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.     

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.     

绿盲蝽气味结合蛋白AlucOBP7的表达及气味结合特性

气味结合蛋白(odorant binding proteins, OBPs) 在昆虫嗅觉识别中起着重要的作用, 尤其是在运输外界脂溶性气味分子通过嗅觉感器淋巴液到达嗅觉受体(olfactory receptors, ORs)的过程中发挥关键作用。明确OBPs在昆虫同外界进行信息交流过程中的作用有利于阐明昆虫嗅觉识别的机制, 同时可为利用干扰昆虫嗅觉识别来进行害虫防治奠定理论基础。本研究克隆了一个绿盲蝽Apolygus lucorum (Meyer-Dür)气味结合蛋白AlucOBP7基因（GenBank登录号: JQ675724）, 并进行了原核表达, 以1-NPN为荧光探针采用荧光竞争结合实验研究了AlucOBP7蛋白和10种棉花挥发物及 6种性信息素类似物的结合能力。结果表明： 在10种棉花挥发物中, AlucOBP7能够和2 己酮及水杨酸甲酯有效结合, 结合常数分别为55.13 μmol/L和28.26 μmol/L。在6种盲蝽性信息素类似物中, 4-氧代-反-2-己烯醛和AlucOBP7 具有较强的结合能力, 结合常数为23.14 μmol/L。丁酸乙酯、 丁酸丁酯及己酸己酯也能够和AlucOBP7 有效结合, 但结合能力中等, 结合常数分别为30.58, 39.26和35.81 μmol/L。初步推测, AlucOBP7 可能是绿盲蝽性信息素结合蛋白(pheromone binding proteins, PBPs), 并在感受性信息素和植物挥发物的过程中发挥双重功能。     

Porcine odorant-binding protein selectively binds to a human olfactory receptor

Odorant-binding proteins (OBPs) represent a highly abundant class of proteins secreted in the nasal mucus by the olfactory neuroepithelium. These proteins display binding affinity for a variety of odorant molecules, thereby assuming the role of carrier during olfactory perception. However, no specific interaction between OBP and olfactory receptors (ORs) has yet been shown and early events in olfaction remain so far poorly understood at a molecular level. Two human ORs, OR 17-209 and OR 17-210, were fused to a Green Fluorescent Protein and stably expressed in COS-7 cell lines. Interaction with OBP was investigated using a highly purified radioiodinated porcine OBP (pOBP) preparation, devoid of any ligand in its binding cavity. No specific binding of the pOBP tracer could be detected with OR 17-209. In contrast, OR 17-210 exhibited specific saturable binding (K(d) = 9.48 nM) corresponding to the presence of a single class of high-affinity binding sites (B(max) = 65.8 fmol/mg prot). Association and dissociation kinetics further confirmed high-affinity interaction between pOBP and OR 17-210. Autoradiographic studies of labeled pOBP to newborn mouse slices revealed the presence of multiple specific binding sites located mainly in olfactory tissue but also in several other peripheral tissues. Our data thus demonstrate a high-affinity interaction between OBP and OR, indicating that under physiological conditions, ORs may be specifically associated with an OBP partner in the absence of odorant. This provides further evidence of a novel role for OBP in the mechanism of olfactory perception.     

Wake up and smell the pheromones

Odorant binding proteins (OBPs) are abundant proteins of unknown function expressed at high levels in insect and vertebrate chemosensory organs. In this issue of Neuron, Xu et al. show that Drosophila OBP76a is necessary for fruit flies to respond to the aggregation pheromone 11-cis vaccenyl acetate. The results suggest a mechanism by which this OBP is intimately involved in pheromone signal transduction.     

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.     

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.     

