Copy Number Variation in TAS2R Bitter Taste Receptor Genes: Structure, Origin, and Population Genetics.

Bitter taste receptor genes (TAS2Rs) harbor extensive diversity, which is broadly distributed across human populations and strongly associated with taste response phenotypes. The majority of TAS2R variation is composed of single-nucleotide polymorphisms. However, 2 closely positioned loci at 12p13, TAS2R43 and -45, harbor high-frequency deletion (Δ) alleles in which genomic segments are absent, resulting in copy number variation (CNV). To resolve their chromosomal structure and organization, we generated maps using long-range contig alignments and local sequencing across the TAS2R43-45 region. These revealed that the deletion alleles (43Δ and 45Δ) are 37.8 and 32.2kb in length, respectively and span the complete coding region of each gene (~1kb) along with extensive up- and downstream flanking sequence, producing separate CNVs at the 2 loci. Comparisons with a chimpanzee genome, which contained intact homologs of TAS2R43, -45, and nearby TAS2Rs, indicated that the deletions evolved recently, through unequal recombination in a cluster of closely related loci. Population genetic analyses in 946 subjects from 52 worldwide populations revealed that copy number ranged from 0 to 2 at both TAS2R43 and TAS2R45, with 43Δ and 45Δ occurring at high global frequencies (0.33 and 0.18). Estimated recombination rates between the loci were low (ρ = 2.7×10(-4); r = 6.6×10(-9)) and linkage disequilibrium was high (D' = 1.0), consistent with their adjacent genomic positioning and recent origin. Geographic variation pointed to an African origin for the deletions. However, no signatures of natural selection were found in population structure or integrated haplotype scores spanning the region, suggesting that patterns of diversity at TAS2R43 and -45 are primarily due to genetic drift.

Comprehensive Analysis of Mouse Bitter Taste Receptors Reveals Different Molecular Receptive Ranges for Orthologous Receptors in Mice and Humans.

One key to animal survival is the detection and avoidance of potentially harmful compounds by their bitter taste. Variable numbers of taste 2 receptor genes expressed in the gustatory end organs enable bony vertebrates (Euteleostomi) to recognize numerous bitter chemicals. It is believed that the receptive ranges of bitter taste receptor repertoires match the profiles of bitter chemicals that the species encounter in their diets. Human and mouse genomes contain pairs of orthologous bitter receptor genes that have been conserved throughout evolution. Moreover, expansions in both lineages generated species-specific sets of bitter taste receptor genes. It is assumed that the orthologous bitter taste receptor genes mediate the recognition of bitter toxins relevant for both species, whereas the lineage-specific receptors enable the detection of substances differently encountered by mice and humans. By challenging 34 mouse bitter taste receptors with 128 prototypical bitter substances in a heterologous expression system, we identified cognate compounds for 21 receptors, 19 of which were previously orphan receptors. We have demonstrated that mouse taste 2 receptors, like their human counterparts, vary greatly in their breadth of tuning, ranging from very broadly to extremely narrowly tuned receptors. However, when compared with humans, mice possess fewer broadly tuned receptors and an elevated number of narrowly tuned receptors, supporting the idea that a large receptor repertoire is the basis for the evolution of specialized receptors. Moreover, we have demonstrated that sequence-orthologous bitter taste receptors have distinct agonist profiles. Species-specific gene expansions have enabled further diversification of bitter substance recognition spectra.

Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception.

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects' genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.

Rubemamine and Rubescenamine, Two Naturally Occurring N-Cinnamoyl Phenethylamines with Umami-Taste-Modulating Properties.

