Elsevier

Gene

Volume 346, 14 February 2005, Pages 195-203
Gene

P450 and P450 reductase cDNAs from the moth Mamestra brassicae: cloning and expression patterns in male antennae

https://doi.org/10.1016/j.gene.2004.11.010Get rights and content

Abstract

The involvement of cytochrome P450 (CYP) enzymes in olfaction has been demonstrated in vertebrates over the past decade. In insects, these enzymes are well known for their role in biosynthesis of endogenous compounds as well as xenobiotic metabolism, but the presence of olfactory cytochrome P450s was poorly investigated. Using a PCR-based strategy, we have isolated cDNAs of two new microsomal P450s from the antennae of the cabbage armyworm Mamestra brassicae, CYP9A13 and CYP4G20 of two new microsomal P450s, as well as their red-ox partner, the cytochrome P450 reductase (CPR). Their distribution through the body and their cellular localization within the antennae were studied by RT-PCR and in situ hybridization. The three genes are strongly expressed in some sensory units of the antennae, the sensilla trichodea, which are tuned to odorants detection. The putative functions of the corresponding enzymes are discussed in regard to their respective expression patterns and to our knowledge on olfactory P450 metabolism in mammals.

Introduction

All organisms live in environments that contain potentially harmful chemicals, natural or anthropic. In vertebrates, extensive studies of detoxification mechanisms, especially in liver, have revealed the great variety of chemical transformation involved, including red-ox reactions catalyzed by cytochrome P450 (CYP) enzymes. Cytochrome P450 monooxygenases (P450s or CYPs) consist in a large superfamily of enzymes that catalyzes the transfer of one atom from O2 into various substrates (monooxygenase reactions), leading to a great diversity of catalytic reactions, such as hydroxylations (for a review, see Mansuy, 1985). P450s play a fundamental role as phase I transformation enzymes during detoxification of exogenous compounds, such as drugs, toxic pollutants or pesticides. The products of phase I reactions are then metabolized by phase II enzymes, such as UDP-glucoronosyl transferases and glutathione S-transferases, leading to the production of hydrophilic compounds that can no longer cross the membranes and are eliminated by secretion. P450-mediated reactions play also a key role in the metabolism of endogenous compounds, such as steroid hormones of vertebrates (Meyer, 1996) or insects (review in Feyereisen, 1999).

In mammals, the CYP1, 2 and 3 families are particularly involved in xenobiotic metabolism, and most of them are expressed in the liver, which plays a dominant role in the first-pass clearance of ingested compounds and controls the systemic levels of drugs and other chemicals substrates (Ding and Kaminsky, 2003). In insects, a great number of P450 genes have been isolated to date in more than 39 species (http://www.drnelson.utmen.edu/cytochromeP450.html). Some of them are involved in fundamental physiological functions, such as growth, development or reproduction through the biosynthesis and the catabolism of key hormones, such as juvenile hormone (JH) or 20-hydroxyecdysone (20E; Feyereisen, 1999). Recent molecular genetic and biochemical approaches in Drosophila and Bombyx mori have lead to the identification of four P450 enzymes (CYP302A1, CYP315A1, CYP314A1 and CYP306A1) involved in hydroxylation steps during 20E biosynthesis (Niwa et al., 2004). Another P450-mediated reaction (26-hydroxylation) is involved in 20E inactivation and the corresponding enzyme could correspond to CYP18A1 in Drosophila (Feyereisen, 1999). In the cockroach Diploptera punctata, CYP15A1, a newly identified microsomal P450, was showed to catalyze the last step of JH biosynthesis via a highly selective epoxidation reaction (Helvig et al., 2004). However, most of the P450s studied to date, especially in agronomical pest insects, were found to be involved in insecticide metabolism and inactivation of plant toxins (Stevens et al., 2000, Li et al., 2002). Among them, the P450s belonging to the CYP4, CYP6, CYP9 and CYP12 families are generally expressed in the digestive tract and fat body (Feyereisen, 1999, Petersen et al., 2001).

