 |
INTRODUCTION |
Organisms are capable of developing minerals and biocomposites
with complex architecture to fulfill important biological functions, such as skeletal support, protection of soft tissues, and food grinding
(1-4). Often, survival of the organism depends on the structure and
strength of these composite materials. Due to the wide range of
mechanical and functional properties of these biocomposites, identifying the organic and inorganic components and understanding the
structure-function relationships have potential industrial and
biotechnological applications (5). The inorganic mineral phase of such
materials is formed over an insoluble organic matrix or mold, and the
mineral phase is intimately associated with organic macromolecules,
such as proteins/glycoproteins, polysaccharides, or proteoglycans (6).
These biomacromolecules are highly acidic in nature and have been
postulated to control nucleation, growth, crystal size, and shape of
the mineral phases (7).
Mann (8) classified the biologically programmed composites into four
types (types I, II, III, and IV), based on matrix intervention in
nucleation and growth processes. Avian eggshells form type II
biocomposites, which is the fastest forming hard acellular composite in
nature. For example, in the case of chicken eggshell, about 5 g of
the mineral phase is produced within 24 h (9). Their calcified
layer consists of ~95% mineral and ~5% organic phase (10). The
mineral phase acts as a mechanical support as well as allows the
diffusion of gases, water, and ions and is, therefore, essential for
survival of the embryo. The organic phase (proteins and proteoglycans)
of the eggshell matrix is believed to be responsible for the nucleation
and directed growth of the calcified layer. So far, several matrix
proteins from chicken eggshells have been purified and characterized
(11-17). These proteins are subdivided into three groups, namely
non-collagenous bone proteins (osteopontin), eggshell-specific proteins
(ovocleidins and ovocallyxins), and egg white proteins (ovalbumin,
ovotransferrin, and lysozyme). Although their presence within the
mineral layer has been demonstrated by immunohistochemistry (11, 12, and 17), the role of these proteins in calcite mineralization is not
clearly understood. Hincke et al. (18) showed that egg white
lysozyme and ovotransferrin influence the morphology of
CaCO3 crystals. Dermatan sulfate, chondroitin sulfate, and
hyaluronic acid were also identified in the chicken eggshell matrix
(19-22). Wu et al. (23) have shown that partially purified
dermatan sulfate proteoglycans obtained from the eggshell reduced the
size of the calcite crystals. Uterine fluid collected at various stages
of eggshell formation (initial, growth, and final stages), reduced the
size, and induced curved faces on the calcite crystals (24). Though
many reports are available on the ultrastructure, composition, and
presence of organic macromolecules in the eggshell matrix (25, 26), information on the role of individual proteins and on the molecular mechanism of avian eggshell mineralization are limited (27). This is
presumably due to problems associated with the separation and
purification of these biomacromolecules (28, 29).
A comparative study of various avian eggshells indicates that the basic
architecture is identical, but their ultrastructure and composition are
different (30). Therefore, one expects the interaction between the
organic macromolecules and the mineral phase to be different in
different avian species. We believe that understanding the structure
and role of the organic matrix would help us to understand the complex
process of biomineralization. Here we report the purification and
characterization of various matrix proteins and peptides present in the
calcified layer of the goose (Anser anser) eggshell. We have
identified a 15-kDa protein (ansocalcin), a major constituent of the
goose eggshell extract and determined its complete amino acid sequence.
For a better understanding of the role of ansocalcin in the eggshell mineralization process, we compared ansocalcin with whole eggshell extract in in vitro crystallization of CaCO3. We
propose that ansocalcin plays an important role in goose eggshell calcification.
 |
EXPERIMENTAL PROCEDURES |
Extraction of Eggshell Matrix Proteins--
Commercially
available fresh goose eggshells were broken and thoroughly washed with
Millipore water. They were decalcified with 1 N HCl for 30 min, filtered, and centrifuged. The supernatant solution was desalted
using an Amicon microconcentrator (YCO5 membrane, 500 Mr cutoff) at 4 °C. The turbid solution was
centrifuged at 4000 rpm for 15 min, and the supernatant liquid was
taken for purification using
RP-HPLC.1
Protein Purification--
The proteins were fractionated on a
Jupiter C18 reversed phase column (5µ, 250 × 10 mm)
using a Vision Work station (PerkinElmer PerSeptive Biosystems). The
column was equilibrated with 0.1% trifluoroacetic acid, and a linear
gradient of acetonitrile was used for elution. The microconcentrated
sample (~15 mg of protein) was injected onto the column and was
eluted at a flow rate of 2 ml/min. The elution of the proteins was
monitored both at 215 and 280 nm.
