Odorant-degrading Enzymes Research Articles

Identification and Characterization of an Antennae-Specific Aldehyde Oxidase from the Navel Orangeworm

Antennae-specific odorant-degrading enzymes (ODEs) are postulated to inactivate odorant molecules after they convey their signal. Different classes of insect ODEs are specific to esters, alcohols, and aldehydes – the major functional groups of female-produced, hydrophobic sex pheromones from moth species. Esterases that rapidly inactive acetate and other esters have been well-studied, but less is known about aldehyde oxidases (AOXs). Here we report cloning of an aldehyde oxidase, AtraAOX2, from the antennae of the navel orangeworm (NOW), Amyelois transitella, and the first activity characterization of a recombinant insect AOX. AtraAOX2 gene spans 3,813 bp and encodes a protein with 1,270 amino acid residues. AtraAOX2 cDNA was expressed in baculovirus-infected insect Sf21 cells as a ≈280 kDa homodimer with 140 kDa subunits. Recombinant AtraAOX2 degraded Z11Z13–16Ald and plant volatile aldehydes as substrates. However, as expected for aldehyde oxidases, recombinant AtraAOX2 did not show specificity for Z11Z13–16Ald, the main constituent of the sex pheromone, but showed high activity for plant volatile aldehydes. Our data suggest AtraAOX2 might be involved in degradation of a diversity of aldehydes including sex pheromones, plant-derived semiochemicals, and chemical cues for oviposition sites. Additionally, AtraAOX2 could protect the insect's olfactory system from xenobiotics, including pesticides that might reach the sensillar lymph surrounding the olfactory receptor neurons.

Odorant Reception in Insects: Roles of Receptors, Binding Proteins, and Degrading Enzymes

Our knowledge of the molecular basis of odorant reception in insects has grown exponentially over the past decade. Odorant receptors (ORs) from moths, fruit flies, mosquitoes, and the honey bees have been deorphanized, odorant-degrading enzymes (ODEs) have been isolated, and the functions of odorant-binding proteins (OBPs) have been unveiled. OBPs contribute to the sensitivity of the olfactory system by transporting odorants through the sensillar lymph, but there are competing hypotheses on how they act at the end of the journey. A few ODEs that have been demonstrated to degrade odorants rapidly may act in signal inactivation alone or in combination with other molecular traps. Although ORs in Drosophila melanogaster respond to multiple odorants and seem to work in combinatorial code involving both periphery and antennal lobes, reception of sex pheromones by moth ORs suggests that their labeled lines rely heavily on selectivity at the periphery.

A Water-Specific Aquaporin is Expressed in the Olfactory Organs of the Blowfly, Phormia regina

The high sensitivity and selectivity of perireceptor events in insect olfactory organs requires the concerted action of odorant-binding proteins (OBPs), odorant receptors (ORs), and odorant-degrading enzymes (ODEs). Sensillum lymph in the sensillum cavity is a physiological saline that not only mediates the olfactory signaling pathway described above, but also protects the olfactory neurons against desiccation. The molecular mechanism of how water balance is maintained in the sensillum cavity still remains to be elucidated. Here, we characterize an aquaporin from the blowfly, Phormia regina (PregAQP1). PregAQP1 possesses six predicted transmembrane domains and two asparagine-proline-alanine (NPA) motifs, and belongs to the Drosophila melanogaster integral protein (DRIP) subfamily. Transcript levels were high in the maxillary palp and moderate in the antenna. PregAQP1 accumulated in accessory cells located underneath a long-grooved hair in the maxillary palp and also in a receptor neuron in a thick-walled sensillum in the antenna. Expression of PregAQP1 in Xenopus oocytes showed water permeability in a mercury-sensitive manner. These results suggest that PregAQP1 plays a role in the maintenance of the aqueous environment of olfactory organs.

