ARTICLE pubs.acs.org/JAFC Establishment of a New Cell-Based Assay To Measure the Activity of Sweeteners in Fluorescent Food Extracts Yasuka Toda, Shinji Okada, and Takumi Misaka* Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan bS Supporting Information ABSTRACT: Taste receptors have been deﬁned at the molecular level in the past decade, and cell-based assays have been developed using cultured cells heterologously expressing these receptors. The most popular approach to detecting the cellular response to a tastant is to measure changes in intracellular Ca2+ concentration using Ca2+-sensitive ﬂuorescent dyes. However, this method cannot be applied to food-derived samples that contain ﬂuorescent substances. To establish an assay system that would be applicable to ﬂuorescent samples, we tested the use of Ca2+-sensitive photoproteins, such as aequorin and mitochondrial clytin-II, as Ca2+ indicators in a human sweet taste receptor assay. Using these systems, we successfully detected receptor activation in response to sweetener, even when ﬂuorescent compounds coexisted. This luminescence-based assay will be a powerful tool to objectively evaluate the sweetness of food-derived samples even at an industry level. KEYWORDS: Cell-based assay, luminescence, ﬂuorescence, aequorin, clytin-II, photoprotein, taste, sweet taste receptor ’ INTRODUCTION Over the past decade, mammalian taste receptors that play a major role in tastant recognition have been found.1 The percep- tion of sweet, umami, and bitter tastants is mediated by G protein- coupled receptors (GPCRs): that is, T1R2/T1R3 for sweet,2 T1R1/T1R3 for umami,3 and T2Rs for bitter tastants.4,5 In vitro assay systems using cultured mammalian cells heterologously expressing each taste receptor together with a chimeric G protein, for example, G16gust44 and G15i3, have been investi- gated not only to characterize the existing ligands for novel taste receptors but also to screen new taste modulators.6,7 After loading taste receptor-expressing cells with Ca2+-sensitive ﬂuor- escent dyes, for example, ﬂuo-3, ﬂuo-4, and fura-2, cellular responses can be easily detected by measuring the changes in the ﬂuorescent intensity produced by those indicators. Although ﬂuorescent indicators are known to be suitable for evaluating various types of natural and artiﬁcial compounds, problems remain. In particular, in the food research ﬁeld, samples contain- ing food-derived compounds or fractions are diﬃcult to evaluate because food often contains a variety of ﬂuorescent substances. Some are endogenous in food materials, such as polyphenols in plants,8 and others are generated during heat processing or fermentation.9 In considering the industrial importance of taste- modulating compounds of food origin, establishing a high- throughput screening system that can be applied to evaluate ﬂuorescent food-derived materials will no doubt be necessary. There are techniques available for measuring the intracellular Ca2+ concentrations of cultured cells without using ﬂuorescent Ca2+ indicators. One method is to utilize jellyﬁsh-derived Ca2+- sensitive photoproteins, such as aequorin,10,11 obelin,12 14 and clytin,15,16 as calcium indicators. The active photoprotein is composed of apophotoprotein and luciferin coelenterazine in the presence of molecular oxygen.17,18 Binding of Ca2+ to the holoprotein induces a conformational change in this protein and triggers the oxidation of coelenterazine to coelenteramide. Because these events induce the subsequent emission of blue light, measuring emission of this light reveals the response mediated by changes in Ca2+ concentration without the use of a ﬂuorescent Ca2+ indicator. Many groups have reported cell-based assay systems that use photoproteins as detectors for several types of GPCRs19 22 and ion channels.23 Because of the essentially undetectable back- ground luminescence of cells, luminescence-based assays provide a high signal-to-noise ratio. Moreover, the localization of photopro- teins to mitochondria results in stronger signals in GPCR assays because the rise in Ca2+ concentration upon IP3-induced Ca2+ mobilization from intracellular stores is more pronounced in the mitochondria than in the cytoplasm.24,25 Although many photoprotein-based functional assays for various types of receptors have been reported, this system has not yet been adapted to analyze taste receptors. To apply a luminescence-based assay for taste receptor evaluation, in this study, four types of photoproteins were transiently transfected into cells that stably coexpressed the human sweet taste receptor, hT1R2/hT1R3, together with a functional chimeric G-protein, hGα16gust44,26 and the cellular responses to sweetener were measured using a luminescence microplate reader. By comparing the luminescence intensities and signal/background ratios ob- tained using each photoprotein with or without mitochondrial localization, we selected the most suitable apophotoprotein and coelenterazine analogue for the taste receptor assay. This lumi- nescence-based assay platform enables us to perform an other- wise impossible assay of evaluating the sweet taste intensity of samples per se containing ﬂuorescent substances. Received: July 25, 2011 Accepted: October 8, 2011 Revised: October 7, 2011 Published: October 08, 2011 r 2011 American Chemical Society 12131 dx.doi.org/10.1021/jf2029835 | J. Agric. Food Chem. 2011, 59, 12131–12138