Electrospray Ionization Mass Spectrometry--
Precise masses of
the proteins and peptides were determined by ESI-MS using a PerkinElmer
Sciex API 300 triple quadrupole instrument equipped with an ion spray
interface. The ion spray voltage was set at 4.6 kV and the orifice
voltage at 30 V. Nitrogen was used as a curtain gas with a flow rate of
0.6 liters/min, while compressed air was utilized as the nebulizer gas.
The sample was injected into the mass spectrometer at a flow rate of 50 µl/min and scanned from a mass to charge (m/z)
ratio of 500-3000. The multiply charged spectrum was deconvoluted into
the mass scale using the Biospec Reconstruct software supplied with the
instrument data system.
Reduction and Alkylation of Native Proteins--
The native
protein (~3 mg) was dissolved in 1000 µl of 130 mM
Tris-HCl, 1 mM EDTA, and 6 M guanidine-HCl (pH
7.5). 2-Mercaptoethanol (20 µl/mg of protein) was added, and the
solution was incubated under nitrogen atmosphere for 2 h at
37 °C. The alkylating agent, 4-vinylpyridine (200 µl/mg of
protein), was subsequently added, and the mixture was incubated under
nitrogen for another 2 h at room temperature. The
S-pyridylethylated protein was separated from the reaction
mixture by RP-HPLC on a Jupiter C18 (250 × 10 mm)
column using a linear gradient of acetonitrile.
Enzymatic and Chemical Cleavage--
The
S-pyridylethylated protein was digested with the enzymes
lysyl endopeptidase and trypsin. In a typical digestion method, 500 µg of the protein was dissolved in 500 µl of 50 mM
Tris-HCl, 4 M urea, 5 mM EDTA (pH 7.5). The
enzymes were added (enzyme/protein ratio of 1:100), and the digestions
were carried out at 37 °C for 18 h. The aspartidyl cleavage was
performed using 2% formic acid (31). The S-pyridylethylated
protein was dissolved in 2% formic acid in a glass vial. The solution
was frozen and thawed completely under vacuum. The vial was then
vacuum-sealed and heated at 108 °C for 2 h. The peptides
generated by chemical and enzymatic cleavage were separated by RP-HPLC
on a Sephasil C18 column (100 × 2.1 mm) using a
linear gradient of acetonitrile.
N-terminal Sequencing--
N-terminal sequencing of the purified
proteins or peptides was performed by automated Edman degradation using
a PerkinElmer Applied Biosystems 494 pulsed-liquid phase protein
sequencer (Procise) with an online 785A PTH-amino acid analyzer.
Size Exclusion Chromatography--
All the standards and
purified protein were dissolved in 7.5 mM CaCl2
solution or in 200 mM Tris-HCl (pH 7.5) solution. The SEC
was performed on a Sepharose CL-6B column (Sigma, 1.5 × 57 cm) at
various concentrations of ansocalcin using CaCl2 (7.5 mM) solution as the mobile phase. SEC experiments were
repeated using 200 mM Tris-HCl (pH 7.5) as the mobile phase
in the absence of calcium ions. Ovalbumin, bovine serum albumin, and
chicken egg lysozyme were used as standards. The sample was dissolved
in eluent and loaded onto the column. Absorbance of the fractions was
measured using a Jasco V-560 uv/vis spectrophotometer.
Circular Dichroism (CD) Experiments--
The secondary structure
of the protein was analyzed using a Jasco J 700 circular dichroism
spectropolarimeter. The instrument was calibrated with 0.05%
(+)-10-camphor sulfonic acid solution. The CD spectra of the protein at
a concentration range of 10-500 µg/ml in water were collected using
a 0.1-mm sample cell. To study the effect of Ca2+ ions,
spectra were also recorded in 7.5 mM CaCl2
solution. The instrument optics were flushed with 30 liters/min
nitrogen gas. A total of three scans was recorded, averaged for each
spectrum, and baseline subtracted. CD spectral data from 195-240 nm
were appended to the reference data set in Convex Constant Analysis program (32). The conformational weight was obtained from 4-component deconvolution, which resulted in best fit with the experimental CD spectrum.
Crystal Growth Experiments--
CaCO3 crystals were
grown on glass cover slips placed inside the CaCl2 solution
kept in a Nunc dish, 4 × 6 wells (33). Typically, the lyophilized
proteins or protein extract was accurately weighed on a microbalance
(Ohaus Analytical Plus, OHAUS Corporation, NJ) and dissolved in a 7.5 mM CaCl2 solution to give a final concentration of 0.1-1000 µg/ml. 1 ml of 7.5 mM CaCl2
solution was introduced into the wells containing the cover slips, and
the whole set up was covered with aluminum foil with a few pinholes on
the top. Crystals were grown inside a closed desiccator for 2 days by
slow diffusion of gases released by the decomposition of ammonium
carbonate placed at the bottom of the desiccator. After 2 days, the
glass slides were carefully lifted from the crystallization wells,
rinsed gently with Millipore water, air-dried at room temperature, and used for characterization.
