Human olfactory receptor responses to odorants

Abstract

Although the human olfactory system is capable of discriminating a vast number of odors, we do not currently understand what chemical features are encoded by olfactory receptors. In large part this is due to a paucity of data in a search space covering the interactions of hundreds of receptors with billions of odorous molecules. Of the approximately 400 intact human odorant receptors, only 10% have a published ligand. Here we used a heterologous luciferase assay to screen 73 odorants against a clone library of 511 human olfactory receptors. This dataset will allow other researchers to interrogate the combinatorial nature of olfactory coding.

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OPEN

SUBJECT CATEGORIES

Behavioural genetics

Olfactory receptors

Received: 22 April 2014

Accepted: 10 December 2014

Published: 03 February 2015

Human olfactory receptor responses to odorants

Joel D. Mainland1,2,3, Yun R. Li2, Ting Zhou2, Wen Ling L. Liu2 & Hiroaki Matsunami2,4 Although the human olfactory system is capable of discriminating a vast number of odors, we do not currently understand what chemical features are encoded by olfactory receptors. In large part this is due to a paucity of data in a search space covering the interactions of hundreds of receptors with billions of odorous molecules. Of the approximately 400 intact human odorant receptors, only 10% have a published ligand. Here we used a heterologous luciferase assay to screen 73 odorants against a clone library of 511 human olfactory receptors. This dataset will allow other researchers to interrogate the combinatorial nature of olfactory coding.

Design Type(s) cellular process design

Measurement Type(s) receptor activityTechnology Type(s) Dual-Luciferase Reporter Assay System Factor Type(s) gene listSample Characteristic(s) Homo sapiens Mus musculus

1Monell Chemical Senses Center, 3500 Market Street, Philadelphia, Pennsylvania 19104, USA. 2Department of

Molecular Genetics and Microbiology, Duke University Medical Center, Research Drive, Durham, North Carolina

27710, USA. 3Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia,

Pennsylvania 19104, USA. 4Department of Neurobiology and Duke Institute for Brain Sciences, Duke University

Medical Center, Research Drive, Durham, North Carolina 27710, USA.

Correspondence and requests for materials should be addressed to J.D.M. (email: [email protected])

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Background & Summary

Previous functional analysis of olfactory receptors (ORs) in olfactory neurons and in heterologous cells found that different odorants are recognized by unique, but overlapping ensembles of ORs14. These ndings suggest that specic patterns of ORs activated by an odorant code for the odorants identity, but there are few, if any, explicit predictions relating OR activity patterns to olfactory perception.

Matching mammalian ORs to ligands has seen limited success, and the picture is even worse when considering human ORs; ligands have been published for only 49 of the approximately 400 intact human ORs521. This lack of data is a critical bottleneck in the eld; matching ligands to ORs is critical for understanding the olfactory system at all levels and is essential for building viable models of olfaction. The characterization of OR responses to ligands in the empty neuron system of Drosophila melanogaster22 has allowed researchers in the eld to choose rationally diverse odorant sets23 and specically manipulate subpopulations of ORs to dissect olfactory coding24,25. Extending this idea by matching odorants to human ORs has the added advantage that humans can directly communicate their perception of odor intensity, pleasantness, and quality.

In addition, understanding the role of a single OR in olfactory perception allows us to look at evolutionary changes in OR genes in a new light. For example, the knowledge that Tas1r2 is a pseudogene in seven of twelve species in the order Carnivora26 is difcult to interpret in isolation. The knowledge that Tas1r2 is the primary mediator of sweet taste in mice, however, suggests that carnivores do not need to taste sweet and therefore there is no selective pressure on the gene. As several genome sequencing projects are examining both genetic variation within humans27 and across species28, understanding the role of OR genes in olfactory perception becomes crucial to the interpretation of how and why genetic changes occur over the course of evolution.

In a recent manuscript we conducted a high-throughput screen of 511 human odorant receptors against 73 odorants15. The resulting screen identied agonists for 27 odorant receptors, including 18 that were previously orphan receptors. We went on to characterize how genetic variation in these receptors alters both in vitro responses and inuences olfactory perception. In this manuscript we present the full screening data to permit wider reuse and reanalysis.

In summary, this dataset addresses a major bottleneck in the eld, namely how the physical stimulus in olfaction is transduced into receptor responses. In addition, the G-protein coupled receptor class accounts for approximately 50% of therapeutic drug targets29. The ORs, being GPCRs, offer the opportunity to examine the strategies employed by this receptor class to recognize a wide variety of ligand structural features and thus will provide insight into fundamental principles of ligand recognition by GPCRs. Matching odorants to ORs will provide a valuable resource to the eld and allow more specic explorations of links between odor, behavior and ecology.

Methods

These methods are expanded from descriptions in our previous work15.