A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in Drosophila

BackgroundInsects respond to the spatial and temporal dynamics of a pheromone plume, which implies not only a strong response to 'odor on', but also to 'odor off'. This requires mechanisms geared toward a fast signal termination. Several mechanisms may contribute to signal termination, among which odorant-degrading enzymes. These enzymes putatively play a role in signal dynamics by a rapid inactivation of odorants in the vicinity of the sensory receptors, although direct in vivo experimental evidences are lacking. Here we verified the role of an extracellular carboxylesterase, esterase-6 (Est-6), in the sensory physiological and behavioral dynamics of Drosophila melanogaster response to its pheromone, cis-vaccenyl acetate (cVA). Est-6 was previously linked to post-mating effects in the reproductive system of females. As Est-6 is also known to hydrolyze cVA in vitro and is expressed in the main olfactory organ, the antenna, we tested here its role in olfaction as a putative odorant-degrading enzyme.ResultsWe first confirm that Est-6 is highly expressed in olfactory sensilla, including cVA-sensitive sensilla, and we show that expression is likely associated with non-neuronal cells. Our electrophysiological approaches show that the dynamics of olfactory receptor neuron (ORN) responses is strongly influenced by Est-6, as in Est-6° null mutants (lacking the Est-6 gene) cVA-sensitive ORN showed increased firing rate and prolonged activity in response to cVA. Est-6° mutant males had a lower threshold of behavioral response to cVA, as revealed by the analysis of two cVA-induced behaviors. In particular, mutant males exhibited a strong decrease of male-male courtship, in association with a delay in courtship initiation.ConclusionsOur study presents evidence that Est-6 plays a role in the physiological and behavioral dynamics of sex pheromone response in Drosophila males and supports a role of Est-6 as an odorant-degrading enzyme (ODE) in male antennae. Our results also expand the role of Est-6 in Drosophila biology, from reproduction to olfaction, and highlight the role of ODEs in insect olfaction.

Degradation of Pheromone and Plant Volatile Components by a Same Odorant-Degrading Enzyme in the Cotton Leafworm, Spodoptera littoralis

BackgroundOdorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant.MethodologyPrevious work has shown that the antennae of the cotton leafworm Spodoptera littoralis, a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants.Conclusion SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.

Characterization of an antennal carboxylesterase from the pest moth Spodoptera littoralis degrading a host plant odorant.

BackgroundCarboxyl/cholinesterases (CCEs) are highly diversified in insects. These enzymes have a broad range of proposed functions, in neuro/developmental processes, dietary detoxification, insecticide resistance or hormone/pheromone degradation. As few functional data are available on purified or recombinant CCEs, the physiological role of most of these enzymes is unknown. Concerning their role in olfaction, only two CCEs able to metabolize sex pheromones have been functionally characterized in insects. These enzymes are only expressed in the male antennae, and secreted into the lumen of the pheromone-sensitive sensilla. CCEs able to hydrolyze other odorants than sex pheromones, such as plant volatiles, have not been identified.MethodologyIn Spodoptera littoralis, a major crop pest, a diversity of antennal CCEs has been previously identified. We have employed here a combination of molecular biology, biochemistry and electrophysiology approaches to functionally characterize an intracellular CCE, SlCXE10, whose predominant expression in the olfactory sensilla suggested a role in olfaction. A recombinant protein was produced using the baculovirus system and we tested its catabolic properties towards a plant volatile and the sex pheromone components.ConclusionWe showed that SlCXE10 could efficiently hydrolyze a green leaf volatile and to a lesser extent the sex pheromone components. The transcript level in male antennae was also strongly induced by exposure to this plant odorant. In antennae, SlCXE10 expression was associated with sensilla responding to the sex pheromones and to plant odours. These results suggest that a CCE-based intracellular metabolism of odorants could occur in insect antennae, in addition to the extracellular metabolism occurring within the sensillar lumen. This is the first functional characterization of an Odorant-Degrading Enzyme active towards a host plant volatile.

Annotation and expression of carboxylesterases in the silkworm, Bombyx mori.

BackgroundCarboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. It plays important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. Previous studies mainly used Dipteran Drosophila and mosquitoes as model organisms to investigate the roles of the insect COEs in insecticide resistance. However, genome-wide characterization of COEs in phytophagous insects and comparative analysis remain to be performed.ResultsBased on the newly assembled genome sequence, 76 putative COEs were identified in Bombyx mori. Relative to other Dipteran and Hymenopteran insects, alpha-esterases were significantly expanded in the silkworm. Genomics analysis suggested that BmCOEs showed chromosome preferable distribution and 55% of which were tandem arranged. Sixty-one BmCOEs were transcribed based on cDNA/ESTs and microarray data. Generally, most of the COEs showed tissue specific expressions and expression level between male and female did not display obvious differences. Three main patterns could be classified, i.e. midgut-, head and integument-, and silk gland-specific expressions. Midgut is the first barrier of xenobiotics peroral toxicity, in which COEs may be involved in eliminating secondary metabolites of mulberry leaves and contaminants of insecticides in diet. For head and integument-class, most of the members were homologous to odorant-degrading enzyme (ODE) and antennal esterase. RT-PCR verified that the ODE-like esterases were also highly expressed in larvae antenna and maxilla, and thus they may play important roles in degradation of plant volatiles or other xenobiotics.ConclusionB. mori has the largest number of insect COE genes characterized to date. Comparative genomic analysis suggested that the gene expansion mainly occurred in silkworm alpha-esterases. Expression evidence indicated that the expanded genes were specifically expressed in midgut, integument and head, implying that these genes may have important roles in detoxifying secondary metabolites of mulberry leaves, contaminants in diet, and odorants. Our results provide some new insights into functions and evolutionary characteristics of COEs in phytophagous insects.

