Positive Charges on the Surface of Thaumatin Are Crucial for the Multi-Point Interaction with the Sweet Receptor.

Thaumatin, an intensely sweet-tasting protein, elicits sweet taste with a threshold of only 50 nM. Previous studies from our laboratory suggested that the complex model between the T1R2-T1R3 sweet receptor and thaumatin depends critically on the complementarity of electrostatic potentials. In order to further validate this model, we focused on three lysine residues (Lys78, Lys106, and Lys137), which were expected to be part of the interaction sites. Three thaumatin mutants (K78A, K106A, and K137A) were prepared and their threshold values of sweetness were examined. The results showed that the sweetness of K106A was reduced by about three times and those of K78A and K137A were reduced by about five times when compared to wild-type thaumatin. The three-dimensional structures of these mutants were also determined by X-ray crystallographic analyses at atomic resolutions. The overall structures of mutant proteins were similar to that of wild-type but the electrostatic potentials around the mutated sites became more negative. Since the three lysine residues are located in 20-40 Å apart each other on the surface of thaumatin molecule, these results suggest the positive charges on the surface of thaumatin play a crucial role in the interaction with the sweet receptor, and are consistent with a large surface is required for interaction with the sweet receptor, as proposed by the multipoint interaction model named wedge model.

Molecular Analysis of the Polymeric Glutenins with Gliadin-Like Characteristics That Were Produced by Acid Dispersion of Wheat Gluten.

We had earlier shown that the dispersion of wheat gluten in acetic acid solution conferred gliadin-like characteristics to the polymeric glutenins. To elucidate the molecular behavior of its polymeric glutenins, the characteristics of gluten powder prepared from dispersions with various types of acid were investigated in this study. Mixograph measurements showed that the acid-treated gluten powders, regardless of the type of acid, had dough properties markedly weakened in both resistance and elasticity properties, as though gliadin was supplemented. The polymeric glutenins extracted with 70% ethanol increased greatly in all acid-treated gluten powders. Size exclusion HPLC and SDS-PAGE indicated that the behavior of polymeric glutenins due to acid treatment was attributed to their subunit composition rich in high molecular weight glutenin subunit (HMW-GS) and not their molecular size. The gluten prepared with the addition of NaCl in acid dispersion had properties similar to those of the control gluten. The results suggest that ionic repulsion induced by acid dispersion made the polymeric glutenins rich in HMW-GS disaggregate, and therefore, act like gliadins.

A Hypersweet Protein: Removal of The Specific Negative Charge at Asp21 Enhances Thaumatin Sweetness.

Thaumatin is an intensely sweet-tasting protein that elicits sweet taste at a concentration of 50 nM, a value 100,000 times larger than that of sucrose on a molar basis. Here we attempted to produce a protein with enhanced sweetness by removing negative charges on the interacting side of thaumatin with the taste receptor. We obtained a D21N mutant which, with a threshold value 31 nM is much sweeter than wild type thaumatin and, together with the Y65R mutant of single chain monellin, one of the two sweetest proteins known so far. The complex model between the T1R2-T1R3 sweet receptor and thaumatin, derived from tethered docking in the framework of the wedge model, confirmed that each of the positively charged residues critical for sweetness is close to a receptor residue of opposite charge to yield optimal electrostatic interaction. Furthermore, the distance between D21 and its possible counterpart D433 (located on the T1R2 protomer of the receptor) is safely large to avoid electrostatic repulsion but, at the same time, amenable to a closer approach if D21 is mutated into the corresponding asparagine. These findings clearly confirm the importance of electrostatic potentials in the interaction of thaumatin with the sweet receptor.

Dispersion in the presence of acetic acid or ammonia confers gliadin-like characteristics to the glutenin in wheat gluten.

