Akiko Shimizu-Ibuka

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)

Purification and Characterization of an α-Amylase of Pichia burtonii Isolated from the Traditional Starter “Murcha” in Nepal

Among more than 20 yeast strains isolated from the traditional starter “murcha” in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an α-amylase. The purified enzyme, named Pichia burtonii α-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 °C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 °C for 30 min. The activity was inhibited by metal ions such as Cd2+, Cu2+, Hg2+, Al3+, and Zn2+.

Effects of Peanut-Skin Procyanidin A1 on Degranulation of RBL-2H3 Cells

Peanut skin contains large amounts of polyphenols having antiallergic effects. We found that a peanut-skin extract (PSE) inhibits the degranulation induced by antigen stimulation of rat basophilic leukemia (RBL-2H3) cells. A low-molecular-weight fraction from PSE, PSEL, also had inhibitory activity against allergic degranulation. A main polyphenol in PSEL was purified by gel chromatography and fractionated by YMC-gel ODS-AQ 120S50 column. Electrospray ionization mass spectrometry (ESI-MS) analysis of the purified polyphenol gave m⁄z 599 [M+Na]+. Based on the results of 1H-NMR, 13C-NMR spectra, and optical rotation analysis, the polyphenol was identified as procyanidin A1. It inhibited the degranulation caused by antigen stimulation at the IC50 of 20.3 μM. Phorbol-12-myristate-13-acetate (PMA) and 2,5,-di(tert-butyl)-1,4-hydroquinone (DTBHQ)-induced processes of degranulation were also inhibited by procyanidin A1. These results indicate that peanut-skin procyanidin A1 inhibits degranulation downstream of protein kinase C activation or Ca2+ influx from an internal store in RBL-2H3 cells.

Hypocholesterolemic Effect of Peanut Skin and Its Fractions: A Case Record of Rats Fed on a High-Cholesterol Diet

Peanut skin (PS) is characterized by almost exclusively consisting of polyphenols and fiber. We fractionated PS into a water-soluble fraction (WSF) and water-insoluble fraction (WIF), and further fractionated WSF into a soluble dietary fiber fraction (DF) and dietary fiber-free, water-soluble fraction (DFF-WSF). Male Sprague-Dawley rats were fed on high-cholesterol diets supplemented with PS and its fractions. PS, WSF, and DFF-WSF decreased the serum lipid and cholesterol levels and increased those in feces. This effect was probably due to the polyphenols that inhibited intestinal cholesterol absorption.

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin

BACKGROUND Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. METHODS Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. RESULTS As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. CONCLUSIONS The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. GENERAL SIGNIFICANCE The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.

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.

Roles of Residues Cys69, Asn104, Phe160, Gly232, Ser237, and Asp240 in Extended-Spectrum β-Lactamase Toho-1

ABSTRACT Toho-1, which is also designated CTX-M-44, is an extended-spectrum class A β-lactamase that has high activity toward cefotaxime. In this study, we investigated the roles of residues suggested to be critical for the substrate specificity expansion of Toho-1 in previous structural analyses. Six amino acid residues were replaced one by one with amino acids that are often observed in the corresponding position of non-extended-spectrum β-lactamases. The mutants produced in Escherichia coli strains were analyzed both for their kinetic properties and their effect on drug susceptibilities. The results indicate that the substitutions of Asn104 and Ser237 have certain effects on expansion of substrate specificity, while those of Cys69 and Phe160 have less effect, and that of Asp240 has no effect on the hydrolysis of any substrates tested. Gly232, which had been assumed to increase the flexibility of the substrate binding site, was revealed not to be critical for the expansion of substrate specificity of this enzyme, although this substitution resulted in deleterious effects on expression and stability of the enzyme.

Molecular Cloning and Characterization of an α-Amylase from Pichia burtonii 15-1

An α-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii α-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis α-amylase, 58% with Saccharomycopsis sp. α-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the Km values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the kcat values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the kcat/Km values of PBA were higher.