Olfactory Binding Proteins Research Articles

Chemical Compositions and Antimicrobial and Mosquito Larvicidal Activities of the Leaf Essential Oils of Goniothalamus yunnanensis W.T.Wang and G. touranensis Ast: Experimental and In Silico Approaches.

The current research describes a phytochemical analysis and antimicrobial activity of essential oils extracted from the leaves of two Vietnamese Annonaceae species Goniothalamus yunnanensis W.T.Wang and G. touranensis Ast. By the GC-FID/MS (gas chromatography-flame ionization detection/mass spectrometry) analyses, sesquiterpene hydrocarbons accounted for the highest percentage of 68.22 % in G. yunnanensis leaf essential oil with bicyclogermacrene (31.03 %) and (E)-caryophyllene (21.12 %) being the main compounds. G. touranensis leaf essential oil was dominated by monoterpene hydrocarbons (57.08 %) with p-cymene (19.95 %) and α-pinene (16.82 %) being the major compounds. By the microdilution broth susceptibility assay, two oil samples showed strong antibacterial effects on the Gram-positive bacteria Enterococcus faecalis ATCC51299, Staphylococcus aureus ATCC29213, and Bacillus cereus ATCC11778 with the MIC values of 16-64 μg/mL. They also inhibited the growth of the yeast Candida albicans ATCC 60193 with the same MIC value of 128 μg/mL. From the vector-based intervention approach, both oil samples showed strong mosquito larvicidal activity against four-instar larvae of Aedes aegypti and Ae. albopictus with the 24 h LC50 values of 16.75-27.60 μg/mL and 24 h LC90 values of 24.31-46.18 μg/mL. Docking results indicated that bicyclogermacrene and p-cymene exhibited the highest ΔG (binding affinity) values of -8.208 and -6.799 kcal/mol with the olfactory binding proteins (OBPs) of Ae. aegypti and Ae. albopictus, respectively.

Food contamination with fipronil alters gene expression associated with foraging in Africanized honey bees.

Taking into consideration that bees can be contaminated by pesticides through the ingestion of contaminated floral resources, we can utilize genetic techniques to assess effects that are scarcely observed in behavioral studies. This study aimed to investigate the genetic effects of ingesting lethal and sublethal doses of the insecticide fipronil in foraging honey bees during two periods of acute exposure. Bees were exposed to fipronil through contaminated honey syrup at two dosages (LD50 = 0.19 µg/bee; LD50/100 = 0.0019 µg/bee) and for two durations (1 and 4 h). Following exposure, we measured syrup consumption per bee, analyzed the transcriptome of bee brain tissue, and identified differentially expressed genes (DEGs), categorizing them functionally based on gene ontology (GO). The results revealed a significant genetic response in honey bees after exposure to fipronil, regardless of the dosage used. Fipronil affected various metabolic, transport, and cellular regulation pathways, as well as detoxification processes and xenobiotic substance detection. Additionally, the downregulation of several DEGs belonging to the olfactory-binding protein (OBP) family was observed, suggesting potential physiological alterations in bees that may lead to disoriented behaviors and reduced foraging efficiency.

Variation of ewe olfactory secretome during a ram effect.

Under temperate latitudes, reproduction in Ovis aries displays a marked seasonality, governed by the photoperiod. In natural conditions, the transition between sexual rest and sexual activity in both sexes is induced by the decrease of day light. Meanwhile, specific odors emitted by a sexually active male are able to reactivate the gonadotropic axis of anovulatory ewes. This physiological effect is called "male effect", precisely ram effect in the ovine species. We have previously shown that the secreted proteins, namely Olfactory Binding Proteins (OBP), contained in the nasal mucus constitute the olfactory secretome (OS), the composition of which is determined by the status of oestrus cycle of females and differs between sexual rest and sexual activity periods. The objective of this study was to test the hypothesis that exposure to sexually active male can also modify the composition of ewes olfactory secretome during a male effect, as well as hormones produced by the reactivation of the oestrus cycle in sexual activity period under natural conditions. We have set up a new non-invasive protocol of nasal mucus sampling and collected it from 12 ewes at different times during a ram effect. We analyzed the composition of their olfactory secretome by proteomics, mainly SDS-PAGE and MALDI-TOF mass spectrometry. As post-translational modifications of OBPs were a hallmark of ewes' sexual activity period, we were looking for glycosylation by western-blot and mass spectrometry. The efficiency of male effect was low in stimulated ewes as only 3 females displayed elevated progesterone levels in their blood. Besides, half of control ewes (non-stimulated ones) were cycled. We noticed a common OS profile in ewes in anoestrus, versus OS of cycled ones. A very clear and important result was the apparition of O-GlcNAcylation, previously detected only in sexual activity, after only 30 min of male introduction into the flock. This exploratory study paves the way for further experiments with larger flock to confirm and reinforce these results, and for eventually exploiting the nasal mucus as an indicator of females' receptivity to male odors.

