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Originally published In Press as doi:10.1074/jbc.M709938200 on March 25, 2008

J. Biol. Chem., Vol. 283, Issue 21, 14815-14825, May 23, 2008
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Hierarchical Role of Fetuin-A and Acidic Serum Proteins in the Formation and Stabilization of Calcium Phosphate Particles*

Alexander Heiss{ddagger}§1, Thomas Eckert, Anke Aretz||, Walter Richtering, Wim van Dorp**, Cora Schäfer{ddagger}, and Willi Jahnen-Dechent{ddagger}

From the {ddagger}Helmholtz Institute for Biomedical Engineering, Biointerface Group, Institute for Physical Chemistry, and ||Central Facility for Electron Microscopy, RWTH Aachen University, 52074 Aachen, Germany, the §Institute for Solid State Research, Research Center Juelich, 52425 Juelich Germany, and **Kennemer Gasthuis, 2000 AK Haarlem, The Netherlands

Received for publication, December 5, 2007 , and in revised form, March 20, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The serum protein fetuin-A is a potent systemic inhibitor of soft tissue calcification. Fetuin-A is highly effective in the formation and stabilization of protein-mineral colloids, referred to as calciprotein particles (CPPs). These particles ripen in vitro in a two-step process, indicated by a morphological conversion from spheres to larger prolate ellipsoids. Using a combined light scattering and electron microscopic imaging approach we determined that the second-stage particles resulted from a highly anisotropic outgrowth of the first-stage particles. Electron microscopy of ascites fluid from a patient with calcifying peritonitis revealed particles reminiscent of secondary CPPs. Thus, CPPs form in the body and undergo the two-step ripening at least in pathological conditions. Unlike in vitro generated CPPs, ascites-derived CPPs contained little fetuin-A but large amounts of albumin. This prompted us to study the role of fetuin-A combined with other serum proteins in CPP formation. Fetuin-A was indispensable for primary CPP formation. Albumin and acidic proteins in general greatly enhanced the fetuin-A triggered formation of secondary CPPs and, thus, substituted substantial amounts of fetuin-A without loss of inhibition of calcium phosphate precipitation. Thus, direct mineral deposition from solute in the body is unlikely even at low fetuin-A serum levels as long as sufficient bulk acidic protein is available. Collectively fetuin-A and other acidic bulk plasma proteins may be considered as mineral chaperones mediating the stabilization, safe transport, and clearance in the body of calcium and phosphate as colloidal complexes, thus, preventing ectopic calcification.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vertebrates all extracellular fluids are supersaturated with respect to the mineral-forming ions calcium and phosphate. Local supersaturation may even be much higher than the ion concentrations in blood indicate, for instance in the bone remodeling compartment or in kidney epithelia. Thus, potent inhibitors of spontaneous salt precipitation that counteract this thermodynamic driving force are essential. Otherwise uncontrolled mineral deposition in the blood or in the soft tissue would inevitably occur causing ectopic calcification. Several different types of calcification inhibitors and mechanisms are known. For example, small molecules and ions like pyrophosphate, citrate, or magnesium (1) slow down crystal growth by interfering with crystal lattice assembly. On the protein level, in vitro binding studies of serum proteins to basic calcium phosphates helped to identify structurally diverse but generally acidic mineral-binding proteins like {alpha}2-Heremans-Schmid glycoprotein/fetuin-A, {gamma}-carboxyglutamate proteins, albumin, fibronectin, or transferrin (2-5). Acidic bone-derived proteins likewise have a high affinity for mineral and prevent their spontaneous precipitation from supersaturated salt solutions (6).

Fetuin-A is a liver-derived plasma protein circulating at 0.7-0.8 mg/ml (~15 µM) in the blood of healthy human individuals (7, 8). Its high affinity for bone mineral (9) and the ability to prevent the precipitation of basic calcium phosphates from supersaturated solutions suggested that fetuin-A was a potent systemic inhibitor of soft tissue calcification (5, 10-12). The decisive role of fetuin-A was confirmed in fetuin-A-deficient mice, which developed general soft tissue calcification (13). Clinical studies indicate an association of fetuin-A serum deficiency and calcification scores in several patient cohorts (8, 14, 15). Biochemical work suggested that fetuin-A "buffered" mineral ion supersaturation by enclosing precursors of mineralization in fetuin-A shielded particles variably termed calciprotein particles (CPPs)2 in analogy to lipoprotein particles (10, 16) or fetuin-mineral complex, FMC (11, 17, 18). In these studies the initial CPPs underwent a two-step ripening process (10, 11, 16). Transmission electron microscopy (TEM) investigations of freshly synthesized CPPs revealed nanometer sized spheres. Subsequent CPP transformation was associated with a morphological change to prolate ellipsoids called secondary CPPs (16). We hypothesized that fetuin-A on the surface of these mineral colloids (i) reduced diffusion of ions to the mineral core and (ii) prevented particle aggregation (16).

The role of albumin as an inhibitor of calcification is ambiguous. Albumin modulates crystal seed growth but is a relatively weak inhibitor of calcium phosphate precipitation from supersaturated solutions. Studies on seeded growth of hydroxyapatite using complete human serum and albumin-depleted human serum indicated that albumin accounted for ~50% of the serum inhibition (3). The overall activity was, however, attributed to the high albumin serum concentration (~40 mg/ml) rather than to a highly specific inhibitor activity (3, 5, 19, 20).

