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Pheromone-binding proteins (PBPs) are specific odorant-binding proteins that can specifically recognize insect pheromones. Through transcriptional analysis of the antennae of adult Endoclita signifer, EsigPBP3 was discovered and identified, and EsigPBP3 was found to be highly expressed in the antennae of male moths. Based on the binding characteristics and ability of EsigPBP3, we can find the key ligands and binding site to consider as a target to control the key wood bore E. signifier. In this study, the fluorescence competitive binding assays (FCBA) showed that EsigPBP3 had a high binding affinity for seven key eucalyptus volatiles. Molecular docking analysis revealed that EsigPBP3 had the strongest binding affinity for the sexual pheromone component, (3E,7E)-4,7,11-trimethyl-1,3,7,10-dodecatetraene. Furthermore, same as the result of FCBA, the EsigPBP3 exhibited high binding affinities to key eucalyptus volatiles, eucalyptol, α-terpinene, (E)-beta-ocimene, (−)-β-pinene, and (−)-α-pinene, and PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 are key sites. In summary, EsigPBP3 exhibits high binding affinity to male pheromones and key volatile compounds and the crucial binding sites PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 can act as targets in the recognition of E. signifier pheromones. [ABSTRACT FROM AUTHOR]

Pheromone-binding proteins (PBPs) play important roles in binding and transporting sex pheromones. However, the PBP genes identified in coleopteran insects and their information sensing mechanism are largely unknown. Cyrtotrachelus buqueti (Coleoptera: Curculionidae) is a major insect pest of bamboo plantations. In this study, a novel PBP gene, CbuqPBP2, from C. buqueti was functionally characterized. CbuqPBP2 was more abundantly expressed in the antennae of both sexes than other body parts, and its expression level was significantly male-biased. Fluorescence competitive binding assays showed that CbuqPBP2 exhibited the strongest binding affinity to dibutyl phthalate (Ki = 6.32 μM), followed by styrene (Ki = 11.37 μM), among twelve C. buqueti volatiles. CbuqPBP2, on the other hand, showed high binding affinity to linalool (Ki = 10.55), the main volatile of host plant Neosinocalamus affinis. Furthermore, molecular docking also demonstrated the strong binding ability of CbuqPBP2 to dibutyl phthalate, styrene, and linalool, with binding energy values of −5.7, −6.6, and −6.0 kcal/mol, respectively, and hydrophobic interactions were the prevailing forces. The knockdown of CbuqPBP2 expression via RNA interference significantly reduced the electroantennography (EAG) responses of male adults to dibutyl phthalate and styrene. In conclusion, these results will be conducive to understanding the olfactory mechanisms of C. buqueti and promoting the development of novel strategies for controlling this insect pest. [ABSTRACT FROM AUTHOR]

Pheromone-binding proteins (PBPs) are mainly responsible for binding and transporting hydrophobic pheromone molecules across the aqueous sensilla lymph to the receptor proteins. The preference of each PBP is believed to be different for each pheromone component within a single species. Significantly higher expression level of PBP1 and PBP2 in the male antennae of Spodoptera frugiperda suggesting that Sfru PBP1 and Sfru PBP2 might play important roles in pheromone perception. However, the preference of these two PBP to the three main pheromone components Z9-14: Ac, Z7-12: Ac and Z11-16: Ac have not been determined. In this study, a fluorescence competitive binding assay revealed that the binding intensities of Sfru PBP1 and Sfru PBP2 to Z9-14: Ac or Z7-12: Ac was comparable. We then used the CRISPR/Cas9 system to individually or simultaneously knock out PBP1 and PBP2 in S.frugiperda. The result of courtship behavior indicated that Sfru PBP1 and Sfru PBP2 were indispensable and played equal roles in perceiving the pheromones Z9-14: Ac and Z7-12: Ac for orientation, wing vibration, and hair-pencil display. Compared with Z9-14:Ac and Z7-12: Ac, Z11-16: Ac showed higher or medium binding intensities with Sfru PBP1 and Sfru PBP2 but played a minor role in inducing the wing vibration behavior. The results of this study are valuable for elucidating the mechanisms involved in sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques. [Display omitted] • Sfru PBP1 and Sfru PBP2 reveals different binding affinity with three sex pheromones. • Sfru PBP1 and Sfru PBP2 play equal role for completing the courtship behavior. • Z11-16:Ac could elicit the wing vibration behavior through Sfru PBP1 and Sfru PBP2. • Z11-16:Ac could not induce the orientation and hair-pencil displaying behavior. [ABSTRACT FROM AUTHOR]

