Characterization of UO₂²⁺binding to osteopontin, a highly phosphorylated protein: insights into potential mechanisms of uranyl accumulation in bones

Abstract

Bones are one of the few organs in which uranyl (UO2(2+)) accumulates. This large dioxo-cation displays affinity for carboxylates, phenolates and phosphorylated functional groups in proteins. The noncollagenous protein osteopontin (OPN) plays an important role in bone homeostasis. It is mainly found in the extracellular matrix of mineralized tissues but also in body fluids such as milk, blood and urine. Furthermore, OPN is an intrinsically disordered protein, which, like other proteins of the SIBLING family, contains a polyaspartic acid sequence and numerous patterns of alternating acidic and phosphorylated residues. All these properties led to the hypothesis that this protein could be prone to UO2(2+) binding. In this work, a simple purification procedure enabling highly purified bovine (bOPN) and human OPN (hOPN) to be obtained was developed. Various biophysical approaches were set up to study the impact of phosphorylations on the affinity of OPN for UO2(2+) as well as the formation of stable complexes originating from structural changes induced by the binding of this metal cation. The results obtained suggest a new mechanism of the interaction of UO2(2+) with bone metabolism and a new role for OPN as a metal transporter.

Phosphate‐Rich Biomimetic Peptides Shed Light on High‐Affinity Hyperphosphorylated Uranyl Binding Sites in Phosphoproteins

“…This effect was also suggested by previous studies with OPN. Indeed, whereas UO 2 2+ interaction is highly dependent on the phosphorylation status of the protein, Ca 2+ binding is not. Therefore, it was proposed that Ca 2+ is bound in an oxygen‐rich site, mostly by acidic residues .…”

“…OPN has 26±1 phosphorylation sites and can complex up to six UO 2 2+ cations at physiological pH. Once dephosphorylated, its affinity toward UO 2 2+ drops 10‐ to 100‐fold . A phosphorylated group was confirmed to be involved in the complexation of UO 2 2+ by an OPN fragment by extended X‐ray absorption fine structure (EXAFS) analyses …”

Evidence of isotopic fractionation of natural uranium in cultured human cells

“…Therefore, we can hypothesize the transport of U(VI)-carbonate complexes into cells through the formation of a complex with a transport protein, involving the exchange of carbonate(s) for stronger ligand(s). Indeed, the high affinity of U(VI) for certain proteins has been recently demonstrated (62)(63)(64). Using the total energies of U(VI) compounds reported in literature for theoretical calculations of fractionation factors associated with an NFS effect for the reduction of [UO 2 L 4 ] 2− into UL 4 (65), we performed calculations for ligand exchange reactions in [UO 2 L 4 ] 2− species (L = F − , Cl − , and Br − ).…”

Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides

“…Although they are known to rapidly circulate and deposit into major organs such as bone, liver, or kidney after contamination (6,(8)(9)(10), the specific molecular mechanisms associated with mammalian uptake of these toxic heavy elements remain largely unexplored. Proposed mammalian actinide acquisition and transport mechanisms have typically focused on proteins that use conserved motifs to directly bind the essential elements iron or calcium (6,8,(10)(11)(12)(13), such as transferrin (14)(15)(16)(17)(18), ferritin (13), osteopontin (19), or fetuin (20). Siderocalin (Scn), an essential antibacterial protein that sequesters iron (21,22), and an important component of iron trafficking (23), is distinct in that it binds ferric iron indirectly, through tight complexes with a siderophore or siderophore-derived chelator.…”