Purification and Characterization of a Novel Calcium-binding Protein from the Extrapallial Fluid of the Mollusc, Mytilus edulis*

Stephen J. HattanDagger , Thomas M. Laue§, and N. Dennis ChasteenDagger

From the Departments of Dagger  Chemistry and § Biochemistry & Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824

Received for publication, July 28, 2000, and in revised form, November 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the bivalve mollusc Mytilus edulis shell thickening occurs from the extrapallial (EP) fluid wherein secreted shell matrix macromolecules are thought to self-assemble into a framework that regulates the growth of CaCO3 crystals, which eventually constitute ~95% of the mature shell. Herein is the initial report on the purification and characterization of a novel EP fluid glycoprotein, which is likely a building block of the shell-soluble organic matrix. This primary EP fluid protein comprises 56% of the total protein in the fluid and is shown to be a dimer of 28,340 Da monomers estimated to be 14.3% by weight carbohydrate. The protein is acidic (pI = 4.43) and rich in histidine content (11.14%) as well as in Asx and Glx residues (25.15% total). The N terminus exhibits an unusual repeat sequence of histidine and aspartate residues that occur in pairs: NPVDDHHDDHHDAPIVEHHD~. Ultracentrifugation and polyacrylamide gel electrophoresis demonstrate that the protein binds calcium and in so doing assembles into a series of higher order protomers, which appear to have extended structures. Circular dichroism shows that the protein-calcium binding/protomer formation is coupled to a significant rearrangement in the protein's secondary structure in which there is a major reduction in beta -sheet with an associated increase in alpha -helical content of the protein. A model for shell organic matrix self-assembly is proposed.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Biomineralization refers to the biological regulation of inorganic mineral deposition (1-3). Generally, organisms use proteins and polysaccharides to regulate mineralization by affecting the nucleation, growth regulation, and growth cessation of the attendant mineral crystals (4-9). Typically, mineralization takes place from a fluid medium, which is biologically regulated in its content, supersaturated with the ions being deposited, and spatially separated from its surroundings (10, 11).

In the mollusc Mytilus edulis, biomineralization manifests itself in exoskeletal shell formation. As a whole, mollusc shells are 95-99.9% by weight CaCO3 with the residual mass being composed of biological macromolecules (11, 12). The shell of M. edulis is not homogeneous in its spatial distribution of CaCO3. Instead, the shell has an outer prismatic and an inner pearl-like nacreous layer, which contain CaCO3 deposited as crystals of calcite and aragonite, respectively. These two mineralized layers are continuous, ultrastructurally unique, and reside one on top of the other along the long axis of the shell (1).

Anatomically, the extrapallial (EP)1 fluid fills the cavity between the most outer visceral organ (the mantle) and the external shell. The EP fluid resides inside of the pallial line (site of mantle attachment near the shell perimeter) and is the medium from which prismatic layer thickening and nacre layer nucleation and growth occur (13-15). The location and contents of the EP fluid implies that it plays an essential role in mineralization/demineralization processes in vivo. Despite this belief, the EP fluid has received little study, because most investigations of shell formation have focused on the shell itself (1).

Molluscan shell elongation and thickening occur throughout the life of the animal and is thought to be an organic matrix-mediated process controlled by a network of macromolecules composed of protein, carbohydrate, and glycoprotein (1, 16-19). In M. edulis these macromolecules are thought to be synthesized in the mantle. Exterior to the pallial line, the mantle is in intimate contact with the shell margin and as such is thought to secrete matrix material directly onto the growing shell edge. Interior to the pallial line, the mantle and shell are spatially separated by the EP cavity and constituent EP fluid. At this location, organic material is secreted from the mantle into the EP fluid where it is thought to self-assemble into a matrix prior to mineral deposition (15, 17, 20).

Qualitative analysis of EP fluid has shown it to contain the biomacromolecular materials found in the mature shell (protein, glycoprotein, carbohydrate, amino acids) (21, 22). The protein component of the EP fluid is heterogeneous, and certain fluid extracts have demonstrated the ability to bind calcium (21, 22) and to inhibit in vitro CaCO3 crystallization in the same manner as fractions of shell matrix protein (13). Studies of the ion content of the EP fluid show that its composition differs from that of the animal blood and the surrounding sea water (23-25). Examination of the pH, [CO2], and [Ca2+] as a function of shell opening and closure reveal the EP fluid to be a dynamic physiological medium, which may play a role in pH regulation within the animal (26).

