Millettia dubia De Wild. (Fabaceae): Structural analysis of the oleanane-type glycosides and stimulation of the sweet taste receptors TAS1R2/TAS1R3.

From the root barks of a Central African tree Millettia dubia De Wild. (Fabaceae), ten previously undescribed oleanane-type glycosides were isolated by various chromatographic protocols. Their structures were elucidated by spectroscopic methods, mainly 2D NMR experiments and mass spectrometry, as mono- and bidesmosidic glycosides of mesembryanthemoidigenic acid, hederagenin and oleanolic acid. The stimulation of the sweet taste receptor TAS1R2/TAS1R3 by these glycosides was evaluated, and structure/activity relationships were proposed. Two of them showed an agonist effect on TAS1R2/TAS1R3.

Modulation of bitter taste receptors by yeast extracts.

Yeast extracts (YEs) are used in foods because of their flavour properties and ability to reduce bitterness. The adenosine 5'-monophosphate (AMP) found in YEs is known to decrease the bitterness of some compounds. This study aimed to investigate the ability of YEs to inhibit bitter taste receptors (TAS2Rs) using in vitro cell-based assays. A screen of TAS2Rs activated by AMP and YEs revealed that AMP and the AMP-rich YE activated more TAS2Rs. The inhibitory effect of the AMP-rich YE on seven TAS2Rs activated by bitter agonists was studied. YE reduced TAS2R activation, increased the EC50 value and decreased the maximum amplitude, demonstrating competitive and non-competitive inhibitions. Amongst the nineteen TAS2Rs tested, seven showed 40 % or greater inhibition after treatment of AMP-rich YE. Our data provide a better understanding of the TAS2R inhibition mechanism of AMP-rich YEs and promote their use as a strategy to reduce bitterness in foods and medicines.

Development of New Models of Oral Mucosa to Investigate the Impact of the Structure of Transmembrane Mucin-1 on the Mucosal Pellicle Formation and Its Physicochemical Properties.

The mucosal pellicle (MP) is a biological film protecting the oral mucosa. It is composed of bounded salivary proteins and transmembrane mucin MUC1 expressed by oral epithelial cells. Previous research indicates that MUC1 expression enhances the binding of the main salivary protein forming the MP, MUC5B. This study investigated the influence of MUC1 structure on MP formation. A TR146 cell line, which does not express MUC1 natively, was stably transfected with genes coding for three MUC1 isoforms differing in the structure of the two main extracellular domains: the VNTR domain, exhibiting a variable number of tandem repeats, and the SEA domain, maintaining the two bound subunits of MUC1. Semi-quantification of MUC1 using dot blot chemiluminescence showed comparable expression levels in all transfected cell lines. Semi-quantification of MUC5B by immunostaining after incubation with saliva revealed that MUC1 expression significantly increased MUC5B adsorption. Neither the VNTR domain nor the SEA domain was influenced MUC5B anchoring, suggesting the key role of the MUC1 N-terminal domain. AFM-IR nanospectroscopy revealed discernible shifts indicative of changes in the chemical properties at the cell surface due to the expression of the MUC1 isoform. Furthermore, the observed chemical shifts suggest the involvement of hydrophobic effects in the interaction between MUC1 and salivary proteins.

Activation of bitter taste receptors by saponins and alkaloids identified in faba beans (Vicia faba L. minor).

Despite their interests, faba beans are characterised by bitterness but little is known about its compounds that activate the 25 human bitter receptors (TAS2Rs). This study aimed to determine the bitter molecules in faba beans, especially saponins and alkaloids. These molecules were quantified by UHPLC-HRMS in flour, starch and protein fractions of 3 faba bean cultivars. The fractions from the low-alkaloid cultivar and the protein fractions exhibited higher saponin content. Vicine and convicine were highly correlated with bitter perception. The bitterness of soyasaponin ßb and alkaloids was studied using a cellular approach. Soyasaponin ßb activated 11 TAS2Rs, including TAS2R42 whereas vicine activated only TAS2R16. The high vicine content should explain the faba bean bitterness considering that concentration of soyasaponin ßb was low. This research provides a better understanding of the bitter molecules in faba beans. Selection of low-alkaloid ingredients or alkaloid removal treatments could improve the faba bean flavour.

Non-Volatile Compounds Involved in Bitterness and Astringency of Pulses: A Review.

