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Dioxin-like molecules have been associated with endocrine disruption and liver disease. To better understand aryl hydrocarbon receptor (AHR) biology, metabolic phenotyping and liver proteomics were performed in mice following ligand-activation or whole-body genetic ablation of this receptor. Male wild type (WT) and Ahr–/– mice (Taconic) were fed a control diet and exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) (61 nmol/kg by gavage) or vehicle for two weeks. PCB126 increased expression of canonical AHR targets (Cyp1a1 and Cyp1a2) in WT but not Ahr–/–. Knockouts had increased adiposity with decreased glucose tolerance; smaller livers with increased steatosis and perilipin-2; and paradoxically decreased blood lipids. PCB126 was associated with increased hepatic triglycerides in Ahr–/–. The liver proteome was impacted more so by Ahr–/– genotype than ligand-activation, but top gene ontology (GO) processes were similar. The PCB126-associated liver proteome was Ahr-dependent. Ahr principally regulated liver metabolism (e.g., lipids, xenobiotics, organic acids) and bioenergetics, but it also impacted liver endocrine response (e.g., the insulin receptor) and function, including the production of steroids, hepatokines, and pheromone binding proteins. These effects could have been indirectly mediated by interacting transcription factors or microRNAs. The biologic roles of the AHR and its ligands warrant more research in liver metabolic health and disease., Graphical abstract The aryl hydrocarbon receptor regulated liver metabolism and endocrine function impacting systemic energy homeostasis and body composition. This regulation appeared to involve both direct and indirect mechanisms.Image 1

Rap/Rgg/NprR/PlcR/PrgX (RRNPP) quorum-sensing systems use extracellular peptide pheromones that are detected by cytoplasmic receptors to regulate gene expression in firmicute bacteria. Rgg-type receptors are allosterically regulated through direct pheromone binding to control transcriptional activity; however, the receptor activation mechanism remains poorly understood. Previous work has identified a disulfide bond between Cys-45 residues within the homodimer interface of Rgg2 from Streptococcus dysgalactiae (Rgg2Sd). Here, we compared two Rgg2Sd(C45S) X-ray crystal structures with that of wild-type Rgg2Sd and found that in the absence of the intermolecular disulfide, the Rgg2Sd dimer interface is destabilized and Rgg2Sd can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2Sd secondary and tertiary structures, but another displayed more extensive rigid-body shifts as well as dramatic secondary structure changes. In parallel experiments, a genetic screen was used to identify mutations in rgg2 of Streptococcus pyogenes (rgg2Sp ) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2Sp*, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2Sd(C45S) crystal structures. The Rgg2Sp* mutations increased Rgg2Sp sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2Sp* mutations invoke shifts in free-energy bias to favor the active state of the protein. Finally, we present evidence for an electrostatic interaction between an N-terminal Asp of the pheromone and Arg-153 within the proposed pheromone-binding pocket of Rgg2Sp.

Helicoverpa armigerautilizes (Z)-11-hexadecenal (Z11-16:Ald) as its major sex pheromone component. Three pheromone binding proteins (PBPs) and two general odorant binding proteins (GOBPs) are abundantly expressed in male antennae ofH. armigera. However, their precise roles in the olfactory detection of Z11-16:Ald remain enigmatic. To answer this question, we first synthesized the antibody against HarmOR13, a pheromone receptor (PR) primarily responding to Z11-16:Ald and mapped the local associations between PBPs / GOBPs and HarmOR13. Immunostaining showed that HarmPBPs and HarmGOBPs were localized in the supporting cells of sensilla trichodea and sensilla basiconica respectively. In particular, HarmPBP1 and HarmPBP2 were colocalized in the cells surrounding the olfactory receptor neurons (ORNs) expressing HarmOR13. Next, using two noninterfering binary expression tools, we heterologously expressed HarmPBP1, HarmPBP2 and HarmOR13 inDrosophilaT1 sensilla to validate the functional interplay between PBPs and HarmOR13. We found that the addition of HarmPBP1 or HarmPBP2 significantly increased the sensitivity of HarmOR13 to Z11-16:Ald. However, the presence of either HarmPBP1 or HarmPBP2 was ineffective to change the tuning breadth of HarmOR13. Taken together, our results support the idea that PBPs are contributors to the peripheral olfactory sensitivity but do not affect the selectivity. Lastly, we discovered that HarmOR13 and theDrosophilaOR67d employed a similar coding mechanism to detect pheromones, suggesting that pheromone detection across different insect orders appears to co-opt a conserved molecular principle to recognize pheromone ligands.

