BmUGT1 was expressed in insect cells using the baculovirus expression system, and a range of compounds belonging to diverse chemical groups were assessed as potential substrates for the
Trang 1Characterization of a novel silkworm ( Bombyx mori ) phenol
UDP-glucosyltransferase
Teresa Luque1, Kazuhiro Okano2and David R O’Reilly1
1
Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK;
2
Laboratory of Molecular Entomology and Baculovirology, Riken, Wako, Japan
Sugar conjugation is a major pathway for the inactivation
and excretion of both endogenous and exogenous
com-pounds We report here the molecular cloning and
func-tional characterization of a phenol UDP-glucosyltransferase
(UGT) from the silkworm, Bombyx mori, which was named
BmUGT1 The complete cDNA clone is 1.6 kb, and the
gene is expressed in several tissues of fifth-instar larvae,
including fat body, midgut, integument, testis, silk gland and
haemocytes The predicted protein comprises 520 amino
acids and has 30% overall amino-acid identity with other
members of the UGT family The most conserved region of
the protein is the C-terminal half, which has been implicated
in binding the UDP-sugar BmUGT1 was expressed in insect
cells using the baculovirus expression system, and a range of compounds belonging to diverse chemical groups were assessed as potential substrates for the enzyme The expressed enzyme had a wide substrate specificity, showing activity with flavonoids, coumarins, terpenoids and simple phenols These results support a role for the enzyme in detoxication processes, such as minimizing the harmful effects of ingested plant allelochemicals This work repre-sents the first instance where an insect ugt gene has been associated with a specific enzyme activity
Keywords: Bombyx mori; detoxication; insect; UDP-glycosyltransferase
The UDP-glycosyltransferases (UGTs) are a superfamily of
enzymes that play a central role in the detoxication and
elimination of a wide range of endogenous and exogenous
compounds Members of this superfamily are present in
animals, plants, bacteria and viruses, suggesting an ancient
origin (reviewed in [1–3]) These enzymes catalyze the
addition of the glycosyl group from a nucleotide sugar to a
variety of small hydrophobic molecules (aglycones),
result-ing in more hydrophilic compounds that are efficiently
excreted The UDP-sugar may be UDP-glucuronic acid,
UDP-galactose, UDP-glucose, or UDP-xylose
The best-characterized UGTs are the mammalian
UDP-glucuronosyltransferases, which use UDP-glucuronic acid
as sugar donor These enzymes catalyze the glucuronidation
of numerous endogenous substrates, such as bile acids,
bilirubin, steroids, thyroxine and fat-soluble vitamins, and a
great number of exogenous compounds, including several
drugs [3] These conjugation reactions are highly important
physiologically, as reflected by the association of several
serious pathologies with altered UGT function [1,4,5]
UDP-glucuronosyltransferases are located in the lumen
of the endoplasmic reticulum and are membrane-bound
The putative transmembrane domain is located near the
C-terminus of the protein and only a small portion of the protein is found in the cytosol [3]
UGTs related to those found in vertebrates have also been found in insects and are likely to play equally important roles However, only limited information is available on UDP-glycosyltransferase activity in insects Insect UGT enzymes typically use UDP-glucose rather than UDP-glucuronic acid as sugar donor [6–8] Similarly to the vertebrates, glucosidation in insects is believed to involve both endogenous and exogenous substrates Thus, the UGTs play an important role in detoxication of plant allelochemicals encountered by many insects in their diet [7] Similarly, UGT-catalyzed biotransformation of xenobiotics has been implicated in some cases of insecticide resistance [9] In addition, glucosidation in insects is known to be involved in cuticle formation, pigmentation and olfaction [10–12] In Drosophila, glucose conjugation of the endoge-nous compound, xanthurenic acid, and several exogeendoge-nous compounds, including 4-nitrophenol, 1-naphthol and 2-naphthol has been reported [8,13] In other insect species, such as Manduca sexta, the presence of multiple enzyme forms has also been suggested [7] Recently, the expression
of some Drosophila ugt genes in the antennae has been reported [12] Similarly, an expressed sequence tag (EST) corresponding to a UGT homologue has been described from a male M sexta antennae cDNA library [14] In all of these cases, the specificity of the enzyme encoded by the gene identified is unknown To date, hardly any information
is available on glucosidation in the silkworm, Bombyx mori This is an economically important species, in particular because of its propagation on a large scale and utilization for silk production
Here, we describe the isolation and characterization of a novel B mori UDP-glucosyltransferase gene, Bmugt1, and analysis of the activity of the enzyme towards a range of
Correspondence to D R O’Reilly, Department of Biology, Imperial
College of Science, Technology and Medicine, Imperial College Road,
London SW7 2AZ, UK Fax: + 44 20 75842056,
Tel.: + 44 20 75945376, E-mail: dor@ic.ac.uk
Abbreviations: UGT, UDP-glycosyltransferase; EST, expressed
sequence tag; EGT, ecdysteroid UDP-glucosyltransferase.