Scanning Electron Microscopy--
Scanning electron
microscopic studies of the CaCO3 crystals were
carried out using a JEOL 2200 scanning electron microscope at 15/20 kV
after coating with gold to increase the conductivity. The crystal
aggregate size distribution was measured by randomly choosing at least
20 aggregates from each experiment, and the size of each aggregate was
measured. The number of crystal aggregates with a particular size at an
accuracy of ± 2 µm was noted, and the percentage was calculated.
SDS-PAGE--
SDS-PAGE was performed on a 4-20% polyacrylamide
gel (Bio-Rad) under non-reducing conditions according to the method of
Laemmli (34). The proteins were stained with Coomassie Brilliant Blue R-250.
Fluorescence Spectroscopy--
The fluorescence emission spectra
were collected on a Shimadzu RF-5301PC spectrofluorometer with the
emission and excitation band passes set at 3 nm. The excitation
wavelength was set at 295 nm (to selectively excite tryptophan
residues), and the spectra were recorded from 300 to 400 nm. Spectra of
the proteins were recorded either in 7.5 mM
CaCl2 solution or in Millipore water.
 |
RESULTS |
Purification and Characterization of Eggshell Proteins--
The
calcified shell was dissolved in 1 N HCl to extract the
entire soluble organic matrix associated with it. After
decalcification, the crude extract was microconcentrated and
fractionated by RP-HPLC on a Jupiter C18 column. Fig.
1A shows a typical
chromatogram of the various eluting fractions (labeled a,
b, c, and d). Using a shallow
gradient, we obtained 3 peptides from fraction b (labeled b1, b2, and
b3) and one peptide from fraction a (a1, data not shown). Fraction c
was separated again on an RP-HPLC column using a shallow gradient into
two subfractions (Fig. 1B, c1 and
c2). Fraction c1 was identified as a 15-kDa protein
(Fig. 2), and the minor component, c2,
was identified as a 16-kDa protein as determined from the positive ion
ESI-MS (data not shown). We named the 15-kDa protein (fraction c1) as
ansocalcin (Anser ovum
calcium-binding protein). Fraction d was found
to contain a 16-kDa protein (Fig. 2F, labeled
d1). This protein is similar to c2 based on its molecular weight
and retention time (data not shown). The RP-HPLC profile showed that
ansocalcin is the major constituent of the eggshell extract.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Purification of ansocalcin from goose
eggshell extract. A, RP-HPLC of goose eggshell extract.
The crude extract was loaded onto the Jupiter C18 column
and eluted using a linear gradient of acetonitrile. The various
fractions were labeled a, b, c, and
d. B, fraction c was pooled and rerun using a
shallow gradient. The dashed lines represent the solvent
gradient applied during elution.
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 2.
ESI-MS of the proteins purified from goose
eggshell extract. Positive ion electrospray ionization mass
spectrograph of the various proteins/peptides purified from goose
eggshell extract. 20 µl of the sample was injected into the
spectrometer using a 1:1 mixture of 0.1% trifluoroacetic acid and
acetonitrile. The multiply charged spectrum was converted using Biospec
Reconstruct software (shown in the inset diagram). The
deconvoluted spectra also indicate the homogeneity of the various
eggshell matrix proteins/peptides. The mass spectra correspond to
fraction a1 (A), fraction b1 (B), fraction b2
(C), fraction b3 (D), fraction c1 (ansocalcin,
E), and fraction d1 (F).
|
|
The mass spectra of the various proteins/peptides purified from the
crude extract are shown in Fig. 2. The mass spectrum of ansocalcin
showed an overlapping envelope of positive ion series from 6+ to 13+
(Fig. 2E). The inset diagram indicates the reconstructed mass of the protein with a value of 15,341.70 ± 1.97 daltons, which confirmed its homogeneity. The mass spectrum of fraction d1, the
second major component in the eggshell extract, showed a charge
distribution of 6+ to 10+. The reconstructed mass of this protein was
found to be 16,278.52 ± 1.36 daltons (Fig. 2F). The
mass spectra of the peptides purified from fractions a and b are shown
in Fig. 2, A-D. Thus the soluble components of the eggshell
extract consist of a number of proteins and peptides with a wide range
of molecular weights. The mass spectral data indicated that all the
proteins/peptides are homogeneous.
