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Identification of key neoculin residues responsible for the binding and activation of the sweet taste receptor.

Koizumi T, Terada T, Nakajima K, Kojima M, Koshiba S, Matsumura Y, Kaneda K, Asakura T, Shimizu-Ibuka A, Abe K, Misaka T - Sci Rep (2015)

Bottom Line: We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities.The mutations had only a slight effect on the pH-dependent functional change.From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Neoculin (NCL) is a heterodimeric protein isolated from the edible fruit of Curculigo latifolia. It exerts a taste-modifying activity by converting sourness to sweetness. We previously demonstrated that NCL changes its action on the human sweet receptor hT1R2-hT1R3 from antagonism to agonism as the pH changes from neutral to acidic values, and that the histidine residues of NCL molecule play critical roles in this pH-dependent functional change. Here, we comprehensively screened key amino acid residues of NCL using nuclear magnetic resonance (NMR) spectroscopy and alanine scanning mutagenesis. We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities. The mutations had only a slight effect on the pH-dependent functional change. These residues should determine the affinity of NCL for the receptor regardless of pH. Their locations were separated from the histidine residues responsible for the pH-dependent functional change in the tertiary structure. From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues. Thus, the receptor activation is induced by local structural changes in the pH-dependent interface.

No MeSH data available.


Production of NCL mutants and evaluation of their sweetness using the cell-based assay.(A,B) Responses of cells expressing hT1R2-hT1R3 and G15Gi3 to fourteen NCL mutants under neutral (pH 7.4) and weakly acidic (pH 6.3) conditions. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Error bars represent the mean ± SE (n = 4–6). *P < 0.05, †P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test). (C–F) Dose-response relationships and pH dependencies of each set of four NCL mutants. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Each point represents the mean ± SE (n = 3–5). *P < 0.05, †P < 0.01, ‡P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test). (G) Evaluation of the antagonistic activities of the four NCL mutants at pH 8.0. Each mutant was applied to cells expressing hT1R2-hT1R3 together with the NBS H11A mutant at pH 8.0. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 8.0. Each point represents the mean ± SE (n = 4-5). *P < 0.05, †P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test).
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f3: Production of NCL mutants and evaluation of their sweetness using the cell-based assay.(A,B) Responses of cells expressing hT1R2-hT1R3 and G15Gi3 to fourteen NCL mutants under neutral (pH 7.4) and weakly acidic (pH 6.3) conditions. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Error bars represent the mean ± SE (n = 4–6). *P < 0.05, †P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test). (C–F) Dose-response relationships and pH dependencies of each set of four NCL mutants. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Each point represents the mean ± SE (n = 3–5). *P < 0.05, †P < 0.01, ‡P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test). (G) Evaluation of the antagonistic activities of the four NCL mutants at pH 8.0. Each mutant was applied to cells expressing hT1R2-hT1R3 together with the NBS H11A mutant at pH 8.0. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 8.0. Each point represents the mean ± SE (n = 4-5). *P < 0.05, †P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test).

Mentions: We evaluated the sweetness of each single-point NCL mutant at weakly acidic and neutral pH using the cell-based assay system established previously21. The human sweet receptor hT1R2-hT1R3 was transiently transfected into HEK293T cells together with G15Gi3 as a chimeric Gα. NCL or its mutant was applied to the cells under neutral (pH 7.4) or weakly acidic (pH 6.3) conditions (Fig. 3A,B). Consistent with the previous results22, replacement of His36 of NAS and His11 and His14 of NBS increased the receptor response at pH 7.4 (Fig. 3C) (the EC50 values for the mutants were: H36A, 1.20 μM; H11A, 2.12 μM; and H14A, 1.20 μM). The activity of the NBS H11A mutant was entirely independent of pH, whereas the activities of the NAS H36A and NBS H14A mutants were slightly lower at neutral pH compared with weakly acidic pH (Fig. 3D). Replacement of Tyr21of NAS also increased the response at neutral pH (EC50 value was 1.03 μM), although the response was slightly lower compared with weakly acidic pH. This result suggests that NAS Tyr21 also contributes to the pH-dependent functional change of NCL. Replacement of Arg48 and Val72 of NBS increased the response compared to the WT at pH 6.3, whereas replacement of Tyr65 and Phe94 of NBS decreased the response (Fig. 3E) (the EC50 values for the WT and mutants were: WT, 1.24 μM; R48A, 0.31 μM; V72A, 0.84 μM; Y65A, 6.90 μM; and F94A, 3.79 μM). Importantly, these changes had only a slight effect on the pH dependency of the response (Fig. 3F), in contrast to the mutations of NAS Tyr21 and His36 and NBS His11 and His14 (Fig. 3D). Next, we evaluated the antagonistic activity of the NBS R48A, Y65A, V72A, and F94A mutants under weakly basic conditions. Each mutant protein was applied to cells expressing hT1R2-hT1R3 together with the NBS H11A mutant at pH 8.0. Interestingly, replacement of Arg48 and Val72 of NBS strongly decreased the cell response induced by NBS H11A compared to the WT, whereas replacement of Tyr65 and Phe94 of NBS weakly decreased the response (Fig. 3G). These results suggest that these four residues in NBS contribute to both the agonist and antagonist potencies without affecting pH-dependency.


Identification of key neoculin residues responsible for the binding and activation of the sweet taste receptor.

