The identification of various isoforms of olfactory binding proteins is of major importance to elucidate their involvement in detection of pheromones and other odors. Here, we report the characterization of the phosphorylation of OBP (odorant binding protein) and Von Ebner's gland protein (VEG) from the pig, Sus scrofa. After labeling with specific antibodies raised against the three types of phosphorylation (Ser, Thr, Tyr), the phosphate-modified residues were mapped by using the beta-elimination followed by Michael addition of dithiothreitol (BEMAD) method. Eleven phosphorylation sites were localized in the pOBP sequence and nine sites in the VEG sequence. OBPs are secreted by Bowman's gland cells in the extracellular mucus lining the nasal cavity. After tracking the secretion pathway in the rough endoplasmic reticulum of these cells, we hypothesize that these proteins may be phosphorylated by ectokinases that remain to be characterized. The existence of such a regulatory mechanism theoretically increases the number of OBP variants, and it suggests a more specific role for OBPs in odorant coding than the one of odorant solubilizer and transporter.

Native porcine odorant-binding protein (OBP) bears eleven sites of phosphorylation, which are not always occupied in the molecular population, suggesting that different isoforms could co-exist in animal tissues. As phosphorylation is a dynamic process resulting in temporary conformational changes that regulate the function of target proteins, we investigated the possibility that OBP isoforms could display different binding affinities to biologically relevant ligands. The availability of recombinant proteins is of particular interest for the study of protein/ligand structure-function relationships, but prokaryotic expression systems do not perform eukaryotic post-translational modifications. To investigate the role of phosphorylation in the binding capacities of OBP isoforms, we produced recombinant porcine OBP in two eukaryotic systems, the yeast, Pichia pastoris, and the mammalian CHO cell line. Isoforms were separated by anion exchange HPLC, and their phosphorylation sites were mapped by MALDI-TOF mass spectrometry and compared to those of the native protein. Binding experiments with ligands of biological relevance in the pig, Sus scrofa, were performed by fluorescence spectroscopy on two isoforms of recombinant OBP expressed in the yeast. The two isoforms, differing only by their phosphorylation pattern, displayed different binding properties, suggesting that binding specificity is driven by phosphorylation.