Odorant binding and conformational changes of a rat odorant-binding protein

Odorant-binding proteins (OBPs) are lipocalins secreted in the nasal mucus of vertebrates, which convey odorants to their neuronal receptors. We compared the binding properties of a recombinant rat OBP (OBP-1F) using a set of six odorants of various chemical structures. We examined the binding properties by both fluorescent probe competition and isothermal titration calorimetry. OBP-1F affinity constants, in the micromolar range, varied by more than one order of magnitude and were roughly correlated to the odorant size. The observed binding stoichiometry was found to be around one odorant per dimer. Using tyrosine differential spectroscopy, the binding of ligand was shown to induce local conformational changes. A three-dimensional structure of OBP-1F, modelled using the known structure of aphrodisin as template, allowed us to suggest the location of the observed structural changes outside of the binding pocket. These results are consistent with one binding site located in one of the two beta-barrels of the OBP-1F dimer and a subtle conformational change correlated with binding of an odorant molecule, which hampers uptake of a second odorant by the other hydrophobic pocket.     

Identification of a third rat odorant-binding protein (OBP3)

From a rat olfactory epithelium cDNA library clones encoding a lipocalin were isolated with sequence identity to the previously described salivary-specific alpha-2u globulin and the N-terminal region of mouse odorant-binding proteins OBP-III and OBP-IV. In situ hybridization showed strong expression in nasal glands displaying a pattern equivalent to rat OBP1. Heterologously expressed protein was evaluated for its binding properties using spectroscopic approaches. The recombinant protein interacted with two fluorescent probes, 1-aminoanthracene (1-AMA) and 1,1'-bis(4-anilino-5-naphthalene)-sulfonic acid. 1-AMA binding was competed by several odorants with high affinity. The thermodynamic parameters of the protein-odorant interaction were determined using isothermal titration calorimetry. Due to its nasal expression and odorant-binding characteristics this protein was designated OBP3.     

Insights into structural features determining odorant affinities to honey bee odorant binding protein 14

Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant.     

Porcine odorant-binding protein: structural stability and ligand affinities measured by fourier-transform infrared spectroscopy and fluorescence spectroscopy

Infrared spectra show that the binding of the odorants 2-isobuthyl-3-methoxypyrazine (PYR) and 3,7-dimethyl-1-octanol (DMO) stabilises the tertiary structure of porcine OBP-I against thermal denaturation. The fluorescence emission spectrum of the single tryptophan shows a lambdamax at 337 nm, indicating that the residue is not directly exposed to the solvent. Tryptophan does not appear to be involved in the odorant binding process and it is not accessible to the fluorescence quenchers NaI, CsCl and acrylamide. The binding of the fluorescent dye 1-aminoanthracene (1-AMA), a strong ligand, does not modify the tryptophan fluorescence spectrum. In contrast, the lambdamax of 1-AMA bound to OBP-I is shifted from 537 to 481 nm, with a lambdamax intensity increase by a factor of 80. Bound 1-AMA is displaced by odorant molecules in competitive binding assays and can be employed in simple and rapid binding assay, avoiding the use of radioactive ligands. The Scatchard plot shows that 1-AMA binds to OBP-I with a dissociation constant of 1.3 microM and an equimolar stoichiometry.     

Internalization of odorant-binding proteins into the mouse olfactory epithelium

The detection of odorants in vertebrates is mediated by chemosensory neurons that reside in the olfactory epithelium of the nose. In land-living species, the hydrophobic odorous compounds inhaled by the airstream are dissolved in the nasal mucus by means of specialized globular proteins, the odorant-binding proteins (OBPs). To assure the responsiveness to odors of each inhalation, a rapid removal of odorants from the microenvironment of the receptor is essential. In order to follow the fate of OBP/odorant complexes, a recombinant OBP was fluorescently labeled, loaded with odorous compounds, and applied to the nose of a mouse. Very quickly, labeled OBP appeared inside the sustentacular cells of the epithelium. This uptake occurred only when the OBP was loaded with appropriate odorant compounds. A search for candidate transporters that could mediate such an uptake process led to the identification of the low density lipoprotein receptor Lrp2/Megalin. In the olfactory epithelium, megalin was found to be specifically expressed in sustentacular cells and the Megalin protein was located in their microvilli. In vitro studies using a cell line that expresses megalin revealed a rapid internalization of OBP/odorant complexes into lysosomes. The uptake was blocked by a Megalin inhibitor, as was the internalization of OBPs into the sustentacular cells of the olfactory epithelium. The results suggest that a Megalin-mediated internalization of OBP/odorant complexes into the sustentacular cells may represent an important mechanism for a rapid and local clearance of odorants.     

