E2 (39-OH), suggesting its preference
UGT1B1 is the most efficient (CLint or CLmax) isoform for
for the 179-OH position of steroids (Fig. 4G). As expected, phenolic aglycone glucuronidation (Table 3). This may be
UGT5A5 also glucuronidates the steroid of testosterone at the partly because of its high affinity toward phenolic com-
corresponding position (179-OH) (Fig. 4H).
pounds. Its apparent Km values for phenolic aglycones (4-MU,
For members of the UGT5B subfamily, we found that 4-NP, 1-naphthol, and MPA) are much lower than those of
UGT5B2 is capable of conjugating 4-MU (Fig. 4J), 4-NP (Fig. the other isoforms (Table 3). In conjunction with its high-
4K), 1-naphthol (Fig. 4L), t-OP (Fig. 4M), and E2 (39-OH) (Fig. level expression in multiple tissues (Fig. 1A), these data
4N). Its activities toward t-OP and E2 (39-OH) are the highest suggest an important role of UGT1B1 in in vivo detoxifi-
among all of the zebrafish UGTs (Table 2). Among members of cation of phenolic compounds. Strikingly, 4-NP glucuroni-
the UGT5C subfamily, only UGT5C3 conjugates 4-MU (Fig. dation by UGT1B1 showed significant substrate inhibition
4O) and t-OP (Fig. 4P).
and autoactivation (Table 3, Hill coefficient 5 1.75), which
Among the five members of the nonclustered Ugt5 genes, was also reported for several human UGTs (Uchaipichat
the extrahepatic UGT5E1 glucuronidates 4-MU (Fig. 4Q), et al., 2008). In contrast to its high efficiency toward
4-NP (Fig. 4R), and t-OP (Fig. 4S) at relatively low rates; phenolic aglycones, UGT1B1 conjugates the steroid hor-
however, its activities toward the 179-OH position of E2 (Fig. mone E2 at very low efficiency, with a CLint value of 0.094,
4T) and testosterone (Fig. 4U) are the highest among all of the which is about two orders of magnitude lower than that for
zebrafish UGTs, suggesting its important role in metabolizing phenolic aglycones (Table 3). The substrate inhibition for
these two sex hormones.
UGT1B1 is also not significant as suggested by the high
Kinetic Analyses. To further investigate the enzymatic ratio of Ki/Km.
properties of the zebrafish UGTs, kinetic analyses were
UGT5 Family. Although UGT5A5 displays the broadest
carried out for six UGT isoforms with high glucuronidation substrate spectrum toward the ten aglycones (Fig. 4, B–I;
activity. These isoforms include three members each of UGT1 Table 2), its glucuronidation efficiency toward 1-naphthol and
and UGT5 families (Table 3). We performed a series of t-OP is very low (1-naphthol CLmax 5 0.0017 and t-OP CLint 5
glucuronidation assays with various concentrations of agly- 0.0084) (Table 3). Its glucuronidation toward 1-naphthol also
cone substrates and fitted the experimental data to the ap- showed both substrate inhibition and sigmoidal kinetics, with
propriate equations (Fig. 5).
a Hill value of 1.11. Furthermore, the Ki value of UGT5A5
At high concentrations of aglycones, substrate inhibition toward 1-naphthol is much lower than the Ks value, suggest-
was observed for all six UGT isoforms with the exception of ing a high substrate inhibition.
t-OP for UGT5A5 (Fig. 5). This may be a phenomenon of the
UGT5B2 conjugates t-OP much more efficiently and at
mammalian expression system. A mechanism based on the a higher turnover rate than UGT5A5, with values of clearance
nonproductive accumulation of a dead-end ternary complex and Vmax two orders of magnitude higher (Table 3). It also
(enzyme-UDP-substrate) has been proposed to explain the shows a strong substrate inhibition (Ki , Km).
substrate inhibition phenomena of glucuronidation reactions
UGT5E1 displays not only the highest turnover rate but
(Luukkanen et al., 2005). Our substrate inhibition data fitted also the highest affinity for 179-OH of E2 and testosterone,
well to the substrate inhibition equation (eq. 2) (Table 3; R2 5 with Km values of 13.4 and 30.6 mM, respectively (Table 3).