Sensory screening of a series of naturally occurring N-cinnamoyl derivatives of substituted phenethylamines revealed that rubemamine (9, from Chenopodium album) and rubescenamine (10, from Zanthoxylum rubsecens) elicit strong intrinsic umami taste in water at 50 and 10 ppm, respectively. Sensory tests in glutamate- and nucleotide-containing bases showed that the compounds influence the whole flavor profile of savory formulations. Both rubemamine (9) and rubescenamine (10) at 10-100 ppm dose-dependently positively modulated the umami taste of MSG (0.17-0.22%) up to threefold. Among the investigated amides, only rubemamine (9) and rubescenamine (10) are able to directly activate the TAS1R1-TAS1R3 umami taste receptor. Moreover, both compounds also synergistically modulated the activation of TAS1R1-TAS1R3 by MSG. Most remarkably, rubemamine (9) was able to further positively modulate the IMP-enhanced TAS1R1-TAS1R3 response to MSG ∼ 1.8-fold. Finally, armatamide (11), zanthosinamide (13), and dioxamine (14), which lack intrinsic umami taste in vivo and direct receptor response in vitro, also positively modulated receptor activation by MSG about twofold and the IMP-enhanced MSG-induced TAS1R1-TAS1R3 responses approximately by 50%. In sensory experiments, dioxamine (14) at 25 ppm in combination with 0.17% MSG exhibited a sensory equivalent to 0.37% MSG.

Variability in Human Bitter Taste Sensitivity to Chemically Diverse Compounds Can Be Accounted for by Differential TAS2R Activation.

The human population displays high variation in taste perception. Differences in individual taste sensitivity may also impact on nutrient intake and overall appetite. A well-characterized example is the variable perception of bitter compounds such as 6-n-propylthiouracil (PROP) and phenylthiocarbamide (PTC), which can be accounted for at the molecular level by polymorphic variants in the specific type 2 taste receptor (TAS2R38). This phenotypic variation has been associated with influencing dietary preference and other behaviors, although the generalization of PROP/PTC taster status as a predictor of sensitivity to other tastes is controversial. Here, we proposed that the taste sensitivities of different bitter compounds would be correlated only when they activate the same bitter taste receptor. Thirty-four volunteers were exposed to 8 bitter compounds that were selected based on their potential to activate overlapping and distinct repertoires of TAS2Rs. Taste intensity ratings were evaluated using the general Labeled Magnitude Scale. Our data demonstrate a strong interaction between the intensity for bitter substances when they activate common TAS2Rs. Consequently, PROP/PTC sensitivity was not a reliable predictor of general bitter sensitivity. In addition, our findings provide a novel framework to predict taste sensitivity based on their specific T2R activation profile.

Oral texture influences the neural processing of ortho- and retronasal odors in humans.

Eating implies mutual interactions between different senses. In the present work we aimed at studying relations between food texture and food odor, using both psychophysical and imaging techniques. Eighteen right-handed healthy human subjects participated to both behavioral and fMRI sessions. Fresh, sweetened milk and a more thickened version were delivered orally; in addition, a buttery-cream aroma was presented ortho- or retronasally. Stimuli were applied using a gustometer and or an air-dilution olfactometer, both computer-controlled. In each session subjects rated separately odor-, taste- and thickness intensities of the stimuli. The behavioral data show that odors, presented through either retro- or orthonasal path, induce a significant flavor enhancement with respect to the no-odor condition. Brain functional data indicated a significant enhancement of the activation of olfactory eloquent areas in favor of ortho-nasal odor presentation while activations of mechanosensory areas were favored by the retro-nasal odor route. As effect of oral stimuli we found a significant correlation between the texture intensity rating vs. the BOLD signal in the supplementary motor area, known to drive subconsciously primed movement, putatively associated in this case with the tongue movement required with the handling of the stimulus. Moreover, we found inhibition of the signal in different sensory specific areas as an effect of the mutual interaction between stimulus qualities. In conclusion, ortho- and retronasal odors differentially affect the neural processing of the texture of oral stimuli.

Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris.

Brazzein is a small, heat-, and pH-stable sweet protein present in the fruits of the West African plant Pentadiplandra brazzeana Baillon. It exists in two forms differing in sweetness intensity. The major form, called pyrE-bra, contains a pyroglutamic acid at its N-terminus, while the minor form, called des-pyrE-bra, lacks this residue. Here we describe the heterologous expression in the methylotrophic yeast Pichia pastoris of two natural forms of brazzein, pyrE-bra and des-pyrE-bra, and an additional form, called Q1-bra, which is not naturally occurring in the fruit. Q1-bra differs from pyrE-bra in having a glutamine residue instead of pyrE at its N-terminus. Over an expression period of 6 days, we obtained approximately 90, 30, and 90 mg/L of purified recombinant pyrE-bra, Q1-bra, and des-pyrE-bra brazzein forms, respectively. Recombinant proteins were purified and submitted to mass spectrometry and (1)H NMR spectroscopy. The data indicate that the recombinant brazzein forms were properly folded. Moreover, they activated the human sweet receptor in vitro and evoked sweetness in vivo with properties similar to those of the two natural brazzein forms.

