A Novel Extrapallial Fluid Protein Controls the Morphology of Nacre Lamellae in the Pearl Oyster, Pinctada fucata

Mollusk shell nacre is known for its superior mechanical properties and precisely controlled biomineralization process. However, the question of how mollusks control the morphology of nacre lamellae remains unresolved. Here, a novel 38-kDa extrapallial fluid (EPF) protein, named amorphous calcium carbonate-binding protein (ACCBP), may partially answer this question. Although sequence analysis indicated ACCBP is a member of the acetylcholine-binding protein family, it is actively involved in the shell mineralization process. In vitro, ACCBP can inhibit the growth of calcite and induce the formation of amorphous calcium carbonate. When ACCBP functions were restrained in vivo, the nacre lamellae grew in a screw-dislocation pattern, and low crystallinity CaCO3 precipitated from the EPF. Crystal binding experiments further revealed that ACCBP could recognize different CaCO3 crystal phases and crystal faces. With this capacity, ACCBP could modify the morphology of nacre lamellae by inhibiting the growth of undesired aragonite crystal faces and meanwhile maintain the stability of CaCO3-supersaturated body fluid by ceasing the nucleation and growth of calcite. Furthermore, the crystal growth inhibition capacity of ACCBP was proved to be directly related to its acetylcholine-binding site. Our results suggest that a "safeguard mechanism" of undesired crystal growth is necessary for shell microstructure formation. Mollusk shell nacre is known for its superior mechanical properties and precisely controlled biomineralization process. However, the question of how mollusks control the morphology of nacre lamellae remains unresolved. Here, a novel 38-kDa extrapallial fluid (EPF) protein, named amorphous calcium carbonate-binding protein (ACCBP), may partially answer this question. Although sequence analysis indicated ACCBP is a member of the acetylcholine-binding protein family, it is actively involved in the shell mineralization process. In vitro, ACCBP can inhibit the growth of calcite and induce the formation of amorphous calcium carbonate. When ACCBP functions were restrained in vivo, the nacre lamellae grew in a screw-dislocation pattern, and low crystallinity CaCO3 precipitated from the EPF. Crystal binding experiments further revealed that ACCBP could recognize different CaCO3 crystal phases and crystal faces. With this capacity, ACCBP could modify the morphology of nacre lamellae by inhibiting the growth of undesired aragonite crystal faces and meanwhile maintain the stability of CaCO3-supersaturated body fluid by ceasing the nucleation and growth of calcite. Furthermore, the crystal growth inhibition capacity of ACCBP was proved to be directly related to its acetylcholine-binding site. Our results suggest that a "safeguard mechanism" of undesired crystal growth is necessary for shell microstructure formation. Many living things have the ability to convert inorganic ions into solid minerals through a process termed biomineralization (1Veis A. The Chemistry and Biology of Mineralized Connective Tissues. 1981; (, Elsevier/North-Holland, New York, Oxford)Google Scholar, 2Simkiss K. Wilbur K.M. Biomineralization: Cell Biology and Mineral Deposition. 1989; (, Academic Press, San Diego, London)Google Scholar). The biogenetic solid minerals, also known as biominerals, form external and internal hard tissues (e.g. otoliths, shells, and bones) that carry out diverse functions. Among these biomineralization products, the aragonite layer of molluscan shell (also called nacre or mother of pearl) is remarkable for its unique microstructure and superior mechanical properties (3Lowenstam H.A. Weiner S. On Biomineralization. 1989; (, Oxford University Press, New York, Oxford)Crossref Google Scholar, 4Weiner S. Gotliv B. Levi-Kalisman Y. Raz S. Weiss I.M. Addadi L. Biomineralization (BIOM2001): Formation, Diversity, Evolution and Application. 