Structure-activity studies with pheromone-binding proteins of the gypsy moth,Lymantria dispar

Pheromone olfaction in the gypsy moth, Lymantria dispar, involves accurate distinction of compounds with similar structure and polarity. The identified sex pheromone is (7R,8S)-2-methyl-7,8-epoxyoctadecane, 1a, and a known antagonist is (7Z)-2-methyloctadec-7-ene, 4a. The first step in pheromone olfaction is binding of odorants by small, soluble pheromone-binding proteins (PBPs), found in the pheromone-sensing hairs. We have studied the molecular determinants recognized by the two PBPs found in the gypsy moth, using three pheromone/PBP binding assays. Results indicate that (i) PBPs bind analogs of the pheromone with some discrimination; (ii) PBPs experience enhancement of binding when presented with 1a or its enantiomer and 4a simultaneously; and (iii) the binding enhancement is also seen at high ligand:PBP ratios. We found no evidence of allostery, so the synergistic binding effects and the concentration effect may only be explained by multimerization of PBPs with each other, which leads to more than one population of binding sites. We suggest that the enhanced ligand binding at high ligand:PBP ratios may serve to sequester excess ligand and thereby attenuate very strong signals.     

A pheromone-binding protein mediates the bombykol-induced activation of a pheromone receptor in vitro

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.     

Structural and functional difference of pheromone binding proteins in discriminating chemicals in the gypsy moth, Lymantria dispar

Pheromone-binding proteins (PBPs) of the gypsy moth, Lymantria dispar L., play an important role in olfaction. Here structures of PBPs were first built by Homology Modeling, and each model of PBPs had seven α-helices and a large hydrophobic cavity including 25 residues for PBP1 and 30 residues for PBP2. Three potential semiochemicals were first screened by CDOCKER program based on the PBP models and chemical database. These chemicals were Palmitic acid n-butyl ester (Pal), Bis(3,4-epoxycyclohexylmethyl) adipate (Bis), L-trans-epoxysuccinyl-isoleucyl-proline methyl ester propylamide (CA-074). The analysis of chemicals docking the proteins showed one hydrogen bond was established between the residues Lys94 and (+)-Disparlure ((+)-D), and л-л interactions were present between Phe36 of PBP1 and (+)-D. The Lys94 of PBP1 formed two and three hydrogen bonds with Bis and CA-074, respectively. There was no residue of PBP2 interacting with these four chemicals except Bis forming one hydrogen bond with Lys121. After simulating the conformational changes of LdisPBPs at pH7.3 and 5.5 by constant pH molecular dynamics simulation in implicit solvent, the N-terminal sequences of PBPs was unfolded, only having five α-helices, and PBP2 had larger binding pocket at 7.3 than PBP1. To investigate the changes of α-helices at different pH, far-UV and near-UV circular dichroism showed PBPs consist of α-helices, and the tertiary structures of PBP1 and PBP2 were influenced at pH7.3 and 5.5. The fluorescence binding assay indicated that PBP1 and PBP2 have similarly binding affinity to (+)-D at pH 5.5 and 7.3, respectively. At pH 5.5, the dissociation constant of the complex between PBP1 and 2-decyl-1-oxaspiro [2.2] pentane (OXP1) was 0.68 ± 0.01 μM, for (+)-D was 5.32 ± 0.11 μM, while PBP2 with OXP1 and (+)-D were 1.88 ± 0.02 μM and 5.54 ± 0.04 μM, respectively. Three chemicals screened had higher affinity to PBP1 than (+)-D except Pal at pH5.5, and had lower affinity than (+)-D at pH7.3. To PBP2, these chemicals had lower affinity than the sex pheromone except Bis at pH 5.5 and pH 7.3. Only PBP1 had higher affinity with Sal than the sex pheromone at pH 5.5. Therefore, the structures of PBP1 and PBP2 had different changes at pH5.5 and 7.3, showing different affinity to chemicals. This study helps understanding the role of PBPs as well as in developing more efficient chemicals for pest control.     

