Coil-to-helix transition and ligand release of Bombyx mori pheromone-binding protein

The transport of hydrophobic insect pheromones through the aqueous medium surrounding their receptors is assisted by pheromone-binding proteins (PBPs). The protein from the silkworm moth Bombyx mori, BmorPBP, exhibits a pH-dependent conformational change postulated to trigger the release of the pheromone bombykol to its receptor. At low pH, an alpha-helix occupies the same binding pocket that houses the pheromone in the BmorPBP-bombykol complex at high pH. We have determined the crystal structure of apo BmorPBP at a resolution of 2.3 angstroms and pH 7.5, which has surprisingly a structure similar to the A-form. These data suggest that BmorPBP undergoes a ligand-dependent conformational change in addition to the previously described pH-dependent conformational change. Analysis of the alpha-helix occupying the binding pocket reveals an amphipathic helix with three acidic residues along one face that are conserved among lepidopteran PBPs and may be involved in a conformational transition of BmorPBP at the receptor membrane.     

Interneurons sensitive to female pheromone in the deutocerebrum of the male silkworm moth, Bombyx mori

ABSTRACT. In response to minute quantities of female sex pheromone, the male silkworm moth, Bombyx mori L., walks upwind to locate the odour source. The axons of antennal receptors specific for the two known components of the pheromone terminate in the deutocerebrum. In this study, single interneurons were recorded extracellularly in the deutocerebrum of the male silkworm moth. Responses were characterized as the antennae were presented with puffs of clean air, or air containing either or both components of the female pheromone, bombykol and bombykal. An apparatus is described which added bombykol or bombykal to a constant air stream flowing over the antenna. Most units (87%) showed qualitatively different responses to bombykol and bombykal. A majority of the pheromone-sensitive units (65%) also showed mechanosensory responses to air puffs. Two units were recorded which were slightly inhibited by either bombykol or bombykal alone, but were excited by a mixture of the two.     

Pheromone discrimination by the pheromone-binding protein of Bombyx mori

Pheromone-binding proteins are postulated to contribute to the exquisite specificity of the insect's olfactory system, acting as a filter by preferentially binding only one of the components of the natural pheromone. Here, we investigated the possible discrimination of the two very similar components of the natural pheromone gland from the silk moth, Bombyx mori, bombykol and bombykal, by the only pheromone-binding protein (BmorPBP) known to be expressed in the pheromone-detecting sensilla. Free-energy calculations and virtual docking indicate that both bombykol and bombykal bind to BmorPBP with similar affinity. In addition, in vitro competitive binding assays showed that both bombykol and bombykal were bound by BmorPBP with nearly the same high affinity. While BmorPBP might filter out other physiologically irrelevant compounds hitting the sensillar lymph, discrimination between the natural pheromone compounds must be achieved by molecular interactions with their cognate receptors.     

A single sex pheromone receptor determines chemical response specificity of sexual behavior in the silkmoth Bombyx mori

In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex pheromones. In most moth species, male moths rely heavily on species-specific sex pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible pheromone system, in which a single pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex pheromone receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex pheromone receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex pheromone receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex pheromone receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol receptor neurons alone is sufficient to trigger full sexual behavior. Thus, a single gene defines behavioral selectivity in sex pheromone communication in the silkmoth. Our findings show that a single molecular determinant can not only function as a modulator of behavior but also as an all-or-nothing initiator of a complex species-specific behavioral sequence.     

Candidate pheromone binding proteins of the silkmoth Bombyx mori

Pheromone reception is thought to be mediated by pheromone binding proteins (PBPs) in the aqueous lymph of the antennal sensilla. Recent studies have shown that the only known PBP of Bombyx mori (BmorPBP1) appears to be specifically tuned to bombykol but not to bombykal, raising the question of whether additional subtypes may exist. We have identified two novel genes, which encode candidate PBPs (BmorPBP2, BmorPBP3). Comparison with PBPs from various moth species have revealed a high degree of sequence identity and the three BmorPBP-subtypes can be assigned to distinct groups within the moth PBP family. In situ hybridization revealed that BmorPBP2 and BmorPBP3 are expressed only in relatively few cells compared to the number of cells expressing BmorPBP1. Double-labeling experiments have shown that the two novel BmorPBPs are expressed in the same cells but are not co-expressed with BmorPBP1. Furthermore, unlike BmorPBP1, cells expressing the newly identified PBPs did not surround neurons containing the BmOR-1 receptor. The results indicate that BmorPBP2 and BmorPBP3 are located in sensilla types, which are different from the long sensilla trichodea.Data deposition: The sequences reported in this paper have been deposited in the EMBL database under accession nos. AM403100 (BmorPBP2) and AM403101 (BmorPBP3).     