Pheromone binding proteins (PBPs) are well studied in lepidopteran moths and are considered to be crucial in detection of sex pheromones as well as some green leaf volatiles. In contrast, evidence that PBPs interact with sex pheromones of hemipteran species is not available. The mirid plant bug, Adelphocoris lineolatus (Goeze), is a notorious hemipteran pest that uses two butyrate esters, trans-2-hexenyl butyrate (E2HB) and hexyl butyrate (HB), and one hexenoic aldehyde trans-4-oxo-2-hexenal (E4O2H), as sex pheromones. In the present study, we report on an odorant binding protein, AlinOBP4, with particular focus on its potential physiological roles in the detection of A. lineolatus sex pheromone components. Phylogenetic analyses indicated that AlinOBP4 and two mirid orthologs clustered in a general phylogenetic clade with the lepidopteran ABX OBPs, the fly LUSH and the OBP83a/b subfamily. Cellular localization by fluorescence in situ hybridization and immunolabeling further demonstrated that AlinOBP4 was strongly expressed in the multiporous sensilla trichodea (str) and middle long sensilla basiconica (mlsba) of male A. lineolatus adults, suggesting a key role associated with sex pheromone and odorant detection. A ligand binding assay revealed that recombinant AlinOBP4 protein highly bound not only to the sex pheromone components E4O2H but also to some host plant volatiles. These findings together with the evidence of insect PBPs available in the literature support the view that AlinOBP4 is involved in sex pheromone detection in male A. lineolatus and provide foundational information for further elucidating the molecular mechanisms of chemosensory based mating behavior in hemipteran mirid bugs.

Insect pheromone-binding proteins (PBPs) play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors (ORs). However, the PBPs of the oriental fruit moth, Grapholita molesta, an important destructive pest of stone fruits worldwide, are not well characterized. In this study, two new putative PBP genes, GmolPBP2 and GmolPBP3, were identified from G. molesta antennae. The deduced amino-acid sequences of these two putative PBP genes are characteristic of the odorant binding protein family, containing six conserved cysteine residues. The genomic DNA sequence of each gene contained two introns. However, the lengths and positions of the introns differed. RT-PCR analyses revealed that the two GmolPBP genes are only expressed in the antennae of female and male moths. Quantitative real-time PCR indicated that the transcription levels of GmolPBP2 are far greater than those of GmolPBP3 in both female and male antennae. GmolPBP3 showed higher transcription levels in female antennae than in male antennae, while GmolPBP2 showed similar transcription levels in both female and male antennae. The transcript levels of both genes were significantly different in premating and post-coitum individuals, implying that mating affects the process of sex pheromone reception. To better understand the functions, two GmolPBPs were expressed in Escherichia coli, and the ligand binding assays were conducted. Results showed that GmolPBP2 has strong binding affinities to two sex pheromone components, E8–12:Ac and Z8–12:Ac, as well as weaker binding affinities to Z8-12:OH and 12:OH. GmolPBP2 also bound some ordinary odor molecules. However, the affinity of GmolPBP3 to both sex pheromones and ordinary odor molecules was very weak. These results show that GmolPBP2 plays the main role in pheromone discrimination and recognition in the oriental fruit moth.

Pheromone-binding proteins (PBPs), a sub-family of odorant-binding proteins, are thought primarily to bind and transport the sex pheromones in moths. Considering multiple components of sex pheromone and multiple PBP genes exist in a single species, PBPs may contribute to the discrimination of different sex pheromone components. However, so far this discrimination is still unclear. Our previous ligand-binding assays showed that Spodoptera litura PBP1 (SlitPBP1) did not exhibit an obvious binding specificity among different sex pheromone components. In this study, binding specificity of the other two PBPs in S. litura (SlitPBP2 and SlitPBP3) was further investigated. As a result, SlitPBP2 was capable of binding all four sex pheromone components with similar affinities; whereas SlitPBP3 showed very weak binding affinities to them except Z9,E12-14:Ac. Similar results were also obtained from studied pheromone analogs, to which SlitPBP2 showed much stronger affinities than SlitPBP3. However, both SlitPBP2 and SlitPBP3 exhibited overall weaker affinities to sex pheromones and their analogs than SlitPBP1. In addition, quantitative real time PCR showed that three SlitPBP genes exhibited a very different sex-biased expression in adult antenna with male-biased for SlitPBP1 and SlitPBP2 while female-biased for SlitPBP3. Finally, ligand-binding assays indicated that the two SlitPBPs showed a similar pH-dependent conformational change as reported SlitPBP1, but these three SlitPBPs showed different behavior across a pH range or something similar. Taken together, our data suggest that in S. litura PBP1 and PBP2 may play critical roles in the perception of female sex pheromones, but do not show an obvious discriminative ability among different sex pheromone components; whereas PBP3 may have other functions.

