Binding specificity of recombinant odorant-binding protein isoforms is driven by phosphorylation.
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.
Brimau
Fanny
F
INRA, UMR8576 CNRS/USTL, UGSF, 59655, Villeneuve d'Ascq, France.
Cornard
Jean-Paul
JP
Le Danvic
Chrystelle
C
Lagant
Philippe
P
Vergoten
Gerard
G
Grebert
Denise
D
Pajot
Edith
E
Nagnan-Le Meillour
Patricia
P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D008024:Ligands; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018035:Receptors, Odorant / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine
D008024:Ligands; D020033:Protein Isoforms; D018035:Receptors, Odorant; D011994:Recombinant Proteins; C045648:odorant-binding protein