From IZKF BIOMAT, University Clinics, RWTH Aachen,
Pauwelsstrasse 30, D-52074 Aachen, Germany,
§ Max-Planck-Institute for Polymer Research, Ackermannweg
10, D-55128 Mainz, Germany, ¶ Institute of Biochemistry,
University of Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany,
Department of Biochemistry, Kinki University School of Medicine,
Osaka 589-8511, Japan, and ** Institute for Clinical
Biochemistry and Pathobiochemistry, Josef-Schneider-Strasse 2, Julius-Maximilians-University, D-97080 Würzburg, Germany
Received for publication, October 24, 2002, and in revised form, January 29, 2003
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ABSTRACT |
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Genetic evidence from mutant mice suggests that
The combination of mineral with an organic matrix called
"biomineral" is commonplace in biology. Biominerals studied in
detail include magnetic crystals in bacteria (1), silica skeletons in
diatomeous algae (2, 3), shells of marine molluscs (4, 5), and
skeletons of vertebrate animals (6). Generally, biominerals form in
close proximity with biomacromolecules. Ultrastructural analyses
suggest that a protein scaffold provides the ordered and spatially
restrained framework for crystal deposition. In mammals, collagen is an
excellent scaffold for calcification. Noncollagenous proteins control
nucleation, growth, shape, and orientation of crystals in the mineral
phase (7, 8). Major mineral ions are equally distributed in the
extracellular space of most living organisms. Extracellular fluids are
especially supersaturated with regard to calcium and phosphate ions.
Therefore, it is surprising that mineralization is restricted to
collagenous matrix of the vertebrate skeleton and that once started
mineralization does not proceed throughout the organism (9). This
suggests that the inhibition of unwanted mineralization is at least as important as the initiation of mineralization. Genetic experimentation with mutant mice indeed suggests that mineralization is the default pathway, which must be actively prevented, not started (10). Unwanted
mineralization resulted from the genetic ablation of mineralization
inhibitors, pyrophosphate (11, 12) and matrix Precipitation Inhibition Assay--
The precipitation inhibition
assay was performed as described (17). Briefly, a buffered salt
solution (50 mM Tris/HCl, pH 7.4, 4.8 mM
CaCl2, 2 × 106 cpm of
[45Ca]Cl2, 1.6 mM
Na2HPO4) containing test proteins as indicated in the figure legends was incubated at 37 °C for 90 min.
Precipitates were collected by centrifugation (15,000 × g, 5 min at room temperature), dissolved in 1% acetic acid,
and quantified by liquid scintillation counting. All incubations were
done in triplicates. Bovine serum albumin (BSA; Roth, Karlsruhe,
Germany) and bovine fetuin/bAhsg (Sigma) were used as negative and
positive control proteins, respectively.
Electron Microscopy and Electron Spectroscopy--
For scanning
electron microscopy, a supersaturated solution of calcium (2.5 mM CaCl2) and phosphate (1.8 mM
KH2PO4) was prepared (18) with and without 200 nM bAhsg added. After a 90-min incubation at 22 °C,
precipitate was spun down, air-dried, and viewed in a Leo series 1400 scanning electron microscope (Leo Electron Microscopy Ltd., Cambridge, UK).
For transmission electron microscopy (TEM) analysis, bAhsg and BSA were
purified by gel filtration (Superdex 200; Amersham Biosciences,
Freiburg, Germany) in 50 mM Tris/HCl, pH 7.4. Monomer-containing fractions were collected, and the protein
concentration was determined using a dye assay (Roti-Nanoquant,
Roth, Karlsruhe, Germany). All solutions were
microfiltered (0.22 µm) before mixing. Following the precipitation
reaction in buffer (5 mM CaCl2, 3 mM Na2HPO4, 50 mM
Tris/HCl) at pH 7.4 and 22 and 37 °C, respectively, samples were
dialyzed against water (MilliQ; Millipore Corp.) using micro dialysis
cartridges. This step was essential for electron microscopy of the
precipitation mixture, which would otherwise have been obscured by
dried salt. The dialyzed samples were cleared by centrifugation for 1 min at 1000 × g. The supernatant and, if formed, the
precipitate were transferred to carbon-coated grids. Excess liquid was
blotted from the side. The samples were viewed directly in TEM without staining. Supporting films of 7-nm thickness were coated onto freshly
cleaved mica using a Balzers BAE 250 vacuum evaporator (Bingen,
Germany), floated onto water, and transferred to 300-mesh copper grids.
For elemental mapping of carbon, supporting films were made of boron.
Energy-filtering transmission electron microscopy was performed on a
Leo 912 Omega instrument (tungsten filament) operated at 120 kV using
an objective aperture of 16.5 millirads. The width of the energy
window controlled by the opening of the energy selector slit was 10 eV.
All images were recorded using a slow scan CCD camera (lateral
resolution 1024 × 1024 pixels, 14-bit gray) and processed using a
SIS-AnalySIS® image processing system. Elemental
distribution images where all obtained by three window potential
background extrapolation (19).
Dynamic Light Scattering--
Dynamic light scattering was
measured using a laser (Spectra Physics 165, Molecular Modeling--
A total of nine cystatin-like protein
domains of fetuin-A/Ahsg, fetuin-B (FETUB), kininogen (KNG), and
histidine-rich glycoprotein (HRG) were modeled comparatively. The
models were generated with Modeler4 software (20) using chicken egg
white cystatin (Protein Data Bank accession code 1CEW) (21) as a
template structure. During iterative refinement steps, the generated
models were evaluated with Procheck and Prosa II (20, 22, 23).