Chemoreception is a key feature in selection of host plants by insects. In this study, preliminary characterization and isolation of cDNA clones from Cnaphalocrocis medinalis antennal libraries identified eight olfactory genes, including two putative general odorant-binding proteins (GOBPs), three pheromone binding proteins (PBPs) and three chemosensory proteins (CSPs). The expression profiles of these eight genes in different tissues (antenna, head (without antennae), thorax, abdomen, leg and wing) were measured by real time qPCR. The results showed that GOBP and PBP genes in C. medinalis seemed to be antenna-specific, but differentially expressed in male and female antennae; while CSP genes were expressed ubiquitously during different developmental stages, but with an extremely elevated transcript level in antennae, legs and wings compared to head, thorax and abdomen. And also, the transcription levels of olfactory genes depended on the age, sex, and mating status of the adults. These findings support the hypothesis that OBPs and CSPs play dynamic roles during development of C. medinalis and are likely to be involved in broader physiological functions.

The two sibling species Helicoverpa armigera and Helicoverpa assulta utilise the same two aldehydes as their sex pheromones, but in opposite ratios. In both species three odorant-binding proteins (OBPs) can be classified as pheromone-binding proteins (PBPs). To investigate the role of these three PBPs in chemical communication between sexes and their mode of action, we have expressed the proteins in bacteria and prepared mutants lacking their C-terminal regions. Using polyclonal antibodies we found that the expression of the three PBPs is basically confined to the antennae of both sexes and both species. Binding experiments with the fluorescent probe N-phenyl-1-naphthylamine across a pH range indicated that, the affinity of wild-type proteins decreases at low pH, while that of the mutants is not or less affected, suggesting that a conformational change of the C-terminus occurs in these proteins, as reported for other lepidopteran OBPs. All three proteins bind with similar strength both pheromone components, as well as their corresponding alcohols and acetates. However, they exhibit significant selectivity to linear alcohols and aldehydes of different length, with optimal affinities to the ligand of 13–15 carbon atoms for PBP1 and 12–14 carbon atoms for PBP2. We suggest that all three PBPs might cooperate to build a unique olfactory image, that could help avoiding cross-mating between the two species and with other noctuids.

Three pheromone-binding proteins of Helicoverpa armigera were cloned and expressed in Escherichia coli. In order to characterize their physiological properties, ligand-binding experiments were performed using five biologically relevant substances including sex pheromones and interspecific signals. The results showed that one of the pheromone-binding proteins, HarmPBP1, binds strongly to each of the two principal pheromone components of H. armigera, (Z)-11-tetradecenal and (Z)-9-hexadecenal, but not to the interspecific signal (Z)-9-tetracecenal. The two remaining pheromone-binding proteins, HarmPBP2 and HarmPBP3, showed only weak affinities with the ligands tested. The 3-D structure of HarmPBP1 was predicted and the docking experiments indicate that the key binding site of (Z)-9-hexadecenal to HarmPBP1 includes Thr112, Lys111, and Phe119 whereas that of (Z)-11-tetradecenal includes Ser9, Trp37, Phe36, and Phe119.

Summary Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. Here, we show that detection of the Drosophila pheromone, 11- cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, LUSH. We show that LUSH undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in LUSH that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, LUSH D118A , produces a dominant-active LUSH protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of LUSH D118A reveals that it closely resembles cVA-bound LUSH. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants.

The yeast pheromone receptor, Ste2p, is a G protein coupled receptor that initiates cellular responses to α-mating pheromone, a 13 residue peptide that carries a net positive charge at physiological pH. We have examined the role of extracellular charged groups on the receptor in response to the pheromone. Substitutions of Asn or Ala for one extracellular residue, Asp275, affected both pheromone binding and signaling, suggesting that this position interacts directly with ligand. The other seven extracellular acidic residues could be individually replaced by polar residues with no detectable effects on receptor function. However, substitution of Ala for each of these seven residues resulted in impairment of signaling without affecting pheromone binding, implying that the polar nature of these residues promotes receptor activation. In contrast, substitution of Ala for each of the six positively charged residues at the extracellular surface of Ste2p did not affect signaling.

