Activity and Stability of a New Sweet Protein with Taste-modifying Action, Curculin

Curculin elicited a sweet taste. After the sweetness of curculindiminished, application of deionized water or an acid to thetongue induced a sweet taste. The maximum sweetness of curculinitself was equivalent to thesweetness of 0.35 M sucrose. Themaximum sweetness induced by 0.02 M citric acid or deionizedwater after curculin dissolved in a buffer of pH 6.0 was heldin mouth for 3 min was also equivalent to that of 0.35 M sucrose.The sweetness induced by deionized water was completely suppressedby the presence of 1 mM CaCl₂ or MgCl₂, while that induced byan acid was not suppressed by the presence of divalent cations.Based on these results, the mechanism of the taste-modifyingactivity was discussed. Stability of curculin was examined undervarious conditions. The taste-modifying activity of curculinwas unchanged when curculin was incubated at 50°C for 1h between pH 3 and 11.     

Molecular Mechanisms for Sweet-suppressing Effect of Gymnemic Acids

Gymnemic acids are triterpene glycosides that selectively suppress taste responses to various sweet substances in humans but not in mice. This sweet-suppressing effect of gymnemic acids is diminished by rinsing the tongue with γ-cyclodextrin (γ-CD). However, little is known about the molecular mechanisms underlying the sweet-suppressing effect of gymnemic acids and the interaction between gymnemic acids versus sweet taste receptor and/or γ-CD. To investigate whether gymnemic acids directly interact with human (h) sweet receptor hT1R2 + hT1R3, we used the sweet receptor T1R2 + T1R3 assay in transiently transfected HEK293 cells. Similar to previous studies in humans and mice, gymnemic acids (100 μg/ml) inhibited the [Ca²⁺]_i responses to sweet compounds in HEK293 cells heterologously expressing hT1R2 + hT1R3 but not in those expressing the mouse (m) sweet receptor mT1R2 + mT1R3. The effect of gymnemic acids rapidly disappeared after rinsing the HEK293 cells with γ-CD. Using mixed species pairings of human and mouse sweet receptor subunits and chimeras, we determined that the transmembrane domain of hT1R3 was mainly required for the sweet-suppressing effect of gymnemic acids. Directed mutagenesis in the transmembrane domain of hT1R3 revealed that the interaction site for gymnemic acids shared the amino acid residues that determined the sensitivity to another sweet antagonist, lactisole. Glucuronic acid, which is the common structure of gymnemic acids, also reduced sensitivity to sweet compounds. In our models, gymnemic acids were predicted to dock to a binding pocket within the transmembrane domain of hT1R3.     

Interaction of gymnemic acid with cyclodextrins analyzed by isothermal titration calorimetry, NMR and dynamic light scattering

The physiological phenomenon that the antisweet taste effect of gymnemic acid (GA) is diminished by application of gamma-cyclodextrin (gamma-CD) to the mouth was evaluated at the molecular level using isothermal titration calorimetry, NMR and dynamic light scattering. These analyses showed that GA specifically binds to gamma-CD. Thermodynamic analysis using isothermal titration calorimetry revealed that the association constant of GA and gamma-CD is 10(5)-10(6) m(-1) with favorable enthalpy and entropy changes. The heat capacity change was negative and large, despite the change in accessible surface area upon binding being small. These thermodynamics indicate that the binding is dominated by hydrophobic interactions, which is in agreement with inclusion complex formation of gamma-CD. In addition, NMR measurements showed that in solution the spectra of GA are broad and sharpened by the addition of gamma-CD, indicating that unbound GA is in a water-soluble aggregate that is dispersed when it forms a complex with gamma-CD. Dynamic light scattering showed that the average diameter of unbound GA is > 30 nm and that of GA and gamma-CD complex is 2.2 nm, similar to unbound gamma-CD, supporting the aggregate property of GA and the inclusion complexation of GA by gamma-CD.     

