Biomedical subjects

Jean-Claude Pernollet

Publications and source records attributed to Jean-Claude Pernollet.

12 recordsLinked to original sources

PubMed2006

[Olfactory receptors and odour coding].

The first step of olfactory detection involves interactions between odorant molecules and neuronal protein receptors. Odour coding results from the combinatory activation of a set of receptors and rests on their clonal expression and olfactory neurone connexion, which lead to formation of a specific sensory map in the cortex. This system, sufficient to discriminate myriads of odorants with a mere 350 different receptors, allows humans to smell molecules that are not natural (new cooking flavours, synthetic chemicals...). The extreme olfactory genome diversity explains the absence of odour semantics. Olfactory receptors are also involved in cellular chemotaxis.

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↗

PubMed2005

Study of Langmuir and Langmuir-Blodgett films of odorant-binding protein/amphiphile for odorant biosensors.

To make ultrathin films for the fabrication of artificial olfactory systems, odorant biosensors, we have investigated mixed Langmuir and Langmuir-Blodgett films of odorant-binding protein/amphiphile. Under optimized experimental conditions (phosphate buffer solution, pH 7.5, OBP-1F concentration of 4 mg L(-1), target pressure 35 mN m(-1)), the mixed monolayer at the air/water interface is very stable and has been efficiently transferred onto gold supports, which were previously functionalized by self-assembled monolayers (SAMs) with 1-octadecanethiol (ODT). Atomic force microscopy and electrochemical impedance spectroscopy were used to characterize mixed Langmuir-Blodgett (LB) films before and after contact with a specific odorant molecule, isoamyl acetate. AFM phase images show a higher contrast after contact with the odorant molecule due to the new structure of the OBP-1F/ODA LB film. Non-Faradaic electrochemical spectroscopy (EIS) is used to quantify the effect of the odorant based on the electrical properties of the OBP-1F/ODA LB film, as its resistance strongly decreases from 1.18 MOmega (before contact) to 25 kOmega (after contact).

Adsorption↗

PubMed2005

Comparison of odorant specificity of two human olfactory receptors from different phylogenetic classes and evidence for antagonism.

Humans are able to detect and discriminate myriads of odorants using only several hundred olfactory receptors (ORs) classified in two major phylogenetic classes representing ORs from aquatic (class I) and terrestrial animals (class II). Olfactory perception results in a combinatorial code, in which one OR recognizes multiple odorants and different odorants are recognized by different combinations of ORs. Moreover, recent data suggest that odorants could also behave as antagonists for other ORs, thus making the combinatorial coding more complex. Here we describe the odorant repertoires of two human ORs belonging to class I and class II, respectively. For this purpose, we set up an assay based on calcium imaging in which 100 odorants were screened using air-phase odorant stimulation at physiological doses. We showed that the human class I OR52D1 is functional, exhibiting a narrow repertoire related to that of its orthologous murine OR, demonstrating than this human class I OR is not an evolutionary relic. The class II OR1G1 was revealed to be broadly tuned towards odorants of 9-10 carbon chain length, with diverse functional groups. The existence of antagonist odorants for the class II OR was also demonstrated. They are structurally related to the agonists, with shorter carbon chain length.

Animals↗

PubMed2005

Specific expression of olfactory binding protein in the aerial olfactory cavity of adult and developing Xenopus.

Olfactory binding proteins (OBP), commonly associated with aerial olfaction, are found in the olfactory mucus of mammals but have never been identified in fish. It is still not clear whether the presence of OBP in aerial olfactory systems is due to phylogenetic or to functional differences linked to the adaptation of the olfactory system to an aerial environment. To test this alternative, the olfactory system of Xenopus offers a unique opportunity because it includes two olfactory cavities, one of which is thought to be devoted to aquatic olfaction and the other to aerial olfaction. We therefore purified and cloned OBPs in two Xenopus species. Xenopus laevis OBP (XlaeOBP) and Xenopus tropicalis OBP (XtroOBP) exhibit 158 and 160 amino acids, respectively, sharing 89 residues. cRNA probes allowed us to demonstrate that XlaeOBP and XtroOBP are expressed at the level of Bowman's gland specifically in the aerial olfactory cavity, as confirmed using anti-XlaeOBP antiserum. OBP mRNA transcription occurs early during metamorphosis, as early as stage 57. This is the first study to demonstrate that OBPs are exclusively present in the aerial chamber and are only expressed as the tadpole becomes an adult in species which possess both aquatic and aerial olfactory organs.

Aging↗

PubMed2004

Aphrodisin, an aphrodisiac lipocalin secreted in hamster vaginal secretions.

Vertebrates communicate through pheromones, which favor biological regulations within each species. Aphrodisin, a protein belonging to the lipocalin superfamily, found in hamster vaginal secretions, is detected by the male accessory olfactory system and induces or facilitates its copulatory behavior. Although much is known about aphrodisin structure, the question of whether aphrodisin bears itself the pheromonal function or is simply a carrier for hydrophobic small pheromones has not been definitely solved. Arguments based on use of recombinant aphrodisin deprived of any natural ligand and its capability to convey hamster pheromonal compounds will be discussed, together with progresses concerning putative natural ligand(s).