0.9631 ∼0.9992) (Fig. 5, A–H, J–L, and O–Q). The conjugation This suggests that UGT5E1 plays an important role in the
of 4-NP by UGT1B1 and 1-naphthol by UGT5A5 showed both homeostasis of these two steroid hormones in vivo. Finally,
substrate inhibition and sigmoidal kinetics (Fig. 5, I and M), the substrate inhibition for these reactions is very weak (Ki/
and the data fitted well to a modified Hill equation (eq. 3)
K
m . 100).
(Table 3; R2 5 0.9959 and 0.9987, respectively). In the case of
In summary, our kinetic analyses demonstrate that diverse
t-OP for UGT5A5, the relatively low catalytic capacity of UGT enzymes have distinct affinity and glucuronidation ef-
UGT5A5 (Tables 2 and 3) and low solubility of t-OP prevented ficiency toward various aglycone substrates.

72
Wang et al.
TABLE 3
Kinetic analyses of six zebrafish UGT enzymes with nine aglycone substrates
Kinetic parameters are shown as mean 6 S.D. of parameter fit according to appropriate models.
UGT
Substrate
Vmax
Km(Ks)
Ki
Vi
n
CLint(CLmax
)
R2
pmol/min/mg
mM
pmol/min/mg
ml/min/mg
1A1 4-NPa
489.8 6 71.6
460.0 6 93.6
26.4 6 2.3
332.1 6 14.3
170.6 6 6.8
855.2 6 74.1
471.2 6 46.0
250.0 6 20.3
659.6 6 43.4
88.3 6 5.6
225.7 6 22.5
8.82 6 0.77
12.7 6 5.2
2.80 6 0.057 334.0 6 16.1
429.7 6 76.2
89.8 6 1.5
2428 6 542.9
159.8 6 58.5
106.7 6 15.8
334.3 6 26.0
901.8 6 64.0
142.4 6 22.2
764.5 6 116.8
17.7 6 2.46
281.9 6 28.4
47.6 6 4.5
6784 6 1852
1845 6 1229
510.9 6 102.9
6573 6 1162
11,051 6 1532
2835 6 1056
2733 6 509.4
94.8 6 15.3
0.20
2.88
0.9727
0.9779
0.9832
0.9985
0.9992
0.9941
0.9958
0.9833
0.9959
0.9910
0.9631
0.9957
0.9987
0.9992
0.9986
0.9988
0.9936
1-Naphthola
BPAa
0.25
MPAa
0.99
Diclofenaca
0.19
1A7 4-MUa
6.01
4-NPa
1B1 4-MUa
4-NPb
0.62
14.1
1931 6 182.8 128.3 6 10.3 1.75 6 0.16
243.0 6 40.4
1.18
1-Naphthola
1.86
MPAa
60.9 6 12.5
93.6 6 14.4
5316 6 2786
594.8 6 135.7
3.71
a,c
E2
1146 6 352.1
0.094
0.0017
0.0084
0.50
5A5 1-Naphtholb
t-OPd
1655 6 612.7
0.78 6 0.11 1.11 6 0.08
5B2 t-OPa
859.3 6 179.4
13.4 6 0.62
30.6 6 2.7
389.4 6 97.0
2579 6 647.7
3422 6 608.9
a,c
5E1 E2
6.70
15.6
Testosteronea 477.1 6 13.7
aSubstrate inhibition model.
bSubstrate inhibition and Hill sigmoidal kinetics model.
cThe kinetic constants of UGT1B1 and UGT5E1 toward estradiol (E2) were tested on the positions 39-OH and 179-OH of estradiol, respectively.
dMichaelis–Menten model.
