Combining In Vivo and In Vitro Approaches To Identify Human

In vivo identification of the pool of olfactory sensory neurons (OSNs), in the ... Traditional in vivo methods take advantage of the topographical map...
0 downloads 0 Views 573KB Size
Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Perspective

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21

Journal of Agricultural and Food Chemistry

1

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

2

responsive to food odorants

3 4

Lucia M. Armelin-Correa1, Bettina Malnic2*

5

1

6

Paulo, São Paulo, Brazil

7

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

1

Journal of Agricultural and Food Chemistry

Page 2 of 21

8

Abstract

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

odorants.

21 22

Keywords:

23

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

24

key food odorants.

ACS Paragon Plus Environment

2

Page 3 of 21

Journal of Agricultural and Food Chemistry

25

Abbreviations

26

Odorant receptor - OR

27

G-protein coupled receptor – GPCR

28

Receptor Transporting Protein – RTP

29

Olfactory Sensory Neuron – OSN

30

Copy Number Variation – CNV

31

Single Nucleotide Polymorphism – SNP

32

Green Fluorescent Protein – GFP

33

phosphorylated ribosomal subunit S6 - pS6

34

phosphorylated ribosomal subunit S6 immunoprecipitation - pS6-IP

35

Reverse Transcription - Polymerase Chain Reaction - RT-PCR

ACS Paragon Plus Environment

3

Journal of Agricultural and Food Chemistry

Page 4 of 21

36

Introduction

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47 8-11

48

OR genes are highly polymorphic in mammals

49

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

50

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

51

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

52

and in the corresponding OR protein expression among individuals

53

polymorphisms generate individual variation within the population, which may

54

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

55

odorants

56

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

57 58

Odorant receptors

ACS Paragon Plus Environment

4

Page 5 of 21

Journal of Agricultural and Food Chemistry

59

Olfactory sensory neurons are the major cell type composing the olfactory

60

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

61

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

62

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

63

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

64

neuron expresses a single odorant receptor gene

65

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

66

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

67

bulb

68

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

69

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

70 71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

challenging task

80

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

81

techniques for ligand-receptor interaction detection in mice

24, 25

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

ACS Paragon Plus Environment

26-28

, in combination

5

Journal of Agricultural and Food Chemistry

Page 6 of 21

82

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

83

that recognize odorants of interest.

84

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

85

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

86

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

87

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

88

orthologous OR sequences may change odorant specificity

89

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

90

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

91

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

92

9, 31

29, 30

, and therefore it is

.

93 94

Odorant receptors and their ligands

95

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

96

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

97

techniques

98

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

99

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

100

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

101

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

102

of the ORs 30.

103

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

104

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

105

the Gαolf interacting guanine nucleotide exchange factor Ric-8B

23, 32, 33

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

ACS Paragon Plus Environment

34-36

and the

6

Page 7 of 21

Journal of Agricultural and Food Chemistry

106

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

107

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

108

49

.

109 110

Ex vivo investigations use olfactory neurons obtained from dissociated olfactory

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

corresponding glomerulus in vivo 51, 52.

23, 32, 50

. The

120 121

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

122

odorant receptors, although they usually involve technically challenging approaches.

123

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

124

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

125

activated neurons will signal their specific glomeruli through their axons projections

126

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

127

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

128

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

129

animals

53-55

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

ACS Paragon Plus Environment

7

Journal of Agricultural and Food Chemistry

Page 8 of 21

130

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

131

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

132

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

133

Recently, three new approaches

134

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

135

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

136

manner.

26-28

have been developed for odorant-receptor

137 27

138

The first one, called Kentucky ligand-receptor assay

139

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

140

dependent on the transcription of the olfactory specific calcium binding protein

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

, uses a transgenic mouse

ACS Paragon Plus Environment

55

. The in vivo

8

Page 9 of 21

Journal of Agricultural and Food Chemistry

55

154

confirming the previous finding

155

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

156

the discovery of new hOR5AN1 ligands 56.

that hOR5AN1 is the human OR responsive to

157 158

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

159

techniques that take advantage of endogenous changes that naturally occur in

160

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

161

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

162

phosphorylated when a neuron is activated

163

exploited

164

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

165

enriched for OR transcripts expressed in the responsive neurons. The

166

immunoprecipitated transcripts are subjected to next-generation sequencing and

167

tested for enrichment in relation to mRNA obtained from unexposed olfactory

168

epithelia.