Sex pheromone-binding proteins (PBPs) play an important role in sex pheromone recognition in Lepidoptera. However, the mechanisms of chemical communication mediating the response to sex pheromones remain unclear in the diurnal moths of the superfamily Zygaenoidea. In this study, Phauda flammans (Walker) (Lepidoptera: Zygaenoidea: Phaudidae) was used as a model insect to explore the molecular mechanism of sex pheromone perception in the superfamily Zygaenoidea. Two novel pheromone-binding proteins (PflaPBP1 and PflaPBP2) from P. flammans were identified. The two pheromone-binding proteins were predominantly expressed in the antennae of P. flammans male and female moths, in which PflaPBP1 had stronger binding affinity to the female sex pheromones Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal, PflaPBP2 had stronger binding affinity only for (Z, Z, Z)-9, 12, 15-octadecatrienal, and no apparent binding affinity to Z-9-hexadecenal. The molecular docking results indicated that Ile 170 and Leu 169 are predicted to be important in the binding of the sex pheromone to PflaPBP1 and PflaPBP2. We concluded that PflaPBP1 and PflaPBP2 may be responsible for the recognition of two sex pheromone components and may function differently in female and male P. flammans. These results provide a foundation for the development of pest control by exploring sex pheromone blocking agents and the application of sex pheromones and their analogs for insect pests in the superfamily Zygaenoidea. [ABSTRACT FROM AUTHOR]

Athetis lepigone Möschler (Lepidoptera, Noctuidae) is a common maize pest in Europe and Asia. However, there is no long-term effective management strategy is available yet to suppress its population. Adults rely heavily on olfactory cues to locate their optimal host plants and oviposition sites. Pheromone-binding proteins (PBPs) are believed to be responsible for recognizing and transporting different odorant molecules to interact with receptor membrane proteins. In this study, the ligand-binding specificities of two AlepPBPs (AlepPBP2 and AlepPBP3) for sex pheromone components and host plant (maize) volatiles were measured by fluorescence ligand-binding assay. The results demonstrated that AlepPBP2 had a high affinity with two pheromones [(Z)-7-dodecenyl acetate, Ki = 1.11 ± 0.1 μM, (Z)-9-tetradecenyl acetate, Ki = 1.32 ± 0.15 μM] and ten plant volatiles, including (-)-limonene, α-pinene, myrcene, linalool, benzaldehyde, nonanal, 2-hexanone, 3-hexanone, 2-heptanone and 6-methyl-5-hepten-2-one. In contrast, we found that none of these chemicals could bind to AlepPBP3. Our results clearly show no significant differences in the functional characterization of the binding properties between AlepPBP2 and AlepPBP3 to sex pheromones and host plant volatiles. Furthermore, molecular docking was employed for further detail on some crucial amino acid residues involved in the ligand-binding of AlepPBP2. These findings will provide valuable information about the potential protein binding sites necessary for protein-ligand interactions which appear as attractive targets for the development of novel technologies and management strategies for insect pests. [ABSTRACT FROM AUTHOR]