2003; (, Tokai University Press, Kanagawa)Google Scholar). Unlike other biogenetic mineral structures, nacre is not embedded in a tissue or layers of cells. Instead, a thin layer of fluid filling the space between the mantle epithelium and the shell, namely the extrapallial fluid (EPF), 4The abbreviations used are: EPF, extrapallial fluid; ACC, amorphous calcium carbonate; ACCBP, amorphous calcium carbonate-binding protein; α7 nAChRs, α7 nicotinic acetylcholine receptors; AChBP, acetylcholine-binding protein; ACh, acetylcholine; rACCBP, recombinant ACCBP; RA, rhodamine; α-Bgt, α-bungarotoxin; TFMS, trifluoromethanesulfonic acid; RACE, rapid amplification of cDNA ends; BSA, bovine serum albumin. is employed as the final medium of nacre calcification (2Simkiss K. Wilbur K.M. Biomineralization: Cell Biology and Mineral Deposition. 1989; (, Academic Press, San Diego, London)Google Scholar, 3Lowenstam H.A. Weiner S. On Biomineralization. 1989; (, Oxford University Press, New York, Oxford)Crossref Google Scholar, 4Weiner S. Gotliv B. Levi-Kalisman Y. Raz S. Weiss I.M. Addadi L. Biomineralization (BIOM2001): Formation, Diversity, Evolution and Application. 2003; (, Tokai University Press, Kanagawa)Google Scholar). In this aqueous microenvironment, inorganic ions, including Na+, K+, Ca2+, Mg2+, Cl-, SO42−, CO32−, and HCO3−, are transported from the external environment and achieve an appropriate supersaturation of CaCO3 necessary for shell crystal growth (5Crenshaw M.A. Biol. Bull. 1972; 143: 506-512Crossref PubMed Google Scholar, 6Pietrzak J.E. Bates J.M. Scott R.M. Hydrobiologia. 1976; 50: 89-93Crossref Scopus (11) Google Scholar). Meanwhile, various organic macromolecules, including proteins, polysaccharides, and lipids, are secreted by mantle cells or transported from elsewhere to the EPF (7Misogianes M.J. Chasteen N.D. Anal. Biochem. 1979; 100: 324-334Crossref PubMed Scopus (48) Google Scholar, 8Marsh M.E. Sass R.L. J. Exp. Zool. 1983; 226: 193-203Crossref Scopus (42) Google Scholar, 9Wilbur K.M. Bernhardt A.M. Biol. Bull. 1984; 166: 251-259Crossref Google Scholar). These macromolecules are thought to also be responsible for transporting the inorganic ions and stabilizing this CaCO3-supersaturated fluid (10Nair P.S. Robinson W.E. Biol. Bull. 1998; 195: 43-51Crossref PubMed Scopus (24) Google Scholar, 11Hattan S.J. Laue T.M. Chasteen N.D. J. Biol. Chem. 2001; 276: 4461-4468Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The shell matrix proteins (the proteins found in the shell) are also in the EPF and self- or co-assemble to form the crystal deposition framework and initiate crystallization (4Weiner S. Gotliv B. Levi-Kalisman Y. Raz S. Weiss I.M. Addadi L. Biomineralization (BIOM2001): Formation, Diversity, Evolution and Application. 2003; (, Tokai University Press, Kanagawa)Google Scholar, 12Sudo S. Fujikawa T. Nagakura T. Ohkubo T. Sakaguchi K. Tanaka M. Nakashima K. Takahashi T. Nature. 1997; 387: 563-564Crossref PubMed Scopus (345) Google Scholar, 13Marin F. Amons R. Guichard N. Stigter M. Hecker A. Luquet G. Layrolle P. Alcaraz G. Riondet C. Westbroek P. J. Biol. Chem. 2005; 280: 33895-33908Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Because all of these complex processes occur in an extracelluar liquid, nacre mineralization provides a useful model of formation of a material with intricate nano-architectures in a mild condition. For a long time, it was believed that the matrix proteins played a dominant role in directing the highly organized nanocomposites of CaCO3 crystals to form the layered structure of nacre (2Simkiss K. Wilbur K.M. Biomineralization: Cell Biology and Mineral Deposition. 1989; (, Academic Press, San Diego, London)Google Scholar, 14Marin F. Luquet G. Comptes Rendus Palevol. 2004; 3: 469-492Crossref Scopus (276) Google Scholar, 15Zhang C. Zhang R. Mar. Biotechnol. (NY). 2006; 8: 572-586Crossref PubMed Scopus (138) Google Scholar). However, although matrix proteins extracted from nacre can induce the formation of aragonite in vitro like that in vivo (16Falini G. Albeck S. Weiner S. Addadi L. Science. 1996; 271: 67-69Crossref Scopus (1290) Google Scholar, 17Belcher A.M. Wu X.H. Christensen R.J. Hansma P.K. Stucky G.D. Morse D.E. Nature. 