Ligand specificity of odorant receptors

Odorant receptors belong to class A of the G protein-coupled receptors (GPCRs) and detect a large number of structurally diverse odorant molecules. A recent structural bioinformatic analysis suggests that structural features are conserved across class A of GPCRs in spite of their low sequence identity. Based on this work, we have aligned the sequences of 29 ORs for which ligand binding data are available. Recent site-directed mutagenesis experiments on one such receptor (MOR174-9) provide information that helped to identify nine amino-acid residues involved in ligand binding. Our modeling provides a rationale for amino acids in equivalent positions in most of the odorant receptors considered and helps to identify other amino acids that could be important for ligand binding. Our findings are consistent with most of the previous models and allow predictions for site-directed mutagenesis experiments, which could also validate our model.     

Adhesion of Pseudomonas aeruginosa to human buccal epithelial cells: evidence for two classes of receptors

The adhesion of Pseudomonas aeruginosa strain 492c to trypsinized and untrypsinized buccal epithelial cells (BECs) was studied. Kinetic analysis of the adhesion data, employing a Langmuir absorption isotherm, indicated the presence of two classes of binding sites on untrypsinized BECs: a high affinity - low copy number site (apparent association constant (Ka) approximately equal to 1.57 X 10(-8) mL/cell with ca. 29 binding sites/cell) and a low affinity - high copy number class of binding sites (Ka approximately equal to 4.78 X 10(-10) mL/cell with ca. 264 binding sites/cell). The low affinity - high copy number class of sites was found to be trypsin sensitive. A single class of binding sites was found on trypsinized BECs exhibiting a high affinity - low copy number (Ka approximately equal to 3.70 X 10(-7) mL/cell with ca. 31 binding sites/cell). Positive cooperativity in binding of P. aeruginosa strain 492c to the low affinity - high copy number class site on untrypsinized BECs was demonstrated by analysis of Hill plots of the adhesion data. Sugar inhibition data using a preincubation methodology showed an inhibition of adhesion to trypsinized BECs in the presence of N-acetylneuraminic acid and D-arabinose, while these same two sugars enhanced adhesion to untrypsinized BECs. D-Galactose and N-acetylglucosamine enhanced adhesion to both types of BECs though the latter did to different extents. D-Fucose only inhibited adhesion to untrypsinized BECs.(ABSTRACT TRUNCATED AT 250 WORDS)     

The location of olfactory receptors within olfactory epithelium is independent of odorant volatility and solubility

Background

Our objective was to study the pattern of olfactory receptor expression within the dorsal and ventral regions of the mouse olfactory epithelium. We hypothesized that olfactory receptors were distributed based on the chemical properties of their ligands: e.g. receptors for polar, hydrophilic and weakly volatile odorants would be present in the dorsal region of olfactory epithelium; while receptors for non-polar, more volatile odorants would be distributed to the ventral region. To test our hypothesis, we used micro-transplantation of cilia-enriched plasma membranes derived from dorsal or ventral regions of the olfactory epithelium into Xenopus oocytes for electrophysiological characterization against a panel of 100 odorants.

Findings

Odorants detected by ORs from the dorsal and ventral regions showed overlap in volatility and water solubility. We did not find evidence for a correlation between the solubility and volatility of odorants and the functional expression of olfactory receptors in the dorsal or ventral region of the olfactory epithelia.

Conclusions

No simple clustering or relationship between chemical properties of odorants could be associated with the different regions of the olfactory epithelium. These results suggest that the location of ORs within the epithelium is not organized based on the physico-chemical properties of their ligands.

     

Expression of mRNAs encoding for two different olfactory receptors in a subset of olfactory receptor neurons

Evidence has accumulated to support a model for odorant detection in which individual olfactory receptor neurons (ORNs) express one of a large family of G protein-coupled receptor proteins that are activated by a small number of closely related volatile chemicals. However, the issue of whether an individual ORN expresses one or multiple types of receptor proteins has yet to be definitively addressed. Physiological data indicate that some individual ORNs can be activated by odorants differing substantially in structure and/or perceived quality, suggesting multiple receptors or one nonspecific receptor per cell. In contrast, molecular biological studies favor a scheme with a single, fairly selective receptor per cell. The present studies directly assessed whether individual rat ORNs can express multiple receptors using single-cell PCR techniques with degenerate primers designed to amplify a wide variety of receptor sequences. We found that whereas only a single OR sequence was obtained from most ORNs examined, one ORN produced two distinct receptor sequences that represented different receptor gene families. Double-label in situ hybridization studies indicated that a subset of ORNs co-express two distinct receptor mRNAs. A laminar segregation analysis of the cell nuclei of ORNs labeled with the two OR mRNA probes showed that for one probe, the histogram of the distribution of the cell nuclei along the depth of the epithelium was bimodal, with one peak overlapping the (unimodal) histogram for the other probe. These results are consistent with co-expression of two OR mRNAs in a population of single ORNs.     

Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes

Porcine odorant binding protein (pOBP) is a monomer of 157 amino acid residues, purified in abundance from pig nasal mucosa. In contrast to the observation on lipocalins as retinol binding protein (RBP), major urinary protein (MUP) or bovine odorant binding protein (bOBP), no naturally occurring ligand was found in the beta-barrel cavity of pOBP. Porcine OBP was therefore chosen as a simple model for structure/function studies with odorant molecules. In competition experiments with tritiated pyrazine, the affinity of pOBP towards several odorant molecules belonging to different chemical classes has been found to be of the micromolar order, with a 1:1 stoichiometry. The X-ray structures of pOBP complexed to these molecules were determined at resolution between 2.15 and 1.4 A. As expected, the electron density of the odorant molecules was observed into the hydrophobic beta-barrel of the lipocalin. Inside this cavity, very few specific interactions were established between the odorant molecule and the amino acid side-chains, which did not undergo significant conformational change. The high B-factors observed for the odorant molecules as well as the existence of alternative conformations reveal a non-specific mode of binding of the odorant molecules in the cavity.     

Distinct molecular mechanisms account for the specificity of two different T-cell receptors

Analysis of the thermodynamics of the interactions between the D3 T-cell receptor (TCR) and its natural ligand, an HIV peptide bound to a HLA-A0201 (HLA-A2) major histocompatibility complex (MHC) protein, shows both similarities and striking differences when compared with the 2B4 TCR binding to its peptide-MHC ligand. The equilibrium thermodynamic parameters of both reactions are consistent with a conformational adjustment at the binding interface during the formation of specific TCR-peptide-MHC complexes. However, osmolytic reagents that dehydrate protein surfaces have profoundly different effects on the strength of the two reactions, indicating that water molecules make very different contributions-enhancing the binding of D3 TCR but weakening the binding of 2B4 TCR. The use of these different mechanisms by TCRs to recognize ligands might be an important means augmenting their inherent cross-reactivity.     

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening

The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.     

Axonal ephrin-As and odorant receptors: coordinate determination of the olfactory sensory map

Olfactory sensory neurons expressing a given odorant receptor (OR) project with precision to specific glomeruli in the olfactory bulb, generating a topographic map. In this study, we demonstrate that neurons expressing different ORs express different levels of ephrin-A protein on their axons. Moreover, alterations in the level of ephrin-A alter the glomerular map. Deletion of the ephrin-A5 and ephrin-A3 genes posteriorizes the glomerular locations for neurons expressing either the P2 or SR1 receptor, whereas overexpression of ephrin-A5 in P2 neurons results in an anterior shift in their glomeruli. Thus the ephrin-As are differentially expressed in distinct subpopulations of neurons and are likely to participate, along with the ORs, as one of a complement of guidance receptors governing the targeting of like axons to precise locations in the olfactory bulb.     

Roles of odorant receptors in projecting axons in the mouse olfactory system

In the mouse olfactory epithelium, there are about ten million olfactory sensory neurons, each expressing a single type of odorant receptor out of approximately 1000. Olfactory sensory neurons expressing the same odorant receptor converge their axons to a specific set of glomeruli on the olfactory bulb. How odorant receptors play an instructive role in the projection of axons to the olfactory bulb has been one of the major issues of developmental neurobiology. Recent studies revealed previously overlooked roles of odorant receptor-derived cAMP signals in the axonal projection of olfactory sensory neurons; the levels of cAMP and neuronal activity appear to determine the expression levels of axon guidance/sorting molecules and thereby direct the axonal projection of olfactory sensory neurons. These findings provide new insights as to how peripheral inputs instruct neuronal circuit formation in the mammalian brain.     

