Introduction of a negative charge at Arg82 in thaumatin abolished responses to human T1R2-T1R3 sweet receptors

Thaumatin, an intensely sweet-tasting protein, elicits a sweet-taste sensation at a level as low as 50 nM. Although previous sensory analyses have suggested that Lys67 and Arg82 are important to the sweetness of thaumatin, the exact effects of each residue on sweet receptors are still unknown. In the present study, various mutants of thaumatin altered at Arg82 as well as Lys67 were prepared and their sweetness levels were quantitatively evaluated by cell-based assays using HEK293 cells expressing human sweet receptors. Mutations at Arg82 had a more deteriorative effect on sweetness than mutations at Lys67. Particularly, a charge inversion at Arg82 (R82E) resulted in an abolishment of the response to sweet receptors even at a concentration as high as 1 mM. These results indicate that Arg82 plays a central role in determining the sweetness of thaumatin. A strict spatial charge location at residue 82 appears to be required for interaction with sweet receptors.     

Pseudogenization of a sweet-receptor gene accounts for cats' indifference toward sugar

Although domestic cats (Felis silvestris catus) possess an otherwise functional sense of taste, they, unlike most mammals, do not prefer and may be unable to detect the sweetness of sugars. One possible explanation for this behavior is that cats lack the sensory system to taste sugars and therefore are indifferent to them. Drawing on work in mice, demonstrating that alleles of sweet-receptor genes predict low sugar intake, we examined the possibility that genes involved in the initial transduction of sweet perception might account for the indifference to sweet-tasting foods by cats. We characterized the sweet-receptor genes of domestic cats as well as those of other members of the Felidae family of obligate carnivores, tiger and cheetah. Because the mammalian sweet-taste receptor is formed by the dimerization of two proteins (T1R2 and T1R3; gene symbols Tas1r2 and Tas1r3), we identified and sequenced both genes in the cat by screening a feline genomic BAC library and by performing PCR with degenerate primers on cat genomic DNA. Gene expression was assessed by RT-PCR of taste tissue, in situ hybridization, and immunohistochemistry. The cat Tas1r3 gene shows high sequence similarity with functional Tas1r3 genes of other species. Message from Tas1r3 was detected by RT-PCR of taste tissue. In situ hybridization and immunohistochemical studies demonstrate that Tas1r3 is expressed, as expected, in taste buds. However, the cat Tas1r2 gene shows a 247-base pair microdeletion in exon 3 and stop codons in exons 4 and 6. There was no evidence of detectable mRNA from cat Tas1r2 by RT-PCR or in situ hybridization, and no evidence of protein expression by immunohistochemistry. Tas1r2 in tiger and cheetah and in six healthy adult domestic cats all show the similar deletion and stop codons. We conclude that cat Tas1r3 is an apparently functional and expressed receptor but that cat Tas1r2 is an unexpressed pseudogene. A functional sweet-taste receptor heteromer cannot form, and thus the cat lacks the receptor likely necessary for detection of sweet stimuli. This molecular change was very likely an important event in the evolution of the cat's carnivorous behavior.     

Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris

Thaumatin, an intensely sweet-tasting protein, was secreted by the methylotrophic yeast Pichia pastoris. The mature thaumatin II gene was directly cloned from Taq polymerase-amplified PCR products by using TA cloning methods and fused the pPIC9K expression vector that contains Saccharomyces cerevisiae prepro alpha-mating factor secretion signal. Several additional amino acid residues were introduced at both the N- and C-terminal ends by genetic modification to investigate the role of the terminal end region for elicitation of sweetness in the thaumatin molecule. The secondary and tertiary structures of purified recombinant thaumatin were almost identical to those of the plant thaumatin molecule. Recombinant thaumatin II elicited a sweet taste as native plant thaumatin II; its threshold value of sweetness to humans was around 50 nM, which is the same as that of plant thaumatin II. These results demonstrate that the functional expression of thaumatin II was attained by Pichia pastoris systems and that the N- and C-terminal regions of the thaumatin II molecule do not -play an important role in eliciting the sweet taste of thaumatin.     

