Purification, Characterization, and in Vitro Mineralization Studies of a Novel Goose Eggshell Matrix Protein, Ansocalcin*

Rajamani LakshminarayananDagger , Suresh ValiyaveettilDagger §, Veena S. Rao, and R. Manjunatha Kini

From the Dagger  Department of Chemistry, National University of Singapore and the  Department of Biological Sciences, National University of Singapore, Singapore 117 543

Received for publication, February 14, 2002, and in revised form, November 7, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Biomineralization is an important process in which hard tissues are generated through mineral deposition, often assisted by biomacromolecules. Eggshells, because of their rapid formation via mineralization, are chosen as a model for understanding the fundamentals of biomineralization. This report discusses purification and characterization of various proteins and peptides from goose eggshell matrix. A novel 15-kDa protein (ansocalcin) was extracted from the eggshell matrix, purified, and identified and its role in mineralization evaluated using in vitro crystal growth experiments. The complete amino acid sequence of ansocalcin showed high homology to ovocleidin-17, a chicken eggshell protein, and to C-type lectins from snake venom. The amino acid sequence of ansocalcin was characterized by the presence of acidic and basic amino acid multiplets. In vitro crystallization experiments showed that ansocalcin induced pits on the rhombohedral faces at lower concentrations (<50 µg/ml). At higher concentrations, the nucleation of calcite crystal aggregates was observed. Molecular weight determinations by size exclusion chromatography and sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed reversible concentration-dependent aggregation of ansocalcin in solution. We propose that such aggregated structures may act as a template for the nucleation of calcite crystal aggregates. Similar aggregation of calcite crystals was also observed when crystallizations were performed in the presence of whole goose eggshell extract. These results show that ansocalcin plays a significant role in goose eggshell calcification.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Theoretical and observed masses of peptides of ansocalcin

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% alpha -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 lambda 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 lambda 346 (wavelength of emission maximum) versus concentration of ansocalcin in water (broken lines) and in CaCl2 solution (continuous lines).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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

At high concentrations of ansocalcin (i.e. in its aggregated state), there is a remarkable increase in the number of crystals nucleated (nucleation density) indicating its ability to trigger calcite crystal nucleation. From amino acid sequence, it is clear that ansocalcin possesses amphiphilic geometry comprising hydrophobic and hydrophilic domains. This amphiphilic character facilitates self-assembly, which can give rise to a hydrophilic exterior surface encompassing acidic and basic side chains. Such a feature may then assist specific interactions with the mineral phase accelerating the crystal nucleation. The decrease in fluorescence intensity and large changes in the CD spectra confirm this prediction. The lack of alignment between individual crystallites in the aggregate grown in the presence of ansocalcin (Fig. 7C) shows that the orientation of crystallites is not well controlled during crystal growth. According to Collier et al. (54) the formation of crystal aggregates in a suspension was controlled by two opposing factors: hydrodynamic forces and crystal growth rate. At lower levels of ansocalcin, fluid shear forces separate the crystals from each other through hydrodynamic forces. At higher concentrations, the growth rate dominates the hydrodynamic forces due to protein aggregation, and nucleation of the crystals takes place in a predefined space resulting in the formation of crystal aggregates. Such morphology is prevalent in rapidly mineralizing organisms such as calcareous algae, pennatulid sea fans, scleractinian corals, and other avian eggshells (55).

Overall, ansocalcin is the most abundant protein in goose eggshell matrix and induces formation of calcite crystal aggregates in the in vitro mineralization experiments. A comparison of the effect of ansocalcin and whole eggshell extract in CaCO3 crystallization indicates that ansocalcin plays a vital role in goose eggshell calcification. The subtle differences observed between the crystals grown in the presence of ansocalcin and whole eggshell extract might be due to the presence of other proteins/peptides found in the eggshell extract. Furthermore, ansocalcin undergoes calcium-independent aggregation in solution, which may be responsible for the nucleation of calcite crystal aggregates. Further studies on the role of other proteins/peptides may help in the understanding of biomineralization in the eggshell as well as in the use of this as a tool toward the development of novel biomaterials.

    ACKNOWLEDGEMENTS

We thank Prof. André Menez, Departmente, d'Ingenierie, d'Etudes des Proteines, C. E. A. Saclay, Gif-sur-Yvette, France for comments and Dr. Seetharama Jois of the Department of Pharmacy, National University of Singapore for his useful suggestions regarding the CD studies. We acknowledge the technical support from the Dept. of Chemistry and financial support from the National University of Singapore.

    FOOTNOTES

* This work was supported by the Department of Chemistry, National University of Singapore.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.

The amino acid sequence reported in this paper has been submitted to the Swiss Protein Database under Swiss-Prot accession no. P83300.

§ To whom correspondence should be addressed: Dept. of Chemistry, National University of Singapore, 3 Science Dr. 3, Singapore 117543. Tel.: 65-6874-4327; Fax: 65-6779-1691; E-mail: chmsv@nus.edu.sg.

Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M201518200

    ABBREVIATIONS

The abbreviations used are: RP-HPLC, reversed phase high performance liquid chromatography; OC-17, ovocleidin-17; CTL, C-type lectins, ESI-MS, electrospray ionization mass spectrometry; SEC, size exclusion chromatography; CD, circular dichroism.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Lowenstam, H. A. (1981) Science 211, 1126-1131[Medline] [Order article via Infotrieve]
2. Krampitz, G., and Graser, G. (1988) Angew. Chem. Int. Ed. 27, 1145-1156
3. Addadi, L., and Weiner, S. (1992) Angew. Chem. Int. Ed. 31, 153-169
4. Weiner, S., and Addadi, L. (1997) J. Mater. Chem. 7, 689-702[CrossRef]
5. Weiner, S., Addadi, L., and Wagner, H. D. (2000) Mat. Sci. Eng. C-Bio. S. 11, 1-8
6. Heuer, A. H., Fink, D. J., Laraia, V. J., Arias, J. L., Calvert, P. D., Kendall, K., Messing, G. L., Blackwell, J., Rieke, P. C., Thompson, D. H., Wheeler, A. P., Veis, A., and Caplan, A. I. (1992) Science 255, 1098-1105[Medline] [Order article via Infotrieve]
7. Linde, A., Lussi, A., and Crenshaw, M. A. (1989) Calcif. Tissue Int. 44, 286-295[Medline] [Order article via Infotrieve]
8. Mann, S. (1989) in Biomineralization: Chemical and Biochemical Perspectives (Mann, S. , Webb, J. , and Williams, R. J. P., eds) , pp. 35-62, VCH Publishers, New York
9. Arias, J. L., Fink, D. J., Xiao, S. Q., Heuer, A. H., and Caplan, A. I. (1993) Int. Rev. Cytol. 145, 217-250[Medline] [Order article via Infotrieve]
10. Simkiss, K. (1961) Biol. Rev. Cambridge Philos. Soc. 36, 321-367
11. Gautron, J., Hincke, M. T., Mann, K., Panhéleux, M., Bain, M., McKee, M. D., Solomon, S. E., and Nys, Y. (2001) J. Biol. Chem. 276, 39243-39252[Abstract/Free Full Text]
12. Hincke, M. T., Gautron, J., Tsang, C. P. W., McKee, M. D., and Nys, Y. (1999) J. Biol. Chem. 274, 32915-32923[Abstract/Free Full Text]
13. Hincke, M. T., Tsang, C. P. W., Courtney, M., Hill, V., and Narbaitz, R. (1995) Calcif. Tissue Int. 56, 578-583[Medline] [Order article via Infotrieve]
14. Hincke, M. T. (1995) Connect. Tissue Res. 31, 227-233
15. Gautron, J., Hincke, M. T., Garcia-Ruiz, J. M., Dominguez-Vera, J. M., and Nys, Y. (1997) in Eggs and Egg Products Quality (Kijowski, J. , and Pikul, J., eds) , pp. 66-72, Pozhaus, Poland, Proceedings VII European Symposium
16. Mann, K., and Siedler, F. (1999) Biochem. Mol. Biol. Int. 47, 997-1007[Medline] [Order article via Infotrieve]
17. Mann, K. (1999) FEBS Lett. 463, 12-14[CrossRef][Medline] [Order article via Infotrieve]
18. Hincke, M. T., Gautron, J., Panhéleux, M., Garcia-Ruiz, J. M., McKee, M. D., and Nys, Y. (2000) Matrix Biol. 19, 443-453[CrossRef][Medline] [Order article via Infotrieve].
19. Carrino, D. A., Rodriguez, J. P., and Caplan, A. I. (1997) Connect. Tissue Res. 36, 175-193[Medline] [Order article via Infotrieve].
20. Baker, J. R., and Balch, D. A. (1962) Biochem. J. 82, 352-361[Medline] [Order article via Infotrieve]
21. Leach, R. M. (1982) Poult. Sci. 61, 2040-2047
22. Heaney, R. K., and Robinson, D. S. (1976) Biochim. Biophys. Acta 451, 133-142[Medline] [Order article via Infotrieve]
23. Wu, T. M., Rodriguez, J. P., Fink, D. J., Carrino, D. A., Blackwell, J., Caplan, A. I., and Heuer, A. H. (1995) Matrix Biol. 14, 507-513[CrossRef][Medline] [Order article via Infotrieve].
24. Dominguez-Vera, J. M., Gautron, J., Garcia-Ruiz, J. M., and Nys, Y. (2000) Poult. Sci. 79, 901-907[Medline] [Order article via Infotrieve]
25. Nys, Y., Gautron, J., McKee, M. D., Garcia-Ruiz, J. M., and Hincke, M. T. (2001) World. Poult. Sci. J. 57, 401-413
26. Arias, J. L., and Fernandez, M. S. (2001) World Poult. Sci. J. 57, 349-357
27. Lavelin, I., Meiri, N., and Pines, M. (2000) Poult. Sci. 79, 1014-1017[Medline] [Order article via Infotrieve].
28. Berman, A., Addadi, L., and Weiner, S. (1988) Nature 331, 546-548[CrossRef]
