Báo cáo khoa học: Role of calcium phosphate nanoclusters in the control of calcification pot

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

Carl Holt1, Esben S Sørensen2and Roger A Clegg1

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 (Pi) 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 chemi-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.

aS1-, aS2- 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

31P-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:

CaR CaHR HðPiÞRPðH2OÞRWðPep  PCÞ1

The average molar ratios of water, Ca and Pito PC are RW, RCa and RP, 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

CaðHPO4ÞyðPO4Þ22y

3 :xðH2OÞ ð2Þ where 3y⁄ (2 + y) is the mole fraction of Pi in the di-anionic form The empirical chemical formula of CaP can then be used to define a type of solubility constant KS as an ion activity product In a dilute solution in which the activity of water is effectively unity:

KS¼ a1

Ca 2þayHPO2

4 að22yÞ=3PO3

4

ð3Þ

KS 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:

rcore  2kDGseq

3AcoreRTlnða1=asÞ

3Vcore 4p

¼ 2kDGseq

3AcoreDGo

core

3Vcore 4p

where Vcore is the empirical formula volume of CaP,

k¼ ð36pV2

coreÞ1=3, Acoreis the core surface area per PC,

DGseqis the free energy of sequestration of the core by the shell of peptides, a1 and as 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

coreis 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 a1> as 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 (a1< as)

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 sequestmatu-ration, 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 Pi can

precipitate as ACP from the initially supersaturated

solution, the condition for thermodynamic stability is

a¼ ½p

f½PPRP

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 aS1-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, aS1-casein; CSN1S2, aS2-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

99– SDESHHSDES -108

120-SADTTQSSED -129 142-SDSKDQDSED -151 161-DSAQDSESEE -170

130- ELSTSEEPVS-139

Non-SCPPs

a Sequence numbers are for the mature peptide chain without the signal sequence.

Conformation of secreted phosphoproteins containing

PCs

The PONDR 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

dictor, metaPrDOS [32] According to PONDR

pre-dictions, the positions of PC sequences in the SPs in

Table 1 were, with the exception of the globular

pro-tein riboflavin-binding propro-tein (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 PONDR 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

PONDR 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 in1H-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 Pi 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

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

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) PONDR predictions for SCPPs in Table 1 (B) PONDR 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.

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 A1 cm

600 nm

of

Fig 2 Kratky plots of the SAXS of OPN (in 20 m M 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.

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)1ÆV)1Æcm 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 mgÆmL)1after 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 nanoclusnanoclus-ters was obtained by subtracting the scattering of the free peptide from the total scat-tering Model calculations used the parameters b = 0.07 nm, Acore= 0.25 nm 2 , ro= 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.

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,

KS= 7.6· 10)10m1.66, 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

KS 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 KS The casein CPN

val-ues for RCa and RP 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.

(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), octacaldi-cal-cium

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 Pi, Ca and

Ca2+ 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 aS1-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

Pi 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 nm2, which is about one-quarter of that for the b-casein 1–25 CPN, and so this alone could account

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 Pi in the fluid For example, the free Ca ion concentra-tions and supersaturation with respect to HA in milk

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.