Biomedical subjects

Claude Nespoulous

Publications and source records attributed to Claude Nespoulous.

4 recordsLinked to original sources

PubMed2006

Odorant binding and conformational dynamics in the odorant-binding protein.

In mammals, the olfactory epithelium secretes odorant-binding proteins (OBPs), which are lipocalins found freely dissolved in the mucus layer protecting the olfactory neurons. OBPs may act as passive transporters of predominantly hydrophobic odorant molecules across the aqueous mucus layer, or they may play a more active role in which the olfactory neuronal receptor recognizes the OBP-ligand complex. To better understand the molecular events accompanying the initial steps in the olfaction process, we have performed molecular dynamics studies of rat and pig OBPs with the odorant molecule thymol. These calculations provide an atomic level description of conformational changes and pathway intermediates that remain difficult to study directly. A series of eight independent molecular dynamics trajectories of rat OBP permitted the observation of a consensus pathway for ligand unbinding and the calculation of the potential of mean force (PMF) along this path. Titration microcalorimetry confirmed the specific binding of thymol to this protein with a strong hydrophobic component. In both rat and pig OBPs we observed lipocalin strand pair opening in the presence of ligand, consistent with potential roles of these proteins in olfactive receptor recognition.

Animals↗

PubMed2006

A single lysyl residue defines the binding specificity of a human odorant-binding protein for aldehydes.

Odorant-binding proteins (OBPs) are small abundant soluble proteins belonging to the lipocalin superfamily, which are thought to carry hydrophobic odorants through aqueous mucus towards olfactory receptors. Human variant hOBP-2A has been demonstrated to bind numerous odorants of different chemical classes with a higher affinity for aldehydes and fatty acids. Three lysyl residues of the binding pocket (Lys62, Lys82 and Lys112) have been suggested as candidates for playing such a role. Here, using site-directed mutagenesis and fluorescent probe displacements, we show that Lys112 is the major determinant for governing hOBP-2A specificity towards aldehydes and small carboxylic acids.

1-Naphthylamine↗

PubMed2002

Evidence of an odorant-binding protein in the human olfactory mucus: location, structural characterization, and odorant-binding properties.

Odorant-binding proteins (OBPs) are small abundant extracellular proteins belonging to the lipocalin superfamily. They are thought to participate in perireceptor events of odor detection by carrying, deactivating, and/or selecting odorant molecules. Putative human OBP genes (hOBP) have recently been described [Lacazette et al. (2000) Hum. Mol. Genet. 9, 289-301], but the presence of the corresponding proteins remained to be established in the human olfactory mucus. This paper reports the first evidence of such expression in the mucus covering the olfactory cleft, where the sensory olfactory epithelium is located. On the contrary, hOBPs were not observed in the nasal mucus covering the septum and the lower turbinate. To demonstrate the odorant binding activity of these proteins, a corresponding recombinant protein variant, hOBP(IIa)(alpha), was secreted by the yeast Pichia pastoris and thoroughly characterized. It appears as a monomer with one disulfide bond located between C59 and C151, a conservative feature of all other vertebrate OBPs. By measuring the displacement of several fluorescent probes, we show that hOBP(IIa)(alpha) is able to bind numerous odorants of diverse chemical structures, with a higher affinity for aldehydes and large fatty acids. A computed 3D model of hOBP(IIa)(alpha) is proposed and reveals that two lysyl residues of the binding pocket may account for the increased affinity for aldehydes. The relatively limited specificity of hOBP(IIa)(alpha) suggests that other human OBPs are expected to take into account the large diversity of odorant molecules.

Alternative Splicing↗

PubMed2002

Characterization of a chemosensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier.

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

Amino Acid Sequence↗