Amino Acid Sequence of Ansocalcin--
The complete amino acid
sequence was determined by sequencing the peptides generated by
chemical and enzymatic digestions of S-pyridylethylated
ansocalcin. The sequences of the overlapping peptides and other details
are shown in Fig. 3. The peptides
generated by formic acid digestion gave more or less the complete
sequence of ansocalcin. The C terminus of the protein was confirmed by the peptides obtained by both formic acid and trypsin digestion. Alanine was identified as the C-terminal residue because it was the
last residue sequenced on peptides A118-A132 and A124-A132 obtained by
formic acid and trypsin digestions, respectively. Table
I shows the observed and calculated
masses of the various peptides generated by chemical and enzymatic
digestions. In all cases, the calculated molecular masses of the
peptides matched the observed molecular weight. Ansocalcin has a total
of 132 amino acid residues with a calculated mass of 15,341.20 daltons.
This matches the observed mass of 15,341.70 ± 1.97. Based on the
amino acid sequence, ansocalcin is rich in Ala (9%), Glu (8%), Ser
(7.6%), and Gly (7.6%) residues. Ansocalcin also has a high content
of tryptophan residues (6%) compared with the natural abundance of 1.3% (35). The amino acid sequence of ansocalcin revealed the presence
of repeat patterns of acidic and basic amino acids. Two quartets
(Glu63-Glu64-Glu65-Asp66
and
Asp93-Asp94-Asp95-Glu96)
and two pairs (Glu47-Glu48 and
Glu116-Asp117) are indicated by
asterisks in Fig. 3. Three basic amino acid pairs
Arg25-Lys26,
Arg61-Arg62, and
His73-His74 and a triplet
Arg86-Arg87-Lys88 are also seen in
the sequence.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Amino acid sequence strategy for
ansocalcin. N-terminal amino acid sequence of intact
S-pyridylethylated ansocalcin. Peptides derived from the
digestion of ansocalcin by formic acid, trypsin, and endopeptidase
Lys-C (Lys-C), are indicated. Solid lines
represent regions whose sequences were determined by Edman degradation
and/or confirmed by mass spectrometry. Broken lines indicate
the regions that were not sequenced.
|
|
Structural Similarity with Other Proteins--
A search of the
NCBI data base revealed that ansocalcin has high identity (31-35%)
and homology (48-52%) to C-type lectin(-like) (CTL) family of
proteins from snake venom (Fig. 4). The
first two proteins are involved in CaCO3 biomineralization
and have CTL-like domains, whereas the next three proteins are CTL
extracted from snake venom. Based on the sequence homology, we propose
intrachain disulfide linkages between cysteines 3 and 14, 31 and 128, and 103 and 120. Ansocalcin lacks the sequence QPD, a sequence motif that was interpreted to account for galactose specificity (36). The
residues that are involved in Ca2+ ion binding sites 1 and
2 of rat mannose-binding protein (37) are not conserved in ansocalcin.
Earlier studies have shown that matrix proteins associated with the
calcium-rich mineral phase (16, 38-40) showed homology to C-type
lectins. However, except for perlucin (39), these proteins lack the
conserved QPD sequence that is required for carbohydrate binding.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 4.
Comparison of amino acid sequence of
ansocalcin with other calcium-binding proteins. Ansocalcin (goose
eggshell, this report, SWISS-PROT no. P83300); OC-17 (ovocleidin 17, uterus-specific chicken eggshell protein, 36% identity, NCBI accession
no. S78596); H-Litho (human lithostathine, 34% identity,
NCBI accession No. 1942639); C. atrox, (Crotalus
atrox, 35% identity NCBI accession no. P21963); B. fasciatus (Bungarus fasciatus, 31% identity NCBI
accession no. AAK43585.1); L. muta (Lachesis
muta, 32% identity, NCBI accession no. AAB49518). The identical
residues with respect to ansocalcin are shaded in black. The
phosphorylated serine residues of OC-17 are represented by
S. The numbers 1 and 2 indicate the
first and second Ca2+ ion binding sites of rat
mannose-binding protein (37).
|
|
The search also indicated ~34% identity and 50% homology with the
amino acid sequence of ovocleidin 17 (OC-17), the eggshell matrix
protein from chicken eggshells (16). Comparison of the sequence of
ansocalcin with OC-17 showed four important differences. Firstly,
ansocalcin has an extra cysteine (Cys38), which is not
present in OC-17. Secondly, the occurrence and number of acidic and
basic amino acids repeats is different. In OC-17, we identified one
acidic triplet
(Asp118-Glu119-Glu120) and four
basic pairs (Arg34-Arg35,
His80-Arg81,
His102-Arg103,
Arg108-Arg109). Thirdly, OC-17 is
phosphorylated at two serine residues (indicated as S in
Fig. 4). On the other hand, ansocalcin is not phosphorylated as the
calculated molecular weight matches the one determined by ESI-MS.
