Intriguing similarities between two novel odorant-binding proteins of locusts

Two novel odorant-binding proteins (OBPs) of locust, LmigOBP2 and LmigOBP3 are very different from each other and from the previously reported LmigOBP1 in their amino acid sequences. Moreover, OBP3 contains three additional cysteines, a fact not previously recorded in standard length OBPs. However, these two proteins exhibit remarkably similar binding affinities to a set of organic compounds. Such behaviour is supported by three-dimensional models, showing very similar folding for LmigOBP2 and LmigOBP3, but clearly different for LmigOBP1. Also several amino acid residues lining the binding pockets of the three proteins appear conserved in LmigOBP2 and LmigOBP3, but not in LmigOBP1. Western blot experiments revealed the presence of LmigOBP2 in antennae, mouth parts and cerci, but could not detected LmigOBP3 in any of these tissues. In immunocytochemistry, antibodies against LmigOBP2 strongly stained the outer lymph of sensilla chaetica of the antennae, in contrast with LmigOBP1, previously reported in sensilla basiconica.     

Odorant binding protein has the biochemical properties of a scavenger for 4-hydroxy-2-nonenal in mammalian nasal mucosa

Odorant binding proteins (OBP) are soluble lipocalins produced in large amounts in the nasal mucosa of several mammalian species. Although OBPs can bind a large variety of odorous compounds, direct and exclusive involvement of these proteins in olfactory perception has not been clearly demonstrated. This study investigated the binding properties and chemical resistance of OBP to the chemically reactive lipid peroxidation end-product 4-hydroxy-2-nonenal (HNE), in an attempt to establish a functional relationship between this protein and the molecular mechanisms combating free radical cellular damage. Experiments were carried out on recombinant porcine and bovine OBPs and results showed that both forms were able to bind HNE with affinities comparable with those of typical OBP ligands (K(d) = 4.9 and 9.0 microm for porcine and bovine OBP, respectively). Furthermore, OBP functionality, as determined by measuring the binding of the fluorescent ligand 1-aminoanthracene, was partially lost only when incubating HNE levels and exposure time to HNE exceeded physiological values in nasal mucosa. Finally, preliminary experiments in a simplified model resembling nasal epithelium showed that extracellular OBP can preserve the viability of an epithelial cell line derived from bovine turbinates exposed to toxic amounts of the aldehyde. These results suggest that OBP, which is expressed at millimolar levels, might reduce HNE toxicity by removing from the nasal mucus a significant fraction of the aldehyde that is produced as a consequence of direct exposure to the oxygen present in inhaled air.     

An overview of odorant-binding protein functions in insect peripheral olfactory reception

Insect olfactory perception involves many aspects of insect life, and can directly or indirectly evoke either individual or group behaviors. Insect olfactory receptors and odorant-binding proteins (OBPs) are considered to be crucial to insect-specific and -sensitive olfaction. Although the mechanisms of interaction between OBPs or OBP/ligand complex with olfactory receptors are still not well understood, it has been shown that many OBPs contribute to insect olfactory perception at various levels. Some of these are numerous and divergent members in OBP family; expression in the olfactory organ at high concentration; a variety of combinational patterns between different OBPs and ligands, but exclusive affinity for one OBP to specific binding ligands; complicated interactions between OBP/ligand complex and transmembrane proteins (olfactory receptors or sensory neuron membrane proteins). First, we review OBPs' ligand-binding property based on OBP structural research and ligand-binding test; then, we review current progress around the points cited above to show the role of such proteins in insect olfactory signal transmission; finally, we discuss applications based on insect OBP research.     

Crystal structure of a novel type of odorant-binding protein from Anopheles gambiae, belonging to the C-plus class

Agam (Anopheles gambiae) relies on its olfactory system to target human prey, leading eventually to the injection of Plasmodium falciparum, the malaria vector. OBPs (odorant-binding proteins) are the first line of proteins involved in odorant recognition. They interact with olfactory receptors and thus constitute an interesting target for insect control. In the present study, we undertook a large-scale analysis of proteins belonging to the olfactory system of Agam with the aim of preventing insect bites by designing strong olfactory repellents. We determined the three-dimensional structures of several Agam OBPs, either alone or in complex with model compounds. In the present paper, we report the first three-dimensional structure of a member of the C-plus class of OBPs, AgamOBP47, which has a longer sequence than classical OBPs and contains six disulfide bridges. AgamOBP47 possesses a core of six α-helices and three disulfide bridges, similar to the classical OBP fold. Two extra loops and the N- and C-terminal extra segments contain two additional α-helices and are held in conformation by three disulfide bridges. They are located either side of the classical OBP core domain. The binding site of OBP47 is located between the core and the additional domains. Two crevices are observed on opposite sides of OBP47, which are joined together by a shallow channel of sufficient size to accommodate a model of the best-tested ligand. The binding sites of C-plus class OBPs therefore exhibit different characteristics, as compared with classical OBPs, which should lead to markedly diverse functional implications.     

