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

Abstract

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.

Human sweet taste receptor mediates acid-induced sweetness of miraculin

“…cDNAs of T1R2, T1R3, and G15Gi3 were transfected into HEK293T cells together with DsRed2 and transfected cells were used for calcium imaging, as previously described (33). For MCL assay, MCL was added to the cells when fura-2 AM was loaded.…”

Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein

“…MD calculations were performed at neutral (pH 7) and acidic (pH 3) conditions for the dimeric structure of neoculin ( Fig 1 ), considering that neoculin elicits its sweetness in an aqueous solution of citric acid (pKa 2.87) [ 10 ]. The temperature and pressure were maintained to be constant.…”

“…The His11Tyr mutant lost most of its sweetness under the neutral condition, while the His11Phe mutant maintained weak sweetness at a level similar to that in the WT [ 10 ]. The two mutants showed sweetness as strong as WT under the acidic condition.…”

“…The mutational experiments reported by Nakajima et al [ 10 ] indicated that His11 plays an important role in the pH dependence of the sweetness-modifying activity of neoculin. Here, we have performed MD calculations on the crystal structure of the WT form and models of several neoculin mutants.…”

“…However, the reduced monomeric form of miraculin also shows no sweetness at any pH. Among the results from several mutational experiments on neoculin, an His11Ala mutant showed sweetness under both neutral and acidic conditions [ 10 ], while a Gln90Lys mutant showed sweetness at neutral conditions but reduced sweetness at low pH [ 11 ]. Thus, these two residues are thought to be important for pH-dependent properties but not for the receptor activation.…”