The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the alpha(2u)-globulin (alpha2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified alpha2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel alpha2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats. This novel alpha2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated de novo sequences which provided additional evidence for the presence of alpha2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to alpha2u. The present investigation confirms the presence of alpha2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat.

Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

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.