Enzyme: UDP-glycosyltransferase (EC 2.4.1.-).
(Received 10 September 2001, revised 28 November 2001, accepted 29
November 2001)
Trang 2structurally different compounds We report that the
enzyme can conjugate a wide range of substrates including
flavonoids, coumarins and other phenolic compounds This
is the first time a specific activity has been ascribed to any
insect ugt gene
M A T E R I A L S A N D M E T H O D S
Identification and sequence analysis ofBmugt1
The B mori ugt1 gene was first identified in the course of an
EST sequencing project A wing disc cDNA library derived
from fifth instar B mori C108 larvae was kindly provided
by Dr Kawasaki (University of Utsunomiya, Japan) A
total of 1000 clones were selected at random and sequenced
from the 5¢ end by automatic sequencing (ABI377XL DNA
sequencer; the EST database based on these sequences is
available on the internet at http://www.ab.a.u-tokyo.ac.jp/
silkbase/) Homology searches revealed that the EST
sequence wdS20142 showed low but significant similarity
to baculovirus ecdysteroid UGTs [15] This clone was
sequenced in its entirety using a Li-Cor model 4000 DNA
sequencer andBASE IMAGIRsoftware The clone was found
to include the complete cDNA The nucleotide sequence of
the Bmugt1 gene has been deposited in the GenBank
database under the accession number AF324465
Analysis of gene expression
B mori (Shuko· Ryuhaku) eggs were obtained from
Katakura Kogyo (Matsumoto, Japan), and the larvae were
reared on an artificial diet as described previously [16]
Tissues were dissected from 5-day-old fifth-instar larvae,
washed twice in NaCl/Pi, snap-frozen in liquid nitrogen, and
stored at)80 °C Haemocytes were isolated from
haemo-lymph by centrifugation at 750 g for 5 min at 4°C and
washed twice in NaCl/Pi before freezing Total cellular
RNA from different tissues (fat body, midgut, integument,
testis, silk gland and haemocytes) was isolated by the
guanidinium thiocyanate method [17] Integument samples
may have contained small amounts of muscle and tracheal
tissue also Bmugt1 expression was studied by RT-PCR
using the internal oligonucleotides 5¢-GATCGCCTTGT
AATTCTG-3¢ (position 798–781 from the ATG start codon)
for cDNA synthesis and 5¢-CCGTGATTGTTGAGTG
GATG-3¢ and 5¢-AAGCAACTCCAGTAGACACG-3¢
(position 386–405 and 769–750, respectively, from the
ATG start codon) for PCR amplification The PCR
conditions were an initial denaturation step of 1 min 30 s
at 94°C and then 35 cycles of 45 s at 94 °C, 45 s at 55 °C,
and 45 s at 72°C
Construction and characterization of a recombinant
baculovirus expressingBmugt1
The Bmugt1 cDNA was cloned into the single SmaI site of
the pSynXIV VI+X3 transfer vector [18] The presence of
the correct insert was confirmed by sequence analysis This
plasmid DNA was cotransfected with Bsu36I-digested
vEGTSyngal+ viral DNA [19] into Spodoptera frugiperda
SF21 insect cells [20] by calcium phosphate coprecipitation
[21] Recombinant viruses were isolated from the
transfec-tion supernatant by plaque purificatransfec-tion Occlusion-positive
plaques, representing recombinant viruses, were picked and plaque purified Single isolated recombinant viruses were amplified to obtain high-titre virus stocks Virus titres were determined by plaque assays [22] Recombinant viral DNA was purified and characterized by restriction mapping following standard procedures [22] The recombinant virus, vSynBmUGT1, containing the Bmugt1 gene, was chosen for expression analyses
Expression of recombinant protein Expression of recombinant protein was characterized by metabolic labelling of infected cells at various times postinfection (p.i.) [22] Cells infected with vSynBmUGT1
or parental viral DNA or mock-infected cells were treated in parallel Briefly, SF21 cells were infected at a multiplicity of infection of 20 plaque-forming units per cell following standard procedures Two hours before the appropriate time point, the medium was removed and replaced by methionine-deficient medium After 1 h, the medium was removed and replaced with methionine-deficient medium containing 25 lCi trans-35S-label (1175 CiÆmmol)1; ICN Biomedicals, Inc.) and incubated at room temperature for
