Characterization of a novel silkworm ( Bombyx mori ) phenol UDP-glucosyltransferase Teresa Luque 1 , Kazuhiro Okano 2 and David R. O’Reilly 1 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 f unc- tional characterization of a phenol UDP-glucosyltransferase (UGT) from the silkworm, Bombyx mori,whichwasnamed 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, t estis, silk gland a nd haemocytes. The pr edicted protein comprises 520 amino acids and has  30% overall amino-acid identity with other members of t he UGT family. T he most conserved r egion 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 s upport a role for t he enzyme in detoxication processes, such as minimizing the harmful effects of ingested plant alle lochemicals. This work repre- sents the first instance where an insect ugt gene has been associated w ith a specific enz yme activity. Keywords: Bombyx mori; detoxication; insect; UDP- glycosyltransferase. The UDP-glycosyltransferases ( UGTs) are a superfamily of enzymes that p lay a central role in the detoxication and elimination of a wide range of endogenous and exogenous compounds. Members o f t his s uperfamily 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 s ugar to a variety of small hydrophobic m olecules (aglycones), result- ing in m ore hydrophilic compoun ds that are e fficiently excreted. The UDP-sugar may be UDP-glucuronic a cid, UDP-galactose, UDP-glucose, or UDP-xylose. The best-characterized UGTs a re the m ammalian 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, inclu ding several drugs [3]. These conjugation r eactions are highly important physiologically, as reflected by the association of s everal serious pathologies with altered UGT function [1,4,5]. UDP-glucuronosyltransferases are located in the lumen of the endoplasmic reticulu m and are membrane-b ound. The putative transmembrane domain is l ocated near the C-terminus of the protein and only a small portion of the protein is found in the cytosol [3]. UGTs r elated to those found in vertebrates have also been found in ins ects and are likely to play e qually 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 a s 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 biotransfo rmation of x enobiotics has been implicated in some cases of insecticide resistance [9]. In addition, glu cosidation in i nsects is known to be involved in cuticle formation, pigmentation and olfaction [10–12]. In Drosophila, glucose conjugation o f t he endoge- nous compound, xanthurenic acid, and several exogenous compounds, including 4-nitrophenol, 1-naphthol and 2-naphthol has been reported [ 8,13]. I n o ther insect species, such as Manduca sexta, t he 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 m ale M. se xta antennae cDNA library [14]. I n all of these cases, the specificity o f 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 s pecies, in particular because of i ts propagation on a large scale a nd 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 S cience, Technology and Med icine, Imperial College Road, London SW7 2AZ, UK. F ax: + 44 20 7 5842056, Tel.: + 44 20 75945376, E-mail: do r@ic.ac.uk Abbreviations:UGT,UDP-glycosyltransferase;EST,expressed sequence tag; EGT, ecdysteroid UDP-glucosyltransferase. Enzyme: U DP-glycosyltransferase (EC 2.4.1 ). (Received 10 September 2001, revised 28 November 2001, accepted 29 November 200 1) Eur. J. Biochem. 269, 819–825 (2002) Ó FEBS 2002 structurally different compounds. We report that t he enzyme can conjugate a wide range o f substrates including flavonoids, coumarins a nd other phenolic compounds. T his is the first time a specific activity has been ascribed to any insect ugt gene. MATERIALS AND METHODS Identification and sequence analysis of Bmugt1 The B. mori ugt1 gene was first identified in the course of an EST s equencing 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 s elected at random and s equenced from the 5¢ end by automatic sequencing (ABI377XL DNA sequencer; the EST datab ase 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 b ut significant similarity to baculovirus ecdysteroid UGTs [15]. This clone was sequenced in its e ntirety using a L i-Cor m odel 4000 DNA sequencer and BASE IMAGIR software. The clone was found to include the complete c DNA. T he nucleotide sequence o f the Bmugt1 gene has been deposited in the GenBank database under the accession number AF324465. Analysis of gene expression B. mori (Shuko · Ryuhak u) eggs were obtained from Katakura Kogyo (Matsumoto, Japan), and the larvae were reared on an artificial diet as d escribed previously [16]. Tissues were dissected f rom 5-day-old fi fth-instar lar vae, washed twice in NaCl/P i , 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 °Cand washed twice in N aCl/P i 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 s tudied by RT-PCR using the internal oligo nucleotides 5¢-GATCGCCTTGT AATTCTG-3¢ (position 798–781 from t he ATG s tart codon) for cDNA synthesis and 5¢-CCGTGATTGTTGAGTG GATG-3¢ and 5¢-AAGCAACTCCAGTAGACACG-3¢ (position 386–405 and 769–750, respectively, from the ATG s tart codon) for PCR amplification. The P CR