Osteopontin is a phosphorylated sialoprotein containing a conserved sequence of contiguous aspartic acid residues. This protein is expressed at high levels in mineralized tissues and has previously been shown to inhibit the in vitro formation of hydroxyapatite (HA). In the present study, protein modification and model compound studies have been used to identify the structural features of osteopontin that are responsible for its crystal-modulating properties. Using metastable calcium phosphate solutions buffered by autotitration, osteopontin caused half-maximal inhibition of HA formation at a concentration (IC50) of 0.06 microgram/ml. The hen egg yolk phosphoprotein phosvitin was a much weaker inhibitor, while dextran sulphate had no effect. The synthetic polypeptide poly(aspartic acid) was almost as effective an inhibitor of HA formation as osteopontin (IC50 0.11 microgram/ml), whereas poly(glutamic acid) was more than a thousand times less potent (IC50 155 micrograms/ml). In a steady-state agarose gel system, much higher polypeptide concentrations were required for inhibition of HA formation, but a similar relative order of inhibitory effectiveness was observed. Treatment of osteopontin with alkaline phosphatase removed 84% of the covalently bound phosphate and reduced its HA-inhibiting activity by more than 40-fold. Treatment with glycine ethyl ester in the presence of carbodi-imide modified 86% of the carboxylate groups in osteopontin and reduced its inhibitory activity by 6-fold. These findings indicate that osteopontin is a potent inhibitor of HA formation. This activity requires phosphate and carboxylate groups, possibly including the conserved sequence of contiguous aspartic acid residues. Osteopontin may act as an inhibitor of phase separation in physiological fluids of high supersaturation.

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