Odor Identification Performance in Idiopathic Parkinson's Disease Is Associated With Gender and the Genetic Variability of the Olfactory Binding Protein.

Deficits in olfaction are among the most frequent non-motor symptoms in Parkinson's disease (PD) and can be detected early compared with motor symptoms. The reason for the early onset, as well as the mechanism involved remains unknown. We aimed to characterize the olfactory performance of patients with PD and age-matched healthy control (HC) participants in association with gender and a specific polymorphism in the odorant-binding protein IIa (OBPIIa) gene, which plays a crucial role in the perception of odors. The olfactory performance was assessed using the odor identification part of the Sniffin' Sticks test in 249 participants (patients with PD: n = 131 and HC participants: n = 118). All participants were genotyped for the rs2590498 polymorphism of the OBPIIa gene, whose major allele A is associated with a higher retronasal perception than the minor allele G. A higher number of men with PD than women with PD exhibited hyposmia. Importantly, OBPIIa gene polymorphism showed an effect on PD-related olfactory deficits only in women. Women with PD carrying two sensitive alleles (AA) showed a better olfactory performance than women with PD with at least one insensitive allele (G); the olfactory scores of the AA genotype women with PD were not different from those of HC participants. In conclusion, our results confirmed a sex effect on the reduced olfactory performance of patients with PD and identified the OBPIIa locus, which may provide a mechanism to determine the risk factor for olfactory deficits in women with PD at the molecular level.

Binding Specificity of Native Odorant-Binding Protein Isoforms Is Driven by Phosphorylation and O-N-Acetylglucosaminylation in the Pig Sus scrofa.

Odorant-binding proteins (OBP) are secreted in the nasal mucus at the vicinity of olfactory receptors (ORs). They act, at least, as an interface between hydrophobic and volatile odorant molecules and the hydrophilic medium bathing the ORs. They have also been hypothesized to be part of the molecular coding of odors and pheromones, by forming specific complexes with odorant molecules that could ultimately stimulate ORs to trigger the olfactory transduction cascade. In a previous study, we have evidenced that pig olfactory secretome was composed of numerous olfactory binding protein isoforms, generated by O-GlcNAcylation and phosphorylation. In addition, we have shown that recombinant OBP (stricto sensu) produced in yeast is made up of a mixture of isoforms that differ in their phosphorylation pattern, which in turn determines binding specificity. Taking advantage of the high amount of OBP secreted by a single animal, we performed a similar study, under exactly the same experimental conditions, on native isoforms isolated from pig, Sus scrofa, nasal tissue. Four fractions were obtained by using strong anion exchange HPLC. Mapping of phosphorylation and O-GlcNAcylation sites by CID-nanoLC-MS/MS allowed unambiguous localization of phosphosites at S13 and T122 and HexNAc sites at S13 and S19. T112 or T115 could also be phosphorylated. BEMAD analysis suggested extra phosphosites located at S23, S24, S41, S49, S57, S67, and T71. Due to the very low stoichiometry of GlcNAc-peptides and phosphopeptides, these sites were identified on total mixture of OBP isoforms instead of HPLC-purified OBP isoforms. Nevertheless, binding properties of native OBP isoforms to specific ligands in S. scrofa were monitored by fluorescence spectroscopy. Recombinant phosphorylated OBP-Pichia isoforms bind steroids and fatty acids with slight differences. Native isoforms, that are phosphorylated but also O-GlcNAcylated show radically different binding affinities for the same compounds, which strongly suggests that O-GlcNAcylation increases the binding specificity of OBP isoforms. These findings extend the role of O-GlcNAc in regulating the function of proteins involved in many mechanisms of metabolic homeostasis, including extracellular signaling in olfaction. Data is available via ProteomeXChange with identifier PXD011371.

Synthetic Ligands of Olfactory Binding Proteins Modulate Aggregation Response of Asian Citrus Psyllid in the Presence of Host-Plant Volatiles.