Effects of gymnemic acid concentration and time since exposure on intensity of simple tastes: A test of the biphasic model for the action of gymnemic acid

At several intervals following exposure to gymnemic acid, subjectsjudged the sweetness, bitterness, saltiness, and sourness ofsimple taste stimuli. The experiment was expressly designedto test Kennedy and Halpern's (1980) biphasic model for theaction of gymnemic acid. The model predicts selective suppressionof sweet taste immediately following exposure to gymnemic acidbut nonselective disruption of tastes with the passage of time.The data show dramatic reductions in sweet taste which recoverwith time but no reductions in bitterness, saltiness, or sournessat any time following exposure to any of a wide range of gymnemicacid concentrations.     

Inhibition of ATPases by gymnemic acid

A specially prepared and partially fractionated preparation of gymnemic acid compounds were found to be inhibitory to the ATPase system from housefly brain and labellum, and fish brain. Enzyme activity in a homogenate fraction from labellum showed greatest sensitivity to the gymnemic acid preparation. Na⁺-K⁺ ATPase activity from the labellum, which contains the sweet taste sensory receptors, was inhibited by over 92% at a concentration of 4 middot; 10^?6M gymnemic acid. Because gymnemic acid compounds are specific suppressants of sweet receptors, the evidence presented in this paper implies that a portion of the ATPase system may be biochemically important in the sweet-sensing mechanism.     

Sweet-taste-suppressing compounds: current knowledge and perspectives of application

Sweet-tasting compounds are recognized by a heterodimeric receptor composed of the taste receptor, type 1, members 2 (T1R2) and 3 (T1R3) located in the mouth. This receptor is also expressed in the gut where it is involved in intestinal absorption, metabolic regulation, and glucose homeostasis. These metabolic functions make the sweet taste receptor a potential novel therapeutic target for the treatment of obesity and related metabolic dysfunctions such as diabetes. Existing sweet taste inhibitors or blockers that are still in development would constitute promising therapeutic agents. In this review, we will summarize the current knowledge of sweet taste inhibitors, including a sweet-taste-suppressing protein named gurmarin, which is only active on rodent sweet taste receptors but not on that of humans. In addition, their potential applications as therapeutic tools are discussed.     

Binding proteins for sweet compounds from gustatory papillae of the cow, pig and rat

The intensely sweet proteins thaumatin and monellin were covalently attached to affinity column supports. Lingual tissue extracts were incubated with the affinity columns which were then eluted with glycine-HCl pH 3.4, the sweet peptide aspartame, or gymnemic acid, which is a sweet taste modifier. SDS-PAGE analysis of eluates from the columns showed that 156 kDa and 47 kDa proteins were the main components from cow fungiform papillae which were specifically bound to thaumatin and monellin. These proteins could be displaced from the column with 0.5 mM aspartame or 0.5 mg/ml gymnemic acid. With circumvallate papillae small amounts of 47 kDa protein were also found. The 47 kDa protein was also the major component bound to a gymnemic acid affinity column and could be displaced from the column with 0.5 mg/ml gymnemic acid. Control experiments with other lingual tissue components indicated that these proteins are localised in the gustatory papillae. Similar protein patterns were also found in extracts of pig fungiform papillae and rat lingual preparations.     

Amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor

In mammals, sweet taste perception is mediated by the heterodimeric G-protein-coupled receptor, T1R2/T1R3. An interesting characteristic of this sweet taste receptor is that it has multiple ligand binding sites. Although there have been several studies on agonists of sweet taste receptors, little is known about antagonists of these receptors. In this study, we constructed a cell line stably expressing the human sweet taste receptor (hT1R2/hT1R3) and a functional chimeric G-protein (hGα16gust44) using the Flp-In system for measuring the antagonistic activity against the receptor. This constructed cell line responded quite intensely and frequently to the compounds applied for activation of hT1R2/hT1R3. In the presence of 3 mM amiloride, the responses to sweet tastants such as sugar, artificial sweetener, and sweet protein were significantly reduced. The inhibitory activity of amiloride toward 1 mM aspartame was observed in a dose-dependent manner with an IC₅₀ value of 0.87 mM. Our analysis of a cell line expressing hT1R3 mutants (hT1R3-A733V or hT1R3-F778A) made us to conclude that the target site of amiloride is distinct from that of lactisole, a known sweet taste inhibitor. Our results strongly indicate that amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor and also that this receptor has multiple inhibitor binding sites.     