Unappealing shriveled fruits are a characteristic of one of the most elusive fruit pests. The perpetrator, Eudocima materna, attacks the fruit at a fully formed stage and, therefore, the antennal transcriptome for this insect was deduced to identify the molecular elicitors involved in the attraction to its host plants. A total of 260 olfactory genes, including 16 odorant-binding proteins (OBPs), four pheromone-binding proteins (PBPs), 40 antennal-binding proteins (ABPs), 178 odorant receptors (ORs), 17 chemosensory proteins (CSPs) and five sensory neuron membrane proteins (SNMPs) were identified. Phylogenetic analysis shows the divergence of E. materna proteins from closely related lepidopterans and provides insights on genes that have exclusively evolved in this insect. STRING network analysis revealed interactions of olfactory proteins among themselves and the proteins of other groups. Interestingly, online tools predicted RNA-editing events in the odorant receptor sequences, suggesting the possibility of multiple protein forms. Transcripts matching transposable element sequences were also detected in the dataset. Thus, the work reported here provides a valuable resource to design molecular methods for pest control. [ABSTRACT FROM AUTHOR]

Abstract Pheromone-binding proteins (PBPs) play a significant role in olfaction and mating. The present work was designed to isolate, extract, and purify the pheromone-binding proteins from the antennae of male Chilo partellus (Swinhoe, 1885) (Lepidoptera: Crambidae). The pheromone-binding proteins extracted from the male antennae were found to be 770 μg in 100 mg of sample. Pheromone-binding protein molecular weight was determined as 17 kDa by SDS-PAGE analysis. Identified proteins were purified through gel extraction with a recovery percentage of proteins up to 95%. Purified protein samples are analyzed on native PAGE gels. Relative mobility of proteins was determined as 0.574 nm in the densitometry analysis. These identified pheromone-binding proteins can be used for identification of novel pheromone compounds in in vitro studies. This study can be helpful in designing integrated pest management programs to control the maize stem borer by mass trapping of male moths.

BACKGROUND Diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most important crucifer pests. Commercial sex attractants have been developed to monitor and control P. xylostella. However, some studies have demonstrated a variety of pheromone responses of P. xylostella in different locations of the world. Soluble pheromone‐binding proteins (PBPs), as a subfamily of odorant‐binding proteins (OBPs), could selectively bind and transport pheromones across aqueous sensillar lymph to the surface of olfactory receptor neurons. It is worthy to study whether the mutation of PxylPBPs is one of the reasons for the different responses of sex attractors in different regions. RESULTS: In this study, P. xylostella males were collected from seven Chinese provinces, including Hainan, Guangdong, Yunnan, Fujian, Hunan, Zhejiang, and Hebei. PxylPBP1, PxylPBP2, and PxylPBP3 were cloned, and 3, 6, and 32 types of mutation pattern were identified, respectively. These mutation patterns were distributed in each province with different frequency. The results of fluorescence displacement binding assay and in silico simulation revealed that the three mutant PxylPBP3 were more sensitive to Z11‐16:Ald than the reference protein (ACI28451). CONCLUSION: This result implied that mutation of PxylPBP3 may have contributed to regional differences in pheromone responses of P. xylostella. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Red palm weevil (Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation.

Abstract: Proteins in the venom of the fire ant Solenopsis invicta have been suggested to function in pheromone binding. Venom from queens and workers contains different isoforms of these proteins, consistent with the differing pheromones they secrete, but questions remain about the venom protein composition and glandular source. We found that the queen venom contains a previously uncharacterized pheromone‐binding protein paralogue known as Sol i 2X1. Using imaging mass spectrometry, we located the main venom proteins in the poison sac, implying that pheromones might have to compete with venom alkaloids for binding. Using the known structure of the worker venom protein Sol i 2w, we generated three‐dimensional homology models of the worker venom protein Sol i 4.02, and of the two main venom proteins in queens and female alates, Sol i 2q and Sol i 2X1. Surprisingly, the models show that the proteins have relatively small internal hydrophobic binding pockets that are blocked by about 10 amino acids of the C‐terminal region. For these proteins to function as carriers of hydrophobic ligands, a conformational change would be required to displace the C‐terminal region, somewhat like the mechanism known to occur in the silk moth pheromone‐binding protein. [ABSTRACT FROM AUTHOR]