Fetuin-A has an exceptionally high affinity for basic calcium phosphate mineral surfaces irrespective of the exact composition and texture, e.g. synthetic apatite, bone mineral, or calciprotein-like particles (4, 17, 18, 20, 21). These studies also suggested that the fetuin-A and albumin co-localized with mineral at ratios varying over a wide range. Here we present a biochemical analysis of CPP-like particles from ascites fluid of a peritoneal dialysis patient. The sample contained albumin as the most abundant protein, whereas the fetuin-A content was low, prompting us to systematically study the effect of serum protein mixtures on the inhibition of calcium phosphate precipitation. Based on the results of these studies, we propose a refined model of systemic calcification inhibition implying cooperative effects of acidic serum proteins and fetuin-A as a key player.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transmission Electron Microscopy—Sample volumes of 50 µl were dialyzed against distilled water at room temperature for 20 min (Mini Slide-A-Lyzer, Perbio Science, Bonn, Germany), and then 10 µl were applied onto Formvar (polyvinyl formal)-coated copper grids (Plano, Wetzlar, Germany). After 5 min of incubation, excess liquid was soaked away, and the grids were dried at room temperature. The mineral particles were visualized without staining using a JEOL JEM 2000 FX II, operated at 200 kV (10, 16).

CPP Analysis—Mature second state CPPs (20 µM fetuin-A, 10 mM CaCl2, 6 mM Na2HPO4, 50 mM Tris, pH 7.4, 140 mM NaCl incubated >12 h at room temperature) were loaded onto a Sephacryl 500 column (GE Healthcare, 36-ml column volume) equilibrated with 10 mM CaCl2, 50 mM Tris, pH 8, 140 mM NaCl. A column void volume V0 of 7.3 ml was determined using 1-µm-sized polystyrene spheres. Phosphate concentration in the eluate was assessed by the molybdate complex method.

To detect CPP-associated fetuin-A, bovine fetuin-A monomer was labeled with N-hydroxysuccinimide biotin following the manufacturer's protocol (Perbio Science). CPPs were synthesized using biotinylated fetuin-A and separated as mentioned above. 5 µl of each eluate fraction was analyzed by SDS-PAGE and immunoblotting. Biotinylated fetuin-A was detected by streptavidin-horseradish peroxidase and enhanced chemiluminescence.

Preparation of Proteins—Bovine fetuin-A, bovine albumin, ovalbumin (all Sigma), and lysozyme (Applichem, Darmstadt, Germany) were purified by gel permeation chromatography using a Superdex 200 16/60 column (GE Healthcare) equilibrated with Tris-buffered saline (20 mM Tris, pH 7.4, 150 mM NaCl). Fetuin-A and albumin concentrations were determined photometrically at 280 nm applying extinction coefficients {epsilon}1% = 5.3 (22) and 6.7 (23), respectively.

Glutathione S-transferase (GE Healthcare) expression was induced in pGEX-2T transformed BL21 (DE3) LysS Escherichia coli (Novagen) with 300 µM isopropyl-1-thio-β-D-galactopyranoside (Applichem). After 2 h of shaking at 37 °C, the bacteria were harvested and lysed. The target protein was isolated from the supernatant by affinity chromatography using glutathione-Sepharose 4B (GE Healthcare) followed by a dialysis against Tris-buffered saline (10).

Protein concentrations of ovalbumin, lysozyme, and recombinant glutathione S-transferase were assessed by the Bradford method (Roth, Karlsruhe, Germany) using albumin as a standard. Purified and lyophilized prothrombin (Enzyme Research Laboratory, Swansea, UK) was dissolved in buffer according to the manufacturer's instructions (20 mM Tris, pH 7.4, 100 mM NaCl).

For protein analysis of the peritoneal dialysate, a 40-µl suspension was centrifuged at 8000 x g for 10 min. The particles were separated from the supernatant, washed once with phosphate-buffered saline, and dissolved in 40 µl of 0.5 M EDTA. 0.2 µl of each sample was separated by SDS-PAGE, stained with colloidal Coomassie (Invitrogen), or blotted onto a nitrocellulose membrane for immunodetection.

Precipitation Inhibition Mix—All stock solutions of NaCl (Roth), Tris buffer (Sigma), CaCl2, and Na2HPO4 (Merck) had analytical quality and were filtered (0.2 µm) before use. A standard inhibition mix contained the inhibitor protein, 10 mM CaCl2, 6 mM Na2HPO4, 50 mM Tris, pH 7.4, and 140 mM NaCl (16). For quantification of mineral formation 45CaCl2 was added to the mix as a tracer (5, 10). All samples were incubated for 9 h at room temperature and centrifuged at 8000 x g for 5 min, and the resulting pellet was dissolved in 200 µl of 1% acetic acid. Precipitates containing 45Ca were measured by scintillation counting after adding 1 ml of scintillation liquid (5, 10).

Turbidimetry—CPP ripening was monitored in 1-ml cuvettes at {lambda} = 400 nm and T = 25 °C in a GE Healthcare 2300 photometer equipped with a six cell changer and operated by a computer. This instrumental setup permitted a parallel assessment of the onsets of CPP transformation and sedimentation covering a time span of 9 h in 5-min intervals.