Sweet taste receptor gene variation and aspartame taste in primates and other species

Aspartame is a sweetener added to foods and beverages as a low-calorie sugar replacement. Unlike sugars, which are apparently perceived as sweet and desirable by a range of mammals, the ability to taste aspartame varies, with humans, apes, and Old World monkeys perceiving aspartame as sweet but not other primate species. To investigate whether the ability to perceive the sweetness of aspartame correlates with variations in the DNA sequence of the genes encoding sweet taste receptor proteins, T1R2 and T1R3, we sequenced these genes in 9 aspartame taster and nontaster primate species. We then compared these sequences with sequences of their orthologs in 4 other nontasters species. We identified 9 variant sites in the gene encoding T1R2 and 32 variant sites in the gene encoding T1R3 that distinguish aspartame tasters and nontasters. Molecular docking of aspartame to computer-generated models of the T1R2 + T1R3 receptor dimer suggests that species variation at a secondary, allosteric binding site in the T1R2 protein is the most likely origin of differences in perception of the sweetness of aspartame. These results identified a previously unknown site of aspartame interaction with the sweet receptor and suggest that the ability to taste aspartame might have developed during evolution to exploit a specialized food niche.     

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.     

Enhanced Expression of the Sweet Taste Receptors and Alpha-gustducin in Reactive Astrocytes of the Rat Hippocampus Following Ischemic Injury

The heterodimeric sweet taste receptors, T1R2 and T1R3, have recently been proposed to be associated with the brain glucose sensor. To identify whether sweet taste signaling is regulated in response to an ischemic injury inducing acute impairment of glucose metabolism, we investigated the spatiotemporal expression of the sweet taste receptors and their associated taste-specific G-protein α-gustducin in the rat hippocampus after ischemia. The expression profiles of both receptor subunits and α-gustducin shared overlapping expression patterns in sham-operated and ischemic hippocampi. Constitutive expression of both receptors and α-gustducin was localized in neurons of the pyramidal cell and granule cell layers, but their upregulation was detected in reactive astrocytes in ischemic hippocampi. Immunoblot analysis confirmed the immmunohistochemically determined temporal patterns of sweet-taste signaling proteins. These results suggest that the expression of sweet taste signaling proteins in astrocytes might be regulated in response to altered extracellular levels of glucose following an ischemic insult.     

The Structure-taste Relationships of Aspartyl Dipeptide Esters

The molecular features common to sweet-tasting dipeptide esters are described. The molecular features of sweet amino acids were represented by the Fischer projection formulas and sweet peptides were related to the sweet amino acids through the Fischer projection formulas of the peptides. It was concluded that a peptide is sweet when it takes the formula 5a, whereas when it takes the formula 5b it is not sweet. It was also concluded that a third binding site (R₁ in 5a) besides the postulated AH–B system in a sweet molecule is necessary for an intense sweetness potency. The location of the site in the molecule relative to the AH–B system is important, as well as the shape and size of this site, because the third binding site is considered to participate in hydrophobic interaction with a similar binding site on the taste receptor. Increased sweetness is observed when these requirements are satisfied.     

Crystal structure of neoculin: insights into its sweetness and taste-modifying activity

Although the majority of sweet compounds are of low molecular mass, several proteins are known to elicit sweet taste responses in humans. The fruit of Curculigo latifolia contains a heterodimeric protein, neoculin, which has both sweetness and a taste-modifying activity that converts sourness to sweetness. Here, we report the crystal structure of neoculin at 2.76A resolution. This is the first well-defined tertiary structure of a taste-modifying protein of this kind. The overall structure is quite similar to those of monocot mannose-binding lectins. However, crucial topological differences are observed in the C-terminal regions of both subunits. In both subunits of neoculin, the C-terminal tails turn up to form loops fixed by inter-subunit disulfide bonds that are not observed in the lectins. Indeed, the corresponding regions of the lectins stretch straight over the surface of another subunit. Such a C-terminal structural feature as is observed in neoculin results in a decrease in subunit-subunit interactions. Moreover, distribution of electrostatic potential on the surface of neoculin is unique and significantly different from those of the lectins, particularly in the basic subunit (NBS). We have found that there is a large cluster composed of six basic residues on the surface of NBS, and speculate that it might be involved in the elicitation of sweetness and/or taste-modifying activity of neoculin. Molecular dynamics simulation based on the crystallography results suggests that neoculin may adopt a widely "open" conformation at acidic pH, while unprotonated neoculin at neutral pH is in a "closed" conformation. Based on these simulations and the generation of a docking model between neoculin and the sweet-taste receptor, T1R2-T1R3, we propose the hypothesis that neoculin is in dynamic equilibrium between open and closed states, and that the addition of an acid shifts the equilibrium to the open state, allowing ligand-receptor interaction.     

Crystal structure of Mabinlin II: a novel structural type of sweet proteins and the main structural basis for its sweetness

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7A resolution by the SIRAS method. The Mab II 3D structure features in an "all alpha" fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54-B64 was found in Face B, which adopts a special sequence, NL-P-NI-C-NI-P-NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model.     