Differential expression of odorant-binding proteins in the mandibular glands of the honey bee according to caste and age

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) mediate both perception and release of chemical stimuli in insects. The genome of the honey bee contains 21 genes encoding OBPs and 6 encoding CSPs. Using a proteomic approach, we have investigated the expression of OBPs and CSPs in the mandibular glands of adult honey bees in relation to caste and age. OBP13 is mostly expressed in young individuals and in virgin queens, while OBP21 is abundant in older bees and is prevalent in mated queens. OBP14, which had been found in larvae, is produced in hive workers' glands. Quite unexpectedly, the mandibular glands of drones also contain OBPs, mainly OBP18 and OBP21. We have expressed three of the most represented OBPs and studied their binding properties. OBP13 binds with good specificity oleic acid and some structurally related compounds, OBP14 is better tuned to monoterpenoid structures, while OBP21 binds the main components of queen mandibular pheromone as well as farnesol, a compound used as a trail pheromone in the honey bee and other hymenopterans. The high expression of different OBPs in the mandibular glands suggests that such proteins could be involved in solubilization and release of semiochemicals.     

Le rôle des protéines liant les odeurs (OBP) dans la transduction olfactive

This paper reviews biochemical and functional properties of a family of proteins involved in the transduction process of chemical signals. Odorant-binding proteins (OBPs) are small soluble proteins highly concentrated in the chemosensory organs of Insects and Vertebrates. They are preferentially expressed in the nasal mucus of Vertebrates and in the sensillar lymph of Insects. They have been found to bind reversibly small hydrophobic molecules detected via the olfactory system. The vertebrate OBPs bind non-specific odorants with low affinities. They belong to the lipocalin family as well as other proteins involved in chemical communication and associated to different organs and functions. Nevertheless, no specific ligand for OBPs has been yet identified in Vertebrates. However, the large microdiversity of OBPs in the same animal suggests that OBPs could be involved in the discrimination of odors. Chemical communication in Noctuid moths was used as a model to study the molecular mechanisms of odor recognition. The pheromonal system is extremely sensitive and specific since the male is able to detect only few molecules of the pheromone and to recognize specific blends of the same molecules. The chemical signals were identified for a large number of Lepidopteran species and the associated behaviours they elicit were fully characterized. The Lepidopteran OBPs are divided into two sub-classes according to their ligands: pheromone-binding proteins (PBP) are expressed in sensilla trichodea responding to pheromonal compounds while general odorant binding proteins (GOBP) are associated with sensilla basiconica tuned to the detection of general odors, such as plant volatiles. The PBPs selectively bind components of the female sex-pheromone with measurable affinities. The ligand binding site was localized in the 40–60 aminoacids region. Substitutions in the binding site of different proteins are correlated with the fixation of different ligands, leading to the hypothesis that the primary structure encodes the ligand specificity. Other proteins expressed in chemosensory organs of other orders of Insects were cloned or purified. In absence of functional data, they were called OBP-like. Some of them were localized in the gustatory organs and could be common carriers of odors in both olfactory and gustatory systems. Many arguments are in accordance with an active role of the OBPs in the early steps of odor discrimination. The heterogeneity of OBPs inside species and between species, the spatial segregation in their expression and the different binding affinities of PBPs towards pheromonal compounds support the hypothesis that the coding of odors is realized as soon as the level of OBPs. More, the complex OBP/odor could be the stimulus for olfactory receptors cloned in Vertebrates and still putative in Insects. This hypothesis suggests that OBPs take part as an essential element of the chemosensory transduction.     

New isoforms of odorant-binding proteins and potential semiochemicals of locusts

To obtain more information on the elements of chemical communication in the migratory locust (Locusta migratoria) (Orthoptera: Acrididae), we have searched for additional odorant-binding proteins (OBPs) and for volatiles in the feces that could represent potential semiochemicals for this species. A two-dimensional electrophoretic (2DE) analysis of an antennal extract showed only three closely positioned spots that were recognized by the antiserum against locust OBP. Three genes were also identified using PCR and 5'RACE-PCR approaches, encoding isoforms differing from each other for a single amino acid substitution. The gas-chromatographic-electroantennogram (GC-EAD) headspace analysis of a feces sample revealed the presence of several compounds that elicited dose-dependent electrophysiological responses in the antennae of both sexes. Most of these compounds are different from those identified in the feces of the desert locust (Schistocerca gregaria) and reported to be behaviorally active. Ligand-binding experiments performed with such volatiles and recombinant OBP did not show affinity, thus indicating that the binding pocket of OBP requires larger molecules than those so far identified.     

