From the Canadian Institutes of Health Research Group
in Skeletal Development and Remodeling, Division of Oral Biology
and Department of Biochemistry, School of Dentistry, University of
Western Ontario, London, Ontario N6A 5C1, Canada and the
§ Canadian Institutes of Health Research Group in Matrix
Dynamics, Faculty of Dentistry, University of Toronto, Toronto,
Ontario M5S 3E2, Canada
Received for publication, November 22, 2002
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ABSTRACT |
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Bone sialoprotein (BSP) is a highly modified,
anionic phosphoprotein that is expressed almost exclusively in
mineralizing connective tissues and has been shown to be a potent
nucleator of hydroxyapatite (HA). Two polyglutamic acid (poly[E])
regions, predicted to be in an Mineralization of the extracellular matrix in bone,
dentin, and cementum is a complex, poorly understood process that is
believed to involve both hydroxyapatite-nucleating and -modulating
noncollagenous proteins. In bone, it has been postulated that
type I collagen acts as a structural matrix, whereas HA nucleation is
mediated by an anionic phosphoprotein (1-3). Of the noncollagenous
proteins, bone sialoprotein
(BSP)1 is the most likely
candidate. The highly anionic nature of BSP and its spatio-temporal
pattern of expression have led investigators to propose a role of this
protein in the mineralization of bone (1-4).
Mammalian BSPs contain an average of 327 amino acids, which includes a
16-residue signal sequence. The protein has a molecular mass of
~33-34 kDa. However, post-translational modifications, including
both N- and O-linked glycosylation, tyrosine
sulfation, and serine and threonine phosphorylation, constitute 50% of
the total mature protein weight of ~75 kDa. Analysis of the mammalian BSP cDNAs reveals a 45% level of sequence identity, plus an
additional 10-23% in conservative replacements. However, identity of
up to 90% is observed in and around two polyglutamic acid sequences (poly[E]); an Arg-Gly-Asp (RGD) cell-binding motif; sites of
phosphorylation, sulfation, and glycosylation; and sequences near the
amino and carboxyl termini, which are rich in tyrosine residues
(5).
Normally BSP expression is limited almost exclusively to mineralized
connective tissues, and its expression is localized to areas of bone
formation. By in situ hybridization, it has been shown that
BSP expression occurs in osteoblasts actively engaged in bone formation
and is found at low or undetectable levels in other regions of
mineralized tissue (6-11). Transfection of BSP into nonmineralizing
MC3T3-E1 osteoblast subclones was shown to restore their ability to
form mineral deposits (12). Overexpression of BSP in a genetically
engineered osteosarcoma cell line, K8, also resulted in an increase in
mineral formation in vitro (13). It has also been observed
that transgenic mice overexpressing BSP demonstrate more rapid healing
of artificially induced wound sites in bone (14). On the basis of this
information, BSP is likely to be involved with early mineral deposition
in bone.
Using a steady-state agarose gel system, BSP was found to nucleate HA
(15, 16). Synthetic homopolymers of glutamic acid have also been shown
to nucleate HA, indicating that the two poly[E] regions found within
BSP may play a role in this function (17). Trypsin digestion of native
porcine BSP produced a series of peptides, two of which contain one of
the poly[E] sequences. Each of these peptides was found to possess
nucleating activity, whereas the tryptic peptides without a poly[E]
sequence were unable to nucleate HA (18). Analysis of recombinant
peptides of the two nucleating domains in porcine BSP revealed that
only the peptide containing the first poly[E] sequence of porcine BSP
was able to nucleate HA (19).
Knowledge of the secondary structure of BSP is limited. Based on the
primary sequence BSP was believed initially to maintain an open,
extended structure with regions that had the ability to form both
The purpose of the present study was to utilize a prokaryotic
full-length rat BSP expression system to further investigate the
domain(s) responsible for the HA nucleating activity of BSP. Site-directed mutagenesis of the poly[E] domains of full-length BSP
was performed, and the importance of charge and conformation to the
nucleating activity of BSP was examined. The conformation of the
wild-type and mutated proteins was studied by circular dichroism and
small angle x-ray scattering. In addition, two recombinant peptides,
each encompassing one of the two poly[E] domains, were expressed and
tested for nucleating activity.
