Journal of Heredity Advance Access published online on July 28, 2007
Journal of Heredity, doi:10.1093/jhered/esm054
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Functional Analysis of a Subset of Canine Olfactory Receptor Genes
From the Laboratory of Genetic and Development, CNRS UMR 6061, Institut de Génétique et Développement de Rennes, Faculté de Médecine, Rennes, F-35043 France (Benbernou, Tacher, Robin, Rakotomanga, Senger, and Galibert) Present address of F. Senger: UMR 6026, Faculté des sciences, Beaulieu, Rennes, France
Address correspondence to F. Galibert at the address above, or e-mail: francis.galibert{at}univ-rennes1.fr.
In this paper, we explored the level complexity of the combinatorial olfactory code that allows mammals with a repertoire of about thousand putatively active olfactory receptors encoded in their genomes to recognize and identify a much larger repertoire of odorant molecules. To that end, we cloned 38 canine OR genes belonging to the same OR gene family and transiently expressed them in a subclone of embryonic human kidney cells (HEK293) permanently expressing the G(olf) subunit. Using a Ca2+ imaging approach, we established for example that as many as 26 out of the 38 cloned OR elicited a Ca2+ response when exposed to octanal, whereas 10 responded to nonanal, other aldehydes providing intermediate responses. Altogether, these results demonstrated that the combinatorial code is quite complex in support to the highly developed sense of olfaction demonstrated by dogs.
Olfaction is a complicated process beginning in the olfactory epithelium with the specific binding of volatile odorant molecules to dedicated olfactory receptors (ORs) expressed by olfactory sensory neurons (OSNs) (Buck 1996; Mombaerts 1999; Firestein 2001; Touhara 2002). OR genes constitute the largest known gene family—the olfactory subgenome. They encode ORs that belong to the G-protein–coupled receptor (Buck and Axel 1991) superfamily of proteins with 7 transmembrane domains (Touhara 2002). OSNs express an olfaction-specific G-protein [G(olf)], (Belluscio et al. 1998), which activates an olfaction-specific adenylate cyclase following the binding of an agonist to the OR. The resulting increase in cyclic adenosine monophosphate concentration leads to the opening of channels, mostly permitting the entry of Ca2+, depolarizing the neuron. This depolarization, amplified by a Ca2+-activated Cl– current, generates an action potential, which is transmitted to the olfactory bulb (Mombaerts 2004). The identification and recognition of odorant molecules following binding to their specific OR has been shown to follow a combinatorial code: a given chemical molecule can bind to several different receptors and receptors can bind several different molecules (Malnic et al. 1999; Gaillard et al. 2002).
Only a few ligand/receptor pairs have been identified in various organisms, including fish, amphibians, and humans, and it is difficult to evaluate the complexity of this combinatorial code (Krautwurst et al. 1998; Zhao et al. 1998; Araneda et al. 2000; Sanz et al. 2005). It therefore remains unclear how many different molecules can bind to a given receptor and how many receptors within a complete OR repertoire bind a specific odorant.
We addressed these issues by cloning a number of canine OR genes (Quignon et al. 2003, 2005) and transiently expressing them in a human embryonic cell line (HEK293) engineered to express the G(olf) subunit constitutively (Belluscio et al. 1998). We then exposed these cells to different odorant compounds.
Our results indicate that the combinatorial code may be highly complex, with some odorants potentially recognized by more than 20 receptors.
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Materials and Methods |
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OR Gene Cloning
Two complementary oligonucleotides (136 mers each) coding a cleavable influenza virus leader peptide and a cMyc epitope were chemically synthesized (Eurogentec, Seraing, Belgium) and cloned in the NheI and EcoRI restriction sites of the plasmid vector pIRES (Clontech, Mountain View, CA) (Gaillard et al. 2002).