Bombyx mori pheromone-binding protein binding nonpheromone ligands: implications for pheromone recognition

Insect pheromone-binding proteins (PBPs) transport sex pheromones through the aqueous layer surrounding G protein-coupled receptors that initiate signaling events leading to mating. This PBP-receptor system strongly discriminates between ligands with subtle structural differences, but it has proved difficult to distinguish the degree of discrimination of the PBP from that of the G protein-coupled receptor. The three-dimensional structures of the PBP of Bombyx mori, the silkworm moth, both with and without its cognate ligand bombykol ([E,Z]-10,12-hexadecadienol), have been determined by X-ray crystallography and NMR. In this paper, the structures of the same binding protein with bound iodohexadecane and bell pepper odorant were determined at 1.9 and 2.0 A, respectively. These structures illustrate the remarkable plasticity in the ligand binding site of the PBP, but suggest the protein might still act as a filter during pheromone signal processing.     

Termination of sex pheromone production in mated females of the silkworm moth

A mating duration of more than 6 h was necessary to permanently terminate the production of the sex pheromone (bombykol) in the silkworm moth, Bombyx mori L. (Lepidoptera: Bombycidae), although the female formed a bursa copulatrix including a spermatophore and laid fertilized eggs even after mating for only 0.5 h. The 6-h mated female again produced bombykol if given an injection of synthetic pheromonotropic neuropeptide (PBAN), which is known to activate pheromone biosynthesis in a virgin female. Extracts of brain-suboesophageal ganglion (SG) complexes, which were removed from 6- and 24-h mated females, showed strong pheromonotropic activities. These results indicated that the pheromone gland of the mated female maintained its ability to biosynthesize bombykol; however, it could not produce pheromone due to a suppression of PBAN secretion from the SG. Furthermore, bombykol titers did not decrease after mating in females with a transected ventral nerve cord, even after the injection of a spermatophore extract, suggesting that the suppression of PBAN secretion was mediated by a neural signal and not by a substance in the spermatophore. The mated females accumulated (10E, 12Z)-10,12-hexadecadienoic acid, a precursor of bombykol biosynthesis, in their pheromone glands as did decapitated females. © 1996 Wiley-Liss, Inc.     

The crystal structure of a cockroach pheromone-binding protein suggests a new ligand binding and release mechanism

Pheromone-binding proteins (PBPs) are small helical proteins found in sensorial organs, particularly in the antennae, of moth and other insect species. They were proposed to solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors, participating thus in the peri-receptor events of signal transduction. The x-ray structure of Bombyx mori PBP (BmorPBP), from male antennae, revealed a six-helix fold forming a cavity that contains the pheromone bombykol. We have identified a PBP (LmaPBP) from the cockroach Leucophaea maderae in the antennae of the females, the gender attracted by pheromones in this species. Here we report the crystal structure of LmaPBP alone or in complex with a fluorescent reporter (amino-naphthalen sulfonate, ANS) or with a component of the pheromonal blend, 3-hydroxy-butan-2-one. Both compounds bind in the internal cavity of LmaPBP, which is more hydrophilic than BmorPBP cavity. LmaPBP structure ends just after the sixth helix (helix F). BmorPBP structure extends beyond the sixth helix with a stretch of residues elongated at neutral pH and folding as a seventh internalized helix at low pH. These differences between LmaPBP and BmorPBP structures suggest that different binding and release mechanism may be adapted to the hydrophilicity or hydrophobicity of the pheromonal ligand.     

Cloning and functional characterization of a fatty acid transport protein (FATP) from the pheromone gland of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone

Sex pheromones of moths are largely classified into two types based on the presence (Type I) or absence (Type II) of a terminal functional group. While Type-I sex pheromones are synthesized from common fatty acids in the pheromone gland (PG), Type-II sex pheromones are derived from hydrocarbons produced presumably in the oenocytes and transported to the PG via the hemolymph. Recently, a fatty acid transport protein (BmFATP) was identified from the PG of the silkworm Bombyx mori, which produces a Type-I sex pheromone (bombykol). BmFATP was shown to facilitate the uptake of extracellular fatty acids into PG cells for the synthesis of bombykol. To elucidate the presence and function of FATP in the PG of moths that produce Type-II sex pheromones, we explored fatp homologues expressed in the PG of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone (Type II). A fatp homologue cloned from E. japonica (Ejfatp) was predominantly expressed in the PG, and its expression is upregulated shortly after eclosion. Functional expression of EjFATP in Escherichia coli enhanced the uptake of long chain fatty acids (C₁₈ and C₂₀), but not pheromone precursor hydrocarbons. To the best of our knowledge, this is the first report of the cloning and functional characterization of a FATP in the PG of a moth producing a Type-II sex pheromone. Although EjFATP is not likely to be involved in the uptake of pheromone precursors in E. japonica, the expression pattern of Ejfatp suggests a role for EjFATP in the PG not directly linked to pheromone biosynthesis.     