Additionally, models were scrutinized for structural clashes by
Ramachandran plotting. No Recombinant Proteins--
The cloning, expression, and
purification of fusion proteins with maltose-binding protein (MBP) has
been described (17). Full-length mAhsg cDNA and deletion mutants
thereof were PCR-amplified and ligated into pRSET-5c vector containing
a Myc tag sequence and an additional EcoRI site downstream
of the cloning sequence. The Myc tag oligonucleotides used for vector
modification are listed in Table I. Using PCR and the primers listed in
Table I, Myc-tagged deletion mutants of mAhsg cDNA were generated
and subcloned by BamHI/EcoRI restriction/ligation
into the vector pGEX-2T (Amersham Biosciences) for the expression of
GST fusion proteins and by BamHI/HindIII
restriction/ligation into the vector pMAL-c2 (New England
Biolabs, Schwalbach, Germany) for MBP fusion protein expression.
Fetuin-B cDNA was cloned by reverse transcription-PCR from rat,
mouse, and human liver mRNA using murine leukemia virus reverse
transcriptase (PerkinElmer Life Sciences) and Pwo polymerase (PeqLab, Erlangen, Germany) into the vector pGEMT (Promega,
Madison, WI). For fusion protein expression, the cDNA
inserts were excised by BamHI/EcoRI restriction
and ligated into the vectors pGEX-2T and pMAL-c2. Using primer
mutagenesis, serine residues Ser120, Ser291,
Ser294, and Ser296 of the mature mouse Ahsg
protein chain were mutated to glutamic acid in order to mimic Ser
phosphorylation at these sites (24, 25). Mutations were achieved by a
three-step overlapping PCR using the primer pairs detailed in Table I.
The mutated mAhsg was ligated into the vector pGEX-2T for subsequent
expression as GST fusion protein (GST-mAhsg/4S>E). Table I lists the
mutated mAhsg constructs and the PCR primers used for their construction.
To clone the cDNA sequences encoding cystatin-domains D1, D2, and
D3 of human kininogen, total RNA was prepared from HepG2 cells
following established protocols. The cystatin domains were cloned by
reverse transcription-PCR using murine leukemia virus reverse
transcriptase (New England Biolabs), Taq polymerase
(Amersham Biosciences), and the primer pairs listed in Table
I. Primers contained additional
restriction sites and stop codons. The amplicons were ligated into
vector pMAL-c2 using BamHI/HindIII (hKNG-D1), XbaI/HindIII (hKNG-D2), or
XbaI/PstI (hKNG-D3), respectively.
All cloning steps were verified by DNA sequencing. Expressed proteins
were probed on the protein level using species-specific antisera and
immunoblotting. Specific antisera at our disposal were originally
raised against human, rat, mouse, and bovine Ahsg and recombinant
human, rat, and mouse FETUB (data not shown).
Protein Expression, Purification, and Refolding--
Bacterial
cultures were inoculated from an overnight preculture 1:500. At an
A600 of 0.5, isopropyl-1-thio-
After centrifugation for 15 min at 4 °C and 30,000 × g, the supernatant was loaded onto an amylose column. After
washing the column with 3 column volumes of amylose column buffer for
MBP fusion proteins and with 3 column volumes of PBS for GST fusion proteins, the MBP-fused proteins were eluted with amylose column buffer
containing 10 mM maltose, and the GST-fused protein was eluted with buffer containing 20 mM Tris, pH 8, and 10 mM reduced glutathione.
Recombinant protein isolated from the bacteria without a
denaturation/refolding cycle was generally inactive in the BCP
precipitation inhibition assay. Therefore, every recombinant protein
had to be denatured and refolded (17). The stability and activity of the fusion proteins depended on the refolding procedure. Following is
an optimized refolding procedure yielding active protein for both GST
and MBP fusion proteins.
All recombinant proteins were solubilized for 2 h at room
temperature in a buffer containing 50 mM Tris/HCl adjusted
to pH 8, 6 M urea, and 50 mM dithiothreitol.
The denatured protein solution was concentrated to 5 mg/ml using the
same buffer with dithiothreitol content reduced to 5 mM.
Refolding was initiated by slow dilution of protein solution (final
concentration less than 50 µg/ml) into a vigorously stirred
redox-refolding buffer (50 mM Tris, pH 8, 2 mM
GSH, 0.2 mM GSSG, 1 M 3-(1-pyridino)-1-propane
sulfonate, NDSB 201). The final yield of functional protein and its
stability was greatly improved by adding NDSB 201 to the
redox-refolding buffer. This solution was incubated for at least 1 day
at room temperature to allow air oxidation of reduced protein. Using
ultrafiltration, the initial buffer was replaced by 50 mM
Tris/HCl, pH 7.4. The formation of aggregates during the refolding
procedure was routinely analyzed by gel filtration chromatography using
Superdex 200 and 75 gel filtration columns (Amersham Biosciences).
Inhibition of Basic Calcium Phosphate Precipitation by
Ahsg--
Our previous work has shown that Ahsg is highly effective
in vitro to inhibit de novo formation of BCP
(17). This inhibition was transient and lasted about 5-6 h at the
conditions of our assay. The precipitation delay seems, however,
sufficient to prevent generalized calcification in vivo. Our
recent finding of severe systemic calcification of most soft
tissues2 in Ahsg-deficient mice validates the biological
relevance of this concept. Here we asked how Ahsg interacts with the
calcium and phosphate ions or the mineral nuclei to achieve inhibition.