草地贪夜蛾三个SfruPBPs对本种及同域种劳氏粘虫性信息素及腺体组分的亲和力

[目的]测定草地贪夜蛾Spodoptera frugiperda 3个信息素结合蛋白(pheromone binding protein,PBP)(SfruPBP)对草地贪夜蛾及同域近缘种劳氏粘虫Leucania loreyi性信息素及腺体组分的结合特性,探究草地贪夜蛾这3个SfruPBPs在两种昆虫不同性信息素组分识...     

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.     

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.     

Novel odorant-binding proteins expressed in the taste tissue of the fly

A taste tissue cDNA library of the fleshfly Boettcherisca peregrina was screened with a subtracted cDNA probe enriched with taste-receptor-tissue-specific cDNA. Seven genes were identified with sequence similarity to insect odorant-binding protein (OBP) genes. The predicted amino acid sequences of the genes contain the putative signal peptide sequence at the N-terminal and most of them conserve the six cysteines common to known insect OBPs. These genes show a high degree of sequence divergence with approximately 20% amino acid identity. The most striking feature was that all seven of these genes are expressed mainly in the taste tissues, such as the labellum and tarsus, unlike the known insect OBP genes expressed in olfactory tissue. The predicted amino acid sequences had the highest degree of sequence similarity to the Drosophila melanogaster OBPs named pheromone binding protein-related proteins (PBPRPs). These gene products are here referred to as gustatory PBP-related proteins (GPBPRPs) 1-7. Homologous GPBPRP genes were found also in D. melanogaster by database search and are shown to be expressed in Drosophila taste tissues.     

Mammalian odorant binding proteins

Odorant binding proteins (OBPs) pertain to one of the most abundant classes of proteins found in the olfactory apparatus. OBPs are a sub-class of lipocalins, defined by their property of reversibly binding volatile chemicals, that we call 'odorants'. Numerous sequences of OBPs are now available, derived from protein sequencing from nasal mucus material, or from DNA sequences. The structural knowledge of OBPs has been improved too in recent years, with the availability of two X-ray structures. The physiological role of OBPs remains, however, essentially hypothetical, and most probably, not linked to a function of odor transport. The present knowledge on OBP biochemistry, sequence and structure will be examined here in relation to the different functional hypotheses proposed for OBPs.     

Source and cyclic release pattern of (Z)-7-dodecenyl acetate, the pre-ovulatory pheromone of the female Asian elephant

Female Asian elephants (Elephas maximus) release a pre-ovulatory urinary pheromone, (Z)-7-dodecenyl acetate (Z7--12:Ac), to signal males of their readiness to mate. Z7--12:Ac is quantitatively elevated during the follicular stage of estrus, reaching maximum concentrations just prior to ovulation, as demonstrated by two complementary headspace techniques: (i) evacuated canister capture followed by cryogenic trapping; (ii) solid phase microextraction (SPME), used prior to gas chromatography/ mass spectrometry (GC/MS). These patterns were coincident with observed male behaviors and were consistent with biochemical and binding properties of the active ligand, including optimal binding pH. To release maximum amounts of Z7--12:Ac for quantitation, serum and urine samples from three mature female Asian elephants in their luteal and follicular stages of several estrous cycles were subjected to heat and pH changes and were then treated with protease prior to SPME-GC/MS analyses. When the post-luteal serum progesterone concentrations declined to baseline levels, Z7--12:Ac became detectable in the female urine. Throughout the follicular stage pheromone concentrations increased linearly with no apparent relationship to the two serum luteinizing hormone peaks. Pre-ovulatory urine also contained related compounds, including (Z)-7--12-dodecenol. The relative amount of this alcohol increased relative to acetate during long-term storage, with a proportional reduction in bioactivity. Z7--12:Ac was not detected in mucus samples from the urogenital tract. A potential precursor of Z7--12:Ac was identified in liver homogenates from female elephants in the follicular stage.     