169

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

170

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

171

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

172

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

173

allowed for sequence comparison among activated receptors and identification of

174

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

175

receptor pairs identified through these assays were checked in heterologous cells

176

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

177

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

ACS Paragon Plus Environment

9

Journal of Agricultural and Food Chemistry

Page 10 of 21

178

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

179

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

180

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

181

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

182 183

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

184

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

185

indirect correlation between odorant stimulation and activity-induced OR gene

186

transcription

187

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

188

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

189

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

190

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

191

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

192

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

193

included ORs previously described to be activated by these odorants

194

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

195

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

196

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

197

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

198

two last methods

199

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

200

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

201

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

ACS Paragon Plus Environment

10

Page 11 of 21

Journal of Agricultural and Food Chemistry

202 203 204

Future directions

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

taste receptor orthologues have distinct agonist profiles

215

evolutionary diversification and largely different OR repertoire sizes between man

216

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

217

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

218

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

219

orthologues respond to a common ligand

220

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

221

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

222

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

223

and need to be tested.

31

59

. In addition, due to

, indicating that the approach should

224

ACS Paragon Plus Environment

11

Journal of Agricultural and Food Chemistry

Page 12 of 21

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

odorant-receptor interactions and specific regional odor perception peculiarities

236

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

237

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

238

niches.

ACS Paragon Plus Environment

12

Page 13 of 21

Journal of Agricultural and Food Chemistry

239

Author information

240

Corresponding Author

241

e-mail: [email protected]

242 243

Notes

244

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

245

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

246

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

247

The authors declare no competing financial interest.

248

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

249 250

Acknowledgments

251

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

ACS Paragon Plus Environment

13

Journal of Agricultural and Food Chemistry

Page 14 of 21

252

References

253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299

1. Shepherd, G. M., Smell images and the flavour system in the human brain. Nature 2006, 444, 316-21. 2. Buck, L.; Axel, R., A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 1991, 65, 175-187. 3. Godfrey, P. A.; Malnic, B.; Buck, L. B., The mouse olfactory receptor gene family. Proc Natl Acad Sci U S A 2004, 101, 2156-2161. 4. Zhang, X.; Firestein, S., The olfactory receptor gene superfamily of the mouse. Nat. Neurosci. 2002, 5, 124-133. 5. Zozulya, S.; Echeverri, F.; Nguyen, T., The human olfactory receptor repertoire. Genome Biology 2001, 2, 18.1-18.2. 6. Shepherd, G., The Human Sense of Smell: Are We Better Than We Think? PLoS Biology 2004, 2, e146. 7. Shepherd, G. M., Outline of a theory of olfactory processing and its relevance to humans. Chemical senses 2005, 30 Suppl 1, i3-5. 8. Hoover, K. C.; Gokcumen, O.; Qureshy, Z.; Bruguera, E.; Savangsuksa, A.; Cobb, M.; Matsunami, H., Global Survey of Variation in a Human Olfactory Receptor Gene Reveals Signatures of Non-Neutral Evolution. Chemical senses 2015, 40, 481-8. 9. Jiang, Y.; Matsunami, H., Mammalian odorant receptors: functional evolution and variation. Curr Opin Neurobiol 2015, 34, 54-60. 10. Mainland, J. D.; Keller, A.; Li, Y. R.; Zhou, T.; Trimmer, C.; Snyder, L. L.; Moberly, A. H.; Adipietro, K. A.; Liu, W. L.; Zhuang, H.; Zhan, S.; Lee, S. S.; Lin, A.; Matsunami, H., The missense of smell: functional variability in the human odorant receptor repertoire. Nat Neurosci 2014, 17, 114-20. 11. Menashe, I.; Man, O.; Lancet, D.; Gilad, Y., Different noses for different people. Nature Genetics 2003, 34, 143-144. 12. Keller, A.; Zhuang, H.; Chi, Q.; Vosshall, L.; Matsunami, H., Genetic variation in a human odorant receptor alters odour perception. Nature 2007, 449, 468472. 13. Hasin, Y.; Olender, T.; Khen, M.; Gonzaga-Jauregui, C.; Kim, P.; Urban, A.; Snyder, M.; Gerstein, M.; Lancet, D.; Korbel, J., High-resolution copy-number variation map reflects human olfactory receptor diversity and evolution. PLoS genetics 2008, 4, e1000249. 14. Hasin-Brumshtein, Y.; Lancet, D.; Olender, T., Human olfaction: from genomic variation to phenotypic diversity. Trends in genetics : TIG 2009, 25, 17884. 15. Young, J.; Endicott, R.; Parghi, S. W., M; Kidd, J.; Trask, B., Extensive copynumber variation of the human olfactory receptor gene family. The American Journal of Human Genetics 2008, 83, 228-242. 16. Dunkel, A.; Steinhaus, M.; Kotthoff, M.; Nowak, B.; Krautwurst, D.; Schieberle, P.; Hofmann, T., Nature's chemical signatures in human olfaction: a foodborne perspective for future biotechnology. Angew Chem Int Ed Engl 2014, 53, 7124-43. 17. Monahan, K.; Lomvardas, S., Monoallelic expression of olfactory receptors. Annual review of cell and developmental biology 2015, 31, 721-40. 18. Nagai, M. H.; Armelin-Correa, L. M.; Malnic, B., Monogenic and Monoallelic Expression of Odorant Receptors. Molecular pharmacology 2016, 90, 633-639.