Receptor agonism and antagonism of dietary bitter compounds.

Food contains complex blends of structurally diverse bitter compounds that trigger bitterness through activation of one or more of the ∼25 human TAS2 bitter taste receptors. It remains unsolved, however, whether the perceived bitterness of binary bitter-compound mixtures can be considered an additive function of all bitter-inducing chemicals in the mouth, suggesting that little mutual interaction takes place among bitter substances or if mixture suppression and synergism occurs. Here we report on two natural sesquiterpene lactones from edible plants, which stimulate distinct sets of hTAS2Rs in transfected cells. Both chemicals also robustly inhibit different but overlapping subsets of agonist-activated hTAS2Rs. These findings demonstrate that mixtures of bitter compounds, because they normally occur in human foodstuff, likely elicit bitter perception in a complex and not in a merely additive manner. An unexpected implication of this discovery is that, during evolution, the naturally occurring bitter taste receptor antagonists have shaped some of the pharmacological properties of the receptors, such as overlapping recognition profiles and breadth of tuning.

Genomic, genetic and functional dissection of bitter taste responses to artificial sweeteners.

Bitter taste perception is initiated by TAS2R receptors, which respond to agonists by triggering depolarization of taste bud cells. Mutations in TAS2Rs are known to affect taste phenotypes by altering receptor function. Evidence that TAS2Rs overlap in ligand specificity suggests that they may also contribute joint effects. To explore this aspect of gustation, we examined bitter perception of saccharin and acesulfame K, widely used artificial sweeteners with aversive aftertastes. Both substances are agonists of TAS2R31 and -43, which belong to a five-member subfamily (TAS2R30-46) responsive to a diverse constellation of compounds. We analyzed sequence variation and linkage structure in the ∼140 kb genomic region encoding TAS2R30-46, taste responses to the two sweeteners in subjects, and functional characteristics of receptor alleles. Whole-gene sequences from TAS2R30-46 in 60 Caucasian subjects revealed extensive diversity including 34 missense mutations, two nonsense mutations and high-frequency copy-number variants. Thirty markers, including non-synonymous variants in all five genes, were associated (P< 0.001) with responses to saccharin and acesulfame K. However, linkage disequilibrium (LD) in the region was high (D', r(2) > 0.95). Haplotype analyses revealed that most associations were spurious, arising from LD with variants in TAS2R31. In vitro assays confirmed the functional importance of four TAS2R31 mutations, which had independent effects on receptor response. The existence of high LD spanning functionally distinct TAS2R loci predicts that bitter taste responses to many compounds will be strongly correlated even when they are mediated by different genes. Integrative approaches combining phenotypic, genetic and functional analysis will be essential in dissecting these complex relationships.

Investigation of interactions between texture and ortho- and retronasal olfactory stimuli using psychophysical and electrophysiological approaches.

UNLABELLED: Flavor is a result of the complex combination of olfactory, gustatory and trigeminal sensations perceived during oral processing of foods, including thermal, painful, tactile and/or kinesthetic effects. Aim of this study was to better understand interactions between synchronous tactile (texture) and olfactory (odor) sensations, using a psychophysical and an electrophysiological approach. Texture stimuli were aliquots of lean milk and thickened lean milk. A butter aroma was presented either orthonasally or retronasally after oral processing and before swallowing the oral stimulus or in the absence of an oral stimulus. Eighteen subjects (11 women, 7 men, mean age 24 years), naïve to the expected effects, rated both odor and texture intensity of each stimulus. Event-related potentials (ERP) were obtained from five recording positions. For the psychophysical data, the presence of an oral stimulus increased odor intensity, irrespective of odor presentation route. For the electrophysiological data, both early and late chemosensory ERPs were affected by odor conditions, texture conditions, and their respective interaction. IN CONCLUSION: (1) perceptual interactions occurred between food texture and odor, with cross-modal interactions being found for both orthonasal and retronasal odor administration, and (2) these interactions between texture and odor occur at both primary-sensory and cognitive evaluative levels of stimulus processing. The temporal dimension plays then a critical role in the investigation of odor-texture interactions.