Functional evolution of duplicated odorant-binding protein genes, Obp57d and Obp57e, in Drosophila

Odorant-binding proteins (OBPs) are extracellular proteins found in insect chemosensilla, where they participate in the sensing of odors, tastes, and pheromones. Although a large number of OBP genes have been identified in insect genomes, their molecular functions and biological roles have been clarified in limited cases. Two OBP genes, Obp57d and Obp57e, were involved in the evolution of host-plant preference in Drosophila sechellia. Comparative analyses of the Obp57d/e genomic sequences from 27 closely related species suggested that the two genes arose by tandem gene duplication and functionally diverged from each other. In this study, the functional evolution of Obp57d and Obp57e was examined by in vitro binding assays using recombinant proteins synthesized in a bacterial system. Compared to the ancestral Dpse\OBP57de, Dmel\OBP57d was more specialized to tridecanoic acid while Dmel\OBP57e was generalized regarding their binding affinity, suggesting that the two OBP genes underwent subfunctionalization and neofunctionalization. A behavioral analysis using knockout flies supported that the biological role is different between OBP57d and OBP57e in vivo. Site-directed mutagenesis of the evolutionarily conserved amino acids revealed that these residues play an important role in protein folding. These findings provide a clue to understanding how the repertoire of OBP genes is maintained in a genome under natural selection.     

Conformational isomers of insect odorant-binding proteins.

We have identified and cloned the cDNAs encoding odorant-binding proteins (OBPs) from the large black chafer, Holotrichia parallela, and the yellowish elongate chafer, Heptophylla picea. Each species possess two OBPs, the proteins migrating faster in native gels (OBP1) showed high amino acid identity (>88%) to previously identified pheromone-binding proteins (PBPs) from scarab beetles. HparOBP1 and HpicOBP1 have 116 amino acids and six highly conserved cysteine residues. In contrast to OBP1 that gave a single band, both HparOBP2 and HpicOBP2 separated each into two bands in native gels (15%). The N-terminal amino acid sequences for the two bands from each species were indistinguishable, and they had the same molecular masses. Although we sequenced several clones from each species, they all encode only one protein for each species, indicating they are different conformational isomers of the same protein. HparOBP2 and HpicOBP2 have 133 amino acids and cysteine residues are conserved in proteins of the same family.     

Discrimination of alarm pheromone (E)-β-farnesene by aphid odorant-binding proteins

OBPs have been recently demonstrated to be required for odour perception in insects and directly involved in odour discrimination. In aphids they might represent new interesting targets for the control of their population in agriculture. Based on sequence information available in the EST database, we have cloned four genes encoding odorant-binding proteins (OBP) in Acyrthosiphon pisum and homologous genes in other aphid species. Unlike OBPs from other orders of insects, that are greatly divergent, in aphids these proteins have been found to be highly conserved, with differences between species limited to only few amino acid substitutions. On the contrary, similarities between OBP sequences of the same species are poor with 31% or less of identical amino acids. Three selected OBPs (OBP1, OBP3 and OBP8) have been expressed in bacteria and purified. Ligand-binding experiments have shown similar behaviour of the three proteins towards several organic compounds, but also some significant selectivities. In particular, (E)-β-farnesene, the alarm pheromone and its related compound farnesol exhibited good affinity to OBP3, but did not bind the other two proteins. We suggest that OBP3 could mediate response of aphids to the alarm pheromone.     

Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.).

In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is generally thought to be accomplished by odorant-binding proteins (OBPs). We report the structural and functional properties of a honeybee OBP called ASP2, heterologously expressed by the yeast Pichia pastoris. ASP2 disulfide bonds were assigned after classic trypsinolysis followed by ion-spray mass spectrometry combined with microsequencing. The pairing [Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI)] was found to be identical to that of Bombyx mori OBP, suggesting that this pattern occurs commonly throughout the highly divergent insect OBPs. CD measurements revealed that ASP2 is mainly constituted of alpha helices, like other insect OBPs, but different from lipocalin-like vertebrate OBPs. Gel filtration analysis showed that ASP2 is homodimeric at neutral pH, but monomerizes upon acidification or addition of a chaotropic agent. A general volatile-odorant binding assay allowed us to examine the uptake of some odorants and pheromones by ASP2. Recombinant ASP2 bound all tested molecules, except beta-ionone, which could not interact with it at all. The affinity constants of ASP2 for these ligands, determined at neutral pH by isothermal titration calorimetry, are in the micromolar range, as observed for vertebrate OBP. These results suggest that odorants occupy three binding sites per dimer, probably one in the core of each monomer and another whose location and biological role are questionable. At acidic pH, no binding was observed, in correlation with monomerization and a local conformational change supported by CD experiments.     