1996; 381: 56-58Crossref Scopus (1067) Google Scholar), the exceedingly orderly morphology of nacre lamellae cannot be reproduced in vitro. In fact, previous research revealed that once nucleation took place, the growth of the nacre lamellae would start, even in an inorganic environment (18Giles R. Manne S. Mann S. Morse D.E. Stucky G.D. Hansma P.K. Biol. Bull. 1995; 188: 8-15Crossref PubMed Google Scholar). In the absence of the macromolecules of the EPF, nacreous lamellae grow by following the pattern of inorganic aragonite instead of forming the layered structure of nacre. In this case, it is reasonable to predict that besides keeping the stability of the CaCO3-supersaturated microenvironment, some macromolecules of the EPF are also directly involved in modifying the morphology of shell crystals. However, because of the difficulties in protein purification and in vivo functional identification, although some calcium-binding proteins were found in the EPF (11Hattan S.J. Laue T.M. Chasteen N.D. J. Biol. Chem. 2001; 276: 4461-4468Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19Yin Y. Huang J. Paine M.L. Reinhold V.N. Chasteen N.D. Biochemistry. 2005; 44: 10720-10731Crossref PubMed Scopus (77) Google Scholar), how the macromolecules maintain the stability of the EPF and affect nacre lamellae growth was unknown. In the present study, a protein was extracted from the EPF of Pinctada fucata through a new method. A series of in vitro and in vivo experiments showed that this protein can inhibit undesired crystal growth and play a key role in stabilizing the CaCO3-supersaturated body fluid and forming the exceedingly orderly microstructure of nacre. Animals—The oyster, P. fucata, was purchased from Guofa Pearl Farm, Beihai, Guangxi Province, China. Animals were maintained in glass aquariums filled with aerated artificial seawater (Sude Instant Sea Salt, 3% at 20 °C) for 3 days prior to experimentation. Extrapallial Fluid Extraction and Protein Purification—The extrapallial fluid (EPF) was extracted by inserting a 0.8 × 30-mm needle into the central extrapallial space and sucking the fluid gently into a sterile syringe. The needle tip was carefully kept in contact with the inner shell surface to avoid contamination by extraneous water or other body fluids. Samples collected (∼50-100 μl per oyster) were immediately transferred to 15 ml of sterile centrifuge tubes and held on ice. After extraction, fluids were centrifuged for 20 min at 1500 × g and 4 °C. Amorphous calcium carbonate (ACC) was synthesized according to the method of Koga et al. (20Koga N. Nakagoe Y.Z. Tanaka H. Thermochimica Acta. 1998; 318: 239-244Crossref Scopus (190) Google Scholar) with some modification. Equimolar sodium carbonate (0.1 m) and calcium chloride (0.1 m) solutions were mixed with vigorous stirring at 0-4 °C. Sodium hydroxide (1 m) was added to obtain mixed solutions within pH 11.2-11.4. Then the precipitated colloidal phase was centrifuged for 10 min at 2000 × g and 4 °C to separate the ACC. To verify that only pure ACC existed, the precipitate was washed with acetone, dried in a vacuum desiccator for 1 day, and identified via x-ray diffraction. The separated ACC was incubated with EPF at a ratio of 1:10 (w/v) for 3 h at 4 °C. Then the mixture was centrifuged for 15 min at 1500 × g and 4 °C, and washed with a large volume of 10 mm HEPES (pH 7.5) and 0.5 m NaCl. After centrifugation, 0.5 m EDTA (pH 8.0) was added to dissolve the ACC. The supernatant was dialyzed exhaustively against 10 mm Tris-HCl buffer (pH 7.5) and concentrated by ultrafiltration with a Millipore Amicon Ultra-4 (molecular weight cutoff 10 kDa). For further purification, the concentrated supernatant was injected onto an anion exchange column (Protein Pak™ DEAE 8HR AP-1 Mini-Column, Φ5 × 100 mm) and a reversed-phase column (GRACEVYDAC 214TPC4 Column, Φ4.6 × 250 mm) connected to a HPLC system (Waters). N-terminal Sequencing of ACCBP—ACCBP was electrotransferred from the SDS-PAGE gel to a polyvinylidene difluoride membrane, and its N terminus was sequenced via Edman degradation (Applied Biosystem, Procise 491). Identification of the cDNA Sequence of ACCBP—Total RNA was extracted from the pearl oyster P. fucata with a RNAzol RNA Isolation Kit (Biotecx, Houston, TX) and 5′-RACE and 3′-RACE were performed with a SMART RACE cDNA Amplification kit (Clontech) and Advantage 2 cDNA Polymerase Mix (Clontech) according to the