Dynamic Light Scattering—To determine the hydrodynamic radius of CPPs, a three-dimensional cross-correlated dynamic light scattering (DLS) device was used. The three-dimensional DLS allows the assessment of the true hydrodynamic radius despite multiple scattering (turbidity) as described (24-26). Typically sample volumes of 0.3 ml were applied to cuvettes (Hellma, Müllheim, Germany) and measured with 90° geometry using an LS Instruments apparatus with two avalanche photon detectors (APD, PerkinElmer Life Sciences, Type SPCM-AQR-13-FC) and an ALV 5000 correlator. A laser diode module (Koheras A/S, Birkerod, Denmark, {lambda} = 681.3 nm, power = 16 milliwatts, Type LGTC 685-35) formed the light source. Temperature control was achieved by a water bath and a thermostat. The diffusion coefficients and the polydispersity index were obtained from a cumulant analysis (27), and hydrodynamic radii were calculated using the Stokes-Einstein equation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CPP-like Complexes in Chronic Kidney Disease Patient Ascites—Late stage chronic kidney disease patients have a deranged mineral homeostasis and require dialysis. The imbalanced homeostasis is generally associated with vascular and valvular calcifications that result in an increased morbidity and mortality. One particular severe form of dialysis-associated calcification is calcifying peritonitis. These patients have a sterile ascites (Fig. 1A) of milky appearance containing colloidal, rice grain-shaped mineral particles (Fig. 1B). Supernatant and pelleted minerals were both analyzed by SDS-PAGE (Fig. 1C) and human fetuin-A immunoblotting (Fig. 1D), respectively. The supernatant in Fig. 1C, lane 1, had a protein composition similar to serum. Lane 2 in Fig. 1, C and D, shows the protein analysis of the mineral colloid (i.e. the proteins attached to the particles). A side by side comparison of the Coomassie-stained SDS-PAGE gel and a matching immunoblot suggested that a substantial amount of albumin and a comparably small amount of fetuin-A were contained in the mineral phase (Fig. 1, C and D). The CPP-like particles isolated from ascites of the calcifying peritonitis patient (Fig. 1B) showed, despite the low fetuin-A and high albumin concentration, a remarkable similarity to second state CPPs synthesized in vitro in the presence of abundant fetuin-A but without any supplementary proteins (Fig. 2B). This observation raised the question of whether the high fraction of mineral-bound albumin simply reflected the abundance of this protein in human ascites or whether calcification inhibition in vivo also involves albumin and similar acidic serum proteins. To address this issue we synthesized CPPs in vitro employing pure proteins and protein mixtures and studied the formation, stability, and composition of various CPPs thus formed.


Figure 1
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  		FIGURE 1.
Biochemical analysis of a peritoneal dialysate containing protein-calcium phosphate particles in high number density. A, the milky appearance of the sample caused by colloidal mineral particles. B, TEM imaging of the particles. The particles had a rice-grain like morphology with crystalline domains in lamellar orientation (inset with higher magnification). C, protein detection by Coomassie staining of SDS-PAGE. The supernatant in lane 1 had a high protein content and showed a protein pattern similar to serum. Lane 2 shows the mineral-associated proteins. Only a strong albumin band was detected. D, immunoblot detection of human fetuin-A. The supernatant gave rise to a strong signal (lane 1), whereas the particle sample (lane 2) produced only a faint band.

 
Synthesis and Structure of Calciprotein Particles—First we synthesized pure and stable fetuin-A containing CPPs. Fetuin-A buffers mineral ion supersaturation by stabilizing precursors of mineralization as colloids. These colloidal particles are initially amorphous with respect to the mineral phase (Fig. 2A), and the CPPs underwent a transformation process (10, 11, 16). The second-stage CPPs are long-term stable and consist of lamellar aggregates of crystalline needles or platelets (Fig. 2B). In contrast, albumin stabilized nascent mineral nuclei in solution even at high concentrations (here 606 µM) only for a short period of time (Fig. 2C) and never displayed a similar ripening process, confirming the relatively weak inhibitory potential of albumin discovered in earlier studies (5, 19).

We employed gel permeation chromatography to study the formation of CPPs from unlabeled or labeled fetuin-A dissolved in calcium and phosphate-containing buffer. Primary CPPs tended to dissolve, but secondary particles were stable and could be readily analyzed by gel permeation chromatography. A typical elution profile monitored at 280 nm (Fig. 2D, solid line) showed a sharp peak, indicating that the high molecular weight CPPs eluted directly after the void volume. A second broader peak co-eluted with the fetuin-A monomer. The peak integrals did not reflect the true protein amounts, because light absorption and light scattering at the particles both contributed to the signal intensity. At 400 nm, only light scattering of the CPPs was measured, enabling the time-resolved study of CPP formation and stability (Fig. 2D, dashed line). Next, biotinylated fetuin-A was used to synthesize CPPs. The mixture was separated by gel permeation chromatography, and fetuin-A content in the eluate was quantified by streptavidin immunoblotting. We determined that a relatively small amount of fetuin-A eluted at the CPP position (<10% by densitometry), and most of the fetuin-A eluted at the monomer position (Fig. 2E). Thus, a relatively small amount of fetuin-A is sufficient to mediate long-term stable second-stage CPPs even at high supersaturations.

Measuring phosphate in the eluted fractions (Fig. 2F) indicated that the high molecular weight peak, i.e. the CPPs, contained about two-thirds of the phosphate originally present in the precipitation mixture, confirming earlier results obtained with small angle neutron scattering (16). Ultrafiltration of the precipitation mixtures employing a 300-kDa cut-off membrane to separate the CPPs (retentate) from the fetuin-A monomer and non-CPP ions (filtrate) confirmed this value. We found that the retained CPP fraction contained 71 ± 5% of the total phosphate.