ACS Paragon Plus Environment

14

Page 15 of 21

Journal of Agricultural and Food Chemistry

300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347

19. Mombaerts, P.; Wang, F.; Dulac, C.; Chao, S.; Nemes, A.; Mendelsohn, M.; Edmondson, J.; Axel, R., Visualizing an olfactory sensory map. Cell 1996, 87, 675686. 20. Ressler, K. J.; Sullivan, S. L.; Buck, L. B., A molecular dissection of spatial patterning in the olfactory system. Curr. Opin. Neurobiol. 1994, 4, 588-596. 21. Vassar, R.; Chao, S.; Sitcheran, R.; Nunez, J.; Vosshall, L.; Axel, R., Topographic organization of sensory projections to the olfactory bulb. Cell 1994, 79, 981-991. 22. Zapiec, B.; Mombaerts, P., Multiplex assessment of the positions of odorant receptor-specific glomeruli in the mouse olfactory bulb by serial twophoton tomography. Proc Natl Acad Sci U S A 2015, 112, E5873-82. 23. Malnic, B.; Hirono, J.; Sato, T.; Buck, L. B., Combinatorial receptor codes for odors. Cell 1999, 96, 713-23. 24. Malnic, B., Searching for the ligands of odorant receptors. Molecular Neurobiology 2007, 35, 175-181. 25. Peterlin, Z.; Firestein, S.; Rogers, M. E., The state of the art of odorant receptor deorphanization: a report from the orphanage. J Gen Physiol 2014, 143, 527-42. 26. Jiang, Y.; Gong, N. N.; Hu, X. S.; Ni, M. J.; Pasi, R.; Matsunami, H., Molecular profiling of activated olfactory neurons identifies odorant receptors for odors in vivo. Nat Neurosci 2015, 18, 1446-54. 27. McClintock, T. S.; Adipietro, K.; Titlow, W. B.; Breheny, P.; Walz, A.; Mombaerts, P.; Matsunami, H., In vivo identification of eugenol-responsive and muscone-responsive mouse odorant receptors. The Journal of neuroscience : the official journal of the Society for Neuroscience 2014, 34, 15669-78. 28. von der Weid, B.; Rossier, D.; Lindup, M.; Tuberosa, J.; Widmer, A.; Col, J. D.; Kan, C.; Carleton, A.; Rodriguez, I., Large-scale transcriptional profiling of chemosensory neurons identifies receptor-ligand pairs in vivo. Nat Neurosci 2015, 18, 1455-63. 29. Gaillard, I.; Rouquier, S.; Chavanieu, A.; Mollard, P.; Giorgi, D., Amino-acid changes acquired during evolution by olfactory receptor 912-93 modify the specificity of odorant recognition. Human molecular genetics 2004, 13, 771-80. 30. Krautwurst, D.; Yau, K. W.; Reed, R. R., Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 1998, 95, 917-926. 31. Adipietro, K. A.; Mainland, J. D.; Matsunami, H., Functional evolution of mammalian odorant receptors. PLoS genetics 2012, 8, e1002821. 32. Touhara, K.; Sengoku, S.; Inaki, K.; Tsuboi, A.; Hirono, J.; Sato, T.; Sakano, H.; Haga, T., Functional identification and reconstitution of an odorant receptor in single olfactory neurons. Proc Natl Acad Sci U S A 1999, 96, 4040-5. 33. Zhao, H.; Ivic, L.; Otaki, J.; Hashimoto, M.; Mikoshiba, K.; Firestein, S., Functional expression of a mammalian odorant receptor. Science 1998, 279, 237242. 34. Malnic, B.; Gonzalez-Kristeller, D. C., Functional expression of chemoreceptors with the help of a Guanine nucleotide exchange factor. Ann N Y Acad Sci 2009, 1170, 150-2. 35. Von Dannecker, L.; Mercadante, A.; Malnic, B., Ric-8B, an olfactory putative GTP exchange factor, amplifies signal transduction through the