Full-Length rBSP Plasmid Construction--
The first 51 base
pairs of rat cDNA encoding the signal sequence as well as the first
amino acid (Phe) were removed and replaced by two vector derived amino
acids (Met and Val). In addition, two contiguous arginine codons at
positions 9 and 10, which are low usage in Escherichia coli,
were silently mutated to two high usage codons using the overlapping
primer extension method (24). For purification purposes, a
thrombin-cleavable pentahistidine tag
(Pro-Arg-Gly-Ser-His-His-His-His-His) was added to the carboxyl terminus of the cDNA. The resulting rBSP-His cDNA was then
subcloned into the pET28a expression vector (Novagen). The amino acid
sequence of the final construct is shown in Fig.
1.
rBSP Peptide Plasmid Construction--
Two partial-length BSP
polypeptides, corresponding to amino acids 3-100 and 101-314 of the
rBSP-pET28 construct, were cloned by the introduction of novel
restriction sites into the rat BSP sequence by Overlap Extension PCR
(24) using oligonucleotide primers (Invitrogen). The PCR product
was initially subcloned into pGEM-T plasmid (Promega) and subsequently
subcloned into pET28a giving rise to pET28a-rBSP (3-100) and
pET28a-rBSP (101-314).
Recombinant DNA procedures were carried out using methods described by
Sambrook et al. (25). The coding sequence of all plasmids
was confirmed by DNA sequencing.
Site-directed Mutagenesis of rBSP--
The poly[E] domains of
rBSP were mutated by introducing restriction sites as above. The
restriction sites AvaI and AatII were engineered
around the first poly[E] domain (residues 62-69), and the
restriction sites NarI and AccI were engineered
around the second domain (residues 139-148). Six mutants were created
by removing the wild-type polyglutamic acid sequence(s) with
AvaI and AatII or NarI and
AccI and replacing it with an oligonucleotide containing the
desired mutation. rBSP-pE1A and rBSP-pE1D replaced the first poly[E]
domain with either a polyalanine (poly[A]) domain or a polyaspartic
acid (poly[D]) domain while maintaining a glycine residue at amino
acid position 65. rBSP-pE2A and rBSP-pE2D replaced the second poly[E]
domain with poly[A] or poly[D], respectively. rBSP-pE1,2A and
rBSP-pE1,2D had both poly[E] domains replaced by poly[A] and
poly[D], respectively, while maintaining the glycine residue at
position 65. The coding sequence of all mutants was confirmed by DNA
sequencing. A diagram depicting the mutations is shown in Fig.
2.
Protein Expression and Purification--
Native rat bone BSP was
purified from the long bones of adult rats as described previously
(26).
For recombinant proteins, E. coli strain BL21(DE3) cells
were transformed with the expression plasmids described above and grown
in PO4-buffered Super Broth (SB) supplemented with 15 µg/ml kanamycin and 0.4% glucose to an A600
of 0.6-0.9. After induction with 2 mM
isopropyl-
The two expressed and His-bond affinity-purified rBSP peptides,
rBSP-(43-101) and rBSP-(134-206), were incubated with trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated)
at an enzyme:substrate ratio of 1:50 in trypsin digestion buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 7.8)
for 90 min at 37 °C. The reaction was terminated by the addition of
an equal volume of Mono-Q buffer A (50 mM Tris-HCl, 7 M urea, pH 7.4). The trypsin-digested peptides were then
purified by FPLC. All buffers and columns used for FPLC purification
followed established protocols (26). Proteins were purified using a
Q-Sepharose Fast Flow column (Fast Q) followed by size exclusion
purification with a Superdex 200PG column (1.6 × 60 cm) (Amersham
Biosciences). Chromatography buffers contained either 7 M
urea for ion exchange purification or 4 M urea for size
exclusion purification. Protein containing fractions were analyzed using 12.5% or high density gels on the Phastgel system (Amersham Biosciences) and stained with Stains-all and silver nitrate
as described previously (27). The corresponding Stains-all cyan-positive protein fractions were pooled and dialyzed using Spectra/Por 3 (cut-off, 12-14 kDa) dialysis membrane (Spectrum) for
the full-length proteins and Spectra/Por 3 (cut-off, 3.5 kDa) dialysis
membrane for the peptides. The fractions were then aliquoted and
lyophilized. Proteins were analyzed for protein content and purity by
amino acid analysis and mass spectrometry.