Despite the many advantages of pulses, they are characterised by off-flavours that limit their consumption. Off-notes, bitterness and astringency contribute to negative perceptions of pulses. Several hypotheses have assumed that non-volatile compounds, including saponins, phenolic compounds, and alkaloids, are responsible for pulse bitterness and astringency. This review aims to provide an overview highlighting the non-volatile compounds identified in pulses and their bitter and/or astringent characteristics to suggest their potential involvement in pulse off-flavours. Sensorial analyses are mainly used to describe the bitterness and astringency of molecules. However, in vitro cellular assays have shown the activation of bitter taste receptors by many phenolic compounds, suggesting their potential involvement in pulse bitterness. A better knowledge of the non-volatile compounds involved in the off-flavours should enable the creation of efficient strategies to limit their impact on overall perception and increase consumer acceptability.

Activation of a Sweet Taste Receptor by Oleanane-Type Glycosides from Wisteria sinensis.

The phytochemical study of Wisteria sinensis (Sims) DC. (Fabaceae), commonly known as the Chinese Wisteria, led to the isolation of seven oleanane-type glycosides from an aqueous-ethanolic extract of the roots. Among the seven isolated saponins, two have never been reported before: 3-O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranosyl-(1→2)-ß-D-glucuronopyranosyl-22-O-acetylolean-12-ene-3ß,16ß,22ß,30-tetrol, and 3-O-ß-D-xylopyranosyl-(1→2)-ß-D-glucuronopyranosylwistariasapogenol A. Based on the close structures between the saponins from W. sinensis, and the glycyrrhizin from licorice, the stimulation of the sweet taste receptor TAS1R2/TAS1R3 by these glycosides was evaluated.

Detection of Bitterness in Vitamins Is Mediated by the Activation of Bitter Taste Receptors.

Vitamins are known to generate bitterness, which may contribute to an off-taste or aftertaste for some nutritional supplements. This negative sensation can lead to a reduction in their consumption. Little is known about the bitter taste threshold and taste sensing system for the bitter taste detection of vitamins. To better understand the mechanisms involved in bitterness perception, we combined taste receptor functional assays and sensory analysis. In humans, bitter taste detection is mediated by 25 G-protein-coupled receptors belonging to the TAS2R family. First, we studied the bitterness of thirteen vitamins using a cellular-based functional taste receptor assay. We found four vitamins that can stimulate one or more TAS2Rs. For each positive molecule-receptor combination, we tested seven increasing concentrations to determine the half-maximal effective concentration (EC50) and the cellular bitter taste threshold. Second, we measured the bitter taste detection threshold for four vitamins that exhibit a strong bitter taste using a combination of ascending series and sensory difference tests. A combination of sensory and biological data can provide useful results that explain the perception of vitamin bitterness and its real contribution to the off-taste of nutritional supplements.

Determination of the nanoscale electrical properties of olfactory receptor hOR1A1 and their dependence on ligand binding: Towards the development of capacitance-operated odorant biosensors.

The transduction of odorant binding into cellular signaling by olfactory receptors (ORs) is not understood and knowing its mechanism would enable developing new pharmacology and biohybrid electronic detectors of volatile organic compounds bearing high sensitivity and selectivity. The electrical characterization of ORs in bulk experiments is subject to microscopic models and assumptions. We have directly determined the nanoscale electrical properties of ORs immobilized in a fixed orientation, and their change upon odorant binding, using electrochemical scanning tunneling microscopy (EC-STM) in near-physiological conditions. Recordings of current versus time, distance, and electrochemical potential allows determining the OR impedance parameters and their dependence with odorant binding. Our results allow validating OR structural-electrostatic models and their functional activation processes, and anticipating a novel macroscopic biosensor based on ORs.

Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor.

The human sweet taste receptor is a heterodimeric receptor composed of two distinct G-protein-coupled receptors (GPCRs), TAS1R2 and TAS1R3. The TAS1R2 and TAS1R3 subunits are members of a small family of class C GPCRs whose members share the same architecture, comprising a Venus Flytrap (VFT) module linked to the seven transmembrane domains (TMDs) by a short cysteine-rich region (CRR). The VFT module of TAS1R2 contains the primary binding site for most of the sweet-tasting compounds, including natural sugars and artificial and natural sweeteners. However, cellular assays, molecular docking and site-directed mutagenesis studies have revealed that the VFT, CRR and TMD of TAS1R3 interact with some sweeteners, including the sweet-tasting protein brazzein. The aim of this study was to better understand the contribution of TAS1R2-VFT in the binding of sweet stimuli. To achieve this, we heterologously expressed human TAS1R2-VFT (hTAS1R2-VFT) in Escherichia coli. Circular dichroism spectroscopic studies revealed that hTAS1R2-VFT was properly folded with evidence of secondary structures. Using size-exclusion chromatography coupled with light scattering, we found that hTAS1R2-VFT behaves as a monomer. Ligand binding quantified by intrinsic tryptophan fluorescence showed that hTAS1R2-VFT is capable of binding sweet stimuli with Kd values, in agreement with physiological detection. Furthermore, we investigated whether the impact of point mutations, already shown to have deleterious effects on cellular assays, could impact the ability of hTAS1R2-VFT to bind sweet ligands. As expected, the ligand affinities of hTAS1R2-VFT were drastically reduced through the introduction of single amino acid substitutions (D278A and E382A) known to abolish the response of the full-length TAS1R2/TAS1R3 receptor. This study demonstrates the feasibility of producing milligram quantities of hTAS1R2-VFT to further characterize the mechanism of binding interaction and perform structural studies.