Inhibition of Calcification by Acidic Plasma Proteins—Our routine assay to assess the inhibitory potential of serum or individual serum proteins was based on the quantification of mineral sedimentation at 10,000 x g (Fig. 3A) (5, 10). It allowed the identification of fetuin-A as a potent stabilizer of CPPs. However, it lacked time-resolved data and, therefore, was unsuitable for the investigation of CPP formation and stability. Moreover, this assay produced spurious results at low fetuin-A concentrations close to the CPP stability threshold. For example, the IC50 of 7.4 µM fetuin-A derived from the assay did not correspond to the observed macroscopic sample stability (Fig. 3A) in that a precipitate could be collected by centrifugation, but the same solution was long-term stable (yet cloudy) at ambient gravity.


Figure 2
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  		FIGURE 2.
CPP isolation and analysis. A and B, CPP formation mediated by 50 µM fetuin-A. TEM images of CPPs in the first (A) and second state (B), respectively. Initial CPPs are amorphous (10), but small angle neutron scattering experiments (16) and electron diffraction (not shown) suggested that mineral crystallization to OCP precedes transformation to the second state. The high resolution image (inset) visualizes the lattice planes. C, in contrast to fetuin-A, albumin as a low affinity mineral binder inhibited mineral formation even at high serum-like concentration for only a few minutes. Similar to fetuin-A, electron diffraction (inset) suggested an initial amorphous mineral phase. Scale bar in all images 100 nm. D, separation of secondary CPPs (second state, 9 h, room temperature) from the free fetuin-A monomer by S500 size exclusion chromatography monitored at {lambda} = 280 nm (solid line) and 400 nm (dashed line). CPPs were eluted directly after the void volume V0, whereas the fetuin-A monomer was eluted at higher volumes. Because detection in the UV range is more susceptible to turbidity, protein detection at 280 nm interferes with the turbidity caused by the CPPs, whereas the 400-nm absorption curve represents the light scattered at the CPPs. mAU, milliabsorbance units. E, peak fractions from D applied onto a SDS gel. Streptavidin-horseradish peroxidase detection of biotinylated fetuin-A revealed that only a minor fraction of total fetuin-A (20 µM) was enclosed in the CPPs. F, phosphate concentration profile. A semiquantitative analysis indicated that about 70% of the total phosphate was enclosed in the CPPs.

 
We employed two light scattering based methods, turbidimetry and dynamic light scattering, to study the formation and stability of CPPs in real time. The onset of mineral sedimentation at ambient conditions, thus, proved a reproducible indicator of CPP stability. Fig. 3B shows the ripening of typical inhibition mixes containing various fetuin-A concentrations followed by time-resolved turbidimetry. Primary CPPs are small; thus, little incident light is scattered and the measured absorption at 400 nm, i.e. the turbidity, is low. CPP transformation is associated with an increase in particle size, and consequently the absorption increases until the second CPP state is reached. Finally, sedimentation is indicated by a rapid decrease in scattering intensity. We determined that 7 µM fetuin-A was the lower threshold concentration to effect CPP stability for at least 9 h (Fig. 3B). A stepwise increase in fetuin-A concentration up to 50 µM resulted in smaller particles, less light scattering, and a further delay of CPP transformation, suggesting increased stability.

At fetuin-A concentrations below 7 µM the CPP stability inversely correlated with the fetuin-A concentration. Increasing fetuin-A from 3 to 5 µM resulted in a delayed onset of transformation as expected but paradoxically was followed by immediate sedimentation, i.e. decreased stability of the secondary particles. A similar "nucleating effect" at low fetuin-A concentrations indicated by the excessive mineral formation was also observed in the sedimentation based assay of Fig. 3A. The time-resolved analysis of CPP formation suggests that the increase in mineral sedimentation was due to reduced stability of secondary CPPs but not due to accelerated nucleation and growth of primary CPPs.

Next we analyzed the inhibition capacities of mixtures of fetuin-A, albumin, control proteins, sera, and mixtures thereof. Spontaneous precipitation of calcium phosphate within the first 2 min of incubation was observed in the protein-free control samples and in the lowest albumin samples (Fig. 3C). Albumin at the nominal serum concentration of 606 µM (40 mg/ml) did not inhibit mineral sedimentation for more than a few minutes. In contrast, fetuin-A at 1.5 µM (~1/10th of its serum concentration) prevented mineral sedimentation for about 2 h. 7 µM fetuin-A proved to be the lower concentration limit that mediated sedimentation inhibition for at least 2 days. The term long-term inhibition (LTI) shall refer to samples that were stable for at least 2 days, i.e. 2880 min. LTI samples generally showed no defined onset of sedimentation anymore. Instead, precipitation proceeded very slowly over days.


Figure 3
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  		FIGURE 3.
Concerted mineral sedimentation inhibition by the two serum proteins fetuin-A and albumin. A, an IC50 of 7.4 µM was assessed for fetuin-A using the precipitate quantification based inhibition assay (see "Materials and Methods"). B, corresponding time-resolved turbidimetry measurements indicated that the inhibition mixes were stable down to a concentration of 7 µM fetuin-A. Moreover, the onset of CPP transformation, i.e. the stability of primary CPPs, correlated with the fetuin-A concentration. C, investigation of the inhibition of fetuin-A, albumin, and mixtures thereof at various concentrations by turbidimetry. The measurements revealed that supplementary albumin, itself a weak inhibitor, significantly enhances the mediated fetuin-A inhibition. n ≥ 3.