conditions were an initial denaturation step of 1 min 30 s at 94 °C a nd then 35 cycles of 45 s a t 94 °C, 45 s a t 5 5 °C, and 45 s at 72 °C. Construction and characterization of a recombinant baculovirus expressing Bmugt1 The Bmugt1 cDNA was cloned into the single SmaIsiteof the pSynXIV VI + X3 transfer vector [18]. The presence of the correct insert was confirmed by s equence 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 purification. Occlusion-positive plaques, representing recombinant viruses, were picked and plaque purified. S ingle isolated r ecombinant viruses w ere amplified to obtain high-titre virus stocks. V irus titres were determined by plaque assays [22]. Recombinant viral DNA was purified and characterized by restriction mapping following standard procedures [22]. The r ecombinant virus, vSynBmUGT1, containing the Bm u gt1 gene, was chosen for expression analyses. Expression of recombinant protein Expression of recom binant p rotein was c haracterized 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 we re infected at a multiplic ity 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 a nd replaced with methionine-deficient medium containing 25 lCi trans- 35 S-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/P i , pH 6.2. Cells were lysed by incubation on ice for 30minin50lL cell l ysis buffer (1% Nonidet P 40, 150 m M NaCl, 50 m M Tris/HCl, pH 8), and stored at )80 °C. Protease inhibitors were added t o t he cell lysis buffer a t 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 )1 tunicamycin 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 representin g 2 · 10 5 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 we re used for assay o f BmUGT1 activity, because SF21 cells were found to express low levels of endogenous UGT a ctivity t owards several of the substrates t ested (data not shown). Cells were infected with vSynBm UGT1 o r parental virus, vEGTSyngal+, at a multiplicity of infection of 20 a nd 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 t he following: cell lysate containing 0.5 mg total protein; 10 m M MgCl 2 ; 100 m M Tris/malate, pH 7.4; 5 m MD -gluconic a cid lactone; 0.1 m M substrate (purchased from Sigma); 0.25 lCi UDP-[ 3 H]glucose (15.3 CiÆmmol )1 ;Sigma)and 50 l M UDP-glucose. The final reaction volume was 100 lL. Reaction mixtures were incubated for 1 h at 37 °Candthen stopped by the addition of 2 vol. ethanol. The reaction mixture was evaporated, and the products were resuspended in 60% ethano l a nd separated by TLC on silica-gel plates (Merck) as described [26]. T he silica-gel p lates were e xposed to 3 H-sensitive phosphoimager screens and r ead w ith a 820 T. Luque et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Fujifilm 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. RESULTS Isolation and analysis of the Bmugt1 gene In the course of an EST sequencing p roject, a cDNA clone from a B. mori larval cDNA library was i dentified with homology to UGT genes. Sequence analysis o f the cDNA demonstrated that it included the complete BmUGT1 coding sequence p lus 64 nucleotides of upstream and 5 4 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 i dentification of this protein as a member of the UGT superfamily. Figure 1 shows a multiple alignment of BmUGT1 with several r epre sentative mem- bers of the UGT superfamily. Gap pairwise comparisons demonstrated that it has about 30% amino-acid identity overall w ith other members of the family, e.g. 31% and 38% with Drosophila melano gaster 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 the Bmugt1 gene The e xp ression 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 Bm ugt1 that are not conserved a mong UGTs, in order to a void amplification of other putative transcripts from the large UGT family. A single transcriptional product of the expected size (380 bp) was a mplified 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 fi fth-instar larvae. Fig. 1. Alignment of UGT amino-acid sequences. Black and grey indicate identical and similar a mino 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 c onsensus is indicated in the region of the UGT signature se quence. Transmembr do m indicates the transmembrane do main, based on the human sequences. UGT35a (ac cession num ber AF116554) and UGT35b (accession number AF116 555) are two UGTs from D. melanogaster;AcEGT (accession number M22619) is an e cdysteroid UGT from the baculovirus Autographa californica nucleopolyhedrovirus; UGT1A10 ( accession number U89508) and UGT2B7 (accession number NM001074) belong to different families of v ertebrate UGTs. Ó FEBS 2002 B. mori UDP-glucosyltransferase (Eur. J. Biochem. 