Finally, the ratio of basic to acidic residues is 1.3 in ansocalcin and
1.9 in OC-17 (including the phosphorylated serines). The acidic amino
acid residues constitute about 13.6% for ansocalcin and 8% for OC-17.
Thus, ansocalcin and OC-17 do not possess a large number of acidic
amino acids compared with other soluble matrix proteins, which possess
a large amount (35-50%) of acidic amino acids (41-43).
Amino Acid Sequence of Other Peptides--
We have determined the
complete amino acid sequence of fraction b1 (Fig.
5A). The calculated mass of
this peptide from the amino acid sequence (2964.3) matched the observed
mass of 2963.89 ± 0.13 determined by ESI-MS. Fraction b1 showed
85% homology to residues 665-685 of ovocleidin-116 (OC-116, Ref. 12).
Thus, it appears to be a proteolytic product of a protein related to OC-116. The partial amino acid sequence of b3 (Fig. 5B) did
not show significant homology to any known proteins. Sequencing of fractions d1 and a1 showed two N-terminal residues, which indicates that these fractions contain two polypeptides, and, thus, their sequence could not be determined.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 5.
Amino acid sequence of fractions b1 and
b3. A, comparison of the amino acid sequence of
fraction b1 with residues 661-687 of OC-116 (NCBI accession no.
AAF00982). The identical amino acids are shaded in black.
B, amino acid sequence of fraction b3.
|
|
Effect of the Eggshell Extract on CaCO3
Crystallization--
To understand the role of ansocalcin in eggshell
calcification, CaCO3 crystals were grown in the presence of
whole eggshell extract or ansocalcin in different sets of experiments
under identical conditions. The experiments were performed by slow
diffusion of carbon dioxide (produced via the decomposition of solid
ammonium carbonate) into the protein extract dissolved in
CaCl2 solution placed inside a closed desiccator. The
concentration of the extract was varied from 0.1-1000 µg/ml. Under
these conditions, only the calcite phase was nucleated in all crystal
growth experiments regardless of the presence or absence of protein(s).
However, the growth pattern of the calcite crystals varied with the
concentration of the eggshell extract (mixture of proteins). No
significant effects were observed below 50 µg/ml of the protein
extract (Fig. 6B), although
pits were observed on the {10.4} rhombohedral faces (Fig.
6B, inset). At a concentration of 100 µg/ml,
some of the crystals exhibited protrusions parallel to the {10.4}
faces (Fig. 6C). As the protein extract concentration was
increased to 250 µg/ml, calcite crystal aggregates started to appear
along with curved edges for the crystals (Fig. 6D). At 500 µg/ml, calcite crystal aggregates with various shapes (spherical,
ellipsoidal, or dumbbell) were formed (Fig. 6E). At 1000 µg/ml, the highest concentration used in these experiments, the
individual crystal aggregates formed were smaller, but greater in
number and more curved at the corners and edges than the aggregates
grown at other concentrations (Fig. 6F).

View larger version (119K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of eggshell extract on
CaCO3 crystallization. Representative scanning
electron microscopic images of calcite crystals grown in the presence
of eggshell extract obtained from goose eggshell matrix. Concentration
of the extract used: 0.0 µg/ml (control, A), 50 µg/ml
(B), 100 µg/ml (C), 250 µg/ml (D),
500 µg/ml (E), 1000 µg/ml (F). Scale
bar is 50 µm; inset scale bar is 10 µm.
|
|
Effect of Ansocalcin on CaCO3 Crystallization--
The
morphology of the calcite crystals changed gradually as a function of
the concentration of ansocalcin (Fig. 7).
At lower concentrations (0.1-1.0 µg/ml) spiral pits appeared at the
sides of the {10.4} rhombohedral crystals without major change in
size and shape (data not shown). As the concentration was increased to
10 µg/ml, the calcite crystals that formed had terraced structures on
the {10.4} face penetrating toward the center of the crystal (Fig.
7B). At 50 µg/ml ansocalcin, the crystal aggregates
exhibited similar morphology to that obtained at 500 µg/ml whole
eggshell extract (mixture of proteins, compare Figs. 6E and
7C). However, in the case of ansocalcin at a concentration
of 50 µg/ml, the edges and corners remained sharp. The aggregates
formed at 100 µg/ml ansocalcin contained a higher number of
crystallites than at 50 µg/ml without significant change in the
overall aggregate size (Fig. 7D). As the ansocalcin
concentration was increased further, smaller and smaller crystallites
were formed, and these crystallites randomly aggregated into spherical
and ellipsoidal forms (Fig. 7, E and F). Attempts
to break the calcite spherules with a sharp blade to identify the core
were unsuccessful due to the collapse of the aggregates into small
crystallites.