Using scanning electron microscopy, we studied the influence of Ahsg on
the morphology of the mineral precipitate. First, we analyzed the
precipitate formed from a metastable calcium phosphate solution with or
without added native bAhsg (Fig. 1). The
exact nature of this precipitate is uncertain because of phase
transitions. Therefore, we collectively address the precipitate formed
as BCP, regardless of its exact chemical and crystallographic
composition of variable proportions of amorphous calcium phosphate,
octacalcium phosphate, and apatite. In the absence of bAhsg, copious
amounts of BCP formed and appeared as a compact pellet (Fig.
1A). The addition of 200 nM bAhsg to the
precipitation mixture barely reduced the amount of precipitate formed.
However, the morphology of the BCP precipitate formed at this low
concentration of bAhsg was changed into a loose, fluffy precipitate
comprising small, 2-15-µm-sized aggregates (Fig. 1B).
A precipitation mix containing 10 µM bAhsg, the nominal
serum concentration, was stable for many hours at 37 °C without any precipitate formation. Control incubations containing 10 µM BSA or no protein formed a clearly visible precipitate
within 2 h under otherwise identical conditions. The precipitates
of both controls were indistinguishable and were microcrystalline
as judged by TEM (not shown). Next, the dialyzed supernatants were
subjected to TEM analysis. Supernatants of precipitation mixtures,
which had been incubated for 2 h at 22 or at 37 °C,
respectively in the presence of 10 µM bAhsg contained
spherical aggregates with a diameter of 30-150 nm (Fig.
2, A and F). The
size and shape of the aggregates were independent of the order of
CaCl2 and Na2HPO4 addition. The
precipitates were amorphous as judged by the lack of discrete
diffraction patterns in TEM (Fig. 2F, inset). We
termed these soluble, colloidal spheres "calciprotein particles" in
analogy to the well established lipoprotein particles. The supernatant of both controls (with BSA and protein-free, respectively) did not
contain any similar particles (not shown).
After 4 h of incubation at 37 °C, small crystalline needles
started to grow on the surface of the calciprotein particles (Fig. 2G), but not at 22 °C (Fig. 2B). The fact that
a diffraction pattern was obtained from samples incubated for 6 h
at 37 °C indicated that crystallization had started (Fig.
2H, inset). A reduced temperature of 22 °C
resulted in a delayed transformation of morphology (Fig. 2,
A-E). After 23 h at 22 °C, crystallization of
needles on the surface of spheres started to appear (Fig.
2C), similar in appearance to the samples harvested at
4 h and 37 °C (Fig. 2G). After 30 h at
37 °C, a solid BCP precipitate had formed. Small crystalline needles
were present in the supernatant (Fig. 2I), whereas the precipitate consisted of large clusters of radially oriented needles with a diameter of about 450 nm (Fig. 2J). In summary, the
transient inhibition of BCP precipitation by Ahsg relies on the
formation of soluble colloidal spheres, "calciprotein particles,"
which progressively turn into an insoluble crystalline precipitate.
Next, we obtained information on the composition of the calciprotein
particles by elemental mapping (Fig. 3).
This procedure visualizes the electron energy loss at absorption edges
characteristic of each element. Imaging of electrons with an energy
loss corresponding to, for example, a calcium absorption edge will
image the calcium-enriched regions of the specimen (19). Fig. 3
represents the elemental mapping of densely packed calciprotein
particles harvested 2 h after the start of a precipitation
reaction performed with 10 µM bAhsg at 37 °C. We show
the elastically filtered image (mass density, Fig. 3A) and
the phosphorus (Fig. 3B), the carbon (Fig. 3C),
and the calcium (Fig. 3D) net distribution image of the same region. The elemental mapping indicates that all three elements were
evenly distributed within the calciprotein particles.
We analyzed by dynamic light scattering (DLS) the speed of calciprotein
particle formation in solution. First, we studied a solution of bAhsg
without calcium and phosphate. We detected a major fraction (~99.5%)
with a hydrodynamic radius (rh) of 4.2 nm corresponding to
the Ahsg monomer and a small fraction (~0.5%) with
rh = 55.4 nm, which we tentatively assigned to Ahsg
aggregates (not shown). The addition of calcium resulted in an increase
of the gyration radius by about 10%. Compared with the calcium
phosphate- and Ahsg-containing samples (below), the scattering
intensities were very low (Fig. 4). We
observed a sharp rise in light scatter immediately after the addition
of calcium and phosphate (Fig. 4). A slow, strongly scattering colloid
matching the size of the calciprotein particles (rh = 40-50 nm) was detected in the solution, which we assigned to the
emerging calciprotein particles. Within the first 1 h of
incubation, a steep increase in the partial scattering intensity was
measured (Fig. 4). During the following 19 h, the increase in
intensity was moderate, yet continued. The hydrodynamic size still
continued to grow after 1 day, but the intensity decreased (not shown).
This is best explained by the complete sedimentation of the newly
formed insoluble BCP precipitate.