Evidence of an odorant-binding protein in the human olfactory mucus: location,structural characterization,and odorant-binding properties

Odorant-binding proteins (OBPs) are small abundant extracellular proteins belonging to the lipocalin superfamily. They are thought to participate in perireceptor events of odor detection by carrying, deactivating, and/or selecting odorant molecules. Putative human OBP genes (hOBP) have recently been described [Lacazette et al. (2000) Hum. Mol. Genet. 9, 289-301], but the presence of the corresponding proteins remained to be established in the human olfactory mucus. This paper reports the first evidence of such expression in the mucus covering the olfactory cleft, where the sensory olfactory epithelium is located. On the contrary, hOBPs were not observed in the nasal mucus covering the septum and the lower turbinate. To demonstrate the odorant binding activity of these proteins, a corresponding recombinant protein variant, hOBP(IIa)(alpha), was secreted by the yeast Pichia pastoris and thoroughly characterized. It appears as a monomer with one disulfide bond located between C59 and C151, a conservative feature of all other vertebrate OBPs. By measuring the displacement of several fluorescent probes, we show that hOBP(IIa)(alpha) is able to bind numerous odorants of diverse chemical structures, with a higher affinity for aldehydes and large fatty acids. A computed 3D model of hOBP(IIa)(alpha) is proposed and reveals that two lysyl residues of the binding pocket may account for the increased affinity for aldehydes. The relatively limited specificity of hOBP(IIa)(alpha) suggests that other human OBPs are expected to take into account the large diversity of odorant molecules.     

Enhancement of odorant detection sensitivity by the expression of odorant-binding protein

Odorant-binding proteins are low molecular weight, soluble proteins that are secreted by glands of the nasal cavity. Their function is known to be the transport of hydrophobic odorants. This feature is important to artificial olfactory biosensors, which operate in the aqueous phase. In this study, one of rat odorant-binding proteins, OBP3, was inserted into a mammalian expression vector pcDNA3, expressed, and secreted from human embryonic kidney-293 (HEK-293) cells. The his(6) tag and signal peptide of the prelysozyme (plys) were fused with OBP3 for the detection and secretion of the proteins, respectively. The secretion level of OBP3 was maximal at 3h of incubation time. The secreted OBP3 increased the solubility of a hydrophobic odorant, octanal, which is the specific odorant of rat olfactory receptor I7. The secreted OBP3 also bound to olfactory receptor I7. These interactions consequently increased the cellular signal intensity stimulated by the odorant in the cells expressing olfactory receptor I7. Our findings indicate that odorant-binding protein can be effectively used to increase the sensitivity of olfactory receptor-based biosensors.     