environmental toxicants and agents (Carvan et al., 2000; James,
2011). It is vital to systematically characterize the expression
profiles as well as the catalytic functions of the complete
zebrafish Ugt repertoire. Expression profiling analyses revealed
that members of the zebrafish Ugt superfamily are expressed in
a tissue-specific manner. For example, Ugt1a2, Ugt1a5, Ugt2a4,
and Ugt5g2 appear to be specifically expressed in the intestine,
gill, liver, and eye, respectively. In addition, several Ugt genes
(Ugt1b5, Ugt1a7, Ugt2b3, Ugt5a3, and Ugt5b3) are expressed
only in two to three organs, suggesting that they play important
roles in organ-specific toxicity.
Interestingly, multiple zebrafish Ugt genes, especially
members of the Ugt1a, Ugt2a, Ugt5b, Ugt5e, Ugt5f, and
Ugt5g subfamilies, are expressed in the gill, which is the
respiratory organ that interacts directly with the xenobiotics
in the aquatic environment. These Ugt genes may be im-
portant in metabolizing large numbers of chemical agents
from the aquatic environment. In the kidney, most members
of the Ugt2 and Ugt5 families are highly expressed, whereas
most members of the Ugt1 family are expressed at low levels.
In particular, Ugt5e1 is enriched in the testis and gill, and it
has the highest glucuronidation efficiency toward hormone
steroids (E2 and testosterone; Table 3) as demonstrated by
functional analyses. The steroid glucuronides have been
reported to serve as sex pheromones capable of inducing
ovulation or attracting the opposite sex (Van den Hurk et al.,
1987). To investigate the functional significance of the tissue-
specific expression patterns of the Ugt repertoire, we system-
atically characterized the catalytic activities of the encoded
enzymes by expressing each of the 40 recombinant UGTs
and then testing them with ten aglycone substrates in a
glucuronidation assay with UDPGA as the donor. Interest-
ingly, we found that many of the recombinant zebrafish UGTs
are N-glycosylated in HEK293T cells. The glycosylation of
zebrafish UGTs might affect their enzymatic activity or
Discussion
The genomic organization of the mammalian Pcdh and
Ugt1 gene clusters is strikingly similar in that both contain
a tandem array of highly similar variable exons followed by
a common set of downstream constant exons (Zhang et al.,
2004). In contrast to the cell-specific expression of Pcdh
genes in the brain, members of the mouse Ugt1 cluster
display tissue-specific expression patterns in a wide variety
of tissues, especially in the digestive and respiratory systems
(Zhang et al., 2004). In addition, the variable and constant
genomic organizations of both the Pcdh and Ugt1 clusters
are conserved in zebrafish (Wu, 2005; Li and Wu, 2007). The
zebrafish has become an important vertebrate model or-
ganism for developmental biology, physiology, and pathology
studies (Howe et al., 2013). Recently, there has been great
interest in developing the zebrafish as a vertebrate model
organism for drug discovery as well as for environmental
toxicology (Hill et al., 2005; Peterson and Macrae, 2012). The
full complement of the phase I drug-metabolizing cyto-
chrome P450 genes has been identified in zebrafish (Gold-
stone et al., 2010). We recently identified the complete
repertoire of the zebrafish phase II drug-metabolizing Ugt
gene superfamily and found that they are divided into three
families, Ugt1, Ugt2, and Ugt5 (Huang and Wu, 2010). These
zebrafish Ugt genes do not display orthologous relationships
to any of the mammalian or avian Ugts, making it difficult to
predict their substrate specificities. Here, we report the
tissue-specific expression patterns of the complete zebrafish
Ugt repertoire. We also systematically analyzed their sub-
strate specificity and glucuronidation activity toward ten
aglycone substrates and found that most zebrafish UGTs are
true glucuronidating enzymes with UDPGA as the donor.
To develop the zebrafish as a model organism for toxicology
screening in the drug discovery pipeline, elucidating the
mechanisms by which drug-metabolizing enzymes conjugate
aglycone substrates is an important prerequisite (Peterson and substrate specificity.