Spray-dried gluten has unique properties and is commercially available in the food industry worldwide. In this study, we examined the viscoelastic properties of gluten powder prepared by dispersion in the presence of acetic acid or an ammonia solvent and then followed by lyophilization instead of a spray drying. Mixograph measurements showed that the acid- and ammonia-treated gluten powders had marked decreases in the time to peak dough resistance when compared with the control gluten powder. The integrals of the dough resistance and bandwidth for 3 min after peak dough resistance decreased in both treated gluten powders. Similar phenomena were observed when gliadin was supplemented to gluten powders. Basic and acidic conditions were applied to the acid- and ammonia-treated gluten powders, respectively, and the viscoelastic behaviors were found to depend on the pH in the gluten dispersion just before lyophilization. These behaviors suggest that gluten may assume a reversible change in viscoelasticity by a fluctuation in pH during gluten dispersion. SDS-PAGE showed that the extractable proteins substantially increased in some polymeric glutenins including the low molecular weight-glutenin subunit (LMW-GS) when the ammonia-treated gluten powder was extracted with 70% ethanol. In contrast, the extractable proteins markedly increased in many polymeric glutenins including the high molecular weight-glutenin subunit and/or the LMW-GS when the acid-treated gluten powder was extracted with 70% ethanol. It thus follows that the extractability of polymeric glutenin to ethanol increases similarly to gliadin when gluten is exposed to an acidic or a basic pH condition; therefore, glutenin adopts gliadin-like characteristics.

Isolation of lactic acid-tolerant Saccharomyces cerevisiae from Cameroonian alcoholic beverage.

We investigated yeast strains used in Cameroonian microbreweries, and identified a Saccharomyces cerevisiae strain (OCY3) with an excellent capacity for alcoholic fermentation. OCY3 showed higher tolerance to lactic acid and better fermentation performance under acidic conditions than a representative Japanese sake yeast, Kyokai No. 7, and a wine yeast, EC1118.

Five amino acid residues in cysteine-rich domain of human T1R3 were involved in the response for sweet-tasting protein, thaumatin.

Thaumatin, a sweet-tasting plant protein, elicits a sweet taste sensation at 50 nM in humans but not rodents. Although it was shown that the cysteine-rich domain (CRD) of human T1R3 (hT1R3) is important for the response to thaumatin, the amino acid residues within CRD critical for response are still unknown. A comparison of the amino acid sequence (69 amino acid residues) of CRD between hT1R3 and mouse T1R3 (mT1R3) revealed sixteen amino acids that differ. In the present study, we converted each of these sixteen amino acids in hT1R3 to their mouse counterpart and examined the response to thaumatin and sucralose using a cell-based assay. No significant decrease in the response to sucralose was seen among any of the sixteen mutants. However, five mutants (Q504K, A537T, R556P, S559P, and R560K) exhibited a significantly diminished response to thaumatin. The five critical residues involved in the response to thaumatin were dispersed in the CRD of hT1R3 and widely distributed when compared to brazzein. The unique intense sweet-taste of thaumatin might be attributed to the different receptor activation mechanism compared to the small molecule sweetener sucralose.

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change.

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50 nM. Although the sweetness remains when thaumatin is heated at 80 °C for 4h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0Å. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of a Cα atom to be substantially greater in the large disulfide-rich region of domain II, especially residues 154-164, suggesting that a loop region in domain II to be affected by solvent conditions. Furthermore, B-factors of Lys137, Lys163, and Lys187 were significantly affected by pH change, suggesting that a striking increase in the mobility of these lysine residues, which could facilitate a reaction with a free sulfhydryl residue produced via the ß-elimination of disulfide bonds by heating at a pH above 7.0. The increase in mobility of lysine residues as well as a loop region in domain II might play an important role in the heat-induced aggregation of thaumatin above pH 7.0.

Changes in lamina propria dendritic cells on the oral administration of exogenous protein antigens during weaning.

