MODULATION OF CRYSTAL-FORMATION BY BONE PHOSPHOPROTEINS - STRUCTURAL SPECIFICITY OF THE OSTEOPONTIN-MEDIATED INHIBITION OF HYDROXYAPATITE FORMATION

Osteopontin is a phosphorylated sialoprotein containing a conserved se quence of contiguous aspartic acid residues. This protein is expressed at high levels in mineralized tissues and has previously been shown t o inhibit the in vitro formation of hydroxyapatite (HA). In the presen t study, protein modification and model compound studies have been use d to identify the structural features of osteopontin that are responsi ble for its crystal-modulating properties. Using metastable calcium ph osphate solutions buffered by autotitration, osteopontin caused half-m aximal inhibition of HA formation at a concentration (IC50) of 0.06 mu g/ml. The hen egg yolk phosphoprotein phosvitin was a much weaker inh ibitor, while dextran sulphate had no effect. The synthetic polypeptid e poly(aspartic acid) was almost as effective an inhibitor of HA forma tion as osteopontin (IC50 0.11 mu g/ml), whereas poly(glutamic acid) w as more than a thousand times less potent (IC50 155 mu g/ml). In a ste ady-state agarose gel system, much higher polypeptide concentrations w ere required for inhibition of HA formation, but a similar relative or der of inhibitory effectiveness was observed. Treatment of osteopontin with alkaline phosphatase removed 84% of the covalently bound phospha te and reduced its HA-inhibiting activity by more than 40-fold. Treatm ent with glycine ethyl ester in the presence of carbodi-imide modified 86% of the carboxylate groups in osteopontin and reduced its inhibito ry activity by 6-fold. These findings indicate that osteopontin is a p otent inhibitor of HA formation. This activity requires phosphate and carboxylate groups, possibly including the conserved sequence of conti guous aspartic acid residues. Osteopontin may act as an inhibitor of p hase separation in physiological fluids of high supersaturation.