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) play a significant role in olfaction and mating. The present work was designed to isolate, extract, and purify the pheromone-binding proteins from the antennae of male Chilo partellus (Swinhoe, 1885) (Lepidoptera: Crambidae). The pheromone-binding proteins extracted from the male antennae were found to be 770 μg in 100 mg of sample. Pheromone-binding protein molecular weight was determined as 17 kDa by SDS-PAGE analysis. Identified proteins were purified through gel extraction with a recovery percentage of proteins up to 95%. Purified protein samples are analyzed on native PAGE gels. Relative mobility of proteins was determined as 0.574 nm in the densitometry analysis. These identified pheromone-binding proteins can be used for identification of novel pheromone compounds in in vitro studies. This study can be helpful in designing integrated pest management programs to control the maize stem borer by mass trapping of male moths. [ABSTRACT FROM AUTHOR]

The olfactory systems of terrestial animals are designed to trap and sample volatile hydrophobic molecules. Some of these molecules are odorants, such as volatile plant compounds and pheromonesemitted from the other organisms. Insect antennae have a primary function of detecting odors including sex pheromones and plant volatiles. In moths, the organs devoted to olfactory perception have been identified in antennae as the sensilla trichoid and basiconic, the former being tuned to the perception of pheromones. The hydrophobic semiochemicals are solublized by odorant-binding proteins and transported through an aqueous environment (sensillar lymph) to the olfactory receptors, where the signal transduction starts. These proteins, subdivided into pheromone-binding proteins and general odorant-binding proteins, all have a hallmark of six conserved cysteine residues forming three disulfide bridges which are essential for the rigidity of their three-dimensional structures. Pheromones are naturally occuring odorless chemical messenger compounds found in all insects, animals, and humans. They could attract male insectsfrom a long distance exceeding kilometers. Pheromone-binding proteins are small helical proteins (13-17 kDa) present in several sensory organs from moth and other insect species. Odorant-binding proteins are small (15 kDa) soluble proteins, very concentrated in the lymph of chemosensory sensilla. A third class of small proteins has been identified in several sensorial organs from a number of insect orders. They have been separated into a group of chemosensory proteins, characterized by four cystein residues and with low sequence similarity to odorant-binding proteins. They are involved in chemoperception (olfaction and taste) and to play a role in chemical transport from air or water to chemosensitive receptors.

Pheromone-binding proteins (PBPs) are believed to be involved in the recognition of semiochemicals. In the present study, western blot analysis, fluorescence-binding characteristics and immunolocalization of CmedPBP4 from the rice leaffolder, Cnaphalocrocis medinalis, were investigated. Western blot analysis revealed that CmedPBP4 showed obvious antenna-specific expression patterns in female and male antenna, and made a clearly different sex-biased expression. Immunocytochemical labeling revealed that CmedPBP4 showed specific expression in the trichoid sensilla. Competitive fluorescence binding assays indicated that CmedPBP4 could selectively recognize three sex pheromone components (Z13-18:Ac, Z11-16:Al and Z13-18:OH) and eleven rice plant volatiles, including cyclohexanol, nerolidol, cedrol, dodecanal, ionone, (−)-α-cedrene, (Z)-farnesene, β-myrcene, R-(+)-limonene, (−)-limonene, and (+)-3-carene. Meanwhile the CmedPBP4 detection of sex pheromones and host odorants was pH-dependent. Our results, for the first time, provide further evidence that trichoid sensilla might be play an important role in detecting sex pheromones and host plant volatiles in the C. medinalis moth. Our systematic studies provided further detailed evidence for the function of trichoid sensilla in insect semiochemical perception. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