View larger version (120K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of ansocalcin on CaCO3
crystallization. Representative scanning electron microscopic
images of calcite crystals grown in the presence of ansocalcin.
Concentration of ansocalcin used: 0.0 µg/ml (control, A),
10 µg/ml (B), 50 µg/ml (C), 100 µg/ml
(D), 250 µg/ml (E), 500 µg/ml (F).
Scale bar is 50 µm; inset scale bar is 10 µm.
|
|
The size distribution of the calcite aggregates measured (accuracy of
±2 µm) at the concentration range of 50-500 µg/ml ansocalcin is
shown in Fig. 8. The aggregate size
distribution was narrow (32-42 µm) with a maximum around 38.5 µm
at ansocalcin concentrations of 50 and 100 µg/ml. At 250 µg/ml, a
reduction in the size of the calcite crystal aggregates was accompanied
by a wide aggregate size distribution (10-32 µm). At 500 µg/ml
ansocalcin, the size distribution was narrower (10-22 µm) with a
maximum number of crystals of size 20 µm. Thus, an increase in
ansocalcin concentration resulted in a decrease in the size of calcite
crystal aggregates. It is important to note that the nucleation density
(i.e. the number of crystals per unit area) increased
significantly at the highest concentration of ansocalcin tested (500 µg/ml, Fig. 7F).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 8.
Size distribution of the polycrystalline
aggregates grown in the presence of ansocalcin. Concentration of
ansocalcin used: 50 µg/ml (A), 100 µg/ml (B),
250 µg/ml (C), and 500 µg/ml (D).
|
|
Aggregation of Ansocalcin--
To determine the noncovalent
aggregation of ansocalcin, we examined the elution behavior of
ansocalcin by SEC using 7.5 mM CaCl2 solution as eluent (Fig.
9A). At 500 µg/ml
ansocalcin, it was observed that ansocalcin separated into two
components. The profile was characterized by the presence of
predominantly trimeric and a smaller amount of tetrameric species.
However, at 100 µg/ml, ansocalcin elutes as a dimer. At a
concentration of 250 µg/ml, chromatogram was complex and indicated
the existence of a mixture of monomers and multimeric species, such as
dimers, trimers, and tetramers (data not shown). The SEC of ansocalcin
at 100 µg/ml in 200 mM Tris-HCl (pH 7.5) as eluant in the
absence of calcium ions showed a profile similar to that observed in
CaCl2 solution (Fig. 9B). At 500 µg/ml, the
profile was characterized by the presence of predominantly trimeric and
a smaller amount of tetrameric species. However, as observed in the
presence of CaCl2, a different profile was obtained at a
concentration of ansocalcin of 250 µg/ml with a strong peak
corresponding to the formation of trimers and, to a smaller extent, the
presence of higher order multimers (data not shown).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 9.
Aggregation of ansocalcin. Size
exclusion chromatogram of ansocalcin in CaCl2 solution (7.5 mM) (A) and in Tris-HCl (200 mM, pH
7.5) (B) without calcium ions at various concentrations of
ansocalcin, 100 µg/ml (broken line), and 500 µg/ml
(continuous line). The vertical dotted lines
represent the position of the standards: bovine serum albumin (66 kDa)
(a), egg white albumin (44 kDa) (b), and chicken
egg lysozyme (14 kDa) (c).
|
|
Fig. 10 shows SDS-PAGE of ansocalcin
under non-reducing conditions in the presence and absence of
Ca2+ ions. Ansocalcin migrated as a single sharp 17-kDa
(mass of monomer) band at a concentration of 15 µg in 20 µl. At 90 µg of protein in 20 µl, the appearance of an additional band with
an apparent molecular mass of 30 kDa (dimer) was observed. A
10-fold increase in the concentration of ansocalcin (150 µg in 20 µl) resulted in an increase in the population of the dimer and, to a
lesser extent, the intensity of a trimer band (molecular mass of 42 kDa). The SDS-PAGE profile was unaltered in the presence of
Ca2+ ions (Fig. 10). The absence of a change in
electrophoretic mobility of ansocalcin in the presence of
Ca2+ ions indicates that ansocalcin does not require
Ca2+ ions for aggregation. The low amount of trimers may be
due to decreased stability of the trimers in the solution used for this experiment or due to presence of large protein domains that could sterically preclude its formation in the SDS micelles. The
concentration of ansocalcin required to form dimer is higher in
SDS-PAGE than in SEC, indicating that at lower concentration SDS
disrupts dimerization.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 10.