ACS Paragon Plus Environment

15

Journal of Agricultural and Food Chemistry

348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394

Page 16 of 21

olfactory-specific G-protein Gaolf. The Journal of neuroscience : the official journal of the Society for Neuroscience 2005, 25, 3793-3800. 36. Von Dannecker, L.; Mercadante, A.; Malnic, B., Ric-8B promotes functional expression of odorant receptors. Proc. Natl. Acad. Sci. USA 2006, 103, 9310-9314. 37. Saito, H.; Kubota, M.; Roberts, R. W.; Chi, Q.; Matsunami, H., RTP family members induce functional expression of mammalian odorant receptors. Cell 2004, 119, 679-691. 38. Zhuang, H.; Matsunami, H., Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells. Nature protocols 2008, 3, 1402-13. 39. Gonzalez-Kristeller, D. C.; do Nascimento, J. B.; Galante, P. A.; Malnic, B., Identification of agonists for a group of human odorant receptors. Front Pharmacol 2015, 6, 35. 40. Chatelain, P.; Veithen, A.; Wilkin, F.; Philippeau, M., Deorphanization and characterization of human olfactory receptors in heterologous cells. Chemistry & biodiversity 2014, 11, 1764-81. 41. Hatt, H.; Gisselmann, G.; Wetzel, C. H., Cloning, functional expression and characterization of a human olfactory receptor. Cellular and molecular biology 1999, 45, 285-91. 42. Wetzel, C. H.; Oles, M.; Wellerdieck, C.; Kuczkowiak, M.; Hatt, H., Specificity and sensitivity of a human olfactory receptor functionally expressed in human embryonic kidney 293 cells and Xenopus Laevis oocytes. J. Neurosci. 1999, 19, 7426-7433. 43. Sanz, G.; Schlegel, C.; Pernollet, J. C.; Briand, L., Comparison of odorant specificity of two human olfactory receptors from different phylogenetic classes and evidence for antagonism. Chemical senses 2005, 30, 69-80. 44. Jaquier, V.; Pick, H.; Vogel, H., Characterization of an extended receptive ligand repertoire of the human olfactory receptor OR17-40 comprising structurally related compounds. Journal of Neurochemistry 2006, 97, 537-544. 45. Neuhaus, E.; Mashukova, A.; Zhang, W.; Barbour, J.; Hatt, H., A specific heat shock protein enhances the expression of mammalian olfactory receptor proteins. Chem. Senses 2006, 31, 445-452. 46. Schmiedeberg, K.; Shirokova, E.; Weber, H.; Schilling, B.; Meyerhof, W.; Krautwurst, D., Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2. J. Struct. Biol. 2007, 159, 400-412. 47. Saito, H.; Chi, Q.; Zhuang, H.; Matsunami, H.; Mainland, J. D., Odor coding by a Mammalian receptor repertoire. Sci Signal 2009, 2, ra9. 48. Jaeger, S. R.; McRae, J. F.; Bava, C. M.; Beresford, M. K.; Hunter, D.; Jia, Y.; Chheang, S. L.; Jin, D.; Peng, M.; Gamble, J. C.; Atkinson, K. R.; Axten, L. G.; Paisley, A. G.; Tooman, L.; Pineau, B.; Rouse, S. A.; Newcomb, R. D., A Mendelian trait for olfactory sensitivity affects odor experience and food selection. Curr Biol 2013, 23, 1601-5. 49. Busse, D.; Kudella, P.; Gruning, N. M.; Gisselmann, G.; Stander, S.; Luger, T.; Jacobsen, F.; Steinstrasser, L.; Paus, R.; Gkogkolou, P.; Bohm, M.; Hatt, H.; Benecke, H., A synthetic sandalwood odorant induces wound-healing processes in human keratinocytes via the olfactory receptor OR2AT4. J Invest Dermatol 2014, 134, 2823-32.

