Role of calcium phosphate nanoclusters in the control of calcification Carl Holt 1 , Esben S. Sørensen 2 and Roger A. Clegg 1 1 Hannah Research Institute, Ayr, UK 2 Protein Chemistry Laboratory, Department of Molecular Biology, University of A ˚ rhus, Denmark Many biological fluids, including blood, milk, extracel- lular fluid, saliva, urine, synovial fluid and cerebrospi- nal fluid, are usually supersaturated with respect to hydroxyapatite (HA) [1–5], but generally remain stable. Nevertheless, dystrophic calcification does occur, and vascular calcification or stone-forming biofluids, for example, have serious consequences for human health. Genetic ablation and other experiments on individual serum proteins have demonstrated the importance of serum fetuin A (FETUA), osteopontin (OPN) and matrix Gla protein (MGP) for inhibiting the precipita- tion of calcium phosphate (CaP) in serum and prevent- ing ectopic calcification of soft tissues [6–8]. A metastable, colloidal, complex of CaP with FETUA, MGP and secretory phosphoprotein 24 (SPP-24) forms when the serum is destabilized [9,10], but the physio- logical mechanism is still unclear. Milk provides an example of a biofluid that seldom forms CaP precipitates or causes dystrophic calcifica- tion of the mammary gland, even though it may con- tain very much higher concentrations of calcium (Ca) and inorganic phosphorus (P i ) than does serum [11]. In milk, casein micelles sequester CaP through phos- phate centre (PC) sequences, typically pSpSpSEE, in Keywords casein; dentin matrix acidic phosphoprotein 1; fetuin; natively unfolded protein; osteopontin Correspondence C. Holt, 47 Logan Drive, Troon KA10 6PN, UK Tel: +44 1292 317 615 E-mail: cholt002@udcf.gla.ac.uk (Received 21 November 2008, revised 17 January 2009, accepted 11 February 2009) doi:10.1111/j.1742-4658.2009.06958.x Calcium phosphate nanoclusters are equilibrium particles of defined chemi- cal composition in which a core of amorphous calcium phosphate is sequestered within a shell of casein phosphopeptides. Sequence analyses and a structure prediction method were applied to secreted phosphopro- teins of known importance in controlling calcification, and eight noncasein phosphoproteins were identified as containing one or more subsequences capable of forming nanoclusters. Small-angle X-ray scattering was used to confirm that a plasmin phosphopeptide of one of the identified proteins, osteopontin, formed a novel type of calcium phosphate nanocluster in which the radius of the amorphous calcium phosphate core was four times larger than is typical of casein nanoclusters. A thermodynamic treatment of nanocluster formation identified the factors of importance in determin- ing the equilibrium size of the core, and showed how a nanocluster solution could be thermodynamically stable yet supersaturated with respect to the mineral phase of bones and teeth. It is suggested that the ability of some secreted phosphoproteins to form nanoclusters is physiologically important for the control or inhibition of calcification in soft and mineralized tissues, the extracellular matrix and a wide range of biofluids, including milk and blood. Abbreviations ACP, amorphous calcium phosphate; CaP, calcium phosphate; CPN, calcium phosphate nanocluster; DCPD, di-calcium phosphate di-hydrate; DMP1, dentin matrix acidic phosphoprotein 1; FETUA, fetuin A; HA, hydroxyapatite; MGP, matrix Gla protein; OCP, octacalcium phosphate; OPN, osteopontin; PC, phosphate centre; pS, phosphoseryl residue; RBP, riboflavin-binding protein; SAXS, small-angle X-ray scattering; SCPP, secretory calcium-binding phosphoprotein; SP, secreted phosphoprotein; SPP-24, secretory phosphoprotein 24. 2308 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS a S1 -, a S2 - and b-caseins. Understanding the sequestra- tion process has been furthered through studies with short casein phosphopeptides containing a PC. Thus, the 25-residue N-terminal b-casein tryptic phosphopep- tide (b-casein 1–25) sequestered CaP to form a calcium phosphate nanocluster (CPN) [12–14] with a core of amorphous, acidic and hydrated calcium phosphate (ACP) of radius 2.4 nm surrounded by a shell of about 50 phosphopeptides with a thickness of 1.6 nm. Ini- tially, it was thought that the CPNs were metastable particles in a state of arrested precipitation, but it was later shown that they were equilibrium particles with a defined composition, size and structure. Most signifi- cantly, they formed spontaneously when the phospho- peptide was added to a pre-existing precipitate of ACP. There is abundant evidence from infrared spectroscopy, X-ray absorption spectroscopy, X-ray and high-resolution electron diffraction and solid state 31 P-NMR spectroscopy that micellar CaP and the core CaP of CPNs are amorphous. Thus, in terms of size, structure, solubility and dynamics, the micellar CaP and core CaP of CPNs appear to be very similar [12–20]. The primary purposes of this investigation were to provide a deeper understanding of the thermodynamics of CaP sequestration and to define more closely the structural characteristics