Electrophoretic mobility of ansocalcin.
SDS-PAGE (4-20%) of ansocalcin at various concentrations with and
without Ca2+ ions. Lane 1, molecular weight
standards (broad range, Bio-Rad); lanes 2, 4, and
6, ansocalcin without Ca2+ ions; lanes
3, 5, and 7, ansocalcin with
Ca2+ ions. The amounts of ansocalcin loaded are 15 µg
(lanes 2 and 3), 90 µg (lanes 4 and
5), and 150 µg (lanes 6 and 7). The
protein was dissolved in water or incubated in CaCl2 (7.5 mM) overnight. 20 µl of 2× SDS loading buffer was added
to an equal volume of the protein sample, the solution was heated at
95 °C for 5 min and loaded onto the gel. M, monomers;
D, dimers; T, trimers.
|
|
Far UV-CD studies of ansocalcin in the presence and absence of
Ca2+ ions showed significant changes in secondary
structure, especially at high concentrations of ansocalcin (Fig.
11). Analysis of the secondary
structure at 50 µg/ml showed the presence of 26%
-helix, 31%
turn conformation, 6.8% aromatic stacking, and 29% random conformation. There were no significant differences in the CD spectra
of ansocalcin in the presence and absence of Ca2+ ions,
when the experiments were performed up to a concentration of 100 µg/ml ansocalcin. This indicates that Ca2+ ions did not
affect the conformation of the protein at these concentrations. Above
100 µg/ml ansocalcin, some differences in the CD spectra were
observed in the presence and absence of Ca2+ ions. The
spectra exhibited fine structures at 208 nm as the concentration was
increased from 100 to 250 µg/ml, which may be due to the presence of
monomeric and multimeric species (Fig. 11D). Further
increases in the concentration of protein to 500 µg/ml decreased the
amplitude of the peaks accompanied by a strong negative maximum at 230 nm (Fig. 11E). Excessive scattering from the solution
complicated the conformational analysis at higher concentrations (250 and 500 µg/ml).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 11.
CD spectra of ansocalcin. Far UV-CD
spectra of ansocalcin at various concentrations with and without
CaCl2 (7.5 mM). A, 10 µg/ml;
B, 50 µg/ml; C, 100 µg/ml; D, 250 µg/ml; E, 500 µg/ml. The spectra showed
significant changes in the secondary structure at higher concentrations
of ansocalcin. The black lines represent CD spectra of
ansocalcin in water, and the gray lines represent CD spectra
recorded in 7.5 mM CaCl2. The instrument
settings used were: scan range, 190-260 nm; scan rate, 50 nm/min;
sensitivity, 10 millidegrees; response time, 1 s.
|
|
Ansocalcin contains eight tryptophan residues. To investigate the
changes in the microenvironment around tryptophan residues, we recorded
the emission spectrum of ansocalcin at
exc of
295 nm (to selectively excite tryptophan). Fig.
12, A and B show
the tryptophan fluorescence of ansocalcin in water and
CaCl2 solution. The spectra were characterized by smooth
Lorentzian curves indicating more or less homogeneous environments for
all of the tryptophan residues. An emission maximum of 346 nm was observed at all concentrations of ansocalcin. The emission spectra were
not significantly affected by the presence of Ca2+ ions
(Fig. 12B). Fig. 12C indicates the variation in
fluorescence emission intensity at 346 nm at various concentration of
ansocalcin in water and in CaCl2 solution. The increase in
fluorescence intensity is linear up to 100 µg/ml, reaches a maximum
at 250 µg/ml and decreases with further increases in the
concentration of ansocalcin. A reduction in fluorescence intensity was
observed at 500 µg/ml without any shift in the emission maximum and
it was greater in the absence of Ca2+ ions.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 12.
Tryptophan fluorescence emission spectra of
ansocalcin. Fluorescence spectra were recorded in water
(A) and in 7.5 mM CaCl2
(B) at various concentrations of ansocalcin: 5 µg/ml
(1); 10 µg/ml (2); 50 µg/ml (3);
100 µg/ml (4); 250 µg/ml (5); 500 µg/ml
(6); 1 mg/ml (7). Emission intensity
(F) is expressed in arbitrary units. C, plot of
fluorescence intensity at 346 (wavelength of emission
maximum) versus concentration of ansocalcin in water
(broken lines) and in CaCl2 solution
(continuous lines).
|
|
 |
DISCUSSION |
Avian eggshell is a unique example of an acellular hard bioceramic
wherein the mineral phase is sequentially assembled over the shell
membrane. Information on the structure-property relationship of
eggshell matrix proteins and their role in the biomineralization process will increase our understanding of the molecular mechanisms of
mineralization and enhance our capability to develop new materials. As
a first step, we have purified various soluble organic matrix proteins
to homogeneity from goose eggshells. The amino acid sequence of
ansocalcin, the major matrix protein, was determined by automated Edman
degradation of the S-pyridylethylated protein and from
peptides obtained by chemical and enzymatic degradation. Ansocalcin has 132 amino acid residues comprising hydrophilic and hydrophobic domains.