 
The inhibitory capacities of albumin/fetuin-A mixtures were, thus, assessed by measuring the onset of sedimentation (Fig. 3C). The LTI threshold concentration of 7 µM fetuin-A could be reduced to ~50% (3-4 µM) when equimolar amounts of albumin were added to the precipitation mixture (Fig. 3C). Further decrease of the fetuin-A concentration and substitution by albumin resulted in unstable CPPs or required unproportionally high amounts of albumin. For example, at 7 µM fetuin-A all three samples showed a LTI effect. Mixtures including 4 µM fetuin-A and 3 µM albumin effected LTI in 3 of 4 samples tested; 4 µM fetuin-A/6 µM albumin mixtures resulted in LTI in all three samples tested. Along these lines, mixtures of 3 µM fetuin-A, 4 µM albumin resulted in LTI in 1 of 4 samples; 3 µM fetuin-A, 8 µM albumin resulted in LTI in all 3 samples. 1.5 µM fetuin-A required the addition of at least 75 µM albumin to attain LTI.

We asked whether this concerted inhibition was confined to the combination of fetuin-A and albumin or if other acidic proteins could complement as well. Replacing albumin with the acidic proteins ovalbumin and prothrombin led to similar results (Table 1), suggesting that acidic proteins like albumin cannot effect LTI on their own (5, 28) but may substitute a certain fetuin-A fraction. Likewise, 5% murine fetuin-A-deficient serum from fetuin-A knock-out mice (C57BL/6 strain) showed a low activity by itself but could be reconstituted by the addition of 3 µM fetuin-A. Previously, we showed that recombinant glutathione S-transferase, a neutral protein, and lysozyme, a basic protein, both proved inactive in the original inhibition assay (5, 10). Both proteins showed no activity in our mixing experiments as well (Table 1), and we concluded that only acidic proteins but not neutral or basic proteins complement fetuin-A to effect LTI.


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  		TABLE 1
Supplementary acidic proteins improve CPP long-term stability

The onsets of sedimentation of several test proteins at a concentration of 10 and 3 µM fetuin-A were assessed by automated turbidity measurements, respectively. Fetuin-A proved to inhibit mineral sedimentation better than the test proteins, but none achieved LTI. Next, the effect of proteins in the presence of fetuin-A was studied in a mixture of 10 µM of the indicated proteins with 3 µM fetuin-A. In the case of a concerted inhibition of fetuin-A and test protein, the inhibition capacity resulted in LTI with an onset of sedimentation ≥2880 min (2 days) and little variation between preparations. If the test protein could not substitute for fetuin-A, the inhibition capacity would stay below the threshold and, thus, lead to mineral sedimentation within the indicated time. Serum from fetuin-A-deficient mice was also tested and reconstituted by adding fetuin-A. Values are the mean ± S.D.; n ≥ 3.

 
To elucidate the mechanism by which acidic proteins amplify the fetuin-A-mediated inhibition, we analyzed the impact of fetuin-A and albumin on the stability of both CPP states. Fig. 4 shows the onset of CPP transformation and the onset of sedimentation as indicators of primary and secondary CPP stability, respectively, plotted against the protein concentration. Fetuin-A dose-dependently delayed both the onset of CPP transformation and sedimentation (Fig. 4A). Titrating increasing amounts of albumin into a fixed amount of fetuin-A over a range of 1.5-4 µM fetuin-A and 0-20 µM bovine serum albumin (Fig. 4, B-D) revealed that the onset of CPP transformation solely depended on the fetuin-A concentration but not on the albumin concentration as indicated by the horizontal lines depicting the onset of transformation. In contrast, both proteins dose-dependently stabilized secondary CPPs as indicated by the delay in the onset of sedimentation. This suggested hierarchical roles of serum proteins in terms of fetuin-A only being able to stabilize primary CPPs, whereas fetuin-A and other acidic proteins like albumin stabilize secondary CPPs. In a following experiment, the procedure was slightly changed. Albumin was not included in the sample from the start but added right after the onset of CPP transformation. The fact that LTI was maintained corroborated the hierarchical and sequential action in that fetuin-A effectively stabilized the first CPP state, whereas acidic proteins or macromolecules at large may stabilize the second state.


Figure 4
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  		FIGURE 4.
Binary sedimentation inhibition model studied by turbidimetry. The protein concentrations were plotted against the recorded onsets of CPP transformation and sedimentation. A, increasing fetuin-A concentrations delayed CPP transformation and sedimentation. 7 µM fetuin-A proved to be the LTI threshold concentration. B-D, at fixed fetuin-A concentrations (1.5, 3, 4 µM), the albumin concentration was plotted against the onsets of CPP transformation and sedimentation. The shallow trend lines through the onsets of transformation indicated that they were independent of the albumin concentration. Instead, increasing the albumin concentration resulted in a sudden long-term stabilization of secondary CPPs.