of the phosphoproteins responsible. A second aim was to identify a group of proteins with the sequence and conformation predicted to be needed for CaP sequestration and to undertake an experimental test of the prediction for one of them. For the experimental work, OPN was selected because, unlike the caseins, it is expressed in a wide range of species, tissues and biofluids [21,22]. A successful dem- onstration would be a step towards establishing the broader physiological importance of CPN formation. OPN is a member of the same paralogous group as the caseins, called the secretory calcium-binding phos- phoproteins (SCPPs) [23,24]. Like the caseins, it has an unfolded conformation [25] and clustered sites of phos- phorylation [26], and among its many recognized func- tions is an involvement in the control of mineralization processes [21,22]. Results Thermodynamics of CPN formation Doc. S1 (see Supporting information) provides addi- tional details of the treatment. The chemical formula of an electroneutral CPN can be written as a multiple of an empirical formula, or ‘monomer’ containing a single PC: Ca R Ca H R H ðP i Þ R P ðH 2 OÞ R W ðPep À PCÞ 1 hi  j ð1Þ The average molar ratios of water, Ca and P i to PC are R W , R Ca and R P , respectively,  j is the average number of PCs in the CPN and Pep is the chemical for- mula of the peptide divided by the number of PCs it contains (f). The formula of the monomer can be further divided into an amorphous hydrated CaP and a sequestering ligand of calcium phosphopeptide. The empirical chemical formula of the electroneutral CaP is CaðHPO 4 Þ y ðPO 4 Þ 2À2y 3 :xðH 2 OÞð2Þ where 3y ⁄ (2 + y) is the mole fraction of P i in the di-anionic form. The empirical chemical formula of CaP can then be used to define a type of solubility constant K S as an ion activity product. In a dilute solution in which the activity of water is effectively unity: K S ¼ a 1 Ca 2þ a y HPO 2À 4 a ð2À2yÞ=3 PO 3À 4 ð3Þ K S can be used, just like the solubility product of a pure bulk phase, to calculate the extent of formation of CPNs. The association of CaP monomers generates an equilibrium distribution of core sizes, and it can be shown by a simple adaptation of the capillary theory of nucleation that an activity distribution results with a modal core radius of: r à core % 2kDG seq 3A core RTlnða 1 =a s Þ  3V core 4p  1=3 ¼ 2kDG seq À3A core DG o core  3V core 4p  1=3 ð4Þ where V core is the empirical formula volume of CaP, k ¼ð36pV 2 core Þ 1=3 , A core is the core surface area per PC, DG seq is the free energy of sequestration of the core by the shell of peptides, a 1 and a s are the activities of a CaP molecule in the nanocluster solution and in a solution saturated with respect to the bulk phase of core material, respectively, and DG  core is the free energy of formation of the bulk core phase. As r* must be a positive real number, two possible solutions exist. In classical nucleation theory, the sur- face energy and bulk free energy terms are positive; precipitation occurs from a supersaturated solution in which a 1 > a s . In the formation of CPNs, the effective surface energy is negative, and hence the solution is undersaturated with respect to the bulk phase of ACP (a 1 < a s ). C. Holt et al. Calcium phosphate sequestration by osteopontin FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2309 Stability and metastability in biofluids and the extracellular matrix Freshly formed ACP can be sequestered by phospho- peptides but, if the rate of ACP formation and matu- ration is faster than the rate of sequestration, the nanoclusters cannot form and a metastable solution results. Certain partial SP sequences have been identi- fied as the starting point of controlled crystal growth in the extracellular matrix of mineralized tissues. These include long phosphorylated sequences in, for example, phosphophoryn, the C-terminal sequence of OPN and the N-terminal sequence of dentin matrix acidic phos- phoprotein 1 (DMP1) [27,28] and long sequences of Glu residues in, for example, integrin-binding sialo- phosphoprotein II [29]. When a sequence that can sequester ACP and a sequence that can accelerate the maturation of ACP into HA are both present in a given SP, the competing reactions of ACP maturation and ACP sequestration may make the formation of CPNs as the equilibrium product more difficult or even impossible. The formation of the nanocluster solution requires not only that maturation of the ACP should be prevented, but also a stoichiometric excess of the phosphopeptide over CaP. If [p] molÆL )1 of P i can precipitate as ACP from the initially supersaturated solution, the condition for thermodynamic stability is a ¼ ½p f ½PPR P 1 ð5Þ where [PP] is the phosphopeptide concentration. Under these conditions, a is also the fraction of reacted PCs. Although a nanocluster solution is stable with respect to the formation of ACP, it remains supersatu- rated with respect to HA (Fig. 7C). HA has never been observed to nucleate directly from solution, but forms by a solution-mediated maturation of ACP [30] and, as the latter