Skip to main content

Advertisement

SpringerLink
Log in

Phosphorylation of Native Porcine Olfactory Binding Proteins

  • Published:
Journal of Chemical Ecology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Blom, N., Gammeltoft, S., and Brunak, S. 1999. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294:1351–1362.

    Article  PubMed  CAS  Google Scholar 

  • Comer, F. I. and Hart, G. W. 2000. O-glycosylation of nuclear and cytosolic proteins. Dynamic interplay between O-GlcNac and O-phosphate. J. Biol. Chem. 275:29179–29182.

    Article  PubMed  CAS  Google Scholar 

  • Dal Monte, M., Centini, M., Anselmi, C., and Pelosi, P. 1993. Binding of selected odorants to bovine and porcine odorant-binding proteins. Chem. Senses 18:713–721.

    Article  CAS  Google Scholar 

  • Ganni, M., Garibotti, M., Scaloni, A., Pucci, P., and Pelosi, P. 1997. Microheterogeneity of odorant-binding proteins in the porcupine revealed by N-terminal sequencing and mass spectrometry. Comp. Biochem. Physiol. 117B:287–291.

    CAS  Google Scholar 

  • Guiraudie, G., Pageat, P., Cain, A. H., Madec, I., and Nagnan-Le Meillour, P. 2003. Functional characterization of olfactory binding proteins for pig appeasing compounds and molecular cloning in the vomeronasal organ of pre-pubertal pigs. Chem. Senses 28:609–619.

    Article  PubMed  CAS  Google Scholar 

  • Hérent, M. F., Collin, S., and Pelosi, P. 1995. Affinities of nutty and green-smelling compounds to odorant-binding proteins. Chem. Senses 20:601–610.

    Article  PubMed  Google Scholar 

  • Jordan, P., Heid, H., Kinzel, V., and Kubler, D. 1994. Major cell surface-located protein substrates of an ecto-protein kinase are homologs of known nuclear proteins. Biochemistry 33:14696–14706.

    Article  PubMed  CAS  Google Scholar 

  • Le Danvic, C., Guiraudie-Capraz, G., Abderrahmani, D., Zanetta, J. P., and Nagnan-Le Meillour, P. 2009. Natural ligands of porcine olfactory binding proteins. J. Chem. Ecol. In press (doi: 10.1007/s10886-009-9645-1)

  • Mann, M., Ong, S. E., Gronborg, M., Steen, H., Jensen, O. N., and Pandey, A. 2002. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. TRENDS Biotechnol. 20:261–268.

    Article  PubMed  CAS  Google Scholar 

  • Nagnan-Le Meillour, P., Lagant, P., Cornard, J. P., Brimau, F., Le Danvic, C., Vergoten, G., and Michalski, J. C. 2009. Phenylalanine 35 and tyrosine 82 are involved in the uptake and release of ligand by porcine odorant binding protein. Biochim. Biophys. Acta. 1794:1142–1150. (doi: 10.1016/j.bbapap.2009.04.012)

  • Nath, D., Maiti, A., and Majumder, G. C. 2008. Cell surface phosphorylation by a novel ecto-protein kinase: a key regulator of cellular functions in spermatozoa. Biochim. Biophys. Acta 1778:153–165.

    Article  PubMed  CAS  Google Scholar 

  • Paolini, S., Scaloni, A., Amoresano, A., Marchese, S., Napolitano, E., and Pelosi, P. 1998. Amino acid sequence, post-translational modifications, binding and labelling of porcine odorant-binding protein. Chem. Senses 23:689–698.

    Article  PubMed  CAS  Google Scholar 

  • Pelosi, P. 2001 The role of perireceptor events in vertebrate olfaction. Cell. Mol. Life Sci. 58:503–509.

    Article  PubMed  CAS  Google Scholar 

  • Tegoni, M., Pelosi, P., Vincent, F., Spinelli, S., Campanacci, V., Grolli, S., Ramoni, R., and Cambillau, C. 2000. Mammalian odorant binding proteins. Biochim. Biophys. Acta 1482:229–240.