manufacturer's instructions. A degenerate sense primer (5′-AAR TGY GAY TAY CCN GAR GC-3′) was designed according to residues 1-7 of ACCBP (KCDYPEA) and used for 3′-RACE in combination with the 3′-RACE primer UPM (Clontech). According to the cDNA sequence acquired from the 3′-RACE, a gene-specific antisense primer (5′-CTT GCC TTT TGA ATG GGG AC-3′) was synthesized and used for 5′-RACE in combination with the 5′-RACE primer UPM (Clontech). Gene Expression Analysis by RT-PCR—Total RNA was isolated from the mantle, viscus, adductor muscle, gill, and hemocytes of adult individuals of P. fucata as described above. An equal quantity (2 μg) of RNA from different tissues was reverse-transcribed into cDNA with Superscript III reverse transcriptase (Invitrogen). GAPDH was used as a positive control for cDNA preparation and amplified with the primer pair of gapdhF (5′-GCC GAG TAT GTG GTA GAA TC-3′) and gap-dhR (5′-CAC TGT TTT CTG GGT AGC TG-3′). ACCBP was amplified with the primer pair of accbpF (5′-AAA TTC CTG CTG GAT GAC TAT G-3′) and accbpR (5′-AAC GGT CCA CCA TCT TTG TTC C-3′). A negative control was carried out in the absence of cDNA template to examine the cross contamination of the samples. PCR consisted of 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 30 s, and 72 °C for 10 min. The PCR products were subcloned and verified by sequencing. Preparation of Wild Type rACCBP—The gene coding sequence of ACCBP was subcloned into the expression vector pPIC9K (without the signal sequence and with a His tag at the C terminus) and overexpressed in yeast, Pichia pastoris GS115 according to the Invitrogen manual. After 2 days of induction, the medium was collected, concentrated, and dialyzed against 10 mm Tris-HCl buffer, pH 7.5, 200 mm NaCl, and 20 mm imidazole. The protein was purified with Ni-NTA agarose following the Qiagen manual and then dialyzed against 5 mm Tris-HCl buffer, pH 7.5. The yield was about 3 mg/liter. Tissue Distribution Analysis by Western Blotting—Polyclonal antibody was prepared by immunizing rabbits with rACCBP and purified with HiTrap Protein A HP by following the Amersham Biosciences manual. Tissues, i.e. mantle, viscus, adductor muscle, and gill, were homogenized and the total soluble proteins were extracted with 50 mm Tris-HCl, 5 mm EDTA, 150 mm NaCl, 0.5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 1% Nonidet P-40. Hemolymph and extrapallial fluid were extracted directly from the pearl oyster P. fucata. Shell powder was treated with 0.5 m EDTA for 3 days and the EDTA soluble and insoluble shell matrix was separated by centrifugation. Protein concentrations were measured with the BCA protein assay kit (Pierce). Protein samples with an equal quantity (50 μg) were run on a 12% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked overnight and treated with a 1:2000 dilution of antibody against ACCBP. It was then washed exhaustively four times, treated with a 1:500 dilution of alkaline phosphatase-labeled goat anti-rabbit antibody, washed exhaustively four times, and finally visualized with NBT/BCIP. Chemical Deglycosylation—ACCBP and rACCBP were lyophilized overnight. Deglycosylation was performed on the freeze-dried sample with a GlycoProfile IV chemical deglycosylation kit (Sigma) according to the manufacturer's instructions. The samples were then extensively dialyzed against 20 mm Tris-HCl buffer, pH 7.5, in dialysis tubing (GeBA) with a molecular size cutoff of 3,500 Da. Western Blotting with Concanavalin A Probe—ACCBP and rACCBP were run on a 12% gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked overnight and treated with 10 μg/ml Biotin-Concanavalin A (Sigma). It was then washed exhaustively four times, treated with a 1:100 dilution of avidin-HRP, washed exhaustively four times, and finally visualized with an enhanced DAB kit. Protein Labeling with Rhodamine-α-bungarotoxin (RA-α-Bgt)—ACCBP or rACCBP was mixed with an equimolar amount of RA-α-Bgt (Sigma) in 10 mm Tris-HCl buffer, pH 7.5. For rACCBP, the mixture was held at 4 °C for more than 1 h. For ACCBP, the mixture was held at 4 °C for over 24 h. Protein Labeling with Rhodamine—1 mg of rACCBP was dialyzed against 0.1 m sodium bicarbonate (pH 8.3) and concentrated to 10 mg/ml. 0.5 mg of 5-TAMRA, S.E. (Promega) was dissolved in 0.05 ml of Me2SO immediately before the reaction. While the protein solution was being stirred, 0.01 ml of the reactive dye solution was added slowly. Then the reaction was incubated for 1 h at room temperature with continuous stirring, and the reaction was stopped by adding 0.01 ml of freshly prepared 1.5 m hydroxylamine, pH 8.5. Finally, dialysis against a large volume of 10 mm HEPES (pH 7.5) was used to separate the conjugate from the unreacted labeling reagent. 