Cloning

OR open reading frames were amplied from genomic DNA using Phusion polymerase and subcloned into pCI expression vectors (Promega) containing the rst 20 residues of human rhodopsin (Rho tag). Human ORs were amplied from the pooled genomic DNA of 20 participants from the International Hapmap Consortium, while mouse ORs were amplied from the genomic DNA of C57/BL6 mice. The sequences of the cloned receptors were veried by sequencing (3100 Genetic Analyzer, Applied Biosystems). Clones that were present in the 1000 Genomes Project, but not cloned from our pooled genomic DNA sample, were created using an overlap extension polymerase chain reaction protocol30.

Luciferase assay

The Dual-Glo Luciferase Assay System (Promega) was used to measure receptor responses as previously described31. Hana3A cells were transfected with 5 ng/well of RTP1S32, 5 ng/well of pRL-SV40, 10 ng/well of CRE-luciferase, 2.5 ng/well of M3 (ref. 33), and 5 ng/well of odorant receptor. 1 M odorant stocks are diluted in DMSO. 24 hours following transfection, transfection media was removed and replaced with the appropriate concentration of odor diluted from the 1 M stocks in CD293 (Gibco). Four hours following odor stimulation luminescence was measured using a Polarstar Optima plate reader (BMG). All luminescence values were divided by the Renilla Luciferase activity to control for transfection efciency in a given well. Data were analyzed with Microsoft Excel, GraphPad Prism 4, and MATLAB.

Primary screen design

Our screen design is outlined in Figure 1. In the primary screen we stimulated 511 human ORs with 73 odorants used in previous psychophysical testing12,34. We applied the majority of odorants at a

concentration of 100 M. All plates in the primary screen included 85 test wells, ve broadly-tuned odorant receptors (Olfr1079, OR2W1, Olfr1377, Olfr73, Olfr1341), and six wells transfected with Or544 which served as a standard. Of the six wells transfected with Olfr544, three were challenged with the diluent (CD293) and three were challenged with 10 M of a known ligand for Olfr544 (nonanedioic acid).

Each screening run consisted of twelve plates where each of the 96-wells were transfected with the same

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511 clones (394 ORs)

1572 odor/receptor pairs

425 hits, 190 clones representing 160 ORs

63 clones (27 ORs)

Clone initial odorant receptor library

Primary Screen:Test all receptors at 100 M with 73 odorants

Secondary Screen: Test the best odor/receptor pairs from the primary screen at 1 M, 10 M and 100 M in triplicate

Dose response curve, seven concentrations in triplicate

Figure 1. Outline of the screening procedure. This gure was reprinted from our previous publication15, where

it was included as Supplementary Figure 1.

set of receptors. One plate had no odor in all test wells and served as a baseline. The other eleven plates were each challenged with a different test odor.

Secondary screen design

To rank hits from the Primary Screen we standardized each plate, setting the mean Olfr544 response to nonanedioic acid minus the mean Olfr544 response to the no-odor control to a value of 1. We then subtracted the baseline response for each receptor from the no-odor plate from the response to the odor challenge and ranked the resulting values. We selected the top 5% of odorant/receptor pairs from the primary screen, although not more than the top ten ligands for a given receptor. We then performed a secondary screen in which each odorant receptor was tested against a no-odor control as well as 1, 10 and 100 M of odor. Each comparison was performed in triplicate, where each measure was collected from separate wells, but each well contained cells from the same parent plate of cells. Note that we began the secondary screen before completion of the entire primary screen, so some odor/receptor combinations outside of the overall top 5% were tested.

Dose-response design

We then constructed dose-response curves using concentrations ranging from 10 nM to 10 mM for the odor/receptor pairs that were signicantly different from baseline in the Secondary Screen. Each odorant receptor-odorant dose was tested in triplicate, where each measure was collected from separate wells, but each well contains cells from the same parent plate of cells. A vector-only control was included for each odorant. We t the data to a sigmoidal curve. We counted an odorant as an agonist if the 95% condence intervals of the top and bottom parameters did not overlap, the standard deviation of the tted log EC50 was less than 1 log unit, and the extra sums-of-squares test conrmed that the odorant activated the receptor signicantly more than the control, which was transfected with an empty vector. This data identied 25 odorant receptors with a signicant response to at least one agonist15 (Figures 2 and 3).

Data Records

The data for this manuscript have been deposited in gshare (Data Citation 1). A summary of the clones tested in each phase of the screen is presented in Supplementary Table 1.

Data record 1primary screen

The raw screening results are presented in a tab-separated values le (Data Citation 1). Each row represents an experiment from a single well.

Plate. A unique ID for a 96-well plate on a given date.

Well. A number assigned to each well of the 96-well plate. The wells are sequentially numbered with the upper-leftmost well assigned as 1 and the lower-leftmost well assigned as 85 (see Figure 4).