Macrae, 2012). Moreover, large efforts have been undertaken
We found that zebrafish UGT1A1, UGT1A3, and UGT1B1
to develop the zebrafish as a sentinel model organism for have high glucuronidation activity toward six important

Glucuronidation Activity of the Zebrafish UGT Superfamily
73
Fig. 5. Kinetic analyses of recombinant zebrafish UGT enzymes toward the aglycone substrates with high glucuronidation activities. Shown are the
glucuronidation kinetic plots (main plots) created by using nonlinear fitting of experimental data of UGT1A1 toward 4-NP (A), 1-naphthol (B), BPA (C),
MPA (D), and diclofenac (E); UGT1A7 toward 4-MU (F) and 4-NP (G); UGT1B1 toward 4-MU (H), 4-NP (I), 1-naphthol (J), MPA (K), and E2 (L); UGT5A5
toward 1-naphthol (M) and t-OP (N); UGT5B2 toward t-OP (O); UGT5E1 toward E2 (P) and testosterone (Q). Data in A–H, J–L, and O–Q were fitted to
the substrate inhibition equation (eq. 2); data in the I and M were fitted to the modified Hill equation (eq. 3); and data in N was fitted to the Michaelis–
Menten equation (eq. 1). The corresponding Eadie–Hofstee plot is shown in the inset of each panel.

74
Wang et al.
phenolic aglycone substrates: 4-MU, 4-NP, 1-naphthol, BPA, phase II glucuronosyltransferases. These results provide in-
t-OP, and MPA. 4-MU, a coumarin derivative, has been used sight into the mechanisms by which zebrafish defend them-
safely for many years as a cholagogue and is a promising selves against a vast number of endobiotics and xenobiotics via
therapeutic agent targeting invasion and metastasis of glucuronidation conjugations and lay the foundation for de-
many kinds of tumors via inhibition of hyaluronan synthesis veloping the zebrafish as a model vertebrate in toxicological,
(Kakizaki et al., 2004). 4-NP is a common metabolite from developmental, and pathologic studies.
a variety of compounds and a biomarker of organophosphate
insecticide exposure (Kaivosaari et al., 2011). 1-Naphthol,
Acknowledgments
The authors thank Dr. Ida Owens for advice on investigating UGT
post-translational modification and members of the Wu laboratory
for discussion.
a prime substrate for human UGT1A6, is a precursor to a
variety of insecticides and a prominent component of Molisch’s
reagent. BPA is a monomer for manufacturing polycarbonate
plastics and epoxy resins and can be released during auto-
claving. It has hormone-like activities that disrupt the endocrine
system and increase the risk of obesity (Howdeshell et al., 1999).
Among environmental alkylphenols, t-OP is the most estrogenic
(Routledge and Sumpter, 1997). MPA is the active metabolite of
mycophenolate mofetil, a prodrug used to prevent tissue
rejection after organ transplantation (Wu et al., 2011).
Steroid hormones are mainly metabolized via glucuronida-
tion (James, 2011). In human, steroid hormones are pre-
dominantly glucuronidated by members of the UGT2 family
(Sten et al., 2009). In zebrafish, however, they are mainly
conjugated by members of the UGT5 family (Tables 2 and 3).
UGT5E1 is the principal enzyme responsible for steroid
conjugations, with high activity and affinity toward testos-
terone and E2. The Ugt5e1 gene is expressed in the ex-
trahepatic tissues of the gill, heart, kidney, testis, and eye
(Fig. 1C).
Bilirubin, a major breakdown product of heme catabolism,
is excreted predominantly as glucuronide conjugates in the
bile. In zebrafish, we found that it is conjugated by several
UGT1 enzymes at very low rates. Among them, UGT1B7 has
the highest glucuronidation activity toward bilirubin, mainly
forming the monoglucuronides (Fig. 3J). In addition, we found
that UGT1B7 is specific toward bilirubin, with no detectable
glucuronidation activity toward any of the other nine aglycone
substrates. Moreover, the Ugt1b7 gene is highly expressed in
the liver, which is responsible for bilirubin catabolism.
