Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor
Abstract
:1. Introduction
2. Results
2.1. Expression and Purification of Human TAS1R2-VFT
2.2. Characterization of hTAS1R2-VFT
2.3. Ligand Binding Properties of hTAS1R2-VFT
3. Discussion
4. Materials and Methods
4.1. Chemicals
4.2. Design of TAS1R2 Expression Constructs
4.3. Protein Expression and Inclusion Bodies Production
4.4. In Vitro Refolding of TAS1R2-VFTs
4.5. Circular Dichroism
4.6. SEC-MALS Analysis
4.7. Intrinsic Tryptophan Fluorescence Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Li, X.; Staszewski, L.; Xu, H.; Durick, K.; Zoller, M.; Adler, E. Human receptors for sweet and umami taste. Proc. Natl. Acad. Sci. USA 2002, 99, 4692–4696. [Google Scholar] [CrossRef] [PubMed]
- Nelson, G.; Chandrashekar, J.; Hoon, M.A.; Feng, L.; Zhao, G.; Ryba, N.J.; Zuker, C.S. An amino-acid taste receptor. Nature 2002, 416, 199–202. [Google Scholar] [CrossRef] [PubMed]
- Nelson, G.; Hoon, M.A.; Chandrashekar, J.; Zhang, Y.; Ryba, N.J.; Zuker, C.S. Mammalian sweet taste receptors. Cell 2001, 106, 381–390. [Google Scholar] [CrossRef]
- Belloir, C.; Neiers, F.; Briand, L. Sweeteners and sweetness enhancers. Curr. Opin. Clin. Nutr. Metab. Care 2017, 20, 279–285. [Google Scholar] [CrossRef]
- DuBois, G.E. Molecular mechanism of sweetness sensation. Physiol. Behav. 2016, 164, 453–463. [Google Scholar] [CrossRef]
- Servant, G.; Kenakin, T.; Zhang, L.; Williams, M.; Servant, N. The function and allosteric control of the human sweet taste receptor. Adv. Pharmacol. 2020, 88, 59–82. [Google Scholar] [CrossRef]
- Behrens, M. Pharmacology of TAS1R2/TAS1R3 Receptors and Sweet Taste. Handb. Exp. Pharmacol. 2022, 275, 155–175. [Google Scholar] [CrossRef]
- Behrens, M.; Meyerhof, W. Vertebrate Bitter Taste Receptors: Keys for Survival in Changing Environments. J. Agric. Food Chem. 2018, 66, 2204–2213. [Google Scholar] [CrossRef]
- Neiers, F.; Belloir, C.; Poirier, N.; Naumer, C.; Krohn, M.; Briand, L. Comparison of Different Signal Peptides for the Efficient Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris. Life 2021, 11, 46. [Google Scholar] [CrossRef]
- Fujiwara, S.; Imada, T.; Nakagita, T.; Okada, S.; Nammoku, T.; Abe, K.; Misaka, T. Sweeteners interacting with the transmembrane domain of the human sweet-taste receptor induce sweet-taste synergisms in binary mixtures. Food Chem. 2012, 130, 561–568. [Google Scholar] [CrossRef]
- Jang, J.; Kim, S.K.; Guthrie, B.; Goddard, W.A., 3rd. Synergic Effects in the Activation of the Sweet Receptor GPCR Heterodimer for Various Sweeteners Predicted Using Molecular Metadynamics Simulations. J. Agric. Food Chem. 2021, 69, 12250–12261. [Google Scholar] [CrossRef] [PubMed]
- Servant, G.; Tachdjian, C.; Li, X.; Karanewsky, D.S. The sweet taste of true synergy: Positive allosteric modulation of the human sweet taste receptor. Trends Pharmacol. Sci. 2011, 32, 631–636. [Google Scholar] [CrossRef] [PubMed]