Combinative stimulation inactivates sex pheromone production in the silkworm moth, Bombyx mori.

Mating results in a strong suppression of sex pheromone (bombykol) production in the female silkworm moth, Bombyx mori. The mechanical stimulation from the insertion of a penis, inflation of the bursa copulatrix (BC), or copulation with the sterile male whose penis was removed in order to prevent ejaculation (pr-male) induced only a partial decline in bombykol production. Artificial insemination stimulates oviposition of fertilized eggs as does normal mating. However, bombykol production did not decline in artificially inseminated females. When females were artificially inseminated before or after mating with pr-males, some females had a small amount of bombykol, similar to females mated with normal males, while other females had a large amount of bombykol similar to virgin females. The former usually laid fertilized eggs, while the latter laid only unfertilized eggs though semen filled their spermatophores and spermathecae. The mechanical stimulation caused by mating with a pr-male could be replaced by covering the abdominal tip with melted paraffin. Neither implantation of the BC obtained from mated females, nor injection of the spermatophore extract, into a female mated with a pr-male could inactivate bombykol production. Injection of hemolymph from a mated female into a virgin also failed to affect bombykol production These results indicate that a combination of both the tactile stimulation of the abdominal tip and the arrival of fertile spermatozoa in the vestibulum trigger a neural inactivation mechanism of bombykol production after mating.     

Dynamic conformational equilibria in the physiological function of the Bombyx mori pheromone-binding protein

The Bombyx mori pheromone-binding protein (BmorPBP) undergoes a pH-dependent conformational transition from a form at basic pH, which contains an open cavity suitable for ligand binding (BmorPBP^B), to a form at pH 4.5, where this cavity is occupied by an additional helix (BmorPBP^A). This helix α7 is formed by the C-terminal dodecapeptide 131-142, which is flexibly disordered on the protein surface in BmorPBP^B and in its complex with the pheromone bombykol. Previous work showed that the ligand-binding cavity cannot accommodate both bombykol and helix α7. Here we further investigated mechanistic aspects of the physiologically crucial ejection of the ligand at lower pH values by solution NMR studies of the variant protein BmorPBP(1-128), where the C-terminal helix-forming tetradecapeptide is removed. The NMR structure of the truncated protein at pH 6.5 corresponds closely to BmorPBP^B. At pH 4.5, BmorPBP(1-128) maintains a B-type structure that is in a slow equilibrium, on the NMR chemical shift timescale, with a low-pH conformation for which a discrete set of ¹⁵N-¹H correlation peaks is NMR unobservable. The full NMR spectrum was recovered upon readjusting the pH of the protein solution to 6.5. These data reveal dual roles for the C-terminal tetradecapeptide of BmorPBP in the mechanism of reversible pheromone binding and transport, where it governs dynamic equilibria between two locally different protein conformations at acidic pH and competes with the ligand for binding to the interior cavity.     

Involvement of calcineurin in the signal transduction of PBAN in the silkworm, Bombyx mori (Lepidoptera)

In several moth species sex pheromone production in the pheromone gland is regulated by a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori it is suggested that PBAN, after binding to the cell-surface receptor, primarily activates a plasma membrane receptor-activated Ca2+ channel to increase cytosolic levels of Ca2+, and Ca2+/calmodulin complex directly or indirectly activates a phosphoprotein phosphatase, which in turn elicits activation of acyl CoA reductase (the key enzyme under PBAN control) through dephosphorylation, resulting in pheromone (bombykol) production. The effect of cyclosporin A (CsA) and FK 506, specific inhibitors of calcineurin (phosphoprotein phosphatase 2B) was studied on the sex pheromone production, in B. mori. The in vitro experiments showed that both chemicals exerted a dose-dependent inhibitory action when they were co-incubated with TKYFSPRL amide (Hez-PBAN fragment peptide). Practically, no difference was detected between the two chemicals in the tested doses (0.025-1250 microM). When effects of CsA or FK 506 were studied on cell-free production of bombykol by using microsomal fraction no inhibition was detected. Since microsomal fraction contains the acyl CoA synthetase, the rate-limiting acyl CoA reductase and the precursor, bombykol is produced if supplied with CoA, ATP and NADPH. Thus, the inhibitory action of CsA and FK506 under in vitro conditions should occur before the step of acyl group reduction and the effect is likely to be attributable to the inhibition of calcineurin in the signal transduction cascade mechanism of PBAN, in B. mori. The existence of calcineurin in the pheromone gland by using Western blot analysis is also demonstrated.     