A major 20-kDa protein is female-specifically expressed in exorbital lacrimal gland (LG) of hamsters and secreted in tears. Here, we identify this female-specific LG protein (FLP) as a lipocalin, having 85% protein sequence identity with male-specific submandibular salivary gland proteins (MSP) secreted in saliva and urine of male hamsters. MSP is also female-specifically expressed in LG and secreted in tears but FLP was undetectable in submandibular gland (SMG). FLP and MSP have similar sex-hormonal regulation in LG, which is different from regulation of MSP in SMG. Female-specific expression of FLP and MSP in LG is due to their incomplete repression by endogenous estrogens and gonadectomy in both sexes and lactation in females resulted in their marked induction, which was prevented by estrogen or androgen treatment. FLP and MSP show best sequence identity with odorant/pheromone-binding lipocalins (58-29%). Maximum identity (58%) is with rat odorant-binding protein (OBP) expressed in lateral nasal glands, followed by aphrodisin of hamster vaginal discharge (39%). Cognate transcript and a cross-reacting 20-kDa protein were detected in nasal glands of rat in both sexes but not in hamsters. Results suggest that two closely related lipocalin genes encode FLP and MSP, which are evolutionarily closer to rat OBP than to hamster aphrodisin and these have evolved different tissue-specificity and sex-hormonal regulation. Possible functions for FLP and MSP are suggested, considering their homology to odorant/pheromone-binding lipocalins, their presence in tears, saliva and urine as well as their sex-specific and lactation-induced expression.

Pheromone-binding proteins (PBPs) located in the antennae of male moth species play an important role in olfaction. They are carrier proteins, believed to transport volatile hydrophobic pheromone molecules across the aqueous sensillar lymph to the membrane-bound G protein-coupled olfactory receptor proteins. The roles of PBPs in molecular recognition and the mechanisms of pheromone binding and release are poorly understood. Here, we report the NMR structure of a PBP from the giant silk moth Antheraea polyphemus. This is the first structure of a PBP with specific acetate-binding function in vivo. The protein consists of nine alpha-helices: alpha1a (residues 2-5), alpha1b (8-12), alpha1c (16-23), alpha2 (27-34), alpha3a (46-52), alpha3b (54-59), alpha4 (70-79), alpha5 (84-100) and alpha6 (107-125), held together by three disulfide bridges: 19-54, 50-108 and 97-117. A large hydrophobic cavity is located inside the protein, lined with side-chains from all nine helices. The acetate-binding site is located at the narrow end of the cavity formed by the helices alpha3b and alpha4. The pheromone can enter this cavity through an opening between the helix alpha1a, the C-terminal end of the helix alpha6, and the loop between alpha2 and alpha3a. We suggest that Trp37 may play an important role in the initial interaction with the ligand. Our analysis also shows that Asn53 plays the key role in recognition of acetate pheromones specifically, while Phe12, Phe36, Trp37, Phe76, and Phe118 are responsible for non-specific binding, and Leu8 and Ser9 may play a role in ligand chain length recognition.

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.

Insect odorant-binding proteins (OBPs) are thought to facilitate the delivery of hydrophobic odorants, such as sex pheromones or food odors, to receptors on sensory neurons. Increasingly, OBP family members are also being found in non-sensory tissues where they might carry other types of small hydrophobic molecules. They are identifiable by four or six conserved Cys residues and contain six α-helices which enclose a hydrophobic ligand-binding pocket. Through exhaustive BLAST searches we have increased the total number of OBPs identified in Drosophila melanogaster to 38, and have amplified the DNA complementary to RNA corresponding to 21 of these by reverse transcriptase polymerase chain reaction. Isoforms frequently share less than 30% amino acid identity and appear to have radically changed since the separation of the major insect orders. However, their sequences are consistent with known OBP structures. Most are located in clusters of between four and 14 genes and several were unusual in that they contained additions, deletions, or fusions. These hexa-helical insect OBPs are structurally unrelated to the functionally analogous lipocalin-like β-barrel OBPs of vertebrates. As only two lipocalin-like proteins have been found in D. melanogaster, these helical proteins appear to be the dominant carrier of small hydrophobic molecules in insects.

