Ken-ichiro Nakajima

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.

Neoculin as a New Taste-modifying Protein Occurring in the Fruit of Curculigo latifolia

A unique taste-modifying activity that converts the sense of sourness to the sense of sweetness occurs in the fruit of the plant Curculigo latifolia, intrinsic to West Malaysia. The active component, known as curculin, is a protein consisting of two identical subunits. We have found a new taste-modifying protein, named neoculin, of the same origin. Both chemical analysis and cDNA cloning characterized neoculin as a heterodimeric protein consisting of an acidic, glycosylated subunit of 113 amino acid residues and a basic subunit that is the monomeric curculin itself.

Extracellular Production of Neoculin, a Sweet-Tasting Heterodimeric Protein with Taste-Modifying Activity, by Aspergillus oryzae

ABSTRACT Neoculin (NCL), a protein with sweetness approximately 500-fold that of sugar, can be utilized as a nonglycemic sweetener. It also has taste-modifying activity to convert sourness to sweetness. NCL is a heterodimer composed of an N-glycosylated acidic subunit (NAS) and a basic subunit (NBS), which are conjugated by disulfide bonds. For the production of recombinant NCL (rNCL) by Aspergillus oryzae, α-amylase with a KEX2 cleavage site, -K-R-, was fused upstream of each of NAS and NBS and the resulting fusion proteins were simultaneously expressed. For accurate and efficient cleavage of the fusion construct by KEX2-like protease, a triglycine motif was inserted after the KEX2 cleavage site. As NBS showed lower production efficiency than did NAS, a larger amount of the NBS expression plasmid than of NAS expression plasmid was introduced during cotransformation, resulting in successful production of rNCL in the culture medium. Moreover, to obtain a higher production yield of rNCL, the active form of hacA cDNA encoding a transcription factor that induces an unfolded protein response was cloned and expressed constitutively. This resulted in a 1.5-fold increase in the level of rNCL production (2.0 mg/liter). rNCL was purified by chromatography, and its NAS was found to be N-glycosylated as expected. The original sweetness and taste-modifying activity of rNCL were comparable to those of native NCL when confirmed by calcium imaging with human embryonic kidney cells expressing the human sweet taste receptor and by sensory tests.

Human sweet taste receptor mediates acid-induced sweetness of miraculin

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.

Acid-induced sweetness of neoculin is ascribed to its pH-dependent agonistic-antagonistic interaction with human sweet taste receptor

Neoculin (NCL) is a sweet protein that also has taste‐modifying activity to convert sourness to sweetness. However, it has been unclear how NCL induces this unique sensation. Here we quantitatively evaluated the pH‐dependent acid‐induced sweetness of NCL using a cell‐based assay system. The human sweet taste receptor, hT1R2‐hT1R3, was functionally expressed in HEK293T cells together with Gα protein. When NCL was applied to the cells under different pH conditions, it activated hT1R2‐hT1R3 in a pH‐dependent manner as the condition changed from pH 8 to 5. The pH‐response sigmoidal curve resembled the imidazole titration curve, suggesting that His residues were involved in the taste‐modifying activity. We then constructed an NCL variant in which all His residues were replaced with Ala and found that the variant elicited strong sweetness at neutral pH as well as at acidic pH. Since NCL and the variant elicited weak and strong sweetness at the same neutral pH, respectively, we applied different proportions of NCL‐variant mixtures to the cells at this pH. As a result, NCL competitively inhibits the variant‐induced receptor activation. All these data suggest that NCL acts as an hT1R2‐hT1R3 agonist at acidic pH but functionally changes into its antagonist at neutral pH.—Nakajima, K., Morita, Y., Koizumi, A., Asakura, T., Terada, T., Ito, K., Shimizu‐Ibuka, A., Maruyama, J., Kitamoto, K., Misaka, T., Abe, K. Acid‐induced sweetness of neoculin is ascribed to its pH‐dependent agonistic‐antagonistic interaction with human sweet taste receptor. FASEB J. 22, 2323–2330 (2008)