50 μg of ACCBP was labeled as above. The dosage of other reagents was reduced accordingly. Injection of Antibody against ACCBP into the Extrapallial Space—Oysters with shells of about 4-6 cm in diameter and 30-40 g in wet weight were held in a tank for 7 days prior to use. The purified antibody against ACCBP (anti-ACCBP) was injected into the center of the extrapallial space through the mantle with a microsyringe at dosages of 0.5 μg and 1.5 μg per gram of wet weight per day. Each group contained 5 specimens. The same dosage of preimmune rabbit serum was used as a control. The oysters were sacrificed after 5 days, and the shells were separated, washed with Milli-Q water, and immersed in 5% NaOH for 24 h to remove the organic components attached to the surfaces. Then the shells were extensively washed with Milli-Q water, air-dried, sealed, and stored at -20 °C until being used. In Vitro Crystal Growth Experiments—Saturated Ca(HCO3)2 solution was prepared by following the method of Xu et al. (21Xu G.F. Yao N. Aksay I.A. Groves J.T. J. Am. Chem. Soc. 1998; 120: 11977-11985Crossref Scopus (302) Google Scholar) with some modification. CO2 gas was bubbled into the mixture of CaCO3 and Milli-Q deionized water for 4 h. Excess solid CaCO3 was removed via filtration, and the filtrate was aerated with CO2 for another hour. The calcium concentration of the solution was about 8.5 mm, as determined by EDTA titration, and the pH 6.2. The experiments were carried out in 96-well COSMO multidishes with cleaned glass slides or silicon wafers on the bottom of each well. Saturated Ca(HCO3)2 solution and protein solution were mixed in each well to a final volume of 100 μl with a calcium concentration of 8 mm. 100 μl of Milli-Q water was introduced to the wells next to the samples to trap the CO2 that diffused from the bicarbonate solution. Water or the same concentration of BSA was chosen as negative controls. The multidishes were placed in a sealed system at room temperature. After 48 h, the glass slides or silicon wafers were lightly rinsed with Milli-Q water, dried, and observed by S.E. and Raman spectrum. For the FTIR spectrum, the precipitate of each well was collected and dried under vacuum. For the crystal growth experiment performed with the mixture of ACCBP and α-Bgt (molar ratio: 1:2), the proteins were incubated 24 h in advance. The mixture of BSA and ACCBP at the same concentration was used as a control. Crystal Growth Inhibition Experiments—The saturated Ca(HCO3)2 solution was prepared as above. The solution, about 200 μl per well, was introduced into a 96-well COSMO multidish with glass slides on the bottom, and 100 μl of Milli-Q water was introduced into the wells next to the samples to trap CO2. After 30 min, the wells were inspected under an optical microscope (Leica DMIRB). When rhombohedral crystal seeds became visible, pictures were taken, and the relative positions were marked. Then 5 μl of protein solution was added to give a final concentration of 0.2 μm, and the multidish was placed into a sealed system at room temperature. Pictures of the crystals were taken at the marked place after 24 h. Crystal Binding Experiments—The CaCO3 crystals were synthesized as in the crystal growth inhibition experiments, except that the multidish was placed in a sealed system at 40 °C to induce the formation of both calcite and aragonite. 