Abstracts - Association for Chemoreception Sciences

P O S T E R S
small sweeteners showed differential preferences for T1R2 or
T1R3 subunits. Acknowledgements: R01 DC009018, R21
DC008805-02, R01 DC008301-01, R01 DC009451-01A2,
and P41 RR02301
#P201 POSTER SESSION IV: CHEMOSENSORY
TRANSDUCTION AND SIGNALING
Suppressing effect of cyclodextrin to taste modifiers
Keisuke Sanematsu 1,2 , Seiji Nakamura 2 , Yuzo Ninomiya 1
1
Section of Oral Neuroscience, Graduate School of Dental Sciences,
Kyushu University Fukuoka, Japan, 2 Section of Oral and
Maxillofacial Oncology, Graduate School of Dental Sciences,
Kyushu University Fukuoka, Japan
Gymnemic acid (GA) and Gurmarin (Gur) are a triterpen
glycoside and a polypeptide that are isolated from the plant
Gymnema sylvestre, respectively. These chemical compounds are
known to selectively suppress taste responses to various sweet
substances. Miraculin (Mir) is a glycoprotein extracted from the
miracle fruit plant. After application of Mir, it modifies taste so
that sour substances taste sweet. Sweet suppressing effect of GA
and sweet modifying effect of Mir are specific to humans, but not
to rodents, whereas Gur inhibits the responses to sweet
compounds in rodents, but not in humans. It has also been known
that sweet suppressing effects of GA and Gur diminished by
g-cyclodextrin (CD) and b-CD, respectively. The molecular
mechanisms between GA, Gur and Mir and each CDs are not
known. In order to examine these interactions, we used the sweet
receptor T1R2+T1R3 assay in transiently transfected HEK293
cells. Similar to previous studies in humans and mice, GA (0.1
mg/ml) and Gur (30 mg/ml) inhibited the [Ca 2+ ]i responses of
HEK293 cells expressing human and mouse sweet receptors to
various sweeteners, respectively. After application of Mir (10
mg/ml), HEK293 cells expressing human sweet receptor showed
the [Ca 2+ ]i responses to 3 mM Citric acid (pH: 5.0). The effects of
GA and Gur rapidly disappeared after rinsing the cells with g-CD
and b-CD, respectively. Interestingly, the taste modifying effect of
Mir is diminished by b-CD. Our present study confirmed the
previous finding and demonstrated that GA and Gur directly
interact with human and mouse sweet receptors on the taste cell
membrane and these interactions are inhibited by g-CD and b-
CD, respectively. We also showed that Mir directly interacts with
human sweet receptor and this interaction is diminished by b-CD.
#P202 POSTER SESSION IV: CHEMOSENSORY
TRANSDUCTION AND SIGNALING
Characterizing the interaction of miraculin, a taste-modifying
protein, with human sweet taste receptor
Ayako Koizumi, Asami Tsuchiya, Ken-ichiro Nakajima,
Keisuke Ito, Tomiko Asakura, Keiko Abe, Takumi Misaka
The University of Tokyo Tokyo, Japan
Miraculin (MCL) is a taste-modifying protein occurring in
miracle fruit, the red berries of Richadella dulcifica. MCL is not
sweet by itself, but it has an unusual property of modifying
sourness into sweetness. This activity holds for 1-2 hours after
MCL is held on the tongue. Since acid-induced sweetness of MCL
is inhibited by human sweet taste receptor (hT1R2-hT1R3)
blocker, lactisole, it has been indicated that MCL interacts with
hT1R2-hT1R3. To elucidate how MCL induces such unique
sensation, we established a cell-based assay system to measure its
taste-modifying activity by calcium imaging analysis. We
transiently expressed hT1R2-hT1R3 in HEK293T cells with
chimeric Ga-protein. Evaluating the cell responses to MCL under
different pH conditions, we found that MCL-induced activation
of hT1R2-hT1R3 increased as pH lowered in the range of 7.4-
5.0. At pH 5.0, the EC50 value was ca. 1 nM, which is less than
that of any other sweetener. At neutral pH, MCL inhibited
hT1R2-hT1R3 activation induced by other sweet proteins such
as thaumatin and neoculin. This suggests that MCL does not
activate hT1R2-hT1R3 but that it binds to the receptor at neutral
pH. Furthermore, we identified MCL binding region on hT1R2-
hT1R3. Accumulating evidence from electrophysiological and
behavioral studies has suggested that MCL shows taste-modifying
effect in man, Catarrhini and Platyrrhini, but not in rodents.
Actually, the mouse receptor (mT1R2-mT1R3) did not respond to
MCL in vitro. Interestingly enough, hT1R2-mT1R3 expressing
cells responded to MCL. This suggests that hT1R2 is required for
MCL response. Further experiments using human/mouse
chimeric receptors, we demonstrated that a certain particular site
in the amino terminal domain of hT1R2 is essential for MCL to
interact with hT1R2-hT1R3. Acknowledgements: This study was
supported by JSPS Research Fellowships for Young Scientists (to
A.K.) and Research and Development Program for New Bioindustry
Initiatives.
#P203 POSTER SESSION IV: CHEMOSENSORY
TRANSDUCTION AND SIGNALING
Structural role of the terminal disulfide bond in the sweetness
of brazzein
Sannali M. Dittli 1 , Hongyu Rao 2 , Emeline Maillet 3 ,
Marianna Max 3 , John Markley 1,2 , Fariba Assadi-Porter 1,2
1
University of Wisconsin-Madison Madison, WI, USA, 2 NMR
Facility at Madison Madison, WI, USA, 3 Mt. Sinai School of
Medicine New York, NY, USA
Brazzein, a 54 residue sweet-tasting protein, is thought to
participate in a multi-point binding interaction with the sweet
taste receptor. The termini, which are connected by a disulfide
bond, and two distantly located surface loops have been proposed
to be sites of interaction; however, the relative importance of these
sites is not well understood. To characterize the structural role of
the termini in the sweetness of brazzein, we altered the
conformation of the disulfide bond connecting the N- and C-
termini of brazzein by inverting the positions of residues K3 and
C4. The activity of the resulting mutant, CKR-brazzein, was
measured by a calcium mobilization assay and found to be
decreased to half that of wild-type (WT) brazzein. The high
resolution structure of CKR-brazzein was determined by nuclear
magnetic resonance (NMR)spectroscopy with a backbone root
mean square deviation of 0.39 angstroms. The structure alignment
of CKR-brazzein with WT-brazzein reveals more extended beta
structure in the terminal strands in CKR-brazzein and increased
dynamics relative to that found in WT-brazzein. These results
support previous mutagenesis studies and further suggest that
while interactions involving the termini are necessary for full
function of brazzein, the termini are not the primary site of
interaction between brazzein and the sweet taste receptor. This
research was supported by NIH grants R01 DC009018, R21
DC008805-02, and P41 RR02301-which funds the National
Magnetic Resonance Facility at Madison.
94 | AChemS Abstracts 2010 Abstracts are printed as submitted by the author(s)