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Combining in vivo and in vitro approaches to identify human odorant receptors responsive to food odorants Lucia M. Armelin-Correa, and Bettina Malnic J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04998 • Publication Date (Web): 05 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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Journal of Agricultural and Food Chemistry

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Combining in vivo and in vitro approaches to identify human odorant receptors

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responsive to food odorants

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Lucia M. Armelin-Correa1, Bettina Malnic2*

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1

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Paulo, São Paulo, Brazil

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2

Department of Biological Sciences, Diadema Campus, Federal University of São

Department of Biochemistry, University of São Paulo, São Paulo, Brazil

ACS Paragon Plus Environment

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Abstract

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Olfactory perception plays an important role in food flavor. Humans have around

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400 odorant receptors, which can be activated by an enormous number of odorants

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in a combinatorial fashion. To date, only a few odorant receptors have been linked

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to their respective odorants, due to the difficulties in expressing these receptor

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proteins in heterologous cell systems. In vivo approaches allow for the analysis of

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odorant-receptor interactions in their native environment and have the advantage

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that the complete OR repertoire is simultaneously tested. Once mouse odorant-

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receptor pairs are defined, one can search for the corresponding human orthologues,

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which can be validated against the odorants in heterologous cells. Thus, the

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combination of in vivo and in vitro methods should contribute to the identification of

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human odorant receptors that recognize odorants of interest, such as key food

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odorants.

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Keywords:

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Odorant receptors, odorants, olfactory sensory neurons, receptor deorphanization,

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key food odorants.


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Abbreviations

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Odorant receptor - OR

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G-protein coupled receptor – GPCR

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Receptor Transporting Protein – RTP

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Olfactory Sensory Neuron – OSN

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Copy Number Variation – CNV

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Single Nucleotide Polymorphism – SNP

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Green Fluorescent Protein – GFP

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phosphorylated ribosomal subunit S6 - pS6

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phosphorylated ribosomal subunit S6 immunoprecipitation - pS6-IP

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Reverse Transcription - Polymerase Chain Reaction - RT-PCR


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Introduction

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Flavor is a critical component of food preference and consumption. Besides taste,

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olfaction plays an important role in flavor perception. When the food is in the oral

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cavity, smell perception during retro nasal olfaction (breathing out) contributes to

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flavor generation 1. Foods release a vast number of volatile odorants that are

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detected and discriminated by the odorant receptors (ORs) expressed in the

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olfactory sensory neurons (OSNs) of the nose 2. The human and mouse genomes,

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contain respectively ~400 and ~1000 intact OR genes 3-5. Even though humans have a

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smaller number of functional OR genes when compared to other species, the human

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sense of smell is very accurate, and in combination with taste and other sensory

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inputs, contributes to a sophisticated sense of flavor 6, 7.

47 8-11

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OR genes are highly polymorphic in mammals

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different odorant-receptor interactions and altered odorant perception 12. OR gene

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loci also display copy number variations (CNVs), which are polymorphic large-scale

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genome deletions or duplications that can lead to differences in gene copy number

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and in the corresponding OR protein expression among individuals

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polymorphisms generate individual variation within the population, which may

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influence human behavior and food consumption. The investigation of how key food

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odorants

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understanding of the role played by olfaction in food preference.

16

. Single base changes can lead to

13-15

. These

are recognized by the diverse ORs, should contribute to the

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Odorant receptors


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Olfactory sensory neurons are the major cell type composing the olfactory

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epithelium located in the dorsal region of the nasal cavity. ORs are G-protein coupled

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receptors (GPCRs) expressed in the cilia of these neurons 2. The olfactory neurons

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make synapses with the mitral cells in the olfactory bulb, forming glomeruli, which

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are responsible for relaying the signal to higher regions of the brain. Each olfactory

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neuron expresses a single odorant receptor gene

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expressing the same odorant receptor gene converge in the same regions in the

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olfactory bulb, so that each OR gene has a corresponding glomerulus in the olfactory

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bulb

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individuals from the same species 22, forming a topographic map where each one of

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the OR genes is represented.