One subtype of the pheromone binding proteins of the silkmoth Antheraea polyphemus (ApolPBP1) has been analysed exploiting the two endogenous tryptophan residues as fluorescent probe. The intrinsic fluorescence exhibited a rather narrow spectrum with a maximum at 336 nm. Site-directed mutagenesis experiments revealed that one of the tryptophan residues (Trp37) is located in a hydrophobic environment whereas Trp127 is more solvent exposed, as was predicted modeling the ApolPBP1 sequence on the proposed structure of the Bombyx mori pheromone binding protein. Monitoring the interaction of ApolPBP1 as well as its Trp mutants with the three species-specific pheromone compounds by recording the endogenous fluorescence emission revealed profound differences; whereas (E6,Z11)-hexadecadienal induced a dose-dependent quenching of the fluorescence, both (E6,Z11)-hexadecadienyl-1-acetate and (E4,Z9)-tetradecadienyl-1-acetate elicited an augmentation of the endogenous fluorescence. These data indicate that although ApolPBP1 can bind all three pheromones, there are substantial differences concerning their interaction with the protein, which may have important functional implications.

The pheromone-binding proteins (PBPs) are 16-kDa abundant proteins in specialized olfactory hairs in insects. The mechanism by which the PBPs remove the pheromone from the inner surface of sensory hairs and deliver it to the sensory cell remains unclear. Existing qualitative models postulate that pheromone is released near the dendrite by a decrease in pH or by a reduced form of the PBP. This study focuses on the two PBPs from the gypsy moth and the enantiomers of the pheromone cis-2-methyl-7,8-epoxyoctadecane. The pH dependence of pheromone binding has revealed three ionizations that are important. The type of ligand influences two of these ionizations. We propose that the (−)-enantiomer of the pheromone interacts with one of the ionizable residues on the protein while the (+)-enantiomer does not. Simultaneous variation of pH and KCl concentration in the physiological range or reduction of disulfide bridges does not change the affinity of PBP for pheromone. We propose a revised model of pheromone transport from the inner surface of the sensory hair to the sensory neuron.

Pheromone-binding proteins (PBPs), located in the sensillum lymph of pheromone-responsive antennal hairs, are thought to transport the hydrophobic pheromones to the chemosensory membranes of olfactory neurons. It is currently unclear what role PBPs may play in the recognition and discrimination of species-specific pheromones. We have investigated the binding properties and specificity of PBPs from Mamestra brassicae (MbraPBP1), Antheraea polyphemus (ApolPBP1), Bombyx mori (BmorPBP), and a hexa-mutant of MbraPBP1 (Mbra1-M6), mutated at residues of the internal cavity to mimic that of BmorPBP, using the fluorescence probe 1-aminoanthracene (AMA). AMA binds to MbraPBP1 and ApolPBP1, however, no binding was observed with either BmorPBP or Mbra1-M6. The latter result indicates that relatively limited modifications to the PBP cavity actually interfere with AMA binding, suggesting that AMA binds in the internal cavity. Several pheromones are able to displace AMA from the MbraPBP1- and ApolPBP1-binding sites, without, however, any evidence of specificity for their physiologically relevant pheromones. Moreover, some fatty acids are also able to compete with AMA binding. These findings bring into doubt the currently held belief that all PBPs are specifically tuned to distinct pheromonal compounds.

Background: Insects use volatile organic molecules to communicate messages with remarkable sensitivity and specificity. In one of the most studied systems, female silkworm moths ( Bombyx mori ) attract male mates with the pheromone bombykol, a volatile 16-carbon alcohol. In the male moth's antennae, a pheromone-binding protein conveys bombykol to a membrane-bound receptor on a nerve cell. The structure of the pheromone-binding protein, its binding and recognition of bombykol, and its full role in signal transduction are not known. Results : The three-dimensional structure of the B. mori pheromone-binding protein with bound bombykol has been determined by X-ray diffraction at 1.8 A resolution. Conclusions : The pheromone binding protein of B. mori has six helices, and bombykol binds in a completely enclosed hydrophobic cavity formed by four antiparallel helices. Bombykol is bound in this cavity through numerous hydrophobic interactions, and sequence alignments suggest critical residues for specific pheromone binding.

