Many proteins found in mineralized tissues have been proposed to function as regulators of the mineralization process, either as nucleators or inhibitors of hydroxyapatite (HA) formation. We have studied the HA-nucleating and HA-inhibiting properties of proteins from bone [osteocalcin (OC), osteopontin (OPN), osteonectin (ON) and bone sialoprotein (BSP)], dentine [phosphophoryn (DPP)] and calcified cartilage [chondrocalcin (CC)] over a wide range of concentrations. Nucleation of HA was studied with a steady-state agarose gel system at sub-threshold [Ca] x [PO4] product. BSP and DPP exhibited nucleation activity at minimum concentrations of 0.3 microgram/ml (9 nM) and 10 micrograms/ml (67 nM) respectively. OC, OPN, ON and CC all lacked nucleation activity at concentrations up to 100 micrograms/ml. Inhibition of HA formation de novo was studied with calcium phosphate solutions buffered by autotitration. OPN was found to be a potent inhibitor of HA formation [IC50 = 0.32 microgram/ml (0.01 microM)] whereas OC was of lower potency [IC50 = 6.1 micrograms/ml (1.1 microM)]; BSP, ON and CC all lacked inhibitory activity at concentrations up to 10 micrograms/ml. The effect of OPN on HA formation de novo is mainly to inhibit crystal growth, whereas OC delays nucleation. These findings are consistent with the view that BSP and DPP may play roles in the initiation of mineralization in bone and dentine respectively. OPN seems to be the mineralized tissue protein most likely to function in the inhibition of HA formation, possibly by preventing phase separation in tissue fluids of high supersaturation.

Bone sdaloprotein (BSP) and osteopontin, the major phosphorylated proteins of mammalin bone, have been proposed to finctIon in the initiation of mineralization. To test this hypothes, the effects ofBSP and osteopontin on hydroxyaatite crystdal formation were determied by usig a steadystate agarose gel system. At low calcium phosphate concentrations, no accmulatio of calcium and phosphate ocurred in control gels or gels cotinin osteopontin. Gels containing BSP at 1-5 pg/mn, however, exhibited a vidble precipitation band and sg ay elevated Ca + P04 contents. By powder x-ray diffrcion, the precipitate formed in the presence of BSP was shown to be hydroxyapatite. These ings es that bone ialoprotein may be involved in the nucleation of hydroxyapatite at the mineralzation front of bone.The mineralization of bone occurs by deposition of carbonated hydroxyapatite (HA) crystals in an extracellular matrix consisting of type I collagen and a variety of noncollagenous proteins. The means by which bone mineralization is achieved is not understood, but several authors have suggested that HA could be nucleated by a complex composed of a polyanionic protein, probably a phosphoprotein, and the large highly ordered fibrils oftype I collagen (1-4). The major phosphoproteins of mammalian bone are bone sialoprotein (BSP) and osteopontin (5). Both are phosphorylated sialoproteins containing tyrosine sulfates, regions enriched in acidic amino acids, and an Arg-Gly-Asp cell attachment sequence. Although osteopontin and BSP do not appear to be evolutionarily related, they have similar amino acid compositions and post-transcriptional modifications. However, BSP contains more sialic acid and sulfate, whereas osteopontin contains more phosphate (5).These proteins also differ in their biological properties. By immunocytochemistry, in situ hybridization, and Northern blotting, it has been shown that osteopontin is expressed at high levels in mineralized connective tissues but also at lower levels in other tissues, including kidney, nervous tissue, and uterus; BSP, in contrast, is specific to mineralized tissues (6-8). During embryogenesis, BSP is first expressed at the onset of bone formation, whereas osteopontin expression is a later event (7). In mineralizingbone cell and organ cultures, BSP associates with the osteoid matrix, whereas osteopontin is released into the culture medium (9, 10). For these reasons, Sodek and coworkers (7) have suggested that BSP is a strong candidate for a role in mineral nucleation.In the present study, the effects of BSP and osteopontin on HA formation have been studied by using a steady-state agarose gel system. METHODSPurfication of BSP and Osteopontin from Porcine Calvaria.BSP and osteopontin were purified from adult porcine cal- varia by fast protein liquid chromatography using ionexchange, HA, and gel-fltration columns as previously described (11). The identity and purity of the protein preparations used were ascertained by gel electrophoresis, Western blotting using affimity-purified antibodies,...

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.

Bone sialoprotein (BSP) is a bone-specific glycoprotein containing phosphoserine and sulphotyrosine residues and regions of contiguous glutamic acid residues. Recent studies in this laboratory have shown that BSP is capable of nucleating the bone mineral hydroxyapatite in a steady-state agarose gel system. We show here that chemical modification of carboxylate groups abolishes the nucleation activity of BSP, but enzymic dephosphorylation has no effect. Formation of hydroxyapatite is also induced by poly(L-glutamic acid) and poly(D-glutamic acid), but not by poly(L-aspartic acid) or poly(L-lysine). Calreticulin, a muscle protein with short sequences of contiguous glutamic acid residues, also lacks nucleation activity. These findings suggest that the nucleation of hydroxyapatite by BSP involves one or both of the glutamic acid-rich sequences. Based on these findings and others, we propose that polycarboxylate sequences represent a general site for growth-modulating interactions between proteins and biological crystals.

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