Rat Odorant-binding Protein Research Articles

Electrochemical detection of the 2-isobutyl-3-methoxypyrazine model odorant based on odorant-binding proteins: The proof of concept

We developed an electrochemical assay for the detection of odorant molecules based on a rat odorant-binding protein (rOBP3). We demonstrated that rOBP3 cavity binds 2-methyl-1,4-naphtoquinone (MNQ), an electrochemical probe, as depicted from the decrease of its electrochemical signal, and deduced the dissociation constant, KdMNQ=0.5(±0.2)μM. The amount of MNQ displaced from rOBP3 by 2-isobutyl-3-methoxypyrazine (IBMP), a model odorant molecule, was measured using square-wave voltammetry. The release of MNQ by competition led to an increase of the electrochemical response. In addition, this method allowed determination of the dissociation constant of rOBP3 for IBMP, KdIBMP=0.5(±0.1)μM. A negative control was performed with a non-binding species, caffeic acid (CA). The determined binding affinity values were confirmed using a fluorescent competitive binding assay and isothermal titration microcalorimetry. This electrochemical assay opens the way for designing robust, reliable and inexpensive odorant biosensors.

Characterization of Rat Odorant Binding Protein Variants and its Post-Translational Modifications (PTMs): LC-MS/MS analyses of Protein Eluted from 2D-Polyacrylamide Gel Electrophoresis

Identification of odorant-binding protein (OBP) variants in mammalian system is of great importance to an understanding of their role in the binding of pheromones and other volatiles during chemical communication. Our previous proteomics studies have revealed the presence of OBP as well as confirming that, in the preputial gland of Indian commensal rat, the bound form of the protein associates with various farnesols. In addition to this, a recent mass spectrometry based proteomics analysis has shown that rat recombinant OBP undergoes post-translational modification (PTM) and there are a number of different variants in terms of phosphorylation. This suggests that the phosphorylation of OBP may affect its binding properties and the binding properties may change in the presence of phosphorylation. Thus, in the present investigation we have investigated these OBP variants. The variants were separated by 2DE-PAGE and protein identification was done using mass spectrometry. The results indicated that OBP has ten variants. Further, we employed anti serine phosphorylation immunoblotting in association with 2DE- PAGE to confirm that three spots were phosphorylation at a serine. In addition to immunoblotting, we also employed structural analysis, by multiple sequence alignment, secondary structural analysis, and a three dimensional analysis of the OBP’s using known lipocalin members. To the best of our knowledge, this is the first report demonstrating PTM variants of an OBP from the preputial gland of the Indian commensal rat.

Rapid Odorant Release in Mammalian Odour Binding Proteins Facilitates Their Temporal Coupling to Odorant Signals

We have measured the effect of rat odorant-binding protein 1 on the rates of ligand uptake and liquid-to-air transfer rates with a set of defined odorous compounds. Comparison of observed rate constants ( k obs) with data simulated over a wide range of different kinetic and thermodynamic regimes shows that the data do not agree with the previously held view of a slow off-rate regime ( k off < 0.0004 s − 1 ). We propose that a rapid k off would be a necessary requirement for such a system, since slow odorant-release rates would result in significant decorrelation between the olfactory world and odour perception.

Structure and Stability of a Rat Odorant-Binding Protein: Another Brick in the Wall

The effect of temperature on the structure of the rat odorant-binding protein was investigated by spectroscopic and in silico methodologies. In particular, in this work, we examined the structural features of the rat OBP-1F by Fourier-transform infrared spectroscopy and molecular dynamics investigations. The obtained spectroscopic results were analyzed using the following three different methods based on the unexchanged amide hydrogens of the protein sample: (1) the analysis of difference spectra; (2) the generalized 2D-IR correlation spectroscopy; (3) the phase diagram method. The three methods indicated that at high temperatures the rOBP-1F structure undergoes a relaxation process involving the protein tertiary organization before undergoing the denaturation and aggregation processes, suggesting the presence of an intermediate state such as a molten globule-like state. Importantly, the proposed analyses represent a general approach that could be applied to the study of protein stability.

