Proteomic Analysis of Pig (Sus scrofa) Olfactory Soluble Proteome Reveals O-Linked-N-Acetylglucosaminylation of Secreted Odorant-Binding Proteins

Abstract

The diversity of olfactory binding proteins (OBPs) is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry, and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT) was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

Identification of Post-translational Modifications on Odorant-Binding Protein Isoforms from Pig Olfactory Secretome by High-Resolution Mass Spectrometry: O-β-N-acetylglucosaminylation and Phosphorylation

“…We have shown in a previous work (Nagnan-Le Meillour et al, 2014) that around 30 OBP-, VEG-, and SAL-isoforms compose the pig olfactory secretome. Among them, only OBP sensu stricto isoforms were labeled with specific antibodies raised against phosphorylation and O-GlcNAcylation.…”

“…Whether the odorant molecule or the complex OBP/odorant acts as a receptor ligand is still an open question, as none of the two hypotheses have received direct experimental evidence. The recent discovery of OBP isoform diversity in pig (Sus scrofa) nasal mucus (NagnanLe Meillour et al, 2014) favors the hypothesis that OBPs could be more than passive carriers of odorants, being more likely involved in the selection of chemical structures to be presented to a given olfactory receptor. Proteomic analysis of pig nasal mucus revealed that the olfactory secretome (Nagnan-Le Meillour et al, 2014) is mainly composed of around 30 OBP-, VEG-(Von Ebner's Gland protein), and SAL-(Salivary Lipocalin) isoforms, resulting from post-translational modifications (PTMs) of these three gene products and variants.…”

“…The recent discovery of OBP isoform diversity in pig (Sus scrofa) nasal mucus (NagnanLe Meillour et al, 2014) favors the hypothesis that OBPs could be more than passive carriers of odorants, being more likely involved in the selection of chemical structures to be presented to a given olfactory receptor. Proteomic analysis of pig nasal mucus revealed that the olfactory secretome (Nagnan-Le Meillour et al, 2014) is mainly composed of around 30 OBP-, VEG-(Von Ebner's Gland protein), and SAL-(Salivary Lipocalin) isoforms, resulting from post-translational modifications (PTMs) of these three gene products and variants. The pig genome contains three genes coding for odorant-binding proteins: (i) the OBP gene sensu stricto, (ii) a second OBP transcript encoded by the porcine VEG (Von Ebner's gland protein) gene, LCN1, (iii) a third OBP, the salivary lipocalin (SAL) encoded by SAL1 gene.…”

“…OBP and LCN1 genes have two transcripts (OBPX1 and OBPX2, VEG-VNO and VEG-RM, respectively), whereas SAL1 leads to a single transcript. In addition to their splicing variants, protein isoforms for these three OBP genes vary between tissues, and in the nasal mucus, around 9 protein isoforms have been previously identified for VEG, 7 for SAL and 12 for OBP (Nagnan-Le Meillour et al, 2014), due to post-translational modifications targeting the secreted proteins. Immunodetection with specific antibodies indicated that only OBP sensu stricto isoforms are modified by phosphorylation and O-linked-β-N-acetylglucosaminylation (O-GlcNAcylation).…”

“…The new enzyme has-for obvious reasons-been named "EGF-domain specific O-GlcNAc transferase, " even if its substrate specificity has not been fully investigated. Although the EOGT gene is well conserved throughout evolution (Nagnan-Le Meillour et al, 2014), the role of extracellular O-GlcNAcylation in Mammals remains to be elucidated, despite identification of an increasing number of modified proteins (Alfaro et al, 2012;Hoffman et al, 2012). A first opening on possible roles of this process is the report of a mutation in the EOGT gene associated with a phenotype of Adams-Oliver syndrome, a human congenital disorder (Shaheen et al, 2013).…”