Diclofenac, a nonsteroidal anti-inflammatory drug widely
used for reducing inflammation and relieving pain, is
primarily eliminated as acyl-glucuronide in mammals (King
et al., 2001). Its acyl-glucuronide forms covalent drug-protein
adducts, possibly contributing to its immunogenicity and
toxicity (King et al., 2001). In zebrafish, UGT1A1 is the only
isozyme that has a high rate of diclofenac glucuronidation
(Table 2). However, kinetic analyses revealed a very high Km
value of UGT1A1 for diclofenac, indicating its low affinity.
In summary, members of the zebrafish Ugt superfamily are
widely expressed in various tissues. In addition, some mem-
bers of the zebrafish UGT5 family have glucuronidation ac-
tivities toward multiple aglycones. Specifically, the zebrafish
UGT5A5 and UGT5E1 predominantly glucuronidate the hor-
mone steroids of E2 and testosterone, and the zebrafish
UGT5B2 glucuronidates t-OP at the highest rate and several
phenols at a lower rate. Thus, UGT5 is a novel UGT family
using UDPGA as the donor. Moreover, some members of the
zebrafish UGT1 family conjugate a wide range of compounds,
including simple phenols, bilirubin, carboxylic acids, coumar-
ins, mycophenolic acid, and bisphenol A. Importantly, each of
these lipophilic compounds is conjugated by at least one
member of the UGT superfamily, confirming that these
diverse 40 UGTs are the entire complement of the zebrafish
Authorship Contributions
Participated in research design: Wang, Huang, Wu.
Conducted experiments: Wang, Huang.
Performed data analysis: Wang, Huang, Wu.
Wrote or contributed to the writing of the manuscript: Wang,
Huang, Wu.
References
Carvan MJ, 3rd, Dalton TP, Stuart GW, and Nebert DW (2000) Transgenic zebrafish
as sentinels for aquatic pollution. Ann N Y Acad Sci 919:133–147.
Clarke DJ, Burchell B, and George SG (1992) Functional and immunochemical
comparison of hepatic UDP-glucuronosyltransferases in a piscine and a mamma-
lian species. Comp Biochem Physiol B 102:425–432.
Dutton GJ (1980) Glucuronidation of Drugs and Other Compounds, CRC Press, Boca
Raton, FL.
Eadie GS (1942) The inhibition of cholinesterase by physostigmine and prostigmine.
J Biol Chem 146:85–93.
Emi Y, Ikushiro S, and Iyanagi T (1995) Drug-responsive and tissue-specific alter-
native expression of multiple first exons in rat UDP-glucuronosyltransferase
family 1 (UGT1) gene complex. J Biochem 117:392–399.
Goldstone JV, McArthur AG, Kubota A, Zanette J, Parente T, Jönsson ME, Nelson
DR, and Stegeman JJ (2010) Identification and developmental expression of the
full complement of Cytochrome P450 genes in Zebrafish. BMC Genomics 11:643.
Harada H, Endo T, Momose Y, and Kusama H (2009) A liquid chromatography/
tandem mass spectrometry method for detecting UGT-mediated bioactivation of
drugs as their N-acetylcysteine adducts in human liver microsomes. Rapid Com-
mun Mass Spectrom 23:564–570.
Hill AJ, Teraoka H, Heideman W, and Peterson RE (2005) Zebrafish as a model
vertebrate for investigating chemical toxicity. Toxicol Sci 86:6–19.
Hill AV (1910) The possible effects of the aggregation of the molecules of haemoglobin
on its dissociation curves. J Physiol 40 (Suppl):4–7.
Hofstee BH (1959) Non-inverted versus inverted plots in enzyme kinetics. Nature
184:1296–1298.