In Vitro Nucleation Assay--
Hydroxyapatite-nucleating
activity was assayed with a modification of the steady-state agarose
gel system described previously (15, 16). Steady-state buffers
contained either 7.1 mM of Ca(NO3)2
or 4.3 mM Na2HPO4. Proteins were
studied in triplicate over a range of concentrations, with the lowest
concentration of protein capable of nucleating HA used as a means to
compare nucleating activity between the different proteins. Total
mineral formation was determined by measuring calcium and phosphate
contents within the gels after ashing and were expressed as weight of
Ca + PO4 per gel. Total phosphate content was quantified
using the ammonium molybdate method (28), and calcium was quantified by atomic absorption spectrophotometry using a Varian SpectAA 30/40. The
experimental data were compared with the negative control (no protein
added) data using one-way analysis of variance.
Circular Dichroism Spectroscopy--
The far-UV spectra of rBSP,
rBSP-pE1,2D, rBSP-pE1,2A, rBSP-(43-101), and rBSP-(134-206) were
recorded in quartz cells of 1-mm optical path length using a Jasco-J810
spectropolarimeter between 190 and 260 nm, in 0.5-nm steps. Proteins
were studied at 0.2 mg/ml concentrations in 5 mM Tris-HCl,
150 mM NaCl, pH 7.4. A base line with buffer only was
recorded separately and subtracted from each spectrum. The effect of
calcium on the conformation of each protein was determined by the
addition of 5 and 10 mM CaCl2.
CaCl2 in buffer alone did not give measurable spectra
within the 190-260 nm range. All spectra were recorded at room
temperature. The molar ellipticity (
Estimates of protein secondary structure from the CD data were made
using the Dicroprot package (29), which incorporates the Contin (30),
K2D (31, 32), VARSLC (33) and Selcon (34, 35) methods, as well as a
calculation from [ Small Angle X-ray Scattering--
All measurements were made at
the European Molecular Biology Laboratory Outstation at the Deutsches
Elektronen-Synchrotron (Hamburg, Germany), beamline X33, at 15 °C
using radiation with a wavelength of 0.15 nm and a path length of 1 mm.
A sample detector distance of 3 m (low angle) was used to cover
the range of momentum transfer (S)2 sin Expression and Purification of Recombinant BSP--
Rat BSP was
expressed in E. coli BL21(DE3) cells and purified to an
apparent 99%+ purity. The recombinant rat BSP with the thrombin
cleavage site and 5×His tag was detected by electrospray mass
spectrometry as a single peak at 34802 Da, corresponding to the
theoretical mass of 34796 Da based on amino acid composition. SDS-PAGE
showed a single band at 67 kDa. The mutant proteins and recombinant
peptides were similarly assessed for purity by SDS-PAGE, amino acid
analysis, and mass spectrometry (data not shown).
Effects of Full-length rBSP on HA Nucleation--
To determine
whether the bacterially expressed BSP was capable of nucleating HA and
to examine the involvement of post-translational modifications in HA
nucleation, various concentrations of native and recombinant BSP were
incorporated into steady-state agarose gels, and the lowest level of
protein required to induce nucleation was determined (Fig.
3). Although both native and recombinant forms of BSP were capable of nucleating HA, native BSP was found to
nucleate HA at concentrations as low as 0.0025 nmol (0.087 µg/ml),
whereas rBSP required 0.05-0.1 nmol (1.7-3.4 µg/ml).
To examine the contribution that each domain makes to the nucleating
activity of full-length rBSP, the single-domain mutants (rBSP-pE1D,
rBSP-pE2D, rBSP-pE1A, and rBSP-pE2A) were tested at various
concentrations to determine the minimum concentration required for HA
nucleation (Table I). Each of these
single domain mutants, as well as the double domain mutant rBSP-pE1,2D,
was found to have nucleating activity at ~0.1 nmol, comparable with unmutated rBSP (Table I). A decrease in activity was seen, however, with rBSP-pE1,2A, where both domains were mutated to poly[A]. rBSP-pE1,2A required 0.5 nmol of protein (Table I).
To more clearly establish the nucleating activity of each poly[E]
domain, two peptides were expressed, each incorporating one of the
Glu-rich domains. The rBSP-(134-206) peptide was shown to have
nucleating activity at a concentration of 0.25 nmol; however, the
rBSP-(43-101) peptide did not promote nucleation at concentrations as
high as 5 nmol (Fig. 4). This finding is
consistent with our previous study on porcine BSP peptides, which
showed different activities for each domain.