Inhibition of BCP Precipitation by Proteins of the Cystatin
Superfamily--
To further analyze the structural requirements for
efficient inhibition of BCP precipitation by Ahsg, we conducted a
structure-function analysis of Ahsg-related proteins. We produced a
series of mutated Ahsg fusion proteins as well as recombinant proteins
related to Ahsg and measured their ability to inhibit BCP
precipitation. To this end, we employed an established assay measuring
co-precipitation of 45Ca in a buffered solution containing
calcium, phosphate, and test protein (17). In this initial work, we had
mapped the basal structural motif mediating the inhibition of
spontaneous BCP precipitation to the amino-terminal cystatin-like Ahsg
domain D1. Hence, we tested several related proteins of the cystatin
superfamily containing structurally related cystatin-like protein
domains. KNG contains three cystatin-like domains, HRG contains two,
and Ahsg as well as its relative, the recently discovered FETUB (27),
both contain two cystatin-like domains. We previously determined that
HRG could inhibit precipitation, albeit with a 2-fold lower molar
efficiency than Ahsg (28). We scanned for additional structural
features that might contribute to the inhibition of calcification by
Ahsg. It is known that post-translational modifications of mineral
binding proteins, notably phosphorylation, influence their binding
properties (7, 29). We and others reported that human Ahsg and rat Ahsg are transiently serine-phosphorylated (24, 25, 30). To assess the
contribution of the resulting additional negative charge of mAhsg in
the precipitation inhibition assay, we constructed the phosphorylation-mimicking mAhsg mutant mAhsg/4S>E by a site-directed mutagenesis of putative Ser (positions 120, 291, 294, and 296) phosphorylation sites with Glu. We expressed mAhsg, mAhsg/4S>E, human,
mouse, and rat FETUB, and the three cystatin-like domains of hKNG in
Escherichia coli as untagged or as FLAG-tagged proteins (data not shown) and as Myc-tagged fusion proteins with GST or with
MBP. Generally, untagged or FLAG-tagged cystatin-like domains in our
hands did not yield sufficient starting material for subsequent purification and functional testing. GST-fused proteins were more highly expressed (~15 mg of GST fusion protein/liter of LB medium) but tended to form insoluble inclusion bodies. Therefore, all recombinant proteins had to undergo an unfolding/refolding cycle in
redox buffer before functional testing in the precipitation assay.
The top section of Fig.
5 shows the inhibition of BCP
precipitation by negative control proteins, BSA and GST, and the
positive control protein, bAhsg, at a concentration of 3 µM. Native serum bAhsg was most active in this assay,
whereas BSA, GST, and MBP (17) did not significantly inhibit BCP
precipitation under identical conditions.
Recombinant GST/MBP full-length mAhsg fused to GST or MBP proved active
inhibitors of BCP precipitation like native bAhsg (Fig. 5). A
comparison of the GST/MBP fusion proteins of mAhsg with the GST/MBP
fusion proteins of mFETUB revealed that the FETUB was a much weaker
inhibitor. Similarly, this observation applies to the GST fusions of
human and rat FETUB. Likewise, none of the three MBP-fused
cystatin-like domains of KNG proved to be an efficient inhibitor of BCP
precipitation. We conclude that Ahsg, but not the related cystatin
family members FETUB and KNG, is an efficient inhibitor of BCP
precipitation. We observed no considerable difference between the
activity of the GST-mAhsg/4S>E mutant and the wild type GST-mAhsg
protein in their ability to inhibit BCP precipitation (Fig. 5).
Therefore, serine phosphorylation of Ahsg does not seem to influence
the inhibitory activity.
Next, we asked the question which minimal sequence within the
cystatin-like Ahsg domain D1 confers the inhibition of BCP
precipitation. To this end, we generated deletion mutants of the Ahsg
domain D1 fused to GST. The resulting fusion proteins GST-mAhsg
1-81-Myc and GST-mAhsg 15-70-Myc were fully active in the BCP
precipitation assay when compared with the full-length fusion proteins
GST-mAhsg-Myc and MBP-mAhsg-Myc, respectively (Fig. 5). Shortening from
the amino terminus (GST-mAhsg 42-81-Myc) and additionally from the C
terminus (GST-mAhsg 42-70-Myc) caused a progressive loss of inhibitory
activity (Fig. 5), which is completely lost in the mutant MBP-mAhsg
1-52 (17).
Structural Requirement for the Inhibition of BCP Precipitation by
Cystatin-like Domains--
To interpret the results obtained by the
precipitation assay in a three-dimensional protein structure context,
we aligned the protein sequences of 16 cystatin-like domains, namely of
chicken egg white cystatin, human, mouse, and bovine Ahsg, human and
mouse fetuin-B, human histidine-rich glycoprotein, and human kininogen using ClustalW software (Fig. 6). The
alignments were manually adjusted to match the secondary structure
elements identified in the crystal structure of chicken egg white
cystatin (21) (Fig. 6A).
Based on the multiple sequence alignment (Fig. 6D), we
generated models of each domain by comparative structure modeling using the published crystal structure of chicken egg white cystatin (21) as a
template. Table II summarizes the
sequence identity and similarity with the template sequence, the
G-factors calculated by Procheck software (22), and the Z
scores obtained by Prosa II software (23) as indicators of the accuracy
of the modeled structures. According to these parameters, all models
were free of steric clashes and well within the confines of theoretical structure prediction.