Given the implied significance of the EP fluid in shell mineralization, the present study was undertaken to purify and characterize its major protein component. We show here for the first time that the major protein component of the EP fluid is a 56,000 molecular weight glycoprotein that is a homodimer composed of 14.3% carbohydrate. This primary EP fluid protein reversibly binds calcium in a manner that leads to significant changes in its secondary structure and to the formation of distinct multimeric species composed of the constituent monomers. The major EP fluid protein appears to be a building block of the soluble organic matrix of the shell. A hypothesis for the mode of matrix self-association is presented.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

EP Fluid Extraction-- Mussels were obtained from coastal New Hampshire waters or from Great Eastern Mussel aquaculture (Tenants Harbor, ME). Live animals were kept on ice for <1 h during extrapallial fluid extraction. The shell cavity was accessed by slicing the adductor muscle with a scalpel. With the shell cavity open, excess water was removed by tilting the animal upright and letting it drain onto a paper towel. The needle of a 500-µl gas-tight syringe was placed bevel down on the shell margin and slid beneath the mantle at the pallial line into the extrapallial space for EP fluid extraction. Care was taken not to disturb the mantle/shell attachment. Once extracted, the EP fluid was placed on ice in a polypropylene vial. To ensure that the EP fluid was not contaminated by blood from the animal, fluid was also extracted directly from the extrapallial space by the careful drilling of a hole through the shell. Identical protein banding patterns on PAGE were observed for fluid obtained by either extraction method. Individual animals yielded an average of 300 µl of fluid; however, variation from practically nothing to in excess of 500 µl was observed.

Unless otherwise mentioned, the protein buffer solution used throughout was 20 mM (3-(N-morpholine)propanesulfonic acid (MOPS), pH 7.5, in 0.1 M KCl, and all steps were performed at 4 °C. The buffer was passed through a 1.5- × 25-cm column of Chelex 100 metal ion chelation resin (Sigma) and then filtered/degassed by suction filtration through a 0.45-µm pore Whatman filter.

Immediately after extraction, the EP fluid was centrifuged (3500 × g) and the supernatant was retained and dialyzed against 1 liter of buffer 85% saturated with ammonium sulfate (Sigma) for at least 4 h using Spectra/Por 6000-8000 molecular weight cut off cellulose dialysis tubing. Precipitated protein was collected by centrifugation at 12,500 × g for 10 min and dissolved in a minimum volume of buffer followed by dialysis against buffer to remove residual (NH4)2SO4.

Chromatography-- Cation exchange chromatography was performed on a 2- × 50-cm column of Sephadex CM-50 resin (Sigma) using a protein buffer mobile phase and a gravity flow rate of 0.5 ml/min without a salt gradient. Anion exchange chromatography was then performed on three 0.7- × 2.5-cm Amersham Pharmacia Biotech HiTrap Q columns connected in series using a buffer mobile phase with a flow rate of 1.5 ml/min and a 200-ml, 0.1-0.5 M KCl linear salt gradient for protein elution. Finally, size exclusion chromatography was performed on 1.5- × 57-cm Sepharose 6B-CL resin (Sigma) with a buffer mobile phase and gravity flow rate of 15 ml/h. All chromatography was performed at room temperature with column effluent monitored at 280 nm. Fractions of 5 ml were collected.

Protein Analysis-- Electrophoresis was carried out on a Hoefer Mighty Small II electrophoresis unit using electrophoresis grade reagents from Sigma, reagent grade ammonium persulfate from J. T. Baker Chemical, low molecular weight SDS-PAGE markers from Bio-Rad, and ampholines from Amersham Pharmacia Biotech. Electrophoretic protocols (native PAGE, SDS-PAGE, isoelectric focusing) using Coomassie Brilliant Blue R-250 protein staining were taken from the Hoefer Scientific Instruments Manual 1992-1993. Glycoprotein-specific gel staining was done by the method of Rauchsen (27). Gels were 1.5 mm thick, and experiments were conducted using a limiting current of 20 mA/gel. Gel scanning was performed on a Hoefer Scientific GS 300 transmittance/reflectance scanning densitometer interfaced to a MINC-23 computer (Digital Equipment Corp.). Photographs of gels were scanned using a UMAX UC300 color scanner interfaced to a MacIntosh Quadra 700 computer. Adobe Photoshop 3.0 was used for image processing.

Protein concentration was estimated by a Bio-Rad protein assay using bovine serum albumin as a standard following the method of Bradford (28). Determination/quantitation of carbohydrate moieties was done by the method of Dubois et al. (29) using D-galactose in buffer to construct 5- to 30-µg standard calibration curves and SDS-PAGE analysis of EP protein before and after digestion with peptide-N4-(N-acetyl-beta -glucosaminyl)asparagine amidase F. A value of 14.3% carbohydrate was determined (30) and confirmed by mass spectrometry (31). The N-linked glycan has a mass of ~4000 Da and is composed of hexoses (30, 31).