Structural Determination--
The CD spectrum of rBSP at pH 7.4 is
shown in Fig. 5A. It has a
pattern typical of an unfolded protein, as generally observed by a
denatured protein in urea or guanidine hydrochloride. Because of the
strong electrostatic repulsion of the high degree of negatively charged
residues, 5 and 10 mM CaCl2 were added to
determine whether rBSP could be transformed into a partially folded
conformation (Fig. 5A). The presence of calcium did not
significantly alter the spectrum of rBSP.
The two full-length, double domain mutants (rBSP-pE1,2A and
rBSP-pE1,2D) were also studied by CD (Fig. 5B). Although the
rBSP and rBSP-pE1,2D spectra were different in intensity, they
exhibited similar shape. The rBSP-pE1,2A, however, appears to have a
different conformation; most noticeably, the minimum is shifted from
~195 nm for rBSP and rBSP-pE1,2D to ~204 nm for rBSP-pE1,2A. The
addition of 5 and 10 mM CaCl2 did not effect
the spectra of either rBSP-pE1,2A or rBSP-pE1,2D (data not shown).
The CD spectra (Fig. 5C) of the two peptides, rBSP-(43-101)
and rBSP-(134-206), although differing in intensity, were similar in
shape and were unchanged with the addition of 5 and 10 mM
CaCl2 (data not shown).
All CD spectra were analyzed for estimates of secondary structure using
the Dicroprot program. Only the rBSP-pE1,2A mutant exhibited a spectrum
similar enough to those found in the data bases for secondary structure
estimates to be given. It is estimated that this protein exhibits
13.8%
Small angle x-ray scattering (SAXS) is a useful tool for investigating
the conformation, shape, and dimensions of proteins in solution. The
degree of unfolding of a protein is best viewed with a Kratky plot,
S2I(S)
versus (S is scattering wave vector;
I(S) is scattering profile intensity), which
emphasizes the high angle part of the scattering profile (37). Globular
proteins scatter as S Studies on the structure and function of BSP have been hindered by
difficulties in protein expression. Although full-length BSP has been
successfully expressed eukaryotically, expression of full-length
prokaryotic BSP has been problematic (40). A successful expression
system for full-length prokaryotic rat BSP was established recently
(41, 42). This expression system was used to investigate the domains
responsible for the nucleating activity of BSP and additionally to
examine the contribution of charge and conformation to this nucleating activity.
Using the bacterially expressed full-length rat rBSP, the nucleating
activities of native BSP and rBSP were compared. Although the rBSP was
capable of nucleating HA, it required approximately a 40-fold increase
in protein concentration over native BSP. This decrease in activity is
in agreement with previous studies on porcine peptides derived from
both native bone extracts and prokaryotic expression, which indicated
that although post-translational modifications are not necessary for
nucleation, lack of these modifications does decrease the potency of
the protein (19). In this study it was shown that a native porcine BSP
peptide encompassing residues 42-125 was capable of nucleating HA at
concentrations as low as 0.3 nmol, whereas the recombinant form of the
peptide required 1.6 nmol or higher to nucleate HA. The exact role of
the post-translational modifications on BSP in HA nucleation remains
unclear. Although it has been proposed that the post-translational
modifications on BSP may stabilize a particular conformation for the
protein, studies have shown that at least for the amino-terminal half
of the eukaryotically expressed BSP, there is no defined secondary structure (22, 23). However, it is possible that the phosphate groups
on the post-translationally modified BSP may provide a higher charge
density, which allows for calcium ion accumulation at lower protein
concentrations. Future studies utilizing eukaryotically expressed BSP
will examine the role(s) the post-translational modifications may have
in HA nucleation.
Because chemical modification of the carboxylate groups has been shown
to abolish the nucleation activity of BSP, and synthetic homopolymers
of glutamic acid have demonstrated HA nucleating activity (17), the
nucleation activity of BSP is believed to reside primarily in two
poly[E] domains found in the amino-terminal half of the molecule.
This belief has also been reinforced through the investigation of
poly[E]-containing tryptic peptides and recombinant peptides of BSP
(18, 19). To further define the contribution of the poly[E] sequences
to nucleation, either one or both of the poly[E] domains were
replaced with either poly[D] or poly[A]. Poly[D] was chosen
because aspartic acid residues possess the same charge as glutamic
acid, and poly[A] was chosen because it shares the same propensity as
poly[E] to form a helical structure (43). To alleviate any anomalies
in which one domain may be compensating for the loss of activity in the
other domain, two peptides, rBSP-(43-101) and rBSP-(134-206), were
expressed so that each domain could be studied independently.