An Antennal Circadian Clock and Circadian Rhythms in Peripheral Pheromone Reception in the Moth Spodoptera littoralis

Circadian rhythms are observed in mating behaviors in moths: females emit sex pheromones and males are attracted by these pheromones in rhythmic fashions. In the moth Spodoptera littoralis, we demonstrated the occurrence of a circadian oscillator in the antenna, the peripheral olfactory organ. We identified different clock genes, period (per), cryptochrome1 (cry1) and cryptochrome2 (cry2), in this organ. Using quantitative real-time PCR (qPCR), we found that their corresponding transcripts cycled circadianly in the antenna as well as in the brain. Electroantennogram (EAG) recordings over 24 h demonstrated for the first time a circadian rhythm in antennal responses of a moth to sex pheromone. qPCR showed that out of one pheromone-binding protein (PBP), one olfactory receptor (OR), and one odorant-degrading enzyme (ODE), all putatively involved in the pheromone reception, only the ODE transcript presented a circadian rhythm that may be related to rhythms in olfactory signal resolution. Peripheral or central circadian clock control of olfaction is then discussed in light of recent data.

Identification of candidate aldehyde oxidases from the silkworm Bombyx mori potentially involved in antennal pheromone degradation

Signal inactivation is a crucial step in the dynamic of olfactory process and involves various Odorant-Degrading Enzymes. In the silkworm Bombyx mori, one of the best models for studying olfaction in insects, the involvement of an antennal-specific aldehyde oxidase in the degradation of the sex pheromone component bombykal has been demonstrated over the three past decades by biochemical studies. However, the corresponding enzyme has never been characterized at the molecular level. Bioinformatic screening of B. mori genome and molecular approaches have been used to isolate several candidate sequences of aldehyde oxidases. Two interesting antennal-expressed genes have been further characterized and their putative functions are discussed in regard to their respective expression pattern and to our knowledge on aldehyde oxidase properties. Interestingly, one gene appeared as specifically expressed in the antennae of B. mori and associated in males with the bombykal-sensitive sensilla, strongly suggesting that it could encode for the previously biochemically characterized enzyme.

A new aldehyde oxidase selectively expressed in chemosensory organs of insects

Signal termination is a crucial step in the dynamic of the olfactory process. It involves different classes of odorant-degrading enzymes. Whereas aldehyde oxidase enzymatic activities have been demonstrated in insect antennae by previous biochemical studies, the corresponding enzymes have never been characterized at the molecular level. In the cabbage armyworm Mamestra brassicae, we isolated for the first time an aldehyde oxidase partial cDNA specifically expressed in chemosensory organs, with the strongest expression in antennae of both sexes. In these organs, expression was restricted to the olfactory sensilla. Our results suggest that the corresponding enzyme could degrade aldehyde odorant compounds, such as pheromones or plant’s volatiles.

Genomics spawns novel approaches to mosquito control.

In spite of advances in medicine and public health, malaria and other mosquito-borne diseases are on the rise worldwide. Although vaccines, genetically modified mosquitoes and safer insecticides are under development, herein we examine a promising new approach to malaria control through better repellents. Current repellents, usually based on DEET, inhibit host finding by impeding insect olfaction, but have significant drawbacks. We discuss how comparative genomics, using data from the Anopheles genome project, allows the rapid identification of members of three protein classes critical to insect olfaction: odorant-binding proteins, G-protein-coupled receptors, and odorant-degrading enzymes. A rational design approach similar to that used by the pharmaceutical industry for drug development can then be applied to the development of products that interfere with mosquito olfaction. Such products have the potential to provide more complete, safer and longer lasting protection than conventional repellents, preventing disease transmission by interrupting the parasite life cycle.

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

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 Gly 195-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 Gly 214-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.

