Cloning of putative odorant-degrading enzyme and integumental esterase cDNAs from the wild silkmoth, Antheraea polyphemus
Introduction
While navigating en route to a pheromone source, a moth encounters intermittent chemical signals with short bursts of flux, separated by periods during which the flux is zero. The average duration of spikes within bursts is in the millisecond scale and it decreases as the animal comes closer to the pheromone source (Murlis et al., 2000). There is growing evidence in the literature that this remarkable temporal precision of the insect olfactory system is determined by early events (peripheral interactions) rather than by intracellular signaling processes (signal transduction) (Kaissling, 2001), but the literature is also dichotomous with respect to deactivation of chemical signals. One school favors the hypothesis that rapid inactivation is an enzymatic process regulated by odorant-degrading enzymes (ODEs) (Vogt et al., 1985), whereas the other school favors the hypothesis that chemical signals are first inactivated by odorant-binding proteins (OBPs) (Kaissling, 2001), with odorant-degrading enzymes participating afterwards in the catabolism (slow process) of pheromone degradation.
While recent structural studies unveiled some features of OBPs, in particular details of the protection (Sandler et al., 2000), binding, and release of pheromones (Horst et al., 2001), the molecular basis of pheromone inactivation is still terra incognita. Insect antenna-specific esterases from the wild silkmoth, Antheraea polyphemus, have been known for more than two decades (Klein, 1987, Vogt and Riddiford, 1981) and their ability to degrade pheromone has been demonstrated (Vogt et al., 1985). While sensillar esterase(s) is (are) specific to male antennae (Fig. 1), integumental esterase(s) is(are) present in male and female antennae and legs. Interestingly, each male antenna of the wild silkmoth has ca. 60,000 pheromone-sensitive sensilla trichodea and 10,000 sensilla basiconica (Keil, 1984, Meng et al., 1989), whereas the female antennae lack pheromone-detecting sensilla and have ca. 12,000 sensilla basiconica (Boeckh et al., 1960). Despite their suggested role in olfaction, these proteins have never been isolated nor have their cDNAs been cloned. Unlike pheromone-binding proteins, which are highly expressed (ca. 10 mM) in the sensillar lymph (Klein, 1987), odorant-degrading enzymes are estimated to occur in concentrations at least four orders of magnitude below that of pheromone-binding proteins (Vogt and Riddiford, 1986). The scanty amounts of odorant-degrading enzymes have prevented their isolation and characterization. Because of the high expression levels of PBPs, protein-based approaches played a critical role in molecular studies. For example, the pheromone-binding protein of Bombyx mori was first isolated and identified (Maida et al., 1993), its cDNA was cloned (Krieger et al., 1996), a functional expression was achieved (Wojtasek and Leal, 1999), and these researches laid the foundation for structural biology studies (Horst et al., 2001, Sandler et al., 2000).
An understanding of the molecular basis of pheromone inactivation is essential if we are to understand how moths perceive and orient in the environment. Here, we describe a bioinformatics approach that led us to cloning of the cDNAs encoding two esterases from A. polyphemus, one specifically expressed in the sensilla of male moth (putative odorant-degrading enzyme) and the other occurring in legs and male and female antennae (putative integumental esterase).
Section snippets
Tissue collection
Cocoons of the wild silkmoth, A. polyphemus, were purchased from various breeders. They were kept at 4 °C for two months, then transferred to 26 °C, 75% relative humidity, and 16L:8D photoregime and used three days after eclosion. For protein extraction, adults were anesthetized on ice and their antennae and legs were collected. Homogenization was performed in an ice-cold glass Dounce tissue grinder in 10 mM Tris-HCl, pH 8. Homogenized samples were centrifuged twice at 12,000 r.p.m (4 °C, 5
Results and discussion
Our bioinformatics approach was based on sequences of carboxylesterases and juvenile hormone esterases previously identified from insects. Although these esterases showed only moderate identity (28–40%), three short conserved regions have been identified (Table 1). PCR screening using templates from antennae or legs and with combinations of the six degenerate primers and universal primer mix (UPM) led to cloning and sequencing of a dozen partial cDNA sequences. The partial sequences were used
Acknowledgements
This research was supported in part by direct financial support from the department, college, and Chancellor’s office at UCD and by USDA grant No. 01-8500-0506-GR. We thank George Kamita and Andrew C. Hinton for their critique of an earlier version of the manuscript.
References (27)
- et al.
Identification of a juvenile homone esterase gene by matching its peptide mass fingerprint with a sequence from the Drosophila genome project
Insect Biochem. Molec. Biol.
(2001) - et al.
Spruce budworm (Chorisfoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning
Mol. Cell. Endocrinol.
(1999) - et al.
Isolation and sequencing of cDNA clones coding for juvenile hormone esterase from Heliothis virescens. Evidence for a catalytic mechanism for the serine carboxylesterases different from that of the serine proteases
J. Biol. Chem.
(1989) - et al.
Characterization of affinity-purified juvenile hormone esterase from Trichoplusia ni
J. Biol. Chem
(1987) - et al.
Purification of juvenile hormone esterase and molecular cloning of the cDNA from Manduca sexta
Insect Biochem. Molec. Biol.
(2001) Sensillum-lymph proteins from antennal olfactory hairs of the moth Anthearea polyphemus (Saturniidae)
Insect Biochem.
(1987)- et al.
Binding proteins from the antennae of Bombyx mori
Insect Biochem. Molec. Biol.
(1996) - et al.
The pheromone binding protein of Bombyx mori: Purification, characterization and immunocytochemical localization
Insect Biochem. Molec. Biol.
(1993) - et al.
cDNA cloning, baculovirus-expression and kinetic properties of the esterase, E3, involved in organophosphorus resistance in Lucilia cuprina
Insect Biochem. Molec. Biol.
(1997) - et al.
Sexual attraction in the silkworm moth: structure of the pheromone-binding protein-bombykol complex
Chem. Biol.
(2000)
Differential glycosylation produces heterogeneity in elevated esterases associated with insecticide resistance in the brown planthopper Nilaparvata lugens Stal
Insect Biochem. Molec. Biol.
Isolation of juvenile horomone esterase and its partial cDNA clone from the beetle, Tenebrio molitor
Insect Biochem. Molec. Biol.
Mosquito carboxylesterase Est 21 (A2). Cloning and sequence of the full-length cDNA for a major insecticide resistance gene worldwide in the mosquito Culex quinquefasciatus
J. Biol. Chem.
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