All four single poly[E] domain full-length mutants were found to
possess similar nucleation activities to rBSP, because nucleation activity was observed at a concentration of ~0.1 nmol (Table I). No
variation was observed between the poly[A] and poly[D] mutants. These analyses indicate that both poly[E] domains in rBSP appear to
have distinct and similar nucleating activity. These results are in
contrast to what is seen with the individual peptides. In this study,
it was found that the first domain peptide, rBSP-(43-101) is inactive
at concentrations as high as 5.0 nmol, whereas the second domain
peptide, rBSP-(134-206), is almost as effective a nucleator as
full-length rBSP, showing activity at 0.25 nmol (Fig. 4). It is
interesting that in previous studies using recombinant porcine BSP, the
reverse was found to be true. The peptide encompassing the first domain
was found to be active, and the second domain peptide was inactive
(19). Analysis of the amino acid sequences for these two species
reveals that the first poly[E] domains are quite similar (rat,
EEEGEEEE; porcine, EEEEEEEE), with the only difference being the
presence of a glycine residue in the middle of the rat sequence. The
second domain exhibits a similar trend (rat, EEEEEEEEEE; porcine,
EDEEEEEENEE); in this case an aspartic acid and an asparagine residue
disrupt the poly[E] sequence. From these sequences, it may be
hypothesized that the nucleating activity of BSP requires a minimum
number of contiguous glutamic acid residues.
When both poly[E] domains of rBSP were simultaneously mutated
(rBSP-pE1,2D and rBSP-pE1,2A), it was found that nucleating activity
was retained, although a higher concentration of protein was required
for the poly[A] mutant compared with unmutated rBSP or poly[D]
mutant (Table I). Aspartic acid and alanine residues were used in the
mutagenesis to deduce the relative contribution of charge and structure
to the nucleating activity of BSP. Although synthetic homopolymers of
poly[D] have been shown not to possess nucleating activity and have
in fact been shown to inhibit HA formation (44, 45), other nucleating
proteins such as dentin phosphophoryn (16) and an aspartic acid-rich
protein from mollusk shell (46, 47) have been shown to nucleate HA and
calcium carbonate crystals, respectively, via their aspartic acid-rich sequences. It is therefore not surprising that the poly[D] BSP mutant
is capable of nucleating HA. Poly[D] is not known, however, to adopt
a helical conformation but has a more random coil conformation (18) and
can, under acidic conditions (or in presence of calcium), form a
The fact that some nucleating activity is still evident after both
domains have been mutated to poly[A] indicates that perhaps another
region or domain may also be involved in nucleation. This notion is
supported by HA binding studies, which have shown that BSP binds to HA,
at least in part, via the contiguous poly[E] sequences. Synthetic
homopolymers of poly[E], however, did not completely inhibit this
binding (48). It was therefore suggested that additional domain(s), or
specific conformational motifs on BSP, are involved in HA binding and
perhaps in HA nucleation. Examination of the rat BSP amino acid
sequence reveals that there are a number of glutamic acid residues
downstream of each of the poly[E] domains (first,
EEENNEDSEGNEDQEAEAE; second, ENEEAEVDENE). Although these glutamic
acid-rich regions do not appear to possess independent nucleating
activity, as evidenced by the lack of activity shown by the rat first
domain peptide and the porcine second domain peptide (19), these
regions may be working cooperatively in the altered full-length rBSP
sequence to compensate for the mutation of both of the poly[E] domains.
Previous structural studies on native and eukaryotic, fully modified
BSP have been conflicting. Fisher et al. (22) found by
one-dimensional proton NMR that BSP was flexible along its entire
length with no significant structural regions. Structural studies on
native and eukaryotic BSP by Wuttke et al. (23), however,
showed that BSP is 5% The three proteins were studied in the presence of 5 and 10 mM calcium chloride in an attempt to mimic in
vivo conditions whereby calcium ions may induce folding of the
protein by neutralizing the negative charges. The calcium did not
appear to have any effect on protein folding and no ordered structure
was evident.