Background: THP12 is an abundant and extraordinarily hydrophilic hemolymph protein from the mealworm Tenebrio molitor and belongs to a group of small insect proteins with four highly conserved cysteine residues. Despite their sequence homology to odorant-binding proteins and pheromone-binding proteins, the function of these proteins is unclear. Results: The first three-dimensional structure of THP12 has been determined by multidimensional NMR spectroscopy. The protein has a nonbundle helical structure consisting of six α helices. The arrangement of the α helices has a ‘baseball glove' shape. In addition to the hydrophobic core, electrostatic interactions make contributions to the overall stability of the protein. NMR binding studies demonstrated the binding of small hydrophobic ligands to the single hydrophobic groove in THP12. Comparing the structure of THP12 with the predicted secondary structure of homologs reveals a common fold for this new class of insect proteins. A search with the program DALI revealed extensive similarity between the three-dimensional structure of THP12 and the N-terminal domain (residues 1–95) of recoverin, a member of the family of calcium-binding EF-hand proteins. Conclusions: Although the biological function of this new class of proteins is as yet undetermined, a general role as α -helical carrier proteins for small hydrophobic ligands, such as fatty acids or pheromones, is proposed on the basis of NMR-shift perturbation spectroscopy.

The pheromone-binding protein (PBP) from Bombyx mori was expressed in Escherichia coli periplasm. It specifically bound radiolabeled bombykol, the natural pheromone for this species. It appeared as a single band both in native and SDS-polyacrylamide gel electrophoresis and was also homogeneous in most chromatographic systems. However, in ion-exchange chromatography, multiple forms sometimes appeared. Attempts to separate them revealed that they could be converted into one another. Analysis of the protein by circular dichroism and fluorescence spectroscopy demonstrated that its tertiary structure was sensitive to pH changes and that a dramatic conformational transition occurred between pH 6.0 and 5.0. This high sensitivity to pH contrasted markedly with its thermal stability and resistance to denaturation by urea. There was also no significant change in CD spectra in the presence of the pheromone. The native protein isolated from male antennae displayed the same changes in its spectroscopic properties as the recombinant material, demonstrating that this phenomenon is not an artifact arising from the expression system. This conformational transition was reproduced by interaction of the protein with anionic (but not neutral) phospholipid vesicles. Unfolding of the PBP structure triggered by membranes suggests a plausible mechanism for ligand release upon interaction of the PBP-pheromone complex with the surface of olfactory neurons. This pH-linked structural flexibility also explains the heterogeneity reported previously for B. mori PBP and other members of this class of proteins.

In the fruit fly Drosophila, odorant-binding proteins are secreted into the fluid that bathes olfactory neurons. Laughlin et al. (2008) now challenge the assumption that the odorant-binding protein LUSH passively transports its pheromone to a specific olfactory receptor. Instead, LUSH undergoes a conformational change upon pheromone binding that is sufficient for neuronal activation.

Despite the remarkable diversity of the sex pheromone chemistry in scarab beetles, various species utilize a common type of γ-lactones in their chemical communication channels. These compounds differ primarily in length of the alkenyl side chain and the stereochemistry at the chiral center. Two species, Anomala osakana and Popillia japonica , utilize the opposite enantiomers of japonilure as sex pheromones. Each species produces only one of the enantiomers that functions as its own sex pheromone and as a behavioral antagonist to the alloreceiver. Pheromone binding proteins (PBPs) have been characterized, which are present in these and several other scarab species. In most cases there was only one class of PBP, which was expressed in both sexes. A. osakana and P. japonica possess each one single PBP with high homology to each other. In each species the same PBP seems to recognize both enantiomers of japonilure, i. e., the pheromonal and the “stop” signals. Based on the N-terminal sequences, the antennae-specific proteins from various other species were highly conserved within the family and showed moderate homology to putative odorant binding protein from Drosophila melanogaster (47%), Lygus lineolaris (45∼50%) and the ABPX protein from Bombyx mori (30∼35%). From analysis of extracts of soluble antennal proteins from several species, significant degradation of the γ-lactones (buibuilactone, japonilure) was detected, essentially in all of them, even in species that do not use these compounds as pheromones. Recently a peculiar pheromone with a diamide moiety [1, 3-dimethyl-2, 4-(1 H , 3 H )-quinazolinedione] was isolated from Phyllopertha diversa , which was rapidly degraded by antennal enzymes from this species. Beetles that utilize lactones as their pheromones possess little or no ability to metabolize this compound.