Amino Acid Sequence↗

PubMed2004

Natural ligands of hamster aphrodisin.

The chemical nature of vertebrate pheromones remains largely to be deciphered. Hamster aphrodisin is a rare instance of mammal proteinaceous sexual pheromone. This protein, found in vaginal secretions, facilitates the mounting behaviour of males via activation of a specialized sensory structure named the vomeronasal organ, which activates the accessory olfactory bulb. Since it might carry small pheromonal ligands due to its lipocalin structure, we analysed organic extracts from natural aphrodisin. We identified five predominant compounds specifically bound onto natural aphrodisin as 1-hexadecanol (44.7%), 1-octadecanol (19.5%), Z-9-octadecen-1-ol (18.2%), E-9-octadecen-1-ol (15.4%) and hexadecanoic acid (2.2%). Interestingly, these compounds are also described as part of insect pheromone blends, disclosing the continuing story of amazing coincidences of chemical communication shared by mammals and insects.

Animals↗

PubMed2003

Sulfur single-wavelength anomalous diffraction crystal structure of a pheromone-binding protein from the honeybee Apis mellifera L.

Pheromone binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in several sensory organs from moth and other insect species. They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors. We report here the crystal structure of a PBP (Amel-ASP1) originating from the honey-bee (Apis mellifera) antennae and expressed as recombinant protein in the yeast Pichia pastoris. Crystals of Amel-ASP1 were obtained at pH 5.5 using the nano-drops technique of crystallization with a novel optimization procedure, and the structure was solved initially with the single-wavelength anomalous diffraction technique using sulfur anomalous dispersion. The structure of Amel-ASP1 has been refined at 1.6-A resolution. Its fold is roughly similar to that of other PBP/odorant binding proteins, presenting six helices and three disulfide bridges. Contrary to the PBPs from Bombyx mori (Sandler, B. H., Nikonova, L., Leal, W. S., and Clardy, J. (2000) Chem. Biol. 7, 143-151) and Leucophea maderae (Lartigue, A., Gruez, A., Spinelli, S., Riviere, S., Brossut, R., Tegoni, M., and Cambillau, C. (2003) J. Biol. Chem. 278, 30213-30218), the extended C terminus folds into the protein and forms a wall of the internal hydrophobic cavity. Its backbone groups establish two hydrogen bonds with a serendipitous ligand, n-butyl-benzene-sulfonamide, an additive used in plastics. This mode of binding might, however, mimic that used by one of the pheromonal blend components and illustrates the binding versatility of PBPs.

Animals↗

PubMed2003

Optimization of crystals from nanodrops: crystallization and preliminary crystallographic study of a pheromone-binding protein from the honeybee Apis mellifera L.

Pheromone-binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in various sensory organs from moths and other insect species. They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors. Here, crystals of a PBP (Amel-ASP1) originating from honeybee (Apis mellifera L.) antennae and expressed as recombinant protein using the yeast Pichia pastoris are reported. Crystals of Amel-ASP1 have been obtained by the sitting-drop vapour-diffusion method using a nanodrop-dispensing robot under the following conditions: 200 nl of 40 mg ml(-1) protein solution in 10 mM Tris, 25 mM NaCl pH 8.0 was mixed with 100 nl of well solution containing 0.15 M sodium citrate, 1.5 M ammonium sulfate pH 5.5. The protein crystallizes in space group C222(1), with unit-cell parameters a = 74.8, b = 85.8, c = 50.2 A. With one molecule in the asymmetric unit, V(M) is 3.05 A(3) Da(-1) and the solvent content is 60%. A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble). The nanodrop crystallization technique used with a novel optimization procedure made it possible to consume small amounts of protein and to obtain a unique crystal per nanodrop, suitable directly for data collection in-house or at a synchrotron-radiation source.

Amino Acid Sequence↗

PubMed2003

Corpus luteum derived copper, zinc-superoxide dismutase serves as a luteinizing hormone-release inhibiting factor in sheep.

In the present study, we report the purification and characterization of a polypeptide from the sheep corpus luteum of pregnancy with a potent luteinizing hormone-release inhibiting factor (LH-RIF) bioactivity that stained as a single band in SDS-PAGE with an apparent molecular mass of 16000 Da. The amino acid sequences obtained after sequence analysis of peptides derived from the trypsin digestion of LH-RIF were subjected to a protein data bank search and were found to be identical with regions of sheep copper, zinc-superoxide dismutase (Cu,Zn-SOD). The measured mass of LH-RIF (15604.2+/-1.9 Da) was found to be similar to the theoretical mass of sheep Cu,Zn-SOD (15603.5 Da), with a disulfide bond and N acetylated alanine at the N-terminus. The inhibitory action of Cu,Zn-SOD on pulsatile LH secretion would suggest that this antioxidant may play an important role, either independently or in concert with some neurotransmitters, in the neuroendocrine regulation of sheep female reproductive function.

Animals↗

PubMed2002

Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.

Acylation↗

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↗