Human sweet taste receptor mediates acid-induced sweetness of miraculin.
ABSTRACT: Miraculin (MCL) is a homodimeric protein isolated from the red berries of Richadella dulcifica. MCL, although flat in taste at neutral pH, has taste-modifying activity to convert sour stimuli to sweetness. Once MCL is held on the tongue, strong sweetness is sensed over 1 h each time we taste a sour solution. Nevertheless, no molecular mechanism underlying the taste-modifying activity has been clarified. In this study, we succeeded in quantitatively evaluating the acid-induced sweetness of MCL using a cell-based assay system and found that MCL activated hT1R2-hT1R3 pH-dependently as the pH decreased from 6.5 to 4.8, and that the receptor activation occurred every time an acid solution was applied. Although MCL per se is sensory-inactive at pH 6.7 or higher, it suppressed the response of hT1R2-hT1R3 to other sweeteners at neutral pH and enhanced the response at weakly acidic pH. Using human/mouse chimeric receptors and molecular modeling, we revealed that the amino-terminal domain of hT1R2 is required for the response to MCL. Our data suggest that MCL binds hT1R2-hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH, and we conclude this may cause its taste-modifying activity.
Project description:Neoculin (NCL) is a heterodimeric protein isolated from the edible fruit of Curculigo latifolia. It exerts a taste-modifying activity by converting sourness to sweetness. We previously demonstrated that NCL changes its action on the human sweet receptor hT1R2-hT1R3 from antagonism to agonism as the pH changes from neutral to acidic values, and that the histidine residues of NCL molecule play critical roles in this pH-dependent functional change. Here, we comprehensively screened key amino acid residues of NCL using nuclear magnetic resonance (NMR) spectroscopy and alanine scanning mutagenesis. We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities. The mutations had only a slight effect on the pH-dependent functional change. These residues should determine the affinity of NCL for the receptor regardless of pH. Their locations were separated from the histidine residues responsible for the pH-dependent functional change in the tertiary structure. From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues. Thus, the receptor activation is induced by local structural changes in the pH-dependent interface.
Project description:Acidification of the glycoprotein, miraculin (MCL), induces sweet taste in humans, but not in mice. The sweet taste induced by MCL is more intense when acidification occurs with weak acids as opposed to strong acids. MCL interacts with the human sweet receptor subunit hTAS1R2, but the mechanisms by which the acidification of MCL activates the sweet taste receptor remain largely unexplored. The work reported here speaks directly to this activation by utilizing a sweet receptor TAS1R2?+?TAS1R3 assay. In accordance with previous data, MCL-applied cells displayed a pH dependence with citric acid (weak acid) being right shifted to that with hydrochloric acid (strong acid). When histidine residues in both the intracellular and extracellular region of hTAS1R2 were exchanged for alanine, taste-modifying effect of MCL was reduced or abolished. Stronger intracellular acidification of HEK293 cells was induced by citric acid than by HCl and taste-modifying effect of MCL was proportional to intracellular pH regardless of types of acids. These results suggest that intracellular acidity is required for full activation of the sweet taste receptor by MCL.
Project description:One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2-hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein-coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2-hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling-based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.
Project description:Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor-taste substance in particular.
Project description:Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.
Project description:Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn(2+)-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K(+) current. We identify KIR2.1 as the acid-sensitive K(+) channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of Kir2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K(+) current in amplifying the sensory response, entry of protons through the Zn(2+)-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K(+) channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.
Project description:Miraculin is a promising protein with taste-modifying properties. Focusing on the unique function and potential of miraculin, recombinant miraculin production has been explored with the use of heterologous expression systems, but the activities of recombinant miraculins were much lower than those of native miraculin, probably due to the difference in post-translational modification, especially N-glycosylation. For practical use therefore, the differences between N-glycan of recombinant miraculin compared to that of native miraculin should be minimized. Here, to establish the platform for functional miraculin production, we expressed miraculin in tomato plants with the same taste-modifying activity as native miraculin purified from miracle fruit, and we compared the N-glycan structures with those of native miraculin. Our N-glycan structural analysis using purified miraculin, followed by hydrazynolysis, 2-pyridylamine (PA)-labeling, high-performance liquid chromatography, and a liquid chromatography tandem-mass spectrometry analysis revealed that both the native and recombinant miraculins carried an M3 structure as a predominant structure and that most of the N-glycan structures on the miraculins were pauci-mannosidic structures with a smaller amount of plant-specific ?1,3-fucosylated and/or ?1,2-xylosylated N-glycans and without a Lewis a epitope. These results indicate that the N-glycoform of native miraculin from miracle fruit and recombinant miraculin expressed in tomato plants are almost identical to each other with similar ratios and that, therefore, plant-specific N-glycans are essential for showing the full taste-modifying activity of miraculin.