269) 821 VSynBmUGT1, a recombinant baculovirus expressing BmUGT1 The baculovirus expression system was chosen to express BmUGT1 in i nsect cells. The par ental viral DNA used lacked the b aculovirus ecdysteroid UDP-glucosyltransfer- ase ( egt) gene, to allow the characte rization 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 i nfected insect cells at different times after infection. Proteins were separated by SDS/PAGE and revealed by autoradiography (Fig. 3). As expected for a bacu lovirus i nfection, 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 m ass of 59.5 kDa based on translation of the complete ORF, including the p utative signal sequence. After treatment with tunicamycin, a glycosylation inhibitor, BmUGT1 migrated more rapidly, with an estima te d molecular mass o f 55 k Da, indicating that it was N-glycosylated. As expected, the recombinant v SynBmUGT1, an occlusion-positive virus, expressed polyhedrin normally (Fig. 3). In contrast, no polyhedrin expression was observed with t he parental virus, which is occlusion negative. Characterization of BmUGT1 enzymatic activity In an attempt t o identify t he enzyme activity of BmUGT1, its activity towards different potential substrates belonging to diverse chemical groups was analysed. It has been previously reported t hat other members o f the U GT superfamily are a ctive o ver 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 a dded to the re action mixture to inhibit low levels of endogenous b-glucosidase activity in the cell culture preparations. Representative assays are s hown in Fig. 4, and the complete s et of data is presented in Table 1. BmUGT1 catalyzed the glucosidation of a range of compounds, mostly phenolics o r phenol- derived compounds. The highest glucosylation rates were observed with t he flavonoids naringenin and quercetin, and with p-hydroxybiphenyl and p-nitrophenol. Other sub- strates included t he 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 R NA with Bmugt1-specific oligonucleotides. N, no DNA, PCR negative control; I, i ntegume nt; M, m idgut; F, f at body; H, haemocyte; S, silk gland; T, testis; L, 100-bp ladder. Fig. 3 . BmUGT1 recombinan t 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-infect ed cells; P, parental virus (vEGTSyngal+); BmUGT1, vSynBmUGT1; T, 48 h p.i. cells t reate d with tunicamycin. The mo lecular masses of g lycosy- lated and no nglycosylated BmUGT 1 a re in dicated by a rrows o n the right side of the gel. The position of the polyhedrin protein (polh) is also sho wn. The sizes of the m olecular-mass markers are shown in kDa onthelefthandsideofthegel. Fig. 4. BmUGT1 co njugation o f some representative aglycones. UDP- [ 3 H]glucose and the indicated aglyco nes 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. 822 T. Luque et al.(Eur. J. Biochem. 269) Ó FEBS 2002 detectable 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 )1 Æ(mg total protein) )1 for all substrates. DISCUSSION This study describes the identification a nd characterization of BmUGT1, a phenol UGT encoded b y 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 r egulation of insect moulting and metamorphosis. The baculoviruses are a large group of viruses t hat infect i nsects, and expression of egt enables them to r egulate the development of their host [28]. Baculoviruses are assumed to have acquired egt from an insect host. However, our data have demonstrated that Bm ugt1 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. T his is thought to reflect the fact t hat t he protein c omprises two major functional domains [30]. The N-terminal half is believed to b e responsible for binding the aglycone. These are highly diverse, explaining the relative lack of conserva- tion in the N -terminal region. In addition, 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 i s proposed to bind the UDP- sugar, e xplaining the similarity of this region among different enzymes [3]. This general pattern is observe d with BmUGT1; the e xtreme N-terminus is hydrophobic a nd 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 o f sequence c onservation in t he N-terminal half of the protein is the r egion 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 i s highly s imilar to other UGTs in this region and therefore is likely to b e a n o ligomer in its n ative state. As mentioned above, the mammalian UGTs are typically present in t he lumen of t he endoplasmic reticulum and possess a hydrophobic C-terminal transmembrane domain, followed by a basic cytoplasmic anchor sequence (Fig. 1 ; [ 32]). I n c ontrast, b aculovirus EGT p roteins, wh ich are secreted, lack the C-terminal anchor motif (Fig. 1). BmUGT1 possesses a C-terminal hydrophobic s equence followed by a basic motif, suggesting that it is also likely to be anchored in the e ndoplasmic reticulum. This is s imilar 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 m ammalian UGTs a re known to be glycosylated, and it seems likely t hat n ative B mUGT1 i s 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) f rom three independent experiments. Specific activities in parental virus-infected cells were less than 0.04 nmolÆh )1 Æ(mg total protein) )1 . ND, Glucoside formation was not de tectable. Aglycone UGT activity [nmolÆh )1 Æ(mg total protein) )1 ] Flavonoids Naringenin 5.30 ± 0.49 Quercetin 2.85 ± 0.37 3-Hydroxyflavone ND Coumarins Umbelliferone 0.28 ± 0.06 4-Hydroxycoumarin ND Scopoletin ND Phenolic compounds Monosubstituted phenols a-Naphthol 0.48 ± 0.06 Phenol ND o-Disubstituted phenols Guaiacol 0.38 ± 0.18 Salicyl aldehyde 0.28 ± 0.14 Catechol ND Salicyl alcohol ND Salicylic acid ND L -Tyrosine ND 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 Pyrogallol ND 3-Hydoxyanthranilic acid ND 1,2,4-Trisubstituted phenols Vanillin 0.67 ± 0.06 Eugenol 0.55 ± 0.11 Dopamine ND L-DOPA ND N-Acetyldopamine ND Protocatechuic acid ND Terpenoids S-(-)-b-Citronellol 0.18 ± 0.06 (+)-Isomenthol 0.13 ± 0.03 (-)-Borneol ND Geraniol ND Steroids Cholesterol ND Ecdysone ND 3-Hydroxyecdysone ND Others Cis-7,8-epoxy-2-methyl octadecane ND Cis-9-tetradeceryl acetate ND Trans-2-hexanal ND Xanthurenic acid ND Ó FEBS 2002 B. mori UDP-glucosyltransferase (Eur. J. Biochem. 