1 h At the time point, cells were rinsed three times with NaCl/Pi, pH 6.2 Cells were lysed by incubation on ice for
30 min in 50 lL cell lysis buffer (1% Nonidet P40, 150 mM NaCl, 50 mM Tris/HCl, pH 8), and stored at )80 °C Protease inhibitors were added to the cell lysis buffer at the following concentrations: pepstatin, 1 lgÆmL)1; leupeptin,
1 lgÆmL)1; aprotinin, 1 lgÆmL)1; phenylmethanesulfonyl fluoride, 100 lgÆmL)1; E64, 0.35 lgÆmL)1 Selected 48 h p.i samples were exposed to 5 lgÆmL)1tunicamycin for 12 h before harvesting (i.e from 36 to 48 h p.i) and then processed in parallel with the rest of the samples The protein samples, each representing 2· 105 cells, were separated by SDS/PAGE (10% gel) [23] The gels were then stained with Coomassie blue, dried, and exposed to X-ray film
Activity assays Trichoplusia ni TN368 [24] cells were used for assay of BmUGT1 activity, because SF21 cells were found to express low levels of endogenous UGT activity towards several of the substrates tested (data not shown) Cells were infected with vSynBmUGT1 or parental virus, vEGTSyngal+, at a multiplicity of infection of 20 and incubated at 27°C After
48 h, the cells and overlying medium were harvested and cells were lysed by several strokes of a Dounce homogenizer Mock-infected cell cultures were treated in parallel Total protein concentration was determined by the Bradford method [25] The standard incubation mixture included the following: cell lysate containing 0.5 mg total protein; 10 mM MgCl2; 100 mMTris/malate, pH 7.4; 5 mM D-gluconic acid lactone; 0.1 mM substrate (purchased from Sigma); 0.25 lCi UDP-[3H]glucose (15.3 CiÆmmol)1; Sigma) and
50 lMUDP-glucose The final reaction volume was 100 lL Reaction mixtures were incubated for 1 h at 37°C and then stopped by the addition of 2 vol ethanol The reaction mixture was evaporated, and the products were resuspended
in 60% ethanol and separated by TLC on silica-gel plates (Merck) as described [26] The silica-gel plates were exposed
to 3H-sensitive phosphoimager screens and read with a
Trang 3Fujifilm BAS-1500 phosphoimager The amount of
radio-labelled sugar conjugated was quantified, and UGT activity
expressed as nmol of glucose conjugated per h per mg of
total protein
R E S U L T S
Isolation and analysis of theBmugt1 gene
In the course of an EST sequencing project, a cDNA clone
from a B mori larval cDNA library was identified with
homology to UGT genes Sequence analysis of the cDNA
demonstrated that it included the complete BmUGT1
coding sequence plus 64 nucleotides of upstream and 54
nucleotides of downstream untranslated sequence A
con-sensus polyadenylation signal (AATAAA) was identified 30
nucleotides downstream of the TAA stop codon The
cDNA clone included a single ORF that could encode a
protein of 520 amino acids The deduced amino-acid
sequence showed homology to a range of UGTs and
included the UGT signature:
[FVA]-[LIVMF]-[TS]-
[HQ]-[SGAC]-G-x(2)-[STG]-x(2)-[DE]-x(6)-P-[LIVMFA]-[LIVMFA]-x(2)-P-[LMVFIQ]-x(2)-[DE]-Q (all amino acids
that can occur at a given position are listed inside square
brackets; reviewed in [2]) The presence of this sequence
strongly supports the identification of this protein as a member of the UGT superfamily Figure 1 shows a multiple alignment of BmUGT1 with several representative mem-bers of the UGT superfamily Gap pairwise comparisons demonstrated that it has about 30% amino-acid identity overall with other members of the family, e.g 31% and 38% with Drosophila melanogaster UGT35a and UGT35b [12], respectively, and 30% with the human UDP-glucur-onosyltransferase UGT1A10 [27] BmUGT1 has been designated UGT35C1 by the Nomenclature Committee for UGTs [2]
