Abstract
We study the effect of isoforms of osteopontin (OPN) on the nucleation and growth of crystals from a supersaturated solution of calcium and phosphate ions. Dynamic light scattering is used to monitor the size of the precipitating particles and to provide information about their concentration. At the ion concentrations studied, immediate precipitation was observed in control experiments with no osteopontin in the solution, and the size of the precipitating particles increased steadily with time. The precipitate was identified as hydroxyapatite by X-ray diffraction. Addition of native osteopontin (nOPN) extracted from rat bone caused a delay in the onset of precipitation and reduced the number of particles that formed, but the few particles that did form grew to a larger size than in the absence of the protein. Recombinant osteopontin (rOPN), which lacks phosphorylation, caused no delay in initial calcium phosphate precipitation but severely slowed crystal growth, suggesting that rOPN inhibits growth but not nucleation. rOPN treated with protein kinase CK2 to phosphorylate the molecule (p-rOPN) produced an effect similar to that of nOPN, but at higher protein concentrations and to a lesser extent. These results suggest that phosphorylations are critical to OPN’s ability to inhibit nucleation, whereas the growth of the hydroxyapatite crystals is effectively controlled by the highly acidic OPN polypeptide. This work also demonstrates that dynamic light scattering can be a powerful tool for delineating the mechanism of protein modulation of mineral formation.
Highlights
Biomineralization is the process by which living organisms produce the minerals that make up hard tissues such as shell, bone, and teeth [1]
We have used dynamic light scattering to study the effects of OPN and related proteins on the precipitation of calcium phosphate particles from solution
We found that native osteopontin (nOPN) strongly inhibits the nucleation of calcium phosphate particles and induces a delay in the appearance of precipitate which increases with increasing protein concentration
Summary
Biomineralization is the process by which living organisms produce the minerals that make up hard tissues such as shell, bone, and teeth [1]. The details remain incompletely understood, but certain proteins are believed to play central roles both in the initial nucleation of biomineral crystals and in the regulation of their subsequent growth, with some proteins acting to inhibit nucleation and growth and others to enhance it [2,3,4]. There is substantial motivation for the development of a more complete understanding of the biomineralization process and of the regulatory roles played by specific proteins. The extracellular fluid in vertebrates is supersaturated with respect to hydroxyapatite and other calcium phosphates, but spontaneous precipitation is inhibited by regulatory proteins. Biomineralized HA can form both plate-like and rod-like crystals, depending on the environment, with the crystal structure again controlled by proteins [3]. Natural bone mineral includes a significant fraction of ionic substitutions and the corresponding structural imperfections, HA itself is typically used as a model for biological mineralization
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