The protein molecular weight was determined by matrix-assisted, laser desorption ionization, time-of-flight mass spectrometry on a Perceptive Biosystems, Voyager-Elite biospectrometry workstation. Instrument calibration was achieved using an insulin mass standard. Analysis of the NH4HCO3 lyophilized EP fluid protein sample was performed using an accelerating voltage of 30,000 V, a pressure of 9.50 × 10-8 torr, and a low mass cutoff of 500.0.

Amino acid analysis samples were dialyzed against 5.0 mM MOPS, pH 7.5, 0.1 M KCl and then hydrolyzed for 20 h in 6 N HCl, 0.05% mercaptoethanol, 0.02% phenol, at 115 °C. At AAA Laboratory (Mercer Island, WA), amino acid separation and quantitation were performed on a Beckman 7300 analyzer using System Gold software and Beckman buffers following the ion-exchange method of Moore and Stein (32). At the University of Michigan laboratory, the analysis was performed on an Applied Biosystems 420H amino acid analyzer. The sample for N-terminal amino acid sequence analysis was prepared by dialysis of purified protein against a 10% (v/v) solution of acetic acid. The analysis was done on an Applied Biosystems Inc., 475A protein sequencer, by the method of automated Edman degradation (33).

Ultracentrifugation Analysis-- Sedimentation equilibrium and velocity experiments were performed on a Beckman XLA analytical centrifuge equipped with Rayleigh interference optics (34). Experiments were conducted at 20 °C using a four-hole titanium rotor spinning at 40,000 rpm. The sample cells were two-channel, charcoal-filled, and equipped with Epon centerpieces and sapphire windows. The buffer used in all sedimentation velocity analyses was 20 mM Tris, pH 7.5, 0.1 M KCl; where mentioned, samples were analyzed with the addition of CaCl2 to the buffer solution. Protein concentrations ranging from 0.25 to 2.0 mg/ml were employed to investigate possible mass action effects in the centrifugation data. The method of Stafford (35) was used to obtain sedimentation coefficient distributions (g(s*)) from the time derivative of the concentration distributions (dc/dt), and sedimentation and diffusion coefficients were calculated from the line widths and positions of Gaussian fits to the g(s*) versus s data (35). The partial specific volume was calculated from the amino acid (vide infra) composition of the protein using the software program Sednterp (36) and assuming a glycan composed of hexoses (30, 31).

Circular Dichroism-- Circular dichroism (CD) experiments were done on a JASCO J700 circular dichroism spectropolarimeter calibrated using a 0.06% (+)-10-camphorsulfonic acid solution (Aldrich). CD spectra of EP fluid protein (0.65 mg/ml) in buffer with and without 10 mM Ca2+, along with matching buffers, were obtained using a 0.05-mm sample cell. Instrument optics and sample chamber were continually flushed with 30 liters/min of dry N2 gas. The instrument settings were; scan range, 180-260 nm; scan rate, 1 nm/min; wavelength step, 0.5 nm; sensitivity, 5 millidegrees; response time, 16 s. Protein secondary structural analysis of the CD spectra (182-260 nm) was done using the Self-Consistent Method (SELCON) program of Sreerama and Woody (37) using the fitting constraints recommended by the authors.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Raw EP Fluid Analysis and Purification of Primary Protein Component-- Protein and carbohydrate concentrations in untreated EP fluid samples were determined for nine batches of fluid yielding an average of 4.3 ± 1.8 mg/ml for protein and 3.8 ± 1.2 mg/ml for carbohydrate. The distribution of fluid proteins in samples taken from both batch extracts and individual animals was studied by native and SDS-PAGE. Fig. 1A shows the consistent distribution of at least six protein bands with the persistent appearance of a major protein band (marked by an arrow in lanes 1-6, Fig. 1A), corresponding to 56 ± 15% (n = 3) of the total fluid protein based on the integrated band intensity. This major protein fraction, hereafter designated the EP protein, was the component targeted for purification.



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Fig. 1.   A, 15% native PAGE gel of fluid samples extracted in October, August, July 1993. Lanes in A: 1, 20 µl of Raw Fluid (RF) Oct.; 2, 10 µl of RF Oct.; 3, 20 µl of RF Aug.; 4, 10 µl of RF Aug.; 5, 20 µl of RF Jul.; 6, 10 µl of RF Jul.; 7, protein standards (from top to bottom), transferrin (80,000); albumin (67,000); carbonic anhydrase (34,000); myoglobin (17,500). The arrow denotes the major protein band. B, 15% native PAGE gel stained for glycoprotein (left) and indiscriminately for protein (right). Lanes in B: 1 and 10, after cation exchange; 2 and 9, Raw fluid; 3 and 8, after anion exchange; 4 and 7, after size exclusion (most purified protein); 5 and 6, protein standards (same as in A).