The segment of the mAhsg domain D1 model (Fig. 6, B and
C), which adopts a
Next we scanned the modeled three-dimensional protein structure of each
domain for features that would explain their differential activity in
the BCP precipitation assay. Fig. 7 shows a compilation of identical
views facing the extending Fetuin/Ahsg is a potent inhibitor of BCP precipitation in
vitro and in vivo (17, 33).2 According to
published literature, bovine fetuin/bAhsg binds one calcium ion tightly
and five calcium ions more weakly (34). Based on a sequence alignment,
it was proposed that Ahsg domain D1 harbors an EF-hand-like calcium
binding motif, 92EGDCDFQLLK101 (35). The amino
acid positions 92-101 would, however, be located within 2-HS glycoprotein/fetuin-A (Ahsg) is a
systemic inhibitor of precipitation of basic calcium phosphate
preventing unwanted calcification. Using electron microscopy and
dynamic light scattering, we demonstrate that precipitation inhibition
by Ahsg is caused by the transient formation of soluble, colloidal
spheres, containing Ahsg, calcium, and phosphate. These "calciprotein
particles" of 30-150 nm in diameter are initially amorphous and
soluble but turn progressively more crystalline and insoluble in a
time- and temperature-dependent fashion. Solubilization in
Ahsg-containing calciprotein particles provides a novel conceptual
framework to explain how insoluble calcium precipitates may be
transported and removed in the bodies of mammals. Mutational analysis
showed that the basic calcium phosphate precipitation inhibition
activity resides in the amino-terminal cystatin-like domain D1 of Ahsg.
A structure-function analysis of wild type and mutant forms of
cystatin-like domains from Ahsg, full-length fetuin-B, histidine-rich
glycoprotein, and kininogen demonstrated that Ahsg domain D1 is most
efficient in inhibiting basic calcium phosphate precipitation. The
computer-modeled domain structures suggest that a dense array of acidic
residues on an extended
-sheet of the cystatin-like domain Ahsg-D1
mediates efficient inhibition.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxyl glutamic
acid (GLA)1-containing
protein (MGP) (13). We showed that the lack of
2-HS glycoprotein/fetuin-A (Ahsg) results in
severe systemic calcification in mice and humans
(15).2 Of note, Ahsg is the
only protein inhibitor of calcification known so far that is systemic
and present throughout the extracellular space in mammals. Due to its
high affinity for the mineral phase of bone, Ahsg accumulates
about 100-fold over other serum proteins in bones and teeth (16). This
seems paradoxical, considering that Ahsg is an efficient inhibitor of
calcification both in vitro and in vivo. Here we
studied how Ahsg inhibits the formation of basic calcium phosphate
(BCP). Using electron microscopy and dynamic light scattering, we
determined that the inhibition is effected by a transient formation of
colloidal spheres containing Ahsg, calcium, and phosphate, which we
call "calciprotein particles." Further, the structure-function
relationship of recombinant forms of Ahsg-like proteins from the
cystatin superfamily, fetuin-B (FETUB), histidine-rich glycoprotein
(HRG), and kininogen (KNG) suggests that the inhibition of unwanted
calcification by Ahsg involves binding of BCP nuclei to an array of
acidic amino acid residues on an extended
-sheet of the
cystatin-like Ahsg domain D1. We suggest that the resulting diffusion
barrier limits further growth of the crystal nuclei and thus delays
their precipitation. This proposed mechanism of the transient
inhibition of BCP precipitation by Ahsg is fundamentally different from
previous concepts, namely sequestration of calcium ions by negatively
charged proteins like serum albumin or calcium binding through an
EF-hand motif.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
= 514 nm, 300 milliwatts). The scattered intensity was recorded at Q = 90° (VV geometry) and analyzed by an ALV 5000 autocorrelator. The
square light scattering cell was equilibrated to T = 20 °C. Two-exponential fitting gave the best accuracy for all
autocorrelation functions. The intensities given are the absolute intensities due to scattering of the corresponding component. The
scattering intensity caused by pure water and the cell was approximately 7 kHz (toluene standard 55 kHz).
and
angles that are located in the
forbidden areas of the Ramachandran plot and no unnatural bond lengths
were observed. All structures proposed formed a compact core.
Energetically intolerable interactions between C-
atoms did not occur.
Oligonucleotide primers used for cloning of cystatin domain-containing
fusion proteins of GST or MBP and mouse Ahsg, mouse, rat, and human
FETUB, and human KNG
-D-galactopyranoside was added to a
final concentration of 300 µM. After a 2-h incubation at
37 °C, the bacteria were harvested by centrifugation for 10 min at
2500 × g. The bacterial pellet was resuspended in buffer containing 20 mM Tris, pH 7.4, 1 mM EDTA, and
200 mM NaCl (MBP fusion protein, amylose column buffer) or
in PBS (GST fusion protein, glutathione-Sepharose column buffer). In
the case of small MBP-fused fragments of Ahsg D1, the salt
concentration was adjusted to 300 mM to improve amylose
binding. The suspension was frozen overnight at
20 °C. The thawed
ice-cold suspension was pulse-sonicated three times for 20 s. Then
1% Triton-X100, a protease inhibitor mixture, DNase, and RNase to a
final concentration 10 µg/ml were added, and the suspension
was mixed for 10 min at 4 °C. Nuclease digestion of crude protein
preparations was critically important for the reproducibility of
precipitation assays, because DNA and RNA are potent inhibitors of BCP
precipitation (26). Protein preparations, which proved active in the
BCP precipitation inhibition assay, were routinely treated with
proteinase K to ensure that the inhibitory activity indeed resided with
the protein fraction of each preparation and not with residual
contaminating nucleic acids or low molecular weight inhibitors.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ahsg-induced changes in morphology of BCP
precipitate. Scanning electron microscopy of BCP precipitate
formed from a supersaturated solution of calcium (2.5 mM
CaCl2) and phosphate (1.8 mM
KH2PO4) without (A) and with
(B) added bovine Ahsg. The addition of 200 nM
Ahsg induced a change in morphology of the precipitate from a compact
to a brittle appearance indicating a strong interaction between the
forming mineral phase and Ahsg.
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Fig. 2.