Extracellular loop 2 of G protein-coupled olfactory receptors is critical for odorant recognition.

G protein-coupled olfactory receptors (ORs) enable us to detect innumerous odorants. They are also ectopically expressed in nonolfactory tissues and emerging as attractive drug targets. ORs can be promiscuous or highly specific, which is part of a larger mechanism for odor discrimination. Here, we demonstrate that the OR extracellular loop 2 (ECL2) plays critical roles in OR promiscuity and specificity. Using site-directed mutagenesis and molecular modeling, we constructed 3D OR models in which ECL2 forms a lid over the orthosteric pocket. We demonstrate using molecular dynamics simulations that ECL2 controls the shape and volume of the odorant-binding pocket, maintains the pocket hydrophobicity, and acts as a gatekeeper of odorant binding. Therefore, we propose the interplay between the specific orthosteric pocket and the variable, less specific ECL2 controls OR specificity and promiscuity. Furthermore, the 3D models created here enabled virtual screening of new OR agonists and antagonists, which exhibited a 70% hit rate in cell assays. Our approach can potentially be generalized to structure-based ligand screening for other G protein-coupled receptors that lack high-resolution 3D structures.

3-oxo-C12:2-HSL, quorum sensing molecule from human intestinal microbiota, inhibits pro-inflammatory pathways in immune cells via bitter taste receptors.

In the gut ecosystem, microorganisms regulate group behaviour and interplay with the host via a molecular system called quorum sensing (QS). The QS molecule 3-oxo-C12:2-HSL, first identified in human gut microbiota, exerts anti-inflammatory effects and could play a role in inflammatory bowel diseases where dysbiosis has been described. Our aim was to identify which signalling pathways are involved in this effect. We observed that 3-oxo-C12:2-HSL decreases expression of pro-inflammatory cytokines such as Interleukine-1ß (- 35%) and Tumor Necrosis Factor-α (TNFα) (- 40%) by stimulated immune RAW264.7 cells and decreased TNF secretion by stimulated PBMC in a dose-dependent manner, between 25 to 100 µM. Transcriptomic analysis of RAW264.7 cells exposed to 3-oxo-C12:2-HSL, in a pro-inflammatory context, highlighted JAK-STAT, NF-κB and TFN signalling pathways and we confirmed that 3-oxo-C12:2-HSL inhibited JAK1 and STAT1 phosphorylation. We also showed through a screening assay that 3-oxo-C12:2-HSL interacted with several human bitter taste receptors. Its anti-inflammatory effect involved TAS2R38 as shown by pharmacologic inhibition and led to an increase in intracellular calcium levels. We thus unravelled the involvement of several cellular pathways in the anti-inflammatory effects exerted by the QS molecule 3-oxo-C12:2-HSL.

Plasmalogens Regulate Retinal Connexin 43 Expression and Müller Glial Cells Gap Junction Intercellular Communication and Migration.

Plasmalogens are a specific glycerophospholipid subtype characterized by a vinyl-ether bound at their sn-1 moiety. Their biosynthesis is initiated in the peroxisome by dihydroxyacetone phosphate-acyltransferase (DHAPAT), which is encoded by the DAPAT gene. Previous studies have shown that plasmalogen-deficient mice exhibit major physiological dysfunctions including several eye defects, among which abnormal vascular development of the retina and a reactive activation of macroglial Müller cells. Interestingly, plasmalogen deficiency in mice is also associated with a reduced expression of brain connexin 43 (Cx43). Cx43 is the main connexin subtype of retinal glial cells and is involved in several cellular mechanisms such as calcium-based gap junction intercellular communication (GJIC) or cell migration. Thus, the aim of our work was 1) to confirm the alteration of Cx43 expression in the retina of plasmalogen-deficient DAPAT-/- mice and 2) to investigate whether plasmalogens are involved in crucial functions of Müller cells such as GJIC and cell migration. First, we found that plasmalogen deficiency was associated with a significant reduction of Cx43 expression in the retina of DAPAT-/- mice in vivo. Secondly, using a siRNA targeting DHAPAT in vitro, we found that a 50%-reduction of Müller cells content in plasmalogens was sufficient to significantly downregulate Cx43 expression, while increasing its phosphorylation. Furthermore, plasmalogen-depleted Müller cells exhibited several alterations in ATP-induced GJIC, such as calcium waves of higher amplitude that propagated slower to neighboring cells, including astrocytes. Finally, in vitro plasmalogen depletion was also associated with a significant downregulation of Müller cells migration. Taken together, these data confirm that plasmalogens are critical for the regulation of Cx43 expression and for characteristics of retinal Müller glial cells such as GJIC and cell migration.