Chromatographically separated protein fractions (see "Experimental Procedures") were analyzed by electrophoresis as a means of tracking the protein of interest during purification. Fig. 1B shows a 15% native PAGE gel loaded with duplicate samples of protein, at the various stages of purification, and a duplicate loading of protein standards. (A 15% acrylamide gel is shown in Fig. 1B, where diffusion of the EP glycoprotein is minimized; however, similar band patterns are seen in 7 and 10% gels.) Upon completion of electrophoresis, the gel was sliced in half and one half (lanes 6-10) was stained for protein, while the other half (lanes 1-5) was stained specifically for glycoprotein. The right half of the gel in Fig. 1B demonstrates the effective purification of the major EP protein to a single band (lane 7), and the left half (lanes 1-4) shows that the major EP protein stains positive for glycoprotein. Of the standard proteins used (lanes 5 and 6), only transferrin, which is ~5% by weight carbohydrate, stained in both gel halves. The purified EP protein was determined to be 14.3% carbohydrate (see "Experimental Procedures").

N-terminal Amino Acid Sequence and Trypsin Digest-- The 20-amino acid sequence of the N terminus of the EP protein reveals an interesting repeat pattern of histidine and aspartate residues, viz.: NPVDDHHDDHHDAPIVEHHD~.

The sequence was entered into several protein data bases,2 none of which produced a protein match containing a compatible sequence. Mass fragments of 943.8, 1056.9, 1093.8, 1112.2, 1150.1, 1241.3, 1299.6, 1337.7, 1429.8, 1584.0, 1700.0, 1731.4, 1805.8, 1984.6, 2131.2, 2164.7, 2201.9, 2260.2, 2685.2, and 3047.3 were produced by trypsin digestion.

Amino Acid Analysis-- The averaged results from the amino acid analyses of three separate EP protein purifications are shown in Table I. The data are presented as percent molar composition for each residue, as well as in residues/protein based on a protein molecular weight of 28,340 (vide infra) containing 14.3% carbohydrate. Table I shows that EP protein contains a significant amount of acidic (Asx, Glx) residues. This finding is consistent with of those of shell-soluble organic matrix protein fractions (18, 38-40). The protein is also rich in histidine content (11.14%), a property also reflected in the N-terminal sequence where 6 of the 20 amino acids are histidine residues that occur in pairs (vide supra).


                              
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Table I
Amino acid composition

Isoelectric Focusing-- Results from isoelectric focusing showed the EP protein to be acidic with at least six distinguishable isoforms, which are focused as a tight stack of bands with isoelectric point (pI) values ranging from 4.08 to 4.67 with a median pI value of 4.43. Although the source of this charge heterogeneity is not known, such occurrences are common for glycoproteins (41).

Molecular Weight Determination-- Fig. 2 shows the time-of-flight mass spectrum of an EP protein sample. The peak centered at 28,340 mass units is the ionized EP protein (M/1+). The peak centered at 14,200 mass units is the doubly ionized protein species (M/2+). The peak breadth indicates microheterogeneity with minor peaks at 27,000, 27,840, and 28,960 Da, in addition to the major 28,340-Da peak. The observation of heterogeneity in the extent of glycosylation is common for glycoproteins (41), and the addition or deletion of monosaccharide moieties may account for the observed mass heterogeneity of the EP protein. The same protein electrophoretic migration was observed on 15% SDS-PAGE under either reducing or nonreducing conditions (data not shown), indicating a single type of subunit and lack of intersubunit disulfide bonds.3



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Fig. 2.   Time-of-flight mass spectrum of purified EP fluid protein.

Fig. 3 shows an overlay of a size exclusion chromatogram of purified EP protein with that of a set of protein standards. A plot of the log molecular weight versus the size exclusion elution coefficient Kav is shown in the Fig. 3 (inset) from which a molecular weight of 52,600 ± 1100 (n = 8) is estimated for the native protein. The resultant molecular weight is approximately double the value from mass spectrometry, a result suggesting that the EP protein in its native state is a homodimer of the 28,350-Da subunits.



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Fig. 3.   Overlay of a size exclusion chromatogram of purified EP protein and size exclusion standards. Peak 1, thyroglobulin (670,000); peak 2, gamma globulin (158,000); peak 3, ovalbumin (44,000); peak 4, myoglobin (17,000); peak 5, vitamin B-12 (1,350). Inset, plot of log molecular weight versus elution coefficient Kav for protein standards. EP protein (open circle) estimated to have molecular weight of 52,600.