19-21

17, 18

. Axons from neurons

. The positions of the different glomeruli in the bulb are highly similar in

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Each OR is thought to recognize more than one odorant and each odorant can be

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recognized by more than one OR so that each odorant activates a specific

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combination of ORs 23. Consequently, a given odorant will activate a specific group of

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glomeruli in the olfactory bulb and the resulting signaling will ultimately lead to

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odorant discrimination. Thus, defining which odorant activates a given OR, and in

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which concentration, is crucial for the comprehension of the olfactory coding

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mechanisms. However, the existence of many ORs and the enormous number of

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possible ligands makes the deorphanization (ligand matching) of these receptors a

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challenging task

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ORs has been linked to their ligands. Recently developed in vivo high throughput

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techniques for ligand-receptor interaction detection in mice

24, 25

. Consequently, so far only a very small fraction of the human


26-28

, in combination

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with heterologous approaches, should contribute to the identification of human ORs

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that recognize odorants of interest.

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In this case, mouse ORs responsive to the odorant of interest would be first

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identified by using in vivo approaches. Then, the corresponding human OR

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orthologue(s) would be identified, and tested against the odorant in heterologous

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system (Figure 1). It is important to note that amino acid changes in some

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orthologous OR sequences may change odorant specificity

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possible that some of the orthologous ORs will not respond to the same ligands. A

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high-throughput study indicated, however, that ~80% of the analyzed human-mouse

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OR orthologues responded to a common ligand, albeit with differences in sensitivity

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9, 31

29, 30

, and therefore it is

.

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Odorant receptors and their ligands

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Even though odorant receptors were discovered in the early nineties, the

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deorphanization of these receptors began only in the late nineties with in vivo

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techniques

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hampered because these receptors are not efficiently targeted to the cell surface.

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One of the first in vitro functional expression of an OR library in an heterologous

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system was achieved by using the addition of a rho tag, the 20 first N-terminal amino

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acids of rhodopsin, to the N-terminus of ORs, which facilitates cell surface expression

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of the ORs 30.

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In vitro OR expression in heterologous cell systems have improved with the

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employment of accessory proteins that are endogenous olfactory molecules, such as

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the Gαolf interacting guanine nucleotide exchange factor Ric-8B

23, 32, 33

. Until then the in vitro OR expression in heterologous cells was


34-36

and the

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Receptor Transporting Proteins (RTP1 and RTP2)37, 38. Different versions of these

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heterologous systems have largely contributed for human OR deorphanization 10, 39-

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.

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Ex vivo investigations use olfactory neurons obtained from dissociated olfactory

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epithelium and take advantage of the fact that these cells are the best OR expression

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system. The activation of endogenous ORs can be detected by calcium imaging and

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the expressed OR can be defined either by OR gene targeting with fluorescent

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reporters or by identification of the OR transcript in one given neuron

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drawbacks of ex vivo techniques are that only a few odorant receptors can be

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analyzed in each assay and the odorant molecules are not dissolved in the mucus,

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which contains odorant binding proteins, P450 enzymes and other factors, what

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could explain differences between the ligand specificity of an OR in vitro and its

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corresponding glomerulus in vivo 51, 52.

23, 32, 50

. The

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In vivo assays provide the most realistic environment in which to deorphanize the

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odorant receptors, although they usually involve technically challenging approaches.

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The olfactory system is kept intact; odorant molecules are delivered in a vapor phase

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and dissolve in the mucus to activate odorant receptors in the neuronal cilia. Also,

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activated neurons will signal their specific glomeruli through their axons projections

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to the brain. Traditional in vivo methods take advantage of the topographical map of

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OR genes in the olfactory bulb and use imaging techniques of the bulb glomeruli to

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detect odorant-induced activity of olfactory neurons, sometimes using transgenic

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animals

53-55

. However, these in vivo methods are still time-consuming and only a


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few receptors can be evaluated at a time. The lack of high-throughput in vivo

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mapping methods has made it difficult to estimate the correspondence between in

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vitro and in vivo results across a large number of ORs activated by a given odor.

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Recently, three new approaches

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binding analysis in live, freely breathing and freely behaving mice. These assays have

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also the advantage to cover the entire OR repertoire simultaneously in a multiplex

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manner.

26-28

have been developed for odorant-receptor

137 27

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The first one, called Kentucky ligand-receptor assay

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model (S100a5–tauGFP) where Green Fluorescent Protein (GFP) expression is

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dependent on the transcription of the olfactory specific calcium binding protein

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S100a5 gene in OSNs, which is up-regulated after odorant stimulation. In these mice,

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odorant-activated OSNs show robust expression of GFP in the olfactory epithelium.