Pheromone-binding proteins ( PBPs) are distributed widely on the antennae of insects, and they are believed to be involved in the process of chemical signal transduction, but their interaction with chemicals is largely unknown. Here, we present our findings on the key amino acid residues of PBPs in the gypsy moth, Lymantria dispar. Potential key residues were screened with the Calculate Mutation Energy program and molecular docking methods. Mutated proteins were obtained by mutating residues to alanine via site-directed mutagenesis. Circular dichroism ( CD) spectroscopy showed that the mutated proteins formed α-helix, and the stability of protein structure was influenced due to mutations. Fluorescence binding assays were further conducted with the mutated proteins, sex pheromones and analogues. Results showed that to PBP 1, tyrosine at position 41 and phenylalanine at position 76 could be the key amino acid residues influencing the stability of structure; in addition, phenylalanine at 36 and lysine at position 94 could be key amino acid residues interacting with chemicals. To PBP 2, glycine at position 49, phenylalanine at position 76 and lysine at position 121 could be the key amino acid residues in the structural stability. These results shed light on the relationship between the specific amino acids and functions of PBPs in transmitting the chemical signals. [ABSTRACT FROM AUTHOR]

Pine caterpillar moths, Dendrolimus spp. (Lepidoptera: Lasiocampidae), are serious economic pest of pines. Previously, phylogenetic analyses of Dendrolimus using different methods yielded inconsistent results. The chemosensory systems of insects may play fundamental roles in promoting speciation. Odorant-binding proteins (OBPs) participate in the first step of odor detection. Studying the evolution of OBPs in closely related species may help us to identify their role in speciation. We identified three OBPs - one pheromone-binding protein and two general odorant-binding proteins - from male antennae of four Dendrolimus species, D. superans (Butler), D. punctatus (Walker), D. kikuchii Matsumura, and D. houi Lajonquiere, the olfactory recognition systems of which had not been previously investigated. We analyzed their molecular characteristics and compared their sequences to those of OBPs in D. tabulaeformis Tsai et Liu. Ka/Ks ratio analyses among the five Dendrolimus species indicate that PBP1 genes experienced more evolutionary pressure than the GOBPs. Phylogenetic relationships of PBP1 and GOBP1 both indicated that D. houi was the basal species, then branched D. kikuchii, while D. tabulaeformis, D. punctatus, and D. superans evolved more recently. These relationships are consistent with the changes in sex pheromone components of these five species. Dendrolimus tabulaeformis and D. punctatus are closely related sister species. However, the distances among GOBP2 sequences in the five Dendrolimus were very short, and the relationships of D. houi and D. kikuchii could not be resolved. Integrating our results with those of previous studies, we hypothesized that D. kikuchii, D. punctatus and D. superans evolved from the basal ancestor because of sex pheromone mutations and environmental pressure. [ABSTRACT FROM AUTHOR]

Pheromone-binding proteins (PBPs) are thought to contribute to the specificity of the pheromone detection system through an initial selective binding with pheromone molecules. Here, we report different expression levels of PBP transcripts in the antennae of two populations of the stemborer Sesamia nonagrioides (Lepidoptera: Noctuidae), one collected in Europe and one in sub-Saharan Africa. The three PBP transcripts previously identified in this species were found to be expressed in both male and female antennae. Whereas PBP3 did not show any differential expression, PBP1 and PBP2 appeared to be expressed differently according to the population origin and sex. Simultaneously, we measured and compared the ratio of the three components of the S. nonagrioides pheromone blend (Z11-16:Ac; Z11-16:OH; Z11-16:Ald) in females of the two populations. The ratio of Z11-16:OH and Z11-16:Ald varied significantly according to the population origin of this species. Cluster analyses revealed similar differentiation patterns between PBP1 and PBP2 expression levels and the ratios of Z11-16:OH and Z11-16:Ald. Different female sexual signals may thus correspond to different male reception systems, which are adjusted by the PBP expression levels, thereby ensuring optimal communication within populations. [ABSTRACT FROM AUTHOR]