24 h later, the glass slides were lightly rinsed with Milli-Q water and air-dried. The crystal polymorphs were determined by Renishaw Raman Spectroscopy. The labeled proteins were diluted with CaCO3-saturated Tris-HCl buffer (10 mm pH 7.5, prepared by mixing 1 g of reagent grade calcite with 100 ml of Tris-HCl buffer and stirring for 24 h) to a final concentration of 0.1 μm and incubated with the glass slides for 1 h at room temperature. Then the glass slides were rinsed with CaCO3-saturated Tris-HCl buffer and Milli-Q water and air-dried. Slides were observed under a fluorescence microscope (Leica DMR). Images were collected with a Leica CCD 300F camera. The exposure time for the fluorescence images was set to 6.5 s. To confirm the binding patterns of different proteins, the crystal binding experiments were repeated at least three times for each kind of protein. When the interaction of shell nacre and rhodamine-labeled ACCBP (RA-ACCBP) was tested, the inner surface of the shell nacre was mechanically cleaved into small pieces (approx. 8 × 8 × 1 mm). In the group of normal nacre, the nacre pieces were incubated in the RA-ACCBP solution together with some synthesized calcite (Sigma). The incubation lasted until fluorescence appeared on all the synthesized calcite. Before observing the inner surface of the nacre under the fluorescence microscope, some of the calcite crystals were carefully dropped on it as positive controls. The nacre pieces from all the specimens of each group were tested. The total examined surface area of normal nacre (and other groups) is more than 6 cm2. The exposure time for these fluorescence images was set to 600 ms. Raman Spectroscopy—The glass slides or silicon wafers were first observed under reflected white light with a Leica microscope at a magnification of ×500. After focusing on a single crystal, the light source of the microscope was transferred to 514-nm diode laser. The spectra were scanned 3 × 20 s in the range of 100-1200 cm-1 with a Renishaw Raman imaging microscope. Fourier Transform Infrared (FTIR) Spectroscopy Analysis—Precipitate was powdered and mixed with anhydrous KBr. The mixture was pressed into a 13-mm diameter pellet. FTIR spectra were obtained with a Nicolet560 Fourier transform infrared spectrometer. Scanning Electron Microscopy—The silicon wafers or incised shells were sputter-coated with gold and analyzed with a FEI Sirion2000 scanning electron microscope, which was operated at 10 kV and equipped with a Kevex energy dispersive x-ray spectrometer for element analysis of crystals. Extraction and Characterization of ACCBP—The stability of the EPF is demonstrated by its CaCO3 crystalline inhibition ability. It has been shown that the organic components of the EPF, even at high dilution, can effectively decrease the rate of CaCO3 crystallization (9Wilbur K.M. Bernhardt A.M. Biol. Bull. 1984; 166: 251-259Crossref Google Scholar). During our research on EPF, we found that EPF can retard further crystallization of the synthesized amorphous calcium carbonate (ACC), which is the most unstable form of CaCO3 (22Ogino T. Suzuki T. Sawada K. Geochim. Cosmochim. Acta. 1987; 51: 2757-2767Crossref Scopus (482) Google Scholar, 23Clarkson J.R. Price T.J. Adams C.J. J. Chem. Soc. Faraday Trans. 1992; 88: 243-249Crossref Google Scholar). To investigate the abnormal stability of ACC, we separated ACC from the EPF by centrifugation and decalcified the ACC with EDTA. SDS-PAGE analysis of the EDTA-soluble component revealed an obvious ∼38-kDa protein band that could not be observed in the SDS-PAGE of the EPF (Fig. 1). This specifically enriched protein was named ACCBP (amorphous calcium carbonate-binding protein). When the HPLC-purified ACCBP was run on a SDS-PAGE gel under unreduced conditions, a band with an apparent molecular mass of ∼76 kDa could be observed (Fig. 1). This result suggested that under physiological conditions ACCBP might exist as a homodimer. Partial N-terminal amino acid sequence of ACCBP (KCDYPEAKLLKFLLDDYEKLVRPVP) was obtained by Western blotting and Edman degradation. On the basis of the N-terminal sequence, 3′-RACE and 5′-RACE were performed to reveal the cDNA sequence of ACCBP (GenBank™ accession no. DQ473430). The 930-base pair cDNA sequence encoded a single protein of 240 amino acids and a typical transcription termination signal (AATAAA), which confirmed the integrity of the cDNA sequence (Fig. 2). The mature protein starts with Lys at position 25 and has a predicted molecular weight of 24,389. The sequence analysis