CDNA cloning and regulation of two sex-hormone-repressed hamster tear lipocalins having homology with odorant/pheromone-binding proteins

A major 20-kDa protein is female-specifically expressed in exorbital lacrimal gland (LG) of hamsters and secreted in tears. Here, we identify this female-specific LG protein (FLP) as a lipocalin, having 85% protein sequence identity with male-specific submandibular salivary gland proteins (MSP) secreted in saliva and urine of male hamsters. MSP is also female-specifically expressed in LG and secreted in tears but FLP was undetectable in submandibular gland (SMG). FLP and MSP have similar sex-hormonal regulation in LG, which is different from regulation of MSP in SMG. Female-specific expression of FLP and MSP in LG is due to their incomplete repression by endogenous estrogens and gonadectomy in both sexes and lactation in females resulted in their marked induction, which was prevented by estrogen or androgen treatment. FLP and MSP show best sequence identity with odorant/pheromone-binding lipocalins (58–29%). Maximum identity (58%) is with rat odorant-binding protein (OBP) expressed in lateral nasal glands, followed by aphrodisin of hamster vaginal discharge (39%). Cognate transcript and a cross-reacting 20-kDa protein were detected in nasal glands of rat in both sexes but not in hamsters. Results suggest that two closely related lipocalin genes encode FLP and MSP, which are evolutionarily closer to rat OBP than to hamster aphrodisin and these have evolved different tissue-specificity and sex-hormonal regulation. Possible functions for FLP and MSP are suggested, considering their homology to odorant/pheromone-binding lipocalins, their presence in tears, saliva and urine as well as their sex-specific and lactation-induced expression.

Elaboration of odorant biosensors based on Langmuir-Blodgett technique

The advent of electronic noses opened a new kind of analytical approach, which offers an easy, quick, and cheap measurement comparing to conventional analytical approaches. It has potential applications on many different fields, such as assessment of various foodstuffs and beverages, environmental contamination monitoring and medical diagnostics etc. In this study, we used odorant binding protein (OBP) as odor-sensing material for developing such kind of artificial odorant biosensor. And biological films of OBP-1F (rat OBP) were deposited by Langmuir-Blodgett (LB) technique on gold electrode previously functionalised with 1-octadecanethiol (ODT) by self-assembled monolayers (SAMs). It is known that successful immobilization of the Langmuir-Blodgett films depends strongly on the characteristics, particularly stability, of the corresponding Langmuir films at the air/water interface. Hence the characteristics of monolayer of amphiphile octadecylamine (ODA) and the mixed monolayer of ODA/OBP were studied. Electrochemical impedance spectroscopy (EIS) was used to monitor the response of the system to a specific odorant molecule, isoamyl acetate.

Odorant binding and conformational changes of a rat odorant-binding protein.

Odorant-binding proteins (OBPs) are lipocalins secreted in the nasal mucus of vertebrates, which convey odorants to their neuronal receptors. We compared the binding properties of a recombinant rat OBP (OBP-1F) using a set of six odorants of various chemical structures. We examined the binding properties by both fluorescent probe competition and isothermal titration calorimetry. OBP-1F affinity constants, in the micromolar range, varied by more than one order of magnitude and were roughly correlated to the odorant size. The observed binding stoichiometry was found to be around one odorant per dimer. Using tyrosine differential spectroscopy, the binding of ligand was shown to induce local conformational changes. A three-dimensional structure of OBP-1F, modelled using the known structure of aphrodisin as template, allowed us to suggest the location of the observed structural changes outside of the binding pocket. These results are consistent with one binding site located in one of the two beta-barrels of the OBP-1F dimer and a subtle conformational change correlated with binding of an odorant molecule, which hampers uptake of a second odorant by the other hydrophobic pocket.

Identification of a third rat odorant-binding protein (OBP3).

From a rat olfactory epithelium cDNA library clones encoding a lipocalin were isolated with sequence identity to the previously described salivary-specific alpha-2u globulin and the N-terminal region of mouse odorant-binding proteins OBP-III and OBP-IV. In situ hybridization showed strong expression in nasal glands displaying a pattern equivalent to rat OBP1. Heterologously expressed protein was evaluated for its binding properties using spectroscopic approaches. The recombinant protein interacted with two fluorescent probes, 1-aminoanthracene (1-AMA) and 1,1'-bis(4-anilino-5-naphthalene)-sulfonic acid. 1-AMA binding was competed by several odorants with high affinity. The thermodynamic parameters of the protein-odorant interaction were determined using isothermal titration calorimetry. Due to its nasal expression and odorant-binding characteristics this protein was designated OBP3.

Ligand-binding properties and structural characterization of a novel rat odorant-binding protein variant

Ligand-binding properties and structural characterization of a novel rat odorant-binding protein variant

Abundant secretory lipocalins displaying male and lactation-specific expression in adult hamster submandibular gland. cDNA cloning and sex hormone-regulated repression.