Molecules and macromolecules involved in chemical communication of scarab beetles

Abstract Chemical communication involves the production and release of specific chemicals (pheromones and other semiochemicals) by the emitter, and the detection and olfactory processing of these signals leading to appropriate behavioral responses in the receiver. In contrast to most of the scarab species investigated to date, the Japanese and Osaka beetles have the ability to detect the allospecific pheromone, which plays a pivotal role in the isolation mechanism between these two species. Each species produces a single enantiomer of japonilure [(Z)-5-(dec1-enyl)oxacyclopentan-2-one], but they have evolved the ability to detect both enantiomers, one as an attractant and the other as a behavioral antagonist (stop signal). There is growing evidence in the literature that the inordinate sensitivity and selectivity of the insect olfactory system is achieved by a combination of various olfactory-specific proteins, namely, odorant-binding proteins (OBPs), odorant receptors (ORs), and odorant-degrading enzymes. The relationship between the pheromone structures and the primary sequences of the proteins suggest that OBPs play a part in the selectivity of the olfactory system in scarab beetles by "filtering" chemical signals during the early olfactory processing (perireceptor events). Nevertheless, it is unlikely that pheromone-binding proteins are "chiral filters" as the Japanese and Osaka beetles each possess only one single binding protein. Upon interaction with negatively charged membranes, OBPs undergo conformational changes that may lead to the release of the ligands.

A photoaffinity-labeled green leaf volatile compound 'tricks' highly selective and sensitive insect olfactory receptor neurons.

The sex pheromone of the scarab beetle, Phyllopertha diversa, is emitted by females and specifically detected by olfactory receptor neurons in the male and female antennae. Single sensillum recordings showed that, in contrast to the less sensitive pheromone sensilla in females, olfactory receptor neurons in the male antennae had a low threshold (1 ng), which rivals those of moths. The male and female antennae also possessed olfactory receptor neurons specific for the detection of floral and green leaf volatile compounds. Detectors for the green leaf volatile (Z)-3-hexenyl acetate had a threshold (10 pg) far below the sensitivity of the pheromone-detecting machinery. In addition, these neurons showed a remarkable selectivity even when challenged with related compounds at 10000-fold higher concentrations. Surprisingly, a diazo analog, (Z)-3-hexenyl diazoacetate, elicited slightly higher nervous activity than the natural ligand in the neurons specific and selective for (Z)-3-hexenyl acetate. The inability of the green leaf volatile-detecting machinery to discriminate the photoaffinity-labeled compound from the natural product indicates that the synthetic ligand interacts with odorant-binding protein, odorant receptor and odorant-degrading enzyme as does the cognate ligand.

Perireceptor events in olfaction.

Before reaching olfactory receptor neurons, odorant molecules have to cross an aqueous interface: the nasal mucus in vertebrates and the sensillar lymph in insects. Biochemical interactions taking place between odorants and the elements of these phases are called perireceptor events. Main protein constituents of these media, in both insects and vertebrates, are OBPs (odorant-binding proteins). Another class of proteins active in the olfactory perireceptor area includes odorant-degrading enzymes. The structure and the properties of these major proteins, with particular reference to OBPs, are reviewed and their role in olfactory transduction is discussed.

Ecdysteroid regulation of olfactory protein expression in the developing antenna of the tobacco hawk moth, Manduca sexta

During adult metamorphosis, the moth olfactory neurons and their glia-like support cells pass through a coordinated and synchronous development. By 60% of development, the olfactory system is anatomically complete, but functional maturation does not occur until about 90% of development. Maturation is characterized by the onset of odorant sensitivity in the sensory neurons and the expression of certain antennal-specific proteins including odorant binding proteins (OBPs) and odorant degrading enzymes (ODEs). The OBPs have been cloned and sequenced, and are thus useful models for investigating the molecular mechanisms coordinating final maturation of the developing olfactory system. The ecdysteroid hormones have been observed to regulate many cellular level neuronal changes during adult metamorphosis. In particular, the late pupal decline in ecdysteroids is known to influence programmed death of nerves and muscles at the end of metamorphosis. Experiments are presented here which indicate that this decline in ecdysteroids also induces the expression of the OBPs. Normal OBP expression occurs 35-40 h before adult emergence. In culture, OBP expression could be induced at least 90 h before adult emergence by the premature removal of ecdysteroid. This premature expression was blocked by culturing tissue in the presence of the biologically active ecdysteroid 20-hydroxyecdysone. These findings suggest that maturation of the olfactory system is regulated by the decline in ecdysteroids, and support the view that olfactory development, in general, may be coordinated by changing levels of pupal ecdysteroids.