Growth and transformation of soluble
calciprotein particles. The inhibition of BCP precipitation by
Ahsg is achieved by the formation of soluble colloidal complexes
containing calcium, phosphate, and Ahsg. The calciprotein particles
have a diameter of 30 to 150 nm (A, B, and
F). Diffraction analysis indicated the temperature and time
dependent transition of amorphous into crystalline BCP precipitate
(insets in F and H). Radial growth of
crystalline needles on the surface of the particles after 23 h at
22 °C (C) or after 4 h at 37 °C (G).
After 30 h at 37 °C, crystalline but soluble needles were found
in the supernatant (I). Electron dense crystals accumulated
in the precipitate (J). The scale bar
represents 100 nm, if not depicted otherwise.
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Fig. 3.
Electron filtering transmission electron
microscopy analysis of the calciprotein particles.
A-D, the identical region of a representative sample.
A, elastic bright field image; B, phosphorus
elemental distribution image using the L2, 3-absorption edge at 200 eV;
C, carbon elemental distribution image using the potassium
absorption edge at 283 eV; D, calcium elemental distribution
image using the L2, 3-absorption edge; both the Ca-L2 edge at 346 eV
and the L3 edge at 350 eV contribute to this calcium mapping recorded
at 350 eV. The three elements measured were evenly distributed within
the calciprotein particles.
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Fig. 4.
Monitoring calciprotein particle formation by
DLS. At a temperature of 20 °C a rapid DLS signal increase
mirrored the formation and growth of amorphous calciprotein particles
in Fig. 2, A-B. The slower increase of DLS signal observed
thereafter reflects the subsequent crystallization depicted in Fig. 2,
C-E.
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Fig. 5.
Inhibition of BCP formation by natural and
mutated forms of cystatin-like domains of cystatin protein family
members. Recombinant forms of mAhsg; mFETUB, rFETUB, and hFETUB;
and hKNG domains D1, D2, and D3 were expressed in E. coli as
fusion proteins with MBP or GST as detailed under "Experimental
Procedures." All proteins were tested at 3 µM final
concentration. Results represent triplicate measurements ± S.E.
GST-mAhsg/4S>E-myc denotes a mutant form of
mouse Ahsg with serine residues 120, 291, 294, and 296 mutated to
glutamic acid to mimic serine phosphorylation at these positions.
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Fig. 6.
Sequence comparison of cystatin-like
domains. A, schematic diagram of
chicken egg white cystatin (21). B and C,
schematic diagram of mouse Ahsg domain D1 (mAhsg
D1) modeled after A. C, a side view of
B illustrating the arrangement of acidic amino acids on the
four-pleated -sheet. D, protein sequences of chicken egg
white cystatin and the cystatin-like domains D1-3 of human, mouse, and
bovine Ahsg (h, m, bAhsg-D1 and
-D2), human and mouse fetuin-B (h,
mFETUB-D1 and -D2), human histidine-rich
glycoprotein (hHRG-D1 and -D2), and human
kininogen (hKNG-D1-D3) were aligned using ClustalW software
(50). Alignments were manually corrected to match the boundaries of
secondary structural elements (linearized schematic
diagram and color code derived from
A) and disulfide bridges (brackets) depicted
above the sequence alignment. Positive charges are
blue, and negative charges are red. Note that
acidic amino acid residues Asp or Glu occupy nearly every exposed
residue in the central
-strands 2 and 3 in human/mouse/bovine Ahsg
domain D1 but not in any other cystatin-like domain depicted
(C and D).
Quality assessment of modeled cystatin-like domains of mAhsg, mFETUB,
hHRG, and hKNG
-C
interactions in each model. Low Z scores indicate a
favorable conformation. Procheck software returned overall G-factors as
an indicator of stereochemical quality of models. Both chemical bond
angles and lengths were considered. All G-factors scored within the
theoretically tolerated limits or better. High G-factors indicate
favorable overall stereochemical geometry.
-sheet conformation when folded
according to the known chicken egg white cystatin template structure
(Fig. 6A) or when predicted by the sequence analysis
software packages PSIPRED and PHD (31, 32) (data
not shown), contains a remarkably high number of acidic amino acids,
causing an extended negative surface charge (red
residues in Figs. 6C and
7). In mAhsg D1, 6 out of 7 exposed
residues in
-strands 2 and 3 of the four-pleated
-sheet are Asp
or Glu, forming a contiguous acidic surface. These residues alternate
with hydrophobic amino acids in a regular fashion, resulting in an
asymmetric distribution of charge on opposing faces of the
-strands.
Charged amino acids cluster on the exposed surface of the
-sheet
facing the external milieu, whereas hydrophobic or uncharged amino
acids cluster on the core side pointing toward the amino-terminal
-helix. No other cystatin-like domain analyzed showed a similarly
regular pattern of charge distribution like mAhsg domain D1.
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Fig. 7.
Surface charge distribution of cystatin-like
protein domains. Using the x-ray structure of chicken egg white
cystatin (Protein Data Bank accession code 1CEW) (21) as a template, we
modeled the cystatin-like domains of mAhsg, mFETUB, hHRG, and hKNG
using Modeler4 software (20). The structures are orientated in order to
allow a top view on the four-pleated -sheets containing an extended
acidic array depicted in red and basic residues in
blue. Note that the accumulation of negative charge on the
extended
-sheet of mAhsg D1 is absent in comparable cystatin-like
domains.