Although secondary structure predictions were attempted on the CD
spectra of all of the proteins, predictions were possible only for the
rBSP-pE1,2A mutant. The difficulty in estimating secondary structure
elements for the BSP proteins is due to the differences in our spectra
and those found in the program data bases. These programs, along with
secondary structure prediction programs based on amino acid sequence,
are biased toward globular proteins and tend to be inaccurate for more
extended, unordered proteins and peptides, often over-estimating
ordered conformations for these proteins. The CONTIN program used by
Wuttke et al. (23) is one of the better programs for
estimating If it is only the poly[E] and downstream glutamic acid-rich
regions that adopt an Normally, proteins need a specific three-dimensional structure to
perform their particular function. In the case of BSP, however, it has
been suggested that the unstructured, flexible nature of BSP may be
advantageous to its function (22). The amino-terminal half of BSP is
known to have a strong affinity for HA (48, 52, 53), whereas a
carboxyl-terminal RGD sequence allows for binding to
In conclusion, we have used a prokaryotic expression system to produce
full-length recombinant rat BSP and have created mutations of desired
domains to investigate its function. We have also tested the
hypotheses that the polyglutamic acid domains of BSP are responsible for the nucleation of HA and that these domains adopt an -helical conformation and located in
the amino-terminal half of the molecule, are believed to be responsible for this activity. Using a prokaryotic expression system, full-length rat BSP was expressed and tested for HA nucleating activity in a
steady-state agarose gel system. The unmodified protein is less potent
than native bone BSP, indicating a role for the post-translational modifications in HA nucleation. Site-directed mutagenesis of the poly[E] regions in full-length BSP was performed, replacing the poly[E] with either polyaspartic acid (poly[D]) or polyalanine (poly[A]) to examine role of charge and conformation, respectively, in HA nucleation. Replacement of single domains with either poly[A] or poly[D] did not alter nucleating activity nor did replacement of
both domains with poly[D]. Replacement of both domains with poly[A], however, significantly decreased nucleating activity. In
addition, two recombinant peptides, each encompassing one of the two
poly[E] domains, were expressed and tested for nucleating activity.
Whereas the peptide encompassing the second poly[E] domain was
capable of nucleating HA, the first domain peptide showed no activity.
The conformation of the wild-type and mutated proteins and peptides
were studied by circular dichroism and small angle x-ray scattering,
and no secondary structure was evident. These results
demonstrate that a sequence of at least eight contiguous glutamic acid
residues is required for the nucleation of HA by BSP and that this
nucleating "site" is not
-helical in conformation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical and
-sheet structure (20). However, nuclear magnetic
resonance (NMR) studies have indicated a loose open structure for a
55-residue peptide containing the RGD cell attachment sequence (21),
whereas more recent studies on full-length, fully modified BSP by
one-dimensional NMR also showed an unstructured, flexible conformation
in solution, with no
-helical and
-sheet structure present (22).
In contrast, Wuttke et al. (23) describe BSP as a
globule linked to a thread-like structure, with 5%
-helix, 32%
-sheet, 17%
-turn, and 46% random coil. They propose that the
carboxyl-terminal part of BSP, which is devoid of glycans, is globular
in nature, whereas the highly glycosylated amino-terminal part of the
protein is thread-like (23).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Rat bone sialoprotein amino acid
sequence. Solid underline, first domain peptide
rBSP-(43-101); broken underline, second domain peptide
rBSP-(134-206); 1
1, first
poly[E] domain; 2
2, second poly[E]
domain; 3
3, thrombin-cleavable His
tag.
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Fig. 2.
Full-length rat BSP mutations. Six
mutants of full-length rBSP were created by removing the wild-type
polyglutamic acid (poly[E]) sequence(s) and replacing them with
polyalanine (poly[A]) or polyaspartic acid (poly[D]) sequences.
rBSP-pE1A and rBSP-pE1D have the first poly[E] sequence replaced by
either poly[A] or a poly[D] while maintaining a glycine residue at
amino acid position 65. rBSP-pE2A and rBSP-pE2D have the second
poly[E] sequence replaced with poly[A] or poly[D], respectively.
rBSP-pE1,2A and rBSP-pE1,2D had both poly[E] sequences altered to
poly[A] and poly[D], respectively, while maintaining the glycine
residue at position 65.