The olfactory system of terrestrial animals, which is specialized in the detection of volatile hydrophobic molecules, has a similar organization in mammals and insects. The dendrites of the olfactory neurons, which carry the olfactory receptors, are bathed in an aqueous medium, the mucus layer in vertebrates or the sensillum lymph in insect antennae, medium that odorants must cross before interaction with receptors. In both cases, the sensory neurons are thus indirectly in contact with the external environment and therefore exposed to odorants but also to volatile xenobiotics. In mammals, olfactory and respiratory tissues, which are exposed to both inhaled and blood-borne compounds, were found to express xenobiotic metabolism P450s (Lazard et al., 1990, Ding and Coon, 1990, Peng et al., 1993). The nasal mucosa is considered as a “metabolic hot spot” (Ding and Kaminsky, 2003) and many P450s found in liver, as well as other biotransformation enzymes, such as UDP-glucoronosyltransferases, were also found in the respiratory tract (Lazard et al., 1990), leading to a tissue-selective response to chemical exposure. These biotransformation enzymes have been proposed to participate in the termination of odorant signals, as well as the protection of the sensory neurons against potentially harmful compounds (Lazard et al., 1990).

In insects, the presence of P450s in olfactory tissues was demonstrated only recently. One cytochrome P450 cDNA belonging to the CYP6 family was found to be expressed in the antennae and chemosensory organs of fruit fly Drosophila melanogaster (Wang et al., 1999) and ESTs encoding P450s were reported from the antennae of the tobacco hornworm Manduca sexta (Robertson et al., 1999). In addition, a biochemical study of the degradation of the pheromone from the scarab beetle Phyllopertha diversa demonstrated the involvement of antennal P450 metabolism (Wojtasek and Leal, 1999). More recently, two P450 cDNAs belonging to the CYP4 family were characterized in the antennae of a Lepidoptera, the cabbage armyworm Mamestra brassicae, and were shown to be expressed in the olfactory sensilla trichodea, devoted to odorant detection (Maïbèche-Coisne et al., 2002). Microsomal P450-mediated monooxygenase activity is supported by NADPH and thus requires an interaction with NADPH-cytochrome P450 reductase (CPR). As CPR plays an essential role in the transfer of reducing equivalents from NADPH to cytochrome P450 enzymes, its presence in olfactory tissues is a necessity. In mammals, CPR is widely expressed in all tissues examined, including the olfactory mucosa, but its expression in insect antennae has been reported only once in D. melanogaster (Hovemann et al., 1997).

In this study, we characterized two new P450 cDNAs strongly expressed in the antennae of M. brassicae, in association with a CPR cDNA, leading to a total of four antennal P450s in the same species. The expression of all these genes in olfactory sensilla is strongly suggestive of a role in odorant and/or xenobiotic clearance.

Section snippets

Animals and tissue collection

Insects were purchased as pupae from Domaine du Magneraud (INRA, France) and were maintained at 20 °C and 60% relative humidity until emergence. Dissected tissues from sexual mature 3-day-old males (antennae, proboscis, brains, legs, thorax and abdomens) and females (antennae, ovipositors) were stored at −80 °C until use for total RNA isolation. For in situ hybridization experiments, male antennae were immediately fixed in 4% paraformaldehyde.

RNA isolation and cDNA synthesis

Total RNAs were extracted with TRI-Reagent

Cloning of two new antennal cytochrome P450 genes, CYP4G20 and CYP9A13

Two cDNA products of 312 and 523 bp were amplified after RT-PCR on male antennal RNA by 3′ RACE, using a degenerate primer deduced from the conserved heme-binding region of cytochrome P450s. After cloning and sequencing, the deduced amino-acid sequences appeared to belong to the cytochrome P450 superfamily. They were then extended to the 5′ region by 5′ RACE using specific primers. Two full-length cDNAs of 2155 and 1779 bp were then obtained and designated by the P450 Nomenclature Committee as

Discussion

In the present work, we described in the noctuid moth M. brassicae the molecular cloning, the tissue distribution and the cellular expression in the antennae of two new members of the cytochrome P450 family, assigned to the CYP4 and CYP9 families, as well as their red-ox partner, the cytochrome P450 reductase. The CYP4 is one of the most ancient and diversified group of P450s; they are found in vertebrates and insects and are related to endogenous as well as xenobiotic metabolism (Feyereisen,

Acknowledgments

The authors would like to thank Dr. D. Nelson for naming the P450 genes. This study was supported by INRA and Université Paris VI.

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