Expression of theBmugt1 gene The expression of Bmugt1 was investigated by carrying out RT-PCR analyses with 5-day-old fifth-instar larval RNA from several tissues Internal oligonucleotides were designed
to regions of Bmugt1 that are not conserved among UGTs,
in order to avoid amplification of other putative transcripts from the large UGT family A single transcriptional product
of the expected size (380 bp) was amplified from all the tissues analysed, including fat body, midgut, integument, testis, silk gland and haemocytes (Fig 2), suggesting that Bmugt1 is widely expressed in 5-day-old fifth-instar larvae
Fig 1 Alignment of UGT amino-acid sequences Black and grey indicate identical and similar amino acids, respectively Multiple sequence alignment was performed with CLUSTALW [45] and amino-acid shading with BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOXform.html).
A consensus is indicated in the region of the UGT signature sequence Transmembr dom indicates the transmembrane domain, based on the human sequences UGT35a (accession number AF116554) and UGT35b (accession number AF116555) are two UGTs from D melanogaster; AcEGT (accession number M22619) is an ecdysteroid UGT from the baculovirus Autographa californica nucleopolyhedrovirus; UGT1A10 (accession number U89508) and UGT2B7 (accession number NM001074) belong to different families of vertebrate UGTs.
Trang 4VSynBmUGT1, a recombinant baculovirus expressing
BmUGT1
The baculovirus expression system was chosen to express
BmUGT1 in insect cells The parental viral DNA used
lacked the baculovirus ecdysteroid
UDP-glucosyltransfer-ase (egt) gene, to allow the characterization of BmUGT1
activity without the interference of EGT, a closely related
enzyme [28] The expression of the recombinant protein was
analysed by metabolic labelling of infected insect cells at
different times after infection Proteins were separated by
SDS/PAGE and revealed by autoradiography (Fig 3) As
expected for a baculovirus infection, there is a dramatic
downregulation of host protein synthesis visible from 24 h
p.i onwards Expression of the recombinant protein was
evident from 24 h p.i in agreement with the fact that
expression was under the control of the very late polyhedrin
promoter BmUGT1 had a molecular mass of about
57 kDa, consistent with the predicted molecular mass of
59.5 kDa based on translation of the complete ORF,
including the putative signal sequence After treatment with
tunicamycin, a glycosylation inhibitor, BmUGT1 migrated more rapidly, with an estimated molecular mass of 55 kDa, indicating that it was N-glycosylated As expected, the recombinant vSynBmUGT1, an occlusion-positive virus, expressed polyhedrin normally (Fig 3) In contrast, no polyhedrin expression was observed with the parental virus, which is occlusion negative
Characterization of BmUGT1 enzymatic activity
In an attempt to identify the enzyme activity of BmUGT1, its activity towards different potential substrates belonging
to diverse chemical groups was analysed It has been previously reported that other members of the UGT superfamily are active over a very wide pH range; for instance, baculovirus EGT activity is detected over the pH range 4–10.5 [29] Thus, enzyme activity was assayed at
pH 7.4.D-Gluconic acid lactone was added to the reaction mixture to inhibit low levels of endogenous b-glucosidase activity in the cell culture preparations Representative assays are shown in Fig 4, and the complete set of data is presented in Table 1 BmUGT1 catalyzed the glucosidation
of a range of compounds, mostly phenolics or phenol-derived compounds The highest glucosylation rates were observed with the flavonoids naringenin and quercetin, and with p-hydroxybiphenyl and p-nitrophenol Other sub-strates included the coumarin umbelliferone, other pheno-lics and some terpenoids In contrast, there was no
Fig 2 Bmugt1 gene expression in different B mori tissues RT-PCR
amplification of total RNA with Bmugt1-specific oligonucleotides.
