Humans are able to perceive five basic tastes which are sweet, umami, bitter, salty and sour. Bitter taste plays a crucial role as a warning sensor against the ingestion of toxic compounds. Bitter taste signaling, in humans, is mediated by a family of 25 G-protein coupled receptors, referred to as T2Rs. Humans prefer bitterness to some extent in certain foodstuffs and beverages, however excessive bitterness decreases their sensory quality. Fermented foods such as cheese, soy sauce, and miso contain numerous peptides derived from material proteins. Enzymatic hydrolysis during the aging process in fermented products frequently results in bitter taste, and bitter peptides formed during the fermentation process were shown to be responsible for the bitter taste of fermented food. The relationship between taste sensation and structures of peptides in fermented food have been widely investigated, however the molecular (sensory) targets of these bitter peptides remained elusive. Some of the peptides isolated from various fermented foods were identified to be inhibitors of the blood pressure regulatory protein, angiotensin-converting enzyme (ACE). A previous study showed that some of the bitter di-peptides isolated from casein hydrolysate can activate some T2Rs, with T2R1-expressing cells activated the most. However, in that study only two dipeptides were tested and the efficacy of the other food-protein derived bitter peptides towards T2Rs was not characterized. The major objective of the study presented here was to functionally characterize the human bitter taste receptor, T2R1, and to elucidate its activation with bitter tasting di- and tri-peptides. Using a heterologous expression system, T2R1 gene was transiently transfected in C6 glial cells and the gene expression was confirmed by reverse transcriptase-PCR analysis. The localization of T2R1 gene was predominantly in the cell membrane of glial cells and was confirmed by immunofluorescence microscopy. Functional assays on T2R1 were carried out by measuring changes in intracellular calcium after stimulating the receptor with increasing concentrations of bitter di- and tri-peptides. The results showed that some of the peptides were very potent in activating T2R1 and causing changes in intracellular calcium levels. Furthermore, molecular modeling was done to elucidate the potential binding sites of these peptides on T2R1. Another objective of the study was to increase the expression of T2R1 using a codon-optimized gene and the HEK293S-TetR inducible mammalian expression system, so that biophysical studies like NMR spectroscopy could be pursued on T2R1. This expression system resulted in 7-8 fold increase in the protein level than what was observed following transient expression of T2R1. In summary, in this work we characterized in vitro a receptor present in the human oral cavity that is activated by some of the food protein derived bitter tasting di- and tripeptides. The results showed that some of the bitter peptides activate the human bitter receptor T2R1, at concentration ranges that humans also perceive as bitter. Among the peptides tested, the tripeptide FFF showed high efficacy with an EC50 in the micromolar range. Some of the peptides with ACE-inhibitory activity were also able to activate the T2R1 receptor. Results from the molecular modeling study of T2R1, identified a small number of amino acid residues in the receptor that might be important for ligand binding, and are potential targets for future structure-function studies on T2R1.
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