Two critical periods of maximum exposure to antigens occur in young mammals, immediately after birth and at weaning, as a result of colonization by commensal bacteria and the ingestion of new diets. At weaning, active immune responses of antibody production against dietary proteins are known to occur, but simultaneously, oral tolerance is acquired for harmless food proteins. However, regulated mechanisms of the immune system at weaning remain to be elucidated although its immune responses may be somewhat similar to those in adulthood. Considering that tolerogenic antigen-presenting cells (APCs) are likely to be a key factor in the acquisition of oral tolerance, in the present study, we examined the changes of dendritic cells (DCs) in the lamina propria (LP) on exposure to food proteins at weaning. C57BL/6 female mice were weaned at the age of 3 weeks and orally administered 10 mg of ovalbumin (OVA) for ten consecutive days after weaning. The administration led to a decrease in the plasma level of immunoglobulin specific for OVA, suggesting the acquisition of oral tolerance. The uptake of fluorescence-labeled OVA was significantly observed for CD11c(+)LPDCs. When we analyzed the changes of two types of LPDCs, PDCA-1(+) MHC II(+) DCs and CD103(+) MHC II(+) DCs, ten consecutive gavages of OVA marginally, but not significantly, augmented only the frequency of PDCA-1(+) MHC II(+) DCs. Considering that the change of APCs likely appears immediately on the response to antigen intake, we found the statistically significant increase in the frequency of PDCA-1(+) DCs, but not in that of CD103(+) DCs, even after two treatments, indicating PDCA-1(+) DCs to be recruited in the LP within 2 days of exposure to food proteins. These results suggest that the behavior of tolerogenic PDCA-1(+) DCs may change at weaning with the removal of the immunoprotective components of maternal milk.

Bacterial heat shock protein 60, GroEL, can induce the conversion of naïve T cells into a CD4 CD25(+) Foxp3-expressing phenotype.

Recent publications report that heat shock proteins (HSPs) can endow regulatory responses to the systemic immune system when administered via the mucosal route, leading to an amelioration of atherosclerosis and allergy. However, it remains poorly understood if HSP antigens exist in the luminal contents of the gastrointestinal tract and which types of HSP induce regulatory responses. Here we addressed these problems, considering that numerous gut microflora and foods are natural sources of HSPs. SDS-PAGE and immunoblotting with the anti-HSP60 antibody demonstrated the intact and degraded forms of HSP60 mainly in appendix and large intestine of the gastrointestinal tract. No reactivity with this antibody was observed for any of the luminal contents derived from germ-free animals, suggesting gut microflora to be a source of the intestinal HSPs because of lack of HSPs in animal chow diet. GroEL, a typical member of bacterial HSP60, showed a tendency to stimulate splenocytes in germ-free mice, compared to that in conventional mice, suggesting that resident commensal bacterial GroEL may stimulate HSP-reactive T cells as regulatory cells in conventional animals. Importantly, GroEL, but not mouse-derived HSP60, caused naïve T cells to differentiate into CD4(+) CD25(+) Foxp3(+) T cells, indicating that the production of regulatory T cells depends on the type of HSP. Thus, HSPs derived from commensal microbes can be utilized to stimulate immunoregulatory pathways for the maintenance of intestinal homeostasis.

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 1mM. 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.

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 Å. 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.

High-resolution structure of the recombinant sweet-tasting protein thaumatin I.

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at a concentration of 50 nM. The crystal structure of a recombinant form of thaumatin I produced in the yeast Pichia pastoris has been determined to a resolution of 1.1 Å. The model was refined with anisotropic B parameters and riding H atoms. A comparison of the diffraction data and refinement statistics for recombinant thaumatin I with those for plant thaumatin I revealed no significant differences in the diffraction data. The R values for recombinant thaumatin I and plant thaumatin I (F(o) > 4σ) were 9.11% and 9.91%, respectively, indicating the final model to be of good quality. Notably, the electron-density maps around Asn46 and Ser63, which differ between thaumatin variants, were significantly improved. Furthermore, a number of H atoms became visible in an OMIT map and could be assigned. The high-quality structure of recombinant thaumatin with H atoms should provide details about sweetness determinants in thaumatin and provide valuable insights into the mechanism of its interaction with taste receptors.

The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin.

Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2-T1R3. Chimeric human- mouse sweet receptors were constructed and their responses to sweeteners were investigated. The human (h) T1R2- mouse (m) T1R3 combination responded to sucralose but not to thaumatin, clearly indicating that a T1R3 subunit from humans is necessary for the interaction with thaumatin. Furthermore, results obtained from using chimeric T1R3s showed that the cysteine-rich domain (CRD) of human T1R3 is important for the interaction with thaumatin. The CRD of T1R3 would be a prominent target for designing new sweeteners.

Changes in textural properties of Japanese tenobe somen noodles during storage.