N, no DNA, PCR negative control; I, integument; M, midgut; F, fat
body; H, haemocyte; S, silk gland; T, testis; L, 100-bp ladder.
Fig 3 BmUGT1 recombinant protein expression SDS/PAGE of total
proteins extracted from metabolically labelled infected insect cells at
different times after infection (10, 24, 48 h p.i) M, mock-infected cells;
P, parental virus (vEGTSyngal+); BmUGT1, vSynBmUGT1; T, 48 h
p.i cells treated with tunicamycin The molecular masses of
glycosy-lated and nonglycosyglycosy-lated BmUGT1 are indicated by arrows on the
right side of the gel The position of the polyhedrin protein (polh) is
also shown The sizes of the molecular-mass markers are shown in kDa
on the left hand side of the gel.
Fig 4 BmUGT1 conjugation of some representative aglycones UDP-[ 3 H]glucose and the indicated aglycones were incubated with total proteins extracted from cells infected with vSynBmUGT1 (BmUGT1)
or vEGTSyngal+ (P) (parental virus), and the products separated by TLC Unreacted UDP-[ 3 H]glucose ( 3 H-gluc) was also run.
Trang 5detectable conjugation of the steroids ecdysone,
20-hydroxy-ecdysone or cholesterol under our conditions Specific
activities in parental virus-infected cells were less than
0.04 nmolÆh)1Æ(mg total protein))1for all substrates
D I S C U S S I O N
This study describes the identification and characterization
of BmUGT1, a phenol UGT encoded by the silkworm
B mori The hypothesis that the Bmugt1 gene encodes a UGT was prompted by its homology to baculovirus egt genes The egt gene encodes an ecdysteroid UGT that conjugates ecdysteroids with UDP-glucose or UDP-galac-tose [28,29] The ecdysteroids are involved in the regulation
of insect moulting and metamorphosis The baculoviruses are a large group of viruses that infect insects, and expression of egt enables them to regulate the development
of their host [28] Baculoviruses are assumed to have acquired egt from an insect host However, our data have demonstrated that Bmugt1 is not an egt gene There was no detectable conjugation of the ecdysteroids tested (Table 1) Nonetheless, BmUGT1 is similar to EGT and other members of the UGT family in a variety of respects Previous alignments of UGTs from diverse sources have demonstrated that the C-terminal half of the protein tends
to be more highly conserved than the N-terminal half This
is thought to reflect the fact that the protein comprises two major functional domains [30] The N-terminal half is believed to be responsible for binding the aglycone These are highly diverse, explaining the relative lack of conserva-tion in the N-terminal region In addiconserva-tion, the extreme N-terminus of the protein comprises the signal sequence for import into the endoplasmic reticulum These signal sequences are all hydrophobic in nature but do not share extensive sequence identity with each other On the other hand, the C-terminal region is proposed to bind the UDP-sugar, explaining the similarity of this region among different enzymes [3] This general pattern is observed with BmUGT1; the extreme N-terminus is hydrophobic and likely to represent a signal sequence, whereas the C-terminal half is clearly more similar to other UGTs than the remainder of the N-terminal half (Fig 1) An exception to the lack of sequence conservation in the N-terminal half of the protein is the region immediately after the putative signal sequence In the mammalian UGTs, this region has been identified as an oligomerization domain [3] Many of these proteins, including a baculovirus EGT, have been demonstrated to be present as oligomers in their native state [31] BmUGT1 is highly similar to other UGTs in this region and therefore is likely to be an oligomer in its native state As mentioned above, the mammalian UGTs are typically present in the lumen of the endoplasmic reticulum and possess a hydrophobic C-terminal transmembrane domain, followed by a basic cytoplasmic anchor sequence (Fig 1; [32]) In contrast, baculovirus EGT proteins, which are secreted, lack the C-terminal anchor motif (Fig 1) BmUGT1 possesses a C-terminal hydrophobic sequence followed by a basic motif, suggesting that it is also likely to