Mammalian sweet taste receptors

The sense of taste provides animals with valuable information about the quality and nutritional value of food. Previously, we identified a large family of mammalian taste receptors involved in bitter taste perception (the T2Rs). We now report the characterization of mammalian sweet taste receptors. First, transgenic rescue experiments prove that the Sac locus encodes T1R3, a member of the T1R family of candidate taste receptors. Second, using a heterologous expression system, we demonstrate that T1R2 and T1R3 combine to function as a sweet receptor, recognizing sweet-tasting molecules as diverse as sucrose, saccharin, dulcin, and acesulfame-K. Finally, we present a detailed analysis of the patterns of expression of T1Rs and T2Rs, thus providing a view of the representation of sweet and bitter taste at the periphery.     

Reduced sweetness of a monellin (MNEI) mutant results from increased protein flexibility and disruption of a distant poly-(L-proline) II helix

Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding.     

The mechanism of interaction of sweet proteins with the T1R2-T1R3 receptor: evidence from the solution structure of G16A-MNEI

The mechanism by which sweet proteins elicit a response on the T1R2-T1R3 sweet taste receptor is still mostly unknown but has been so far related to the presence of "sweet fingers" on the protein surface able to interact with the same mechanism as that of low molecular mass sweeteners. In the search for the identification of sweet fingers, we have solved the solution structure of G16A MNEI, a structural mutant that shows a reduction of one order of magnitude in sweetness with respect to its parent protein, MNEI, a single-chain monellin. Comparison of the structures of wild-type monellin and its G16A mutant shows that the mutation does not affect the structure of potential glucophores but produces a distortion of the surface owing to the partial relative displacement of elements of secondary structure. These results show conclusively that sweet proteins do not possess a sweet finger and strongly support the hypothesis that the mechanism of interaction of sweet-tasting proteins with the recently identified T1R2-T1R3 GPC receptor is different from that of low molecular mass sweeteners.     

Sweetness determinant sites of brazzein, a small, heat-stable, sweet-tasting protein

Brazzein, originally isolated from the fruit of the African plant Pentadiplandra brazzeana Baillon, is the smallest, most heat-stable and pH-stable member of the set of proteins known to have intrinsic sweetness. These properties make brazzein an ideal system for investigating the chemical and structural requirements of a sweet-tasting protein. We have used the three-dimensional structure of the protein (J. E. Caldwell et al. (1998) Nat. Struct. Biol. 5, 427-431) as a guide in designing 15 synthetic genes in expression constructs aimed at delineating the sweetness determinants of brazzein. Protein was produced heterologously in Escherichia coli, isolated, and purified as described in the companion paper (Assadi-Porter, F. M., Aceti, D., Cheng, H., and Markley, J. L., this issue). Analysis by one-dimensional (1)H NMR spectroscopy indicated that all but one of these variants had folded properly under the conditions used. A taste panel compared the gustatory properties of solutions of these proteins to those of sucrose and brazzein isolated from fruit. Of the 14 mutations in the des-pGlu1-brazzein background, four exhibited almost no sweetness, six had significantly reduced sweetness, two had taste properties equivalent to des-pGlu1-brazzein (two times as sweet as the major form of brazzein isolated from fruit which contains pGlu1), and two were about twice as sweet as des-pGlu1-brazzein. Overall, the results suggest that two regions of the protein are critical for the sweetness of brazzein: a region that includes the N- and C-termini of the protein, which are located close to one another, and a region that includes the flexible loop around Arg43.     

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.     

Identification of novel sweet protein for nutritional applications

The prevalence of obesity and diabetes has increased exponentially in recent years around the globe, especially in India. Sweet proteins have the potential to substitute the sugars, by acting as natural, good and low calorie sweeteners. They also do not trigger a demand for insulin in diabetic patients unlike sucrose. In humans, the sweet taste perception is mainly due to taste-specific G protein-coupled heterodimeric receptors T1R2-T1R3. These receptors recognize diverse natural and synthetic sweeteners such as monelin, brazzein, thaumatin, curculin, mabinlin, miraculin and pentadin. Structural modeling of new sweetener proteins will be a great leap in further advancement of knowledge and their utility as sweeteners. We have explored the fingerprints of sweetness by studying the aminoacid composition and structure properties of the above proteins. The structural analysis of monellin revealed that the individual A or B chains of monellin are not contributing to its sweetness. However, the native conformation and ionic interaction between AspB7 of monellin with active site of T1R2-T1R3 receptor, along with hydrogen bonding stability of IleB6 and IleB8 are responsible for the sweet taste. Based on structural similarity search, we found a new hypothetical protein from Shewanella loihica, which has the presence of Asp(32) with adjacent isoleucine residues. Further, we examined the lead protein by two-step docking for the study of interaction of functionally conserved residues with receptors. The identified protein showed similar ionic and hydrophobic interactions with monelin. This gives a promising opportunity to explore this protein for potential health application in the low calorie sweetener industry viz., soft drinks, snacks, food, chocolate industries etc.     