To confirm this finding, sedimentation velocity and equilibrium measurements were also carried out on the native protein. Fig. 4 shows the sedimentation distribution coefficient, g(s), versus the apparent sedimentation coefficient, s, for the protein in Tris buffer. Two components are present, a major species at s approx  4.7 and a minor one near s approx  10.5 that we ascribe to an assembly of the s approx  4.7 species (vide infra). The minor species typically represented only 7-20% of the total, depending on the protein preparation. Lack of mass action effects over a 16-fold concentration range of protein indicated that equilibration between the major and minor species is slow during the 24-h time period between dilution of the sample and acquisition of the centrifugation data. That both species are derived from a single type of subunit is evidenced by the presence of a single band in SDS-PAGE over a wide range of sample loadings on the gel.



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Fig. 4.   Distribution g(s*) versus s of the EP protein in the absence of added calcium. Experimental data (solid line); Gaussian fit (dotted line). Inset, apparent molecular weight versus protein concentration. Conditions: 0.45 mg of protein/ml, 20 mM Tris, 0.1 M KCl, pH 7.5.

A sedimentation coefficient of 4.7 is incompatible with a protein of Mr approx  28,000. Accordingly, the apparent molecular weight of the 4.7-s component was determined. Gaussian curve fitting of the data (e.g. Fig. 4) for protein concentrations ranging from 0.055 to 0.9 mg/ml produced a diffusion coefficients for the s = 4.7 species of D = 7.3 × 10-11 to 8.1 × 10-11 m2/s. Apparent molecular weights were calculated from the Svedberg equation using the apparent diffusion coefficients, D, at each protein concentration and the partial specific volume v = 0.7089 cm3/g for the protein (see "Experimental Procedures"). Over a wide range of concentrations (Fig. 4, inset), the molecular weights determined from the 4.7-s peak were ~52,000. Although there appears to be a very slight decrease in the apparent molecular weight with decreasing concentration, the uncertainties in the fitting of the lower concentration data preclude interpretation of this observation. Thus, the molecular weights determined in this manner agree with the value from size exclusion chromatography and are in accord with the protein being a dimer in its native state. (A similar determination of the molecular weight of the s approx  10.5 component by sedimentation velocity was not possible due to variability in the experimental value of D obtained from curve fitting of the small peak of this minor species.)

Sedimentation equilibrium experiments produced Z average apparent molecular weights that were observed to decrease with increasing rotor speed due to the presence of the two nonequilibrating species. Nevertheless, at higher rotor speeds, where the heavier s = 10.5 component sediments out, the apparent molecular weight reached a value of Mr ~ 52,000 (Fig. 5). This value, although clearly less accurate, corresponds well with the molecular weights derived from sedimentation velocity and size exclusion chromatography. Therefore, we conclude that the s approx  4.7 species is a dimer.



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Fig. 5.   Apparent molecular weight from sedimentation equilibrium as a function of rotor speed. Conditions are the same as in Fig. 4.

Protein Interaction with Calcium-- Protein behavior in the presence of calcium was examined by electrophoresis experiments wherein all solutions used in the production and running of gels were brought to 10 mM Ca2+, the physiological calcium concentration in the EP fluid (22). Fig. 6 shows a 10% native gel (gel A) with the EP protein migrating two-thirds of the way through the gel as a single band; gel B corresponds to an identical set of samples run on a Ca2+-doped gel. Gel B shows that in the presence of the Ca2+ the single major EP fluid protein band in gel A is converted into several more slowly migrating protein bands, which remain in the 3% stacking gel of gel B. The gels demonstrate that, in the presence of calcium, the single EP protein band is converted into several larger, more slowly migrating species. Other divalent metal ions, including Mg2+, Mn2+, and Cd2+, have similar effects to those of Ca2+.



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Fig. 6.   Native PAGE gels in the absence (A) and presence (B) of 10 mM Ca2+. All aspects of the gels are identical. Lanes (for both gels): 1, protein standards (from top to bottom, transferrin (80,000); albumin (67,000); carbonic anhydrase (34,000); myoglobin (17, 500)); 2-9, EP fluid protein. SG and RG denote the 3% stacking and 10% running gels, respectively.

