Taste-modifying sweet protein, neoculin, is received at human T1R3 amino terminal domain

doi:10.1016/j.bbrc.2007.04.171

Biochemical and Biophysical Research Communications

Volume 358, Issue 2, 29 June 2007, Pages 585-589

https://doi.org/10.1016/j.bbrc.2007.04.171 Get rights and content

Abstract

This study examines taste reception of neoculin, a Curculigo latifolia sweet protein with taste-modifying activity which converts sourness to sweetness. Neoculin tastes sweet to humans, but not to mice, and is received by the human sweet taste receptor hT1R2–hT1R3. In the present study with calcium imaging analysis of HEK cells expressing human and mouse T1Rs, we demonstrated that hT1R3 is required for the reception of neoculin. Further experiments using human/mouse chimeric T1R3s revealed that the extracellular amino terminal domain (ATD) of hT1R3 is essential for the reception of neoculin. Although T1R2–T1R3 is known to have multiple potential ligand-binding sites to receive a wide variety of sweeteners, the present study is apparently the first to identify the ATD of hT1R3 as a new sweetener-binding region.

Section snippets

Materials and methods

Sample preparation. Purification of native neoculin from the fruits of C. latifolia was carried out as previously described [4]. Briefly, raw samples of the fruits were lyophilized and soaked in 0.05 N H₂SO₄ to extract a sensory-active fraction. The extract was loaded onto an Amberlite IRC-50 column (Organo, Tokyo, Japan), and the eluate was brought to 60% saturation with (NH₄)₂SO₄. The resulting precipitate was desalted through a Sephadex G-25 column (GE Healthcare, Piscataway, New Jersey,

hT1R3 is necessary for neoculin reception

Using a sweetness-assay system with HEK293T cells expressing hT1R2–hT1R3, we have demonstrated that the original sweetness and taste-modifying activity of neoculin are recognized by the receptor [9], [10]. This result is consistent with assays showing that the sweetness of neoculin is recognized by human sensory panelists [3]. In contrast, our preliminary phenomenological observations confirmed that neoculin is not recognized by mice (data not shown). Thus, we began to look at the differences

Discussion

The positive response of hT1R2–hT1R3 and the negative response of hT1R2–mT1R3 to neoculin indicate that hT1R3 is necessary for the reception of neoculin (Fig. 1). Additionally, assays using several different T1R3 chimeras, revealed that the ATD of hT1R3 is indispensable for the reception of neoculin (Fig. 3). Although the ATD of hT1R3 shares approximately 70% amino acid sequence similarity with that of mT1R3 (Fig. 2), our experimental evidence indicates that the 30% dissimilarities exert a

Acknowledgments

This study was partially supported by Grant-in-Aid for Scientific Research (S) (to K.A.), Grant-in-Aid for Scientific Research (C) (to T.A.), Young Scientists (A) (to T.M.) and Young Scientists (B) (to A.S.-I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Society for the Promotion of Science (JSPS) (to K.N.).

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Taste-modifying sweet protein, neoculin, is received at human T1R3 amino terminal domain

Abstract

Section snippets

Materials and methods

hT1R3 is necessary for neoculin reception

Discussion

Acknowledgments

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J. Biol. Chem.

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J. Biol. Chem.

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Gene