Tenobe somen (TS) noodles are traditional Japanese wheat-based noodles that are produced manually using a sophisticated method called tenobe (literally, "hand stretched"). In the tension test, both the tensile strength and extensibility of TS noodles were greater than those of machine-made (MS) noodles. In the biting test, the chewy texture of TS noodles was realized in the analysis of the force-deformation curves of each type of somen noodles. The creep test indicated a clear difference between the external and internal elasticity of TS noodles. The texture of TS noodles appeared to change dramatically during storage in the rainy season. In addition, firm and chewy textures of TS noodles stored for 20 mo were observed. Similar to the results from our previous study, TS noodles exhibited significantly higher dityrosine content than the flour used for their manufacture. However, the increase in the dityrosine content during the manufacturing process was not observed in the case of MS noodles. Although clear textural differences were observed between TS noodles stored for 0 and 20 mo, the dityrosine contents of TS noodles at each stage were not largely different.

Lymphoid neoplastic P388D1 cells express membrane protein candidates that discriminate among the C-terminal phylogenetic diversity in heat shock protein 70 sequences.

We previously found that mouse inducible Hsp72 bound more extensively to lymphoblast-like lymphoid neoplastic P388D1 cells than to RAW264.7 monocyte-macrophages. In the present study, we analyzed the characteristics of the binding to P388D1 cells of recombinant HSP70 derived from different species. Recombinant mouse inducible-type Hsp72 bound extensively to P388D1 cells in a saturable manner, but not to P815 mastocytoma or EL4 thymoma. Spinach Hsc70-1, highly homologous with mouse Hsp72 in the C-terminal region, also bound to P388D1 cells. In contrast, significant binding was not observed for bacterial DnaK derived from Lactobacillus acidophilus and Escherichia coli which have relatively little homology in this region. Analyses of surface antigens showed that B220, and CD19, but not CD91, LOX-1, and CD40, the HSP70 receptors reported so far, were present on P388D1 cells, suggesting a B-cell lineage for this cell line. A similar discrimination of the diversity between mouse Hsp72 and bacterial DnaK occurred for CD19(+) B cells derived from mouse spleen, Peyer's patches, and mesenteric lymph nodes. The binding of HSP70 to P388D1 cells was partially, but significantly, antagonized by fucoidan and maleylated BSA, implying a few types of scavenger receptors to be responsible for the binding of HSP70 to this cell line. Furthermore, photo-affinity labeling revealed several membrane protein candidates larger than 110kDa to be involved in the recognition of HSP70 molecules. Using a NF-κB-luciferase reporter assay, we found that exogenous HSP70 did not stimulate NF-κB-dependent signal transduction in P388D1 cells. It thus follows that lymphoid neoplastic P388D1 cells express membrane protein candidates that discriminate among the C-terminal sequences of the HSP70 family. The present results indicate that several types of cells in the innate immune system may distinguish among the phylogenetically specific signals in protein molecules.

Rheological properties of somen noodles--a traditional Japanese wheat product.

We investigated the rheological properties of the Japanese wheat product tenobe somen noodles manufactured using a unique traditional process-"Te-nobe (hand-stretched)." In an extension test, the maximum resistance to extension (R(max)) and extensibility until rupture (Erup) of boiled somen noodles were measured on a Texture Analyzer in the tension test mode and compared with those of machine-made somen noodles. The R(max) and Erup values per unit cross-sectional area were significantly higher for boiled tenobe somen noodles than for machine-made somen noodles, clearly indicating the higher resistance to extension and extensibility of the former. A compression test performed using the Texture Analyzer in the biting-test mode revealed that although the maximum force of compression (F(max)) was lower for boiled tenobe somen noodles than for machine-made somen noodles, the former had more characteristic texture than the latter, which was shown by comparing the force-deformation curve of each somen noodle. Scanning electron microscopy revealed differences between dried tenobe and machine-made somen noodles, which may reflect their rheological differences. Lateral and sectional micrographs of tenobe somen noodles showed regular arrays of starch granules and gluten networks, and some air spaces. Tenobe somen noodles exhibited significantly higher dityrosine content than the flour used for their manufacture, indicating that tyrosine residues in gluten proteins cross-link during the manufacturing process; however, the dityrosine content in tenobe somen noodles were not higher than that in machine-made somen noodles.