- Assadi-Porter, F.M.; Maillet, E.L.; Radek, J.T.; Quijada, J.; Markley, J.L.; Max, M. Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor. J. Mol. Biol. 2010, 398, 584–599. [Google Scholar] [CrossRef] [PubMed]
- Maillet, E.L.; Cui, M.; Jiang, P.; Mezei, M.; Hecht, E.; Quijada, J.; Margolskee, R.F.; Osman, R.; Max, M. Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor. Chem. Senses 2015, 40, 577–586. [Google Scholar] [CrossRef] [PubMed]
- Masuda, K.; Koizumi, A.; Nakajima, K.; Tanaka, T.; Abe, K.; Misaka, T.; Ishiguro, M. Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds. PLoS ONE 2012, 7, e35380. [Google Scholar] [CrossRef]
- Nie, Y.; Vigues, S.; Hobbs, J.R.; Conn, G.L.; Munger, S.D. Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli. Curr. Biol. CB 2005, 15, 1948–1952. [Google Scholar] [CrossRef]
- Xu, H.; Staszewski, L.; Tang, H.; Adler, E.; Zoller, M.; Li, X. Different functional roles of T1R subunits in the heteromeric taste receptors. Proc. Natl. Acad. Sci. USA 2004, 101, 14258–14263. [Google Scholar] [CrossRef]
- Zhang, F.; Klebansky, B.; Fine, R.M.; Liu, H.; Xu, H.; Servant, G.; Zoller, M.; Tachdjian, C.; Li, X. Molecular mechanism of the sweet taste enhancers. Proc. Natl. Acad. Sci. USA 2010, 107, 4752–4757. [Google Scholar] [CrossRef]
- Maitrepierre, E.; Sigoillot, M.; Le Pessot, L.; Briand, L. Recombinant expression, in vitro refolding, and biophysical characterization of the N-terminal domain of T1R3 taste receptor. Protein Expr. Purif. 2012, 83, 75–83. [Google Scholar] [CrossRef]
- Jiang, P.; Cui, M.; Zhao, B.; Snyder, L.A.; Benard, L.M.; Osman, R.; Max, M.; Margolskee, R.F. Identification of the cyclamate interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3. J. Biol. Chem. 2005, 280, 34296–34305. [Google Scholar] [CrossRef]
- Winnig, M.; Bufe, B.; Kratochwil, N.A.; Slack, J.P.; Meyerhof, W. The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor. BMC Struct. Biol. 2007, 7, 66. [Google Scholar] [CrossRef] [PubMed]
- Cai, C.; Jiang, H.; Li, L.; Liu, T.; Song, X.; Liu, B. Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine. PLoS ONE 2016, 11, e0160079. [Google Scholar] [CrossRef] [PubMed]
- Servant, G.; Tachdjian, C.; Tang, X.Q.; Werner, S.; Zhang, F.; Li, X.; Kamdar, P.; Petrovic, G.; Ditschun, T.; Java, A.; et al. Positive allosteric modulators of the human sweet taste receptor enhance sweet taste. Proc. Natl. Acad. Sci. USA 2010, 107, 4746–4751. [Google Scholar] [CrossRef] [PubMed]
- Zhao, M.; Xu, X.-Q.; Meng, X.-Y.; Liu, B. The Heptahelical Domain of the Sweet Taste Receptor T1R2 Is a New Allosteric Binding Site for the Sweet Taste Modulator Amiloride That Modulates Sweet Taste in a Species-Dependent Manner. J. Mol. Neurosci. 2018, 66, 207–213. [Google Scholar] [CrossRef]
- Assadi-Porter, F.M.; Radek, J.; Rao, H.; Tonelli, M. Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties. Molecules 2018, 23, 2531. [Google Scholar] [CrossRef] [PubMed]