Proteins extracted form the antennae of Mamestra brassicae (L.) (Lepidoptera: Noctuidae) adults were biochemically characterized as pheromone-binding proteins (PBP) and general odorant-binding proteins (GOBP). PBP and GOBP were purified by two successive and different HPLC (high performance liquid chromatography) systems and native polyacrylamide gel electrophoresis (native-PAGE). Their N-terminal sequence was determined by Edman microsequencing. The combined results showed evidence for three different PBPs in males, and two different PBPs in females. In addition, one GOBP was characterized in both males than in females antennae. In the males, two isoforms of PBP have the same N-terminal sequence, but different apparent mobilities and hydrophobicities: they could be separated by electrophoresis and reverse phase-HPLC (RP-HPLC). The other PBP sequence (SQEIM) showed particularly high homology (88%) with the PBP of Heliothis virescens, another noctuid moth. The existence of several forms of PBP in the same animal strongly supports the hypothesis of the specificity of binding between the proteins and their odorant ligands, the pheromonal compounds. The observed microdiversity at the soluble proteins level could provide a good model for studying their involvement in the initial stages of odor discrimination.

This paper reviews the characteristics of pheromone and odorant-binding proteins (OBP) in insects, with particular reference to Lepidoptera. They are small (15 kDa) soluble proteins, very concentrated in the lymph of chemosensory sensilla and belonging to two major classes, pheromone-binding proteins (PBP) and general odorant-binding proteins. They represent the insect equivalent of vertebrate OBP. The main unsolved question with OBP of insects and vertebrates regards their physiological role in olfactory transduction. The recent discovery of several types of OBP in the same animal species suggests that these proteins may be involved in the discrimination of odours.

Four genes expressed in the olfactory system of Drosophila melanogaster have been identified by subtractive hybridization. Two of these genes, OS-E and OS-F, are related to genes encoding moth pheromone-binding proteins. The OS-E and OS-F genes are tightly linked and are expressed in a subregion of the antenna (the primary olfactory organ). A protein sequence analysis suggests the possibility that pheromone-binding proteins are members of a larger class of proteins, extending beyond the olfactory system. The predicted product of a third gene, OS-D, shares features common to vertebrate odorant-binding proteins, but has a primary structure unlike odorant-binding proteins. The fourth gene, OS-C, encodes a novel 13-kDa protein that contains a putative nuclear import sequence and an acid-rich region. The expression patterns of these genes differ within the antenna; their transcript distributions support the notion of specialized roles for different olfactory sensilla. The functions of the OS gene products have not been demonstrated; however, the potential identification of pheromone-binding proteins in Drosophila, a species with well characterized genetics, may offer a means of analyzing the function of these molecules that is not available in other systems.

Many mammalian social odors do not elicit an observable specific response in the recipient and therefore strictly cannot be considered to be pheromones. The pheromones now known in mammals are mostly transferred by contact and detected by accessory olfaction, which further indicates that pheromones in mammals should not be considered to be even a subclass of social odors. Aphrodisin, a female hamster pheromone that elicits sexual behavior in male hamsters, is a member of the lipocalycin family of 20 kDa extracellular proteins, and it is most closely related to rat odorant binding protein. Homologous proteins occur in the urine and scent glands of mice, rats and possibly voles, where they may serve as pheromone binding proteins. A 20 kDa protein, pheromaxein, binds the known pheromones androstenol and related steroids in boar saliva, and uncharacterized small proteins have been found in monkey and human skin gland secretions. Thus it appears that proteins may generally be associated with mammalian pheromones.

Genomic DNA from the silk moth Antheraea pernyi bearing the gene of a pheromone binding protein has been isolated from a partial genomic library using specific cDNA probes. The DNA spans 3.5 kilobases, contains three exons and two intervening sequences that interrupt the protein coding region of the gene. A DNA fragment of a second gene was isolated and the complete primary structure of a corresponding cDNA clone was unravelled. The expression of two different genes, giving rise to different pheromone binding proteins, implies a more specific function of these proteins than was hitherto assumed.