Discrimination of pheromone enantiomers by two pheromone binding proteins from the gypsy moth Lymantria dispar

The gypsy moth, Lymantria dispar, uses (7R, 8S)-cis-2-methyl-7, 8-epoxyoctadecane, (+)-disparlure, as a sex pheromone. The (-) enantiomer of the pheromone is a strong behavioral antagonist. Specialized sensory hairs, sensillae, on the antennae of male moths detect the pheromone. Once the pheromone enters a sensillum, the very abundant pheromone binding protein (PBP) transports the odorant to the sensory neuron. We have expressed the two PBPs found in gypsy moth antennae, PBP1 and PBP2, and we have studied the affinity of these recombinant PBPs for the enantiomers of disparlure. To study pheromone binding under equilibrium conditions, we developed and validated a binding assay. We have addressed the two major problems with hydrophobic ligands in aqueous solution: (1) concentration-dependent adsorption of the ligand on vial surfaces and (2) separation of the protein-bound ligand from the material remaining free in solution. We used this assay to demonstrate for the first time that pheromone binding to PBP is reversible and that the two PBPs from L. dispar differ in their enantiomer binding preference. PBP1 has a higher affinity for the (-) enantiomer, while PBP2 has a higher affinity for the (+) enantiomer. The PBP from the wild silk moth, Antheraea polyphemus (Apol-3) bound the disparlure enantiomers more weakly than either of the L. dispar PBPs, but Apol-3 was also able to discriminate the enantiomers. We have observed extensive aggregation of both L. dispar PBPs and an increase in pheromone binding at high (>2 microM) PBP concentrations. We present a model of disparlure binding to the two PBPs.     

Insights into genome differentiation: pheromone-binding protein variation and population history in the European corn borer (Ostrinia nubilalis)

Examination of sequence variation at nuclear loci can give insights into population history and gene flow that cannot be derived from other commonly used molecular markers, such as allozymes. Here, we report on sequence variation at a single nuclear locus, the pheromone-binding protein (PBP) locus, in the European corn borer (Ostrinia nubilalis). The European corn borer has been divided into three races in New York State on the basis of differences in pheromone communication and life history. Previous allozyme data have suggested that there is a small but significant amount of genetic differentiation between these races. The PBP does not appear to be involved in the pheromone differences between these races. Examination of variation at the PBP locus in the three races reveals no fixed differences between races despite high levels of polymorphism. There also appears to have been considerable recombination in the history of the pheromone-binding protein alleles. Observation of both recombination between alleles and lack of significant nucleotide or insertion/deletion divergence between races leads us to suggest that these populations are either recently diverged or have continued to exchange genetic material subsequent to divergence in pheromone communication and life history.     

Effects of aromatic compounds on antennal responses and on the pheromone-binding proteins of the gypsy moth (Lymantria dispar)

Female gypsy moths emit a pheromone, (+)-disparlure, which the males follow until they locate the emitter. The male moths' antennae are covered with innervated sensory hairs, specialized in detection of the pheromone. The neurons in these sensory hairs are bathed by a solution rich in pheromone-binding protein (PBP). PBPs are soluble proteins that bind the pheromone and other odorants reversibly with variable thermodynamic and kinetic selectivity and are essential for olfactory responses. Here, we have studied the interaction between 2 gypsy moth PBPs with aromatic compounds that modulate the responses of male moth antennae to (+)-disparlure. The aromatic compounds do not elicit responses by themselves, but when administered together with pheromone, they inhibit, enhance, or prolong the electrophysiological response to the pheromone. Three interactions between the compounds and PBPs were studied: 1) the equilibrium binding of the compounds by themselves to the PBPs, 2) the equilibrium binding of the compounds in the presence of pheromone or a fluorescent reporter ligand, and 3) the effect of the compounds on the conformation of the pheromone-PBP complex. A subset of compounds causes a prolongation of the electroantennogram response, and from this study, we conclude that these compounds follow a structure-activity pattern and stabilize a particular conformer of the PBPs that appears to activate the olfactory response.     

CRISPR/Cas9‐mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis

Pheromone-binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo.In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target-gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBPl-sgRNA injected eggs and 19.5% for PBP3-sgRNA injected eggs. Second, by an in-crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA-injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.     

