Cloning of putative odorant-degrading enzyme and integumental esterase cDNAs from the wild silkmoth, Antheraea polyphemus

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Abstract

Odorant-degrading enzymes have been postulated to participate in the fast deactivation of insect pheromones. These proteins are expressed specifically in the sensillar lymph of insect antennae in such low amounts that, hitherto, isolation and protein-based cDNA cloning has not been possible. Using degenerate primers based on conserved amino acid sequences of insect carboxylesterases and juvenile hormone esterases, we were able to amplify partial cDNA fragments, which were then used for the design of gene-specific primers for RACE. This bioinformatics approach led us to the cloning of cDNAs, encoding a putative odorant-degrading enzyme (Apol-ODE) and a putative integumental esterase (Apol-IE) from the wild silkmoth, Antheraea polyphemus. Apol-ODE had a predicted molecular mass of 59,994 Da, pI of 6.63, three potential N-glycosylation sites, and a putative catalytic site Ser characterized by the sequence Gly195-Glu-Ser-Ala-Gly-Ala. Apol-IE gave calculated molecular mass of 61,694 Da, pI of 7.49, two potential N-glycosylation sites, and a putative active site with the sequence Gly214-Tyr-Ser-Ala-Gly. The transcript of Apol-ODE was detected by RT-PCR in male antennae and branches (sensillar tissues), but not in female antennae and other control tissues. Apol-IE was detected in male and female antennae as well as legs.

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)

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