be anchored in the endoplasmic reticulum This is similar to the recently identified D melanogaster ugt genes [12] Our data have also shown that BmUGT1 is N-glycosylated when it is expressed in SF21 insect cells (Fig 3) Baculovirus EGT proteins and several mammalian UGTs are known to
be glycosylated, and it seems likely that native BmUGT1 is also a glycoprotein
There have been several previous reports of UGT activity
in insect-derived samples UGTs active against several plant phenolics and tyrosine have been described in M sexta
Table 1 Substrate specificity of BmUGT1 expressed in insect cells.
Conjugation activity is expressed as the mean ± (SEM) from three
independent experiments Specific activities in parental virus-infected
cells were less than 0.04 nmolÆh)1Æ(mg total protein))1 ND, Glucoside
formation was not detectable.
Aglycone
UGT activity [nmolÆh)1Æ(mg total protein))1] Flavonoids
3-Hydroxyflavone ND
Coumarins
Umbelliferone 0.28 ± 0.06
4-Hydroxycoumarin ND
Phenolic compounds
Monosubstituted phenols
o-Disubstituted phenols
Salicyl aldehyde 0.28 ± 0.14
p-Disubstituted phenols
p-Hydroxybiphenyl 3.92 ± 0.39
p-Nitrophenol 1.97 ± 0.47
p-Coumaric acid 0.80 ± 0.35
Hydroquinone 0.45 ± 0.05
p-Methoxyphenol 0.23 ± 0.04
1,2,3-Trisubstituted phenols
3-Hydoxyanthranilic acid ND
1,2,4-Trisubstituted phenols
N-Acetyldopamine ND
Protocatechuic acid ND
Terpenoids
S-(-)-b-Citronellol 0.18 ± 0.06
(+)-Isomenthol 0.13 ± 0.03
Steroids
3-Hydroxyecdysone ND
Others
Cis-7,8-epoxy-2-methyl
octadecane
ND Cis-9-tetradeceryl acetate ND
Xanthurenic acid ND
Trang 6[7,33] UGT activity against p-nitrophenol, 1-naphthol,
2-naphthol and xanthurenic acid has been reported in
D melanogaster[13,34], and activity against p-nitrophenol
has also been reported in the housefly, Musca domestica
[35] As discussed already, the insect baculovirus EGT
enzymes are UGTs specific for various ecdysteroids [36]
Ecdysteroid-glucosides have also been isolated from
M sexta, suggesting the existence of an EGT-like activity
in this insect [37,38] UGT activity has also been implicated
in other processes such as cuticle formation, pigmentation
and olfaction [10–12] With the exception of the baculovirus
EGTs, the ugt genes responsible for any of the activities
described above have not yet been identified It was thus of
particular interest to try to assign a specific activity to the
Bmugt1gene identified in this study Because of the low
conservation of the N-terminal aglycone-binding region of
the ugt genes, it is not possible to predict substrate specificity
based on sequence data We therefore elected to express the
Bmugt1 gene and assay the expressed enzyme against a
range of potential substrates, chosen on the basis of
UGT-like activities already reported or postulated in insects
The baculovirus expression system represented an
attrac-tive approach to the expression of Bmugt1 for a number of
reasons First, the system has an excellent track record for
the expression of biologically active higher eukaryotic
proteins [39] Secondly, in this case these viruses had the
added advantage of naturally infecting lepidopteran insect
cells Thus, the expressed protein would be produced in an
environment very similar to its normal environment,
maximizing the probability of obtaining active enzyme
The only caveat to the use of a baculovirus system was the
fact that they express EGT, which could cause false-positive
assay results with ecdysteroid substrates It was therefore
necessary to generate the recombinant virus expressing
Bmugt1using an egt-parent virus [19]