-sheets of all cystatin-like domains. The
modeled structures are orientated in order to allow a top view of the
four-pleated
-sheets. They illustrate striking differences in charge
density on the extended
-sheets, which can also be detected in the
protein sequence alignment (Fig. 6D). The alternating
pattern of charges, which leads to a uniformly negative charge flanked
by positive charges on the extended
-sheet of Ahsg D1 is absent or
grossly distorted in the cystatin-like domains of chicken egg white
cystatin, Ahsg domain D2, FETUB, and KNG (Fig. 7). The two
cystatin-like domains of HRG show a coherent charged surface like Ahsg
D1, but to a lesser extent. This may explain why hHRG inhibited the
precipitation of BCP, albeit with a 2-fold lower molar efficiency than
Ahsg (28), whereas none of the remaining cystatin-like domains
inhibited BCP precipitation in this study (Fig. 5).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-strand 3 in our model of Ahsg domain D1 (Fig. 6), a configuration incompatible
with a functional EF-hand. Therefore, EF-hand-like calcium binding is
unlikely to mediate the efficient inhibition of precipitation of BCP
caused by Ahsg. Also, on theoretical grounds, the simple binding of
ionic calcium by Ahsg cannot be responsible for the inhibition of BCP
precipitation. Considering the ion concentrations of the precipitation
assay and assuming that one Ahsg molecule would bind six calcium ions,
one can calculate that 3 µM Ahsg (the nominal
concentration present in the precipitation mixture) would reduce the
free calcium concentration to 6 × 3 = 18 µmol at best.
This is insignificant compared with the millimolar concentration of
calcium contained in the precipitation mixture and indeed most extracellular fluids of living animals. The solution would still be
supersaturated, and consequently, BCP precipitation would not be
delayed. Therefore, the inhibition of BCP precipitation by Ahsg cannot
be explained by a reduction of the ion product through calcium ion
binding as in the case of albumin (36). Albumin does not have any
extended
-sheet structure but is exclusively
-helical in nature
(37). In the case of Ahsg, the arrangement of acidic amino acids in
domain D1 and their folding into a defined array of charges on an
extended
-sheet is, however, a crucial feature of effective
inhibition of BCP precipitation. Based on the amount of calcium
neutralized by Ahsg binding, we suggest that Ahsg binds BCP, not ionic
calcium like serum albumin. Thus, both Ahsg and serum albumin
contribute to extracellular calcium homoeostasis, albeit on a different
level of complexity. In summary, extracellular proteins can bind
calcium in three different ways illustrated in Fig.
8. Ahsg binds BCP, whereas albumin and
the EF-hand protein SPARC/osteonectin bind free calcium with low and high affinity, respectively.
View larger version (26K):
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Fig. 8.
Calcium (phosphate) binding mechanisms of
extracellular proteins Ahsg, serum albumin, and SPARC/osteonectin.
A, in Ahsg domain D1, surface binding of calcium is mediated
by negative charges on the extended -sheet of domain D1
(top) that might occupy PO4 positions on the
(001) face of apatite crystals (bottom), resulting in high
affinity binding despite the relatively low serum concentration of Ahsg
(10 µM). B, serum albumin binds calcium as
counterions to numerous negatively charged amino acids (red)
facing the external milieu. The high serum concentration of albumin (1 mM) causes high capacity calcium binding. C,
high affinity binding is achieved by a functional EF-hand conformation.
The calmodulin-like mode of calcium binding depicted here is
established for few extracellular proteins (e.g.
SPARC/osteonectin) (14).
Our structure-function study of cystatin-like domains of Ahsg, FETUB,
HRG, and KNG suggests that the -sheet in Ahsg domain D1 is
critically important for the inhibition of BCP precipitation. It forms
a contiguous negatively charged, almost planar structure that is
missing in Ahsg domain D2 and in all other modeled cystatin-like domains (Fig. 7). It is tempting to speculate that the acidic residues
might occupy the phosphate lattice positions in apatite-like mineral
through epitactic lattice matching. We hypothesize that the regularly
spaced pattern of charges, characteristic of the extended
-sheet in
Ahsg domain D1, is well suited to bind hydroxyapatite, especially at
lattice planes with a high calcium content like the (001) plane.
This binding mode is reminiscent of globular antifreeze proteins (38),
which bind ice through several hydrogen bonding Asn and Gln residues
arranged in a flat plane similarly arranged like the acidic residues in
the extended
-sheet of Ahsg domain D1.
Structure-function information on the mechanism of calcification
inhibition exists also for matrix-GLA protein, MGP. Targeted deletion
of the MGP gene in mice results in lethal calcification of the
aorta (13). Blocking of MGP glutamic acid -carboxylation by warfarin causes vascular calcification in young rats (39). Therefore, glutamate
-carboxylation is regarded as critical for the
function of MPG. The low solubility of MGP (<10 µg/ml) hampers structural research on this important inhibitor molecule, but the
related bone-GLA protein/BGP/osteocalcin (40) has been analyzed by CD
spectroscopy (41). In the
-helical conformation determined in this
study, the spacing of the GLA residues in human BGP
((GLA)PRR(GLA)VC(GLA)) results in the clustering of GLA residues on one
side of the
-helix. This configuration may also exist in human MGP,
which harbors two comparable sequence motifs ((GLA)RIR(GLA) and
(GLA)LNR(GLA)). Intriguingly, the interval of an
-helix of 540 pm
corresponds to the distance of the Ca(I) ions in the (001) plane of the
apatite structure permitting a tight binding of the GLA residue pairs to the Ca(I) ions on the mineral surface. Besides direct calcium binding, MGP may interfere with osteogenesis by regulating BMP-2 (42).