Houston JB and Kenworthy KE (2000) In vitro-in vivo scaling of CYP kinetic data not
consistent with the classical Michaelis-Menten model. Drug Metab Dispos 28:
246–254.
Howdeshell KL, Hotchkiss AK, Thayer KA, Vandenbergh JG, and vom Saal FS (1999)
Exposure to bisphenol A advances puberty. Nature 401:763–764.
Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins JE,
Humphray S, McLaren K, and Matthews L et al. (2013) The zebrafish reference
genome sequence and its relationship to the human genome. Nature 496:498–503.
Huang H and Wu Q (2010) Cloning and comparative analyses of the zebrafish Ugt
repertoire reveal its evolutionary diversity. PLoS ONE 5:e9144.
James MO (2011) Steroid catabolism in marine and freshwater fish. J Steroid Bio-
chem Mol Biol 127:167–175.
Kaivosaari S, Finel M, and Koskinen M (2011) N-glucuronidation of drugs and other
xenobiotics by human and animal UDP-glucuronosyltransferases. Xenobiotica 41:
652–669.
Kakizaki I, Kojima K, Takagaki K, Endo M, Kannagi R, Ito M, Maruo Y, Sato H,
Yasuda T, and Mita S et al. (2004) A novel mechanism for the inhibition of hyal-
uronan biosynthesis by 4-methylumbelliferone. J Biol Chem 279:33281–33289.
King C, Tang W, Ngui J, Tephly T, and Braun M (2001) Characterization of rat and
human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation
of diclofenac. Toxicol Sci 61:49–53.
Li C and Wu Q (2007) Adaptive evolution of multiple-variable exons and structural
diversity of drug-metabolizing enzymes. BMC Evol Biol 7:69.
LiCata VJ and Allewell NM (1997) Is substrate inhibition a consequence of allostery
in aspartate transcarbamylase? Biophys Chem 64:225–234.
Luukkanen L, Taskinen J, Kurkela M, Kostiainen R, Hirvonen J, and Finel M
(2005) Kinetic characterization of the 1A subfamily of recombinant human
UDP-glucuronosyltransferases. Drug Metab Dispos 33:1017–1026.
Mackenzie PI, Bock KW, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, Miners
JO, Owens IS, and Nebert DW (2005) Nomenclature update for the mammalian
UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenet Genomics 15:
677–685.
Maitland ML, Grimsley C, Kuttab-Boulos H, Witonsky D, Kasza KE, Yang L, Roe BA,
and Di Rienzo A (2006) Comparative genomics analysis of human sequence vari-
ation in the UGT1A gene cluster. Pharmacogenomics J 6:52–62.
Meech R, Miners JO, Lewis BC, and Mackenzie PI (2012) The glycosidation of
xenobiotics and endogenous compounds: versatility and redundancy in the UDP
glycosyltransferase superfamily. Pharmacol Ther 134:200–218.

Glucuronidation Activity of the Zebrafish UGT Superfamily
75
Ménard V, Girard H, Harvey M, Pérusse L, and Guillemette C (2009) Analysis of
inherited genetic variations at the UGT1 locus in the French-Canadian population.
Hum Mutat 30:677–687.
Michaelis L and Menten ML (1913) Die kinetik der invertinwirkung. Biochem Z 49:
333–369.
Uchaipichat V, Galetin A, Houston JB, Mackenzie PI, Williams JA, and Miners JO
(2008) Kinetic modeling of the interactions between 4-methylumbelliferone,
1-naphthol, and zidovudine glucuronidation by UDP-glucuronosyltransferase 2B7
(UGT2B7) provides evidence for multiple substrate binding and effector sites. Mol
Pharmacol 74:1152–1162.
Michaelis L, Menten ML, Johnson KA, and Goody RS ( 2011) The original Michaelis
constant: translation of the 1913 Michaelis-Menten paper. Biochemistry 50:
8264–8269.