The sequence showed structural similarity to OC-17 from chicken
eggshell matrix and CTL from snake venom (Fig. 4).
At lower concentration, ansocalcin exists in monomeric form (based on
ESI-MS, Fig. 2E). As the concentration increases, it oligomerizes to dimers and trimers, as observed in the size exclusion chromatogram of ansocalcin at various concentrations. It is interesting to note that ansocalcin aggregates are stable in 2% SDS solution indicating that the protein-protein interactions are most likely through hydrophobic forces. The aggregation was not influenced by
Ca2+ ions at lower concentrations (<100 µg/ml). The
presence of multimers severely hindered the conformational analysis.
The appearance of a negative maximum at 230 nm in the CD spectra
indicates the probable role of tryptophan-tryptophan interactions in
the aggregation of ansocalcin (44). The observation that the wavelength
of the emission maxima is 346 nm at all concentrations of ansocalcin indicates that tryptophan residues are exposed to the solvent (45). The
presence of multimers in solution is further confirmed from the
concentration-dependent fluorescence intensity at 346 nm
(Fig. 12C). At lower concentrations, the fluorescence
intensity increases linearly with concentration and is maximal at 250 µg/ml. However, a significant decrease in intensity was observed in
water and in CaCl2 solution at higher concentrations of
ansocalcin. The decrease was greater in water than in CaCl2
solution (Fig. 12C). Such a reduction in the fluorescence
intensity of ansocalcin (in the absence of an external quencher) may be
due to the quenching of the tryptophan fluorescence emission by
polarizable groups (e.g. -COOH, -COO
,
-NH2, -SH, imidazole, and others) present in close vicinity (46). In CaCl2 solution, calcium ions would bind to some of these polar groups, which reduces the fluorescence quenching and, thus,
results in a higher fluorescence intensity.
It is interesting to note the similarities and differences of
ansocalcin aggregation under both denaturing (SDS-PAGE) and non-denaturing (SEC) conditions. Gregoire et al. (47)
reported that lithostathine protein, a CTL-like protein, in its
proteolytic S1 form oligomerizes to dimers and tetramers even under
denaturing conditions (47). Thus, we believe that ansocalcin
aggregation behavior is similar to that of the S1 form of
lithostathine, but ansocalcin forms dimers and trimers instead of
tetramers at high concentration. Hattan et al. (48) observed
that a glycoprotein purified from the extrapallial fluid of molluscan
shell showed Ca2+ ion-dependent oligomerization
(48). However, our results from SEC, SDS-PAGE, CD spectra, and
fluorescence studies show that the aggregation of ansocalcin does not
require Ca2+ ions.
Acidic and basic amino acid multiplets are present in soluble matrix
proteins associated with calcium ions in biominerals (Table
II and Refs. 49-52). However, their
exact role in the mineralization process has not yet been fully
elucidated. Molecular modeling studies indicated that such repetitive
arrangements might be responsible for the nucleation and growth
regulation of crystals (50). The existence of acidic amino acid
multiplets in the amino acid sequence of ansocalcin highlights its
influence in CaCO3 crystallization. In vitro
crystal growth experiments showed that individual crystals transformed
into polycrystalline aggregates as the concentration of ansocalcin or
the eggshell extract was increased. Purified ansocalcin was about 5-10
times more effective than whole eggshell extract toward inducing the
aggregation of calcite crystals (Figs. 6 and 7). The formation of
spiral pits at lower concentration of ansocalcin (0.1 µg/ml)
indicates strong interaction between the acidic groups present in the
protein and the growing crystal nuclei. Similar effects were observed
in the presence of highly acidic polyaspartic acid molecules (53). As
the concentration of ansocalcin was increased (>50 µg/ml), calcite
crystal aggregates were formed. The concentration-dependent
aggregation of ansocalcin may provide templates for calcite crystal
aggregates.
View this table:
[in this window]
[in a new window]
|
Table II
Occurrence of acidic and basic amino acid multiplets in the amino acid
sequence of proteins associated with CaCO3 biomineralization
|
|