Regulatory factors in the vertebrate olfactory mucosa

Access to and clearance of ligands from binding sites on olfactorycilia are regulated by a complex interplay of molecular, physicaland cellular factors. Nasal/olfactory glands secrete mucus thatcontains many proteins, among them odorant-binding proteins(OBP) that may solubilize lipophilic odorants in the aqueousmucous phase and subsequently transport them to receptor sites.The rate of transport of the ligand–OBP complex or unboundodorant is a function of the diffusion coefficient that, underphysiological conditions, is determined largely by the molecularsize of the complex or unbound odorant, the viscosity of mucusand the tortuosity factor. The binding constants must favorassociation of the ligand with the binding protein, dissociationof the complex and possible reassociation of the ligand withthe odorant receptor. Neural regulation of secretion determinesthe properties of the olfactory mucus that affect ligand accessand clearance, including viscosity, water content and depth.Extrinsic autonomic (adrenergic, cholinergic) and peptidergic(substance P/CGRP, VIP) neurons innervate olfactory glands andregulate both secretory granule release and electrolyte/waterbalance. Extrinsic peptidergic (substance (P/CGRP, VIP) neuronsterminate near the epithelial surface in close apposition tosustentacular cells and olfactory receptor neurons. The substanceP/CGRP fibers, in addition to functioning as sensory fibers,appear to regulate secretion from sustentacular cells througha secretomotor reflex and to neuromodulate the sensitivity ofolfactory receptor neurons to odorant stimulation. The actionof regulatory factors in the olfactory mucosa is an emergingtopic of research focused on molecular, physical and cellularfactors that affect sensory transduction.     

Specificity of odorant-binding proteins: a factor influencing the sensitivity of olfactory receptor-based biosensors

Odorant-binding proteins (OBPs) primarily function in the transport of hydrophobic odorants. In this study, OBPs originating from rat and pig were cloned into a mammalian expression vector, pcDNA3, and expressed in HEK-293 cells, and their specificity for odorants and olfactory receptors was examined. Results suggest that OBPs have a high affinity for the olfactory receptors when both the OBP and receptor originate from the same species. The rat OBPs were bound not only to the rat olfactory receptor I7 but also to the odorant specific to I7. The solubility of the odorant was increased by both OBP2 and OBP3, which originate from rat, but with different efficiencies. These results demonstrate that OBPs specifically interact with odorants as well as olfactory receptors, and these interactions can influence the sensitivity of olfactory receptor-based biosensors.     

Molecular and functional characterization of an odorant binding protein of the Asian elephant,Elephas maximus: implications for the role of lipocalins in mammalian olfaction

The sex pheromone present in the pre-ovulatory urine of female Asian elephants is the simple lipid (Z)-7-dodecen-1-yl acetate (Z7-12:Ac). Using radiolabeled probes, we have identified a pheromone binding protein that is abundant in the mucus of the trunk; this protein is homologous to a class of lipocalins known as odorant binding proteins (OBPs). To test five previously proposed roles for the OBP in chemosensory perception, we determined the equilibrium dissociation constant of the OBP-pheromone complex, as well as the association and dissociation rates. Using a mathematical model in conjunction with experimental data, we suggest that the binding and release of the pheromone by the OBP are too slow for the OBP to function in transporting the pheromone through the mucus that covers the olfactory sensory epithelium. Our data indicate that the elephant OBP only modestly increases the solubility of the pheromone in the mucus. Our results are most consistent with the notion that elephant OBP functions as a scavenger of the pheromone and possibly other ligands, including odorants. In light of these findings, and published results for other mammalian OBP-ligand complexes, a general model for the role of OBPs in mammalian olfaction is proposed. Moreover, the potential implications of these findings for interaction of Z7-12:Ac with insect antennal proteins are discussed.     

Numerical modeling of odorant uptake in the rat nasal cavity

An anatomically accurate 3-dimensional numerical model of the right rat nasal cavity was developed and used to compute low, medium, and high flow rate inspiratory and expiratory mucosal odorant uptake (imposed patterning) for 3 odorants with different mucus solubilities. The computed surface mass flux distributions were compared with anatomic receptor gene expression zones identified in the literature. In general, simulations predicted that odorants that were highly soluble in mucus were absorbed dorsally and medially, corresponding roughly to receptors from one of the gene expression zones. Insoluble odorants tended to be absorbed more peripherally in the rat olfactory region corresponding to the other 2 zones. These findings also agreed in general with the electroolfactogram measurements and the voltage-sensitive dye measurements reported in the literature. This numerical approach is the first to predict detailed odorant flux information across the olfactory mucosa in the rat nasal cavity during inspiratory and expiratory flow and to relate it to anatomic olfactory receptor location, physiological function, and biochemical experiment. This numerical technique can allow us to separate the contributions of imposed and inherent patterning mechanisms on the rat olfactory mucosa.     