Do Pheromone Binding Proteins Converge in Amino Acid Sequence When Pheromones Converge?

Convergence in amino acid sequences between proteins can be strong evidence for selection. Here, I look for evidence of convergence in the amino acid sequences of pheromone binding protein (PBP) in response to convergence in pheromones. PBPs are involved in sex pheromone reception by the antennae of male moths. In this role PBPs may selectively bind pheromone components and experience convergent selection in response to convergence in pheromone components. However, examination of the PBPs of the taxa that have converged upon the use of (E)- or (Z)-11-tetradecenyl acetate as their major pheromone component reveals little evidence for convergence in the PBPs identified from these taxa. A few sites show a pattern consistent with convergence or parallelism; however, it cannot be ruled out that these sites share the ancestral state. Two of these sites fall within the proposed binding region of PBPs. These results suggest that PBPs either have not converged in sequence or have converged at very few sites in response to convergence on the same pheromone component. Received: 29 July 1999 / Accepted: 8 November 1999     

Identification and Characterization of Pheromone Receptors and Interplay between Receptors and Pheromone Binding Proteins in the Diamondback Moth,Plutella xyllostella

Moths depend on olfactory cues such as sex pheromones to find and recognize mating partners. Pheromone receptors (PRs) and Pheromone binding proteins (PBPs) are thought to be associated with olfactory signal transduction of pheromonal compounds in peripheral olfactory reception. Here six candidate pheromone receptor genes in the diamondback moth, Plutella xyllostella were identified and cloned. All of the six candidate PR genes display male-biased expression, which is a typical characteristic of pheromone receptors. In the Xenopus-based functional study and in situ hybridization, PxylOR4 is defined as another pheromone receptor in addition to the previously characterized PxylOR1. In the study of interaction between PRs and PBPs, PxylPBPs could increase the sensitivity of the complex expressing oocyte cells to the ligand pheromone component while decreasing the sensitivity to pheromone analogs. We deduce that activating pheromone receptors in olfactory receptor neurons requires some role of PBPs to pheromone/PBP complex. If the chemical signal is not the pheromone component, but instead, a pheromone analog with a similar structure, the complex would have a decreased ability to activate downstream pheromone receptors.     

Pheromone-binding proteins contribute to the activation of olfactory receptor neurons in the silkmoths antheraea polyphemus and Bombyx mori

The sensilla trichodea of the silkmoth Antheraea polyphemus are innervated by three types of receptor neurons each responding specifically to one of three pheromone components. The sensillum lymph of these sensilla surrounding the sensory dendrites contains three different types of pheromone-binding proteins (PBPs) in high concentrations. The sensilla trichodea of the silkmoth Bombyx mori are supplied by two receptor neurons each tuned specifically to one of the two pheromone components bombykol and bombykal, but only one type of PBP has been found so far in these sensilla. Recombinant PBPs of both silkmoth species in various combinations with pheromone components were applied to the receptor neurons via tip-opened sensilla during electrophysiological recordings. Over a fairly broad range of pheromone concentrations the responses of the receptor neurons depended on both, the pheromone component and the type of the PBP. Therefore, the PBPs appear to contribute to the excitation of the receptor neurons. Furthermore, bombykal in combination with the expressed PBP of B. mori failed to activate the corresponding receptor neuron of B. mori, but did so if combined with one of the PBPs of A. polyphemus. Therefore, a still unknown binding protein involved in bombykal transport might be present in B. mori.     