Concentration. The concentration of the odorant applied in uM. A 9999 indicates no odor was applied (DMSO was diluted 1:10,000 in CD293).

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Figure 2. Normalized dose-response curves of the receptor encoded by the most common functional allele for 25 receptors. The responses of cells transfected with either a plasmid encoding the indicated odorant receptor or an empty vector to the indicated odorants. Responses have been normalized such that each receptor has a minimum response of zero and a maximum response of one. Error bars, s.e.m. over three replicates. Abbreviations for the odorants are as follows: +CAR are shown, (+)-carvone; LIN, linalool; GA, geranyl acetate; COUM, coumarin; OTHI, octanethiol; C3HEX, cis-3-hexen-1-ol; EUG, eugenol; EUGME, eugenol methyl ether; ANIS, anisaldehyde; ANDI, 4,16-androstadien-3-one; AND, 5-androst-16-en-3-one; DMHDMF, caramel furanone; +MEN, (+)-menthol; 3PPP, 3-phenyl propyl propionate; VAN, vanillin; LYR, lyral; 2EF, 2-ethyl fenchol; IVA, isovaleric acid; APA, allyl phenyl acetate. This gure was modied from our previous publication15, where it was included as Figure 1.

Luc. The number of photons counted by the plate reader when the well was treated with the luciferase substrate. This is the cAMP reporter, and therefore correlates with receptor responses to odorants.

RL. The number of photons counted by the plate reader when the well was treated with the Renilla luciferase substrate. This is the constitutively active reporter, which serves as a control for cell death and transfection efciency.

OR. A unique ID for each olfactory receptor clone.

Odor. A unique ID for the odorant applied to the well.

Date. The date the experiment was run in MM/DD/YY format.

Data record 2secondary screen

The raw screening results are presented in a tab-separated values le (Data Citation 1). Each row represents an experiment from a single well.

Date. The date the experiment was run in MM/DD/YY format.

OR. A unique ID for each olfactory receptor clone.

Odor. A unique ID for the odorant applied to the well.

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Figure 3. Dose-response curves of the receptor encoded by the most common functional allele for 25 receptors. The responses of cells transfected with either a plasmid encoding the indicated odorant receptor or an empty vector to the indicated odorants. Error bars, s.e.m. over three replicates. Abbreviations for the odorants are as follows: +CAR are shown, (+)-carvone; LIN, linalool; GA, geranyl acetate; COUM, coumarin; OTHI, octanethiol; C3HEX, cis-3-hexen-1-ol; EUG, eugenol; EUGME, eugenol methyl ether; ANIS, anisaldehyde; ANDI, 4,16-androstadien-3-one; AND, 5-androst-16-en-3-one; DMHDMF, caramel furanone;

+MEN, (+)-menthol; 3PPP, 3-phenyl propyl propionate; VAN, vanillin; LYR, lyral; 2EF, 2-ethyl fenchol; IVA, isovaleric acid; APA, allyl phenyl acetate. This gure was modied from our previous publication15, where it was included as Figure 1.

Concentration. The concentration of the odorant applied in uM. A 0 indicates no odor was applied (CD293 only). Note that rows for the no odor condition will contain an Odor label to facilitate pairing controls with the matched experiments at other concentrations.

NormalizedLuc. The Luc/RL ratio from each well.

Data record 3dose-response

The Luc/RL ratios are presented in a tab-separated values le (Data Citation 1). Each row represents an experiment from a single well. The EC50 for each odor/receptor pair that passed this phase of screening is listed in Table 1 (available online only).

Concentration. The molarity applied to the well. Note that the no-odor condition was coded as 12 in this column.

NormLuc. The Luc/RL ratio from each well.

OR. A unique ID for each olfactory receptor clone.

Odor. A unique ID for the odorant applied to the well.

Date. The date the experiment was run in MM/DD/YY format.

Data record 4receptors

The receptor information is presented in a tab-separated values le (Data Citation 1). Each row represents a single olfactory receptor.

OR. A unique ID for the olfactory receptor, used in Data Records 13.

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Transfection Master Plate

Each master transfection plate contains new receptors in the test wells, but the same standard and broadly-tuned controls were used across all master plates.

Test wells

Broadly tuned ORs

Standard, no odor baseline

Standard, with odor

Each 96-well plate received a different odor.

The 12th plate is a no-odor control.

Figure 4. Plate layout for the primary screen. Screens were set up with a master transfection plate for each day. The master transfection plate was used to transfect twelve plates. Each plate was then stimulated with a different odor. Eleven wells were reserved for broadly tuned receptors and a standard to validate the protocol.

