Phosphorylation of Native Porcine Olfactory Binding Proteins

Abstract

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.

Binding Specificity of Recombinant Odorant-Binding Protein Isoforms is Driven by Phosphorylation

“…In addition, we demonstrated recently that a dynamic mechanism involving PTM could be responsible for OBP diversity and specificity (Le Danvic et al 2009;Nagnan-Le Meillour et al 2009b). Indeed, porcine OBP can be posttranslationally modified by phosphorylation, and we suggested that this PTM could generate a diversity of OBP isoforms with specific binding properties (Nagnan-Le Meillour et al 2009b). PTM is known to regulate signalling pathways, and phosphorylation could be a mechanism of regulation of OBP specificity towards odorant ligands.…”

“…Typically, samples of 500 μl in buffer A were injected, and resulting fractions were collected and extensively dialyzed against MilliQ water. OBP isoforms were identified in HPLC fractions by Western blot with anti-OBP antibodies (Nagnan-Le Meillour et al 2009b). Their identity was confirmed by peptide mapping, followed by MALDI-TOF mass spectrometry as already described Immunodetection of Phosphorylations with Specific Antibodies HPLC fractions (OBP-Pichia and OBP-CHO) and total OBP-Pichia were suspended in sample buffer, to be separated by SDS-PAGE (Nagnan-Le Meillour et al 2009a).…”

“…An aliquot of crude nasal tissue extract (NagnanLe Meillour et al 2009b) was added as a migration control of OBP, together with molecular weight markers (Precision Plus Protein All Blue, Bio-Rad, Marnes-la-Coquette, France). Western blots with specific antibodies directed against phosphoserine, phosphotyrosine, and phosphothreonine (Invitrogen, Paysley, UK) were performed exactly as described in Nagnan-Le Meillour et al (2009b). Detection of signal was carried out with an ECL Plus Western Blotting Detection kit (GE Healthcare, Orsay, France).…”

“…Our goal was to validate the use of recombinant proteins to study further the role of phosphorylation in OBP binding to pheromone components. We purified OBP isoforms by anion-exchange HPLC and we mapped their phosphorylation sites, which were compared with those previously characterized in the native form of OBP (Nagnan-Le Meillour et al 2009b). Binding experiments with ligands of biological relevance to S. scrofa (male and female pheromones) were conducted by a fluorescent probe displacement assay on two isoforms of recombinant OBP expressed in the yeast.…”

Reverse chemical ecology: Olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles

“…1 and 2. Phosphorylation of mammalian OBPs has been reported in the past (41) and suggested to be a way of modifying the binding specificity of the protein. However, the function of this modification on OBPs still remains to be experimentally shown.…”