 
Given the fact that fetuin-A could be substituted for by other negatively charged proteins, we asked how much fetuin-A was actually contained in second-stage CPPs (LTI) formed in protein mixtures. We ultra-filtered (300-kDa cutoff) the secondary CPPs matured for 1 day at room temperature. At 7 µM fetuin-A, i.e. the LTI threshold concentration, 3.8 ± 0.3 µM of the initial fetuin-A was contained in the CPPs. Repeating the mineralization reaction in the presence of 20 µM fetuin-A similarly resulted in 3.6 ± 0.8 µM fetuin-A bound to CPPs. Thus, even at the fetuin-A threshold concentration ~60% of the total fetuin-A was bound to secondary CPPs. Moreover, these data suggested that at a given supersaturation, the amount of CPP bound fetuin-A contained in the second-stage CPPs was independent of the initial fetuin-A concentration.

CPP Ripening Studied by Time-resolved Dynamic Light Scattering and {zeta}-Potential Measurement—Selected samples were analyzed by DLS covering a time span of at least 16 h. The values for the hydrodynamic radius Rh were obtained via the Stokes-Einstein relation taking the friction coefficient of spherical particles,

Formula(Eq.1)

Formula(Eq.2)
with Boltzmann constant kB and solvent viscosity {eta}.

At 296 K (23 °C), fetuin-A concentration (7 to 50 µM) correlated inversely with the size of the CPPs formed, i.e. 38 ± 9 to 63 ± 6 nm for the first state and 72 ± 8 to 103 ± 13 nm for the second state. As in the turbidimetry measurements, increasing fetuin-A delayed the onset of CPP transformation in a concentration-dependent manner (Fig. 5A). To study the combined effects of fetuin-A concentration and temperature on CPP ripening, three concentrations were tested at three temperatures. Increasing the temperature shortened the onset of CPP transformation greatly but changed CPP size slightly. For example, Fig. 5, B-D, illustrates that raising the temperature from room temperature (23 °C, 296 K) to body temperature (37 °C, 310 K) accelerated CPP ripening roughly 6-7-fold regardless of the fetuin-A concentration. This was associated with a low CPP size variation by comparison of 11-20% (across all fetuin-A concentrations and temperatures).


Figure 5
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  		FIGURE 5.
Time-resolved DLS analysis of CPP ripening. A, the formation of CPPs was monitored by dynamic light scattering. The inhibition kinetics closely resembled the turbidimetry measurement shown in Fig. 3B. B-D, CPP ripening was measured at the concentrations and temperatures indicated. Increasing temperature and decreasing fetuin-A concentration were associated with reduced stability of primary CPPs, indicated by a left-shift of curves. Small changes in CPP sizes were also detected. All samples were stable within the time, and temperature range was tested.

 
The particles in the first CPP state were spherical as shown in Fig. 2A, whereas the secondary CPPs were elongated ellipsoids with a prolate shape. The friction coefficient of prolate ellipsoids depends on the size and ratio of their semi-axes a and b (29). One obtainsFormula

From electron micrographs (e.g. Fig. 2B) the ratio of the semi-axes was estimated to b/a = 0.3 so that the semi-axes can be calculated from the Rh values (Table 2). We found that the size of the shorter semi-axis b of the secondary CPPs was very close to the radius of the primary CPPs, suggesting that the secondary CPPs were formed by anisotropic growth of the primary particles, probably induced by crystallization. These results confirm our recent neutron scattering experiments (16).


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  		TABLE 2
Hydrodynamic radii Rh of selected CPP samples assessed by DLS

CPP transformation is accompanied by a reduction in polydispersity as indicated by the polydispersity index (PDI). Regarding the prolate ellipsoidal shape of secondary CPPs, the length of the semi-axes a and b was derived from the initial Rh according to Equation 2. The analysis of electron micrographs (Fig. 2B) led to a b/a semi-axes ratio of approximately 0.3; hence, a = 1.96 Rh. The shorter semi-axis b of the elongated secondary CPPs matches the Rh of primary, spherical CPPs, indicating an anisotropic growth during transformation. Values are the mean ± S.D.

 
Primary CPPs were notoriously unstable. Even at 50 µM fetuin-A (4-5-fold serum concentration in humans) primary CPPs lasted no longer than 7 h. Fetal calf serum is known to contain up to 13 g/liter (260 µM) fetuin-A (30). We reasoned that this should bestow a very high inhibitory capacity to fetal calf serum. Indeed CPP synthesis in 85% fetal calf serum at 37 °C resulted in the smallest primary CPPs observed throughout this study with a Rh of 18 nm (Table 2). Nevertheless, even in the presence of this exceedingly high amount of fetuin-A and supplementary acidic proteins, the primary CPPs were transiently stable for only 11 h, after which they transformed into secondary CPPs. This suggested that the observed LTI (>48 h) basically results from an efficient stabilization of secondary CPPs.

Sub-threshold fetuin-A concentrations, like 3 µM, resulted in a short-term stabilization of the first CPP state followed by transformation, rapid particle aggregation and growth, and finally mineral sedimentation. LTI reconstitution by supplementing 3 µM fetuin-A with 10 µM albumin, ovalbumin, or prothrombin resulted in particles with hydrodynamic radii of 67-84 nm for the first state and of 125-286 nm for the second state. These hydrodynamic radii of protein mixtures were intermediate between 7 µM pure fetuin-A samples (Rh 62 nm, first state; 96 nm, second state) and 3 µM pure fetuin-A samples (Rh 84 first state, Table 2), suggesting that additional acidic protein could not fully replace fetuin-A as a stabilizer but, nevertheless, mediated LTI. CPPs tended to be monodisperse as indicated by the low polydispersity indices listed in Table 2 ranging from 0.1 to 0.3 for primary CPPs and from 0.05 to 0.1 for secondary CPPs, indicating a narrowing of the size distribution during CPP transformation.