Sedimentation Velocity in the Presence of Calcium-- Fig. 7A shows traces of EP protein sample (0.9 mg/ml) analyzed before and immediately after the addition of 10 mM calcium. The formation of multiple protomeric species is evident. It is curious that peak A from the dimer shifts from 4.7 to ~4.0 s in the presence of calcium, an observation suggesting that Ca2+ binding produces a less compact structure in the intermediate ~4.0-s species prior to its assembly into protomeric species as shown (Fig. 7B). Fig. 7B also shows the result of the multiple Gaussian curve fit to the species distribution pattern of a sedimentation velocity cell containing 0.6 mg/ml protein in 20 mM Tris, pH 7.5, brought to 10 mM Ca2+ and allowed to stand for 24 h. Both Figs. 7A and 7B clearly demonstrate the presence of at least four, distinguishable, protomeric species having s values of ~10, 15, 19, and 22 (labeled B-E) as well as the intermediate ~4.0-s species. Also, these figures show that the stoichiometry of protomer formation is finite, leading to species that are distinguishable and reasonably uniform in molecular size and that, given sufficient time and a physiological concentration of Ca2+, the parent peak (peak A) fully converts protomers. It is also apparent from the data in these figures that the breadth of the peaks from B through E is increasing, which is opposite of what would be expected if these peaks represented pure components. Because there was no definitive evidence for mass action equilibrium between species (vide infra), the excess peak spreading probably reflects increased microheterogeneity in the structures of the more rapidly sedimenting components. Such heterogeneity with respect to self-association may reflect the trapping of stable or mesostable protomer conformation states upon calcification of the protein. That some of the protomeric species represent different structural assemblies involving the same number of subunits is also a possibility. The anomalous peak widths of the protomers preclude the determination of molecular weights of these species by sedimentation velocity measurements.



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Fig. 7.   Distribution g(s*) versus s, the apparent sedimentation coefficient. A, sedimentation velocity analysis of samples containing 0.9 mg/ml without calcium (dotted line) and in the presence of 10 mM Ca2+ (solid line) added just prior to analysis. B, 0.65 mg/ml protein sample in 10 mM Ca2+ equilibrated for 24 h prior to analysis. Solvent for all cells was 20 mM Tris, pH 7.5, 0.1 M KCL. Gaussian components are shown.

Although protomer assembly is calcium-dependent, analysis at higher calcium concentration (100 or 500 mM) did not change the species distribution profile from that in Fig. 7B. Additionally, Fig. 8 shows that the relative abundances of protomers, analyzed from samples taken from a single preparation and run at different concentrations, remains essentially constant regardless concentration (see Fig. 8 legend). Repeated attempts to demonstrate simple mass action behavior through diluting and reconcentrating samples failed to clearly exhibit the reversible redistribution of species expected. Therefore, although there may be some small portion of the protein capable of participating in reversible association, our evidence indicates that the bulk of the material forms stable complexes on the time scale of our experiments. However, some variation in protomer species distribution was seen between different protein preparations for reasons that are not understood at this time.



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Fig. 8.   Distribution g(s*) versus s, the apparent sedimentation coefficient as a function of protein concentration. The protein concentrations were 0.25, 0.5, 1.0, and 2.0 mg/ml in 20 mM Tris, 0.1 M KCl, pH 7.5, with 10 mM CaCl2. From Gaussian curve fitting, the percentages of the A, B, C, and D components, respectively, are: 2.6 ± 0.3, 34.7 ± 0.2, 30.4 ± 0.3, and 35.0 ± 0.5% for the 2.0 mg/ml sample; 2.1 ± 0.1, 32.4 ± 0.1, 35.4 ± 0.2, and 33.2 ± 0.2% for the 1.0 mg/ml sample; and 2.5 ± 0.4, 29.0 ± 0.3, 28.8 ± 0.6, and 44.0 ± 0.7% for the 0.5 mg/ml sample.

Fig. 8 also shows a pronounced dependence of the sedimentation coefficient on the concentration of the protomeric species. The apparent values of the sedimentation coefficient s as a function of protein concentration for species A through D is illustrated in Fig. 9 from which s20,w0 values of 4.71 ± 0.06, 11.8 ± 0.3, 17.4 ± 0.3, and 25.2 ± 0.3 for the four species were obtained after correcting for the density and viscosity of the buffer. The data for the A species were obtained in the absence of calcium and for the B through D species in the presence of calcium. (The drop in sedimentation coefficients at lowest concentration points in Fig. 9 reflect the difficulty in accurately measuring s values of the most dilute solutions; in other experiments a small increase in s is seen at the lowest concentrations.)



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Fig. 9.   Apparent sedimentation coefficient s as a function of protein concentration for the A, B, C, and D species. Conditions are given in Fig. 8. The data for species A is in the absence of 10 mM CaCl2; the others contain 10 mM calcium.

The rather large variation in s with protein concentration (Fig. 9) implies that the protomeric species have elongated structures, more so than the parent A species. For spherical structures, the dependence of s on concentration is predicted to be small, <1%/mg/ml (42), contrary to what is observed. The dependence of s on concentration for the C and D species is rather pronounced, namely -7.0 ± 1.2 and -6.4 ± 0.8%/mg/ml for the two components, respectively (Fig. 9). The slopes of the lines for the A and B species are zero within experimental error. If we assume that the A species dimer has a molecular weight of 56,000 (twice the value from mass spectrometry) and a partial specific volume of 0.7089 cm3/g, we calculate a Stoke's radius of 3.05 ± 0.10 nm. For hydrations of 0.3 to 0.4 H2O/g protein, an axial ratio r <=  3 is predicted for either a prolate or oblate ellipsoid. We conclude that the A species is probably somewhat asymmetric but much less than the C and D protomers.