Cross-adaptation between olfactory responses induced by two subgroups of odorant molecules

It has long been believed that vertebrate olfactory signal transduction is mediated by independent multiple pathways (using cAMP and InsP3 as second messengers). However, the dual presence of parallel pathways in the olfactory receptor cell is still controversial, mainly because of the lack of information regarding the single-cell response induced by odorants that have been shown to produce InsP3 exclusively (but not cAMP) in the olfactory cilia. In this study, we recorded activities of transduction channels of single olfactory receptor cells to InsP3-producing odorants. When the membrane potential was held at -54 mV, application of InsP3-producing odorants to the ciliary region caused an inward current. The reversal potential was 0 +/- 7 mV (mean +/- SD, n = 10). Actually, InsP3-producing odorants generated responses in a smaller fraction of cells (lilial, 3.4%; lyral, 1.7%) than the cAMP-producing odorant (cineole, 26%). But, fundamental properties of responses were surprisingly homologous; namely, spatial distribution of the sensitivity, waveforms, I-V relation, and reversal potential, dose dependence, time integration of stimulus period, adaptation, and recovery. By applying both types of odorants alternatively to the same cell, furthermore, we observed cells to exhibit symmetrical cross-adaptation. It seems likely that even with odorants with different modalities adaptation occurs completely depending on the amount of current flow. The data will also provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2

An interaction of odorants with olfactory receptors is thought to be the initial step in odorant detection. However, ligands have been reported for only 6 out of 380 human olfactory receptors, with their structural determinants of odorant recognition just beginning to emerge. Guided by the notion that amino acid positions that interact with specific odorants would be conserved in orthologs, but variable in paralogs, and based on the prediction of a set of 22 of such amino acid positions, we have combined site-directed mutagenesis, rhodopsin-based homology modelling, and functional expression in HeLa/Olf cells of receptors OR1A1 and OR1A2. We found that (i) their odorant profiles are centred around citronellic terpenoid structures, (ii) two evolutionary conserved amino acid residues in transmembrane domain 3 are necessary for the responsiveness of OR1A1 and the mouse ortholog Olfr43 to (S)-(-)-citronellol, (iii) changes at these two positions are sufficient to account for the differential (S)-(-)-citronellol responsiveness of the paralogs OR1A1 and OR1A2, and (iv) the interaction sites for (S)-(-)-citronellal and (S)-(-)-citronellol differ in both human receptors. Our results show that the orientation of odorants within a homology modelling-derived binding pocket of olfactory receptor orthologs is defined by evolutionary conserved amino acid positions.     

An update on carotenoid biosynthesis in algae: phylogenetic evidence for the existence of two classes of phytoene synthase

Carotenoids play crucial roles in structure and function of the photosynthetic apparatus of bacteria, algae, and higher plants. The entry-step reaction to carotenoid biosynthesis is catalyzed by the phytoene synthase (PSY), which is structurally and functionally related in all organisms. A comparative genomic analysis regarding the PSY revealed that the green algae Ostreococcus and Micromonas possess two orthologous copies of the PSY genes, indicating an ancient gene duplication event that produced two classes of PSY in algae. However, some other green algae (Chlamydomonas reinhardtii, Chlorella vulgaris, and Volvox carteri), red algae (Cyanidioschyzon merolae), diatoms (Thalassiosira pseudonana and Phaeodactylum tricornutum), and higher plants retained only one class of the PSY gene whereas the other gene copy was lost in these species. Further, similar to the situation in higher plants recent gene duplications of PSY have occurred for example in the green alga Dunaliella salina/bardawil. As members of the PSY gene families in some higher plants are differentially regulated during development or stress, the discovery of two classes of PSY gene families in some algae suggests that carotenoid biosynthesis in these algae is differentially regulated in response to development and environmental stress as well.     

Comparison of human alkaline phosphatase isoenzymes. Structural evidence for three protein classes.

The structural relationships among human alkaline phosphatase isoenzymes from placenta, bone, kidney, liver and intestine were investigated by using three criteria. 1. Immunochemical characterization by using monospecific antisera prepared against either the placental isoenzyme or the liver isoenzyme distinguishes two antigenic groups: bone, kidney and liver isoenzymes cross-react with anti-(liver isoenzyme) serum, and the intestinal and placental isoenzymes cross-react with the anti-(placental isoenzyme) antiserum. 2. High-resolution two-dimensional electrophoresis of the 32P-labelled denatured subunits of each enzyme distinguishes three groups of alkaline phosphatase: (a) the liver, bone and kidney isoenzymes, each with a unique isoelectric point in the native form, can be converted into a single form by treatment with neuraminidase; (b) the placental isoenzyme, whose position also shifts after removal of sialic acid; and (c) the intestinal isoenzyme, which is distinct from all other phosphatases and is unaffected by neuraminidase digestion. 3. Finally, we compare the primary structure of each enzyme by partial proteolytic-peptide 'mapping' in dodecyl sulphate/polyacrylamide gels. These results confirm the primary structural identity of liver and kidney isoenzymes and the non-identity of the placental and intestinal forms. These data provide direct experimental support for the existence of at least three alkaline phosphatase genes.     