Expression pattern analysis of odorant‐binding proteins in the pea aphid Acyrthosiphon pisum

Odorant‐binding proteins (OBPs) are soluble proteins mediating chemoreception in insects. In previous research, we investigated the molecular mechanisms adopted by aphids to detect the alarm pheromone (E)‐β‐farnesene and we found that the recognition of this and structurally related molecules is mediated by OBP3 and OBP7. Here, we show the differential expression patterns of 5 selected OBPs (OBP1, OBP3, OBP6, OBP7, OBP8) obtained performing quantitative RT‐PCR and immunolocalization experiments in different body parts of adults and in the 5 developmental instars, including winged and unwinged morphs, of the pea aphid Acyrthosiphon pisum. The results provide an overall picture that allows us to speculate on the relationship between the differential expression of OBPs and their putative function. The expression of OBP3, OBP6, and OBP7 in the antennal sensilla suggests a chemosensory function for these proteins, whereas the constant expression level of OBP8 in all instars could suggest a conserved role. Moreover, OBP1 and OBP3 are also expressed in nonsensory organs. A light and scanning electron microscopy study of sensilla on different body parts of aphid, in particular antennae, legs, mouthparts, and cornicles‐cauda, completes this research providing a guide to facilitate the mapping of OBP expression profiles.     

Specificity of odorant-binding proteins: a factor influencing the sensitivity of olfactory receptor-based biosensors

Odorant-binding proteins (OBPs) primarily function in the transport of hydrophobic odorants. In this study, OBPs originating from rat and pig were cloned into a mammalian expression vector, pcDNA3, and expressed in HEK-293 cells, and their specificity for odorants and olfactory receptors was examined. Results suggest that OBPs have a high affinity for the olfactory receptors when both the OBP and receptor originate from the same species. The rat OBPs were bound not only to the rat olfactory receptor I7 but also to the odorant specific to I7. The solubility of the odorant was increased by both OBP2 and OBP3, which originate from rat, but with different efficiencies. These results demonstrate that OBPs specifically interact with odorants as well as olfactory receptors, and these interactions can influence the sensitivity of olfactory receptor-based biosensors.     

Insights into structural features determining odorant affinities to honey bee odorant binding protein 14

Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant.     

Developmental expression of odorant‐binding proteins and chemosensory proteins in the embryos of Locusta migratoria

We have investigated the development of chemosensilla and the secretion of odorant‐binding proteins (OBPs) and chemosensory proteins (CSPs) in the embryo of Locusta migratoria manilensis. We first report the changes of each sensillum in embryo just preceding hatch in detail and show that different sensilla have different developmental processes. Trichogen cells are first involved in forming the structure of pegs, and then, after retraction, they start secreting OBPs and CSPs in the sensillar lymph. The synthesis of LmigOBP1 starts during the embryogenesis about 0.5 h preceding hatching, specifically in sensilla trichodea and basiconica of the antenna. LmigOBP2, instead, was only found in the outer sensillum lymph (oSl) of sensilla chaetica of the antenna, while we could not detect LmigOBP3 in any type of sensilla of the antenna. The ontogenesis of CSPs in the embryos is similar to that of OBPs. Expression of CSPI homolog in Locusta migratoria is detected using the antiserum raised against SgreCSPI. CSPI is specifically expressed in the outer sensillum lymph of sensilla chaetica of the antenna, and anti‐LmigCSPII dose not label any sensilla of the embryos. These data indicate that in locusts, OBPs and CSPs follow different temporal expression patterns, and also that OBPs are expressed in different types of sensilla. © 2009 Wiley Periodicals, Inc.     

Odorant-binding proteins and chemosensory proteins in pheromone detection and release in the silkmoth Bombyx mori

The genome of the silkmoth Bombyx mori contains 44 genes encoding odorant-binding proteins (OBPs) and 20 encoding chemosensory proteins (CSPs). In this work, we used a proteomic approach to investigate the expression of proteins of both classes in the antennae of adults and in the female pheromone glands. The most abundant proteins found in the antennae were the 4 OBPs (PBP, GOBP1, GOBP2, and ABP) and the 2 CSPs (CSP1 and CSP2) previously identified and characterized. In addition, we could detect only 3 additional OBPs and 2 CSPs, with clearly different patterns of expression between the sexes. Particularly interesting, on the other hand, is the relatively large number of binding proteins (1 OBP and 7 CSPs) expressed in the female pheromone glands, some of them not present in the antennae. In the glands, these proteins could be likely involved in the solubilization of pheromonal components and their delivery in the environment.