Large amounts of an odorant-binding protein have been isolated from submaxillary glands of mature male pig. This polypeptide molecule is sex-specific, being absent in females. On electrophoretic gels under denaturing conditions it migrated as a broad band with an apparent molecular mass of around 20 kDa. Electrospray mass spectrometry revealed the presence of three main components, whose mass differences are not interpretable as result of any common post-translational modifications, indicating the presence of distinct polypeptide chains. N-terminal Edman degradation yielded a single sequence of 29 amino acids. It includes the lipocalin signature (-G-X-W-) and shows clear homology with a subclass of odorant-binding proteins present in mouse saliva, nasal mucus and urine. The purified protein still retained small ligands tightly bound ; among them 5A-androst-16-en-3-one and 5A-androst-16-en-3A-ol, both known sex pheromones for the pig, were identified. The protein also binds 2-isobutyl-3-methoxypyrazine, a good ligand for most odorant-binding proteins, with a dissociation constant of 5 µM.

An odorant-binding protein, migrating in SDS-PAGE with an apparent molecular weight of 22 kDa and an isoelectric point of 4.2, has been purified from pig nasal mucosa. Its complete amino acid sequence was determined by a combination of mass spectrometry and Edman degradation procedures. The protein consists of a single polypeptide chain of 157 amino acids, presenting at the N-terminus a pyroglutamic acid residue. The two cysteine residues, occurring in the primary structure at positions 63 and 155, are involved in an intramolecular disulphide bridge. Sequence comparison with other lipocalins revealed a good similarity with bovine odorant-binding protein, the only member of this class which does not contain disulphide bonds and of which the three-dimensional structure recently has been resolved. Nine out of the 1 6 residues lining the binding pocket in bovine OBP are conserved in the porcine protein, suggesting structural similarities in this region of the molecule. The synthesis of a fluorescent photoaffinity labelling agent and of two tin-containing thymol analogues is also described. These compounds together with other ligands were able to bind the protein as revealed by competitive binding experiments.

Odorant-binding proteins (OBP) in the mucus of the olfactory epithelium are thought to transfer the hydrophobic odorous compounds through the aqueous barrier towards the chemo-sensory cells. To evaluate their binding properties, two distinct OBP subtypes of the rat were expressed as N-terminal Histagged fusion proteins in Escherichia coli, thus allowing an efficient purification. Based on gel chromatography and CD spectroscopy analysis the recombinant OBP subtypes seem to share several structural features with other members of the lipocalin family. Approaches to elucidate whether heterologous expressed OBPs interact with odorous compounds revealed that OBP1 specifically binds 2-[3 H]-isobutyl-3-methoxypyrazine whereas OBP2 did not shown any specific binding to this compound. In contrast, the chromophore 1-anilinonaphthalene 8-sulfonic acid (1,8-ANS) specifically interacted with OBP2 but not with OBP1. Displacement experiments monitored by the relative fluorescence intensity revealed that fatty acids with appropriate chain length act as efficient competitors. Some odorous compounds, notably lilial (p-tert-butyl-A-methyl dihydrocinnamic aldehyde) and citralva (3,7-dimethyl-2,6-octadienenitrile), also displaced efficiently the chromophore, whereas pyrazine derivatives including 2-isobutyl-3-methoxypyrazine and other odorants did not. These results indicate that rat OBPs have distinct ligand specificities.Keywords : olfaction; odorant-binding protein; procaryotic expression ; competitive binding assay; rat.Odorant-binding proteins (OBPs) of vertebrates are ex-two cloned rat OBP genes, purify the proteins to homogeneity and assess their ligand specificity. pressed in the nasal glands, released into the mucus and are thought to transfer the hydrophobic odorous molecules through the aqueous mucus barrier towards the olfactory sensory neurons [1]. These proteins are members of the lipocalin family, which MATERIALS AND METHODS are classified primarily as transporters for lipophilic molecules Materials. Oligodeoxyribonucleotides were synthesized by in the body fluids [2] and share some common structural features Roth. The TRIzol reagent was from Gibco BRL Life Technol-[3Ϫ5]. The first vertebrate OBP was isolated from bovine nasal ogies. The pGEM-T vector was purchased from Promega. mucus and characterized as a pyrazine-binding protein using 2-Escherichia coli BL21(DE3) was from Novagen. The expression isobutyl-3-methoxypyrazine as ligand [6, 7]. Subsequently, vectors pTrcHisA, B and C were obtained from Invitrogen. The OBPs have been identified in a variety of species, including pig, Ni-nitrilotriacetic acid agarose came from Qiagen. Isopropylrabbit, mouse and rat [8Ϫ11]. Two rat OBPs have already been thio-β-D-galactoside was from BTS. Enterokinase was obtained cloned and the cDNA sequence is known [11, 12]. Different from Boehringer. Tritiated 2-[ 3 H]isobutyl-3-methoxypyrazine subtypes of OBPs were discovered first in rat [12, 13] and subsewas prepared at the Amersham Laboratories by catalytic hydroquently also in othe...