Before a pheromone molecule of Antheraea polyphemus can stimulate a dendrite of a sensillum trichodeum, it has to pass through an aqueous solution, the “sensillum lymph”. Furthermore, after such a stimulation it has to be rapidly inactivated to prevent continuous stimulation of the dendrite. In the sensillum lymph a pheromone binding protein in mM concentrations is present. The literature describes two hypotheses concerning the function of the binding protein: one hypothesis claims that the latter serves to carry the pheromone through the sensillum lymph, while the other assumes a migration of the pheromone to the dendrite through the pore tubuli and an inactivator function of the binding protein. By means of a perfusion technique, the dendrites of sensilla trichodea were bathed in a solution of pheromone [(E,Z)-6,11-hexadecadienyl acetate] in Ringer with and without binding protein or bovine serum albumin for comparison. The responses of the cells were measured electrophysiologically. To elicit nerve impulses, a pheromone concentration of 180 nM in Ringer was necessary. Addition of binding protein or bovine serum albumin decreased the threshold concentration by a factor of 100. It was calculated that with such a pheromone concentration (1.8 nM) plus binding protein less than 1000 pheromone molecules within the hair produced a strong cell response, although in equilibrium 99.96% of these molecules are bound to the binding protein. It was therefore concluded that pheromone binding protein can act as a solubilizer or carrier.

Using recombinant DNA approaches a cDNA clone was isolated from antennal libraries of Antheraea pernyi which encodes the complete sequence of a putative pheromone or odorant binding protein. The deduced protein sequence consist of a signal peptide of 19 amino acids and a mature protein of 141 amino acid residues. It shares a number of structural features with previously identified pheromone binding proteins although the predicted primary structures show only moderate homologies; Furthermore, the identified clone is preferentially expressed in female antennae.

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431-residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell.

The soluble proteins in the antennae of the male and female saturniid moth Antheraea polyphenus were analyzed by isoelectric focusing in miniature ultrathin-layer polyacrylamide gels and by microgradient PAGE (μPAGE). Different preparations of sensillum lymph from male antennal olfactory hairs (isolated sensillum-lymph droplets, rinsing solution from isolated hairs) and homogenates of male and female antennae were compared. The pheromone-binding protein, PBP, and the pheromone-degrading sensillar esterase, SE, were detected only in male antennal preparations. Isolated sensillum lymph exhibited two proteins in Brilliant Blue staining: (1) the PBP appeared as a double band with a relative molecular mass of M r (α-lactalbumin); (2) an oligopeptide was detected as a faint band at a very small molecular mass ( M r ⪡ 14,000 ). The PBP in isolated sensillum lymph was identical to the PBP in the rinsing solution of isolated hairs and in male branch homogenates. Purified PBP from branch homogenates showed a third slightly smaller band. In reducing SDS-μPAGE, the denatured PBP from all preparations showed only one band at a M r of 14,400. The isoelectric point (pI) of the PBP was 4.7. The PBP appeared to be a non-glycosylated protein. Glycoproteins were present in the hemolymph in different patterns in both sexes. The concentration of PBP in sensillum-lymph droplets was ≈ 10 mM, corresponding to a total amount of ≈9 μg/55 μl sensillum lymph in one antenna. A third protein in the sensillum lymph, the sensillar esterase, was only visible by enzyme staining and had a M r of 55,000 and a pI of 3.0. The integumental esterases found in male and female antennae had a M r of 65,000, pI 5.85, and M r 90,000, pI 5.0. From body scales of both sexes, esterases in the range of M r 100,000 were solubilized by 0.01% Triton buffer and could be demonstrated in μPAGE.

Sexual reproduction in the eukaryotic fungi Achlya is controlled by two steroid pheromones. Antheridiol is the steroid released by female cells that induces male sexual differentiation. The antheridiol-induced response of male cells has been shown to be influenced by the composition of the culture medium. The present study was designed to determine if the composition of the culture media might also affect the levels of antheridiol binding protein in the cytosol of male cells. The mycelial content of cytosolic steroid pheromone binding sites in Achlya ambisexualis E87 males was measured at daily intervals during 6 days of suspension culture in media containing different nitrogen sources. Levels of binding sites increased during the first 2 days in culture to a plateau that was maintained for the next 2–3 days. During the first 3 days in culture, levels were much lower in mycelia cultured in an enriched medium containing lactalbumin hydrolysate compared to mycelia cultured in defined media containing glutamic acid as the nitrogen source. The level of binding sites increased rapidly when mycelia were transferred from an enriched medium to a nutrient-free salt solution and decreased when mycelia were transferred from a defined to an enriched medium. The relative differences in cytosolic binding measured by in vitro radioligand saturation analysis were confirmed by in vivo uptake studies. It is concluded that the mycelial content of antheridiol binding sites can be experimentally manipulated by variations in the composition of the culture medium and/or the time period of incubation in the medium.