The substrates chosen for assay with BmUGT1
repre-sented a wide range of chemistries and potential functions
for the enzyme in vivo Of the 38 substrates tested,
conjugation activity was detected with 16 diverse
com-pounds, suggesting that BmUGT1 has a wide substrate
specificity This is a common feature of many members of
the UGT family [40,41] Under our conditions, BmUGT1
can catalyze the glucosylation of a range of phenolics and
phenol-derived compounds, including flavonoids (e.g
naringenin and quercetin), terpenoids [e.g
S-(–)-b)citronel-lol] and simple phenols (e.g p-nitrophenol and eugenol) As
noted already, the steroids cholesterol, ecdysone and
20-hydroxyecdysone did not act as substrates for the
enzyme Similarly, there was no detectable conjugation of
other endogenous compounds tested, such as the tyrosine/
dopamine-related compounds Thus, BmUGT1 is unlikely
to be involved in processes such as ecdysteroid metabolism
or cuticle formation In contrast, many of the substrates
conjugated are plant allelochemicals, suggesting a major
role for the enzyme in detoxication responses For instance,
flavonoids are abundant in fruits, vegetables, seeds and
roots [42] It is known that many plant phenolics can act as
toxins or feeding deterrents to insects and thus play an
important role in plant defence against herbivorous insects
The detoxication of ingested plant phenolics is believed to be
one of the principle functions of insect UGT enzymes [7]
Analysis of the presence of Bmugt1-specific transcripts
suggested that the enzyme is widely expressed in final-instar
B mori larvae (Fig 2) This is consistent with a role in detoxication, which is known to occur in many different organs However, it is important to note that the RT-PCR technique used is highly sensitive and not very quantitative Thus, it is not possible to draw firm conclusions about the relative levels of Bmugt1 expression in the tissues assayed
It is interesting to note that the compounds identified here
as substrates of BmUGT1 include a number of odorants, such as vanillin, eugenol, b-citronellol, isomenthol, p-hydroxybiphenyl, and guaiacol There is accumulating evidence for a role for some vertebrate UGTs in olfaction [41,43] In addition, expression of insect UGTs in the antennae has been reported [12,14] It is possible that BmUGT1 is also involved in olfaction, although we have not yet examined whether it is expressed in olfactory organs
It is likely that a large number of insect ugt genes will be identified in the near future as a result of cDNA and genome sequencing projects Sequence analysis of the Drosophila genome has already demonstrated that it contains 32 ugt genes [44] Biochemical evidence and comparisons with mammalian systems point to a range of important functions for these genes A critical step in understanding the functions of these genes will be to identify the activity of the enzyme encoded by each gene In this paper, we describe the first instance where this has been achieved, demon-strating that the Bmugt1 gene encodes a phenol UGT that is probably involved in the detoxication of plant allelochemicals
A C K N O W L E D G E M E N T S
We thank Dr L Vilaplana and Dr J Olszewski (Imperial College of Science, Technology and Medicine) for helpful comments on the manuscript, Dr T Shimada (University of Tokyo) and Dr K Mita (National Institute of Radiological Science) for the provision of the EST clone, and Dr Canoira (Universidad Politecnica de Madrid) and
Dr S Hadfield (Syngenta) for advice on the substrates assayed This work was supported by the Leverhulme Trust (F/58/AS) to D O’R., by the Special Postdoctoral Researchers Program, Riken (to K O.) and by
a Grant-in-Aid for Scientific Research (A1-12306002) from the Ministry of Education, Science and Culture of Japan (to K O.).
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