Chemical Synthesis, Pharmacokinetic Properties and Biological Effects of JM-00266, a Putative Non-Brain Penetrant Cannabinoid Receptor 1 Inverse Agonist.

Targeting cannabinoid 1 receptors (CB1R) with peripherally restricted antagonists (or inverse agonists) shows promise to improve metabolic disorders associated with obesity. In this context, we designed and synthetized JM-00266, a new CB1R blocker with limited blood-brain barrier (BBB) permeability. Pharmacokinetics were tested with SwissADME and in vivo in rodents after oral and intraperitoneal administration of JM-00266 in comparison with Rimonabant. In silico predictions indicated JM-00266 is a non-brain penetrant compound and this was confirmed by brain/plasma ratios and brain uptake index values. JM-00266 had no impact on food intake, anxiety-related behavior and body temperature suggesting an absence of central activity. cAMP assays performed in CB1R-transfected HEK293T/17 cells showed that the drug exhibited inverse agonist activity on CB1R. In addition, JM-00266 counteracted anandamide-induced gastroparesis indicating substantial peripheral activity. Acute administration of JM-00266 also improved glucose tolerance and insulin sensitivity in wild-type mice, but not in CB1R-/- mice. Furthermore, the accumulation of JM-00266 in adipose tissue was associated with an increase in lipolysis. In conclusion, JM-00266 or derivatives can be predicted as a new candidate for modulating peripheral endocannabinoid activity and improving obesity-related metabolic disorders.

Ligand Binding Properties of Odorant-Binding Protein OBP5 from Mus musculus.

Odorant-binding proteins (OBPs) are abundant soluble proteins secreted in the nasal mucus of a variety of species that are believed to be involved in the transport of odorants toward olfactory receptors. In this study, we report the functional characterization of mouse OBP5 (mOBP5). mOBP5 was recombinantly expressed as a hexahistidine-tagged protein in bacteria and purified using metal affinity chromatography. The oligomeric state and secondary structure composition of mOBP5 were investigated using gel filtration and circular dichroism spectroscopy. Fluorescent experiments revealed that mOBP5 interacts with the fluorescent probe N-phenyl naphthylamine (NPN) with micromolar affinity. Competitive binding experiments with 40 odorants indicated that mOBP5 binds a restricted number of odorants with good affinity. Isothermal titration calorimetry (ITC) confirmed that mOBP5 binds these compounds with association constants in the low micromolar range. Finally, protein homology modeling and molecular docking analysis indicated the amino acid residues of mOBP5 that determine its binding properties.

Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line.

Sweet taste perception is mediated by a heterodimeric receptor formed by the assembly of the TAS1R2 and TAS1R3 subunits. TAS1R2 and TAS1R3 are class C G-protein-coupled receptors whose members share a common topology, including a large extracellular N-terminal domain (NTD) linked to a seven transmembrane domain (TMD) by a cysteine-rich domain. TAS1R2-NTD contains the primary binding site for sweet compounds, including natural sugars and high-potency sweeteners, whereas the TAS1R2-TMD has been shown to bind a limited number of sweet tasting compounds. To understand the molecular mechanisms governing receptor-ligand interactions, we overexpressed the human TAS1R2 (hTAS1R2) in a stable tetracycline-inducible HEK293S cell line and purified the detergent-solubilized receptor. Circular dichroism spectroscopic studies revealed that hTAS1R2 was properly folded with evidence of secondary structures. Using size exclusion chromatography coupled to light scattering, we found that the hTAS1R2 subunit is a dimer. Ligand binding properties were quantified by intrinsic tryptophan fluorescence. Due to technical limitations, natural sugars have not been tested. However, we showed that hTAS1R2 is capable of binding high potency sweeteners with Kd values that are in agreement with physiological detection. This study offers a new experimental strategy to identify new sweeteners or taste modulators that act on the hTAS1R2 and is a prerequisite for structural query and biophysical studies.