To estimate the surface charge of ripening CPP (20 µM fetuin-A), we measured time-resolved {zeta} potentials. A slightly negative potential of -9(±4) and -10.5 (±4) mV, respectively, was assessed for the first and the second CPP state, respectively. Apparently, the resulting weak Coulomb interaction and steric repulsion between the particles was sufficient to prevent particle aggregation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Physiologic Relevance of CPPs in Mineral Transport and Clearing—Serum-derived mineral-binding proteins are long known to exist in bone. Fetuin-A is in fact the most abundant non-collagenous protein in mineralized bone matrix, and the reason for this strong accumulation has been somewhat enigmatic. We propose that fetuin-A and other acidic serum proteins like albumin play an important role in the formation and stabilization of mineral complexes under conditions of local supersaturation as in transport epithelia, e.g. in the kidney or in the bone remodeling compartment. In bone, osteoclasts degrade mineralized bone using acid to dissolve mineral and proteolytic enzymes to cleave matrix proteins. Osteoclasts internalize dissolved bone components by endocytosis and transport and deliver these contents by vesicular transcytosis (31-33) to basolateral membrane sites and then to the bone marrow compartment, whose highly permeable vascular sinusoids are continuous with other vascular elements and the blood. The continuum of these processes begs the question of why calcium phosphate mineral does not instantly re-precipitate at the surface of the cell or extracellularly once mineral ions leave the acidified vesicles of the osteoclasts. This example illustrates that the mineralization "risk" is probably the highest in the organ that regularly mineralizes and re-mineralizes; that is, the skeleton (34, 35). Given this, it seems reasonable to propose a powerful cargo system to prevent hydroxyapatite from spontaneously precipitating at sites not normally destined for mineralization. This system elaborates on a largely hypothetical concept of "bone solubility" (36) based on the original finding that bone mineral is highly soluble in the presence of non-collagenous bone proteins (37, 38), notably acidic {gamma}-carboxyglutamate (GLA) proteins like osteocalcin/bone GLA protein or phosphoproteins like osteopontin (6) and SPP 24 (20).

Increasing evidence suggests that soluble mineral complexes like the CPPs described here actually operate in vivo. Treatment of rats with high doses of etidronate or vitamin D caused a massive surge of calciprotein-like particles in the blood. Protein-mineral particle isolation and subsequent protein analysis by SDS-PAGE led to the identification of fetuin-A as the most abundant protein in the "fetuin mineral complex" (17, 18). Fetuin-A was also found to stabilize calcium phosphate in CPP-like particles in endocytic transport vesicles of vascular smooth muscle cells challenged with high extracellular calcium and phosphate (39). In the absence of fetuin-A the cells underwent apoptosis and subsequently calcified. The mineral containing colloids from human ascites presented here likewise seemed very similar in structure and composition to CPPs generated in vitro (Fig. 2 and Heiss et al. (16)). We speculate that under physiological conditions (i) primary CPPs form spontaneously at sites of elevated mineral ion supersaturation, (ii) subsequently may redissolve spontaneously, be (re-) integrated into bone, be taken up by phagocytic cells, (iii) or otherwise finally transform to secondary CPPs. A reduced fetuin-A serum level or increased mineral supersaturation, e.g. kidney failure in humans, would result in reduced stability of the first CPP state, increased CPP size, and possibly a higher number density of secondary CPPs. We conclude that the blood is well buffered against the risk of direct mineral deposition. Hypercalcemia and hyperphosphatemia induced apoptosis as well as defective CPP clearing by the reticuloendothelial system may, however, result in the local accumulation of protein-mineral complexes at sites of tissue injury causing dystrophic tissue calcification.

Hierarchical, Concerted Action of Acidic Serum Proteins in the Formation of CPPs—Fetuin-A is the protein inhibitor with the highest capacity in the general circulation when both the serum concentration and the activity are taken into account (5, 10, 11, 13, 14, 17). In comparison, acidic plasma proteins albumin (3, 5), ovalbumin (5), and prothrombin (28) proved to be relatively weak inhibitors of mineral deposition in vitro when used alone. The surprising finding that large amounts of albumin were nevertheless contained in CPPs isolated from ascites of a calcifying peritonitis patient prompted us to reconsider the respective roles of acidic serum proteins in the solubilization of calcium phosphate and, hence, in the prevention of spontaneous mineral deposition.

Various methods to identify mineralization-inhibiting proteins and to assess their activity have been published, e.g. solvent constant composition (28), mineral affinity (40), and precipitate quantification (5, 10). Here we introduce time-resolved light scattering methods for in situ measurements of CPP formation. According to our light scattering experiments, 7 µM fetuin-A proved to be the threshold concentration for LTI. Of a total of 7 µM concentrations of protein, up to ~40% fetuin-A could be substituted by roughly equimolar amounts of other acidic protein. A further decrease in the fetuin-A concentration required disproportionately more auxiliary protein to maintain LTI. A maximum of 80% of fetuin-A could be substituted by adding a 14-fold molar excess of albumin. Even though LTI was maintained macroscopically, the stability of the first state decreased and the hydrodynamic radii increased for both CPP states (Fig. 3C, Table 2).