Gene. The gene name of the olfactory receptor encoded in a given plasmid, followed by the amino acid changes from the hg19 reference sequence for the gene. For example, OR6Y1 V252I encodes the gene OR6Y1, but while the hg19 reference sequence has a V as the 252nd amino acid, this clone encodes an I at position 252. Note that some plasmids were cloned from older builds of the genome, which may start at a different methionine than the current model. These differences from h19 reference may not appear here. Please consult the nucleotide sequence for a more thorough description of differences from the reference sequence.

NucleotideSeq. The nucleotide sequence for the olfactory receptor encoded in a given plasmid. Note that the rhodopsin tag is not included in this eld.

Data record 5odors

The odor information is presented in a tab-separated values le (Data Citation 1). Each row represents a single odor. Further synonyms can be found in a le which correlates all of the CIDs in PubChem with submitted synonyms (ftp://ftp.ncbi.nlm.nih.gov/pubchem/Compound/Extras/CID-Synonym-ltered.gz).

Odor. A unique ID for the odorant, used in Data Records 13.

CASRegistryNum. The Chemical Abstracts Service number for the odorant, when available. OdorName. Common name for the odorant applied to the well.

CID. PubChem Compound Identication number, a non-zero integer PubChem accession identier for a unique chemical structure, when available.

SMILES. Simplied Molecular-Input Line-Entry System string, an ASCII string identier for a unique chemical structure.

Technical Validation

The screen included two types of negative controls. Cells transfected with each receptor clone were challenged with a no-odor stimulation (CD293 alone) to control for baseline receptor activity. Cells transfected with an empty vector were challenged with all of the tested odorants to control for nonspecic activation. All plates in the primary screen included ve broadly-tuned odorant receptors and six wells transfected with Olfr544 (also known as MOR42-3 or S6) which served as a standard. Of the six wells transfected with Olfr544, three were challenged with the diluent (CD293), and three were challenged with 10 M of nonanedioic acid. Rankings from the primary screen consistently predicted results from later screens (Figure 5). The ultimate validation of this assay is prediction of behaviour, and previous results from similar in vitro assays have been shown to predict human olfactory perception12,15,35,36.

Usage Notes

We have included R37 scripts to facilitate analysis of the data. The included R scripts, Supplementary Files 1 and 2, have been implemented as a hosted Shiny38 application (http://www.monell.org/ supplemental_les/jmainland/jm0714) to facilitate browsing the data3941. The R markdown le,

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1

85

2 3

All plates on a given date were transfected with the same master.

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1.00

True Positive Rate (Sensitivity)

0.75

0.50

0.25

AUC=0.72 (95%CI 0.74 0.71) line of no discrimination

0.00

0.00 0.25 0.50 0.75 1.00

False Positive Rate (1Specificity)

1.00

True Positive Rate (Sensitivity)

0.75

0.50

0.25

AUC=0.64 (95%CI 0.67 0.61) line of no discrimination

0.00

0.00 0.25 0.50 0.75 1.00 False Positive Rate (1Specificity)

1.00

True Positive Rate (Sensitivity)

0.75

0.50

0.25

AUC=0.7 (95%CI 0.74 0.66) line of no discrimination

0.00

0.00 0.25 0.50 0.75 1.00 False Positive Rate (1Specificity)

Figure 5. Validation of the screen. An ROC curve indicates that (a) the primary screen predicts odor/receptor pairs that pass the secondary screen, (b) the primary screen predicts odor/receptor pairs that pass the dose response lter, and (c) the secondary screen predicts odor/receptor pairs that pass the dose response lter.

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Supplementary File 3, includes code to carry out routine normalization of the primary screen, t an ANOVA to data from the secondary screen, t a sigmoid to the dose-response data, and create Figures 2, 3 and 542,43.

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Data Citation

1. Mainland, J. D., Yun, R. L., Zhou, T., Liu, W. L. L. & Matsunami, H. Figshare http://dx.doi.org/10.6084/m9.gshare.979135 (2014).

Acknowledgements

This work was supported by R01 DC005782, R01 DC012095, R03 DC011373, R01 DC013339, the Defense Advanced Research Project Agency RealNose Project, and an NRSA postdoctoral fellowship F32 DC008932 to J.D.M.

Author Contributions

J.D.M. conceived and designed the project, supervised the screen, carried out the analysis and wrote the paper. Y.R.L. carried out portions of the screen and analysis. T.Z. carried out portions of the screen. W.L.L.L. carried out portions of the screen. H.M. conceived, designed, and supervised the project.

Additional information

Table 1 is only available in the online version of this paper.

Supplementary information accompanies this paper at http://www.nature.com/sdata

Competing nancial interests: The authors declare no competing nancial interest.

How to cite this article: Mainland, J. D. et al. Human olfactory receptor responses to odorants. Sci. Data 2:150002 doi: 10.1038/sdata.2015.2 (2015).

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