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The live and freely behaving transgenic mice are exposed to the odorant of interest,

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and after that their olfactory epithelia are dissociated, and GFP-fluorescent OSNs are

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separated from non-fluorescent cells. The transcripts extracted from olfactory

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epithelia of odorant-exposed mice are compared with vehicle-exposed mice through

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microarray investigation. The stimulated OSNs are enriched in transcripts of ORs

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that are responsive to the tested odorant. Through this assay three of the four

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known eugenol-responsive ORs were identified, validating the method 27. In addition,

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five mouse ORs responsive to muscone, were identified, including olfr1440

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(MOR251-1), which was also identified through a different method

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findings of mouse ORs responsive to muscone allowed the search for human

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orthologues based on sequence similarity. The in silico prediction was tested in vitro,

, uses a transgenic mouse


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. The in vivo

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confirming the previous finding

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musk. Extending in vitro tests with odorants structurally similar to musk resulted in

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the discovery of new hOR5AN1 ligands 56.

that hOR5AN1 is the human OR responsive to

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While the Kentucky assay requires the use of transgenic mice, two additional in vivo

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techniques that take advantage of endogenous changes that naturally occur in

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activated olfactory neurons have been recently developed. One of these approaches

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is based on the previous observation that the S6 ribosomal subunit is

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phosphorylated when a neuron is activated

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exploited

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phosphorylated S6 (pS6) in odorant-activated OSNs 26. The purified mRNAs should be

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enriched for OR transcripts expressed in the responsive neurons. The

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immunoprecipitated transcripts are subjected to next-generation sequencing and

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tested for enrichment in relation to mRNA obtained from unexposed olfactory

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epithelia.

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identification of ORs responsive to acetophenone (48 ORs) and trimethylthiazoline

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(TMT, 21 ORs). There was a high correlation of in vivo and in vitro results indicating

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that detection of odorant-responsive ORs through the pS6-IP method is efficient.

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Interestingly, the identification of several ORs activated by acetophenone and TMT

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allowed for sequence comparison among activated receptors and identification of

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conserved amino acid residues probably involved in odorant recognition. Odorant –

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receptor pairs identified through these assays were checked in heterologous cells

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reinforcing the use of in vitro systems as important tools to test the results obtained

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with the in vivo methods. However, it is important to note that the absence of

to

immunoprecipitate

OR

57

mRNAs

. This neuronal peculiarity was which

are

associated

with

This approach, denominated the pS6-IP method, allowed for the


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responses in vitro does not necessarily prove that receptors are unable to respond to

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a given odorant. As previously mentioned, there are several factors affecting ligand-

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receptor recognition in the cilia, such as mucus-associated proteins and receptor

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localization in the membrane, which are missing in the heterologous systems.

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The third approach, denominated DREAM (Deorphanization of Receptors based on

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Expression Alterations of Messenger RNA levels) is based on the fact that there is an

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indirect correlation between odorant stimulation and activity-induced OR gene

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transcription

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due to adaptation of the sensory neuron to a continuous stimulus 28. The transcripts

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from olfactory epithelia of animals exposed to the tested odorant and to control

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vehicle are extracted, subjected to deep sequencing and compared. Only OR genes

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showing a highly significant reduction in mRNA expression are taken as positive

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results. By using this approach, 26 ORs were detected after mouse exposure to

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acetophenone, and 11 after mouse exposure to ethyl butyrate 28. These results also

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included ORs previously described to be activated by these odorants

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DREAM was applied to rats exposed to ethyl isobutyrate it revealed rat ORs that are

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orthologous to some of the ethyl isobutyrate responsive mouse ORs, indicating again

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that the search for orthologous receptors may help with OR deorphanization.

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Comparison of the results obtained for the same odorant (acetophenone) by the

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two last methods

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overlapping, some of the ORs were identified only by one of the two approaches 26,

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indicating that slightly different responsive OR profiles can be obtained when using

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different methods.