Owens IS, Basu NK, and Banerjee R (2005) UDP-glucuronosyltransferases: gene
structures of UGT1 and UGT2 families. Methods Enzymol 400:1–22.
Peterson RT and Macrae CA (2012) Systematic approaches to toxicology in the
zebrafish. Annu Rev Pharmacol Toxicol 52:433–453.
Ritter JK, Crawford JM, and Owens IS (1991) Cloning of two human liver bilirubin
UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells. J Biol Chem
266:1043–1047.
Routledge EJ and Sumpter JP (1997) Structural features of alkylphenolic chemicals
associated with estrogenic activity. J Biol Chem 272:3280–3288.
Saeki M, Saito Y, Jinno H, Sai K, Ozawa S, Kurose K, Kaniwa N, Komamura K,
Kotake T, and Morishita H et al. (2006) Haplotype structures of the UGT1A gene
complex in a Japanese population. Pharmacogenomics J 6:63–75.
Stegeman JJ, Goldstone JV, and Hahn ME (2010) Perspectives on zebrafish as
a model in environmental toxicology, in Zebrafish (Perry SF, Ekker M, Farrell AP,
and Brauner CJ eds), pp 367–439, Academic Press, Amsterdam.
Sten T, Bichlmaier I, Kuuranne T, Leinonen A, Yli-Kauhaluoma J, and Finel M
(2009) UDP-glucuronosyltransferases (UGTs) 2B7 and UGT2B17 display converse
specificity in testosterone and epitestosterone glucuronidation, whereas UGT2A1
conjugates both androgens similarly. Drug Metab Dispos 37:417–423.
Thomas SS, Li SS, Lampe JW, Potter JD, and Bigler J (2006) Genetic variability,
haplotypes, and htSNPs for exons 1 at the human UGT1A locus. Hum Mutat 27:
717.
Uchaipichat V, Mackenzie PI, Guo XH, Gardner-Stephen D, Galetin A, Houston JB,
and Miners JO (2004) Human UDP-glucuronosyltransferases: isoform selectivity
and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of
organic solvents, and inhibition by diclofenac and probenecid. Drug Metab Dispos
32:413–423.
van den Hurk R, Schoonen WGEJ, van Zoelen GA, and Lambert JGD (1987) The
biosynthesis of steroid glucuronides in the testis of the zebrafish, Brachydanio
rerio, and their pheromonal function as ovulation inducers. Gen Comp Endocrinol
68:179–188.
Wu B, Kulkarni K, Basu S, Zhang S, and Hu M (2011) First-pass metabolism via
UDP-glucuronosyltransferase: a barrier to oral bioavailability of phenolics.
J Pharm Sci 100:3655–3681.
Wu Q (2005) Comparative genomics and diversifying selection of the clustered ver-
tebrate protocadherin genes. Genetics 169:2179–2188.
Yang J, Cai L, Huang H, Liu B, and Wu Q (2012) Genetic variations and haplotype
diversity of the UGT1 gene cluster in the Chinese population. PLoS ONE 7:e33988.
Zhang H, Patana AS, Mackenzie PI, Ikushiro S, Goldman A, and Finel M (2012)
Human UDP-glucuronosyltransferase expression in insect cells: ratio of active to
inactive recombinant proteins and the effects of a C-terminal his-tag on glucu-
ronidation kinetics. Drug Metab Dispos 40:1935–1944.
Zhang T, Haws P, and Wu Q (2004) Multiple variable first exons: a mechanism for
cell- and tissue-specific gene regulation. Genome Res 14:79–89.
Address correspondence to: Qiang Wu, Shanghai Jiao Tong University, 800
Dongchuan Rd, Minhang, Shanghai 200240, China. E-mail: qwu123@gmail.
com
Tokarz J, Möller G, de Angelis MH, and Adamski J (2013) Zebrafish and steroids:
what do we know and what do we need to know? J Steroid Biochem Mol Biol 137:
165–173.