Sex pheromone-binding proteins (PBPs) play an important role in sex pheromone recognition in Lepidoptera. However, the mechanisms of chemical communication mediating the response to sex pheromones remain unclear in the diurnal moths of the superfamily Zygaenoidea. In this study, Phauda flammans (Walker) (Lepidoptera: Zygaenoidea: Phaudidae) was used as a model insect to explore the molecular mechanism of sex pheromone perception in the superfamily Zygaenoidea. Two novel pheromone-binding proteins ( PflaPBP1 and PflaPBP2 ) from P. flammans were identified. The two pheromone-binding proteins were predominantly expressed in the antennae of P. flammans male and female moths, in which PflaPBP1 had stronger binding affinity to the female sex pheromones Z -9-hexadecenal and ( Z , Z , Z )-9, 12, 15-octadecatrienal, PflaPBP2 had stronger binding affinity only for ( Z , Z , Z )-9, 12, 15-octadecatrienal, and no apparent binding affinity to Z -9-hexadecenal. The molecular docking results indicated that Ile 170 and Leu 169 are predicted to be important in the binding of the sex pheromone to PflaPBP1 and PflaPBP2. We concluded that PflaPBP1 and PflaPBP2 may be responsible for the recognition of two sex pheromone components and may function differently in female and male P. flammans . These results provide a foundation for the development of pest control by exploring sex pheromone blocking agents and the application of sex pheromones and their analogs for insect pests in the superfamily Zygaenoidea.

Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins, using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila odorant-binding protein that binds the pheromone 11- cis vaccenyl acetate ( cVA). Refolding of LUSH expressed in Escherichia coli was assessed by measuring N-phenyl-1-naphthylamine ( NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 ( W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin- cVA dissociation constant, gives the LUSH- cVA dissociation constant: ∼100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ∼200 nM for the silk moth pheromone bombykol and ∼90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the Laughlin, Ha, Jones and Smith model of pheromone reception are discussed. [ABSTRACT FROM AUTHOR]

Our knowledge of the molecular basis of odorant reception in insects has grown exponentially over the past decade. Odorant receptors (ORs) from moths, fruit flies, mosquitoes, and the honey bees have been deorphanized, odorant-degrading enzymes (ODEs) have been isolated, and the functions of odorant-binding proteins (OBPs) have been unveiled. OBPs contribute to the sensitivity of the olfactory system by transporting odorants through the sensillar lymph, but there are competing hypotheses on how they act at the end of the journey. A few ODEs that have been demonstrated to degrade odorants rapidly may act in signal inactivation alone or in combination with other molecular traps. Although ORs in Drosophila melanogaster respond to multiple odorants and seem to work in combinatorial code involving both periphery and antennal lobes, reception of sex pheromones by moth ORs suggests that their labeled lines rely heavily on selectivity at the periphery. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Binding properties of six heterologously expressed pheromone-binding proteins (PBPs) identified in the silkmoths Antheraea polyphemus and Antheraea pernyi were studied using tritium-labelled pheromone components, (E,Z)-6,11-hexadecadienyl acetate (3H-Ac1) and (E,Z)-6,11-hexadecadienal (3H-Ald), common to both species. In addition, a known ligand of PBP and inhibitor of pheromone receptor cells, the tritium-labelled esterase inhibitor decyl-thio-1,1,1-trifluoropropanone (3H-DTFP), was tested. The binding of ligands was measured after native gel electrophoresis and cutting gel slices. In both species, PBP1 and PBP3 showed binding of 3H-Ac1. In competition experiments with 3H-Ac1 and the third unlabelled pheromone component, (E,Z)-4,9-tetradecadienyl acetate (Ac2), the PBP1 showed preferential binding of Ac1, whereas PBP3 preferentially bound Ac2. The PBP2 of both species bound 3H-Ald only. All of the six PBPs strongly bound 3H-DTFP. Among unlabelled pheromone derivatives, alcohols were revealed to be the best competitors for 3H-Ac1 and 3H-Ald bound to PBPs. No pH influence was found for 3H-Ac1 binding to, or its release from, the PBP3 of A. polyphemus and A. pernyi between pH 4.0 and pH 7.5. The data indicate binding preference of each of the three PBP-subtypes (1–3) for a specific pheromone component and support the idea that PBPs contribute to odour discrimination, although to a smaller extent than receptor activation. [ABSTRACT FROM AUTHOR]

Red palm weevil (Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation. [ABSTRACT FROM AUTHOR]