cannot form, the nanocluster solution is stable with respect to this phase also. Identification of sequestering phosphoproteins Identification of PCs in secreted phosphoproteins (SPs) The canonical PC used in the search was derived from the known casein PCs, and comprised a sequence of 10 or fewer consecutive residues containing at least three sites of phosphorylation, no Cys and fewer than three hydrophobic residues. Example PC sequences found in SPs with known involvement in mineralization are shown in Table 1, and aligned sequences of their orthologues are given in Doc. S2 (see Supporting infor- mation). Most of the identified SPs and all of the proven CPN-forming SPs are members of the SCPP paralogous group. Most PCs contain a block of consecutive phos- phorylation sites, followed by the primary recognition site of the casein kinase 2 or Golgi kinase. The longest block of consecutive sites of phosphorylation in a casein PC is in rat a S1 -casein with eight, with a ninth close by. Longer sequences of phosphorylated residues, such as those found in phosphophoryn and the C-terminal half of OPN and N-terminal part of DMP1, have been shown to promote the maturation of ACP into more crystalline phases, and so were discounted as CPN-forming sequences. A minor PC pattern involves three or more repeats of a primary kinase recognition triplet SXE (MGP) or SD[E,pS] (OPN). When the aligned orthologue sequences were examined (Doc. S2), it was found that not all PCs were conserved, particu- larly when a protein contained more than one PC. For example, the N-terminal half of bovine OPN contained all three PCs coded by exons 3, 5 and 6. The last two were not as highly conserved as the first, but none of the orthologues had fewer than two PCs. Table 1. Identified PC sequences formed by the action of the Golgi kinase and casein kinase 2 on selected secreted phosphoproteins. CSN1S1, a S1 -casein; CSN1S2, a S2 -casein; CSN2, b-casein; IBSP-II, integrin-binding sialophosphoprotein II; MEPE, matrix extracellular bone phosphoglycoprotein. Potential sites of phosphorylation are shown in bold. Protein Species Swiss-Prot No. PC a SCPPs OPN Cow P31096 6- TSSGSSEEKQ -15 42- QNSVSSEETD -51 99– SDESHHSDES -108 DMP1 Mouse O55188 8- NTESESSEER -17 28- PTNSESSEES -37 49- HTHSSESGEE -58 120-SADTTQSSED -129 142-SDSKDQDSED -151 161-DSAQDSESEE -170 CSN1S1 Guinea pig P04656 19- SSSSSSSEER -28 54- IISESTEERE -63 65- SSISSSEEV -73 CSN1S2 Pig P39036 5- EHVSSSEESI -14 54- ASSSSSEESV -63 130- ELSTSEEPVS-139 CSN2 Human P05814 5- ESLSSSEESI -14 IBSP-II Human P21815 55- GDDSSEEEEE -64 MEPE Human Q9NQ76 498- DS GSSSESDG -507 Non-SCPPs FETUA Human P02765 307- SLGSPSGEVS -316 SPP-24 Human Q13103 108-SSSTSESYSS -117 MGP Human P08493 2- ESHESMESYE -11 PRB4 Human P10163 2- SSSEDVSQEE -11 RBP Chicken P02752 192-ESSSMSSSEE -201 a Sequence numbers are for the mature peptide chain without the signal sequence. Calcium phosphate sequestration by osteopontin C. Holt et al. 2310 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS Conformation of secreted phosphoproteins containing PCs The PONDRÒ predictor is the oldest and most thor- oughly tested of the predictors of partial or complete disorder in proteins. It continues to perform well in comparative tests with more recent methods [31], and is one of the components in the most recent meta pre- dictor, metaPrDOS [32]. According to PONDRÒ pre- dictions, the positions of PC sequences in the SPs in Table 1 were, with the exception of the globular pro- tein riboflavin-binding protein (RBP), disordered, and had disordered flanking sequences (Fig. 1A,B). The PC motif of RBP was disordered and is undefined in the crystal structure [33], but its N-terminal flanking sequence was correctly predicted to be ordered. The prediction for FETUA indicated a folded N-terminal sequence containing the two cystatin-like domains, but a flexible C-terminal half in which the PC lies. The result for SPP-24 was the least clear-cut with only short disordered sequences flanking the PC. Essentially the same results were obtained by the top-idp predic- tor [34], with the notable exception that SPP-24 was borderline stable near the PC and stable in its flanking sequences (Fig. 1C), but the metaPrDOS predictor [32] agreed better with the PONDRÒ result for this protein (Fig. 1D). All methods were in agreement in showing that OPN has little or no stable conformation, and hence can be described as a worm-like, or rheomorphic [35], chain. With the exception of proline-rich protein 1, all other members of the SCPP paralogous group identi- fied by Kawasaki and Weiss [24,36] were predicted by PONDRÒ to be flexible over a substantial fraction of their total sequence (results not shown). Characterization of OPN and OPN 1–149 in free solution Small-angle X-ray scattering (SAXS) of OPN and OPN 1–149 Both OPN and OPN 1–149 showed the scattering pat- tern expected of a flexible but non-Gaussian chain with short, rod-like segments (Fig. 2). The average of three determinations of the radii of gyration of OPN and OPN 1–149 in the concentration range 5–15 mgÆmL )1 were 5.50 ± 0.17 and 2.17 ± 0.24 nm, respectively. The worm-like chain model fitted to the OPN SAXS gave b = 1.74 nm, which could correspond, for exam- ple, to an average of five to six residues temporarily arranged in a poly-l-proline II local helix. The lower chain stiffness of OPN 1–149 (Fig. 2) is possibly a result of the higher proportion of Pro residues in this part of the sequence (eight of the total of 13), each of which produces a sharp change in chain direction in the cis configuration, and of Gly residues (four of four), which allow markedly more chain flexibility than other residues because of their short side-chain. Apart from Asp, the other residues are present in similar pro- portions in the two halves of OPN. It is possible, therefore, that both OPN and OPN 1–149 contain sim- ilarly sized runs of local poly-l-proline II structure but, in the latter, the frequency of hinge residues is greater. Microcalorimetry of OPN 1–149 The thermogram shown in Fig. 3 shows an almost per- fectly smooth increase in specific heat with temperature in accord with the SAXS observations of a worm-like chain and consistent with the low chemical shift dispersion in 1 H-NMR spectra of OPN [25]. Binding of Ca ions to OPN 1–149 Three pK values and three Ca ion association con- stants were allowed to vary during the fitting to the experimental isotherms of the b-casein 1–25 peptide, and the resulting fitted curves are shown in Fig. 4. The three Ca ion association constants obtained were 3000, 400 and 30 m )1 . The single phosphoseryl residue (pS) had an effective pK value of 6.0 and the cluster of three pS residues ionized with a pK value of 7.2. The OPN 1–149 isotherm, also shown in Fig. 4, was fitted by two Ca ion association constants of 3000 (dianionic phosphate) and 30 m )1 but, because it does not have the triplet of pS residues, two pK values of 6.4 and 5.0 were required. Formation of OPN 1–149 nanoclusters OPNmix and OPN 1–149 were able to sequester CaP to form nanoclusters, but OPN could not, suggesting that the extended phosphorylated sequences in the C-terminal half either were too large to form PCs or the sequence catalysed the maturation of ACP into more crystalline phases. Using the simple mixing method at a peptide concentration of 30 mgÆmL )1 of OPNmix, there was no initial precipitation, even with a single addition of the P i stock, provided that it was added slowly with good stirring. The initial turbidity slowly disappeared over about 1 week to give a slightly opalescent solution, comparable to that of CPNs pre- pared by the urea ⁄ urease method. When the peptide concentration was reduced to below 10 mgÆmL )1 , an initial precipitate or turbid colloidal suspension C. Holt et al. Calcium phosphate sequestration by osteopontin FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2311 developed which did not fully redisperse on standing. If, however, further peptide was added to a final con- centration of 30 mgÆmL )1 , soon after the development of the initial precipitate, the solution clarified com- pletely over about 1 week. However, if the addition of the phosphopeptide was delayed, or if the initial pep- tide concentration was below 5 mgÆmL )1 , complete redispersion was not achieved, even after 4 months. These experiments demonstrated that, like the casein CPNs, the OPN 1–149 CPNs can be formed by either a forward reaction from a supersaturated solution or by a back reaction from a two-phase system containing a precipitate of ACP and sufficient sequestering pep- tide to convert all the ACP to CPNs. Neither casein nor OPN phosphopeptides could form the nanoclusters from partially matured ACP. Characterization of OPN nanoclusters SAXS of OPN 1–149 nanoclusters prepared by the urea ⁄ urease method The results of the SAXS measurements on CPN subs- amples, measured as a function of time after the addi- tion of urease, are summarized in Fig. 5A,B. The first AB C D Fig. 1. Prediction of disorder as a function of residue position in SPs having known or potential PC sequences. The positions of known or predicted PCs in the sequence are shown as full lines. (A) PONDRÒ predictions for SCPPs in Table 1. (B) PONDRÒ predictions for the other secreted phosphoproteins in Table 1. (C) TOP-IDP predictions for h-OPN and h-SPP-24 plotted as the midpoint of a window of 51 residues. (D) metaPrDOS predictions for h-OPN and h-SPP-24. Calcium phosphate sequestration by osteopontin C. Holt et al. 2312 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS two subsamples were taken after 17 min, when the pH was 6.82, and after 50 min, when the pH was 6.87, but, by the third sample, the pH was essentially con- stant and close to 7.0. Strongly scattering spherical particles formed from an initial state dominated by the scattering of a statistical polymer but, after about 2 days, the scattering profile showed hardly any fur- ther change, as demonstrated by a measurement 5 months later. SAXS of the matured system was modelled as a mixture of free peptide and CPNs, as shown in Fig. 5C. The worm-like chain representation of the free peptide was used with the