Triterpenoid Saponins from the Cultivar "Green Elf" of Pittosporum tenuifolium.

Four oleanane-type glycosides were isolated from a horticultural cultivar "Green Elf" of the endemic Pittosporum tenuifolium (Pittosporaceae) from New Zealand: three acylated barringtogenol C glycosides from the leaves, with two previously undescribed 3-O-ß-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-ß-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, 3-O-ß-d-galactopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-ß-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and the known 3-O-ß-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-ß-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C (Eryngioside L). From the roots, the known 3-O-ß-d-glucopyranosyl-(1→2)-ß-d-galactopyranosyl-(1→2)-ß-d-glucuronopyranosyloleanolic acid (Sandrosaponin X) was identified. Their structures were elucidated by spectroscopic methods including 1D- and 2D-NMR experiments and mass spectrometry (ESI-MS). According to their structural similarities with gymnemic acids, the inhibitory activities on the sweet taste TAS1R2/TAS1R3 receptor of an aqueous ethanolic extract of the leaves and roots, a crude saponin mixture, 3-O-ß-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-ß-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and Eryngioside L were evaluated.

Perspectives on Astringency Sensation: An Alternative Hypothesis on the Molecular Origin of Astringency.

Flavor is one of the main drivers of food consumption and acceptability. It is associated with pleasure feels during eating. Flavor is a multimodal perception corresponding to the functional integration of information from the chemical senses: olfaction, gustation, and nasal and oral somatosensory inputs. As a result, astringency, as a sensation mediated by the trigeminal nerves, influences food flavor. Despite the importance of astringency in food consumer acceptance, the exact chemosensory mechanism of its detection and the nature of the receptors activated remain unknown. Herein, after reviewing the current hypotheses on the molecular origin of astringency, we proposed a ground-breaking hypothesis on the molecular mechanisms underpinning this sensation as a perspective for future research.

Comparison of Different Signal Peptides for the Efficient Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris.

Brazzein is a small sweet-tasting protein found in the red berries of a West African evergreen shrub, Pentadiplandra brazzeana Baillon. Brazzein is highly soluble and stable over a large pH range and at high temperatures, which are characteristics that suggest its use as a natural sweetener. However, Pentadiplandra brazzeana culture is difficult at a large scale, limiting the natural source of brazzein. Heterologous expression of brazzein has been established in numerous systems, including bacteria, yeast, and transgenic plants. Brazzein requires four disulfide bonds to be active in eliciting an intense sweet taste, and the yeast Pichia pastoris appears to be one of the best options for obtaining functional brazzein in high quantities. Employing yeast secretion in the culture medium allows us to obtain fully active brazzein and facilitate purification later. To increase yeast secretion, we compared seven different signal peptides to successfully achieve brazzein secretion using the yeast P. pastoris. The brazzein proteins corresponding to these signal peptides elicited activation of the sweet taste receptor functionally expressed in a cellular assay. Among these tested signal peptides, three resulted in the secretion of brazzein at high levels.

Bacterial expression and purification of vertebrate odorant-binding proteins.

Vertebrate odorant-binding proteins (OBPs) are small soluble proteins abundantly secreted in the olfactory mucus of many animal species, including humans. Vertebrate OBPs reversibly bind odorant molecules with micromolar range affinities. Although their physiological role is not clearly understood, OBPs are proposed to carry airborne odorants toward membrane olfactory receptors through the nasal mucus. Measurements of odorant-OBP interactions and structural studies require a large amount of pure OBPs devoid of ligands. The bacterial expression system is the first choice for expressing vertebrate OBPs used in our laboratory and others. This system generally produces OBPs in large amounts without major problems. In this chapter, we describe the milligram-scale production of recombinant pig OBP1 (pOBP1) in E. coli. The different steps of expression and purification are presented and discussed. Protocols for secondary structures investigation by circular dichroism and binding properties of the recombinant protein are also provided. More generally, these approaches can be used to produce and characterize any vertebrate OBPs for use in functional and structural studies.

Novel scaffold of natural compound eliciting sweet taste revealed by machine learning.

Sugar replacement is still an active issue in the food industry. The use of structure-taste relationships remains one of the most rational strategy to expand the chemical space associated to sweet taste. A new machine learning model has been setup based on an update of the SweetenersDB and on open-source molecular features. It has been implemented on a freely accessible webserver. Cellular functional assays show that the sweet taste receptor is activated in vitro by a new scaffold of natural compounds identified by the in silico protocol. The newly identified sweetener belongs to the lignan chemical family and opens a new chemical space to explore.