- Belloir, C.; Brulé, M.; Tornier, L.; Neiers, F.; Briand, L. Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line. Sci. Rep. 2021, 11, 22238. [Google Scholar] [CrossRef]
- Belloir, C.; Savistchenko, J.; Neiers, F.; Taylor, A.J.; McGrane, S.; Briand, L. Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli. PLoS ONE 2017, 12, e0187051. [Google Scholar] [CrossRef]
- Maitrepierre, E.; Sigoillot, M.; Le Pessot, L.; Briand, L. An efficient Escherichia coli expression system for the production of a functional N-terminal domain of the T1R3 taste receptor. Bioengineered 2013, 4, 25–29. [Google Scholar] [CrossRef]
- Behrens, M.; Briand, L.; de March, C.A.; Matsunami, H.; Yamashita, A.; Meyerhof, W.; Weyand, S. Structure-Function Relationships of Olfactory and Taste Receptors. Chem. Senses 2018, 43, 81–87. [Google Scholar] [CrossRef]
- Harrison, S.K.; Bernhard, R.A. Time-Intensity Sensory Characteristics of Saccharin, Xylitol and Galactose and Their Effect on the Sweetness of Lactose. J. Food Sci. 1984, 49, 780–786. [Google Scholar] [CrossRef]
- Schiffman, S.S.; Gatlin, C.A. Sweeteners: State of knowledge review. Neurosci. Biobehav. Rev. 1993, 17, 313–345. [Google Scholar] [CrossRef]
- Galindo-Cuspinera, V.; Winnig, M.; Bufe, B.; Meyerhof, W.; Breslin, P.A. A TAS1R receptor-based explanation of sweet ‘water-taste’. Nature 2006, 441, 354–357. [Google Scholar] [CrossRef] [PubMed]
Sweetener | hTAS1R2-VFT (Kd) | EC50 | Sweetness Potency * |
---|---|---|---|
Lactose | >70 mM | n.d. | 0.3 f |
Glucose | 1.5 ± 0.3 mM | n.d. | 0.6 f |
Fructose | 0.9 ± 0.2 mM | 8 ± 4 mM b | 0.7 e |
Sucrose | 4.2 ± 0.9 mM | 11 ± 3 mM b | 1 e |
Cyclamate | n.b. | 767 ± 83 μM c | 26 e |
Acesulfame-K | 0.9 ± 0.2 μM | 213 ± 59 μM c | 200 e |
Aspartame | 17.2 ± 3.1 μM | 75 ± 11 μM d | 250 e |
Saccharin | 0.06 ± 0.02 μM | 190 ± 7 μM d | 500 e |
Sucralose | 7.0 ± 1.3 μM | 36 ± 2 μM b | 600 e |
Perillartine | n.b. | 2.54 ± 0.48 µM c | 1000 g |
Alitame | 0.15 ± 0.03 μM | 13.8 ± 0.3 μM a | 4500 e |
Neotame | 0.09 ± 0.02 μM | 0.90 ± 0.09 μM c | 11,000 g |
Sweetener | hTAS1R2-VFT | hTAS1R2-VFT-D278A | hTAS1R2-VFT-E382A |
---|---|---|---|
Sucralose | 7.0 ± 1.3 μM | n.b. * | 3.5 ± 0.8 μM |
Acesulfame-K | 0.9 ± 0.2 μM | n.d. * | n.b. |
Aspartame | 17.2 ± 3.1 μM | 222.8 ± 27.7 μM | n.d. |
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Laffitte, A.; Belloir, C.; Neiers, F.; Briand, L. Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor. Int. J. Mol. Sci. 2022, 23, 9216. https://doi.org/10.3390/ijms23169216
Laffitte A, Belloir C, Neiers F, Briand L. Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor. International Journal of Molecular Sciences. 2022; 23(16):9216. https://doi.org/10.3390/ijms23169216
Chicago/Turabian StyleLaffitte, Anni, Christine Belloir, Fabrice Neiers, and Loïc Briand. 2022. "Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor" International Journal of Molecular Sciences 23, no. 16: 9216. https://doi.org/10.3390/ijms23169216