Tài liệu Báo cáo Y học: Characterization of a novel silkworm (Bombyx mori ) phenol

Characterization of a novel silkworm (Bombyx mori ) phenol

UDP-glucosyltransferase

Teresa Luque1

, Kazuhiro Okano2 and 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 exogenous

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: [email protected]

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)

Eur. J. Biochem. 269, 819–825 (2002) Ó FEBS 2002