Evidence for antagonism between odorants at olfactory receptor binding in humans

W.L.P. Bredie and M.A. Petersen (Editors) Flavour Science: Recent Advances and Trends 9 2006 Elsevier B.V. All rights reserved.

Evidence for antagonism between odorants at olfactory receptor binding in humans G. Sanz, C. Schlegel, J.-C. Pernollet and L. Briand Biochimie de l'Olfaction et de la Gustation, Neurobiologie de l'Olfaction et de la Prise Alimentaire, INRA, Domaine de Vilvert, Bdtiment 526, F 78352 Jouy-en-Josas Cedex France

ABSTRACT The odorant repertoire of two human olfactory receptors (ORs) belonging to two major phylogenetic classes representing ORs from aquatic (class I) and terrestrial animals (class II) were elucidated. For this purpose, a new biomimetic screening assay based on calcium imaging on HEK293 cells expressing an OR and the promiscuous G protein Gin6 was developed. Class I OR52D1 is functional, exhibiting a narrow repertoire related to that of its orthologous murine OR, demonstrating that this class I OR is not an evolutionary relic. In contrast, class II OR1G 1 was broadly tuned towards odorants of 9 to 10 carbons chain length, with diverse functional groups. The existence of antagonism between odorants at level of OR binding was demonstrated. OR1G1 antagonists were observed to be OR specific and structurally related to its agonists, with a shorter size. 1. I N T R O D U C T I O N Humans are able to detect and discriminate myriads of structurally diverse odorants owing to approximately 350 olfactory receptors (ORs). Functional studies have demonstrated that odorant perception results in a combinatory code. One OR recognises multiple odorants and different odorants are recognised by different combinations of ORs [ 1,2]. Moreover, recent data revealed that, in addition to their agonist role, odorants could also inhibit or antagonise other ORs [3-5]. This dual agonist/antagonist combinatorial coding is in good agreement with psychophysical observations of mixture perception, designated as odour masking or counteraction phenomenon [6,7]. The human OR genes, as other mammals, have been classified according to two major phylogenetic classes named class I (fish-like) and class II (terrestrial-type) ORs. Class I ORs have been originally identified in fish and subsequently found in vertebrate species to be intermixed with class II ORs. Whereas human class I ORs have been first

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suggested to be evolutionary relics [8,9], the pseudogene fraction among the human class I ORs (52%) is considerably lower than that observed for human class II ORs (77%) [ 10], suggested than human class I receptors could be functional. Here we report the odorant repertoire of two human ORs. The class I OR52D1 was chosen because it is the orthologue of the known mouse OR S19, for which some agonists have been described [2], whereas the class II OR1G1 was studied because it has been proved to be expressed in olfactory epithelium [ 11 ]. 2. M A T E R I A L S AND M E T H O D S

2.1. Vector constructions and cell culture Human OR genes were amplified by PCR from human genomic DNA (Novagen) using gene-specific primers. In order to help ORs to translocate to the plasma membrane, OR52D1 and OR1G1 receptors were fused at their amino terminal end to the first 36 amino acids of bovine rhodopsin. HEK293 cells (Human Embryo Kidney cells) that stably express G~6 and ORs were transfected and cultured as described elsewhere [12]. The G protein subunit, G~16, w a s co-expressed because it has been shown to couple OR to intracellular Ca 2+ release [ 13].

Figure 1. Cells seeded in a 96-well tissue-culture plate were loaded with Ca 2~ sensitive Fluo-4 fluoprobe and covered with 60 lal of calcium assay buffer. Wells were sealed with a transparent adhesive plastic film. MeOH diluted odorant was applied using a 10 lal Hamilton syringe as a 1-~tl drop hanging beneath the inner face of the plastic film. The MeOH drop evaporated freely in a few s leading to progressive stimulation of cells with the odorant. When antagonist odorants were screened, agonist and antagonist were mixed into MeOH and co-applied as a l-~tl drop. 2.2. Calcium imaging HEK293 derivative cells were seeded onto a poly-L-lysine-coated 96-well tissue-culture plate. Twenty-four hours post-seeding, cells were washed and loaded 30 min at 37 ~ with 2.5 ~tM of the Ca2+-sensitive fluorescent dye Fluo-4 acetoxymethyl ester (Molecular Probes), as described [12]. Calcium imaging was carried out at 28 ~ using

11 an inverted epifluorescence microscope (CK40 Olympus) equipped with a digital camera (ORCA-ER, Hamamatsu Photonics).

2.3. Agonist and antagonist screening using VOFA Agonist and antagonist screening was achieved using a n e w method of odorant application called volatile-odorant functional assay (VOFA) (Figure 1). 3. R E S U L T S Using OR1GI/Gal6 expressing cells, we tested 95 odorants individually by VOFA at a concentration of 10 gM in 1 gl-drop. We found that various odorants belonging to different chemical classes differently elicited OR1 G1 Ca 2+ responses. As illustrated in Figure 2, most active odorants are 8-, 9- and 10-carbon molecules, with an optimum for 9-carbon length. Among the 5 strong agonists, which exhibited aliphatic chains, we found 2 alcohols (2-ethyl-l-hexanol, 1-nonanol), 1 ester (ethyl isobutyrate), 1 lactone (y-decalactone), and 1 aldehyde (nonanal). Medium agonists were thioesters, ketones, one aliphatic acid, and diverse cyclic molecules such as pyrazines or thiazoles. /

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Figure 2. Structural comparison of OR1 G1 ligands and antagonists. Strong agonists are located in the circle, while medium agonists are outside and gathered by chemical classes. Antagonists are shown in grey boxes. Arrows outline the chemical relationships between agonist and antagonist molecules. Peculiar structure features shared by active molecules are also highlighted in grey.

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Studying OR1G1 responses, we observed that co-applications o f equimolar odorant mixtures were less active than pure odorants applied at an identical concentration. By testing odorant couples, we revealed that OR1G1 antagonists were all 6-carbon molecules (Figure 2), with a functional group in common with agonists. In contrast to OR1G1, the fish-like OR52D1 receptor was observed to be functional with a more limited repertoire than the class II OR1G1, which was globally different from OR1G1 agonists (data not shown). Moreover, we observed that OR52D 1 activation was not as size-dependent as observed with OR1G1, suggesting a different mode of interaction with its agonists. 4. D I S C U S S I O N A N D C O N C L U S I O N For the first time the odorant repertoire of two human ORs, belonging to different phylogenetic classes were identified. Interestingly, we found that fish-like OR52D1 odorant spectrum includes the reported agonists o f its murine orthologous OR S19, which was shown to respond to C7 to C9 aliphatic acids and alcohols [2]. We also revealed antagonists against OR1G1 sharing common features with its agonists. While well documented in other G-protein coupled receptors, antagonists at receptor binding level were also recently reported for rodent ORs [3-5] and for human spermatozoa ORs [14]. Future investigations on structure-activity relationships o f human ORs using molecular modelling and mutagenesis might help understanding how aroma perception occurs at the first level o f sensory detection. References

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14.

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