assumption that the PCs on the same peptide all react together to give either fully bound or fully free peptide, so that the fraction of free peptide equals the fraction of unreacted PCs. The weighted subtraction produced a scattering curve which is characteristic of spherical, but polydisperse, particles with a corona of statistical scattering elements. The Gaussian copolymer micelle model of Pedersen and Gerstenberg [37] with a log-normal size distribution produced a reasonably close representation of the scattering of the CPNs, although the OPN peptide chains in free solution deviated from true Gaussian behaviour. Electrophoretic light scattering by nanoclusters The maturation of a CPN solution prepared by the rapid urea ⁄ urease method using the b-casein (f1-25) phosphopeptide is shown in Fig. 6A. At pH 5.5, before the urease was added and below the point at which CPN formation begins, the intensity of scattered light was low and the solution was apparently unchanged. Nevertheless, inversion of the correlation function gave an intensity-weighted size distribution of colloidal particles, almost certainly CaP formed at the time of mixing, as the solution is undersaturated with respect to ACP at pH 5.5. All other results in Fig. 6A were recorded after the final pH value of 7.0 was attained. A progressive loss of colloidal particles at the expense of the CPN component occurred as the solution matured. The intensity distribution of a similar solu- tion that had been stored at ambient temperature and pH 7 for 1 day showed that the colloidal particles were nearly absent. In another experiment, CPNs prepared with a mixture of casein phosphopeptides [38] by the simple mixing method were compared with those made by the urea ⁄ urease method. The turbidity A 1 cm 600 nm ÀÁ of Fig. 2. Kratky plots of the SAXS of OPN (in 20 mM P i buffer, pH 7.0, ionic strength 80 m M) and of OPN 1–149 (in the CaP dilu- tion buffer used in the nanocluster experiments). Fitted curves are from the worm-like chain model. Each set of results has been scaled by the mean square radius of gyration determined by the fitting procedure. Fig. 3. Normalized differential scanning calorimetry thermogram of OPN 1-149 at pH 7.0. Fig. 4. Ca-binding isotherms of b-casein 1–25 as a function of pH and of OPN 1–149 at pH 7.0. C. Holt et al. Calcium phosphate sequestration by osteopontin FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2313 the CPN solution made by the first method fell from 0.017 to 0.003 over 5 days to equal that of the CPN solution prepared by the urea ⁄ urease method, which showed no change in absorbance over time. The hydrodynamic radii of filtered solutions after equilibration for 5 days were 6.05 and 6.75 nm for the first and second methods, respectively (results not shown). The OPN 1–149 CPN had a hydrodynamic radius of 21.9 nm after 2 days of equilibration, whether pre- pared by the urea ⁄ urease method or the simple mixing method, although the mixing method produced an initial slight precipitate which quickly dispersed, confirming that an equilibrium size was attained. The hydrodynamic radius is comparable with the radius of gyration determined by SAXS. In the intensity- weighted size distribution of the unfiltered OPN 1–149 CPN (Fig. 6B), there was a very small peak of much larger particles which could be removed by filtration through a 0.2 lm filter. The origin of these larger par- ticles may have been the result of a very small amount of cross-linking between nanoclusters produced by the trifunctional peptides or of unequilibrated colloidal CaP particles. Another peak, contributing 8.5% to the total scattered intensity, on the low side of the main CPN peak, corresponded to the hydrodynamic size of the free peptide. The electrophoretic mobility of the OPN 1–149 CPN was 1.4 lmÆs )1 ÆV )1 Æcm. According to the Henry equation [39], it corresponds to a f potential of )15.4 mV. A B C D Fig. 5. Study by SAXS of the maturation of nanoclusters prepared with OPN 1–149 by the urea ⁄ urease method. (A) Effect of time on the radius of gyration determined by the Guinier method. (B) Normalized, q 2 -weighted SAXS of the nanoclusters diluted to 5 mg Æ mL )1 after the given times. (C) Model of the scattering of the matured nanocluster solution as a mixture of scattering from copolymer micelle-like nanoclus- ters and free peptide. The scattering of the nanoclusters was obtained by subtracting the scattering of the free peptide from the total scat- tering. Model calculations used the parameters b = 0.07 nm, A core = 0.25 nm 2 , r o = 12.5 nm, b = 0.35. (D) Representation of an OPN 1–149 nanocluster. An eighth section of the spherical core of ACP is shown. Surrounding the core is a shell of OPN 1–149 molecules, each anchored to the core through its three PCs. For clarity, only one phosphopeptide molecule is shown. The mesh illustrates the position of the surface of shear, which determines the hydrodynamic radius of the nanocluster. The diagram is scaled to give approximately correct impres- sions of the relative magnitudes of A core , r g,peptide and r h for a core