Two distinct classes of lipocalin isoforms (OBP-IIs and OBP-IIIs) were purified and identified from porcine nasal mucosa of male and female individuals. Using primers designed on their N-terminal sequence, the complete primary structures of the mature polypeptides were determined. Mass spectrometry analysis confirmed the identity of the cDNA-derived sequences and provided information regarding their post-translational modifications. These species strongly resemble a lipocalin expressed by von Ebner's gland and salivary lipocalins carrying sex-specific pheromones secreted only by the boar's submaxillary glands. Both OBP-IIs and OBP-IIIs present two cysteines paired in a disulphide bond; the remaining residues occur in a reduced form. In addition, OBP-IIIs are heavily glycosylated and markedly different in their glycan moiety from the salivary lipocalins. A three-dimensional model is proposed based on protein species with known structure. Like salivary lipocalins, OBP-IIIs bind a number of odorant molecules, with highest affinity for the specific pheromone 5alpha-androst-16-en-3-one. The high similarity between OBPs from the nasal area and lipocalins from secretory glands suggests a common function in binding the same pheromonal ligands, the latter carrying chemical messages into the environment the former delivering them to specific receptors.

No abstract

Chemosensory proteins (CSPs) are a class of small, soluble proteins present at high concentrations in chemosensory organs of different insect species. Several pieces of evidence suggest their involvement in carrying chemical messages from the environment to chemosensory receptors. However, a structural description of the mechanism of delivery has not been reported. In order to provide the first detailed conformational characterization of these molecules, we cloned a specific isoform (CSP-sg4) from Schistocerca gregaria and expressed it in Escherichia coli. The product was obtained with yields of more than 20 mg per L of culture, all in its soluble form. The recombinant protein was identical to the native one with respect to pairing of the disulfide bridges, aggregative state and secondary structure elements. Structural investigations revealed a significantly stable polypeptide with respect to variations in temperature and acidity. CD analysis, preliminary NMR data and secondary structure prediction pointed to a correctly folded structure where helical regions and loops are alternated in a similar fashion as that observed for other classes of odorantand pheromone-binding proteins presenting no sequence similarity to CSPs.Keywords: chemosensory proteins; pheromone; Schistocerca gregaria; heterologous expression.A new class of soluble proteins expressed in chemoreception organs of insects has recently received increasing interest. Since the first member was discovered in the antennae of Drosophila melanogaster [1,2], homologous proteins have been reported in most orders of insects, Phasmatodea [3][4][5], Orthoptera [6,7], Blattoidea [8], Lepidoptera [9 -11] and Hymenoptera [12]. In the absence of a clear functional characterization, different names have been proposed for these proteins. We have adopted the general term of chemosensory proteins (CSPs) to indicate that their occurrence is generally associated to chemosensory structures; thus, for example, in Schistocerca gregaria they are specifically present in the outer lymph of contact chemosensilla [6]. However, we found that the expression of these polypeptides is not limited to the antennae, but includes other chemosensory organs, such as tarsi and palpi [6]. The expression of CSPs in other sites of the insect body, such as the ejaculatory bulb of D. melanogaster [13] and cells underlying the cuticle in Phasmatodea and Orthoptera (S. Angeli, S. Marchese & P. Pelosi, unpublished results), could be related to the production and release of pheromones. On the other hand, biochemical data support the hypothesis that CSPs are involved in chemoreception. In fact, CSPs from the lepidopteran Mamestra brassicae have been shown to specifically bind the D. melanogaster pheromone vaccenyl acetate [10].However, CSPs do not bear any structural similarity to other classes of odorant-and pheromone-binding proteins (OBPs and PBPs) from vertebrates and insects [6,14]. They are polypeptides of 100-110 amino acids, containing two disulfide bridges between cysteine residues close i...

No abstract