Interestingly, Ahsg can likewise regulate osteogenesis by sequestering
transforming growth factor-
and BMP-2 (43).
Here, we present a time-resolved TEM morphological study of direct
physical calcification inhibition by Ahsg in vitro (Fig. 2)
in the absence of osteogenic cells. Our study details the growth and
transformation of the soluble precursors instead of the precipitate, which have been described (6, 26, 44, 45). The most important discovery
of this study is that Ahsg forms transiently soluble, colloidal
complexes with calcium and phosphate, which we termed calciprotein
particles. At 37 °C and 10 µM Ahsg, which correspond to the normal serum temperature and Ahsg concentration, a BCP precipitate formed with a delay of at least 6 h. In biological terms, this suggests that Ahsg coating of BCP nuclei will delay the
growth of insoluble crystals long enough to assure the mobilization and
removal of otherwise insoluble calcium salts in the form of Ahsg
containing calciprotein particles. We hypothesize that phagocytotic cells of the reticuloendothelial system (namely macrophages in spleen
and liver and osteoclasts in bone marrow) will clear the calciprotein
particles, thus mediating the recycling of extracellular calcium and
phosphate from BCP. Corroborating this view, the total absence of Ahsg
causes severe ectopic calcification of almost every soft tissue in
Ahsg/
knockout mice,2 and Ahsg deficiency is an
independent predictor of vascular calcification in long term dialysis
patients (15).
It remains to be determined whether the formation of calciprotein particles also plays a role in the dissolution of bone mineral. The removal of osteoclast bone resorption products involves transcytosis of vesicles containing bone mineral and bone matrix proteins (46, 47). Ahsg is a major noncollagenous protein in bone and teeth (48) and could therefore prevent the precipitation of calcium salts during the transcytosis and thereafter. If this is so, calciprotein particles should be detectable in the bone remodeling compartment (49) and perhaps in the circulation. Our attempts to isolate Ahsg-, calcium-, and phosphate-containing calciprotein particles from blood of normo- and hypercalcemic mice were up until now unsuccessful. However, a high molecular weight complex containing calcium, phosphate, Ahsg/fetuin-A, and matrix-GLA protein was recently isolated in large amounts from the serum of etidronate-treated rats (51). It is very possible that this complex is identical with the calciprotein particles described in this study.
Our data suggest a novel mechanistic concept on the inhibition of
generalized calcification by serum protein, namely by stabilization of
soluble, colloidal particles containing calcium, phosphate, and the
mineral-binding protein, Ahsg. We present a model calciprotein particle
in Fig. 9. We arrived at this model by
comparing the hydrodynamic radius of the Ahsg molecule (4.2 nm)
estimated by dynamic light scattering (see Fig. 4) with the radius of
25 nm for an early calciprotein particle (Fig. 2A). A rough
estimate of the number of globular Ahsg molecules required to fill the volume of a calciprotein particle arrives at ~100 Ahsg molecules. We
adapted the stoichiometry of Ahsg-containing calciprotein from published literature (51), reporting an Ahsg/phosphate ratio of 7.6 mg/mg for soluble high molecular weight complex (in our model equal to
early, soluble calciprotein particles (Fig. 2, A and
F) and 3.4 mg/mg for the pelleted complex, respectively (late calciprotein particles-precipitate (Fig. 2, I and
J)). Assuming a molecular mass of 50,000 for
Ahsg and 1,000 for one apatite unit cell, this predicts a ratio of
roughly 10 apatite unit cells (Mr 10,000)
per Ahsg molecule for soluble, early calciprotein particles and ~22
apatite unit cells per Ahsg molecule for precipitating late
calciprotein particles. Thus, on average, 9-12 apatite unit cells
(Ca10(PO4)6(OH)2),
corresponding to 90-120 calcium atoms and 54-72 phosphate ions,
could be sequestered by one Ahsg molecule into a soluble complex aiding
the transport of potentially insoluble BCP. This model is
mechanistically similar to the well established lipoprotein particles,
a colloid of lipid and protein. It should stimulate more research into
the formation and recycling of BCP precipitates observed in many
calcified lesions as well as novel approaches to the management of
common calcification disorders for which there is currently no adequate
therapy.
|
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ACKNOWLEDGEMENTS |
---|
We thank Jaçek Gapinski for DLS analysis and Hermann Götz and Heinz Duschner for help with scanning electron microscopy.
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FOOTNOTES |
---|
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to W. J-D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: IZKF BIOMAT,
University Clinics, Pauwelsstrasse 30, D-52074 Aachen, Germany.
Tel.: 49-241-80-80163; Fax: 49-241-80-82573; E-mail:
willi.jahnen@rwth-aachen.de.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M210868200
2 C. Schäfer, A. Heiss, A. Schwarz, R. Westenfeld, M. Ketteler, J. Floege, W. Müller-Esterl, T. Schinke, and W. Jahnen-Dechent, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
GLA, -carboxyl
glutamic acid;
MGP, matrix GLA-containing protein;
Ahsg,
2-HS glycoprotein/fetuin-A;
BCP, basic calcium
phosphate;
bAhsg and mAhsg, bovine and mouse Ahsg, respectively;
BSA, bovine serum albumin;
TEM, transmission electron microscopy;
FETUB, fetuin-B;
hFETUB and mFETUB, human and mouse FETUB, respectively;
HRG, histidine-rich glycoprotein;
hHRG, human HRG;
KNG, kininogen;
hKNG, human KNG;
MBP, maltose-binding protein;
GST, glutathione
S-transferase;
HS,
2-Heremans-Schmid.
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