Alteration of sweet taste in high-fat diet induced obese rats after 4 weeks treatment with exenatide

Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is effective in inducing weight loss. The exact mechanisms are not fully understood. Reduced appetite and food intake may play important roles. Sweet taste contributes to food palatability, which promotes appetite. Interestingly, GLP-1 and its receptor are expressed in the taste buds of rodents and their interaction has an effect on mediating sweet taste sensitivity. Our aim was to investigate whether sweet taste will be changed after long term treatment with exenatide. The results showed that high-fat diet induced obese rats (HF-C) presented metabolic disorders in food intake, body weight, blood glucose and lipid metabolism compared with long term exenatide treated obese rats (EX) and normal chow fed control rats (NC). Meanwhile, greater preference for sweet taste was observed in HF-C rats but not in EX rats. Compared with NC rats, brain activities induced by sweet taste stimulation were stronger in HF-C rats, however these stronger activities were not found in EX rats. We further found reduced sweet taste receptor T1R3 in circumvallte taste buds of HF-C rats, while GLP-1 was increased. Besides, serum leptin was evaluated in HF-C rats with decreased leptin receptor expressed in taste buds. These changes were not observed in EX rats, which suggest them to be the underlying hormone and molecular mechanisms responsible for alterations in sweet taste of HF-C rats and EX rats. In summary, our results suggest that long term treatment with exenatide could benefit dietary obese rats partially by reversing sweet taste changes.     

ON THE GUSTATORY EFFECTS OF GYMNEMIC ACID AND MIRACULIN IN DOG, PIG AND RABBIT

The effects of gymnemic acid, 1 mg/ml, and miraculin, 1 mg/mlor in a tablet (Miralin^TM) on the taste response to sucrose,citric acid, NaCl and quinine have been studied in dog, pigand rabbit. In the dog gymnemic acid suppressed the responseto sucrose for about 5 min in two out of three animals. It alsoaffected the response to NaCl. Miraculin caused a slight short-lastingincrease in the response to acid. No effects of the taste modifierswere observed in the pig. In the rabbit gymnemic acid did notsuppress the response to sucrose while miraculin enhanced theresponse to citric acid in one out of three animals for a fewminutes.     

Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce

Taste-modifying proteins are a natural alternative to artificial sweeteners and flavor enhancers and have been used in some cultures for centuries. The taste-modifying protein, miraculin, has the unusual property of being able to modify a sour taste into a sweet taste. Here, we report the use of a plant expression system for the production of miraculin. A synthetic gene encoding miraculin was placed under the control of constitutive promoters and transferred to lettuce. Expression of this gene in transgenic lettuce resulted in the accumulation of significant amounts of miraculin protein in the leaves. The miraculin expressed in transgenic lettuce possessed sweetness-inducing activity. These results demonstrate that the production of miraculin in edible plants can be a good alternative strategy to enhance the availability of this protein.     

Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants

Miraculin is a taste-modifying protein isolated from the red berries of Richadella dulcifica , a shrub native to West Africa. Miraculin by itself is not sweet, but it is able to turn a sour taste into a sweet taste. This unique property has led to increasing interest in this protein. In this article, we report the high-yield production of miraculin in transgenic tomato plants. High and genetically stable expression of miraculin was confirmed by Western blot analysis and enzyme-linked immunosorbent assay. Recombinant miraculin accumulated to high levels in leaves and fruits, up to 102.5 and 90.7 µg/g fresh weight, respectively. Purified recombinant miraculin expressed in transgenic tomato plants showed strong sweetness-inducing activity, similar to that of native miraculin. These results demonstrate that recombinant miraculin was correctly processed in transgenic tomato plants, and that this production system could be a good alternative to production from the native plant.     