Molecular mechanisms underlying sex pheromone production in the silkmoth, Bombyx mori: characterization of the molecular components involved in bombykol biosynthesis

Many species of female moths produce sex pheromones to attract conspecific males. To date, sex pheromones from more than 570 moth species have been chemically identified. Most moth species utilize Type I pheromones that consist of straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester and usually with several double bonds. In contrast, some moth species use unsaturated hydrocarbons or hydrocarbon epoxides, classified as Type II lepidopteran pheromones, as sex pheromones. Studies over the past three decades have demonstrated that female moths usually produce sex pheromones as multi-component blends where the ratio of the individual components is precisely controlled, thus making it possible to generate species-specific pheromone blends. As for the biosynthesis of Type I pheromones, it is well established that they are de novo synthesized in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways. However, as many of the molecular components within the PG cells (i.e., enzymes, proteins, and small regulatory molecules) have not been functionally characterized, the molecular mechanisms underlying sex pheromone production in PG cells remain poorly understood. To address this, we have recently characterized some of the molecules involved in the biosynthesis of the sex pheromone bombykol in the silkmoth, Bombyx mori. Characterization of these, and other, key molecules will facilitate our understanding of the precise mechanisms underlying lepidopteran sex pheromone production.     

Sex pheromone production in the silkworm, Bombyx mori, is mediated by store-operated Ca2+ channels

In most female moths, pheromone biosynthesis activating neuropeptide (PBAN) regulates sex pheromone production by stimulating an influx of extracellular Ca(2+). Little is known about the plasma membrane channel or how the PBAN stimulus is communicated to the channel. Fluorescent Ca(2+) imaging techniques confirmed PBAN-induced Ca(2+) influx in the silkworm, Bombyx mori, and showed that the PBAN response is reduced with repeated stimulation. Compounds known to impact Ca(2+) signaling were examined for their effects on sex pheromone production. These experiments demonstrated that the PBAN signal is likely mediated by a store-operated channel (SOC). SOC blockers, SKF-96365 and 2-aminoethoxydiphenyl borate, abolished sex pheromone production, as did flufenamic acid, a blocker of transient receptor potential (TRP) channels. Thapsigargin mimicked the pheromonotropic effects of PBAN. Similar results were seen when PBAN-induced lipase activity was assayed. Conversely, 1-oleoyl-2-acetyl-sn-glycerol and arachidonic acid, activators of diacylglycerol-dependent Ca(2+) channels, had no effect on bombykol production.     

Decyl-thio-trifluoropropanone, a competitive inhibitor of moth pheromone receptors

An earlier study (Pophof 1998) showed that the esterase inhibitor decyl-thio-trifluoropropanone inhibited the responses of two receptor neurons of the moth Antheraea polyphemus tuned to straight-chain pheromone components, an acetate and an aldehyde, respectively. Here we report that decyl-thio-trifluoropropanone also inhibited the responses of two pheromone receptor neurons of Bombyx mori to bombykol and bombykal. In contrast, decyl-thio-trifluoropropanone activated receptor neurons of the moth Imbrasia cytherea tuned to the pheromone component (Z)-5-decenyl 3-methyl-butanoate. However, decyl-thio-trifluoropropanone did not affect the responses of two receptor neurons of B. mori females specialized to the plant volatiles benzoic acid and linalool, respectively. These results indicate that decyl-thio-trifluoropropanone, besides inhibiting the sensillar esterase, interferes with proteins involved specifically in the excitation of pheromone receptor neurons. In binding studies with radiolabelled decyl-thio-trifluoropropanone, the inhibitor was bound by the pheromone-binding protein of A. polyphemus. However, the amount of decyl-thio-trifluoropropanone causing response inhibition was 300 times lower than the amount of pheromone-binding protein present in the sensilla. Since the amount of decyl-thio-trifluoropropanone adsorbed corresponded to about the maximum number of receptor molecules calculated per sensillum, we expect that decyl-thio-trifluoropropanone, probably in complex with pheromone-binding protein, competitively inhibits the pheromone receptor molecules. Accepted: 8 January 2000     