There is interest in using ligands of chemosensory binding proteins (CBP) to augment an insect’s responsiveness to chemosensory cues. We showed previously that combining a synthetic ligand of a CBP with limonene, a common citrus volatile, enhanced the probing response of Asian citrus psyllid (Diaphorina citri). Here, we determined whether synthetic compounds, which were ligands of D. citri olfactory binding protein (OBP) DCSAP4, influenced the settling and aggregation levels of psyllids on young citrus shoots. The test ligands and Cmac scent were dispensed from a droplet of an emulsified wax product (SPLAT) placed on the bottom of each vial. The shoots were presented: (1) alone (shoot + blank SPLAT), (2) with a mixture of citrus volatiles (“Cmac scent”) (shoot + SPLAT with Cmac scent), or (3) with different concentrations of test ligands (shoot + SPLAT with test ligand at concentration 1, shoot + SPLAT with test ligand at concentration 2, etc.). Depending on the availability of test ligands, sprigs, and psyllids, each test included from two to four replicates of each treatment (i.e., shoot only, shoot + Cmac scent, shoot + test ligand at concentration 1, shoot + test ligand at concentration 2, etc.); only a single test ligand was presented in each test. For each test, 200 D. citri were released in the test area and the numbers of psyllids on each sprig were counted 24 h later. Sprigs with ≥7 psyllids were considered to be an aggregation. A total of seven ligands were tested individually. Four of the ligands (654, 717, 784, and 861) modulated psyllid settling and aggregation response, causing greater settling and aggregation to sprigs presented with the Cmac scent than to those sprigs with blank SPLAT. Presentation of one of the ligands (019) resulted in an opposite effect in which psyllid settling and aggregation levels were lower on sprigs with Cmac scent than on those with blank SPLAT. There were no differences in settling levels in the different treatment vials in the Ligand 905 experiment. In the Ligand 937 experiment, settling levels did not vary significantly between treatment vials although settling levels were relatively high in all treatment vials and there was a significant treatment effect. Increased settling and aggregation levels were largely not observed with in the vials with only the test ligands, and there was little effect of ligand concentration on psyllid response levels. This suggests that the test ligands themselves did not attract the psyllids but rather modulated the psyllid’s response to the Cmac scent. The results suggest that synthetic ligands of D. citri CBPs can be used to increase the effectiveness of citrus scent lures used to attract psyllids to monitoring traps and attract and kill devices.

The genome of an underwater architect, the caddisfly Stenopsyche tienmushanensis Hwang (Insecta: Trichoptera).

BackgroundCaddisflies (Insecta: Trichoptera) are a highly adapted freshwater group of insects split from a common ancestor with Lepidoptera. They are the most diverse (>16,000 species) of the strictly aquatic insect orders and are widely employed as bio-indicators in water quality assessment and monitoring. Among the numerous adaptations to aquatic habitats, caddisfly larvae use silk and materials from the environment (e.g., stones, sticks, leaf matter) to build composite structures such as fixed retreats and portable cases. Understanding how caddisflies have adapted to aquatic habitats will help explain the evolution and subsequent diversification of the group.FindingsWe sequenced a retreat-builder caddisfly Stenopsyche tienmushanensis Hwang and assembled a high-quality genome from both Illumina and Pacific Biosciences (PacBio) sequencing. In total, 601.2 M Illumina reads (90.2 Gb) and 16.9 M PacBio subreads (89.0 Gb) were generated. The 451.5 Mb assembled genome has a contig N50 of 1.29 M, has a longest contig of 4.76 Mb, and covers 97.65% of the 1,658 insect single-copy genes as assessed by Benchmarking Universal Single-Copy Orthologs. The genome comprises 36.76% repetitive elements. A total of 14,672 predicted protein-coding genes were identified. The genome revealed gene expansions in specific groups of the cytochrome P450 family and olfactory binding proteins, suggesting potential genomic features associated with pollutant tolerance and mate finding. In addition, the complete gene complex of the highly repetitive H-fibroin, the major protein component of caddisfly larval silk, was assembled.ConclusionsWe report the draft genome of Stenopsyche tienmushanensis, the highest-quality caddisfly genome so far. The genome information will be an important resource for the study of caddisflies and may shed light on the evolution of aquatic insects.

30 Years Old: O-GlcNAc Reaches the Age of Reason - Regulation of Cell Signaling and Metabolism by O-GlcNAcylation.

30 Years Old: O-GlcNAc Reaches the Age of Reason - Regulation of Cell Signaling and Metabolism by O-GlcNAcylation.

Proteomic Analysis of Pig (Sus scrofa) Olfactory Soluble Proteome Reveals O-Linked-N-Acetylglucosaminylation of Secreted Odorant-Binding Proteins.