29. Weiner, S., and Addadi, L. (1991) Trends Biochem. Sci. 16, 252-256[CrossRef][Medline] [Order article via Infotrieve]
30. Panhéleux, M., Bain, M., Fernandez, M. S., Morales, I., Gautron, J., Arias, J. L., Solomon, S. E., Hincke, M. T., and Nys, Y. (1999) Br. Poult. Sci. 40, 240-252[CrossRef][Medline] [Order article via Infotrieve]
31. Inglis, A. S. (1983) Methods Enzymol. 91, 324-332[Medline] [Order article via Infotrieve]
32. George, D. (1985) Adv. Prot. Chem. 37, 1-97[Medline] [Order article via Infotrieve]
33. Albeck, S., Aizenberg, J., Addadi, L., and Weiner, S. (1993) J. Am. Chem. Soc. 115, 11691-11697
34. Laemmli, U. K. (1970) Nature 227, 680-682[Medline] [Order article via Infotrieve].
35. Robinson, A. B., and Robinson, L. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8880-8884[Abstract]
36. Drickamer, K. (1988) J. Biol. Chem. 263, 9557-9660[Free Full Text]
37. Weis, W. I., Dirckamer, K., and Hendrickson, W. A. (1992) Nature 360, 127-134[CrossRef][Medline] [Order article via Infotrieve]
38. Killian, C. E., and Wilt, F. H. (1996) J. Biol. Chem. 271, 9150-9159[Abstract/Free Full Text]
39. Mann, K., Weiss, I. M., André, S., Gabius, H. -J., and Fritz, M. (2000) Eur. J. Biochem. 267, 5257-5264[Abstract/Free Full Text]
40. Bertrand, J. A., Pignol, D., Bernard, J. P., Verdier, J. M., Dagorn, J. C., and Fontecilla-Camps, J. C. (1996) EMBO J. 15, 2678-2684[Abstract]
41. Weiner, S. (1983) Biochemistry 22, 4139-4145
42. Greenfield, E. M., Wilson, D. C., and Crenshaw, M. A. (1984) Am. Zool. 24, 925-932
43. Halloran, B. A., and Donachy, J. E. (1995) Comp. Biochem. Physiol. 111, 221-231
44. Woody, R. W., and Dunker, A. K. (1996) in Circular Dichroism and the conformational analysis of Biomolecules (Fasman, G. D., ed) , p. 123, Plenum Press, New York
45. Pain, H. R. (1995) in Current Protocols in Protein Science (Coligan, J. E. , Dunn, B. M. , Ploegh, H. I. , Speicher, D. W. , Wingfield, P. T. , and Chanda, V. B., eds) , pp. 7.7.1-7.7.19, Wiley, New York
46. Demchenko, A. P. (1994) in Topics in Fluorescence Spectroscopy Vol. 3 (Lackowicz, J. R., ed) , p. 77, Plenum Press, New York
47. Gregoire, C., Marco, S., Thimonier, J., Duplan, L., Laurine, E., Chauvin, J. P., Michel, B., Peyrot, V., and Verdier, J. M. (2001) EMBO J. 20, 3313-3321[Abstract/Free Full Text]
48. Hattan, S. J., Laue, T. M., and Chasteen, N. D. (2001) J. Biol. Chem. 276, 4461-4468[Abstract/Free Full Text]
49. Samata, T., Hayashi, N., Kono, M., Hasegawa, K., Horita, C., and Akera, S. (1999) FEBS Lett. 462, 225-229[CrossRef][Medline] [Order article via Infotrieve]
50. George, A., Bannon, L., Sabsay, B., Dillon, J. W., Malone, J., Veis, A., Jenkins, N. A., Gilbert, D. J., and Copeland, N. G. (1996) J. Biol. Chem. 271, 32869-32873[Abstract/Free Full Text]
51. Sarashina, I., and Endo, K. (2001) Mar. Biotechnol. 3, 362-369[Medline] [Order article via Infotrieve]
52. Testeniere, O., Hecker, A., Le, Gurun, S., Quennedey, B., Graf, F., and Luquet, G. (2002) Biochem. J. 361, 327-335[CrossRef][Medline] [Order article via Infotrieve]
53. Gower, L. A., and Tirrell, D. A. (1998) J. Cryst. Growth 191, 153-160[CrossRef]
54. Collier, A. P., Hetherington, C. J. D., and Hounslow, M. J. (2000) J. Cryst. Growth 208, 513-519[CrossRef]
55. Lowenstam, H. A., and Weiner, S. (1989) On Biomineralization , Oxford University Press, New York


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.



This Article
Abstract
Full Text (PDF)
All Versions of this Article:
278/5/2928    most recent
M201518200v1
Purchase Article
View Shopping Cart
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Copyright Permissions
Google Scholar
Articles by Lakshminarayanan, R.
Articles by Kini, R. M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Lakshminarayanan, R.
Articles by Kini, R. M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   Biochemistry and Molecular Biology Education 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.