269) 823 [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 b een reported i n 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, sugge sting the existence of an EGT-like activity in this insect [37,38]. UGT ac tivity has also been i mplicated in other p rocesses 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 a bove h ave not yet b een i dentified. I t w as thus of particular interest to try to assign a specific activity to the Bmugt1 gene identified i n this study. Because o f the low conservation of the N-terminal aglycone-binding region of the ugt genes, it is not possible to predict substrate specificity based on s equence data. We therefore elected to express the Bmugt1 gene and assay the expressed enzyme against a range of potential substrates, chosen on th e basis of UGT- like activities already reported or postulated in insects. The b aculovirus expression system represented an attrac- tive approach to the expression o f Bmugt1 for a number o f 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 o f obtaining active enzyme. The only caveat t o the use o f a baculovirus system was the fact that they express EGT, which could cause false-positive assay results with ecdyste roid substrates. It was therefore necessary to generate the recombinant virus expressing Bmugt1 using an egt-parent virus [19]. The substrates chosen for a ssay with BmUGT1 r epre- sented a wide range of chemistries and potential functions for t he enz yme in vivo. O f t he 38 substrates tested, conjugation activity was detected with 16 diverse com- pounds, suggesting that B mUGT1 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 a nd phenol-derived compounds, including flavonoids (e.g. naringenin and quer cetin), terpenoids [e.g. S-(–)-b)citronel- lol] and s imple phenols (e.g. p-nitrophenol and e ugenol). 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 t he tyrosine/ dopamine-related compounds. Thus, BmUGT1 is unlikely to be invo lved in processes s uch a s ecdysteroid metabolism or cuticle f ormation. In contrast, many of the substrates conjugated are plant allelochemicals, suggesting a m ajor role for the enzyme in detoxication re sponses. F or instance, flavonoids are abundant in fruits, vegetables , seeds a nd roots [ 42]. I t is known that m any p lant phenolics can act as toxins or feeding deterrents to i nsects and thus play an important role in plant d efence 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 w idely expressed in final-instar B. mori larvae (Fig. 2). This is c onsistent with a r ole in detoxication, which is known to occur in many different organs. However, it is important to n ote that the R T-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 g uaiacol. There is accumulating evidence for a role for some vertebrate UGTs in o lfaction [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 h ave 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 t hese genes. A c ritical s tep 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. ACKNOWLEDGEMENTS We thank Dr L. Vilaplana and D r J. Olszewski (I mperial College of Science, Technology and Medicine) f or helpful co mments on the manuscript, Dr T. Shimada (University of Tokyo) a nd Dr K. Mita (National Institute of Radiological Science) for the provision of the EST c lone, and Dr Canoira (Unive rsidad Politecnica de Madrid) and Dr S. Hadfield (Syngenta) for advice on the subst rates assayed. This work was supported by the Leverhulme Trust (F/58/AS) to D. O’R., by the Spec ial Po stdoctoral Researchers Program, Riken (to K. O.) a nd by a Grant-in-Aid for Scientific Research (A1-12306002) from the Ministry of Edu cation, Science a nd Culture of J apan (to K . O .). REFERENCES 1. Burchell, B. & Coughtrie, M. (1989) UDP -glucuro nosyltrans- ferases. Ph armacol. Ther. 43 , 261–289. 2. Mackenzie, P.I., O wens, I.S., B urchell, B., Bock, K.W., B airoch, A.,Belanger,A.,Fournel-Gigleux,S.,Green,M.,Hum,D.W., Iyanagi, T., et al. (1997) The UDP glycosyltransferase g ene superfamily: recommended nomenclatu re u pdate based on evo- lutionar y divergen ce. Pharmacogenetics 7, 255–269. 3. Meech, R. & Mackenzie, P.I. 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(1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Re s. 22, 4673– 4680. Ó FEBS 2002 B. mori UDP-glucosyltransferase (Eur. J. Biochem. 269) 825 . 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. Characterization of a novel silkworm ( Bombyx mori ) phenol UDP-glucosyltransferase Teresa Luque 1 , Kazuhiro Okano 2 and David R. O’Reilly 1 1 Department