Odorant binding initially occurring at the central pocket in bovine odorant-binding protein

Why bovine odorant-binding protein (OBPb), among OBP family, assumes a dimeric structure has been unclear. Here we clarified, by measuring the fluorescence of intrinsic tryptophan and tyrosine residues of intact OBPb and OBPb whose C-terminal 10 amino acids were deleted, that odorant enters the central pocket formed by the dimerization when OBPb first encounters odorant, and odorant with high affinity with OBPb subsequently enters the internal cavity (suggested binding site), releasing the pre-bound odorant. The internal cavity-bound odorant can be released by the binding of other odorants at another internal cavity or at the central pocket, depending on the binding odorants. Due to this mechanism enabled by the dimerization, OBPb is more reactive than other monomeric OBPs.     

Molecular cloning,expression profile,odorant affinity,and stability of two odorant‐binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

The polyembryonic endoparasitoid wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is deployed successfully as a biocontrol agent for corn pest insects from the Lepidopteran genus Ostrinia in Europe and throughout Asia, including Japan, Korea, and China. The odorants are recognized, bound, and solubilized by odorant‐binding protein (OBP) in the initial biochemical recognition steps in olfaction that transport them across the sensillum lymph to initiate behavioral response. In the present study, we examine the odorant‐binding effects on thermal stability of McinOBP2, McinOBP3, and their mutant form that lacks the third disulfide bonds. Real‐time PCR experiments indicate that these two are expressed mainly in adult antennae, with expression levels differing by sex. Odorant‐binding affinities of aldehydes, terpenoids, and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes in the thermal stability of the proteins upon their affinities to odorants. The obtained results reveal higher affinity of trans‐caryophelle, farnesene, and cis‐3‐Hexen‐1‐ol exhibits to both wild and mutant McinOBP2 and McinOBP3. Although conformational flexibility of the mutants and shape of binding cavity make differences in odorant affinity between the wild‐type and mutant, it suggested that lacking the third disulfide bond in mutant proteins may have chance to incorrect folded structures that reduced the affinity to these odorants. In addition, CD spectra clearly indicate proteins enriched with α‐helical content.     

Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.).

In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is generally thought to be accomplished by odorant-binding proteins (OBPs). We report the structural and functional properties of a honeybee OBP called ASP2, heterologously expressed by the yeast Pichia pastoris. ASP2 disulfide bonds were assigned after classic trypsinolysis followed by ion-spray mass spectrometry combined with microsequencing. The pairing [Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI)] was found to be identical to that of Bombyx mori OBP, suggesting that this pattern occurs commonly throughout the highly divergent insect OBPs. CD measurements revealed that ASP2 is mainly constituted of alpha helices, like other insect OBPs, but different from lipocalin-like vertebrate OBPs. Gel filtration analysis showed that ASP2 is homodimeric at neutral pH, but monomerizes upon acidification or addition of a chaotropic agent. A general volatile-odorant binding assay allowed us to examine the uptake of some odorants and pheromones by ASP2. Recombinant ASP2 bound all tested molecules, except beta-ionone, which could not interact with it at all. The affinity constants of ASP2 for these ligands, determined at neutral pH by isothermal titration calorimetry, are in the micromolar range, as observed for vertebrate OBP. These results suggest that odorants occupy three binding sites per dimer, probably one in the core of each monomer and another whose location and biological role are questionable. At acidic pH, no binding was observed, in correlation with monomerization and a local conformational change supported by CD experiments.     