Spinophilin as a novel regulator of M3 muscarinic receptor-mediated insulin release in vitro and in vivo

Spinophilin (SPL), a multidomain scaffolding protein known to modulate the activity of different G‐protein‐coupled receptors, regulates various central nervous system (CNS) functions. However, little is known about the role of SPL expressed in peripheral cell types including pancreatic β cells. In this study, we examined the ability of SPL to modulate the activity of β‐cell M3 muscarinic acetylcholine receptors (M3Rs), which play an important role in facilitating insulin release and maintaining normal blood glucose levels. We demonstrated, by using both in vitro and in vivo approaches (mouse insulinoma cells and SPL‐deficient mice), that SPL is a potent negative regulator of M3R‐mediated signaling and insulin release. Additional biochemical and biophysical studies, including the use of bioluminescence resonance energy transfer technology, suggested that SPL is able to recruit regulator of G‐protein signaling 4 (RGS4) to the M3R signaling complex in an agonist‐dependent fashion. Since RGS4 is a member of the RGS family of proteins that act to reduce the lifetime of activated G proteins, these findings support the concept that the inhibitory effects of SPL on M3R activity are mediated by RGS4. These data suggest that SPL or other G‐protein‐coupled receptor‐associated proteins may serve as novel targets for drug therapy aimed at improving β‐cell function for the treatment of type 2 diabetes.—Ruiz de Azua, I., Nakajima, K.‐I., Rossi, M., Cui, Y., Jou, W., Gavrilova, O., Wess, J. Spinophilin as a novel regulator of M3 muscarinic receptor‐mediated insulin release in vitro and in vivo. FASEB J. 26, 4275–4286 (2012). www.fasebj.org

Surface plasmon resonance analysis on interactions of food components with a taste epithelial cell model.

A new device for evaluating the continuity of taste was developed with the use of surface plasmon resonance (SPR). The model of lingual cells was constructed with liposomes immobilized onto an L1 sensor chip for SPR. Using this device, we classified food components into three categories according to the sensorgram pattern and residual ratio on lipid bilayer. Samples in group A strongly interacted with lipid bilayer, those in group B poorly interacted, and those in group C belong to neither group A nor group B. Sweet proteins and gymnemic acids that prolonged sweet perception were categorized in group A. Almost all the carbohydrates investigated and aspartame, of which the taste perception does not continue, belonged to group B. This device made it possible to detect the interaction with lipid bilayer and dissected the mechanism of taste continuity.

Identification and Modulation of the Key Amino Acid Residue Responsible for the pH Sensitivity of Neoculin, a Taste-Modifying Protein

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.

pH-Dependent Structural Change in Neoculin with Special Reference to Its Taste-Modifying Activity

Neoculin has pH-dependent taste-modifying activity. This study found that neoculin changed pH-dependently in its tryptophan- and ANS-derived fluorescence spectra, while no such change occurred in a neoculin variant whose histidine residues were replaced with alanine. These results suggest that the sweetness of neoculin depends on structural change accompanying the pH change, with the histidine residues playing a key role.

Biochemical and Genomic Analysis of Neoculin Compared to Monocot Mannose-Binding Lectins

Neoculin occurring in an edible tropical fruit is a heterodimeric protein which has both sweetness and a taste-modifying activity that converts sourness to sweetness. Both the primary and the overall tertiary structures of neoculin resemble those of monocot mannose-binding lectins. This study investigated differences in biochemical properties between neoculin and the lectins. Structural comparison between the mannose-binding sites of lectins and the corresponding regions of neoculin showed that there is at least one amino acid substitution at each site in neoculin, suggesting a reason for the lack of its mannose-binding ability. This was consistent with hemagglutination assay data demonstrating that neoculin had no detectable agglutinin activity. DNA microarray analysis indicated that neoculin had no significant influence on gene expression in Caco-2 cell, whereas kidney bean lectin (Phaseolus vulgaris agglutinin) greatly influenced various gene expressions. These data strongly suggest that neoculin has no lectin-like properties, encouraging its practical use in the food industry.