ACS Paragon Plus Environment

16

Page 17 of 21

Journal of Agricultural and Food Chemistry

395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431

50. Bozza, T.; Feinstein, P.; Zheng, C.; Mombaerts, P., Odorant receptor expression defines functional units in the mouse olfactory system. J. Neuroscience 2002, 22, 3033-3043. 51. Heydel, J. M.; Coelho, A.; Thiebaud, N.; Legendre, A.; Le Bon, A. M.; Faure, P.; Neiers, F.; Artur, Y.; Golebiowski, J.; Briand, L., Odorant-binding proteins and xenobiotic metabolizing enzymes: implications in olfactory perireceptor events. Anatomical record 2013, 296, 1333-45. 52. Nagashima, A.; Touhara, K., Enzymatic conversion of odorants in nasal mucus affects olfactory glomerular activation patterns and odor perception. The Journal of neuroscience : the official journal of the Society for Neuroscience 2010, 30, 16391-8. 53. Bozza, T.; McGann, J. P.; Mombaerts, P.; Wachowiak, M., In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse. Neuron 2004, 42, 9-21. 54. Oka, Y.; Katada, S.; Omura, M.; Suwa, M.; Yoshihara, Y.; Touhara, K., Odorant receptor map in the mouse olfactory bulb: in vivo sensitivity and specificity of receptor-defined glomeruli. Neuron 2006, 52, 857-69. 55. Shirasu, M.; Yoshikawa, K.; Takai, Y.; Nakashima, A.; Takeuchi, H.; Sakano, H.; Touhara, K., Olfactory receptor and neural pathway responsible for highly selective sensing of musk odors. Neuron 2014, 81, 165-78. 56. Sato-Akuhara, N.; Horio, N.; Kato-Namba, A.; Yoshikawa, K.; Niimura, Y.; Ihara, S.; Shirasu, M.; Touhara, K., Ligand Specificity and Evolution of Mammalian Musk Odor Receptors: Effect of Single Receptor Deletion on Odor Detection. The Journal of neuroscience : the official journal of the Society for Neuroscience 2016, 36, 4482-91. 57. Knight, Z. A.; Tan, K.; Birsoy, K.; Schmidt, S.; Garrison, J. L.; Wysocki, R. W.; Emiliano, A.; Ekstrand, M. I.; Friedman, J. M., Molecular profiling of activated neurons by phosphorylated ribosome capture. Cell 2012, 151, 1126-37. 58. Noe, F.; Polster, J.; Geithe, C.; Kotthoff, M.; Schieberle, P.; Krautwurst, D., OR2M3: A Highly Specific and Narrowly Tuned Human Odorant Receptor for the Sensitive Detection of Onion Key Food Odorant 3-Mercapto-2-methylpentan-1-ol. Chemical senses 2016. 59. Lossow, K.; Hubner, S.; Roudnitzky, N.; Slack, J. P.; Pollastro, F.; Behrens, M.; Meyerhof, W., Comprehensive Analysis of Mouse Bitter Taste Receptors Reveals Different Molecular Receptive Ranges for Orthologous Receptors in Mice and Humans. The Journal of biological chemistry 2016, 291, 15358-77.

ACS Paragon Plus Environment

17

Journal of Agricultural and Food Chemistry

Page 18 of 21

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

ACS Paragon Plus Environment

or

antagonists.

18

Page 19 of 21

Journal of Agricultural and Food Chemistry

443

ACS Paragon Plus Environment

19

Journal of Agricultural and Food Chemistry

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)

ACS Paragon Plus Environment

Page 20 of 21

Page 21 of 21

Journal of Agricultural and Food Chemistry

TOC graphic 334x188mm (100 x 100 DPI)

ACS Paragon Plus Environment