Koizumi T, Terada T, Nakajima K, Kojima M, Koshiba S, Matsumura Y, Kaneda K, Asakura T, Shimizu-Ibuka A, Abe K, Misaka T - Sci Rep (2015)

Production of NCL mutants and evaluation of their sweetness using the cell-based assay.(A,B) Responses of cells expressing hT1R2-hT1R3 and G15Gi3 to fourteen NCL mutants under neutral (pH 7.4) and weakly acidic (pH 6.3) conditions. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Error bars represent the mean ± SE (n = 4–6). *P < 0.05, †P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test). (C–F) Dose-response relationships and pH dependencies of each set of four NCL mutants. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Each point represents the mean ± SE (n = 3–5). *P < 0.05, †P < 0.01, ‡P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test). (G) Evaluation of the antagonistic activities of the four NCL mutants at pH 8.0. Each mutant was applied to cells expressing hT1R2-hT1R3 together with the NBS H11A mutant at pH 8.0. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 8.0. Each point represents the mean ± SE (n = 4-5). *P < 0.05, †P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test).
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f3: Production of NCL mutants and evaluation of their sweetness using the cell-based assay.(A,B) Responses of cells expressing hT1R2-hT1R3 and G15Gi3 to fourteen NCL mutants under neutral (pH 7.4) and weakly acidic (pH 6.3) conditions. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Error bars represent the mean ± SE (n = 4–6). *P < 0.05, †P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test). (C–F) Dose-response relationships and pH dependencies of each set of four NCL mutants. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 7.4. Each point represents the mean ± SE (n = 3–5). *P < 0.05, †P < 0.01, ‡P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test). (G) Evaluation of the antagonistic activities of the four NCL mutants at pH 8.0. Each mutant was applied to cells expressing hT1R2-hT1R3 together with the NBS H11A mutant at pH 8.0. The number of responsive cells was normalised relative to the maximum response to aspartame (6.7 mM) at pH 8.0. Each point represents the mean ± SE (n = 4-5). *P < 0.05, †P < 0.001 vs. WT (one-way ANOVA followed by Dunnett’s test).
Mentions: We evaluated the sweetness of each single-point NCL mutant at weakly acidic and neutral pH using the cell-based assay system established previously21. The human sweet receptor hT1R2-hT1R3 was transiently transfected into HEK293T cells together with G15Gi3 as a chimeric Gα. NCL or its mutant was applied to the cells under neutral (pH 7.4) or weakly acidic (pH 6.3) conditions (Fig. 3A,B). Consistent with the previous results22, replacement of His36 of NAS and His11 and His14 of NBS increased the receptor response at pH 7.4 (Fig. 3C) (the EC50 values for the mutants were: H36A, 1.20 μM; H11A, 2.12 μM; and H14A, 1.20 μM). The activity of the NBS H11A mutant was entirely independent of pH, whereas the activities of the NAS H36A and NBS H14A mutants were slightly lower at neutral pH compared with weakly acidic pH (Fig. 3D). Replacement of Tyr21of NAS also increased the response at neutral pH (EC50 value was 1.03 μM), although the response was slightly lower compared with weakly acidic pH. This result suggests that NAS Tyr21 also contributes to the pH-dependent functional change of NCL. Replacement of Arg48 and Val72 of NBS increased the response compared to the WT at pH 6.3, whereas replacement of Tyr65 and Phe94 of NBS decreased the response (Fig. 3E) (the EC50 values for the WT and mutants were: WT, 1.24 μM; R48A, 0.31 μM; V72A, 0.84 μM; Y65A, 6.90 μM; and F94A, 3.79 μM). Importantly, these changes had only a slight effect on the pH dependency of the response (Fig. 3F), in contrast to the mutations of NAS Tyr21 and His36 and NBS His11 and His14 (Fig. 3D). Next, we evaluated the antagonistic activity of the NBS R48A, Y65A, V72A, and F94A mutants under weakly basic conditions. Each mutant protein was applied to cells expressing hT1R2-hT1R3 together with the NBS H11A mutant at pH 8.0. Interestingly, replacement of Arg48 and Val72 of NBS strongly decreased the cell response induced by NBS H11A compared to the WT, whereas replacement of Tyr65 and Phe94 of NBS weakly decreased the response (Fig. 3G). These results suggest that these four residues in NBS contribute to both the agonist and antagonist potencies without affecting pH-dependency.

Bottom Line: We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities.The mutations had only a slight effect on the pH-dependent functional change.From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Neoculin (NCL) is a heterodimeric protein isolated from the edible fruit of Curculigo latifolia. It exerts a taste-modifying activity by converting sourness to sweetness. We previously demonstrated that NCL changes its action on the human sweet receptor hT1R2-hT1R3 from antagonism to agonism as the pH changes from neutral to acidic values, and that the histidine residues of NCL molecule play critical roles in this pH-dependent functional change. Here, we comprehensively screened key amino acid residues of NCL using nuclear magnetic resonance (NMR) spectroscopy and alanine scanning mutagenesis. We found that the mutations of Arg48, Tyr65, Val72 and Phe94 of NCL basic subunit increased or decreased both the antagonist and agonist activities. The mutations had only a slight effect on the pH-dependent functional change. These residues should determine the affinity of NCL for the receptor regardless of pH. Their locations were separated from the histidine residues responsible for the pH-dependent functional change in the tertiary structure. From these results, we concluded that NCL interacts with hT1R2-hT1R3 through a pH-independent affinity interface including the four residues and a pH-dependent activation interface including the histidine residues. Thus, the receptor activation is induced by local structural changes in the pH-dependent interface.

No MeSH data available.