-D-thiogalactopyranoside, cultures were grown for a further 4 h. The bacterial cells were then fractionated by
sonication in denaturing binding buffer (5 mM imidazole,
0.5 M NaCl, 0.02 M Tris/HCl, 6 M
urea, pH 7.9). The protein extract was loaded onto Poly-prep
chromatography columns (Bio-Rad) packed with 1.5 ml His-bind resin
(Novagen) that had previously been charged with 50 mM
NiSO4. Proteins were eluted by competition with 0.5 M imidazole-containing elution buffer. Nickel affinity elution fractions were then pooled, and the full-length proteins were
immediately subjected to fast protein liquid chromatography (FPLC) purification.
) expressed in degrees
cm2 dmol
1 was calculated on the basis of mean
residue molecular mass.
]220 nm and a simple least squares
method based on the Gauss-Jordan elimination.
/
,
where 2
is the scattering angle) from 0.003 to 0.085 Å-1. Fifteen
successive 1-min exposures were recorded for each sample. Each protein
sample was preceded and followed by recording of the buffer alone to
ensure the cleanliness of the cell between readings of protein
solutions. Averaging of frames, corrections for detector response and
beam intensity, and buffer subtraction were done using the programs
SAPOKO2 and OTOKO (36).
Protein samples were dialyzed against 5 mM Tris-HCl,
150 mM NaCl, pH 7.0. Samples were run in the
presence and absence of 5 mM CaCl2. Protein
concentrations were rBSP, 4.7 mg/ml, and rBSP-pE1,2A, 7.4 mg/ml.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 3.
Effect of native and recombinant BSP on HA
nucleation in vitro. Prokaryotic recombinant BSP
and native BSP extracted from rat long bones were tested for HA
nucleating activity in steady-state agarose gels at concentrations of
calcium (7.1 mM) and phosphate (4.3 mM) below
the threshold for spontaneous precipitation. A negative control
containing no protein was included. Native BSP was shown to nucleate HA
at concentrations of 0.0025-0.025 nmol, whereas rBSP was shown to
require a minimum concentration of between 0.05-0.10 nmol of protein
to nucleate HA. *, statistical significance compared with negative
control value determined by one-way analysis of variance,
p < 0.05.
Effects of mutations in the polyglutamic acid regions of BSP on
HA-nucleation in vitro
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[in a new window]
Fig. 4.
Effect of rBSP peptides on HA nucleation
in vitro. rBSP-(43-101), a peptide encompassing
the first Glu-rich domain of BSP, and rBSP-(134-206), a peptide
incorporating the second Glu-rich domain, were tested for their ability
to nucleate HA at subthreshold concentrations of calcium (7.1 mM) and phosphate (4.3 mM). A negative control
containing no protein was included. The rBSP-(43-101) peptide did not
have HA nucleating activity at 1.0-5.0 nmol. The rBSP-(134-206)
peptide induced nucleation at a minimum concentration of 0.25 nmol. *,
statistical significance compared with negative control value
determined by one-way analysis of variance, p < 0.05.
View larger version (14K):
[in a new window]
Fig. 5.
Circular dichroism spectra of rBSP peptides
and mutants. All proteins were studied at 0.2 mg/ml in 5 mM Tris-HCl, 150 mM NaCl. A,
rBSP with 0 mM CaCl2 ( -), 5 mM
CaCl2 (- - - -), or 10 mM CaCl2
(-·-·-). B, rBSP (
-), rBSP-pE1,2D (- - -), and
rBSP-pE1,2A with no calcium added (-·-·-). C,
rBSP-(43-101) (
-) and rBSP-(134-206) with no calcium added
(- - -).
-helix, 43.5%
-sheet, 10.8%
-turn, 8.1% poly-pro
(II) helix, and 18.9% unordered structure.
4 at high S
values, yielding a Kratky plot that is proportional to
S
2 (38), whereas a random coil scatters as
S
1 at high angles, yielding a plot with
S1 dependence (39). Therefore, a Kratky plot of
a native globular protein will have a characteristic maximum that is
dependent on its radius of gyration (Rg),
whereas unfolded and partially folded proteins give a plateau and then
rise (37). Analysis of the x-ray scattering of rBSP and rBSP-pE1,2A in
the form of a Kratky plot shows that neither has a well developed
globular structure under any condition studied (Fig.
6). The profile of the Kratky plots for
both proteins at neutral pH, pH 4, and in the presence of 5 mM CaCl2 (data not shown) is typical of a
random coil.
View larger version (16K):
[in a new window]
Fig. 6.
