Yuji Morita

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.

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)

Bimanual coordination of force enhances interhemispheric inhibition between the primary motor cortices.

The purpose of this study was to elucidate whether bimanual coordination of force affects interhemispheric inhibition (IHI) between the primary motor cortices (M1s). IHI with the index fingers isometrically abducted against a fixed plate (AAP task) was compared with IHI with the index fingers isometrically abducted against each other (AAF task). The index fingers were held stationary at the midline and activity levels of the first dorsal interosseous muscles were equalized between the tasks. The abduction force of each index finger was individually controlled during the AAP task, and bimanually coordinated during the AAF task. IHI during the AAF task was significantly higher than that during the AAP task. IHI between the M1s is related not only to the suppression of unwanted activity of the M1 contralateral to the active M1 but also to bimanual coordination of force.

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.

Non-Acidic Compounds Induce the Intense Sweet Taste of Neoculin, a Taste-Modifying Protein

Neoculin, a sweet protein found in the fruit of Curculigo latifolia, has the ability to change sourness into sweetness. Neoculin turns drinking water sweet, indicating that non-acidic compounds may induce the sweetness. We report that ammonium chloride and certain amino acids elicit the intense sweetness of neoculin. Neoculin can thus sweeten amino acid-enriched foods.