Discrimination of cis-trans sex pheromone components in two sympatric Lepidopteran species

Pheromone-binding proteins (PBPs) play an important role in the recognition of pheromones by insects. However, the abilities of these PBPs to discriminate pheromone components and recognize the isomers are unclear. Dendrolimus houi and Dendrolimus kikuchii are two sympatric coniferous pests whose pheromones have cis-trans isomers. We used these insect species to detect the precise recognition abilities of PBPs. The four PBPs examined showed male-biased antenna-intensive expression patterns, whereas PBP1 showed higher expression than PBP2 in the antenna. DhouPBP1 only bound to a minor interspecific pheromone component, whereas DhouPBP2 bound to all three intraspecific components and another minor interspecific component. DkikPBP1 and DkikPBP2 could recognize all three intraspecific components with affinities negatively correlated with their ratios, and they bound to interspecific pheromones with affinity that was positively correlated with the ratios. The four PBPs have different cis-trans isomer discrimination abilities, i.e., DhouPBP1 and DkikPBP1 could not discriminate the two cis-trans isomer pairs of pheromones from the two species, whereas DhouPBP2 could discriminate between both pairs, and DkikPBP2 could only discriminate one pair. Overall, PBPs from D. houi and D. kikuchii use different strategies to help the moths to discriminate the intra- and interspecific pheromone components. Our work will contribute to better understanding of the sex pheromone recognition mechanism in these two sister species of moths and provide insights into more effective management practices of these pest species.     

Pheromone‐binding proteins in the Asian gypsy moth females,Lymantria dispar,recognizing the sex pheromone and plant volatiles

Lepidopterans are known to have different pheromone‐binding proteins with differential expression patterns that facilitate specific signal transduction of semiochemicals. Two PBPs of the Asian gypsy moth, Lymantria dispar, were reported to express in both females and males, but their physiological functions were unknown. Results showed that LdisPBP1 and LdisPBP2 were expressed in the sensilla trichodea of males and the s. trichodea and s. basiconica of females. When LdisPBP1 gene was targeted by RNA interference (RNAi) in males, the expression of LdisPBP1 and LdisPBP2 decreased by 69 and 76%, respectively, and when LdisPBP2 gene was targeted by RNAi, they decreased by 60 and 42%, respectively. In females, after treatment with LdisPBP1 dsRNA, LdisPBP1 and LdisPBP2 levels were reduced by 26 and 69%, respectively, and LdisPBP2 dsRNA reduced the relative expression of them by 4 and 62%, respectively. The expression of LdisPBP1 and LdisPBP2 was interdependent. Electroantennogram (EAG) recordings showed that LdisPBPs participate in the recognition of the sex pheromone in males, and the sex pheromone and plant volatiles in females. The function of LdisPBPs represents the sex‐specific roles.     

Olfaction in the gypsy moth, Lymantria dispar: effect of pH, ionic strength, and reductants on pheromone transport by pheromone-binding proteins

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.     

Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1). Functional and structural characterization.

Pheromone binding proteins (PBPs) are small proteins (17 kDa on average) present at high concentrations ( approximately 10 mM) in the sensillum lymph of Lepidoptera antennae, where they play a key role in the perception of pheromones. By expression in Escherichia coli, we have obtained large quantities (2-3 mg.L-1) of pure, soluble, Mamestra brassicae PBP1 (MbraPBP1). These quantities are compatible with the requirements of X-ray and NMR studies. The recombinant protein has been characterized by native-polyacrylamide gel electrophoresis, Western blotting, N-terminal sequencing, mass spectrometry, gel filtration, circular dichroism, and NMR. Moreover, the recombinant MbraPBP1 has been shown to be able to bind the specific pheromone and a structural analogue, Z11-16:TFMK (cis-11-hexadecenyl trifluoromethyl ketone), in displacement experiments. Our results on MbraPBP1 confirm and extend previous findings on PBPs. MbraPBP1 and two PBPs from different species have been found to exist as dimers under nondenaturing conditions. The CD and structural prediction data confirm a markedly helical structure for insect PBPs rather than the beta-barrel fold found in vertebrates odorant binding proteins. We have tentatively identified the location of the helices and the short beta-strands with respect to the binding site. Currently we have obtained small diffracting crystals of the recombinant MbraPBP1 and determined their space group and molecular content.     

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.     