The sweet taste quality is linked to a cluster of taste fibers in primates: lactisole diminishes preference and responses to sweet in S fibers (sweet best) chorda tympani fibers of M. fascicularis monkey

Background

Psychophysically, sweet and bitter have long been considered separate taste qualities, evident already to the newborn human. The identification of different receptors for sweet and bitter located on separate cells of the taste buds substantiated this separation. However, this finding leads to the next question: is bitter and sweet also kept separated in the next link from the taste buds, the fibers of the taste nerves? Previous studies in non-human primates, P. troglodytes, C. aethiops, M. mulatta, M. fascicularis and C. jacchus, suggest that the sweet and bitter taste qualities are linked to specific groups of fibers called S and Q fibers. In this study we apply a new sweet taste modifier, lactisole, commercially available as a suppressor of the sweetness of sugars on the human tongue, to test our hypothesis that sweet taste is conveyed in S fibers.

Results

We first ascertained that lactisole exerted similar suppression of sweetness in M. fascicularis, as reported in humans, by recording their preference of sweeteners and non- sweeteners with and without lactisole in two-bottle tests. The addition of lactisole significantly diminished the preference for all sweeteners but had no effect on the intake of non-sweet compounds or the intake of water. We then recorded the response to the same taste stimuli in 40 single chorda tympani nerve fibers. Comparison between single fiber nerve responses to stimuli with and without lactisole showed that lactisole only suppressed the responses to sweeteners in S fibers. It had no effect on the responses to any other stimuli in all other taste fibers.

Conclusion

In M. fascicularis, lactisole diminishes the attractiveness of compounds, which taste sweet to humans. This behavior is linked to activity of fibers in the S-cluster. Assuming that lactisole blocks the T1R3 monomer of the sweet taste receptor T1R2/R3, these results present further support for the hypothesis that S fibers convey taste from T1R2/R3 receptors, while the impulse activity in non-S fibers originates from other kinds of receptors. The absence of the effect of lactisole on the faint responses in some S fibers to other stimuli as well as the responses to sweet and non-sweet stimuli in non-S fibers suggest that these responses originate from other taste receptors.     

Molecular mechanisms of the action of miraculin,a taste-modifying protein

Miraculin (MCL) is a homodimeric protein isolated from the fruits of Richadella dulcifica, a shrub native to West Africa. Although it is flat in taste at neutral pH, MCL has taste-modifying activity in which sour stimuli produce a sweet perception. Once MCL enters the mouth, strong sweetness can be detected for more than 1 h each time we taste a sour solution. While the human sweet taste receptor (hT1R2–hT1R3) has been identified, the molecular mechanisms underlying the taste-modifying activity of MCL remain unclear. Recently, experimental evidence has been published demonstrating the successful quantitative evaluation of the acid-induced sweetness of MCL using a cell-based assay system. The results strongly suggested that MCL binds hT1R2–hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH. Since sweet-tasting proteins may be used as low-calorie sweeteners because they contain almost no calories, it is expected that MCL will be used in the near future as a new low-calorie sweetener or to modify the taste of sour fruits.     

Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2

Although the five basic taste qualities—sweet, sour, bitter, salty and umami—can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5’-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.     

A gene‐driven recovery mechanism: Drosophila larvae increase feeding activity for post‐stress weight recovery

Recovery from weight loss after stress is important for all organisms, although the recovery mechanisms are not fully understood. We are working to clarify these mechanisms. Here, we recorded enhanced feeding activity of Drosophila melanogaster larvae from 2 to 4 h after heat stress at 35°C for 1 h. During the post‐stress period, expression levels of sweet taste gustatory receptor genes (Grs), Gr5a, Gr43a, Gr64a, and Gr64f, were elevated, whereas bitter taste Grs, Gr66a, and Gr33a, were decreased in expression and expression of a non‐typical taste receptor Gr, Gr68a, was unchanged. Similar upregulation of Gr5a and downregulation of Gr66a was recorded after cold stress at 4°C. Expression levels of tropomyosin and ATP synthase ß subunit were significantly increased in larval mouth parts around 3 to 5 h after the heat stress. We infer that up‐regulation of post‐stress larval feeding activity, and weight recovery, is mediated by increasing capacity for mouth part muscular movements and changes in taste sensing physiology. We propose that Drosophila larvae, and likely insects generally, express an efficient mechanism to recover from weight loss during post‐stress periods.     