Variation in the human TAS1R taste receptor genes

We have performed a comprehensive evaluation of single-nucleotide polymorphisms (SNPs) and haplotypes in the human TAS1R gene family, which encodes receptors for sweet and umami tastes. Complete DNA sequences of TAS1R1-, TAS1R2-, and TAS1R3-coding regions, obtained from 88 individuals of African, Asian, European, and Native American origin, revealed substantial coding and noncoding diversity: polymorphisms are common in these genes, and polymorphic sites and SNP frequencies vary widely in human populations. The genes TAS1R1 and TAS1R3, which encode proteins that act as a dimer to form the umami (glutamate) taste receptor, showed less variation than the TAS1R2 gene, which acts as a dimer with TAS1R3 to form the sweet taste receptor. The TAS1R3 gene, which encodes a subunit common to both the sweet and umami receptors, was the most conserved. Evolutionary genetic analysis indicates that these variants have come to their current frequencies under natural selection during population growth and support the view that the coding sequence variants affect receptor function. We propose that human populations likely vary little with respect to umami perception, which is controlled by one major form of the receptor that is optimized for detecting glutamate but may vary much more with respect to sweet perception.     

The liaison of sweet and savory

The sense of taste provides humans with necessary information about the composition and quality of food. For humans, five basic tastes are readily distinguishable and include sweet, bitter, salty, sour, and savory (or umami). Although each of these qualities has individualized transduction pathways, sweet and umami tastes are believed to share a common receptor element, the T1R3 receptor subunit. The two G-protein-coupled heteromer receptors that comprise an umami stimulus receptor (T1R1-T1R3) and a sweetener receptor (T1R2-T1R3) constitute a potential link between these two qualities of perception. While the role of the individual monomers in each human heteromer has been examined in vitro, very little is known of the implication of this research for human perception, or specifically, how sweet and savory taste perceptions may be connected. Using a psychophysical approach, we demonstrate that lactisole, a potent sweetness inhibitor that binds in vitro to hT1R3, also inhibits a significant portion of the perception of umami taste from monosodium glutamate. Following the molecular logic put forward by Xu et al. (2004, Proc. Natl Acad. Sci. USA, 101, 14258-14263), our psychophysical data support the in vitro hypothesis that the shared T1R3 monomer moderates the activation of both T1R2 and T1R1 in humans and impairs suprathreshold perception, respectively, of sweetness and, to a lesser degree, umaminess in the presence of lactisole.     

Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds

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.     

The cysteine-rich region of T1R3 determines responses to intensely sweet proteins

A wide variety of chemically diverse compounds taste sweet, including natural sugars such as glucose, fructose, sucrose, and sugar alcohols, small molecule artificial sweeteners such as saccharin and acesulfame K, and proteins such as monellin and thaumatin. Brazzein, like monellin and thaumatin, is a naturally occurring plant protein that humans, apes, and Old World monkeys perceive as tasting sweet but that is not perceived as sweet by other species including New World monkeys, mouse, and rat. It has been shown that heterologous expression of T1R2 plus T1R3 together yields a receptor responsive to many of the above-mentioned sweet tasting ligands. We have determined that the molecular basis for species-specific sensitivity to brazzein sweetness depends on a site within the cysteine-rich region of human T1R3. Other mutations in this region of T1R3 affected receptor activity toward monellin, and in some cases, overall efficacy to multiple sweet compounds, implicating this region as a previously unrecognized important determinant of sweet receptor function.     

Crystal structure of the sweet-tasting protein thaumatin II at 1.27Å

Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 ?. Overall structure of thaumatin II is similar to thaumatin I, but a slight shift of the Cα atom of G96 in thaumatin II was observed. Furthermore, the side chain of residue 67 in thaumatin II is highly disordered. Since residue 67 is one of two residues critical to the sweetness of thaumatin, the present results suggested that the critical positive charges at positions 67 and 82 are disordered and the flexibility and fluctuation of these side chains would be suitable for interaction of thaumatin molecules with sweet receptors.