The diversity of olfactory binding proteins (OBPs) is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry, and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT) was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

Roborovskin, a Lipocalin in the Urine of the Roborovski Hamster, Phodopus roborovskii

Many rodents are now known to exhibit an obligate proteinuria that delivers urine-mediated chemosignals. In this paper, we explore the urinary proteins of the Roborovski hamster (Phodopus roborovskii). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of urine from individual male and female Roborovski hamsters revealed 2 proteins, with approximate masses of 6 and 17 kDa, the expression pattern of which showed little variation between individuals or between sexes. Peptide mass fingerprints obtained from these 2 proteins revealed a number of features: 1) the proteins of a given mass were the same in all individuals regardless of sex, 2) the 6 kDa protein was not a fragment of the 21 kDa protein, and 3) neither protein was a fragment of a larger, conserved protein such as serum albumin. Electrospray mass spectrometry of purified protein preparations established the mass of the larger protein as invariant, at 17144 ± 2 Da in all samples. This protein has been termed roborovskin. The primary structure of roborovskin was determined by tandem mass spectrometry of peptides derived from independent and overlapping digestion with 3 proteases, supported by Edman degradation of the protein N-terminus. Roborovskin shared significant homology with olfactory-binding proteins from Myodes glareolus (bank vole) and with aphrodisin and submandibular protein from the golden hamster Mesocricetus auratus, all of which belong to the lipocalin superfamily. Lower levels of homology were also indicated between a variety of other lipocalins including the major urinary proteins from house mice and Norway rats. A model of the tertiary structure of roborovskin was constructed from the primary sequence by homology modeling. This model structure resembled other 8-stranded beta barrel lipocalins. Thus, the Roborovski hamster may demonstrate another variant of urinary lipocalin expression, as for the animals studied here, there appears to be no polymorphism in expression either between sexes or individuals.

Phosphorylation of Native Porcine Olfactory Binding Proteins

The identification of various isoforms of olfactory binding proteins is of major importance to elucidate their involvement in detection of pheromones and other odors. Here, we report the characterization of the phosphorylation of OBP (odorant binding protein) and Von Ebner's gland protein (VEG) from the pig, Sus scrofa. After labeling with specific antibodies raised against the three types of phosphorylation (Ser, Thr, Tyr), the phosphate-modified residues were mapped by using the beta-elimination followed by Michael addition of dithiothreitol (BEMAD) method. Eleven phosphorylation sites were localized in the pOBP sequence and nine sites in the VEG sequence. OBPs are secreted by Bowman's gland cells in the extracellular mucus lining the nasal cavity. After tracking the secretion pathway in the rough endoplasmic reticulum of these cells, we hypothesize that these proteins may be phosphorylated by ectokinases that remain to be characterized. The existence of such a regulatory mechanism theoretically increases the number of OBP variants, and it suggests a more specific role for OBPs in odorant coding than the one of odorant solubilizer and transporter.

Natural Ligands of Porcine Olfactory Binding Proteins

Knowledge of endogenous ligands of olfactory binding proteins is a prerequisite for studying their role in odor and pheromone transduction. Here, we report the extraction, derivatization, and characterization by gas chromatography-mass spectrometry of the natural ligands of pig, Sus scrofa (L.), Von Ebner's Gland protein (VEG) and odorant binding protein (OBP). We identified two isoforms (VEG1 and VEG2), which differed only by the linkage of an O-N-acetylglucosamine (O-GlcNac) group on VEG1. The natural ligands of VEG1 were characterized as two isomers of testosterone, whereas ligands of VEG2 and OBP were fatty acids or their derivatives. Our findings suggest that the binding specificity of VEG1 for steroids is governed by the presence of an O-GlcNac moiety on the protein. This specificity was confirmed by the binding of radiolabeled testosterone only by VEG1 in an in-gel binding assay. This is the first evidence for a post-translational modification in the process of odorant discrimination by olfactory binding proteins.

Scent of a Fly

Sexual courtship is a highly ritualized behavior in many animals. Recent work in the vinegar fly, Drosophila melanogaster, has illuminated how the pheromone cis-vaccenyl acetate modulates sexual behavior in the fly. Chemosensory receptors and a sexually dimorphic circuit activated by this pheromone have been identified. This minireview highlights recent advances in the field of fly courtship.