Kratky plots of scattering curves from rBSP
and rBSP-pE1,2A. A, rBSP at pH 7 ( ) and pH 4 (
).
B, rBSP-pE1,2A at pH 7 (
) and pH 4 (
).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet. If a helical conformation were required for nucleation, the
poly[A] mutant would likely be a more potent nucleator of HA
(i.e. require lower protein concentration) than the
poly[D] mutant. This, however, was not observed, and it
appears that the charge of the contiguous domains, rather than ordered
secondary structure, is critical in maintaining the nucleating activity of BSP.
-helix, 32%
-sheet, 17%
-turn, and
46% random coil; they proposed that the carboxyl-terminal part of the
protein, which is free of glycans, forms a globular structure, whereas
the highly glycosylated regions are thread-like due to lack of
secondary structure. Here, we have studied the conformation of
full-length rBSP, as well as the double-domain mutants (rBSP-pE1,2D and
rBSP-pE1,2A), by circular dichroism and small angle x-ray scattering.
-helix,
-sheet, and
-turn conformations from CD
data. However, the method suffers from the choice of proteins in the
data base of standards (49). The inclusion of denatured proteins in the
data base has been shown to significantly improve the estimates for
unordered proteins (50); however, these have not been included in these
programs at this time. Even the structural estimates obtained for the
rBSP-pE1,2A mutant appear to be inflated when considering the small
angle x-ray scattering data. A protein with such high secondary
structure content would likely appear as more of a globular protein in
the Kratky plot. The rBSP and rBSP-pE1,2A proteins, however, are both lacking the typical maximum, which is indicative of its
Rg, and show the characteristic plateau of
unordered, unfolded proteins.
-helical conformation, it may be possible that
these regions are being masked by the unstructured nature of the bulk
of the protein. Because the poly[E] and glutamic acid-rich regions
make up a large part of the peptides, it should be possible to observe
any
-helical conformation by CD. Although the rBSP-(134-206) peptide is predicted by Consensus Secondary Structure Prediction (51)
to be 37% helical, and the rBSP-(43-101) is predicted to have 22%
helical character, virtually no
-helical content was detected even
in the presence of calcium. Although a small difference in the shape of
the two spectra was seen, it was difficult to determine how great the
structural differences in these two proteins may be without any
reliable means of ascertaining their structure. The fact that no
ordered secondary structure was observed by CD between the nucleating
rBSP-(134-206) peptide and the nonnucleating rBSP-(43-101) peptide
indicates once again that ordered secondary structure does not appear
to play an important role in the nucleating activity of BSP.
v
3 integrin (19, 21, 54). The flexibility
seen with BSP would thus allow it to serve as a bridge for attachment
of cells to HA. In the case of mineralization, the flexibility of the
protein may allow binding to type I collagen in the hole zones, as proposed by Fujisawa et al. (55), followed by initiation of mineral formation. Ongoing studies on the localization of the collagen-binding domain(s) of BSP and nucleation studies within collagen gels will provide further insight into this idea.
-helical conformation. Our results have revealed that a sequence of at least
eight contiguous glutamic acid residues followed by a glutamic acid-rich region may be requisite for the nucleation of HA by wild-type
BSP and that this region is not
-helical in conformation. Our
secondary structure results are in agreement with those in the current
literature and support the idea that the highly flexible nature of BSP
may be advantageous to its function.
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ACKNOWLEDGEMENTS |
---|
We thank T. M. Underhill, B. S. Shilton, and D. M. Currie for insights and assistance.
![]() |
FOOTNOTES |
---|
* Circular dichroism experiments were carried out at the University of Western Ontario Biomolecular Interactions and Conformations Facility, which is supported by a Multi-user Equipment and Maintenance Grant from the Canadian Institutes of Health Research (CIHR). This work was supported by the CIHR and the Canadian Arthritis Network Centers of Excellence.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: School of Dentistry, Dental Sciences Bldg., University of Western Ontario, London, Ontario N6A 5C1, Canada. Tel.: 519-661-2182; Fax: 519-850-2316; E-mail: hagoldbe@uwo.ca.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M211915200
2 D. I. Svergun and M. H. J. Koch, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: BSP, bone sialoprotein; rBSP, recombinant BSP; HA, hydroxyapatite; poly[E], polyglutamic acid; poly[D], polyaspartic acid; poly[A], polyalanine; FPLC, fast protein liquid chromatography.
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