This suggests that substantial amounts of fetuin-A could be replaced by albumin on the basis of an increasingly distorted molar exchange ratio. However, without any fetuin-A in the precipitation mixtures, stable secondary CPPs mediating LTI could never be formed. Furthermore, the stabilization of secondary CPPs was independent of protein structure, as it could be achieved with fetuin-A as well as with other acidic but structurally unrelated proteins. Further studies will focus on the molecular topologies to elucidate why this strong impact of acidic macromolecules is confined to the stabilization of secondary CPPs.

Our in vitro model system was useful in that it enabled the time-resolved study of CPP formation and maturation at high resolution and over a wide concentration range of fetuin-A. Furthermore, the CPPs synthesized in vitro may serve as a model for entities present in human pathology like nanoscopic calcifications (41) or colloidal protein-mineral composites from the ascites of a dialysis patient (Fig. 1B) that are similar to secondary CPPs (Fig. 2B). The former particles were initially described as calcified nanobacteria (41). A recent biochemical analysis, however, challenged the "calcifying nanobacteria theory" in stating that the alleged nanobacteria are actually fetuin-mineral complexes (42). This re-classification of a pathologic calcification mechanism further corroborates our view that CPPs may be a vehicle for mineral transport involved in physiological and pathological tissue remodeling (35).

Critical Fetuin-A Serum Concentration—Fetuin-A serum deficiency was tightly associated with calcification and cardiovascular mortality in several patient cohorts (8, 14, 15). This allows one to address the issue of the fetuin-A serum concentration critically required to prevent calcification from solution. Our study suggests that fetuin-A concentrations as low as 1.5 µM (~0.1 mg/ml) are sufficient to stabilize CPPs as long as sufficient other acidic serum protein is present. In agreement with this value, our own patient study showed that the highest mortality risk existed in patients with fetuin-A serum concentrations below 0.2 mg/ml (8). Dialysis patients often suffer, however, combined deficiency in fetuin-A and albumin, which may help to explain why these patients are at much higher risk of cardiovascular calcification-associated morbidity and mortality than the general population (8). In light of this study fetuin-A serum levels should always be judged against the albumin serum concentration and the so-called serum calcium-phosphate product. Furthermore, the large variability between fetuin-A assays needs to be addressed, because it prevents a meaningful comparison of individual studies.

A Putative Clearing Pathway for CPPs—Small angle neutron scattering analysis with contrast variation revealed that secondary CPPs consist of an octacalcium phosphate core covered by a dense negatively charged fetuin-A monolayer (16). {zeta}-Potential measurements in this study showed that CPP transformation did not affect the surface charge, suggesting that a dense protein layer also covered the primary CPPs. The negatively charged protein layer on the surface of both primary and secondary CPPs intriguingly suggests that a potential clearing of CPPs from circulation may rely on this charge. Nagayama et al. (43) recently showed that the uptake of fetuin-A-coated 50-nm latex particles by hepatic cells was mediated by scavenger receptors, which have a propensity to bind negatively charged foreign material. A more general role of fetuin-A in apoptotic cell, debris, and vesicle clearing has also been suggested (39, 44). Alternative clearing mechanisms may involve annexin 2 (45) or the asialoglycoprotein receptor (46).

Inflammation may impede efficient clearing and promote calcification. Chronic inflammation is commonly found in dialysis patients and causes a reduction in serum fetuin-A, which is a negative acute phase protein (47, 48). A recent publication investigated how the properties of mineralization precursors may influence vascular calcification. It was reported that hydroxyapatite microcrystals, depending on their crystal size, triggered a pro-inflammatory tumor necrosis factor-{alpha} response in macrophages. Tumor necrosis factor-{alpha} is a known promoter of vascular calcification, thus starting a destructive cycle (49). The inflammatory capacity of small primary and larger secondary CPPs, respectively, as soluble colloidal precursors in bone remodeling and vascular calcification is unknown and merits further study.

Mineral Chaperones—In conclusion, the data presented here provide further evidence for the capacity of fetuin-A in calcification inhibition, which is outstanding among serum proteins. Time-resolved measurements indicated that this effect is based on the efficient stabilization of nascent mineral nuclei, i.e. primary CPPs by fetuin-A. Fetuin-A stabilized secondary CPPs equally well but could be partially substituted by acidic serum proteins. Acidic serum proteins may be regarded as "mineral chaperones" mediating the formation of CPPs and the transport of calcium phosphate as colloids. This notion should be viewed as a minor but critically important alternative to the well established homeostasis of calcium and phosphate ions in the body.


   FOOTNOTES
 
* This work was funded by the priority program of the Deutsche Forschungsgemeinschaft "Principles of Biomineralization."; The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Helmholtz Institute for Biomedical Engineering, Biointerface Group, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany. Tel.: 49-241-8088719; Fax: 49-241-8082573; E-mail: alexander.heiss{at}post.rwth-aachen.de.

2 The abbreviations used are: CPP, calciprotein particle; DLS, dynamic light scattering; LTI, long-term inhibition; TEM, transmission electron microscopy. Back


   ACKNOWLEDGMENTS
 
We thank J. Mayer for supporting TEM analysis, S. Brincker and S. Gräber for excellent technical assistance, C. Shanahan for providing prothrombin, and D. Schwahn for helpful discussions.



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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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