Project description:A growing body of behavioral and genetic information indicates that taste perception and food sources are highly coordinated across many animal species. For example, sweet taste perception is thought to serve to detect and motivate consumption of simple sugars in plants that provide calories. Supporting this is the observation that most plant-eating mammals examined exhibit functional sweet perception, whereas many obligate carnivores have independently lost function of their sweet taste receptors and exhibit no avidity for simple sugars that humans describe as tasting sweet. As part of a larger effort to compare taste structure/function among species, we examined both the behavioral and the molecular nature of sweet taste in a plant-eating animal that does not consume plants with abundant simple sugars, the giant panda (Ailuropoda melanoleuca). We evaluated two competing hypotheses: as plant-eating mammals, they should have a well-developed sweet taste system; however, as animals that do not normally consume plants with simple sugars, they may have lost sweet taste function, as has occurred in strict carnivores. In behavioral tests, giant pandas avidly consumed most natural sugars and some but not all artificial sweeteners. Cell-based assays revealed similar patterns of sweet receptor responses toward many of the sweeteners. Using mixed pairs of human and giant panda sweet taste receptor units (hT1R2+gpT1R3 and gpT1R2+hT1R3) we identified regions of the sweet receptor that may account for behavioral differences in giant pandas versus humans toward various sugars and artificial sweeteners. Thus, despite the fact that the giant panda's main food, bamboo, is very low in simple sugars, the species has a marked preference for several compounds that taste sweet to humans. We consider possible explanations for retained sweet perception in this species, including the potential extra-oral functions of sweet taste receptors that may be required for animals that consume plants.
Project description:The relationship between taste-intensity patterns and 5-year change in adiposity-related health measures was determined. Participants were members of the Beaver Dam Offspring Study, a study of the adult children of participants in the population-based Epidemiology of Hearing Loss Study. There were 1,918 participants (mean baseline age=48.8 years; range=22 to 84 years) with baseline taste (2005 to 2008) and follow-up (2010 to 2013) data. Outcomes included 5-year change in body mass index, waist circumference, blood pressure, non-high-density lipoprotein cholesterol, and glycosylated hemoglobin A1c, and hedonic ratings of specific foods. Cluster analysis with Ward's minimum variance method identified the following 5 patterns of the suprathreshold taste intensities of salt, sweet, sour, and bitter: salt and sweet intensities slightly above population averages, average sour and bitter intensities; salt, sour, and bitter intensities above population average, average sweet intensity; salt, sour, and bitter intensities above population average, sweet intensity substantially above average; all intensities below population averages; and all intensities close to population average. The General Linear Model procedure was used for testing cluster differences in the outcomes. With covariate adjustment, the group with all intensities close to population averages had a significantly lower average increase in body mass index compared with the group with above-average intensities for salt, sour, and bitter (+0.4 vs +0.9), and in glycosylated hemoglobin A1c compared with the group with above-average intensities for all tastes (+0.20% vs +0.34%). Clusters differed in the hedonics of foods representing sweetness and saltiness. The study's findings provide evidence that perceived taste intensity might be related to changes in adiposity-related health.
Project description:Five tastes have been identified, each of which is transduced by a separate set of taste cells. Of these sour, which is associated with acid stimuli, is the least understood. Genetic ablation experiments have established that sour is detected by a subset of taste cells that express the TRP channel PKD2L1 and its partner PKD1L3, however the mechanisms by which this subset of cells detects acids remain unclear. Previous efforts to understand sour taste transduction have been hindered because sour responsive cells represent only a small fraction of cells in a taste bud, and numerous ion channels with no role in sour sensing are sensitive to acidic pH. To identify acid-sensitive conductances unique to sour cells, we created genetically modified mice in which sour cells were marked by expression of YFP under the control of the PKD2L1 promoter. To measure responses to sour stimuli we developed a method in which suction electrode recording is combined with UV photolysis of NPE-caged proton. Using these methods, we report that responses to sour stimuli are not mediated by Na(+) permeable channels as previously thought, but instead are mediated by a proton conductance specific to PKD2L1-expressing taste cells. This conductance is sufficient to drive action potential firing in response to acid stimuli, is enriched in the apical membrane of PKD2L1-expressing taste cells and is not affected by targeted deletion of the PKD1L3 gene. We conclude that, during sour transduction, protons enter through an apical proton conductance to directly depolarize the taste cell membrane.