Characterization of osteopontin-uranyl interaction: role of multiple phosphorylations

While some metals are essential for Life, other ones are only toxicants for living organisms, tolerated below well-definite concentrations. This is the case for uranium, a natural element which has no known biological function. It is a low α emitter and its chemical toxicity rather than its radiological toxicity is a subject of concern. Once in the body, this metal reaches the blood and accumulates in the bones under the action of unknown mechanisms. Uranium mainly exists in form of uranyl ion (UO22+) in aqueous media and particularly reacts with carboxylates, phenolates and phosphates of the proteins. Previous studies have highlighted that UO22+ modulates the SPP1 expression, a gene which codes for osteopontin (OPN). This highly phosphorylated glycoprotein plays an important role in bone homeostasis. This role and its biochemical properties led us to hypothesize that OPN might be a potential target of UO22+ and involved in its accumulation in bones. A simple and original purification process was optimized to produce very highly purified OPN starting from human and bovine milk. Various biophysical approaches were set up and confirmed that both bovine and human OPN display very high affinity for UO22+. Moreover, the formation of stable UO2-protein complexes originating from structural changes was evidenced. The major role of phosphorylations, both on the OPN's affinity for UO22+ and the stability of the UO2-protein complexes, was confirmed. These results demonstrate that OPN presents all the characteristics to be a major UO22+ binding-protein in vitro, and they open new insights in the understanding of the UO22+ mineralization process mechanisms. (author)