Attempts to separate the multimeric species, B through E, were made by incubating the purified EP protein in 10 mM Ca2+ buffer and then running the sample over a Sepharose 6B-CL size exclusion chromatography column using a 10 mM Ca2+ buffered mobile phase. Analysis on 5% native PAGE gels of the peaks produced by this separation showed that, although enrichment of a given multimer band could be observed, isolation of distinct multimers was never realized. This result may be due to the inability to fully separate the protomers on size exclusion, because of their elongated structures or because of heterogeneity in the associated species (vide supra).

Circular Dichroism-- CD spectra of EP fluid protein in the presence and absence of calcium demonstrate a significant change in secondary structure accompanying Ca2+ binding (Fig. 10). Analysis of secondary structure rearrangement is summarized in Table II and shows that Ca2+ binding causes an 18% increase in the estimated amount of alpha -helix with concurrent reduction in the beta -sheet. The increase in a negative absorption at 222 nm and positive adsorption at 190 nm are hallmark features of alpha -helix formation. Thus the protomeric forms (B through E) contain more alpha -helical structure than the parent ~4.7-s species (A species).



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Fig. 10.   CD spectra of a 0.65 mg/ml protein solution of 20 mm MOPS, pH 7.5, 0.1 M KCl in the absence (dotted line), and in the presence (solid line) of 10 mM Ca2+ after incubating the sample for more than 24 h. The 10 mM Ca2+ sample represents complete conversion to protomeric species B through E.


                              
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Table II
Secondary structure composition from CD spectral analysis



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study reinforces and extends previous work (22, 24, 25) in finding the EP fluid rich in macromolecular content (~4 mg/ml in both protein and carbohydrate). Inventory of the fluid proteins (Fig. 1) was consistent, showing a single, dominant glycoprotein (14.3% carbohydrate) in all preparations. This glycoprotein was purified to homogeneity on the basis of PAGE and a single N-terminal amino acid sequence. The native state of the EP glycoprotein in the absence of calcium is a dimer (A species) of the 28,340-Da monomers (Figs. 2-5) and has a pI of 4.43.

Sedimentation velocity analysis in the absence of added calcium shows that the EP protein species A associates to form a limited amount of B (Fig. 4). However, upon addition of calcium, the EP protein forms a finite number of larger protomers, B through E, which appear to have a discrete stoichiometry in their assembly (Figs. 6 and 7). Circular dichroic spectra (Fig. 10) of the EP protein taken in calcium-free and calcium-containing (10 mM, the physiological concentration) buffer solutions show that the protein undergoes a significant structural change upon calcium binding and that the protein has a much greater alpha -helical content with concurrent reduction in the amount of beta sheet (Table II).

Interestingly, although calcium binding is critical to significant protomer assembly, a fixed concentration of protein (0.6 mg/ml) in a 10, 100, or a 500 mM Ca2+ solution, respectively, produced the same species distribution profile as that seen in Fig. 7B, indicating that a 50-fold increase in calcium concentration does not affect the multimeric distribution pattern. This phenomenon implies that the formation of higher order multimers (peaks C, D, and E) is not the result of additional calcium-protein interaction but rather the result of protein-protein interactions (Fig. 7B). Similarly, the distribution of protomer species at a fixed Ca2+ concentration of 10 mM is relatively unaffected by the protein concentration (Fig. 8).

The ability to self-assemble is one of the most intriguing properties ascribed to the macromolecules of the organic matrix. Little is known about how newly secreted matrix materials assemble into a functioning, precisely arranged network, capable of directing the construction of mineral formations with exacting detail. The novel EP fluid glycoprotein described here may provide important clues regarding how the organic matrix is developed. Soluble EP protein secreted into the 10 mM calcium environment of the EP fluid take on a definitive secondary structure as a result of the calcium binding as demonstrated by the CD measurements (Fig. 10). This characteristic structural arrangement enables the Ca-protein complexes to form protein-protein associations allowing the protein to organize into large protomeric assembles (Fig. 7). Furthermore, the centrifugation data (Fig. 7) implies that Ca2+ binding is largely involved in the conversion of the A species to the B species. Taken together, these results suggest that the alteration in secondary structure that results in, or accompanies, the conversion from A to B also allows for B-B or A-B (protein-protein) interactions, resulting in the formation of the C, D, and E protomers. This hypothesis is supported by the fact that removal of Ca2+ by dialysis results in protomer conversion back to the A species. Much further work is needed to elucidate the structural properties of these very interesting and complex protein assemblies and their interaction with calcium. Such studies are currently underway.