indicated that ACCBP is an acidic protein with a theoretical pI of 4.56 and is rich in acidic residues (Asp + Glu nearly 16%). However, compared with many matrix proteins purified from the shell matrix of bivalves, such as caspartin (13Marin F. Amons R. Guichard N. Stigter M. Hecker A. Luquet G. Layrolle P. Alcaraz G. Riondet C. Westbroek P. J. Biol. Chem. 2005; 280: 33895-33908Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), ACCBP is not a typical Asx-rich protein, and the mol percentage of positively charged residues is high (Arg + His + Lys nearly 11%). In fact, except for the most abundant residues (Asp and Leu, nearly 11%), the amino acid composition of ACCBP is not significantly biased to any residue. The BLAST search against GenBank™ revealed that ACCBP has 30% sequence identity with the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptors (nAChR) of Drosophila melanogaster (Fig. 2). ACCBP has the residues which are conserved in nAChRs, especially those considered to be involved in acetylcholine (ACh)-binding, as well as the characteristically conserved cysteine loop region between Cys133 and Cys145 and the typical disulfide bond between adjacent cysteine residues Cys195 and Cys196 (24Corringer P.J. Le Novere N. Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (708) Google Scholar). There have been reports about the soluble analogues of the extracellular domain of the α7 nAChR (25Smit A.B. Syed N.I. Schaap D. van Minnen J. Klumperman J. Kits K.S. Lodder H. van der Schors R.C. van Elk R. Sorgedrager B. Brejc K. Sixma T.K. Geraerts W.P. Nature. 2001; 411: 261-268Crossref PubMed Scopus (467) Google Scholar, 26Hansen S.B. Talley T.T. Radic Z. Taylor P. J. Biol. Chem. 2004; 279: 24197-24202Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 27Celie P.H. Klaassen R.V. van Rossum-Fikkert S.E. van Elk R. van Nierop P. Smit A.B. Sixma T.K. J. Biol. Chem. 2005; 280: 26457-26466Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), such as the ACh-binding protein (AChBP) isolated from the glia cells of mollusk Lymnaea stagnalis (25Smit A.B. Syed N.I. Schaap D. van Minnen J. Klumperman J. Kits K.S. Lodder H. van der Schors R.C. van Elk R. Sorgedrager B. Brejc K. Sixma T.K. Geraerts W.P. Nature. 2001; 411: 261-268Crossref PubMed Scopus (467) Google Scholar). AChBP is believed to actively modulate neuronal synaptic transmission. Unlike the α7 nAChR and AChBP, ACCBP can form a homodimer via an intermolecular disulfide bond between Cys2 residues (Figs. 1 and 2) and has two potential glycosylation sites at Asn29 and Asn184. Western blotting of ACCBP in different tissues reveals that ACCBP mainly exists in the hemolymph and extrapallial fluid, and some is detected in the adductor muscle (Fig. 3A). Although RT-PCR analysis of ACCBP gene expression demonstrates that ACCBP is expressed in the mantle, the adductor muscle, and the viscus (Fig. 4), no positive signal was detected in the mantle and the viscus by Western blotting of soluble extracts of the tissue (Fig. 3A). However, ACCBP was weakly detected on Western blots of extracts of mantle and viscus, which were enriched for ACCBP with ACC (data not shown). This phenomenon might be explained in this way: immediately after ACCBP is synthesized in these tissues, it is secreted into the body fluid; this makes its content in these tissues too low to be detected on Western blots with equal quantities of total extract proteins.FIGURE 4RT-PCR analysis of ACCBP in different tissues. RT-PCR was performed on RNA prepared from viscus, mantle, gill, adductor muscle, and hemocytes. GAPDH was used as the positive control. Specific primer sets for ACCBP and GAPDH are presented under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Effect of ACCBP on CaCO3 Crystallization in Vitro and Its Interaction with Calcite—To examine whether ACCBP is responsible for the enhanced stability of ACC in the EPF, in vitro crystal growth experiments were performed. We found that just 0.2 μm ACCBP can totally inhibit the formation of calcite. The infrared spectra of all precipitates show broad v2 and v1 absorption bands of ACC at 864 and 1070 cm-1, respectively, and the split of the v3 band around 1450 cm-1, which indicates that