Interactions of the sweet-tasting proteins thaumatin and lysozyme with the human sweet-taste receptor.

This study investigated the sweetness of the sweet-tasting protein thaumatin and lysozyme by both an in vitro cell-based assay and an in vivo sensory analysis to elucidate the differences between in vitro and in vivo response profiles. Hek293 cells were constructed that stably expressed the human T1R2+T1R3 sweet-taste receptor, and their responses to thaumatin and lysozyme were analyzed by monitoring the levels of intracellular cAMP. The results indicated that thaumatin and lysozyme as well as aspartame induced a decrease in the intracellular cAMP accumulation of the T1R2+T1R3-transfected cells and that EC(50) values of thaumatin and lysozyme determined by cell-based assay are well-consistent with the results of the sweetness threshold value determined by sensory analysis in the presence of 140 mM NaCl. The results of both in vitro and in vivo experiments confirmed that the sweetness inhibitor lactisole significantly suppressed the sweetness of thaumatin and lysozyme.

Surface expression of a C-terminal alpha-helix region in heat shock protein 72 on murine LL/2 lung carcinoma can be recognized by innate immune sentinels.

Surface expression of Hsp70 members has been previously reported on human tumor cell lines. Here we examined how the inducible mouse Hsp72 can be expressed on the surface of two types of murine tumor cell lines in response to non-lethal heat shock. Exposure to 42 degrees C for 2h led to the intracellular production of Hsp72 for both murine LL/2 lung carcinoma and B16 melanoma cells. Flow cytometric analyses showed that living LL/2 carcinoma, but not B16 melanoma, transported a fraction of inducible Hsp72 to the cell-surface membrane. Induction of the surface expression of Hsp72 occurred upon non-lethal heat shock only when Hsp72 expression was forced to be elevated in B16 transfectants. Hsp72 expressed on the LL/2 cell surface was detected by the monoclonal antibody that recognized the epitope of 504-617 amino acid residues, but not by another antibody with the epitope of 122-264 residues. When we analyzed the binding of recombinant full-length Hsp72 to mouse splenocytes, significant binding was observed for innate immune cells such as CD11b(+)-, CD11c(+)-, or NK1.1(+)-cells. The recombinant variants obtained by truncation of the C-terminal helical region of Hsp72 exhibited more robust binding to these innate immune cells in a similar fashion, however, further deletion offered less binding to those immunocytes. Two fragment variants lacking the N-terminal nucleotide-binding domain were found to extensively bind to peritoneal macrophages. Taken together with these results, it thus follows that the sentinels in an innate immune system, macrophages, dendritic cells and NK cells, can be involved in the surveillance of functionally aberrant cells through the recognition of a specific C-terminal structure of Hsp70 as a danger signal in living cells.

Effects of CpG-oligodeoxynucleotides on dendritic cell development.

Dendritic cell (DC) development begins in the bone marrow and immature progenitors reach their sites of residence in lymphoid organs. The mechanism of DC development in the bone marrow and in peripheral lymphoid organs is poorly understood. Here, we examined the effects of synthetic oligodeoxynucleotides containing a CpG motif (CpG-ODNs) on the development of DC from the bone marrow cells. Approximately 15% of bone marrow cells expressed CD11c surface antigen in the in vitro culture for 8 days in the presence of IL-3. However, the addition of a phosphorothioate-modified CpG-ODN (PTO-CpG-ODN), but not a phosphodiester-modified ODN (PO-CpG-ODN), suppressed the expression of CD11c surface antigen. Also, we examined the effects of CpG-ODNs on the maintenance of DCs resident in the gastrointestinal lymphoid tissues, where immature progenitors may be challenged by a variety of ODN derived from microflora. The population of CD11c(+)B220(int)Gr-1(low) cells decreased by the addition of PTO-CpG-ODN when the lymphocytes from mesenteric lymph nodes as well as spleen are cultured for 2 days in the presence of GM-CSF. In contrast, this population increased in the case of the lymphocytes from Peyer's patches. It thus follows that PTO-CpG-ODN plays a regulatory role in the differentiation of bone marrow cells into DCs and in the functional maintenance of DCs at peripheral lymphoid organs.

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I.

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.