Evolution of noctuid pheromone binding proteins: identification of PBP in the black cutworm moth,Agrotis ipsilon

Male black cutworm moths (Agrotis ipsilon, Lepidoptera, Noctuoidea, Noctuidae), which are attracted by a three-component pheromone blend ((Z)-7-dodecenyl acetate, Z7-12:Ac; (Z)-9-tetradecenyl acetate, Z9-14:Ac; (Z)-11-hexadecenyl acetate, Z11-16:Ac), express diverse antennal pheromone binding proteins (PBPs). Two PBP isoforms (Aips-1 and Aips-2) that show 46% identity were cloned from antennal cDNA of male A. ipsilon. The protein Aips-1 displays a high degree of identity (70-95%) with PBPs of other noctuiids, but shows only 42-65% identity with the PBPs of more phylogenetically distant species. The other protein, Aips-2, represents a distinct group of PBP that includes proteins from Sphingidae and Yponomeutidae. These differences observed suggest that each of the two PBPs may be tuned to a specific pheromone ligand.     

The tryptophan phosphorescence of porcine and mutant bovine odorant-binding proteins: a probe for the local protein structure and dynamics

Vertebrate odorant-binding proteins (OBPs) are small extracellular proteins belonging to the lipocalin superfamily. They have been supposed to play a role in events of odorant molecules detection by carrying, deactivating, and/or selecting odorant molecules. The OBPs share a conserved folding pattern, an eight-stranded beta-barrel flanked by an alpha-helix at the C-terminal end of the polypeptide chain. The beta-barrel creates a central nonpolar cavity whose role is to bind and transport hydrophobic odorant molecules. These proteins reversibly bind odorant molecules with dissociation constants ranging from nanomolar to micromolar range. In this work, we have studied the structural features of the OBP from pig and from cow by phosphorescence spectroscopy. The obtained results demonstrate that the indolic phosphorescence of the two studied proteins can be readily detected at ambient temperature solutions and that it is owed exclusively to the internal tryptophan residue located next to the ligand binding cavity, which is generally conserved in the mammalian OBPs. In addition, while both the phosphorescence spectrum and the lifetime yield a picture of the fold of the studied protein in good agreement with the protein crystallographic structures, the triplet probe points out that in solution the polypeptide structure of the both investigated OBPs exists as a multiplicity of slowly interconverting protein conformations. Finally, this work also demonstrates that it is possible to directly detect the binding of the ligands to OBPs as variations of the protein luminescence features, thus, representing the very first observation reported in the literature so far that a fast and direct assay can be used for monitoring the binding of ligands to OBPs.     

Evidence of an odorant-binding protein in the human olfactory mucus: location,structural characterization,and odorant-binding properties

Odorant-binding proteins (OBPs) are small abundant extracellular proteins belonging to the lipocalin superfamily. They are thought to participate in perireceptor events of odor detection by carrying, deactivating, and/or selecting odorant molecules. Putative human OBP genes (hOBP) have recently been described [Lacazette et al. (2000) Hum. Mol. Genet. 9, 289-301], but the presence of the corresponding proteins remained to be established in the human olfactory mucus. This paper reports the first evidence of such expression in the mucus covering the olfactory cleft, where the sensory olfactory epithelium is located. On the contrary, hOBPs were not observed in the nasal mucus covering the septum and the lower turbinate. To demonstrate the odorant binding activity of these proteins, a corresponding recombinant protein variant, hOBP(IIa)(alpha), was secreted by the yeast Pichia pastoris and thoroughly characterized. It appears as a monomer with one disulfide bond located between C59 and C151, a conservative feature of all other vertebrate OBPs. By measuring the displacement of several fluorescent probes, we show that hOBP(IIa)(alpha) is able to bind numerous odorants of diverse chemical structures, with a higher affinity for aldehydes and large fatty acids. A computed 3D model of hOBP(IIa)(alpha) is proposed and reveals that two lysyl residues of the binding pocket may account for the increased affinity for aldehydes. The relatively limited specificity of hOBP(IIa)(alpha) suggests that other human OBPs are expected to take into account the large diversity of odorant molecules.