Requirement for Drosophila SNMP1 for Rapid Activation and Termination of Pheromone-Induced Activity

Pheromones are used for conspecific communication by many animals. In Drosophila, the volatile male-specific pheromone 11-cis vaccenyl acetate (cVA) supplies an important signal for gender recognition. Sensing of cVA by the olfactory system depends on multiple components, including an olfactory receptor (OR67d), the co-receptor ORCO, and an odorant binding protein (LUSH). In addition, a CD36 related protein, sensory neuron membrane protein 1 (SNMP1) is also involved in cVA detection. Loss of SNMP1 has been reported to eliminate cVA responsiveness, and to greatly increase spontaneous activity of OR67d-expressing olfactory receptor neurons (ORNs). Here, we found the snmp1¹ mutation did not abolish cVA responsiveness or cause high spontaneous activity. The cVA responses in snmp1 mutants displayed a delayed onset, and took longer to reach peak activity than wild-type. Most strikingly, loss of SNMP1 caused a dramatic delay in signal termination. The profound impairment in signal inactivation accounted for the previously reported “spontaneous activity,” which represented continuous activation following transient exposure to environmental cVA. We introduced the silk moth receptor (BmOR1) in OR67d ORNs of snmp1¹ flies and found that the ORNs showed slow activation and deactivation kinetics in response to the BmOR1 ligand (bombykol). We expressed the bombykol receptor complex in Xenopus oocytes in the presence or absence of the silk moth SNMP1 (BmSNMP) and found that addition of BmSNMP accelerated receptor activation and deactivation. Our results thus clarify SNMP1 as an important player required for the rapid kinetics of the pheromone response in insects.     

Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms

Olfaction plays an essential role in many important insect behaviors such as feeding and reproduction. To detect olfactory stimuli, an odorant receptor co-receptor (Orco) is required. In this study, we deleted the Orco gene in the Lepidopteran model insect, Bombyx mori, using a binary transgene-based clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. We initially generated somatic mutations in two targeted sites, from which we obtained homozygous mutants with deletion of a 866 base pair sequence. Because of the flight inability of B. mori, we developed a novel method to examine the adult mating behavior. Considering the specialization in larval feeding, we examined food selection behavior in Orco somatic mutants by the walking trail analysis of silkworm position over time. Single sensillum recordings indicated that the antenna of the homozygous mutant was unable to respond to either of the two sex pheromones, bombykol or bombykal. An adult mating behavior assay revealed that the Orco mutant displayed a significantly impaired mating selection behavior in response to natural pheromone released by a wild-type female moth as well as an 11:1 mixture of bombykol/bombykal. The mutants also exhibited a decreased response to bombykol and, similar to wild-type moths, they displayed no response to bombykal. A larval feeding behavior assay revealed that the Orco mutant displayed defective selection for mulberry leaves and different concentrations of the volatile compound cis-jasmone found in mulberry leaves. Deletion of BmOrco severely disrupts the olfactory system, suggesting that BmOrco is indispensable in the olfactory pathway. The approach used for generating somatic and homozygous mutations also highlights a novel method for mutagenesis. This study on BmOrco function provides insights into the insect olfactory system and also provides a paradigm for agroforestry pest control.     

Representation of a mixture of pheromone and host plant odor by antennal lobe projection neurons of the silkmoth Bombyx mori

Pheromone-source orientation behavior can be modified by coexisting plant volatiles. Some host plant volatiles enhance the pheromonal responses of olfactory receptor neurons and increase the sensitivity of orientation behavior in the Lepidoptera species. Although many electrophysiological studies have focused on the pheromonal response of olfactory interneurons, the response to the mixture of pheromone and plant odor is not yet known. Using the silkmoth, Bombyx mori, we investigated the physiology of interneurons in the antennal lobe (AL), the primary olfactory center in the insect brain, in response to a mixture of the primary pheromone component bombykol and cis-3-hexen-1-ol, a mulberry leaf volatile. Application of the mixture enhanced the pheromonal responses of projection neurons innervating the macroglomerular complex in the AL. In contrast, the mixture of pheromone and cis-3-hexen-1-ol had little influence on the responses of projection neurons innervating the ordinary glomeruli whereas other plant odors dynamically modified the response. Together this suggests moths can process plant odor information under conditions of simultaneous exposure to sex pheromone.