We have previously identified massively expressed 24- and 20.5-kDa male-specific proteins in submandibular salivary glands (SMG) of adult hamsters. Here we report the cloning of the cDNA encoding the 24-kDa protein which we have now found to be a heterogenously N-glycosylated form of the 20.5-kDa protein. The deduced amino acid sequence indicated that the protein is a member of the lipocalin family, the two most related lipocalins being rat odorant-binding protein of nasal mucosa and aphrodisin, a pheromonal protein present in vaginal discharge and saliva of female hamsters. Northern blot analysis showed that cognate mRNA is expressed in hamster SMG and lacrimal gland (LG) displaying marked sex-hormonal repression. The sex-hormonal repression patterns showed similarities and dissimilarities between SMG and LG but they were, respectively, similar to the sex-hormonal repression pattern noted for the SMG 24/20.5-kDa male-specific proteins and for an abundant female-specific 20-kDa LG secretory protein. These SMG and LG proteins were found to be immunologically similar and secretion of the SMG proteins in saliva and their excretion in urine was detected. The male-specific and abundant expression of the SMG proteins were seen at and after sexual maturity but was not dependent on androgens. Surprisingly, a temporary male-like expression of these SMG proteins was seen in lactating females which was obliterated by oestrogen administration. Our results show that despite differences in their repression by sex hormones, the gene for SMG 24/20.5-kDa proteins is similar or identical to that of LG 20-kDa protein and their marked repression by both androgens and oestrogens might be at the transcriptional level. Moreover, they might be excellent models with which to study sex hormone repression of gene expression at the molecular level. The results of homology search and the male- and lactation-specific pressure of the SMG proteins in adult saliva and urine suggests a possibility of their involvement in olfaction-mediated chemical communication between hamsters.

Expression of major urinary protein genes in the nasal glands associated with general olfaction.

Gene expression of major urinary protein (MUP) mRNAs was examined in the mouse nasal tissue. By polymerase chain reaction, we identified two cDNA segments encoding MUP 4 and MUP 5 genes in the nose. The expression level of both MUP 4 and 5 mRNAs in the nasal tissue was very high and exceeded that of the liver. Liver MUPs are excreted into the urine and are known to play an important role in pheromonal communication. We showed that nose and liver MUPs were composed of different subtypes of MUPs and that nose MUP mRNAs was detected in prepubescent periods when liver MUP mRNAs had not yet been transcripted. In situ hybridization revealed that nose MUP mRNAs are localized in the lateral wall and nasal septum and their expression pattern is identical to that of rat odorant-binding protein (OBP)-I. We also identified cDNA of mouse OBP-II gene from the nasal tissue and showed that the expression pattern of MUP gene was identical to that of OBP-II gene in the nose. These histological data indicate that nose MUPs are favorable for catching odorant molecules rather than pheromones, and may share their function with OBPs.

CDNA sequence and expression pattern of the putative pheromone carrier aphrodisin.

The cDNA sequence for aphrodisin, a lipocalin from hamster vaginal discharge which is involved in pheromonal activity, has been determined. Corresponding genomic clones were isolated and the promoter region was identified. Primer extension analysis revealed an adenosine residue as the main transcription initiation site, located 50 bp upstream of the translation start codon ATG, which is surrounded by a typical Kozak sequence. However, data from polymerase chain reaction analysis suggest the existence of at least one alternative transcription initiation site. The aphrodisin cDNA is 732 bp long and codes for the mature 151-aa aphrodisin and an additional N-terminal 16-aa secretory signal peptide. The 3' nontranslated region is 228 bp long. Among the known sequences, the aphrodisin cDNA shares the highest homology with the rat odorant-binding protein cDNA (45%), which verifies the protein data. Vaginal tissue and Bartholin's glands are the main aphrodisin gene-expressing tissues of the female hamster genital tract, as demonstrated by Northern blot analysis. Under less stringent hybridization conditions, RNA isolated from rat Bartholin's glands also showed a signal, indicating the occurrence of aphrodisin-related mRNA in this species.

Odorant-binding protein and its mRNA are localized to lateral nasal gland implying a carrier function.

Odorant-binding protein selectively binds various odorants and is discretely concentrated in nasal mucosa and secretions. We have localized rat odorant-binding protein mRNA to the lateral nasal gland by in situ hybridization histochemistry and have also localized the protein to this gland by immunohistochemistry and by tritiated-odorant autoradiography. The lateral nasal gland extends a long duct toward the external nares. Odorant-binding protein, released from this duct, may transport odorants to olfactory receptor neurons.