Olfactory neuron responsiveness and pheromone blend preference in hybrids between Ostrinia furnacalis and Ostrinia nubilalis (Lepidoptera: Crambidae)

The olfactory receptor neuron (ORN) and behavioral responses of hybrids between the Asian corn borer (ACB), Ostrinia furnacalis, and the E-strain European corn borer (ECB(E)), Ostrinia nubilalis were examined and compared to the parental populations. In hybrids and both parents, the large-spike-size ORN was capable of responding to all four pheromone components of ACB and ECB, despite differences in which compounds elicited the greatest spike frequency in each population. There was a small-spiking ORN more narrowly tuned to the minor pheromone components in both ACB and ECB(E). In hybrids the homologous small-spiking ORN was tuned primarily to the ECB(E) minor pheromone component, with some responsiveness to the ACB minor component. Both species and all the hybrids had an intermediate spike-size ORN tuned primarily to their common behavioral antagonist. Dominance of responsiveness to the ECB(E) versus the ACB minor pheromone component on the small-spiking ORN may explain the greater tendency of hybrids to fly upwind to the ECB(E) pheromone blend than the ACB blend. This finding points toward a distinct evolutionary role for this ORN in allowing a pheromone shift.     

Relatedness of penicillin-binding proteins from various Listeria species

The heterogeneity of penicillin-binding proteins (PBPs) of five Listeria species was investigated. Similarities in the overall PBP pattern were found between those of L. welshimeri and L. innocua, and between L. ivanovii and L. seeligeri, and all were distinct from the PBPs of L. monocytogenes. In all species, however, the primary target for beta-lactam antibiotics, as identified in L. monocytogenes recently, appeared highly conserved. In addition, the low-Mr PBP 5 was biochemically very similar in all strains and contained identical binding properties to beta-lactam compounds, suggesting that this protein may play an important role. All other PBPs varied considerably in their penicilloyl-peptide pattern, indicating differences in their amino acid sequences.     

Evidence for directional selection acting on pheromone-binding proteins in the genus Choristoneura

Patterns of nucleotide variation consistent with the action of natural selection have been discovered at a number of different gene loci. Here, pheromone-binding proteins (PBPs) are examined to determine if selection has acted to fix amino acid changes in PBPs in lineages in which pheromone changes have occurred. PBPs from five different species of moths in the genus Choristoneura were sequenced, along with the PBP of Argyrotaenia velutinana, which serves as an outgroup. Three independent major pheromone changes are represented within this group of five Choristoneura species. Two different lineages show evidence for selection based on polymorphism and divergence comparisons and comparisons of rates of replacement evolution to silent and noncoding evolution. Along one of these lineages, leading to Choristoneura fumiferana, there has been a change to an aldehyde pheromone from an acetate pheromone. The second branch does not appear to be associated with a major pheromone change. Other branches in the tree show a trend toward greater replacement fixation than expected under neutrality. This trend could reflect undetected selective events within this group of PBPs. Selection appears to have acted to fix amino acid changes in the PBP of moths from the genus Choristoneura, but it is not clear that this selection is due to pheromone changes between species.     

Different Penicillin-Binding Protein Profiles in Amoxicillin-Resistant Helicobacter pylori

Background. The β-lactam group of antibiotics kills bacteria by inhibiting the terminal stages of peptidoglycan metabolism. We have recently identified amoxicillin-resistant Helicobacter pylori , none of which expressed β-lactamase. Penicillin-binding proteins (PBPs) represent a group of target enzymes for the β-lactam antibiotic family, and alterations in PBPs have been described in other penicillin-resistant bacteria. The amoxicillin-resistant phenotype characteristically was lost after freezing but could be restored by consecutive transfers into gradient plates.
Materials and Methods. To determine whether amoxicillin resistance in H. pylori was related to alterations in any of the H. pylori PBPs, five H. pylori strains resistant to amoxicillin and three amoxicillin-sensitive strains were tested. PBPs were extracted from bacteria grown to logarithmic phase, labeled in vivo with ³H-benzylpenicillin, and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Four main PBPs were separated from all amoxicillin-sensitive H. pylori strains.
Results. Only three of the four main PBPs were found in the amoxicillin-resistant H. pylori strains. The differentially detectable PBP (PBP D) had an apparent molecular weight of 30 to 32 kD.
Conclusion. These results suggest that PBP D might play a role in the amoxicillin-resistant phenotype of H. pylori strains lacking β-lactamase activity.