28

. OSN activation leads to reduction in the cell’s OR mRNA, probably

26, 28

50

. When

indicated that while the group of the ORs identified are


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Future directions

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Human OR deorphanization should profit from the new high throughput in vivo

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techniques 26-28 (Figure 1). As novel mouse ORs are deorphanized in vivo, and these

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ligand-receptor associations are validated in vitro, human OR orthologues can be

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identified by using bioinformatic tools and be analyzed in vitro for responsiveness to

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specific odorants. It is important to note however that, as mentioned above, some

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human-mouse OR orthologues may not respond to the same odorants. For example,

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orthologues (including the mouse orthologue) of the human OR OR2M3, which

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specifically responds to the onion key food odorant 3-mercapto-2-methylpentan-1-ol,

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do not respond to this same odorant 58. It was also shown that human-mouse bitter

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taste receptor orthologues have distinct agonist profiles

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evolutionary diversification and largely different OR repertoire sizes between man

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and mouse, there are species-specific receptors, i.e. many mouse ORs have no clear

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human homologue, and vice versa. Still, human and mouse share many OR gene

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subfamilies 3, and as mentioned above about 80% of the human-mouse OR

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orthologues respond to a common ligand

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work for many of the ORs. It is important to be aware though that there are other

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limitations to the approach, for example some ORs may not be functionally

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expressed in heterologous systems, or too many mouse ORs are identified in vivo

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and need to be tested.

31

59

. In addition, due to

, indicating that the approach should

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Once an odorant is associated with a specific human OR, new ligands and

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antagonists can be searched by structural similarity. Key receptors affecting flavor

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perception can be further analyzed for population genotypic variation, such as the

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presence of SNPs. For example, a SNP in OR5A1, a human OR that responds to β-

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ionone, a key aroma in food and beverages, is correlated with odor sensitivity and

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perception 48. This SNP leads to the exchange of an asparagine to an aspartic acid in

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the second extracellular loop of the receptor 48. These results show that genotypic

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variation in OR genes may explain aspects of food preferences, human nutritional

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behavior, and health. In addition, in an increasingly competitive market where food

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flavor must be exploited as a selling tool, the knowledge of new, unexploited

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odorant-receptor interactions and specific regional odor perception peculiarities

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may represent a food industry strategic differential. These studies should contribute

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to the development of new odorant compounds of interest for specific consumer

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niches.


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Author information

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Corresponding Author

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e-mail: [email protected]

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Notes

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This study was supported by grants from Fundação de Amparo à Pesquisa do Estado

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de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

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The authors declare no competing financial interest.

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Author contributions: L. A.-C. and B.M. wrote the paper.

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Acknowledgments

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We thank Cleiton Fagundes Machado for help with elaboration of the figure.


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432

Figure 1. A strategy for the identification of human odorant receptors responsive

433

to food odorants. In vivo identification of the pool of olfactory sensory neurons

434

(OSNs), in the olfactory epithelium (OE), activated by a specific food odorant allows

435

for the deorphanization of mouse odorant receptors (ORs). Identified odorant-

436

receptor pairs are validated in vitro through OR cloning, expression and activation in

437

heterologous cells. After in vitro validation, bioinformatic tools are used to search for

438

corresponding human orthologous ORs, which in turn must also be validated in vitro.

439

Studies examining receptor genotypic variants in the population may help to

440

understand differences in human olfactory perception elicited by the food odorants.

441

In addition, the definition of odorant-receptor pairs will allow for the identification

442

of

structurally

similar

agonists


or

antagonists.

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443


19


Figure 1. A strategy for the identification of human odorant receptors responsive to food odorants. In vivo identification of the pool of olfactory sensory neurons (OSNs), in the olfactory epithelium (OE), activated by a specific food odorant allows for the deorphanization of mouse odorant receptors (ORs). Identified odorantreceptor pairs are validated in vitro through OR cloning, expression and activation in heterologous cells. After in vitro validation, bioinformatic tools are used to search for corresponding human orthologous ORs, which in turn must also be validated in vitro. Studies examining receptor genotypic variants in the population may help to understand differences in human olfactory perception elicited by the food odorants. In addition, the definition of odorant-receptor pairs will allow for the identification of structurally similar agonists or antagonists. 297x209mm (300 x 300 DPI)


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TOC graphic 334x188mm (100 x 100 DPI)


Combining In Vivo and In Vitro Approaches To Identify Human

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