radius of 12.5 nm. Calcium phosphate sequestration by osteopontin C. Holt et al. 2314 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS Calculation of the ionic equilibria and partition of salts in OPN 1–149 and OPNmix nanocluster solutions An invariant ion activity product in the ultrafiltrates was found for a TCP stoichiometry (y =0, K S = 7.6 · 10 )10 m 1.66 , results not shown). This is a more basic ACP than was found in the casein CPNs, which have y = 0.4 [13]. Below pH 5.97, no CPNs could form because the ion activity product was below K S . Above pH 5.97, the extent of reaction of PCs with ACP was found which allowed the ion activity product in the CPN solution to equal K S . The casein CPN val- ues for R Ca and R P were used, and peptide binding was calculated on the assumption that all the peptides in the OPNmix sample had the same binding isotherm as OPN 1–149. The complete model of ionic equilibria was then used to calculate the composition of an equi- librium diffusate, so that it could be compared with the composition of the experimental ultrafiltrate A B Fig. 6. Intensity distribution curves derived from the dynamic light scattering measurements. (A) Unfiltered nanocluster solution prepared with b-casein (f1–25) by the urea ⁄ urease method from an initial pH of 5.5 to a final pH of 7.0. The larger particles observed at pH 5.5 are probably colloidal ACP formed during mixing, which gradually dissolve at the expense of the nanoclusters formed above pH 6. (B) Mature OPN 1 149 nanoclusters. A B C Fig. 7. Calculated properties of OPNmix nanocluster solutions. (A) Comparison of calculated ultrafiltrate concentrations of P i , Ca and free Ca 2+ with experimental values shown as symbols. (B) Calcu- lated fraction of reacted PCs. (C) Log of the saturation index versus pH for DCPD, OCP and HA. C. Holt et al. Calcium phosphate sequestration by osteopontin FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2315 (Fig. 7A). The general agreement of the model with experiment is evident. Figure 7B shows how the calcu- lated extent of reaction of the PCs varied with pH. The saturation indices, defined as the ratio of the ion activity product to the solubility product, for di-cal- cium phosphate di-hydrate (DCPD), octacalcium phos- phate (OCP) and HA are shown in Fig. 7C. Above pH 5.97, the CPN solutions were undersaturated or only slightly supersaturated with respect to DCPD and OCP, but over the entire pH range, the nanocluster solution was highly supersaturated with respect to HA. In addition to the work with the OPNmix sample, a single determination was made of the partition of salts in a CPN solution at pH 7.0 prepared with the pure OPN 1–149 peptide. The experimental and, in paren- theses, model, ultrafiltrate concentrations of P i , Ca and Ca 2+ were 12.1 (10.5), 1.4 (1.1) and 1.1 (0.65) mm, respectively, which compare quite closely with the values obtained with the OPNmix nanoclusters. Discussion Structure of CPN-forming phosphopeptides and phosphoproteins Detailed structural studies on CPNs have been made using purified short peptides of lengths between 21 and 42 residues, namely a S1 -casein 59–79 and b-cas- eins 1–25 and 1–42. The results from the present work utilized a peptide of 149 residues, and it is most likely that the individual micellar CaP particles comprise the core of equilibrium complexes formed from proteins of more than 200 residues. It may be concluded that the length of the peptide or protein is not an important consideration. The OPN plasmin peptide has no signif- icant sequence similarity to any casein sequence out- side of the PCs. Flanking sequences of all the SPs in Table 1 are deficient in hydrophobic residues and Cys, and so they tend to have a low degree of sequence complexity and favour an unfolded conformation. On the larger scale, all PC-containing SCPPs and the non-SCPPs proline-rich basic phosphoprotein 4 and MGP are known or predicted to be unfolded over most or all of their length. The absence of a globular structure close to the surface of the core allows a higher density of PCs to bind to the surface, and so clearly a fully globular protein is at a disadvantage. The unfolded conformation may also allow a faster rate of CaP sequestration, which may be of importance when the rate of maturation of ACP nuclei is compa- rable with the rate of sequestration. Nevertheless, it can be envisaged that a globular domain, if it has an extended, flexible, linker sequence connecting it to a PC, could be just as effective as a natively unfolded protein or short peptide. FETUA, with two cystatin- like domains in the N-terminal half, and SPP-24, with one, are predicted to have part of their sequence remote from the PC in a more stable globular confor- mation. If it can be demonstrated that these proteins are also able to sequester CaP through their PCs, the requirement for an unfolded flexible conformation could be limited to a more restricted region adjacent to the PC. Thermodynamic stability of the OPN 1–149 nanocluster solution CPNs could be prepared with OPN 1–149 by either the urea ⁄ urease