Journal of Agricultural and Food Chemistry Figure 4. Comparison of coelenterazine and its derivatives in the mitochondrial clytin-II assay using hT1R2/hT1R3-expressing cells. The cells were loaded with native coelenterazine or each of eight analogues and stimulated with 0.25, 1, and 10 mM aspartame. Each bar indicates the mean ( SEM (n = 5 6). ﬂuo-4 assay (Figure 1C and Table 1). In this study, we adopted mitochondrial clytin-II for further validation. Comparison between Coelenterazine and Its Derivatives as Luminescent Cofactors. Because of the differences in both Ca2+ sensitivities and luminescence properties, several types of coelenterazine derivatives have been synthesized29,30 and used in photoprotein luminescence-based GPCR assays.20,23,31 To select a suitable cofactor for our functional taste receptor assay, the luminescence intensities obtained from native coelenterazine and eight analogues were examined using mitochondrial clytin- II as the photoprotein. The luminescence intensities in response to the application of aspartame varied among the coelenterazine derivatives used (Figure 4). The strongest signals were observed using native or coelenterazine f, and the signals obtained exhibited a dose- dependent response to aspartame. The cp, h, and ip derivatives also manifested dose-dependent responses to aspartame but yielded signals that were about half as intense as those from native and coelenterazine f. For this reason, native coelenterazine was selected for further experimentation. Application of the Luminescence Assay in the Presence of an Autofluorescent Food-Derived Substance. The response of human sweet taste receptor-expressing cells was also measured by the mitochondrial clytin-II luminescence-based assay in the presence of fluorescent substances. First, we confirmed that this luminescence-based assay functioned as effectively as the aequor- in assay in the presence of the model fluorescent substance BCECF (Figure 5A). Next, the cellular response was measured in the presence of a food-derived fluorescent substance. For this, we selected riboflavin (vitamin B2) as a food-derived fluorescent substance, which is excited and detected at the wavelength used for fluo-4 measurements (Ex. 485 nm/Em. 525 nm). When ﬂuo-4-loaded cells were treated with 0.25 1.0 μg/mL of riboﬂavin and the ﬂuorescence intensity was monitored, robust and dose-dependent rectangular ﬂuorescence was de- tected immediately after the addition of the riboﬂavin solution (Figure 5B), similar to when BCECF was added (Figure 2A). The ﬂuorescence derived from riboﬂavin was suﬃciently strong to prevent detection of the sweet taste receptor response to aspartame (Figure 5C,D). In contrast, when the mitochondrial clytin-II luminescence- based assay was used, the addition of riboﬂavin did not produce any nonspeciﬁc luminescence signals, and the time response ARTICLE curves were nearly the same as the curves obtained using the assay buﬀer alone (Figure 5E). Furthermore, the luminescence intensities in response to aspartame did not change in the presence of any of the riboﬂavin concentrations added (Figure 5F,G). ’ DISCUSSION In this study, we have established a luminescence-based assay system applicable to human sweet taste receptor-expressing cells. This assay system can be used to measure the sweet taste intensities of not only aspartame but also natural sweeteners, for example, sucrose (Figure S1 in the Supporting Information), which is about 100 200 times less sweet than aspartame.32 Moreover, our results strongly indicate that photoprotein lumi- nescence-based assays are suitable for the taste evaluation systems because these assays can overcome the limitation of ligands that contain ﬂuorescent compounds (Figures 2 and 5). Many researchers have developed cell-based assays for taste receptors that use ﬂuorescent dyes as intracellular Ca2+ indicators.26,33,34 However, these assays are limited because the cellular responses remain subject to interference from the surroundings (e.g., temperature, pH, and osmotic pressure). Moreover, detection of the ﬂuorescent signals was often dis- turbed by the ligand because of the unsuitable characteristics of ligand (e.g., cytotoxicity, absorbance, and autoﬂuorescence). Autoﬂuorescence has been a serious limitation in previous assay systems, especially when samples derived from food materials were examined, because various ﬂuorescent substances, for example, chlorophyll, vitamins, and Maillard reaction products, are con- tained in foods that can disturb the detection of the ﬂuorescent signals produced by the ﬂuorescent dyes. To address ligand autoﬂuorescence, an electrophysiological assay, such as patch- clamp recording or the two-electrode voltage-clamp method using Xenopus oocytes, could be used. However, taste receptor- mediated responses are not necessarily converted to changes in membrane current. Therefore, the development of a high- throughput assay for taste receptors that is not aﬀected by ligand autoﬂuorescence is required. In this study, we have succeeded in establishing a sweet taste evaluation system for ﬂuorescent ligands that uses Ca2+-sensitive photoproteins as intracellular Ca2+ indicators. Ca2+-sensitive photoproteins, such as aequorin, obelin, and clytin, are derived from jellyﬁsh and have been used as intracel- lular Ca2+ indicators in GPCR19 22 and ion channel23 assays. Because it is well-known that luminescent properties vary under the same experimental conditions depending on the type of photoprotein used, it is important to select the best agent for each assay system. Upon comparing the luminescence intensities of four types of photoprotein, the most robust signal was generated by clytin-II (Figure 3), which was isolated from the jellyﬁsh Clytia gregarium as an isotype of clytin.35 It was reported that although the luminescence capacity of clytin-II was almost the same as those of aequorin and clytin, the initial luminescence intensity of clytin-II triggered by Ca2+ was about ﬁve times higher, and thus, the signal-to-noise ratio of clytin-II was higher than those of aequorin and clytin.35 Furthermore, localizing clytin-II and aequorin to the mitochondria resulted in improved signal/back- ground ratios, which were nearly equivalent to the ratio obtained using the ﬂuo-4 ﬂuorescence-based assay (Figure 1C and Table 1). The location of the photoproteins did not result in changes in the EC50 values (Table 1); the mitochondrial photoproteins 12135 dx.doi.org/10.1021/jf2029835 |J. Agric. Food Chem. 2011, 59, 12131–12138