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

Rajkumar R,Alagendran S,Ilayaraja R

doi:10.4172/jpb.1000191

Abstract

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.

Highlights

In rodents, the preputial gland is being considered to be the predominant organ associated with chemo-communication [1,2,3]
34% of the proteins were found to be involved in metabolism, 9% were found to be involved in transport, 5% were found to be involved in proteolysis and 5% were found to be involved in cell motility, 3% were found to be involved in translation, reproduction, apoptosis and responses to stimulus, while 2% were found to be involved in nucleosome assembly, free radicals metabolism, epidermis development, signal transduction, blood coagulation, apoptosis, acrosome reaction, phosphorylation, and endoplasmic reticulum
In terms of cellular localization, 36 % of the proteins were found to be from the cytoplasm, 11% were found to be from secreted, 13% were found to be from the mitochondria, 8% were found to be from the cytosol, 5% were found to be from the melanosome, and 3% were found to be from the endoplasmic reticulum and peroxisome, as is shown in Figure 2, panel B

Summary

Introduction

The preputial gland is being considered to be the predominant organ associated with chemo-communication [1,2,3]. The presence of α2u-globulin in the pub preputial gland of rat indicating that the pheromone carrying/odorant binding protein plays significant a role in adult [15]. This protein have been expressed in most of the mammalian species with the different designation for instance, major urinary protein in mice [16] α2u-globulin (α2u) in rat [17] and aphrodisin in hamster [18] salivary OBP in buffalo [19] and Received August 04, 2011; Accepted October 14, 2011; Published October 20, 2011

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Discussion

Conclusion