title: Osteopontin binds multiple calcium ions with high affinity and independently of phosphorylation status.
abstract: Osteopontin (OPN) is an acidic, intrinsically disordered extracellular matrix protein with a capacity to modulate biomineralization in vitro and in vivo. The role of posttranslational modification of osteopontin has been intensively studied. Phosphorylation of OPN has been demonstrated to play a role in inhibition of biomineral formation and growth in vitro. Here, we used isothermal titration calorimetry (ITC) to investigate the ability of OPN to bind the divalent cations Ca(2+) and Mg(2+), both essential components of inorganic minerals in vivo. We found, that bovine OPN binds ~10 Ca(2+) ions with an apparent affinity ~50-fold tighter than Mg(2+), both regardless of OPN phosphorylation, and with affinities significantly stronger than previously reported. These results were confirmed using human derived OPN. This implies that a majority of the acidic residues within OPN must be engaged in calcium interaction under physiological conditions.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Denmark; Department of Biomedicine, Aarhus University, Denmark.
authors.forename: Eva
authors.initials: E
authors.lastname: Kläning
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Denmark.
authors.forename: Brian
authors.initials: B
authors.lastname: Christensen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Denmark.
authors.forename: Esben S
authors.initials: ES
authors.lastname: Sørensen
authors.affiliation: Department of Biomedicine, Aarhus University, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Vorup-Jensen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Denmark; Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Denmark. Electronic address: jkj@mb.au.dk.
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002151:Calorimetry; D002413:Cations, Divalent / Q000378:metabolism; D002417:Cattle; D004492:Edetic Acid / Q000378:metabolism; D006801:Humans; D007477:Ions; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D053495:Osteopontin / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D013816:Thermodynamics
chemical_list: D002413:Cations, Divalent; D007477:Ions; D053495:Osteopontin; D004492:Edetic Acid; D008274:Magnesium; D002118:Calcium
keywords: Calcium; EDTA; Intrinsically disordered; Magnesium; Metal affinity; Milk protein