Modulation of sweet responses of taste receptor cells

Taste receptor cells play a major role in detection of chemical compounds in the oral cavity. Information derived from taste receptor cells, such as sweet, bitter, salty, sour and umami is important for evaluating the quality of food components. Among five basic taste qualities, sweet taste is very attractive for animals and influences food intake. Recent studies have demonstrated that sweet taste sensitivity in taste receptor cells would be affected by leptin and endocannabinoids. Leptin is an anorexigenic mediator that reduces food intake by acting on leptin receptor Ob-Rb in the hypothalamus. Endocannabinoids such as anandamide [N-arachidonoylethanolamine (AEA)] and 2-arachidonoyl glycerol (2-AG) are known as orexigenic mediators that act via cannabinoid receptor 1 (CB₁) in the hypothalamus and limbic forebrain to induce appetite and stimulate food intake. At the peripheral gustatory organs, leptin selectively suppresses and endocannabinoids selectively enhance sweet taste sensitivity via Ob-Rb and CB₁ expressed in sweet sensitive taste cells. Thus leptin and endocannabinoids not only regulate food intake via central nervous systems but also modulate palatability of foods by altering peripheral sweet taste responses. Such reciprocal modulation of leptin and endocannabinoids on peripheral sweet sensitivity may play an important role in regulating energy homeostasis.     

The receptors for mammalian sweet and umami taste

Sweet and umami (the taste of monosodium glutamate) are the main attractive taste modalities in humans. T1Rs are candidate mammalian taste receptors that combine to assemble two heteromeric G-protein-coupled receptor complexes: T1R1+3, an umami sensor, and T1R2+3, a sweet receptor. We now report the behavioral and physiological characterization of T1R1, T1R2, and T1R3 knockout mice. We demonstrate that sweet and umami taste are strictly dependent on T1R-receptors, and show that selective elimination of T1R-subunits differentially abolishes detection and perception of these two taste modalities. To examine the basis of sweet tastant recognition and coding, we engineered animals expressing either the human T1R2-receptor (hT1R2), or a modified opioid-receptor (RASSL) in sweet cells. Expression of hT1R2 in mice generates animals with humanized sweet taste preferences, while expression of RASSL drives strong attraction to a synthetic opiate, demonstrating that sweet cells trigger dedicated behavioral outputs, but their tastant selectivity is determined by the nature of the receptors.     

Synergistic responses of the chorda tympani to mixtures of umami and sweet substances in rats

It has been known that umami substances such as monosodium L-glutamate (MSG) and 5'-inosine monophosphate (IMP) elicit a unique taste called 'umami' in humans. One of the characteristics of the umami taste is synergism: the synergistic enhancement of the magnitude of response produced by the addition of 5'-ribonucleotides to MSG. In addition to this well-documented synergism, we report here for the first time on another type of synergism between a glutamate receptor agonist, L-AP4, and sweet substances, by analyzing the chorda tympani responses in rats. The results are as follows: (i) when L-AP4 was mixed with one of the sweet substances, such as sucrose, glucose, fructose and maltose, large synergistic responses were observed. (ii) These synergistic responses, except to L-AP4 + sucrose, were not suppressed by sweet taste suppressants, gurmarin and pronase E. (iii) These synergistic responses were not suppressed by either metabotropic or ionotropic glutamate receptor antagonists. (iv) Fibers that responded well to the binary mixtures of L-AP4 and sweet substances also responded well to NaCl and HCl, but very weakly to sucrose. These findings are different from the characteristics of synergism between glutamate and IMP. The multiple transduction mechanisms for the umami taste in rat taste cells are discussed.     

Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli

Animals utilize hundreds of distinct G protein-coupled receptor (GPCR)-type chemosensory receptors to detect a diverse array of chemical signals in their environment, including odors, pheromones, and tastants. However, the molecular mechanisms by which these receptors selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. Here, we report that each of the two subunits in the heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in behaving mice. Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.