A genomic DNA sample prepared from a mongrel dog hepatocytes was used as the starting material for cloning a number of OR genes in frame with the leader peptide and the cMyc epitope. Pairs of specific oligonucleotides, 19–25 bases long, were designed with primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) for the amplification of various OR reading frames by polymerase chain reaction (PCR) (20 ng of mongrel DNA were mixed with 1 µl of primers [3 µM each], 0.8 µl of dNTP [2.5 mM], and 0.1 µl of Phusion enzyme [Finnzymes, Espoo, Finland] in buffer [final volume: 10 µl]). Amplification was performed with a touchdown program (1 min at 98 °C followed by 20 cycles [10 s at 98 °C/15 s at 61 °C {–0.5 °C per cycle}/30 s at 72 °C] and 10 cycles [10 s at 98 °C/15 s at 51 °C/30 s at 72 °C]). A second round of PCR was then carried out, with 31 to 45 mer pairs of oligonucleotides. One oligonucleotide in each pair was designed with a 5' extension of 16 nucleotides (5'-GGAGGACCTGCTCGAG... ...) corresponding to the cloning vector and a 3' sequence overlapping the ATG start codon of each OR open reading frame (ORF). The second oligonucleotide had a common 5' extension corresponding to the vector sequence (5'-TGCATGCTCGACGCGT... ... ...) and a specific 3' sequence overlapping a region downstream from the OR gene stop codon. PCR conditions were as described above starting with one-tenth of the product obtained previously. The products of this second PCR were integrated in frame with the leader peptide and the cMyc epitope by in vitro recombination, according the BD In-Fusion PCR Cloning Kit protocol (Clontech). We recovered up to 5 isolated colonies per cloning experiment for complete sequencing of the ORF and selection of a suitable clone for expression in heterologous cells. (Sequences of the 38 clones and corresponding OR sequences can be visualized at: http://genoweb.univ-rennes1.fr/Dogs/ORpolymorphism.html.)
Cell Culture
All cell culture products, media, and antibiotics were obtained from Invitrogen (Cergy-Pontoise, France). Adherent HEK293 cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 4500 mg/l glucose, 2 mM L-Glutamax, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, supplemented with 50 units/ml penicillin and 50 µg/ml streptomycin, 1x modified Eagle medium, and 10% fetal calf serum (FCS from PAA, Les Mureaux, France), at 37 °C, under a humidified atmosphere containing 5% CO2. For the HEK293 subclone expressing the human G(olf) subunit, G418 sulfate (Sigma, Saint Quentin Fallavier, France) was added to the culture medium and maintained at a concentration of 800 µg/ml.
Construction of a HEK293 Subclone Expressing the Human G(olf) Subunit
The IRAKp961J1091Q2 plasmid encoding the human G(olf) gene (GenBank accession No. BC050021) was obtained from RZPD Deutsches Ressourcenzentrum für Genomforshung GmbH. The human gene was amplified by PCR and inserted into site A of the pIRES vector. The resulting plasmid was used to transfect HEK293 cells using Fugene (Roche Life Sciences, Meylan, France) as a transfection reagent according to the standard protocol. Selection pressure was continually maintained by adding 800 µg/ml of G418. Cell cloning was carried out by limited serial dilution. Finally, the HEK293-3B9 (Figure 1) clone was retained and used for transient OR expression experiments.
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excitation: 546 nm; Transient OR Expression
HEK293-3B9 cells were plated on coverslips or in LabTek vessels (8-well chambers) and incubated for 24 h. They were then transfected with 200 ng of a plasmid encoding a canine OR using Fugene as a transfection reagent, a yield of 30% was regularly achieved. Cell monolayers were almost confluent after 48 h and were exposed to odorants.
cMyc and G(olf) Immunodetection
The cellular expression of cMyc–tagged OR was assessed by incubation with anti-cMyc 9E10 monoclonal antibody for 2 h at room temperature in phosphate-buffered saline (PBS) supplemented with 5% FCS and 0.1% Triton. Bound antibodies were detected by incubation with fluorescein isothiocyanate–labeled donkey anti-mouse antibody (Jackson Immunoresearch, Suffolk, UK) for 1 h. G(olf) was detected by incubation for 2 h with a polyclonal rabbit antibody (C-18, Santa Cruz, Heidelberg, Germany;) and then for 1 h with Tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit antibody (Jackson Immunoresearch). Labeled ORs and G(olf) were visualized under a Leica microscope (DMRXA2). cMyc fluorescence signals were analyzed by deconvolution using Metamorph software (Figure 2).