TreeGraph 2: Combining and visualizing evidence from different phylogenetic analyses

Background  

Today it is common to apply multiple potentially conflicting data sources to a given phylogenetic problem. At the same time, several different inference techniques are routinely employed instead of relying on just one. In view of both trends it is becoming increasingly important to be able to efficiently compare different sets of statistical values supporting (or conflicting with) the nodes of a given tree topology, and merging this into a meaningful representation. A tree editor supporting this should also allow for flexible editing operations and be able to produce ready-to-publish figures.     

Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein alphaolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Galphaolf in odorant-specific signaling out of OR. The employment of the non-typical G protein alpha15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Galpha15 or Galphaolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.     

Odorants of different chemical classes interact with distinct odorant binding protein subtypes.

The ligand profile for three odorant binding proteins (OBPs) of the rat have been determined using a large number of odorous compounds from different chemical classes. To evaluate the binding spectra of distinct subtypes, all OBPs were produces in Escherichia coli as recombinant His-tagged fusion proteins. The individual binding properties of each OBP subtype were analysed using a large array of organic compounds, representing derivatives of aliphatic and aromatic compounds, as well as terpenes, pyrazines and thiazoles, in a competitive spectroscopic binding assay with various fluorescence chromophores as the specific interacting partner for the OBPs. Most of the compounds were identified to interact only with one OBP subtype. But interestingly, a small change, for example in the 2-methyl or 2-ethoxy side chain in the pyrazine and thiazole derivatives to a 2-isobutyl group, caused overlapping binding affinities to rat-OBP1 and rat-OBP3. However, the data strongly support the notion that each OBP subtype displays a characteristic ligand binding profile and interacts with a different subset of exogenous organic compounds in a micromolar range.     

Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor

Odorant receptors (ORs) provide the core determinant of identity for axons of olfactory sensory neurons (OSNs) to coalesce into glomeruli in the olfactory bulb. Here, using gene targeting in mice, we examine how the OR protein determines axonal identity. An OR::GFP fusion protein is present in axons, consistent with a direct function of ORs in axon guidance. When the OR coding region is deleted, we observe OSNs that coexpress other ORs that function in odorant reception and axonal identity. It remains unclear if such coexpression is normally prevented by negative feedback on OR gene choice. A drastic reduction in OR protein level produces axonal coalescence into novel, remote glomeruli. By contrast, chimeric ORs and ORs with minor mutations perturb axon outgrowth. Strikingly, the beta2 adrenergic receptor can substitute for an OR in glomerular formation when expressed from an OR locus. Thus, ORs have not evolved a unique function in axon guidance.     

Antennal expression pattern of two olfactory receptors and an odorant binding protein implicated in host odor detection by the malaria vector Anopheles gambiae

Odor-detection in the malaria mosquito Anopheles gambiae involves large families of diverse proteins, including multiple odorant binding proteins (AgOBPs) and olfactory receptors (AgORs). The receptors AgOR1 and AgOR2, as well as the binding protein AgOBP1, have been implicated in the recognition of human host odors. In this study, we have explored the expression of these olfactory proteins, as well as the ubiquitous odorant receptor heteromerization partner AgOR7, in the thirteen flagellomeres (segments) of female and male antenna. Expressing cells were visualized by adapting a whole mount fluorescence in situ hybridization method. In female mosquitoes, AgOR1-expressing olfactory receptor neurons (ORNs) were almost exclusively segregated in segments 3 to 9, whereas AgOR2-expressing ORNs were distributed over flagellomeres 2 to 13. Different individuals comprised a similar number of cells expressing a distinct AgOR type, although their antennal topography and number per flagellomere varied. AgOBP1-expressing support cells were present in segments 3 to 13 of the female antenna, with increasing numbers towards the distal end. In male mosquitoes, total numbers of AgOR- and AgOBP1-expressing cells were much lower. While AgOR2-expressing cells were found on both terminal flagellomeres, AgOR1 cells were restricted to the most distal segment. High densities of AgOBP1-expressing cells were identified in segment 13, whereas segment 12 comprised very few. Altogether, the results demonstrate that both sexes express the two olfactory receptor types as well as the binding protein AgOBP1 but there is a significant sexual dimorphism concerning the number and distribution of these cells. This may suggest gender-specific differences in the ability to detect distinct odorants, specifically human host-derived volatiles.