Specific expression of olfactory binding protein in the aerial olfactory cavity of adult and developing Xenopus

Olfactory binding proteins (OBP), commonly associated with aerial olfaction, are found in the olfactory mucus of mammals but have never been identified in fish. It is still not clear whether the presence of OBP in aerial olfactory systems is due to phylogenetic or to functional differences linked to the adaptation of the olfactory system to an aerial environment. To test this alternative, the olfactory system of Xenopus offers a unique opportunity because it includes two olfactory cavities, one of which is thought to be devoted to aquatic olfaction and the other to aerial olfaction. We therefore purified and cloned OBPs in two Xenopus species. Xenopus laevis OBP (XlaeOBP) and Xenopus tropicalis OBP (XtroOBP) exhibit 158 and 160 amino acids, respectively, sharing 89 residues. cRNA probes allowed us to demonstrate that XlaeOBP and XtroOBP are expressed at the level of Bowman's gland specifically in the aerial olfactory cavity, as confirmed using anti-XlaeOBP antiserum. OBP mRNA transcription occurs early during metamorphosis, as early as stage 57. This is the first study to demonstrate that OBPs are exclusively present in the aerial chamber and are only expressed as the tadpole becomes an adult in species which possess both aquatic and aerial olfactory organs.

Functional characterization of olfactory binding proteins for appeasing compounds and molecular cloning in the vomeronasal organ of pre-pubertal pigs.

The appeasing behaviour of pre-pubertal pigs appears to result from the perception of maternal odours (fatty acids) and of steroids coming from the male. We have used a ligand-oriented approach to functionally characterize olfactory binding proteins involved in the detection of appeasing compounds in the nasal mucosa (NM) and the vomeronasal organ (VNO) of pre-pubertal pigs. Several proteins were identified, combining binding assay, immunodetection and protein sequencing. Their sites of expression in nasal and vomeronasal tissues were studied by reverse transcription polymerase chain reaction (RT-PCR). The proteins belong to the lipocalin superfamily: Alpha-1-acid glycoprotein (AGP), odorant-binding protein (OBP), salivary lipocalin (SAL) and Von Ebner's gland protein (VEG), and displayed different binding capacities for the appeasing compounds. RT-PCR experiments showed that OBP and VEG are expressed not only in the NM, but also in the VNO and that SAL is only expressed in the VNO. This is the first report of the expression of these lipocalins in the VNO. Different binding affinities between lipocalins and appeasing compounds, together with their different localizations in the olfactory systems, suggest multiple possibilities for the peripheral coding of appeasing signals.

Disulfide pairing and secondary structure of ASP1, an olfactory-binding protein from honeybee (Apis mellifera L).

In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is accomplished by olfactory-binding proteins (CBPs). We report the structural characterization of a honeybee OBP called ASP1 found in workers and drones, previously observed to bind queen pheromone components. A novel method based on ion-spray mass spectrometry analysis of cyanylation-induced cleavage products of partially reduced protein with Tris(2-carboxyethyl)phosphine was needed to determine the recombinant ASP1 disulfide bond pairing. It was observed to be Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI), similar to those already described for other OBPs from honeybee and Bombyx mori suggesting that this pattern occurs commonly throughout the diverse family of insect OBPs. Circular dichroism revealed that ASP1 is an all-alpha protein in accordance with NMR preliminary data, but unlike lipocalin-like vertebrate OBPs.

A semiempirical SCF MO study of the interaction of odorant molecules with a biological substrate

AbstractThe AM1 semiempirical SCF MOmethod developed by Dewar and co‐workers has been employed to compute possible interaction processes of the odorant molecules cocaine, methyl benzoate, and benzaldehyde with the biological substrate lysine. The only process predicted as possible in each case was nucleophilic attack on the carbonyl carbons of each odorant by the nitrogen lone pairs on lysine. The remote amino group of lysine in each case was predicted to react with lower activation enthalpy than did the α‐amino group, a prediction consistent with preliminary experimental data from these laboratories. Although the computed activation enthalpies were high (ca. 40 kcal mol−1) for the gas‐phase processes, it is conceivable that such processes could occur by alternate pathways with considerably lower ctivation energies in solvated media such as exists in the presence of mucus‐containing olfactory binding protein in vertebrate olfactory reception. © 1992 John Wiley & Sons, Inc.

A semiempirical theoretical study of the molecular interaction of cocaine with the biological substrate glycine

AbstractSemiempirical SCFMO computations of the MNDO and AM1 varieties have been employed to model possible interaction processes for cocaine with the biological substrate glycine. It was found for the gas‐phase species that the most likely interactions occurred as nucleophilic attack of the nitrogen lone pair of glycine on the two carbonyl groups of cocaine. These reactions led to intermediates which further decomposed exothermically to amide and alcohol species. The activation enthalpies for the gas‐phase reactions were predicted to be high (39–46 kcal/mol), but it is believed that such processes could possibly occur by reaction pathways with considerably lower activation energies in the liquid state in the presence of mucus containing olfactory binding protein in vertebrate olfactory reception.