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Odorant Exposure and Calcium Imaging
Odorant compounds of the highest purity available were purchased from Sigma. Odorant stock solutions were prepared immediately before use, at a concentration of 0.5 M, in dimethylsulfoxide (DMSO). Serial dilutions of stock solutions in PBS were prepared before use. Cells were washed twice with PBS and loaded by incubation for 30 min at 37 °C with the fluorescent Ca2+ indicator Fluo4 from Invitrogen (5 µM final concentration) dissolved in DMSO, with 0.02% Pluronic as an adjuvant, in the presence of 2.5 mM probenicid (Invitrogen). Cells were washed twice in PBS and incubated for a further 15 min at 37 °C. Labtek vessels were placed on the stage of a Leica DMIRB microscope equipped with Metamorph software, and odorant solutions were added. The cells were observed with a 10x oil immersion objective. Images were acquired every 3 s for 90 s to record the fluorescence emitted due to the binding of the odorant and the increase in intracellular Ca2+ concentration. Signal/background ratio >2 was considered as positive. All experiments were repeated several times (3–6 times).
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Results |
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RnORI7 has been the subject of numerous studies, and its recognition of octanal is well documented (Krautwurst et al. 1998; Zhao et al. 1998; Araneda et al. 2000). Based on sequence comparison and position in the genome (Lindblad-Toh et al. 2005), we selected CfOR12A07 as the most likely candidate for its canine ortholog and used this molecule as the starting point for our study.
Subclone HEK293-3B9 (HEK293 cells constitutively expressing G(olf)) 48 h after transfection with a plasmid encoding CfOR12A07 displayed an increase in intracellular Ca2+ concentration in response to exposure to 10–11 M octanal. This result provides functional confirmation that CfOR12A07 is the true ortholog of RnORI7.
CfOR12A07 belongs to canine OR family 6. Based on sequence comparison and the phylogenetic tree, this family comprises 88 genes plus 46 pseudogenes (Quignon et al. 2005). We investigated the complexity of the combinatorial code by selecting the 38 canine OR genes constituting the 17 subfamilies within the phylogenetic tree sharing a root with subfamily 6U, to which CfOR12A07 belongs (Figure 3). All these genes were inserted into pIRES containing cMyc, as described in Materials and Methods and used to transfect clone HEK293-3B9. We then analyzed expression of the cMyc epitope at the cell membrane. Figure 2 provides an illustration of cMyc epitope immunodetection for the transient expression of 6 OR genes. All were correctly expressed 48 h after transfection, and at least some of the corresponding proteins had been translocated to the cell membrane, a prerequisite for odorant binding and signal processing.
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HEK293-3B9 subclones expressing the 38 ORs were exposed to octanal at concentrations of 10–11, 10–8, and 10–6 M. We found that 28 of the 38 ORs tested responded to octanal by inducing an increase in calcium concentration, and 14 of these 28 ORs responded to concentrations as low as 10–11 M.
HEK293-3B9 cells expressing the same set of OR were also exposed to various concentrations of the entire collection of aliphatic aldehydes from C6 to C12. Results presented in Table 1 showed that all but 2 of the ORs (CfOR0334 and CfOR0274) responded to between 1 and 6 compounds, 3 on average (Table 1; Figure 4). The various aldehydes bound to different numbers of OR, with nonanal binding to 14 ORs and octanal to 28. However, no 2 aldehydes bound to the same set of OR, reflecting the evolution of a highly specific odorant recognition function in dogs.
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We then tested 13 ORs, 5 of which had bound to octanal in the previous experiment with another set of chemical compounds: vanillin, limonene, cyclohexanone, and lyral. These ORs were far less sensitive to these nonaldehyde compounds (Table 2).
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Discussion |
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Only a handful of the many thousands of possible ligand/receptor pairs have been identified for technical reasons and due to the lack of simple, reliable methods. Two main approaches have been used. The first is based on the exposure of fresh epithelium explants to an odorant and subsequent isolation of the responding neurons by tissue microdissection followed by reverse transcribed-PCR, cloning, and sequencing of the corresponding OR mRNA (Malnic et al. 1999; Touhara et al. 1999). The second, more widely used approach is based on the expression of OR by transiently transformed cells (Krautwurst et al. 1998; Zhao et al. 1998; Wetzel et al. 1999; Gaillard et al. 2002; Spehr et al. 2003). However, the difficulties encountered in obtaining correctly expressed receptors translocated to the cell membrane have limited the use of this technique to a small number of OR. Based on published results (Gaillard et al. 2002), we constructed a vector based on the pIRES backbone plasmid that worked fairly well, with substantial expression of the encoded OR at the cell surface for all the OR genes that we attempted to clone.