method or simple mixing and, after a few days of maturation, during which the turbidity decreased to a constant, low value, achieved an equi- librium size which did not change in the following 5 months of storage. The results shown in Fig. 6A and the changes in turbidity with time show that particles larger than the equilibrium size were produced during mixing and, to a lesser extent, by the urea ⁄ urease method, but during maturation, the larger particles disappeared at the expense of CPNs (Fig. 6A); the same equilibrium size was achieved whichever method was employed to make CPNs. Although the nanoclus- ter solution was stable, the ion equilibria calculations showed that it was highly supersaturated with respect to HA; however, as this phase can only form via solu- tion-mediated transformation of ACP, there is no means by which it can be generated when there is a sufficient excess of the sequestering peptide. Core shell structure of the OPN 1–149 nanocluster The radius of gyration of the peptide on the core sur- face was about one-third of its value in free solution, and this can be understood qualitatively if it is assumed that the peptide is attached to the surface through the three PCs (Fig. 5D). Compared with the casein CPNs, the core CaP is more basic, correspond- ing to the empirical chemical formula of TCP, and nearly four times larger, but the molar ratios of Ca or P i to PC were calculated to be the same. Most proba- bly, the core is simply more hydrated. According to Eqn (4), the size is determined mainly by the ratio of the free energy of sequestration to the free energy of formation of the bulk core phase and the core surface area per PC. The latter was found to be 0.25 nm 2 , which is about one-quarter of that for the b-casein 1–25 CPN, and so this alone could account Calcium phosphate sequestration by osteopontin C. Holt et al. 2316 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS for the difference. It is more probable that the differ- ence in chemical composition and hydration in the core affects the two free energy terms equally, so that their ratio is unchanged. Notwithstanding the difference in hydration in the core, it is most probable that the core is amorphous, similar to CaP in casein micelles and the core CaP of casein CPNs, otherwise the particles would not have equilibrated to a path-independent constant size. Moreover, highly phosphorylated OPN, like casein, is a very powerful inhibitor of ACP maturation, even at much lower concentrations than those employed here [40]. Nonequilibrium, pathway and time-dependent phe- nomena are commonly observed in CaP precipitation from solution at near-neutral or alkaline pH, and the usual product is a poorly crystalline HA or OCP phase (Fig. 8A). Numerous reports exist of the effects of phosphoproteins or phosphopeptides on the lag time before precipitation, the rate and extent of precipita- tion and rate of conversion of ACP into more crystal- line phases (recently reviewed by George and Veis [41]). Invariably, the studies have been made under conditions in which there is a large molar excess of CaP over the peptide [in Eqn (5), a ) 1], so that the results involve metastable phases or metastable colloi- dal solutions, some with very long lifetimes (Fig. 8C). When much higher concentrations of phosphopeptide are employed, such that 0 < a £ 1, the maturation of ACP may be completely inhibited and, provided that the free energy of sequestration by the phosphopeptide is sufficiently high, it can form the equilibrium com- plexes called CPNs (Fig. 8B). Is CaP sequestration to form equilibrium nanocl- usters of broad physiological importance? The properties of nanocluster solutions that can be exploited in biofluids are, firstly, that they are ther- modynamically stable, so that mineralization of soft tissues should not occur. Second, when a fresh ecto- pic deposit of ACP does form, it can be removed by an excess of the sequestering protein or peptide. Third, in contact with hard tissue, the nanocluster solution cannot cause demineralization and could indeed act as a reservoir of CaP for crystal growth or tissue remineralization. Fourth, Eqn (5) places no upper limit on the concentrations of Ca and P i in the fluid. For example, the free Ca ion concentra- tions and supersaturation with respect to HA in milk A BC Fig. 8. Schematic drawing of the alternative fates of ACP nuclei formed from a supersaturated solution. (A) In the absence of a competent sequestering peptide [i.e. a in Eqn (5) is infinite], ACP nuclei grow and mature into a crystalline or poorly crystalline calcium phosphate; under physiological conditions, the final state is usually poorly crystalline OCP or HA or, in the case of tooth enamel, highly crystalline HA. (B) In the presence of a stoichiometric excess or equivalence of PCs (0 < a £ 1), a thermodynamically stable solution of CPNs may form if all the CaP is sequestered by the competent SPs. The CPNs have a defined composition and size at equilibrium. If some of the nuclei escape sequestration to grow and mature to a poorly crystalline state, they cannot subsequently form the equilibrium nanoclusters. (C) In the presence of a substoichiometric concentration of competent SPs (1 < a < ¥), the growth and maturation of the ACP nuclei may be slo- wed to give a metastable colloidal suspension or precipitate of complexes of variable stoichiometry, size and degree of crystallinity. C. Holt et al. Calcium phosphate sequestration by osteopontin FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2317 [...]