Similarities between the properties of the EP protein described here and general characteristics of soluble matrix proteins from other systems are also noted. It is largely accepted that the soluble organic matrix (SOM) is comprised of proteins rich in aspartic acid and glutamic acid (18, 38-40) with acidic glycoproteins considered to be a prominent matrix component (17, 20, 43). Asx and Glx are the most abundant residues in the EP protein (Table I) and based on the N-terminal amino acid sequence data and the acidic isoelectric point of 4.43, both residues seem to be present in predominantly acidic form.

Analyses for phosphorylation and sulfation of the EP protein were both negative, indicating that the EP protein is not likely to be a homologue of the calcium-sequestering phosphoprotein particles found at the inner shell lamella of some bivalve species. This finding is in agreement with prior studies on this subject (44, 45).

A SOM primary protein sequence based on amino acid analysis which has received much attention is the repeat: DXDXDX (X = glycine or serine). This sequence is proposed to act as a template for CaCO3 crystal growth by epitaxy (46, 47). Although not an exact match, the N-terminal sequence of the EP fluid protein (see "Results") also shows a pattern of consistently spaced acidic residues. The fact that the two sequences are not present in sequence data banks but seem to occur in SOM proteins and now in the EP fluid protein strengthens the notion that the EP fluid protein ultimately functions as a matrix protein. This idea is consistent with the anatomical location of the protein, its relatively large abundance, and by its behavior in the presence of calcium. Work is currently underway in an attempt to identify the EP protein within the SOM; however, the cross-linked multicomponent nature the SOM makes it a difficult material to study.

The EP protein resembles, in some aspects, a recently reported blood glycoprotein from M. edulis (48) in that both are rich in histidine, have similar dimer molecular weights (~56 versus ~61 kDa), bind Ca2+, and are acidic proteins. Their amino acid compositions are also similar. However, the blood protein is composed of two types of subunits of molecular masses, ~29 and ~35-39 kDa, whereas the EP protein consists of only one type of 26-kDa subunit. The blood protein has been postulated to be a metal ion transporter involved in the accumulation of metals by the animal; it binds Cd2+ in addition to Ca2+ (48), as does the EP protein (vide supra).

In summary, the extrapallial fluid of M. edulis contains a structurally unique dimeric glycoprotein (A), which has been purified and partially characterized. This glycoprotein binds calcium to form a protomer (B), a process that is accompanied by a significant secondary structural rearrangement in the protein. This structural rearrangement appears to trigger a self association into higher order protomers (C, D, and E) that follows a stoichiometric pattern yet to be determined and is fully reversible upon the removal of calcium. Based on its characteristic features, inherent similarities with soluble organic matrix proteins and its anatomical location, this study suggests that the EP protein is a precursor or building block to the soluble organic matrix of the shell.


    ACKNOWLEDGEMENTS

We thank Dr. Paul Matsudaira for his assistance with the protein sequence determination, Drs. Robert MacColl and John Osterhout for their help with the circular dichroism measurements, John K. Grady and Kari L. Hartman for preparing some of the figures and helping with the centrifuge data analysis, and Dr. Robert Trimble for comments on the manuscript.


    FOOTNOTES

* This work was supported by Grant R37-GM20194 from the National Institute of General Medical Sciences (to N. D. C) and by Grants BIR-9314040 and DBI-9876582 from the National Science Foundation (to T. M. L.).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: Dept. of Chemistry, Parsons Hall, University of New Hampshire, Durham, NH 03824. Tel./Fax: 603-862-2520; E-mail: ndc@cisunix.unh.edu.

Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M006803200

2 Data bases of NCBI, Rutgers Protein Data Bank, Swiss-Prot, TrEMBL, Genpept, GenBankTM, and Kabat Sequences.

3 An apparent molecular weight of 37,300 ± 1,800 was obtained from SDS-PAGE using the protein standards: lysozyme (14,400), trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (45,000), and serum albumin (66,200). Because glycans do not bind SDS, erroneously high glycoprotein molecular weights are commonly observed by SDS-PAGE (41).


    ABBREVIATIONS

The abbreviations used are: EP, extrapallial; PAGE, polyacrylamide gel electrophoresis; pI, isoelectric point; g(s*), sedimentation coefficient distribution; s, apparent sedimentation coefficient; s20, w0 sedimentation coefficient corrected for density, viscosity, and solute concentration; CD, circular dichroism; SOM, soluble organic matrix; MOPS, (3-(N-morpholine)propanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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