Article Figures & Data
Figures
- Figure 1.
Phylogenetic analysis and responsiveness of the muscone OR family. A, Phylogeny and muscone responses of the muscone OR family in six mammalian species. Left, Phylogenetic tree of OR genes in three OGGs (Niimura et al., 2014). The scale bar indicates the number of amino acid substitutions per site. Bootstrap values obtained from 500 resamplings are shown on the tree. Right, Responses of ORs expressed in HEK293 cells to 100 μm muscone in the luciferase reporter gene assay. The ORs with colored letters showed relatively strong responses to muscone. Normalized value was calculated by dividing the fold increase upon stimulation with muscone in OR-expressing cells by the fold increase in empty vector control cells. The broken line indicates the value of empty vector control, 1.0. Error bars indicate SEM (n = 3). OR5AN1 and OR5AN2 (an inferred functional version of OR5AN2P, see Materials and Methods) in human; Patr_5AN2 (PatrOR11R.6.1) in chimpanzee; Poab_5AN1 (PoabORc2797.1) and Poab_5AN2 (PoabORc2797.5) in orangutan; Mamu_5AN1 (MamuOR14.1.9) and Mamu_5AN2 (MamuOR14.1.12) in macaque; Caja_5AN2 (CajaORc7026.1) in marmoset; MOR215-1 (Olfr1440), MOR214-1 (Olfr262), MOR214-2 (Olfr1436), MOR214-3 (Olfr235), MOR214-4 (Olfr1433/1434), MOR214-5 (Olfr1431), and MOR214-6 (Olfr1437) in mice. B, Mouse cluster on chromosome 19 corresponded to marmoset clusters on chromosome 11, macaque clusters on chromosome 14, orangutan clusters on chromosome 11, chimpanzee clusters on chromosome 11, and human clusters on chromosome 11. Each horizontal line represents a chromosome. The position of each OR gene is represented by a colored vertical bar above or below a horizontal line, the latter indicating the opposite transcriptional direction to the former. Each bar is colored according to the OGG to which the OR gene belongs. The genes in the same OGG are connected by a dashed line. C, Dose–response curves of muscone ORs. The squares show normalized luciferase values. The normalized luciferase value was calculated by the following formula: (Fn − Fno odor) * 100/(Fmax − Fno odor), where Fn represents the fold increase at a concentration of muscone, Fno odor represents the fold increase for no odor, and Fmax represents the fold increase for muscone at a concentration that showed a maximal response. EC50 and 95% confidential intervals (in parentheses) are shown in each graph. Error bars indicate SEM (n = 3).
- Figure 2.
Structure–activity relationships of the muscone OR family for muscone-related compounds. A, Chemical structures of 18 musk odorants and seven related compounds used in structure–activity relationship studies grouped into five types: MCM (pink): 1. muscone [C16H30O], 2. muscenone [C16H28O], 3. cyclopentadecanone [C15H28O], 4. ambretone [C16H28O], 5. globanone [C16H28O], 6. cosmone [C15H26O], 7. 3-methylcyclotetradecanone [C15H28O], 8. ethylene brassylate [C15H26O4], 9. ambrettolide [C16H28O2], 10. exaltolide [C15H28O2], and 11. habanolide [C15H26O2]); NM (blue): 12. musk xylol [C12H15N3O6] and 13. musk ketone [C14H18N2O5]); PCM (yellow): 14. galaxolide [C18H26O], 15. tonalide [C18H26O], 16. celestolide [C17H24O], and 17. cashmeran [C14H22O]); ACM (orange): 18. helvetolide [C17H32O3]; and others (gray): 19. cyclopentadecane [C15H30], 20. cyclopentadecanol [C15H30O], 21. cyclohexanone [C6H10O], 22. cyclodecanone [C10H18O], 23. cycloundecanone [C11H20O], 24. 2-pentadecanone [C15H30O], and 25. 8-pentadecanone [C15H30O]). B, Response patterns of musk ORs to various musk-related compounds at 10 μm in the HEK293 luciferase assay. The upper dendrogram indicates the result of cluster analysis based on odor response patterns. The normalized values used for the cluster analysis were calculated by the following formula: (Fn−Fno odor) × 100/∑n=125Fn − Fno odor where Fn and Fno odor represent the fold increase for the nth odor and no odor, respectively. When Fn is smaller than Fno odor, the normalized value is regarded to be zero. Only positive values are shown. The empty boxes indicate that the value is <0.1.
- Figure 3.
Dose–responses of MOR215-1 and OR5AN1 to NMs and muscone enantiomers. A, Dose–response curves of MOR215-1 and OR5AN1 to muscone, musk xylol, and musk ketone. The EC50 values (μm) of MOR215-1 to muscone were 0.50; to musk xylol, 34.9; and to musk ketone, 2.9. The EC50 values (μm) of OR5AN1 to muscone were 12.5; to musk xylol,16.7; and to musk ketone, 0.79. The calculation method of fold increase was described in the Materials and Methods. Error bars indicate SEM (n = 3). The data for muscone are the same as in Figure 1C. B, Dose–response curves of MOR215-1 and OR5AN1 to racemic-muscone, l-muscone, and d-muscone. The EC50 values (μm) of MOR215-1 to l-muscone were 0.63 and to d-muscone were 2.2. The EC50 values (μm) of OR5AN1 to l-muscone were 13.3 and to d-muscone were 11.3. Response (%) of y-axis is similar to the normalized luciferase value in Figure 1C, but Fmax represents the fold increase for racemic-muscone at a concentration that showed the maximal response. Error bars indicate SEM (n = 3). The data for racemic-muscone are the same as in Figure 1C.
- Figure 4.
Screening of human ORs for muscone and NMs. A, Screening of musk receptors from the human chemosensory receptor repertoire. A total of 405 ORs, seven TAARs, and five VNRs expressed in HEK293 cells were stimulated with 100 μm muscone, musk xylol, or musk ketone, and the luciferase assay was performed. Numbers (#1–14, #51, #52, #56) with color bars on the x-axis indicate OR families. T, TAARs; V, VNRs. Fold increase was calculated using the same method as in Figure 3 (see Materials and Methods). Mean values of two screening replicates are shown. B, Structure–activity relationships of OR5AN1, OR1A1, and OR2J3 for musk-related compounds shown in Figure 2A. The blue and orange bars show the responses to 10 and 100 μm compounds, respectively. The normalized value was calculated for each compound by the following formula: Fn/Fempty vector, where Fn and Fempty vector represent the fold increase of each OR and empty vector, respectively. Error bars indicate SEM (n = 3).
- Figure 5.
Behavioral responses to muscone in MOR215-1 KO mice. A, Time to find the odor source in WT C57BL/6 mice (W) and MOR215-1 KO mice (K). Each triangle depicts a single individual. Animals that failed to find muscone (left) or eugenol (right) in 5 min were plotted at 5. PG, Propylene glycol. Muscone and eugenol were serially diluted from 10−1 (10%) to 10−7 (0.00001%) in PG and from 10−1 (10%) to 10−8 (0.000001%) in mineral oil. B, Summary of odor source-exploring experiments shown in Figure 5A. Vertical axis represents the percentage of the animals that found the odor sources within 5 min. The number of animals that found the odor sources and the number of total animals examined are shown inside each bar.
