Following the purification of an odorant-binding protein (OBP) from rabbit nasal mucosa, two additional OBPs have been identified, purified and partially characterized from the nasal tissue of the same animal species. OBP-II is a monomer of 21 kDa and isoelectric point 4.2; OBP-III is a dimer with subunits of 23 kDa and isoelectric point 4.8. Like OBP-I, both these new members bind the odorant 2-isobutyl-3-methoxypyrazine. The partial amino acid sequences of the three OBPs, determined by Edman degradation, confirm that they are members of the OBP family, but reveal little similarity between them. However, higher similarity is found between each OBP and other members of the lipocalin family. In particular, OBP-I is most similar to bovine OBP (55% identity in the N-terminal region); OBP-II is > 50% identical (limited to its first 18 amino acids) to mouse OBP-I and porcupine OBP-II, while OBP-III shares 26 out of the first 40 amino acids with major urinary protein (MUP) 4, a member of the mouse salivary proteins (Garibotti, 1997).

After the isolation of two odorant-binding proteins (OBP-I and OBP-II) from mouse nasal tissue, two additional OBPs were isolated that bind tritiated 2-isobutyl-3-methoxypyrazine. OBP-III is a homodimer with subunits of M(r) 22,000 and pI 4.2. OBP-IV is a homodimer with subunits of M(r) 21,000 and pI 4.85. N-terminal amino acid sequences indicate that OBP-III is identical in its first 40 amino acids to the mouse urinary protein, MUP-5. OBP-IV is > 90% identical in its first 30 amino acids to the MUP-4; OBP-II is nearly 80% similar in its first 40 amino acids to OBP-I of the rat, and both subunits of OBP-I are > 50% identical with hamster aphrodisin (Pes, 1995).

One odorant-binding protein purified from male mouse nasal epithelium, named OBP-I, appears to be a heterodimer whose subunits Ia and Ib show significant similarity to hamster aphrodisin in their N-terminal amino acid sequences. The complete amino acid sequences of these two polypeptide chains is reported, as deduced from nucleotide sequences of their relative cDNA. These data confirm the high similarity of both proteins to hamster aphrodisin. A comparison with the sequences of other known OBPs indicates that the two subunits are more closely related to members of class I, including bovine OBP, rat OBP-I and pig OBP-I. A putative odorant-binding site is indicated by the presence of amino acid residues conserved with respect to the bovine protein, whose three-dimensional structure has been recently resolved. In-situ hybridization has revealed identical expression patterns for the two proteins, further supporting the heterodimeric structure of these proteins in the nasal mucus (Pes, 1998).

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 His-tagged 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 reveal that OBP1 specifically binds 2-[3H]-isobutyl-3-methoxypyrazine whereas OBP2 does not shown any specific binding to this compound. In contrast, the chromophore 1-anilinonaphthalene 8-sulfonic acid (1,8-ANS) specifically interacts with OBP2 but not with OBP1. Displacement experiments monitored by the relative fluorescence intensity reveal that fatty acids with appropriate chain length act as efficient competitors. Some odorous compounds, notably lilial (p-tert-butyl-alpha-methyl dihydrocinnamic aldehyde) and citralva (3,7-dimethyl-2,6-octadienenitrile), also displace efficiently the chromophore, whereas pyrazine derivatives including 2-isobutyl-3-methoxypyrazine and other odorants do not. These results indicate that rat OBPs have distinct ligand specificities (Lobel, 1998).

Specific binding of 125I-labelled bovine odour-binding protein (OBP) to isolated membranes from nasal mucosa has been demonstrated. The interaction reaches equilibrium within 30 min at 37 degrees C and is reversible. A Scatchard analysis of the equilibrium binding reveals a single population of binding sites, with the calculated equilibrium dissociation constant and maximum number of binding sites being 2.25 +/- 0.5 microM and 18.5 +/- 2 pmol/mg of membrane protein respectively (n = 2). Receptor activity is decreased on digestion by trypsin, proteinase K or endoglycosidase H, is heat labile and is sensitive to thiol-group-specific reagents. With the exception of rat and mouse major urinary proteins, which exhibit a high degree of structural similarity with OBP and bind similar ligands, other members of the lipocalin family, such as retinol-binding protein and beta-lactoglobulin, fail to inhibit the binding of 125I-labelled OBP to its receptor. The receptor seems not to be restricted to olfactory tissues, as it is detected in a variety of other tissues. This suggests that OBP is unlikely to play a role only in olfactory signal transduction. It might have a much broader role within the body; possibilities include a role in detoxification or signaling (Boudjelal, 1996).

Pregnancy block in mice requires exposure of recently mated females to urinary pheromones of a strange male. When working with inbred strains, this invariably requires urine from an outbred line. The pheromones, which induce oestrus and early puberty in mice, have been identified as the brevicomins and dihydrothiazoles. Since the same vomeronasal, neural and neuroendocrine pathways are also activated in pregnancy block, these compounds are likely candidates for pregnancy blocking pheromones. However, these relatively simple chemicals lack the capacity to code for differing mouse strains. Since large quantities of the polymorphic major urinary proteins from the lipocalin family found in urine serve as transporters for the dihydrothiazoles and brevicomins, and differ across strains, these proteins must participate in pheromone recognition in the context of pregnancy block (Keverne, 1998).

Odorant binding protein (OBP) is the major odorant binding component of mammalian nasal mucosa. The two structures of bovine OBP reported in this paper (one crystallized as purified and one soaked in the presence of a selenium-containing odorant) show that: (1) the OBP dimer is composed of two compact domains related by an approximate two-fold axis of symmetry; (2) between residues 122 and 123 the polypeptide chains cross from one domain to the other, such that each domain is formed by residues from both monomers; (3) purified OBP already contains two bound odorant molecules (one per monomer)-odorant binding occurs by replacement of these molecules with the added odorant, and (4) the structure of the odorant binding site can explain OBP's extraordinarily broad odorant specificity (Bianchet, 1996).

In mammals, odorant binding proteins may play an important role in the transport of odors toward specific olfactory receptors on sensory neurons across the aqueous compartment of the nasal mucus. The X-ray structure of such a transport protein, bovine odorant binding protein (OBP) has been solved at 2.0 A resolution. The beta-barrel of OBP is similar to that of lipocalins, but OBP dimer association results from domain swapping, an observation unique among the lipocalins. The alpha-helix of each monomer stacks against the beta-barrel of the other monomer. Contrary to previous reports, each monomer has an internal buried cavity that could accommodate a naturally occurring molecule. Besides this cavity, an open cavity is located at the dimer interface. Data in solution suggest that this central cavity may be a binding site created by domain swapping (Tegoni, 1996).

The X-ray structure of the porcine odorant binding protein (OBPp) was determined at 2.25 A resolution. This lipocalin is a monomer and is devoid of naturally occurring bound ligand, contrary to what was observed in the case of bovine OBP. In this latter protein, a dimer without any disulfide bridges, domain swapping was found to occur between the beta- and alpha-domains. A single Gly (121) insertion was found in OBPp when it was compared to OBPb, which may prevent domain swapping from taking place. The presence of a disulfide bridge between the OBPp beta- and alpha-domains (cysteines 63 and 155) may lock the resulting fold in a nonswapped monomeric conformation. Comparisons with other OBPs indicate that the two cysteines involved in the OBPp disulfide bridge are conserved in the sequence, suggesting that OBPp may be considered a prototypic OBP fold, and not OBPb (Spinelli, 1998).