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Roleofcalciumphosphatenanoclustersinthecontrol 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 ofcalciumphosphate (CaP) in serum and prevent-
ing ectopic calcificationof 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 ofthe mammary gland, even though it may con-
tain very much higher concentrations ofcalcium (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 phosphatenanoclusters are equilibrium particles of defined chemi-
cal composition in which a core of amorphous calciumphosphate 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 ofthe identified proteins,
osteopontin, formed a novel type ofcalciumphosphate nanocluster in
which the radius ofthe amorphous calciumphosphate 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 ofthe 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 thecontrol or inhibition ofcalcificationin 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, calciumphosphate 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 ofthe thermodynamics
of CaP sequestration and to define more closely the
structural characteristics ofthe 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 ofthe 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 ofthe 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 inthecontrolof mineralization
processes [21,22].
Results
Thermodynamics of CPN formation
Doc. S1 (see Supporting information) provides addi-
tional details ofthe 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 inthe CPN and Pep is the chemical for-
mula ofthe peptide divided by the number of PCs it
contains (f). The formula ofthe monomer can be
further divided into an amorphous hydrated CaP and a
sequestering ligand ofcalcium phosphopeptide. The
empirical chemical formula ofthe 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 ofthe capillary theory
of nucleation that an activity distribution results with
a modal core radius of:
r
Ã
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 ofthe core by
the shell of peptides, a
1
and a
s
are the activities of a
CaP molecule inthe 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 ofthe 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
. Inthe 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. Calciumphosphate 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 ofthe nanocluster solution
requires not only that maturation ofthe 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 ½PPR
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 inthe 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 ofthe identified SPs and all ofthe proven
CPN-forming SPs are members ofthe SCPP paralogous
group. Most PCs contain a block of consecutive phos-
phorylation sites, followed by the primary recognition
site ofthe 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 ofthe 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 ofthe predictors of partial or complete
disorder in proteins. It continues to perform well in
comparative tests with more recent methods [31], and
is one ofthe components inthe most recent meta pre-
dictor, metaPrDOS [32]. According to PONDRÒ pre-
dictions, the positions of PC sequences inthe SPs in
Table 1 were, with the exception ofthe 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 ofthe 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 ofthe radii of gyration of OPN and
OPN 1–149 inthe 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 ofthe higher proportion of Pro residues in this
part ofthe sequence (eight ofthe 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 inthe 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, inthe 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 ofthe 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 ofthe 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. Calciumphosphate 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 ofthe 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 inthe 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 ofthe matured system was modelled as a
mixture of free peptide and CPNs, as shown in Fig. 5C.
The worm-like chain representation ofthe 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 ofthe 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 ofthe 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 ofthe 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 inthe 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. Calciumphosphate 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. Inthe intensity-
weighted size distribution ofthe 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 ofthe 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 ofthe maturation ofnanoclusters 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 ofthenanoclusters diluted to 5 mg Æ mL
)1
after the
given times. (C) Model ofthe scattering ofthe matured nanocluster solution as a mixture of scattering from copolymer micelle-like nanoclus-
ters and free peptide. The scattering ofthenanoclusters was obtained by subtracting the scattering ofthe 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 ofthe 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 ofthe nanocluster. The diagram is scaled to give approximately correct impres-
sions ofthe 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 ofthe ionic equilibria and partition of salts
in OPN 1–149 and OPNmix nanocluster solutions
An invariant ion activity product inthe 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 inthe 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 ofthe 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 ofthenanoclusters 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 ofthe saturation index versus
pH for DCPD, OCP and HA.
C. Holt et al. Calciumphosphate sequestration by osteopontin
FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2315
(Fig. 7A). The general agreement ofthe model with
experiment is evident. Figure 7B shows how the calcu-
lated extent of reaction ofthe PCs varied with pH.
The saturation indices, defined as the ratio ofthe 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 ofthe 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 ofthe peptide or protein is not an important
consideration. The OPN plasmin peptide has no signif-
icant sequence similarity to any casein sequence out-
side ofthe 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 ofthe 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 inthe 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 ofthe 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 inthe 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 ofthe sequestering peptide.
Core shell structure ofthe OPN 1–149
nanocluster
The radius of gyration ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe alternative fates of ACP nuclei formed from a supersaturated solution. (A) Inthe 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, inthe case of tooth enamel, highly crystalline HA.
(B) Inthe 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 ofthe 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 ofthe 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. Calciumphosphate sequestration by osteopontin
FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2317
[...]... hence the composition ofthe 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 inthe 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 ofthe pH Further FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2319 Calciumphosphate sequestration by osteopontin C Holt et al details ofthe model are provided in Doc S3 (see Supporting information) The Ca2+ binding isotherm of OPN 1–149 was determined... degree of phosphorylation ofthe 28 potential phosphorylation sites inthe whole protein was 79% The N-terminal fragment analysis was consistent with an average of about 60–65% phosphorylation ofthe 16 sites inthe 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 ofthe 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 ofthe intensity autocorrelation function by means of. .. Identification of PCs in secreted phosphoproteins Searches for PCs were made by manual and automated methods inthe 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) ofthe 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 inthe 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 ofthe 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 ofthe Kuhn segment length b, which is the. .. precise number of moles of CaP inthe 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 ofthe target pH is followed by several hours during which thenanoclusters 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 ofcontrolof mineralization through the degree of phosphorylation ofthe 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