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

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Abstract

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.

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Acknowledgments

The authors thank the University of Lille1 (USTL), the French INRA (Institut National de la Recherche Agronomique), and CNRS (Centre National de la Recherche Scientifique) for funding.

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Authors and Affiliations

  1. INRA, UMR8576 CNRS/USTL, UGSF, 59655, Villeneuve d’Ascq, France

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

  2. UMR8516 CNRS/USTL, LASIR, 59655, Villeneuve d’Ascq, France

    Jean-Paul Cornard

  3. CNRS, UMR8576 CNRS/USTL, UGSF, 59655, Villeneuve d’Ascq, France

    Philippe Lagant & Gerard Vergoten

  4. INRA, UR1197 NOeMI, 78350, Jouy-en-Josas, France

    Denise Grebert & Edith Pajot

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  1. Fanny Brimau
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Correspondence to Patricia Nagnan-Le Meillour.

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Supplemental Data Fig. S1

Monoisotopic mass spectra of MALDI-TOF MS analysis of carboxymethylated band (SDS-PAGE) containing OBP-Pichia-iso2 after Beta-Elimination followed by Michael Addition of Dithiothreitol (BEMAD, DTT) with T+CT treatment. a) Peptide elution with acetonitrile 25% and b) Peptide elution with acetonitrile 50%. (GIF 123 kb)

High resolution image (TIFF 65 kb)

Supplemental Data Fig. S2

Monoisotopic mass spectra of MALDI-TOF MS analysis of carboxymethylated band (SDS-PAGE) containing OBP-Pichia-iso2 after Beta-Elimination followed by Michael Addition of Dithiothreitol (BEMAD, DTT) with CT treatment. Peptide elution with acetonitrile 50%. (GIF 116 kb)

High resolution image (TIFF 57 kb)

Supplemental Data Fig. S3

Monoisotopic mass spectrum of MALDI-TOF MS analysis of carboxymethylated band (SDS-PAGE) containing OBP-Pichia-iso2 after Beta-Elimination followed by Michael Addition of Dithiothreitol (BEMAD, DTT) with T treatment. a) Peptide elution with acetonitrile 25% and b) Peptide elution with acetonitrile 50%. (GIF 130 kb)

High resolution image (TIFF 66 kb)

Supplemental Data Fig. S4

Monoisotopic mass spectra of MALDI-TOF MS analysis of carboxymethylated band (SDS-PAGE) containing OBP-Pichia-iso3 after Beta-Elimination followed by Michael Addition of Dithiothreitol (BEMAD, DTT)with T+CT treatment. a) Peptide elution with acetonitrile 25% and b) Peptide elution with acetonitrile 50%. (GIF 106 kb)

High resolution image (TIFF 66 kb)

Supplemental Data Fig. S5

Monoisotopic mass spectra of MALDI-TOF MS analysis of carboxymethylated band (SDS-PAGE) containing OBP-Pichia-iso3 after Beta-Elimination followed by Michael Addition of Dithiothreitol (BEMAD, DTT) with CT treatment. a) Peptide elution with acetonitrile 25% and b) Peptide elution with acetonitrile 50%. (GIF 133 kb)

High resolution image (TIFF 64 kb)

Supplemental Data Fig. S6

Monoisotopic mass spectrum of MALDI-TOF MS analysis of carboxymethylated band (SDS-PAGE) containing OBP-Pichia-iso3 after Beta-Elimination followed by Michael Addition of Dithiothreitol (BEMAD, DTT) with T treatment. Peptides elution with acetonitrile 50%.) (GIF 82 kb)

High resolution image (TIFF 54 kb)

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Brimau, F., Cornard, JP., Le Danvic, C. et al. Binding Specificity of Recombinant Odorant-Binding Protein Isoforms is Driven by Phosphorylation. J Chem Ecol 36, 801–813 (2010). https://doi.org/10.1007/s10886-010-9820-4

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  • DOI: https://doi.org/10.1007/s10886-010-9820-4

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