All but 2 of the ORs tested (CfOR0334 and CfOR0274) responded to at least one aldehyde (Tables 1 and 2). The observation that the cMyc epitope, encoded in frame with these 2 ORs, was correctly expressed at the cell membrane rules out the possibility that the failure to recognize any of these aldehydes was due to a lack of correct expression and translocation. Surprisingly, although different aldehydes bound different sets of receptors, they all bound a large number of receptors, ranging from 14 for nonanal to 28 for octanal. These large numbers of bound receptors were consistent with previous estimates of 33–55 octanal-responsive OR in the rat OR repertoire (Araneda et al. 2004) but raised questions concerning the specificity of responses and the number of OR from the entire repertoire likely to recognize these aldehydes. Given the large number of positive responses observed, our main concern was false positives rather than false negatives due to fluorescence emission in the absence of aldehyde binding. Evidence supporting this view is provided by the setup of the experiments and the diversity of the responses. Within this experimental design, HEK293 cells plated in parallel in a large number of Labtek wells were exposed to different odorants at different concentrations. In the context of this setup, it is difficult to imagine why cells in one well would react to octanal but those in neighboring wells would not. Furthermore, the timing of the fluorescence emission, beginning about 25 s after the addition of the odorant and not observed after the addition of solvent alone, clearly demonstrated the specificity of the response. Finally, whereas these OR tended to respond strongly to aldehydes, they gave mostly negative results with other compounds (Table 2).
If the positive results obtained are regarded as specific and we accept that many of the 38 ORs tested recognized one or several aldehydes, then several questions remain to be answered: Was the proportion of positive results high because all 38s OR are members of family 6? How many ORs within the entire repertoire would recognize such compounds? The fact that all these ORs belong to family 6, constituting a group of subfamilies with a common root, may account for the high proportion of positive answers. However, there is evidence to suggest that this explanation is insufficient: 1) phylogenetic trees are based on alignments of the complete amino acid sequence; 2) the ligand-binding pockets are composed of limited numbers of unknown amino acids; and 3) it is therefore possible to envisage the possibility of 2 ORs with limited overall sequence identity, belonging to 2 different families or even classes, retaining a similar or identical ligand-binding pocket. One way to resolve this issue would be to clone and to express all members of the OR repertoire—clearly a difficult task—or to have a thorough knowledge of ligand-binding pockets, for which only partial descriptions are currently available (Lai et al. 2005).
Other intriguing questions remain unanswered. Why are the OR repertoires encoded in mammalian genomes so large? Why is the combinatorial code for odor perception so redundant, with so many receptors recognizing the same odorant molecule? No satisfactory answers have yet been found to these questions, but it has been shown that, in addition to acting as agonists of a number of ORs, odorant molecules may well also act as antagonists. This suggests that the receptor code may not be additive, with the perception of an odorant mixture not resulting from simple addition of the perception of each individual component (Kajiya et al. 2001; Oka et al. 2004; Sanz et al. 2005). Thus, 2 odorants acting as agonists of a number of ORs when applied alone, could, when applied together, be agonists of other ORs or antagonists of a certain number of ORs for which they are agonists when applied alone. For example, in the experiments reported, nonanal acted as an agonist of 14 ORs and octanal acted as an agonist of 28 ORs. Octanal acted as an agonist of 12 of the 14 ORs recognized by nonanal, so only 2 ORs, CfOR5925 and CfOR0093, were recognized by nonanal alone. With an additive receptor code, the difference between octanal alone and a mixture of octanal and nonanal would be marginal. By contrast, with a nonadditive receptor code, the difference between octanal alone and the octanal–nonanal mixture would be expected to be greater, suggesting that a complex combinatorial receptor code combined with a nonadditive receptor code would be an efficient strategy for the perception not only of many individual odorants but also of myriad mixtures.
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Funding |
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Centre National de la Recherche Scientifique, the Université de Rennes 1, and Technical Support Working Group (to F.G.).
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Acknowledgments |
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The technical assistance for microscopy of Stephanie Dutertre was greatly appreciated.
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Footnotes |
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Corresponding Editor: Elaine Ostrander
This paper was delivered at the 3rd International Conference on the Advances in Canine and Feline Genomics, School of Veterinary Medicine, University of California, Davis, CA, August 3–5, 2006.
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