... hence the composition of the ultrafiltrate was obtained from the Donnan equilibrium across the membrane [46] Equation (6) requires the calculation of Ca ion binding to the free peptide at any pH in the range 5.0–8.0 A semi-empirical model was used to describe the binding isotherms obtained previously for the b-casein 1–25 phosphopeptide in this pH range, and the same model was adapted to fit the binding... isotherm of OPN 1–149 measured at pH 7.0 The rescaled model was then used to predict binding at any other value of the pH Further FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2319 Calcium phosphate sequestration by osteopontin C Holt et al details of the model are provided in Doc S3 (see Supporting information) The Ca2+ binding isotherm of OPN 1–149 was determined... degree of phosphorylation of the 28 potential phosphorylation sites in the whole protein was 79% The N-terminal fragment analysis was consistent with an average of about 60–65% phosphorylation of the 16 sites in the N-terminal plasmin peptides All the glycans (three to four sites) were present in both catalyse the reaction, producing the strong base ammonia and weak carbonic acid The amount of urea... the number fraction of free chains was assumed to equal the fraction of unreacted PCs Further details of the scattering models are given in Doc S4 (see Supporting information) Electrophoretic light scattering The intensity-averaged diffusion coefficient was determined with a Malvern Zetasizer Nano instrument (Malvern, Worcestershire, UK) Inversion of the intensity autocorrelation function by means of. .. Identification of PCs in secreted phosphoproteins Searches for PCs were made by manual and automated methods in the sequences of SPs, known to the authors to be involved in CaP mineralization processes, using the UniProt (=Swiss-Prot + TrEMBL) database on the ExPASy server (http://www.expasy.com) of the Swiss Institute of Bioinformatics Alignment of orthologous sequences and the generation of general motifs... that are the hallmark of CPNforming phosphopeptides One or more of these proteins is physiologically important in the tissues of bone, dentine, cementum and osteoid, or is secreted into biofluids, such as blood, milk, saliva and urine Our contention is that among the physiologically important functions of the non-casein SPs of Table 1 are presently unrecognised ones that involve the formation of thermodynamically... and the intercept at q = 0 were determined by a Guinier plot of ln(I) versus q2 Scattering curves were normalized by dividing by the Guinier intercept and weighted by q2 to emphasize the low-intensity features (Kratky plot) The scattering of OPN and OPN 1–149 in free solution was fitted to a worm-like chain model [47,48] Chain stiffness was measured by means of the Kuhn segment length b, which is the. .. precise number of moles of CaP in the presence of an excess concentration of sequestering phosphopeptide, as required by Eqn (5) Although this can be performed by simply mixing together stock solutions containing high concentrations of Ca, Pi and the phosphopeptide, the concentration gradients generated and their persistence during inefficient mixing can allow the inital ACP to mature into a more stable... determines the final pH (typically 6–8) and the amount of enzyme can vary the time taken to approach the target pH from 2 min to 2 h Rapid attainment of the target pH is followed by several hours during which the nanoclusters grow to their equilibrium size [13] Nanoclusters were prepared by the urea ⁄ urease method [12] using either the OPNmix or OPN 1–149 (fraction F3a) and magnesium-free Buffer A [13] The. . .Calcium phosphate sequestration by osteopontin C Holt et al remain comparable with those in blood, even though the total Ca concentration in milk may be two orders of magnitude higher Fifth, there is scope for an exquisite degree of control of mineralization through the degree of phosphorylation of the competent SPs, particularly when, as in OPN and DMP1, they have opposing functional . scattering was used to confirm that a plasmin phosphopeptide of one of the identified proteins, osteopontin, formed a novel type of calcium phosphate nanocluster in which the radius of the amorphous calcium. as the starting point of controlled crystal growth in the extracellular matrix of mineralized tissues. These include long phosphorylated sequences in, for example, phosphophoryn, the C-terminal. sequences in the SPs in Table 1 were, with the exception of the globular pro- tein riboflavin-binding protein (RBP), disordered, and had disordered flanking sequences (Fig. 1A,B). The PC motif of RBP