    PubMed  CAS  Google Scholar 

  • Wells, L., Vosseler, K., Cole, R. N., Cronshaw, J. M., Matunis, M. J., and Hart, G. W. 2002. Mapping sites of O-GlcNac modification using affinity tags for serine and threonine post-translational modifications. Mol. Cell. Proteomics 1:791–804.

    Article  PubMed  CAS  Google Scholar 

  • Whelan, S. A. and Hart, G. W. 2006. Identification of O-GlcNac sites on proteins. Methods Enzymol. 415:113–133.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We warmly thank Dr Frédéric Lévy for his skillful dissection of pig RM, and Professor Paolo Pelosi for his kind gift of anti-VEG antibodies. We are also grateful to Michael Thant and Djamel Abderrahmani for their contribution to preliminary experiments. We thank two anonymous referees and Dr. S.J. Seybold for their help to improve the manuscript.

Author information

Authors and Affiliations

  1. INRA, UMR 8576 CNRS/Université Lille1, Unité de Glycobiologie Structurale et Fonctionnelle, F-59655, Villeneuve d’Ascq, Cedex, France

    Patricia Nagnan-Le Meillour, Chrystelle Le Danvic & Fanny Brimau

  2. UNCEIA, 149, rue de Bercy, F-75595, Paris, Cedex 12, France

    Chrystelle Le Danvic

  3. INRA, UMR 6175 INRA/CNRS/Université François Rabelais/Haras Nationaux, Physiologie de la Reproduction et des Comportements, F-37380, Nouzilly, France

    Fanny Brimau & Philippe Chemineau

  4. INSERM, UMR 8576 CNRS/Université Lille1, Unité de Glycobiologie Structurale et Fonctionnelle, F-59655, Villeneuve d’Ascq, Cedex, France

    Jean-Claude Michalski

Authors
  1. Patricia Nagnan-Le Meillour
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chrystelle Le Danvic
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fanny Brimau
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Philippe Chemineau
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jean-Claude Michalski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Patricia Nagnan-Le Meillour.

Additional information

Chrystelle Le Danvic and Fanny Brimau contributed equally to the work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary data Fig. 1

Monoisotopic mass spectrum of MALDI-TOF MS analysis of carboxymethylated band slice containing pOBP and VEG (SDS-PAGE) of the pig, Sus scrofa, respiratory mucosa (RM) extract after CT treatment and BEMAD. A) Peptides eluting with 25% acetonitrile; B) Peptides eluting with 50% acetonitrile (GIF 6 kb)

Supplementary data Fig. 1

High resolution image file (TIF 6 kb)

Supplementary data Fig. 2

Monoisotopic mass spectrum of MALDI-TOF MS analysis of carboxymethylated band slice containing pOBP and VEG (SDS-PAGE) of the pig, Sus scrofa, respiratory mucosa (RM) extract after T + CT treatment and BEMAD. Peptides eluting with 25% acetonitrile (GIF 6 kb)

Supplementary data Fig. 2

High resolution image file (TIF 6 kb)

Supplementary data Fig. 3

Monoisotopic mass spectrum of MALDI-TOF MS analysis of carboxymethylated band containing pOBP and pVEG (SDS-PAGE) of the pig, Sus scrofa, respiratory mucosa (RM) after T treatment and BEMAD. A) Peptides eluting with 25% acetonitrile. The following peaks correspond to unmodified peptides generated by the digestion with trypsin: OBP- 138-157 (m/z = 2240.0447), VEG- 21–42 (m/z = 2351.1283), and 21–42 MSO:22 (m/z = 2367.1328). DTT-modified peptides are listed in Table 2. B) Peptides eluting with 50% acetonitrile. The following peaks correspond to unmodified peptides generated by the digestion with trypsin: OBP- 121–133 (m/z = 1537.6552), 1–15 PYRR:1 (m/z = 1711.7432), and 138–157 (m/z = 2239.9551). DTT-modified peptides are listed in Table 2 (GIF 6 kb)

Supplementary data Fig. 3

High resolution image file (TIF 6 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nagnan-Le Meillour, P., Le Danvic, C., Brimau, F. et al. Phosphorylation of Native Porcine Olfactory Binding Proteins. J Chem Ecol 35, 752–760 (2009). https://doi.org/10.1007/s10886-009-9663-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10886-009-9663-z

Keywords

Search

Navigation