Porcine odorant-binding protein selectively binds to a human olfactory receptor

Odorant-binding proteins (OBPs) represent a highly abundant class of proteins secreted in the nasal mucus by the olfactory neuroepithelium. These proteins display binding affinity for a variety of odorant molecules, thereby assuming the role of carrier during olfactory perception. However, no specific interaction between OBP and olfactory receptors (ORs) has yet been shown and early events in olfaction remain so far poorly understood at a molecular level. Two human ORs, OR 17-209 and OR 17-210, were fused to a Green Fluorescent Protein and stably expressed in COS-7 cell lines. Interaction with OBP was investigated using a highly purified radioiodinated porcine OBP (pOBP) preparation, devoid of any ligand in its binding cavity. No specific binding of the pOBP tracer could be detected with OR 17-209. In contrast, OR 17-210 exhibited specific saturable binding (K(d) = 9.48 nM) corresponding to the presence of a single class of high-affinity binding sites (B(max) = 65.8 fmol/mg prot). Association and dissociation kinetics further confirmed high-affinity interaction between pOBP and OR 17-210. Autoradiographic studies of labeled pOBP to newborn mouse slices revealed the presence of multiple specific binding sites located mainly in olfactory tissue but also in several other peripheral tissues. Our data thus demonstrate a high-affinity interaction between OBP and OR, indicating that under physiological conditions, ORs may be specifically associated with an OBP partner in the absence of odorant. This provides further evidence of a novel role for OBP in the mechanism of olfactory perception.     

Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects

The olfactory receptors of terrestrial animals exist in an aqueous environment, yet detect odorants that are primarily hydrophobic. The aqueous solubility of hydrophobic odorants is thought to be greatly enhanced via odorant binding proteins (OBP) which exist in the extracellular fluid surrounding the odorant receptors. We have isolated and partially sequenced 14 candidate OBPs from six insect (moth) species. All 14 represent a single homologous family based on conserved sequence domains. The 14 proteins can be divided into three subfamilies based on differences in tissue specific expression and similarities in amino acid sequences. All 14 proteins are specifically expressed in antennal olfactory tissue. Subfamily I represents previously described pheromone binding proteins (PBP), which are male-specific, associate with pheromone-sensitive neurons, and are highly variable in their sequences when compared among species. Subfamilies II and III are expressed in both male and female antennae, appear to associate with general-odorant-sensitive neurons, and are highly conserved when compared among species. The properties of the subfamily II and III proteins suggest these are general-odorant binding proteins (GOBP). The properties of the respective insect OBP subfamilies suggest that they have different odorant binding specificities. The association of different insect OBP subfamilies with distinct classes of olfactory neurons having different odorant specificities suggests that OBPs can act as selective signal filters, peripheral to the actual receptor proteins.     

Combined behavioral and c-Fos studies elucidate the vital role of sodium for odor detection

Salt, known as taste quality, is generally neglected in olfaction, although the olfactory sensory neurons stretch into the salty nasal mucus covering the olfactory epithelium (OE). Using a psychophysical approach, we directly and functionally demonstrate in the awake rat for a variety of structurally diverse odorants that sodium is a critical factor for olfactory perception and sensitivity, both very important components of mammalian communication and sexual behavior. Bathing the olfactory mucus with an iso-osmotic sodium-free buffer solution results in severe deficits in odorant detection. However, sensitivity returns fully within a few hours, indicating continuous mucus production. In the presence of sodium in the mucus covering the OE, all odorants induce odorant-specific c-Fos expression in the olfactory bulb. Yet, if sodium is absent in the mucus, no c-Fos expression is induced as demonstrated for n-octanal. Our noninvasive approach to induce anosmia in mammals here presented--which is fully reversible within hours--opens new possibilities to study the functions of olfactory communication in awake animals.     

Sequence requirements for interaction of human herpesvirus 7 origin binding protein with the origin of lytic replication

As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication (oriLyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBP(H7)) with its cognate sites in the 600-bp HHV-7 oriLyt. We expressed the carboxyl-terminal domain of OBP(H7) and found that amino acids 484 to 787 of OBP(H7) were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBP(H7) has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBP(H6B)), which binds with similar affinity to its two cognate OBP sites in the HHV-6B oriLyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBP(H7) consensus recognition sequence of the 9-bp core, BRTYCWCCT (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBP(H6B) consensus YGWYCWCCY and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBP(H7) interacts along one face of the DNA helix, with the major groove, as do OBP(H6B) and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBP(H7) and OBP(H6B).