A Pheromone-Binding Protein Mediates the Bombykol-Induced Activation of a Pheromone Receptor In Vitro

Institute of Physiology (230), University of Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany.
Chemical Senses (Impact Factor: 3.16). 08/2006; 31(6):547-55. DOI: 10.1093/chemse/bjj059
Source: PubMed


The enormous capacity of the male silkmoth Bombyx mori in recognizing and discriminating bombykol and bombykal is based on distinct sensory neurons in the antennal sensilla hairs. The hydrophobic pheromonal compounds are supposed to be ferried by soluble pheromone-binding proteins (PBPs) through the sensillum lymph toward the receptors in the dendritic membrane. We have generated stable cell lines expressing the candidate pheromone receptors of B. mori, BmOR-1 or BmOR-3, and assessed their responses to hydrophobic pheromone compounds dissolved by means of dimethyl sulfoxide. BmOR-1-expressing cells were activated by bombykol but also responded to bombykal, whereas cells expressing BmOR-3 responded to bombykal only. In experiments employing the B. mori PBP, no organic solvent was necessary to mediate an activation of BmOR-1 by bombykol, indicating that the PBP solubilizes the hydrophobic compound. Furthermore, the employed PBP selectively mediated a response to bombykol but not to bombykal, supporting a ligand specificity of PBPs. This study provides evidence that both distinct pheromone receptors and PBPs play an important role in insect pheromone recognition.

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Available from: Ewald Grosse-Wilde, Oct 07, 2015
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    • "Both protein classes reversibly bind small ligands with dissociation constants in the micromolar range (Pelosi et al. 2006). OBPs are likely involved in chemosensory perception, participating in the solubilization and transfer of odorants through the sensillum lymph (Vogt et al. 1991; Pelosi 1994; Prestwich et al. 1995; Pophof 2004; Tsuchihara et al. 2005; Grosse-Wilde et al. 2006). Additionally, they are supposed to contribute to the sensitivity of the olfactory system (Gomez-Diaz et al. 2013) and could protect odors from enzymatic degradation (Chertemps et al. 2012; Gomez-Diaz et al. 2013). "
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    ABSTRACT: Chemosensory protein (CSP) and gustatory receptor genes have been identified in all major arthropod groups. However, odorant binding proteins (OBP) and olfactory receptor genes are insect specific, suggesting that both gene families originated after the Hexapoda-Crustacea split (~470 million years ago). The seemingly parallel diversification of OBP and olfactory receptors has been suggested as coevolution between these genes after insect terrestrialization. To test this hypothesis we used the recently published transcriptomes of the jumping bristletail Lepismachilis y-signata and the firebrat Thermobia domestica to search for putative OBP and CSP sequences and analyzed their relationship to binding proteins of other insects and crustaceans. Our results suggest an evolution and expansion of OBPs as an adaptation to a terrestrial insect lifestyle, independently from the emergence of olfactory receptors.
    Chemical Senses 09/2015; DOI:10.1093/chemse/bjv050 · 3.16 Impact Factor
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    • "These six cysteines form three disulfide bridges, which play important roles in maintaining the protein tertiary structure. Experimental evidence has demonstrated that OBPs could selectively bind odorants or pheromones (Vogt and Riddiford, 1981; Plettner et al., 2000; Pophof, 2004; Grosse-Wilde et al., 2006; Syed et al., 2006; Gong et al., 2009b; Zhou et al., 2009). "
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    ABSTRACT: In this study, we constructed a high-quality cDNA library from the antennae of the Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). A total of 1,235 colonies with inserts greater than 0.7 kb were sequenced and analyzed. Homology searching coupled with bioinformatics analysis identified 15 and 7 cDNA sequences, respectively, encoding putative odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). A phylogenetic tree of CsupCSPs showed that each CsupCSP has orthologs in Manduca sexta and Bombyx mori with strong bootstrapping support. One CSP was either very specific or more related to the CSPs of another species than to conspecific CSP. The expression profiles of the OBPs and CSPs in different tissues were measured by real-time quantitative PCR. The results revealed that of the 11 OBP genes, the transcript levels of CsupOBP1, CsupOBP5, and CsupOBP7 were higher in both male and female antennae than those in other tissues. And CsupCSP7 was highly expressed in both male and female antennae. Based on these results, the possible physiological functions of CsupOBPs and CsupCSPs were discussed. © 2015 Wiley Periodicals, Inc.
    Archives of Insect Biochemistry and Physiology 02/2015; 89(1). DOI:10.1002/arch.21224 · 1.02 Impact Factor
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    • "These characteristics, as well as other cell properties (i.e., growth rate, adhesion to surfaces) make HEK293 cells an ideal high-throughput option for characterizing ORs and for identifying novel molecules that may affect OR-ligand interactions. Human embryonic kidney cells were first used to characterize insect ORs in 2006 when PR genes from Bombyx mori were transfected into an HEK293 cell line expressing a Ga15 gene, allowing detection of OR activation using fluorescent calcium-sensitive dyes and fluorescent microscopes (Forstner et al., 2009; Grobe-Wilde et al., 2007, 2006). More recently, a modified commercially available expression vector was used to generate HEK293 cell lines with stable and inducible expression of insect Orco and ORs. "
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    ABSTRACT: The development of rapid and reliable assays to characterize insect odorant receptors (ORs) and pheromone receptors (PRs) remains a challenge for the field. Typically ORs and PRs are functionally characterized either in vivo in transgenic Drosophila or in vitro through expression in Xenopus oocytes. While these approaches have succeeded, they are not well suited for high-throughput screening campaigns, primarily due to inherent characteristics that limit their ability to screen large quantities of compounds in a short period of time. The development of a practical, robust and consistent in vitro assay for functional studies on ORs and PRs would allow for high-throughput screening for ligands, as well as for compounds that could be used as novel olfactory-based pest management tools. Here we describe a novel method of utilizing human embryonic kidney cells (HEK293) transfected with inducible receptor constructs for the functional characterization of ORs in 96-well plates using a fluorescent spectrophotometer. Using EposOrco and EposOR3 from the pest moth, Epiphyas postvittana as an example, we generated HEK293 cell lines with robust and consistent responses to ligands in functional assays. Single-cell sorting of cell lines by FACS facilitated the selection of isogenic cell lines with maximal responses, and the addition of epitope tags on the N-termini allowed the detection of recombinant proteins in homogenates by western blot and in cells by immunocytochemistry. We thoroughly describe the methods used to generate these OR-expressing cell lines, demonstrating that they have all the necessary features required for use in high-throughput screening platforms.
    Insect Biochemistry and Molecular Biology 08/2014; 54. DOI:10.1016/j.ibmb.2014.08.005 · 3.45 Impact Factor
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